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Design and Characterisation of Niosomes
for Ocular Delivery of Naltrexone
Hydrochloride
Hamdy Abdelkader Mohamed Abdelkader
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy,
School of Pharmacy,
The University of Auckland
2011
Abstract
Abstract
Background: Recent reports have demonstrated that topical ocular administration of naltrexone
(an opioid growth factor antagonist) is able to reverse the diabetic complications on the cornea
(diabetic keratopathy). The topical administration of naltrexone accelerates corneal wound
healing, restores corneal sensitivity and enhances corneal epithelialisation in diabetic rats, rabbits
and humans. Naltrexone can be considered as a new therapeutic agent for treatment of diabetic
keratopathy. To our best knowledge, an ophthalmic formulation has not yet been developed for
naltrexone hydrochloride (NTX) nor has it been properly formulated in a liquid dosage form.
Niosomes are non-ionic surfactant vesicles and have the same closed bilayer structures and
properties of the well-known phospholipid vesicles, liposomes. Niosomes are considered more
chemically stable and more economically viable alternatives to liposomes. At the same time, they
could offer the same advantages as vesicular ocular delivery systems such as prolonged
precorneal residence time and enhanced corneal penetration. Objective: The aim of this work is to
design and characterise niosomal formulations for ocular delivery of NTX having the
convenience of being delivered as eye drops. Methods: An HPLC method was developed and
validated for NTX; preformulation studies were performed to study the physicochemical
properties of NTX, such as aqueous solubility, lipid solubility, melting behaviour, spectrometric
identification and construction of the Arrhenius plots for predicting NTX stability. Two classes of
non-ionic surfactants, sorbitan fatty acid esters (Span®) and polyoxyethylene alkyl ethers (Brij®),
were selected and investigated for their ability to form niosomes and encapsulate NTX using
different levels of cholesterol (a bilayer membrane stabiliser). Also, different membrane additives
[dicetyl phosphate (DCP), Solulan C24 (C24) and sodium cholate (CH)] were studied to modify
the physical properties of niosomes, such as their percentage entrapment efficiency (EE %), size,
morphology, rheology and spreading ability characteristics. Four different niosomes; F-S60, FDCP, F-C24 and F-CH were prepared and evaluated for ocular irritation, using the hen’s egg
chorioallantoic membranes (HET-CAM) test, bovine eye test and histological corneal
examination. Finally, they were studied for in vitro NTX release; transcorneal permeation of
NTX; and physical stability. Results and discussion: The preformulation studies showed that
NTX is a hydrophilic drug with a log P value of 1.61 at 35oC. The main degradation pathway of
NTX in aqueous solutions was found to be autoxidation. Span 60-based niosomes, consisting of
Span 60: cholesterol 7:3 mol/mol (F-S60), demonstrated superior EE % for NTX. Cryogenic
scanning electron microscope (Cryo-SEM) images of niosomes showed onion-like structures,
indicating multilamellarity. Incorporation of the membrane additives into bilayer membranes
produced both spherical niosomes and discomes (giant disc-like niosomes), as confirmed by
confocal laser scanning microscope (CLSM). The vesicular incorporation of NTX imparted a
protective effect against light-induced oxidation, and the prepared niosomes demonstrated
minimal to no irritation potential; significantly controlled NTX release; enhanced transcorneal
permeation of NTX through excised cow corneas compared with the aqueous NTX solution; and
were physically stable at least for three months at 4oC. Conclusion: The developed niosomes
were able to protect NTX chemically, impart a penetration-enhancing effect and control NTX
corneal permeation through bovine corneas, and demonstrate good ocular tolerability, suggesting
that they are potential ocular delivery systems for NTX.
i
Dedication
Dedicated to
My parents, brother Mohamed Abdelkader,
My wife Zeinab, my son Omar, my daughter Malak
and the 1000 young people who gave up their lives for Egypt’s freedom in 25th
January revolution
ii
Acknowledgment
Acknowledgement
It is with a great moment of pride and relief that I come to the end of a challenging and exciting
project of my PhD Research. The completion of this thesis could not have been possible without
the help, support, encouragement and input of many people.
Foremost, I would like to express my gratitude to the main supervisor Dr Raid Alany for his
thorough guidance, mentorship, editing my manuscripts and financial support of the third year. I
would also like to cordially congratulate him on the post of professor at Kingston University,
London, UK.
I also greatly appreciate the expertise and guidance provided by my co-supervisors Dr Zimei Wu
and Dr Raida Al-Kassas for their keen interest, valuable input and encouragement.
I would like to thank my advisors Prof. Sayed Ismail (Department of Pharmaceutics, Faculty of
Pharmacy, Assiut University, Assiut, Egypt) and Dr Amal Kamal (Department of Pharmaceutics,
Faculty of Pharmacy, Minia University, Minia, Egypt) for their help with scholarship application
to the Egyptian government.
My sincere thanks go to Mr Ahmed Abu Moussa and Dr Moustafa Ibrahim at the Egyptian
Embassy in Canberra for connecting me to the Culture and Mission Bureau, Ministry of Higher
Education, Cairo, Egypt.
I would like to pay my immense gratitude to Professor John Show and the School of Pharmacy
for providing me the travel grant for international conference which was an important part of
my PhD process and the professional career.
A big thank you goes to Mrs Satya Amirapu from Histology Lab. for preparing histological
sections of the bovine corneas for my research project. Another big thank you also goes to Hilary
Holloway (the Biological Imaging Research Unit), who helped me to get niosome images using
confocal laser scanning microscopy. A big thank you also goes to Associate Professor Brian
Palmer (Auckland Cancer Society Research Centre) for help interpret H1-NMR and mass spectra
of naltrexone hydrochloride.
I would like to acknowledge the Association for Research in Vision and Ophthalmology as the
copyright holder to grant me a permission to reprint the figure “the Architecture of human
iii
Acknowledgment
corneal nerves” in my PhD thesis. I also would like thank the head of the English Language SelfAccess Centre Dr Penny Hacker for her great help and assistance and Mr. Ben Jackson who
helped with proofreading.
I have been fortunate to have some of my dearest colleagues during my studies. Thank you guys
Alvin, Darren, Akhlaq, Thilini, Sara and James.
Finally, I would like to take this opportunity to acknowledge my family who have always been
supportive and have never given up on me.
iv
Publications
Publications to date arising from this thesis
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. ( 2 0 1 0 ) . Preparation of niosomes
as an ocular delivery s ystem for naltrexone hydrochloride: Physicochemical
characterization. Die Pharmazie, 65, 811-817.
• Abdelkader, H., Patel, D., McGhee, C., & Alany, R. (2011). New therapeuti c
approaches
in
treatment
of
diabetic
keratopathy. Clinical and Experimental
Ophthalomology, 39, 259-270.
• Abdelkader, H., Wu, Z., Al-Kassas, R., Brown, J. E., & Alany, R. (2011). Preformulation
characteristics of the opioid growth factor antagonist- naltrexone hydrochloride: stability and
lipophilicity studies. Journal of Drug Delivery Science and Technology, 21, 157-163.
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. ( 2 0 1 1 ) . Design and evaluation of
niosomes and discomes for controlled release ocular delivery of naltrexone
h ydrochloride. Journal of Pharmaceutical sciences, 100, 1833-1846.
Conference papers to date arising from this thesis
• Abdelkader, H., McGhee, C., & Alany, R. (2009). Naltrexone from alcoholism to corneal
wound healing: a formulation approach. Paper presented at Australasian Pharmaceutical
Science Association (APSA) conference, Hobart, Australia. (Poster).
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Naltrexone hydrochloride a new
ophthalmic pharmaceutical: physichochemical characterisation. Paper presented at the 12th
Conference on Formulation and Delivery of Bioactives, the University of Otago, Dunedin, New
Zealand (Podium).
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Development of a validated
HPLC-method for naltrexone hydrochloride-encapsulated in niosomal formulations. Paper
presented at the 12th conference on formulation and delivery of bioactives, The University of
Otago, Dunedin, New Zealand. (Poster).
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Effect of selected ionic and nonionic membrane additives on the entrapment efficiency of naltrexone hydrochloride in
niosomes. (2010). Paper presented at the 37th Annual Meeting and Exposition of the controlled
release Society (CRS), Portland, USA. (Poster).
v
Publications
• Abdelkader, H., Kamal, A., Ismail, S., & Alany, R. (2010). Characterization of ocular
naltrexone niosomes using differential scanning calorimetry (DSC) and cryo-scanning electron
microscopy (Cryo-SEM). Paper presented at the 37th Annual Meeting and Exposition of the
CRS CRS, Portland, USA. (Poster).
• Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2010). Evaluation of niosomes and
discomes for ocular delivery of an opioid growth factor antagonist. Paper presented at
Australasian Pharmaceutical Science Association (APSA) conference, Brisbane, Australia.
(Poster).
•
Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). In vitro release, transcorneal
permeation and stability studies of an opioid growth factor antagonist (naltrexoneHCl) niosomes
and discomes. Paper presented at the 38th Annual Meeting & Exposition of the CRS, National
Harbor, Maryland, USA. (Poster)
•
Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). Bovine corneal opacity,
permeability (BCOP) and histological examination for evaluating ocular irritation and toxicity of
non-ionic surfactant vesicles (niosomes). Paper presented at the 38th Annual Meeting &
Exposition of the CRS, National Harbor, Maryland, USA. (Poster).
•
Abdelkader, H., Wu, Z., Al-Kassas, R., & Alany, R. (2011). Niosomes as a potential ocular
delivery system for the opioid growth factor antagonist-naltrexone hydrochloride: influence of
vesicular encapsulation on photoxidation. Paper presented at the 3rd PharmSciFair, Prague,
Czech Republic. (Poster).
vi
Content
Content
Abstract ............................................................................................................................................. i
Acknowledgement .......................................................................................................................... iii
Publications to date arising from this thesis .................................................................................... v
Conference papers to date arising from this thesis .......................................................................... v
Content .......................................................................................................................................... vii
List of Figures ................................................................................................................................ xii
List of Tables ............................................................................................................................... xvii
List of Abbreviations .................................................................................................................... xix
1.
General introduction .............................................................................................................. 1
1.1.
Ocular bioavailability from ophthalmic dosage forms ..................................................... 1
1.1.1.
Anatomical and physiological considerations ........................................................... 2
1.1.2.
Pharmacokinetic considerations ................................................................................ 8
1.1.3.
Drug and formulations factors ................................................................................. 10
1.2.
Drug delivery systems providing controlled and continuous ocular delivery ................ 14
1.2.1.
Ophthalmic inserts ................................................................................................... 15
1.2.2.
Collagen shields ....................................................................................................... 16
1.2.3.
Polymeric gels ......................................................................................................... 17
1.2.4.
Colloidal delivery systems ...................................................................................... 20
1.3.
Diabetic keratopathy and the available treatment options .............................................. 28
1.3.1.
Physiology of the cornea ......................................................................................... 28
1.3.2.
Diabetes mellitus and the cornea ............................................................................. 28
1.3.3.
Corneal innervations ................................................................................................ 29
1.3.4.
Mechanisms of corneal maintenance and healing ................................................... 33
1.3.5.
Treatment of diabetic keratopathy ........................................................................... 34
1.4.
Thesis aims and structure ................................................................................................ 45
vii
Content
2.
Preformulation studies of naltrexone hydrochloride ........................................................ 47
2.1.
Introduction ..................................................................................................................... 47
2.1.1.
Physical properties of naltrexone hydrochloride ..................................................... 48
2.1.2.
Pharmacological uses and dosage for systemic administration of NTX ................. 48
2.1.3.
Pharmacokinetics of NTX ....................................................................................... 48
2.1.4.
Adverse reactions of systemic administration of NTX ........................................... 49
2.1.5.
Warnings on the systemic administration of NTX .................................................. 49
2.1.6.
Naltrexone as a new ophthalmic pharmaceutical .................................................... 50
2.1.7.
Chemical stability of NTX ...................................................................................... 50
2.1.8.
Oxidation ................................................................................................................. 53
2.1.9.
Arrhenius plot and predicting drug stability ............................................................ 56
2.2.
Chapter aims ................................................................................................................... 57
2.3.
Materials and methods .................................................................................................... 58
2.3.1.
Materials .................................................................................................................. 58
2.3.2.
Methods ................................................................................................................... 58
2.3.3.
Statistical analysis ................................................................................................... 64
2.4.
Results and discussion .................................................................................................... 65
2.4.1.
DSC study ................................................................................................................ 65
2.4.2.
Spectrometric analyses of NTX powder.................................................................. 66
2.4.3.
UV-spectrophotometric assay.................................................................................. 70
2.4.4.
HPLC method validation ......................................................................................... 71
2.4.5.
Forced degradation studies ...................................................................................... 74
2.4.6.
Degradation products identification using ESI-MS ................................................ 81
2.4.7.
pH-degradation rate of NTX.................................................................................... 83
2.4.8.
Accelerated stability and chemical kinetic studies .................................................. 85
2.4.9.
Solubility studies of NTX ........................................................................................ 87
viii
Content
2.4.10. Determination of n-octanol/PBS distribution coefficient (D) and partition
coefficient (P) of NTX ........................................................................................................... 87
Conclusion ................................................................................................................................. 89
3.
Preparation of niosomes using biocompatible surfactants ............................................... 90
3.1.
Introduction ..................................................................................................................... 90
3.1.1.
Niosomes ................................................................................................................. 90
3.1.2.
Non-conventional niosomes .................................................................................... 99
3.1.3.
Niosomes as drug delivery systems ....................................................................... 100
3.2.
Chapter aims ................................................................................................................. 103
3.3.
Materials and methods .................................................................................................. 104
3.3.1.
Materials ................................................................................................................ 104
3.3.2.
Methods ................................................................................................................. 104
3.3.3.
Statistical analysis ................................................................................................. 108
3.4.
Results and discussion .................................................................................................. 108
3.4.1.
Niosome formation and imaging using plane /polarised microscopy ................... 108
3.4.2.
Niosome imaging using Cryo-SEM ...................................................................... 111
3.4.3.
Niosome size measurements.................................................................................. 111
3.4.4.
NTX EE% .............................................................................................................. 114
3.4.5.
DSC studies ........................................................................................................... 121
Conclusion ............................................................................................................................... 126
4.
Effect of membrane additives on the physical properties of niosomes.......................... 127
4.1.
Introduction ................................................................................................................... 127
4.1.1.
Bilayer membrane additives .................................................................................. 127
4.1.2.
Surfactant and ocular delivery ............................................................................... 130
4.2.
Chapter aims ................................................................................................................. 131
4.3.
Materials and methods .................................................................................................. 132
ix
Content
4.3.1.
Materials ................................................................................................................ 132
4.3.2.
Methods ................................................................................................................. 132
4.3.3.
Statistical analysis ................................................................................................. 138
4.4.
Results and discussion .................................................................................................. 139
4.4.1.
Effect of selected membrane additives on NTX EE% .......................................... 140
4.4.2.
Effect of preparation methods on NTX EE% ....................................................... 142
4.4.3.
Niosome size and distribution measurements ....................................................... 144
4.4.4.
Morphology and lamellarity .................................................................................. 147
4.4.5.
DSC studies ........................................................................................................... 155
4.4.6.
Surface tension measurements............................................................................... 160
4.4.7.
Contact angle and spreading coefficient measurements ........................................ 161
4.4.8.
Rheological properties measurements ................................................................... 162
4.4.9.
Effect of niosomal encapsulation on NTX oxidation ............................................ 167
Conclusion ............................................................................................................................... 172
5.
Evaluation of niosomal formulations for ocular delivery of naltrexone hydrochloride
...............................................................................................................................................173
5.1.
Introduction ................................................................................................................... 173
5.1.1.
Eye irritation tests .................................................................................................. 174
5.1.2.
Toxicity of niosomes ............................................................................................. 179
5.1.3.
Physical stability of niosomes ............................................................................... 180
5.2.
Chapter aims ................................................................................................................. 181
5.3.
Materials and methods .................................................................................................. 182
5.3.1.
Materials ................................................................................................................ 182
5.3.2.
Methods ................................................................................................................. 182
5.3.3.
Statistical analysis ................................................................................................. 196
5.4.
Results and discussion .................................................................................................. 197
x
Content
5.4.1.
Conjunctival (HET-CAM) test .............................................................................. 197
5.4.2.
Cornea (bovine eye) test ........................................................................................ 200
5.4.3.
Histopathological evaluation of the bovine corneas .............................................. 203
5.4.4.
In vitro release studies ........................................................................................... 216
5.4.5.
In vitro release kinetic studies ............................................................................... 220
5.4.6.
Ex-vivo corneal permeation studies ....................................................................... 221
5.4.7.
Physical stability of the prepared niosomes .......................................................... 227
Conclusion ............................................................................................................................... 234
6.
General discussion, conclusion and direction for future research................................. 235
6.1.
General discussion and conclusion ............................................................................... 235
6.2.
Limitations and future direction ................................................................................... 241
References ................................................................................................................................... 245
Appendix 1 .................................................................................................................................. 286
xi
List of Figures
List of Figures
Figure 1.1 Gross anatomy of the anterior segment of the human eye ............................................. 1
Figure 1.2 Factors limiting ocular bioavailability of an ophthalmic formulation following topical
administration to the eye surface ..................................................................................................... 2
Figure 1.3 Structure of the precornal tear film ................................................................................ 4
Figure 1.4 Photomicrograph of H & E stained bovine cornea ........................................................ 4
Figure 1.5 Human corneal nerves penetration and distribution..................................................... 30
Figure 2.1 Chemical structure of NTX .......................................................................................... 48
Figure 2.2 Schematic of morphine autoxidation in aqueous solutions .......................................... 55
Figure 2.3 DSC thermogram of NTX ............................................................................................ 65
Figure 2.4 APCI mass spectra of NTX .......................................................................................... 66
Figure 2.5 FT-IR spectrum of NTX .............................................................................................. 67
Figure 2.6 1H-NMR spectrum of NTX in CD3SO, 400 MHz ....................................................... 68
Figure 2.7 UV-Spectrum of NTX (100 µg/ml) in PBS pH 7.4 ..................................................... 70
Figure 2.8 Standard UV-spectrophotometric calibration curve of NTX in PBS at pH 7.4 ........... 71
Figure 2.9 Representative HPLC chromatograms of standard NTX (0.4 mg/ml) solution and NTX
resolved from Span 60: cholesterol: DCP-niosomal formulation ................................................. 73
Figure 2.10 PDA- spectra of the NTX peak acquired from 4 different positions throughout the
eluted peak for peak purity assessment ......................................................................................... 74
Figure 2.11 Percentage (%) remaining of NTX after 24 h exposure to various stress conditions 75
Figure 2.12 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)
pH 10 after 24 h exposure to heat at 60oC ..................................................................................... 77
Figure 2.13 PDA spectra of NTX eluted at 4.16 min (A), the degradation product resolved at 3.7
min (B) and the second degradation product resolved at 3.3 min (C) after exposure to pH 10 at 60
o
C ................................................................................................................................................... 78
Figure 2.14 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)
of pH 1.2 after 24 h exposure to heat at 60oC................................................................................ 79
Figure 2.15 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml)
of PBS pH 7.4 after 24 h exposure to heat at 60oC ....................................................................... 79
Figure 2.16 HPLC chromatograms of the blank solution and aquous NTX solution (0.4 mg/ml) of
PBS pH 7.4 after 24 h exposure to H2O2 (2% v/v) ........................................................................ 80
xii
List of Figures
Figure 2.17 HPLC chromatograms of the blank and aquous NTX solution (0.4 mg/ml) of PBS
pH 7.4 after 24 h exposure to artificial daylight illumination (10,000 lux) at 40oC...................... 80
Figure 2.18 ESI mass spectra of product I and Product II of NTX oxidation ............................... 81
Figure 2.19 Schematic of the proposed oxidative degradation pathway for NTX ....................... 82
Figure 2.20 First-order degradation plots for NTX at 60oC in phosphate buffers of various pH
values ............................................................................................................................................. 83
Figure 2.21 pH-rate profile for NTX at 60oC ................................................................................ 84
Figure 2.22 First-order degradation plots for NTX in PBS pH 7.4 at various temperatures......... 86
Figure 3.1 Locations of entrapped hydrophilic, lipophilic and amphipathic drugs in niosomes .. 91
Figure 3.2 Major types of niosomes, MLV (multilamellar vesicles), OLV (oligolamellar vesicles)
and ULV (unilamellar vesicles)..................................................................................................... 91
Figure 3.3 Schematic of a single-chain surfactant ........................................................................ 93
Figure 3.4 Chemical structure of cholesterol................................................................................. 95
Figure 3.5 Suitable niosome sizes for particular routes of administration .................................... 98
Figure 3.6 Representative micrographs of Brij 72:cholesterol 7:3 mol/mol-based niosomes under
plane light and polarised light microscope ................................................................................ 110
Figure 3.7 Niosomal dispersions containing various molar ratios of Brij 52:cholesterol and Span
60:cholesterol ............................................................................................................................. 110
Figure 3.8 Cryo-SEM micrographs of Span 60: cholesterol 7:3 mol/mol and Brij 72: cholesterol
7:3 mol/mol niosomes ................................................................................................................. 111
Figure 3.9 Effect of HLB on Span-based niosomes composed of surfactant:cholesterol at 7:3
molar ratio ................................................................................................................................... 113
Figure 3.10 Effect of gel/liquid transition temperature on EE% for niosomes composed of
surfactant:cholesterol at 7:3 molar ratio ..................................................................................... 115
Figure 3.11 Effect of cholesterol concentration on EE% for Span-based niosomes .................. 117
Figure 3.12 Effect of the total surfactant/lipid content on EE % for Span 60: cholesterol ......... 118
Figure 3.13 Effect of the total surfactant/lipid content on DL % for Span 60: cholesterol ......... 119
Figure 3.14 Effect of the initial amount of NTX on EE % for Span 60: cholesterol .................. 120
Figure 3.15 Effect of the initial amount of NTX on DL % for Span 60: cholesterol .................. 121
Figure 3.16 DSC thermograms of Span 60-based niosomes containing different concentrations of
cholesterol .................................................................................................................................... 124
xiii
List of Figures
Figure 3.17 Effect of NTX on gel/liquid transition temperature of (Span 60: cholesterol 7:3
mol/mol) niosomes ...................................................................................................................... 125
Figure 4.1 Equilibrium between forces acting on a drop of liquid on a solid surface ................. 135
Figure 4.2 KSV-CAM 101 goniometer setup for measuring contact angle ............................... 136
Figure 4.3 Effect of selected membrane additives concentration on NTX EE% for niosomes
prepared using the TFH method ................................................................................................. 140
Figure 4.4 Effect of the preparation method on NTX EE% ........................................................ 143
Figure 4.5 Size-frequency distribution curves of the prepared niosomes ................................... 146
Figure 4.6 Cryo-SEM micrographs of F-S60 and F-DCP prepared using the REV method ...... 148
Figure 4.7 Cryo-SEM micrographs of F-C24 and F-CH prepared using the REVmethod ......... 149
Figure 4.8 CLS micrographs of F-S60 niosomal formulation loaded with CF produced by the
REV method ................................................................................................................................ 151
Figure 4.9 CLS micrographs of F-DCP niosomal formulation loaded with CF produced by the
REVmethod ................................................................................................................................. 152
Figure 4.10 CLS micrographs of F-C24 discomes loaded with CF produced by the REV method
..................................................................................................................................................... 153
Figure 4.11 CLS micrographs of F-CH niosomal formulation loaded with CF produced by the
REV method ................................................................................................................................ 154
Figure 4.12 Effects of different membrane additives on gel/liquid transition temperatures of the
prepared niosomes ....................................................................................................................... 155
Figure 4.13 Hypothetical position occupied by DCP in the bilayer membrane of F-DCP based on
DSC ............................................................................................................................................. 157
Figure 4.14 Hypothetical position occupied by C24 in the bilayer membrane of F-C24 based on
DSC ............................................................................................................................................. 158
Figure 4.15 Hypothetical position occupied by CH in the bilayer membrane of F-CH based on
DSC ............................................................................................................................................. 159
Figure 4.16 Surface tension measurements for the prepared niosomes compared with NTX
aqueous solution .......................................................................................................................... 160
Figure 4.17 Representative rheograms for the aqueous vehicle (PBS) and the prepared niosomes
35oC ............................................................................................................................................. 164
Figure 4.18 Viscosity values for the aqueous solution (PBS) compared with the prepared
niosomes at two different temperatures 25oC and 35oC .............................................................. 165
xiv
List of Figures
Figure 4.19 Effect of niosomes encapsulation on the chemical stability of NTX against
oxidation and daylight illumination............................................................................................. 168
Figure 4.20 First-order degradation kinetics for PBS solution of NTX and NTX encapsulated
niosomes under artificial daylight illumination at 40oC in PBS pH 7.4 ...................................... 170
Figure 4.21 First-order degradation rate constants for PBS solution of NTX and NTX
encapsulated niosomes under artificial daylight illumination at 40oC in PBS pH 7.4 ................ 171
Figure 5.1 Development stages of the growing embryos ............................................................ 183
Figure 5.2 Temperature, percentage relative humidity (% RH) and dew point inside the egg
incubator over the 10-day incubation period ............................................................................... 184
Figure 5.3 Vascular responses used to score the test substances................................................. 187
Figure 5.4 Excised cow eyes immersed in normal saline with silicone O-rings centered on top of
the cornea and incubated a water bath thermostatically equilibrated at 37oC ± 0.5oC ................ 189
Figure 5.5 Degree of corneal opacity and fluorescein permeability used to score the test
substances .................................................................................................................................... 191
Figure 5.6 Degree of corneal opacity and fluorescein permeability used to score the test
substances ................................................................................................................................... 192
Figure 5.7 Excised bovine cornea, modified parts and top view of the final assembly of the Franzdiffusion cell ............................................................................................................................... 195
Figure 5.8 Cumulative HET-CAM scores for the controls and representative examples of the test
substances ................................................................................................................................... 198
Figure 5.9 Cumulative bovine eye scores for the controls and representative examples of the test
substances .................................................................................................................................... 201
Figure 5.10 Photomicrographs of H&E stained corneal sections of negative control treated with
saline for 30 s ............................................................................................................................... 204
Figure 5.11 Photomicrographs of H&E stained corneal sections of acetone-treated cornea for 30 s
..................................................................................................................................................... 206
Figure 5.12 Photomicrographs of H&E stained corneal sections of NaOH (0.5 M)-treated cornea
for 30 s ......................................................................................................................................... 208
Figure 5.13 Photomicrographs of H&E stained corneal sections treated with CH Powder, C24
powder and the prepared niosomes for 30 s ................................................................................ 210
Figure 5.14 Photomicrographs of H&E stained corneal sections treated with the prepared
niosomes for 1 h .......................................................................................................................... 212
xv
List of Figures
Figure 5.15 Photomicrographs of H&E stained corneal sections treated with the prepared
niosomes for 3 h .......................................................................................................................... 213
Figure 5.16 Photomicrographs of H&E stained corneal sections treated with the prepared
niosomes for 8 h .......................................................................................................................... 214
Figure 5.17 In vitro release profiles of NTX from the NTX solution and the prepared niosomes
..................................................................................................................................................... 217
Figure 5.18 Effect of sample volume on the diffusion of NTX from the aqueous solution ........ 218
Figure 5.19 Transcorneal permeation profiles of NTX from the NTX solution and the prepared
niosomes using excised cow corneas ......................................................................................... 223
Figure 5.20 Relationship between NTX release rate and apparent permeability coefficient (Papp)
for NTX from the NTX solution and the prepared niosomes ...................................................... 225
Figure 5.21 Relationship between contact angle and lag time from the NTX solution and four
niosomal formulations ................................................................................................................. 226
Figure 5.22 Effect of storage temperature on NTX retention in the prepared niosomes over three
months ......................................................................................................................................... 232
Figure 5.23 Effect of niosome composition on NTX retention over three months ..................... 233
xvi
List of Tables
List of Tables
Table 1.1 Causes of decreased corneal sensitivity ........................................................................ 32
Table 1.2 Major attributes of insulin ............................................................................................. 37
Table 1.3 Major attributes of murine NGF .................................................................................... 39
Table 1.4 Major attributes of naltrexone ....................................................................................... 43
Table 2.1 Summary for HPLC conditions of NTX determination ................................................ 52
Table 2.2 Assignments of 1H-NMR spectra for NTX ................................................................... 69
Table 2.3 Reverse predicted concentrations, % recovery and regression coefficient (R2)............ 72
Table 2.4 Precision and accuracy data of the QC samples ............................................................ 73
Table 2.5 First–order kinetic parameters and correlation coefficient of NTX degradation in
aqueous solution of PBS pH 7.4 at different temperatures............................................................ 86
Table 2.6 n-octanol-buffer distribution coefficient (D) and partition coefficient (P) and calculated
pKa at different temperatures ....................................................................................................... 88
Table 3.1 Composition of the prepared niosomes using various surfactant: cholesterol molar
ratios ............................................................................................................................................ 105
Table 3.2 Chemical structure, phase transition temperature, HLB and vesicle-forming ability of
the used surfactants ...................................................................................................................... 109
Table 3.3 Effect of surfactant type and cholesterol level on the D [4,3] and EE % of the prepared
niosomes ..................................................................................................................................... 112
Table 3.4 DSC parameters of Span 60-based niosome dispersions containing different
concentrations of cholesterol ....................................................................................................... 123
Table 4.1 Codes and composition of the prepared niosomal formulations ................................. 132
Table 4.2 Chemical structure, molecular weight, CMC and phase transition temperature of the
investigated additives .................................................................................................................. 139
Table 4.3 Effect of selected membrane additives on D [4,3] and span values for niosomes
prepared by the REV method ..................................................................................................... 145
Table 4.4 Contact angle and spreading coefficient measurements for the prepared niosomes .. 162
Table 4.5 Tukey’s pair wise comparison of viscosity values for the prepared niosomes at 25oC
and 35oC ...................................................................................................................................... 166
Table 4.6 Tukey’s pair wise comparison of oxidation and photolysis for the prepared niosomes
..................................................................................................................................................... 169
Table 5.1 Ocular tissue and score for assessing ocular irritation using the Draize test............... 174
xvii
List of Tables
Table 5.2 HET-CAM scoring system .......................................................................................... 186
Table 5.3 Classification of cumulative scores in HET-CAM ...................................................... 186
Table 5.4 Average dimensions of human and cow corneas ........................................................ 188
Table 5.5 Bovine eye scoring system .......................................................................................... 190
Table 5.6 Classification of cumulative scores in bovine eye test ................................................ 190
Table 5.7 Summary of HET-CAM and bovine eye test interpretations ...................................... 202
Table 5.8 Histological corneal lesions scores for controls and the prepared niosomes at different
time points .................................................................................................................................. 215
Table 5.9 In vitro release parameters for NTX from the prepared niosomes compared with NTX
aqueous solution ......................................................................................................................... 218
Table 5.10 Tukey’s pair wise comparison of Q2h, Q6h and DE%-12 for NTX from the aqueous
PBS solution and the prepared niosomal formulations ............................................................... 219
Table 5.11 In vitro release kinetic parameters for NTX from the prepared niosomes ................ 221
Table 5.12 Steady state flux, apparent permeability coefficient (Papp) and tL for NTX from the
aqueous PBS solution and the prepared niosomes though excised cow corneas ....................... 224
Table 5.13 Tukey’s pair wise comparison of steady state flux, Papp and tL for NTX from the
aqueous PBS solution and the prepared niosomal formulations ................................................. 224
Table 5.14 Effect of ageing on the volume diameter (D [4,3]) for the prepared niosomes over
three months at three different temperatures ............................................................................... 229
xviii
List of Abbreviations
List of Abbreviations
ao
Optimal polar head group area of an amphiphile
Abs
Absorbance
ANOVA
Analysis of variance
AUC
Area under the curve
o
C
Degree Celsius
cal
Calorie(s)
CH
Sodium cholate
CMC
Critical micelle concentration
cps
Centipoises
Cryo-SEM
Cryogenic scanning electron microscope
C24
Solulan C24
D
Distribution coefficient
Da
Dalton
δ
Chemical shift
DCP
Dicetyl phosphate
DSC
Differential scanning calorimetry
D [4,3]
Volume diameter
EE%
Entrapment efficiency %
FAT
Freeze and thaw
g
Gram(s)
γ
Surface tension
H&E
Haematoxylin and eosin stain
HET-CAM
Hen’s egg test chorioallanoic membrane
h
Hour(s)
ICH
International Conference on Harmonisation
xix
List of Abbreviations
k
o
K
Kilo
Degree Kelvin
KCl
Potassium chloride
KH 2PO4
Potassium dihydrogen phosphate
λ max
Wave length of maximum drug absorption
lc
critical chain length of an amphiphile
LD%
Loading efficiency %
Lipid vesicles
Liposomes and niosomes
Liposomes
Phospholipid vesicles
LOD
Limit of detection
LOQ
Limit of quantitation
LUV
Large unilamellar vesicles
min
Minute(s)
µl
Microlitre
MLV
Multilamellar vesicles
mm
Millimetre
µm
Micromitre
µg
Microgram(s)
µmol
Micromole(s)
µM
Micromolar
mM
Millimolar
mol/mol
Mole per mole
Mol wt
Molecular weight
mPa.s
MilliPascal-second = 1centipoise
n
Release exponent
NaCl
Sodium chloride
xx
List of Abbreviations
Na2 HPO4
Disodium phosphate
Niosomes
Non-ionic-surfactant vesicles
nm
Nanometre
NTX
Naltrexone hydrochloride
O/W
Oil in water
P
Partition coefficient
Papp
Apparent permeability coefficient
PBS
Phosphate buffer saline
PDA
Photodiode array
pH
Negative log hydrogen ion concentration
R2
Determination (regression) coefficient
®
Registered trade mark
REV
Reverse phase evaporation
rpm
Rotation (s) per minute
s
Second(s)
SUV
Small unilamellar vesicles
TFH
Thin film hydration
θ
Contact angle
tL
Lag time
t1/2
Time required for 50% drug degradation
t90%
Time at which drug decomposed to 90% of its original concentration
U
1 unit of insulin
V
Volt
v
Volume of the hydrocarbon chains of an amphiphile
v/v
Volume per volume
W/O
Water in oil
xxi
List of Abbreviations
w/v
Weight per volume
w/w
Weight per weight
xxii
Chapter 1….Literature Review
1. General introduction: challenges in ocular delivery, controlled
ocular drug delivery systems and new therapeutic approaches in
diabetic keratopathy
1.1. Ocular bioavailability from ophthalmic dosage forms
Topical ocular drug delivery is a commonly used and preferred route for treating disorders that
affect the anterior segment of the eye (the cornea, conjunctiva, sclera, iris and lens) (Figure 1.1).
This is because of the rapid and local effects, accessibility to the ocular tissue, relative safety and
patient acceptability, as well as the relatively lower risk compared with systemic routes of drug
administration (Macha et al., 2003).
Figure 1.1 Gross anatomy of the anterior segment of the human eye
However, there are many anatomical and physiological barriers forming part of the eye’s natural
defence (Lang et al., 2002). These barriers and protective mechanisms prevent the administered
ophthalmic formulations from residing on the eye surface for enough time to allow complete drug
absorption. This prevents a significant portion of the instilled dose from being absorbed by the
ocular tissue (ocular bioavailability). Therefore, the ocular bioavailability of topically applied
drugs from simple solutions is often less than 1% (Urtti, 2006b). The protective barriers and
constraints limiting ocular bioavailability of topically administered drugs are outlined in Figure
1.2 and are discussed in this chapter.
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Chapter 1….Literature Review
Figure 1.2 Factors limiting ocular bioavailability of an ophthalmic formulation following
topical administration to the eye surface
1.1.1. Anatomical and physiological considerations
The corneal and the conjuctival-scleral routes are the two major routes for ocular drug absorption
from topically administered ophthalmic dosage forms (Ahmed & Patton, 1987, 1989).
1.1.1.1. Corneal route
The cornea is the transparent avascular dome-shaped structure at the anterior part of the eye
(Figure 1.1). The anterior corneal surface is covered by the tear film and the posterior surface is
bathed by the aqueous humour. The surface area of the cornea is approximately 1.3 cm2, forming
one-sixth of the surface area of the globe. The cornea is thinnest at the centre and its thickness
gradually increases towards the periphery, with mean thickness of 0.5 mm and 0.7 mm
respectively. Anatomically, it is divided into five layers (Klyce & Beuerman, 1998) (Figure 1.4):
2
Chapter 1….Literature Review
1. Epithelium
2. Bowman’s layer
3. Stroma
4. Descemet’s membrane
5. Endothelium
Although it is not a part of the cornea, the precorneal tear film is intimately associated with the
cornea both anatomically and functionally (Macha et al., 2003) (Figure 1.3).
Precorneal Tear film
The surface of the cornea must be kept moist to prevent damage to the corneal epithelium and to
maintain the optical quality of the cornea. Moisture and smoothing are provided to the ocular
surface by the preocular tear film in conjunction with the spreading function of the eyelids during
blinking. The tear film is approximately 7 µm thick and 6.5 µl in volume. It consists of three
structurally identifiable layers (Figure 1.3) (Ehlers & Hjortdal, 2006; Klyce & Beuerman, 1998):
1. A thin superficial lipid layer (0.1 to 0.5 µm) secreted by meibomian glands retards
evaporation from the preocular tear film, which prevents drying between blinks.
2. A thick (approximately 6 µm) middle aqueous layer supplied by lacrimal glands, into
which a mucin-rich glycocalyx extends.
3. A thin (0.02 to 0.05 µm) inner mucous layer composed mainly of glycoprotein and
produced by the conjuctival goblet cells and the lacrimal gland. The mucin layer ensures
the wetting of the corneal and conjunctival epithelia.
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Chapter 1….Literature Review
Figure 1.3 Structure of the precorneal tear film
Figure 1.4 Photomicrograph of H & E stained bovine cornea showing five
layers [epithelium (EP), Bowman’s membrane (BM), stroma (ST),
Descemet’s membrane (DM) and endothelium (EN)]
4
Chapter 1….Literature Review
Corneal epithelium
The entire epithelium is about 4 to 6 cell layers thick and represents 10 % of the corneal
thickness. The epithelial cells are non-keratinised, stratified squamous and divided
morphologically into three layers (Klyce & Beuerman, 1998):
The outermost layer comprises non-keratinised, stratified squamous epithelial cells. The
innermost layer is a basal cell layer. The basal cells are the only epithelial cells that undergo
mitosis. The daughter cells thus formed push anteriorly and change their shape conforming to the
middle epithelial layer, which is called the wing cell layer. The wing cells are a transition stage
between basal cells and superficial squamous cells. The superficial cells are polygonal in shape
and 40-60 µm in diameter and have unique specialisations that maintain the tear film and the
barriers that separate the extracellular space of the cornea from the tears. The surface membranes
of superficial cells are distinctive in two respects:
1. They have microscopic projections (microvilli, reticulations and microplicae).
2. The outer leaflet is thickened and supports an extensive fibrillar glycocalyx, also called
the buffy coat.
The cells of all epithelial layers interdigitate and are separated by a 10-20 nm intercellular space.
Important properties of the superficial cells include adherent junctions and junctional complexes
formed with laterally adjacent cells. These complexes consist of tight junctions (zonulae
occludens) that surround the entire cell and resist the flow of fluid through the epithelial surface.
If the aqueous humour passes into the stroma due to a defect in the tight epithelial junction, it is
trapped within the epithelium, resulting in epithelial oedema (Ehlers & Hjortdal, 2006).
Basal lamina and bowman’s layer
The basal lamina is an extracellular secretory product of the basal epithelial cells. It forms a
scaffold for the organisation of the epithelium and separates the epithelium from the stroma.
Bowman’s layer is an acellular region lying just under the basal epithelial membrane. It
composed of randomly organised collagen fibrils that merge into more organised anterior stroma.
Bowman’s layer is very thin and functions as a dome-shaped structure anchored to the limbus.
Bowman’s layer is a rigid structure and it shapes the anterior corneal curvature. Once damaged,
its architecture many not be restored, leading to abnormalities in the corneal thickness and optical
properties that could result in permanent vision deficit (Klyce & Beuerman, 1998).
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Chapter 1….Literature Review
Corneal stroma
The stroma constitutes approximately 90% of corneal thickness and it is composed of collagen
fibrils in parallel arrays which make up the 300 to 500 lamellae of the stroma. The lamellae
extend from limbus to limbus. Collagen constitutes approximately 71% of the dry weight of the
cornea and is the structural macromolecule providing tissue transparency and mechanical
resistance to intraocular pressure. Nerve axons and their Schwann cells are found in the anterior
and middle third of the stroma. The stroma can be considered a comparatively open structure
allowing diffusion of solute molecules weighing about 500 kilo Dalton (kDa). However, it acts as
a diffusion barrier to all lipophilic drugs (Klyce & Beuerman, 1998).
Descemet’s membrane
Descemet’s membrane is the thick basal lamina (~5 µm thick) secreted by the endothelium. It is
loosely attached to the stroma. Descemet’s membrane increases in thickness as an inevitable
consequence of ageing. On the other hand, many diseases might damage the endothelial cells
(e.g. interstitial keratitis) and consequently this damage can result in overproduction of
Descemet’s membrane materials (Klyce & Beuerman, 1998). Also, wrinkling of the Descemet’s
membrane is a diabetic complication of the anterior segment of the eye (Schultz et al., 1981 ).
Corneal endothelium
The corneal endothelium forms a single layer, of approximately 4 to 6 µm thick, on the posterior
corneal surface. These cells play a key role in maintaining corneal transparency through their
transporting, synthetic and secretory functions. The endothelium maintains tight apposition with
neighbouring cells, preventing excessive seepage of aqueous humour into the stroma (Klyce &
Beuerman, 1998).
As previously outlined, the corneal route has exclusive tight junctional complexes of epithelial
cells which endow the cornea with tight barriers against chemical and microbial insults. This
makes the corneal epithelium a significant barrier to the penetration of many drugs. However,
paracellular (small ions and hydrophilic molecules) and transcellular (hydrophobic) diffusion is
reported. Most topically applied drugs permeate via the transcellular route (Lee & Li, 1989).
The conjunctival-scleral route is thought to be an alternative portal gate for ocular permeation of
hydrophilic large molecules such as peptides and proteins and its anatomical and physiological
features are discussed in the following section.
6
Chapter 1….Literature Review
1.1.1.2. Conjunctival-scleral route
The conjunctival-scleral route is the major pathway for polar and large-molecular weight drugs to
reach the intraocular tissues (Ahmed & Patton, 1989). For example, high molecular weight drugs
such as inulin (mol wt 5500) can gain access to the iris and ciliary body by diffusion via the
conjuctival-sclera route. Further, drugs permeated through this route gain access directly to the
posterior segment of the eye (choroid, vitreous humour and retina) (Ahmed & Patton, 1987).
Conjunctiva
Conjunctiva is a thin and vascularised mucus membrane. It covers the anterior surface of the eye
globe with the exception of the cornea, where the conjunctiva ends at the limbus bordering the
cornea. The conjunctiva consists of 2-3 layers of non-keratinised epithelial cells but conjunctival
epithelial cells are not tight as corneal epithelial cells. The conjunctiva is permeable to molecules
up to 20 kDa, in contrast to the cornea which is impermeable to molecules larger than 5 kDa
(Greaves & Wilson, 1993). Anatomically, the conjunctiva is divided into three areas: conjunctiva
covering the interior of eyelids, called palpebral conjunctiva; conjunctiva which is folded to
cover the fornix, called fornix conjunctiva; and conjunctiva covering the anterior surface of the
globe, called bulbar conjunctiva. The conjunctiva of the eyelid and globe forms a continuous
surface area of 18 cm2, 17 times larger than the cornea1 surface area (Watsky et al., 1988). The
bulbar conjunctiva, combined with the subsequent layer (sclera), represents the non-corneal
barrier to drugs topically applied to the surface of the eye.
Sclera
The sclera (the white part of the eye) is the outermost layer that covers the anterior surface of the
eye. It protects the sensitive inner parts of the eye. The sclera is a firm and resistant structure
(0.5-1 mm thick) composed of the same collagen fibrils as the corneal stroma. However, the
sclera’s collagen fibres are arranged in an irregular network rather than a lattice structure. This
makes the sclera appear opaque compared with the transparent cornea. The sclera constitutes the
posterior five-sixths of the globe, whereas the cornea comprises the remaining one-sixth (Macha
et al., 2003).
Generally, the sclera is significantly more permeable to solutes than the cornea. Solute size has a
more pronounced effect on the sclera than the cornea whereas drug lipophilicty predominantly
controls drug corneal permeability (Ahmed & Patton, 1989). The conjunctival-scleral route can
be a promising avenue for intra-ocular drug delivery of hydrophilic and/or large molecules. These
7
Chapter 1….Literature Review
molecules can benefit more from the conjunctival-scleral route than the corneal route (Fattal &
Bochot, 2006; Geroski & Edelhauser, 2001). Therefore, large bio-organic compounds such as
proteins and bioactive oligonucleotides drugs can be absorbed via this route and gain access to
the intraocular tissues for safer and more convenient topical treatment of posterior segment
diseases (e.g. diabetic retinopathy) than invasive intravitreous injection route.
1.1.2. Pharmacokinetic considerations
Topical ocular pharmacokinetics studies the main pathways of drug administration, elimination
and ocular bioavailability such as tears dynamics, non-productive absorption and enzymatic
degradation (Urtti, 2006a).
1.1.2.1. Tears and tears dynamics
The precorneal tear film structure, composition and function were previously discussed and
shown in Figure 1.3. Human tears also contain around 0.6 to 2% w/v of proteins. The proteins in
the tear fluid are mainly albumin, globulin, lysozyme and lactoferrin (Mikkelson et al., 1973a;
Nishida, 2005). Emotional stress, irritation and inflammatory diseases greatly influence the
protein content in the tear fluid. The relatively high concentration of proteins coupled with a
relatively rapid tear turnover leads to a considerable loss in the drug activity. For example, the
mitotic activity of pilocarpine hydrochloride was tested in the presence and absence of added
rabbit serum albumin. A 3-fold increase in the mitotic activity was obtained in absence of the
protein compared with that in the presence of 3% w/v albumin. The mitotic activity decreased
gradually depending on the available protein concentration (Mikkelson et al., 1973a).
The normal volume of tears, present in the human eye is approximately 7-10 µl. The average
basal secretion is 1.2-1.5 µl/min and the physiological turnover rate is 0.1-0.15 min-1. Upon
instillation of fluid into the eye, the drainage of instilled solution proceeds at a rapid rate until the
volume is back to normal volume, about 7 µl. Thus as the volume of instilled dose increases, the
volume of solution lost by spillage on the cheek and drainage increases (Chrai et al., 1974; Chrai
et al., 1973). The drop size is responsible for considerable loss of the drug and hence affects the
ocular bioavailability. Furthermore, this drainage contributes to non-productive absorption and
leads to unwanted side effects. Therefore, it is recommended that the size of the eye drop should
be reduced from 50-75 µl/drop to 5-10 µl/drop to maximise drug activity from ophthalmic ocular
dosage forms (Chrai et al., 1973). In support of previous findings, it has been found that the
ocular bioavailability, estimated as the area under the miosis–time curve, in rabbits did not show
8
Chapter 1….Literature Review
any significant differences after the treatment using a 30 µl instillate of 1% w/v pilocarpine
hydrochloride solution and the spray delivery of 5 µl volumes of the 1% w/v solution of
pilocarpine hydrochloride. This study demonstrated that a 6-fold reduction in the administered
volume achieved an equivalent miotic response (Martini et al., 1997).
1.1.2.2. Non-productive absorption
Non-productive absorption is the absorption of topically applied ophthalmic drugs into the
systemic circulation due to absorption from conjunctival blood vessels and dominantly from
nasal mucosa due to the naso-lacrimal drainage (Chang & Lee, 1987; Shell, 1982). Most of the
instilled dose (approx. 70%) is lost due to drainage via the naso-lacrimal duct within the first 15
to 30 s after instillation (Shell, 1982).
Non-productive absorption following ocular drug administration not only leads to decreasing the
ocular bioavailability, but is also seriously responsible for systemic side effects. Systemic side
effects were reported with many drugs topically applied to the eye surface. For instance, topical
ocular administration of atropine (German & Siddiqui, 1970), timolol (Kaila et al., 1986),
betaxolol (Polansky & Alvarado, 1985), epinephrine (Anderson, 1980), oxymetazoline (Duzman
et al., 1983), pilocarpine (Urtti et al., 1985), flurbiprofen (Tang-Liu et al., 1984) and cyclosporine
(Mosteller et al., 1985) has been associated with systemic side effects.
In addition, serious cardiovascular and pulmonary side effects were observed with topical ocular
administration of timolol (a widely used anti-glaucomic drug) (Nelson et al., 1986). It has been
reported that the nasal mucosa accounts for 70% of the timolol systemically absorbed (Chang &
Lee, 1987). This is probably due to the consequence of rapid drainage of the instilled solution
into the naso-lacrimal duct and then the nasal cavity.
The conjunctival systemic absorption of ocularly applied drugs is increased by the relative
leakiness of the membrane, rich blood flow, and large surface area (Urtti & Salminen, 1993). The
nasal cavity has a large surface area of 150-200 cm2 and a total volume of 20 ml in humans. The
nasal cavity surface area is 13 times that of the conjunctiva and almost 200 times that of the
cornea (Lee et al., 2002; Pontiroli et al., 1989; Urtti & Salminen, 1993). The contact area between
the instilled drug solution and nasal mucosa increases with increasing the instilled volume of the
eye drop. The drug can gain access to the systemic circulation via absorption from nasal mucosa
lining the mucosal cavity and/or via the gastrointestinal tract. Therefore, the absorption of the
drug from the nasal region is reproducible and comparable to parenteral drug administration. This
9
Chapter 1….Literature Review
efficient systemic absorption has been utilised as a non-invasive route of systemic drug delivery
of proteins and peptides (Lee et al., 2002).
Punctal occlusion and eyelid closure following topical solution instillation are the only measure
recommended to the patients to minimise systemic drug absorption via the nasolacrimal
apparatus (Kaila et al., 1986; Passo et al., 1984; Zimmerman et al., 1984). However, neither
approach is a satisfactory long-term solution due to patient incompliance. The formulation
approach could be a more effective solution. For example, both charged and neutral multilamellar
liposomes have been shown to decrease the ocular drainage of [111In]indium chloride and [99Tc]
sodium pertechnate
compared with the buffer solution (Fitzgerald et al., 1987b). The two
isotopes encapsulated in liposomes demonstrated prolonged precorneal residence time. The
suspending medium drains away independently of the liposomes. As the size increases the
nasolacrimal drainage decreases due to the larger liposomes restricted at the inner canthus region.
In another study, they concluded that multilamellar liposomes have been found to reduce
effectively the precorneal clearance rate and offer an advantage as a delivery system compared
with small unilamellar vesicles, as the lower entrapment efficiency of the latter negate any
advantages gained due to higher surface area (Fitzgerald et al., 1987a).
1.1.2.3. Enzymatic degradation
A number of enzymes are secreted and detected in the precorneal tear film. These include
carbonic anhydrases (Lonnerholm, 1974), esterases (Lee et al., 1982), peptidases (Stratford &
Lee, 1985), ketone reductase (Lee et al., 1988) and glutathione-conjugating enzyme (Saneto et
al., 1982). Depending on the nature of the applied drugs, these enzymes can adversely affect the
biological activity of the applied active drugs or can activate topically applied prodrugs (Lee &
Li, 1989).
1.1.3.
Drug and formulations factors
1.1.3.1. Physicochemical properties of the drug
The three main drug properties governing corneal drug absorption are:
1. Lipophilicity as reflected by n-octanol/buffer partition coefficient.
2. Dissociation constant (pKa) which determines the proportion of drug in its preferentially
absorbed form at a given pH.
3. Drug molecular size.
10
Chapter 1….Literature Review
Firstly, drug lipid solubility (lipophilicity) is an essential property for drug permeation via the
lipidic epithelial corneal membrane (Schoenwald & Ward, 1978). Drug lipophilicity is related to
its chemical structure and can be assessed by measuring the partition coefficient (P). According
to drug partition coefficients, β-adrenoreceptor blockers are classified into three classes: very
lipophilic (P ranged from 1640 to 14200), lipophilic (P ranged from 76 to 249) and hydrophilic
(P ranged from 1.46 to 59) (Schoenwald & Huang, 1983). Lipophilic β-blockers, such as timolol,
propranolol, atenolol, metoprolol and practolol, are able to lower the intraocular pressure and are
used successfully as antiglaucomic agents, whereas other β-blockers, such as nadolol and sotatol,
appear not to. This could be ascribed to the marked difference in lipid solubility and their ability
to cross corneal barriers. The corneal epithelium is the rate-determining barrier for hydrophilic
drugs whereas it is not for lipophilic drugs. Lipophilic drugs penetrate the cornea more rapidly
(Huang et al., 1983). For instance, dipivalyl epinephrine ester (apparent P = 4.89) has been
found to be about 100 times more effective than epinephrine (apparent P = 0.0081) in the
management of glaucoma and about 100-400 times weaker than epinephrine in affecting the
cardiovascular system in dogs and cats (McClure, 1975; Wei et al., 1978). This is due to
enhancement of lipophilicity of epinephrine through the use of dipivalyl ester which can facilitate
penetration through the lipoidal layers of the cornea (Lee & Li, 1989). Topically administered
drugs, however, must possess adequate aqueous solubility in tear fluid in order to achieve an
adequate penetration rate, to diffuse across the water-filled corneal stroma and to gain access to
deeper ocular tissues (Schoenwald & Huang, 1983). In another report, the optimal P for corneal
drug absorption was found to be in the range of 10-100 (Kaur & Smitha, 2002).
Secondly, ionised drugs, such as weak acids and weak bases, can exist in both ionised and
unionised forms at the pH of the lacrimal fluid. Unionised species usually penetrate more easily
lipophilic barriers of the epithelial layer than ionised ones. Therefore, the degree of drug
ionisation (expressed in pKa) in lacrimal fluid can determine how readily the drug passes
epithelial corneal barriers. For example, the in vitro corneal permeability of pilocarpine base was
2-3 times greater than that of the ionized form (Francouer et al., 1983) and the miotic response of
pilocarpine eye drops was greater at pH 7.0 than at pH 4.5 (Jarvinen et al., 1994). Timolol (weak
base, pKa 9.2) concentrations in ocular tissues increase with increasing the pH of the instilled
solution. pH values ranging from 6.2 to 7.5 are found to be optimum for ocular bioavailability
(Kyyrönen & Urtti, 1990). Also, the charge of the drug molecule may influence its corneal
11
Chapter 1….Literature Review
permeability. Cationic drugs are expected to permeate the cornea more easily than anionic drugs
where the latter encounters electrostatic repulsion from the negatively charged mucin layer on the
eye surface as well as the negatively charged pores present in the corneal epithelium (Jarvinen et
al., 1995; Le Bourlais et al., 1998; Loftssona & Jarvinen, 1999). However, the permeability of the
positively charged drugs can be hampered by ionic interactions with the negatively charged
carboxylic groups of the tight junction proteins (Hornof et al., 2005; Palmgren et al., 2002).
Thirdly, drug molecular size can also affect the amount of drug permeated through the tight
junctions of the corneal epithelium. Drugs of medium molecular weight such as inulin (mol wt
5500) can cross the cornea solely by paracellular mechanism rather than transcellular and in
smaller amounts than uncharged compounds of lower molecular weight (Lee et al., 1986).
However, larger molecules such as insulin (mol wt ~6000) can only cross the cornea in the
presence of permeation enhancers, such as saponin or Brij 78, which loosen the epithelial tight
junctions (Pillion et al., 1991).
1.1.3.2. Formulation factors
Some excipients were found to alter drugs’ ocular permeability, and consequently their ocular
bioavailability from the used ophthalmic dosage forms. For instance, benzalkonium chloride is a
cationic surfactant and is widely used as a preservative in ocular preparations. Benzalkonium
chloride and other cationic surfactants have been found to enhance the ocular absorption of a
number of drugs varying in molecular size and lipophilicity, including pilocarpine (Mikkelson et
al., 1973b), carbachol (Smolen et al., 1973) and prednisolone (Green & Downs, 1974). This
might be attributed to the amphiphilic nature of benzalkonium chloride, which imparts drug
molecules with some penetration-enhancing properties through the corneal epithelium. Other
formulation factors such as drop size, pH and the tonicity of the solution instilled into the surface
of the eye must be physiologically compatible to minimise tissue irritation, reflex tearing and
blinking, and consequently, this may lead to a substantial loss of the administered dose.
As mentioned above, the cul-de-sac of the eye normally holds 7-10 µl, but it can accommodate
up to 30 µl without overflowing. However, most commercially available eye droppers deliver a
drop of approximately 50 µl. This leads to reflex blinking, drainage to naso-lacrimal duct and
12
Chapter 1….Literature Review
spillage of a significant portion of the instilled dose to the cheek until the tear film returns to the
normal volume (Chrai et al., 1974; Chrai et al., 1973; Martini et al., 1997).
The normal pH of the tear fluid is 7.4. Increased blinking rate and lacrimation are associated with
any disturbance of the physiological pH of the tear fluid. Therefore, the pH of the ophthalmic
preparations should be formulated in pH 7 to 7.7 to minimise irritation to the eye surface
(Ludwig, 2005). In some cases, however, the ideal pH for drug solubility and/or stability is
outside this range. Therefore, it is recommended in this case to use the pH which can offer
maximum stability, permeability and solubility of the administered drug with minimal buffer
capacity, in order to allow the tear fluid to regain its normal pH rapidly (Ali & Lehmussaari,
2006; Van Ooteghem, 1993).
Parallel to the ophthalmic formulations’ pH, adjustment of the osmolality of the instilled
formulations is an issue. Lacrimal fluid contains various dissolved electrolytes (Na, K, Ca, Cl,
and HCO3 ions) constituting about 310-350 mOsm/kg (Ludwig, 2005). Therefore, the instilled
formulation must be isotonic with the tear fluid to minimise ocular irritation. The normal isoosmolar range is 270–330 mOsm/kg (Waymouth, 1970). Generally, hypotonic solutions are more
tolerable than hypertonic solutions. Osmolality lower than 260 mOsm/kg or higher than 480
mOsm/kg is considered irritating to the eye (Lang et al., 2002; Ludwig, 2005).
From the previous discussion, there seems to be a need for the development of an effective ocular
delivery system for topically applied drugs, in order to overcome the aforementioned hurdles.
The properties of the proposed ocular delivery system should be tailored to fit the major
anatomical and physiological barriers of the eye.
13
Chapter 1….Literature Review
1.2. Drug delivery systems providing controlled and continuous ocular
delivery
Conventional ocular dosage forms such as simple solutions, suspensions and ointments account
for approximately 90 % of currently marketed ophthalmic pharmaceuticals (Lang, 1995). This
wide-spread use is due mainly to simplicity and patient acceptability. However, the ocular
bioavailability of drugs topically applied from eye drops is typically less than 5%. This is due to
the very short contact time with the ocular surface, normal tear turnover, conjunctival absorption
and rapid drainage through the nasolacrimal duct. This prompts the clinician to recommend a
frequent drug instillation at an extremely high concentration. This typical pulse-type dosing
might pose safety concerns, by subjecting the ocular tissues to a relatively high local drug
concentration and causing several systemic side effects. Therefore, conventional ocular dosage
forms no longer constitute optimal therapy (Bochot et al., 1998; Urtti, 2006a). On the other hand,
controlled release ocular drug delivery systems can offer many advantages compared with the
conventional ones. These include:
•
Reducing the dose regimen with the goal of improving the patient compliance (Kaur et
al., 2004; Kaur & Kanwar, 2002).
•
Decreasing the effects of washout when instilling multiple drops and hence decreasing
systemic side effects (Kaur & Kanwar, 2002).
•
Minimising the preservative burden by decreasing the number of drops administered per
day (Furrer et al., 2002a).
Formulation of topical ocular delivery system with consistent bioavailability and minimal adverse
effects is still a challenging endeavour facing the pharmaceutical formulator (Kaur et al., 2004;
White & Byrne, 2010). The two options currently available to improve ocular drug bioavailability
are firstly, prolonging precorneal residence time and minimising precorneal loss and secondly,
maximising ocular permeability (du Toit et al., 2011; Kaur et al., 2004). There are many
formulation approaches which have been investigated. These formulation approaches are
discussed below.
14
Chapter 1….Literature Review
1.2.1. Ophthalmic inserts
Ocular inserts are solid or semi-solid sterile preparations to be placed in the cul-de-sac of the eye
or on the cornea, in order to prolong residence time (Gurtler & Gurny, 1995; Rathore & Nema,
2009). The size and shape of the inserts are tailored for ophthalmic application. They are
composed of a polymeric support with or without drug(s). The drug is incorporated as dispersion
or solution in the polymeric support. The inserts can be used for topical or systemic therapy
(Gurtler & Gurny, 1995; Rathore & Nema, 2009). Ocular inserts are mostly inserted in the lower
fornix and less frequently in the upper fornix or on the cornea. These solid ocular devices require
minor surgery by which the polymeric system (into which the drug has been incorporated) is
implanted inside the eye (Gurtler & Gurny, 1995). Based on solubility behaviour, ophthalmic
inserts can be classified as insoluble, soluble and bioerodible inserts.
1.2.1.1. Insoluble inserts
Insoluble inserts can be classified into three groups: diffusional, osmotic systems and contact
lenses (Rathore & Nema, 2009). The first two systems include an insoluble membrane
surrounding a drug reservoir to control the drug release rate. The reservoir contains a liquid, gel,
colloid, semisolid or solid matrix containing a drug homogeneously or heterogeneously dispersed
or dissolved therein. Drug-controlling membranes are composed of a wide range of polymers
including hydrophobic, hydrophilic, organic, inorganic, naturally occurring or synthetic materials
(Gurtler & Gurny, 1995). The third class includes contact lenses which are fabricated using
insoluble ophthalmic devices. These classes are likely associated with a high rate of patient
incompliance resulting from foreign body sensation due to insolubility of these ocular devices
(Rathore & Nema, 2009).
1.2.1.2. Soluble inserts
Ocular soluble inserts are completely soluble or biodegradable. This is likely to be an advantage
over insoluble ocular inserts, because the former do not need to be removed from their site of
application. Ocular soluble inserts can be formulated from natural polymers, synthetic or semisynthetic polymers (Gurtler & Gurny, 1995; Rathore & Nema, 2009). For example, a soluble
insert loaded with gentamicin sulfate and dexamethasone phosphate was prepared (Baeyens et al.,
1998). The system not only provided a sustained drug release but also showed a concomitant
release of the two drugs for the first 10 h, followed by a sustained release of gentamicin over a
period of 50 h. However, blurred vision was experienced by patients during treatment. In
addition, bioerodible inserts are composed of a drug homogenously dispersed in monolithic
15
Chapter 1….Literature Review
matrix devices to control the drug release rate. The main components used for the production of
this type of inserts are bioerodible polymers. Bioerodible inserts-based polymers undergo
hydrolysis of chemical bonds and hence dissolve over a prolonged period of time, in response to
the environment in the eye. Further, a soluble bioadhesive ophthalmic drug insert (BODI)
contained 25% w/w gentamicin sulphate was inserted into the lower cul de sac of dogs’ eyes. The
clinical efficacy of the treatment of keratoconjunctivitis was compared with that of the aqueous
gentamicin eye drops. The total clinical recovery outcomes from the BODI and gentamicin eye
drops were obtained after 3 and 7 days respectively. The BODI significantly reduced the
treatment period and improved the patient compliance when compared with the aqueous drug
solution (Baeyens et al., 2002). Acyclovir water-soluble inserts were fabricated from polyvinyl
alcohol and methylcellulose. Both the rate and acyclovir release profile were modified depending
on the additives used. The prepared inserts were stored for 6 months at 25oC and the aged inserts
demonstrated no change in the drug release profiles indicating good physical stability (El Gamal
et al., 2008).
1.2.2. Collagen shields
Collagen shields are ocular devices fabricated from porcine or bovine scleral tissues, which bear
a collagen composition similar to that of the human cornea and sclera. These ocular devices
should be hydrated before insertion to the eye (Lee, 1990; Willoughby et al., 2002). Typically,
the drug is loaded into the collagen shield by soaking it in the drug solution for a period of time
prior to application. Once in the eye, shields are hydrated by tear fluids, soften and form a clear,
pliable, thin film approximately 0.1 mm in thickness with a diameter of 14.5 mm and a base
curve of 9 mm that conforms to the corneal surface and is designed to dissolve slowly within 12,
24, or 72 h (Lee, 1990).
Collagen shields have been utilised as ocular delivery systems for many pharmacological classes
of drugs such as antibiotics, antifungal agents, steroids and immunosuppressive agents (Lee et al.,
1992; Schwartz et al., 1990; Unterman et al., 1988; Vasantha et al., 1988; Willoughby et al.,
2002). Collagen shields pre-soaked in tobramycin produced significantly higher concentrations of
antibiotic in the cornea at one hour than subconjunctival injections of tobramycin (Unterman et
al., 1988). The authors claimed that collagen shields containing antibiotics can serve as a vehicle
for drug delivery and may prove superior to current methods for preoperative and postoperative
antibiotic prophylaxis and the initial treatment of bacterial keratitis.
16
Chapter 1….Literature Review
In addition, amphotericin B (an antifungal agent) was formulated in collagen shields. The
prepared ocular devices were able to maintain ocular drug levels in the anterior segment of rabbit
eyes, as well as frequent-drop delivery with the potential benefit of added convenience and
compliance
(Schwartz
et
al.,
1990).
Further,
a
combination
of
gentamicin
and
methypredinsolone, commonly used drugs in treatment of ocular problems, was formulated in a
collagen shield as prolonged drug release ocular devices. However, severe corneal toxicity was
reported due to sudden rapid drug release from the device (Lee et al., 1992).
In other two reports, collagen shields pre-soaked in aqueous antibiotic solutions were studied for
a potential topical ocular treatment for bacterial endophthalmitis (one of serious complications
after intraocular surgery) (Hariprasad et al., 2005; Haugen et al., 2008). In one study, both 0.3%
w/v gatifoxacin eye drops and collagen shield pre-soaked in 1% w/v gatifloxacin solution
achieved significantly lower endophalmitis incidences than balanced salt solution controls in
rabbits. However, no statistically significant difference was obtained between the presoaked
collagen shields and the eye drops (Haugen et al., 2008). In the other study, collagen shields presoaked in 0.5% aqueous solution of moxifloxacin were studied as a topical ocular treatment
option for prevention of bacterial endophthalmitis in 10 patients prior to ocular surgery
(Hariprasad et al., 2005). The aqueous humour moxifloxacin levels estimated for collagen shields
were lower than those for topical drops. However, the ability to leave the eye batch undisturbed
after surgery and avoid patient manipulation of the eye post surgery, so as to instill topical drops
could be a potential advantage for this route, especially in the immediate post-operative period
(Hariprasad et al., 2005).
1.2.3. Polymeric gels
Polymeric gels can be divided into two groups: preformed hydrogels and in situ forming gels (Le
Bourlais et al., 1998).
1.2.3.1. Preformed or bio-adhesive hydrogels
Bioadhesive hydrogels (hydrocolloids) are hydrophilic polymers which tend to swell, viscolise,
gel and adhere upon contact with physiological mucus membranes by virtue of hydrophilic
functional groups and structures. Bioadhesive polymers are capable of forming strong noncovalent bonds with the mucin coating mucus membranes and reside in place as long as the
17
Chapter 1….Literature Review
mucin is present (Le Bourlais et al., 1998). Over the past three decades bioadhesive hydrogels
have been utilised in topical ocular drug delivery to prolong the ocular residence time and
minimise the frequency of the instilled drops (Robinson & Mlynek, 1995). For instance,
betaxolol, a β-adrenoreceptor antagonist, polyacrylic acid (PAA) gel showed significantly higher
bioavailability than a 0.5% w/v solution of betaxolol in rabbits (Weinreb & Jani, 1992). Also,
acetazolamide (an antiglaucomic agent) formulated in carboxymethylcellulose hydrogel showed a
prolonged effect when compared with the drug solution (Tous & El Nasser, 1992). Moreover,
polycarbophil gel enhanced the ocular delivery of topically applied gentamicin in rabbits. The
polymeric formulation increased the uptake of gentamicin by the bulbar conjunctiva twice,
compared with an aqueous control formulation (Lehr et al., 1994). A three-fold increase of the
precorneal residence time of tobramycin was achieved when formulated in a chitosan gel
compared with the commercial drug solution of the drug (Ludwig, 2005). Xyloglucan [tamarind
seed polysaccharide (TSP)] was tested as a novel vehicle for ophthalmic delivery of timolol
(Burgalassi et al., 2000). The polymer used, in spite of a comparatively low viscosity, showed
high timolol concentrations in the ocular tissues and a low systemic absorption compared with
the commercially available product based on in situ gelling (Timoptic XE®). The results
presented TSP as a potentially useful carrier for ophthalmic delivery systems.
One of the most successful applications of hydrogels in ophthalmology is their use as tear
substitutes due to their commercial availability. For example, celluloses (Lacril®), polyvinyl
alcohol (Liquifilm®), polyacrylic acid (Lacrigel®, Lubrithal®, Gel-Larm®), and hyaluronic acid
(Hy-Drop®) are frequently used as tear substitutes for the treatment of dry eye disease (Tsubota
& Dogru, 2006; Zignani et al., 1995). Bioadhesive polymers, however, are associated with some
pitfalls as an ocular drug delivery system. The drop size or volume of commercial ocular
medication formulated using bioadhesive polymers is not as uniform as conventional ocular
solutions. The amount delivered to the eye, consequently, is generally incorrect. Moreover, the
presence of a viscous vehicle can cause blurred vision and formation of a veil in the corneal area
leading to loss of eyesight (Winfield et al., 1990).
1.2.3.2. In situ activated gel forming systems
The abovementioned problems related to the accuracy of the instilled dose and blurred vision
from the preformed hydrogels could be overcome by in situ activated gel delivery systems. In situ
activated gel-forming systems can be described as viscous liquids that upon exposure to
18
Chapter 1….Literature Review
physiological conditions will shift to a gel phase (Le Bourlais et al., 1998). Three methods
causing phase transition on the eye surface have been reported in the literature: a change in
temperature (Miller & Donovan, 1982), pH (Gurny, 1981; Gurny et al., 1987) or electrolyte
composition (Rozier et al., 1989). Other methods can induce in situ gel activation have been
recently reported in the literature such as UV/visible light-irradiation (photopolymerisable
hydrogels) and enzyme mediated gelation, however, they have not been investigated for ocular
drug delivery (Van Tomme et al., 2008). Here are some applications of various methods used to
induce in situ sol/gel transition on the surface of the eye.
Poloxamers or pluronics are block co-polymers consisting of poly (oxyethylene) and poly
(oxypropylene) units. They rapidly undergo thermal gelation when the temperature is raised to
that of the ocular surface, while they remain liquid at refrigerator temperature. A mixture of
pluronic F127 and pluronic F68 was prepared as an in situ gelling system for ocular delivery of
sparfloxacin (antibacterial agent) for treatment of bacterial keratitis. The developed formulations
provided sustained release of the drug over a 24-h period and better improvement in artificially
induced bacterial conjunctivitis in rats' corneas (Nesseem, 2011). However, Poloxamers exhibit
thermal gelation at high concentrations (usually between 20 and 30% w/v); Pluronic F127 has
been found to be more damaging to the cornea than a physiological saline solution (Furrer et al.,
2000). The relatively high polymer concentration (20-30% w/v) required for in situ thermal
gelation can pose toxicological concerns (Miyazaki et al., 2001).
Cellulose acetophthalate (CAP) latex is defined as a highly unstable system which coagulates
when its native pH of 4.5 is raised by the tear fluid to pH 7.4(Gurny, 1981). This pH-induced
coagulation of CAP occurs at a high polymer concentration (30% w/v) (Gurny, 1981; Kumar et
al., 1994). Also, carbopol is a polyacrylic acid (PAA) polymer, which shows sol-gel transition in
an aqueous solution as the pH is raised above its pKa of about 5.5. As the concentration of
carbopol increases in the vehicle in order to improve its rheological properties, the acidity of the
vehicle increases. Hence, the inherent acidity of the vehicle could induce ocular tissue irritation
and induced lacrimation (Nanjawade et al., 2007).
An example of the ion-activated gelation is gellan gum (an anionic polysaccharide) which
exhibits phase transition with increased ionic strength. Also, it has been found that the extent of
19
Chapter 1….Literature Review
gel-formation of gellan gum increases proportionally with the amount of mono- or divalent
cations present in the tear fluid. Hence, the main triggering effect inducing phase transition is due
to the generous availability of mono- and divalent cations associated with reflex tearing (Greaves
et al., 1990). The dependency of the gel transition of some in situ gelling systems on the uptake
of cations present in the tear film may be disrupted in the presence of dry eye syndromes
(especially with diabetic patients). Moreover, the dependency on electrolytes for gel transition is
likely to hinder the tonicity adjustment and consequently, the final product is not iso-osmotic
with the tear fluid.
1.2.4. Colloidal delivery systems
1.2.4.1. Microemulsions
Microemulsions (MEs) have been utilised as topical ocular drug delivery systems because of their
inherent properties and structures (Vandamme, 2002). The use of MEs for ocular drug delivery is
advantageous because the presence of surfactant and co-surfactant increases membrane
permeability to the applied drug (Lawrence & Rees, 2000). Moreover, these systems offer the
additional advantage of having low viscosity and the convenience of eye drops.
Pilocarpine-based MEs have shown more sustained drug activity than the drug solution where
twice daily instillations of these systems were equivalent to four instillations of conventional eye
drops (Naveh et al., 1994). Recently, ME-based phase transition systems have been prepared and
evaluated as ocular delivery systems using pilocarpine hydrochloride as a model water-soluble
drug (Chan et al., 2007). These systems underwent phase transition from ME to liquid crystalline
(LC) and to coarse emulsion (EM) with a change in viscosity depending on water content. ME
and LC showed higher drug release retarding efficiency than EM. However, toxicity and tissue
irritancy have been observed with microemulsion formulations. For example, ocular application
of ME and LC disrupted the precorneal tear film and the tear evaporation rate increased
compared with the aqueous solution. This is due to the use of relatively high concentrations of
surfactants and co-surfactants within the formulations (Alany et al., 2006; Chan et al., 2008).
1.2.4.2. Nanosuspensions
The ocular formulation of poorly water-soluble drugs is not an easy task. These drugs suffer from
erratic ocular absorption and reduced availability (Kayser et al., 2005). This class of drugs can be
formulated as a nanosuspension in an appropriate dispersing vehicle, in an attempt to reduce the
20
Chapter 1….Literature Review
aforementioned problems. Drug compounds that form crystals with high energy content,
rendering them insoluble in either organic (lipophilic) or hydrophilic media, are good candidates
for formulation as a nanosuspension (Kayser et al., 2005). Thus, the use of nanosuspensions in
ophthalmic pharmaceutical formulations is likely to be an attractive area offering a great
possibility to overcome the inherent difficulties associated with the ocular delivery of poorly
water-soluble drugs. For instance, methyl prednisolone was prepared in a nanosuspension. The
prepared formulations showed localised and controlled ocular anti-inflammatory activity in
rabbits (Adibkia et al., 2007).
1.2.4.3. Polymeric microspheres and nanoparticles
Polymeric microspheres and nanoparticles are polymer-based particulate drug delivery systems.
The difference between these systems is based on their size. Nanoparticles can be defined as
particles with a diameter of less than 1 µm, whereas particles in the micrometre size (> 1 µm) are
called microspheres (Ali & Lehmussaari, 2006). They can be fabricated from various
biodegradable polymers such as natural and synthetic polymers. Drugs can either be dispersed in
the polymeric matrix or physically bound to the surface. Polylactides (PLAs), polycyanoacrylate,
chitosan, gelatin, sodium alginate and albumin were studied for efficient drug delivery to the
ocular tissues (Sahoo et al., 2008).
For instance, Polylactide-based nanoparticles (NPs) encapsulating two flourochromes
(Rhodamine-6G and Nile red) were tested as drug delivery systems targeting retina and retinal
pigment epithelium (RPE) and applied intravitreally (Bourges et al., 2003). NPs were able to give
a high local concentration of the tested compounds at the RPE suggesting the feasibility of
targeting the posterior segment of the eye by NPs. Moreover, chitosan is a cationic
polysaccharide and is obtained by deacetylation of chitin. Chitosan has been shown to promote
intraocular drug penetration with good ocular tolerability (Zambito & D i Colo, 2010). Chitosanbased nanoparticles have been prepared to enhance the ocular bioavailability of indomethacin, an
anti-inflammatory drug used to minimise ocular inflammation after cataract surgeries. The
prepared ocular carriers showed controlled and higher bioavailability than that of the drug
solution (Badawi et al., 2008).
1.2.4.4. Solid lipid nanopraticles
Solid lipid nanoparticles (SLNs) are used as a colloidal carrier system for controlled drug
delivery. SLNs represent an alternative carrier system to emulsions and polymeric nanoparticles.
21
Chapter 1….Literature Review
The main reasons for their development is more stable alternatives to liposomes and the
combination of advantages from different carriers systems like liposomes and polmeric
nanoparticles (Muller et al., 2000).
SLNs have been evaluated for ocular controlled drug delivery (Cavalli et al., 2002; Cavalli et al.,
1995; Gonzalez-Mira et al., 2011). For instance, tobramcin-loaded SLNs produced significantly
higher bioavailability in aqueous humour when compared with an equal dose of tobramycin
administered using standard commercial eye drops (Cavalli et al., 2002). Physical instability and
encapsulating water-soluble drugs into the lipidic core are still unresolved issues. It has been
found that SLN formulations gelled after a relatively short period of storage. Although this can be
prevented by altering the composition and concentration of stabilising surfactants, these
stabilising additives are hampered by toxicological considerations and incompatibilities with the
incorporated drugs due to chemical degradation (Freitas & Muller, 1999). Recently, flurbiprofenloaded SLNs (FB-SLNs) have been prepared from stearic acid, miglyol 812 and castor oil. The
prepared FB-SLNs were evaluated for ocular toxicity using the ocular Draize test. The tested
formulations were found to be minimally irritant (Gonzalez-Mira et al., 2011).
1.2.4.5. Dendrimers
Dendrimers are artificial nano-constructs and as the name suggests, have a tree-like structure.
Their basic units are active chemical moieties, which construct around small molecules or the
core using connectors and branching units. Poly(amidoamine) (PAMAM), poly-lysine, or
poly(oropylenimine)-based dendrimers are amongst the preferred classes of these (Jain &
Asthana, 2007; Tomalia et al., 1985). The final shape of the dendrimers is globular, with hollow
internal cavities and a number of terminal groups. By virtue of the highly branched terminals, a
number of drugs can be encapsulated or attached to the surface or peripheral group and hence,
dendrimers can be utilised as a potential drug delivery system. For instance, many poorly soluble
drugs can be properly encapsulated in the internal cavities of dendritic constructs (Cheng et al.,
2008; Jain & Asthana, 2007; Quintana et al., 2002; Tomalia et al., 1985).
PAMAM dendrimers were prepared for controlled ocular delivery of pilocarpine nitrate and
tropicamide (Vandamme & Brobeck, 2005). The macromolecular carriers showed a prolonged
ocular residence time compared with 0.2% w/v carbopol 980 NF solution. The residence time of
the dendrimers was markedly dependent on the size and molecular weight of the prepared
22
Chapter 1….Literature Review
carriers. The main drawback which hampers the wide use of dendrimers as a drug delivery
system is the lack of sufficient evidence of their safety and toxicity (Jain & Asthana, 2007).
Recent evidence has shown that fatal complication such as disseminated intravascular
coagulopathy was reported after iv injection of small doses (> 10 mg/kg) of positively charged
PAMAM dendrimers generations 3.5 to 7 in sizes of 50 nm in mice. Also, hemobilia
(haemorrhage of the biliary tract) and a preferential toxicity on the intestinal wall in terms of
haemorrhage and intraluminal bleeding were recorded with oral administration of the PAMAM
dendrimers (Greish et al., 2010).
1.2.4.6. Cubosomes
Cubosomes are submicron lipid particles of bicontinous cubic liquid crystalline phase. They have
been proposed as a colloidal delivery system for lipophilic drugs for better solubilisation and a
sustained drug release effect (Boyd, 2003; Spicer, 2005). Research on cubosomes, as drug
carriers for ocular delivery, has been scarce (du Toit et al., 2011).
Cubosomes were prepared from a mixture of monoolein (glycerol mono-oleate) and water by
high pressure emulsification using poloxamer 407 as a dispersing agent (Gan et al., 2010). These
cubosomes were formulated as an ocular delivery system for dexamethasone (DEX). Ex vivo
permeation studies, using excised rabbit corneas showed that the apparent permeability
coefficient calculated for the prepared DEX cubosomes was 3.5-4.5 times greater than the DEX
aqueous solution. Also, the ocular bioavailability for DEX cubosomes was 1.8 times greater than
that for the DEX aqueous solution. Histological examination of the excised rabbit corneas
incubated in DEX cubosomes for 2 h did not show any harmful signs. These findings might
present cubosomes as a new ocular delivery system for water-insoluble drugs (Gan et al., 2010).
1.2.4.7. Vesicular delivery systems
The abovementioned ocular delivery devices can achieve more controlled and effective
therapeutic actions with minimal systemic and ocular side effects associated with pulse-type
conventional dosage forms. However, these systems are still not devoid of pitfalls, including:
•
Poor patient compliance during insertion, as in ocular inserts.
•
Tissue irritation and damage induced by penetration enhancers and co-surfactants
commonly used to stabilise microemulsion and collagen shields (Alany et al., 2006; Kaur
et al., 2004).
23
Chapter 1….Literature Review
•
Ocular toxicity of some polymers used to fabricate microspheres and nanoparticles such
as albumin and polybutylcyanoacrylate (Zimmer & Kreuter, 1991).
•
Toxicological concerns due to relatively high polymer concentrations required for some in
situ thermal gelation (Miyazaki et al., 2001).
•
Failure of drug delivery devices. For example, some in situ gelling systems rely on a
generous supply of cations from the resident tears for sol-to-gel phase transition.
Alteration of the composition of the tear fluid and/or the presence of dry eye symptoms
(common with diabetic patients) (Akinci et al., 2007; Dogru et al., 2001; Kaiserman et al.,
2005) might compromise the sol-to-gel phase transition of these systems. Hence, these
systems might not be optimal for drug delivery in diabetic patients.
Surfactant vesicles (liposomes and niosomes) could overcome the aforementioned hurdles (du
Toit et al., 2011; El-Gazayerly & Hikal, 1997; Gregoriadis & Florence, 1993; Le Bourlias et al.,
1995; Uchegbu & Vyas, 1998). They can offer a prolonged and controlled action at the corneal
surface and a substantial increase of ocular bioavailability, by providing an intimate contact of
the drug with the lipidic corneal barrier and preventing the metabolism of the drug by the enzyme
present at the precorneal tear film (Azeem et al., 2009; Kaur et al., 2004). Moreover, lipid
vesicles offer a promising approach to meet the need for an ophthalmic drug delivery system that
has the convenience of being in the form of eye drops, but will localise and maintain drug activity
at its site of action (Azeem et al., 2009; Kaur et al., 2004). Furthermore, plain liposomes have
been used in a form of spray for topical ocular administration for treatment of dry eye symptoms.
Two commercially available liposome products, clinically indicated for the management of dry
eye, are ClaryMist® manufactured by Savant, UK and Ocusoft® manufactured by Ocusoft, USA.
They are sprayed on the eyelids to improve the stability of the lipid layer of the tear film and
treatment of dry eye symptoms (Craig et al., 2010).
Liposomes
Liposomes are closed bilayer lipid vesicles that can be produced basically from natural non-toxic
phospholipids and a membrane stabilising lipid, most notably cholesterol. Liposomes offer many
advantages as a unique drug delivery system. They are biodegradable and biocompatible; they
can encapsulate both hydrophilic drug moieties in their aqueous milieu and lipophilic moieties
between hydrocarbon chain bilayer membranes; and their properties can be tuned with lipid
24
Chapter 1….Literature Review
composition, size, surface charge and the method of preparation (Kaur et al., 2004; Schaeffer &
Krohn, 1982).
Liposomes encapsulating gancyclovir and iododeoxyuridine (antiviral drugs) were prepared for
more efficient treatment of herpes simplex virus eye infections (Norley et al., 1986). It was found
that these delivery systems could act as improved vehicles for drug delivery in treatment of
ocular herpes simplex virus infection. Also, antisense oligonucleotides which can be used
efficiently to treat ocular diseases like cytomegalovirus retinitis can be encapsulated in the
liposomes and efficiently targeted at the retina (Bochot et al., 2002). Moreover, liposomes
encapsulating mitomycin C (MMC) were prepared in an attempt to minimise the toxicity of
mitomycin on ocular tissues. Liposomes containing 0.2 mg/ml of MMC were tested against
aqueous solutions and tamarind seed polysaccharide (TSP)-based viscous solutions. The results
showed reduced cytotoxicity of MMC in TSP viscous vehicles and liposomes compared with the
drug solution. Only liposomes encapsulating MMC vehicles produced both lower toxicity for a
rabbit corneal epithelial cell line culture and a marked reduction of the corneal healing rate in
vivo (Chetoni et al., 2007). Recently, fluconazole liposomes have been found to be more effective
than the fluconazole aqueous solution in treating Candida keratitis (corneal inflammation due to
fungal infection) in rabbits. The fluconazole liposome-treated group showed complete corneal
healing for 86.4% of the infected rabbits compared with 50% with the fluconazole solution
treated group. Liposomes offer more intimate contact with corneal barriers and can enhance
corneal drug absorption (Habib et al., 2010).
Niosomes
Over the last three decades, liposomes of different compositions used for topical ophthalmic drug
delivery have increased/prolonged the therapeutic effect while minimising toxic effects (Chetoni
et al., 2007; du Toit et al., 2011; Gregoriadis & Florence, 1993; Kaur et al., 2004; Treblay et al.,
1993).
Vesicles consisting of one or more surfactant bilayers enclosing aqueous spaces are called nonionic surfactant vesicles or simply niosomes. Niosomes are considered of particular interest as
they offer several advantages over liposomes:
•
Niosomes are more stable chemically (Kaur et al., 2004; Uchegbu & Florence, 1995).
•
Niosomes incur lower production cost due to the availability of starting materials (Sahin,
2006).
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Chapter 1….Literature Review
•
Niosomes are biodegradable and non-immunogenic (Kaur et al., 2004).
•
Niosomes do not require expensive handling (storing in a freezer and preparation under
nitrogen gas).
Nevertheless, the formulation of niosomes can pose challenges due to possible physical
instability and poor ability to encapsulate a considerable amount of water-soluble drug molecules
(Essa, 2010). Niosomes have been evaluated as ocular vehicles for a wide range of therapeutic
classes such as anticholinergic (cyclopentolate), anti glaucomic (acetazolamide and timolol
maleate) and antibiotics (gentamicin) with minimal signs of ocular irritation as well (Abdelbary
& El-gendy, 2008; Aggarwal et al., 2004; Saettone et al., 1996b; Vyas et al., 1998). Tween 20based niosomes significantly improved the ocular bioavailability of cyclopentolate, with respect
to the reference buffer drug solution and micellar solution, suggesting that it can be used as an
efficient vehicle for ocular drug delivery (Saettone et al., 1996b). Also, niosomes encapsulating
timolol maleate (anti-gluacomic agent) were prepared using the reverse phase evaporation
method and coated with 0.5% w/v chitosan solution. The timolol niosomes achieved a
significantly (1.7 times) higher timolol concentration in the aqueous humour than that of the drug
solution. This was attributed to better improvement of corneal penetration and prolonged
precorneal residence time compared with the timolol aqueous solution (Kaur et al., 2010).
Niosomes should have certain attributes in relation to their potential use as vehicles for ocular
drug delivery. These include:
•
The size of the vesicle should be large enough to resist drainage by reflex tearing and eye
blinking. For example, large multilamellar vesicles (MLV) were found to reside for a
longer period on the ocular surface than small unilamellar liposomes. This was attributed
to the ability of MLV to entrap a relatively higher amount of the drug and resist
nasolacrimal drainage (Fitzgerald et al., 1987a; Hathout et al., 2007). The same argument
applies to niosomes. Ideally, it has been reported that niosome sizes > 10 µm are suitable
for ocular delivery (Sahin, 2006; Uchegbu & Vyas, 1998).
•
The shape of the prepared niosomes should show some irregularities to fit properly into
the cul-de-sac of the eye and lodge on the eye surface (Uchegbu et al., 1992; Uchegbu et
al., 1997; Uchegbu & Vyas, 1998).
•
Ocular niosomes should ideally be thermo-responsive in order to release drug content in a
controlled, yet timely manner before being flushed by blinking and nasolacrimal drainage
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Chapter 1….Literature Review
(Uchegbu et al., 1997; Uchegbu & Vyas, 1998). Complete abolishment of gel/liquid
transition of surfactant forming niosomes could produce niosomes with an extremely low
release rate to water soluble drugs, which does not suit the short residence time of
ocularly administered ophthalmic products.
The abovementioned criteria have typically been attributed to discomes and polyhedral niosomes.
These are modified vesicular structures and are different from conventional spherical niosomes
(Azeem et al., 2009; Uchegbu & Florence, 1995; Uchegbu & Vyas, 1998). Discomes containing a
low cholesterol concentration less than 30% mol/mol were prepared by incubating the preformed
spherical niosomes in cholesteryl poly-24-oxyethylene ether (Solulan C24) at 75oC for 1 h. This
resulted in the formation of large (11-60 µm) and multifaceted vesicular systems (Uchegbu et al.,
1992). Since then, there has been only one report on discomes as an ocular delivery system for
timolol maleate (Vyas et al., 1998). The prepared discomes have been found to entrap a relatively
large quantity of timolol and improve the ocular bioavailability compared with a timolol maleate
solution (Vyas et al., 1998). One possible reason for such scarcity is the need for a relatively high
temperature during discome preparation which might affect the chemical stability of some
thermo-labile therapeutic agents. On the other hand, spherical niosomes have been shown to
possess more stable membranes than polyhedral ones due to the presence of cholesterol
(Arunothayanun et al., 1999).
In conclusion, ophthalmic niosomes could offer prolonged drug release and enhance drug
permeation. Thus, the potential of niosomes as a vehicle for ocular delivery of naltrexone
hydrochloride are studied in this thesis. Naltrexone hydrochloride has been found to be a
promising treatment for diabetic keratopathy. Causes, prevalence and other treatment options of
diabetic keratopathy are discussed in the following sections.
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Chapter 1….Literature Review
1.3. Diabetic keratopathy and the available treatment options
1.3.1. Physiology of the cornea
The physiological functions of the cornea are (Klyce & Beuerman, 1998; Nishida, 2005):
•
Refractive: it transmits and focuses light onto the retina.
•
Protective: it forms a mechanical and chemical barrier to the posterior segment of the eye.
•
Regulatory: it contains the intraocular pressure.
1.3.2. Diabetes mellitus and the cornea
Diabetes mellitus has increasingly attracted the attention of scientists, especially since the
WHO’s announcement that “the worldwide burden of diabetes in adults was around 173 million
in the year 2002”. The incidence of diabetes has risen dramatically in recent decades and a twofold, or greater, increase is expected to occur in the coming decades (Wild et al., 2004).
Diabetes mellitus has a serious impact on the tissues of the entire eye. For instance, diabetic
retinopathy (damage of the retina from diabetes) and cataracts (opacity of the eye lens) are major
ocular complications arising from diabetes. Diabetic retinopathy and cataracts are the leading
causes of vision loss in the industrial world. These disorders account for approximately 6% of the
US population and 17% of Americans ≥ 40 years, respectively (Frank, 2004; Kenny et al., 1995).
However, in the last four decades, diabetic retinopathy has been investigated fully. Advances in
treatment over the past 40 years have greatly reduced the risk of blindness from this disease
(Frank, 2004; McLaughlin et al., 2010).
On the other hand, diabetic effects on the anterior segment of the eye have been reported for
many decades (Herse, 1988; Hyndiuk et al., 1977; McLaughlin et al., 2010; Schwartz, 1974).
Observed diabetic abnormalities in the anterior segment of the eye include:
•
Decreased tear production (Herse, 1988).
•
Presence of glucose in tears (Gasset et al., 1968).
•
Increased risk of infection (Herse, 1988; Schultz et al., 1981 ).
•
Corneal epithelial fragility (Schultz et al., 1981 ).
•
Wrinkling of Descemet’s membrane (Henkind & Wise, 1961).
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Chapter 1….Literature Review
•
Reduced corneal sensation (Ishida et al., 1984; Schwartz, 1974).
•
Delayed wound repair (Lambiase et al., 2000; Lambiase et al., 1998b).
•
Neurotrophic corneal ulcers (Hyndiuk et al., 1977).
Corneal disorders secondary to diabetes (diabetic keratopathy) are increasingly recognized as a
cause of the morbidity associated with diabetes (Cisarik-Fredenburg, 2001; Kaji, 2005). Indeed,
diabetic keratopathy has been estimated to occur in 47-64% of diabetic patients during the course
of their disease (Schultz et al., 1981 ) and diabetics have an increased risk of developing corneal
epithelial fragility, epithelial defects, recurrent epithelial erosions, decreased sensitivity,
abnormal wound healing, increased susceptibility to injury and non-healing or infected corneal
ulceration. Ultimately such corneal complications may lead to visual impairment. Diabetic
keratopathy is mainly attributed to corneal nerve tortuosity and degeneration as part of general
neuropathy of diabetes mellitus (Rosenberg et al., 2000; Ruben, 1994; Schwartz, 1974).
Nevertheless, the available treatment options for diabetic keratopathy are tear substitutes and
antibiotics. Such treatments are symptomatic and conservative to keep the cornea hydrated with
minimal microbial burden. There has not yet been a causative treatment developed for this
serious diabetic condition. However, damage of the corneal nerves due to diabetes has been
thought to be the main culprit (Feman, 2000; McLaughlin et al., 2010; Schultz et al., 1981 ). The
following section sheds light on corneal nerve role and structure.
1.3.3. Corneal innervations
Innervations of the cornea are essential for pain sensation and for tissue repair (Nishida, 2005).
The cornea is the most densely innervated mammalian tissue (Figure 1.5). Human cornea has a
nerve density of 300-600 times greater than that of the skin and 20-40 times greater than that of
the tooth pulp (Muller et al., 1997). Most of the sensory nerves in the cornea are derived from the
ciliary nerves of the ophthalmic branch of the trigeminal nerve (Nishida, 2005). Nerve bundles
enter the corneal mid stroma at the periphery, each containing a few myelinated fibres and a
number of finer unmyelinated fibres. The unmyelinated nerve endings are sensitive to touch,
temperature and chemical stimulation (Muller et al., 1997). Because transparency is of prime
importance, the cornea is avascular and receives necessary nutrients via diffusion from the tear
film, the aqueous humour and from neurotrophins supplied by nerve fibres (Gobbels et al., 1989).
Axons from the trigeminal ganglion and sympathetic ganglion terminate in delicate endings
among the epithelial cells of the cornea.
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Chapter 1….Literature Review
Figure 1.5 Human corneal nerves penetration and distribution, modified from (Muller
et al., 1997) and reproduced with permission of the copyright owner.
Many ocular, neurological and systemic diseases can alter the typical corneal nerve architecture
and impair corneal sensitivity (Table 1.1) (Lambiase et al., 1999). A major complication of that is
the neurotrophic corneal ulcer. It is a degenerative corneal disease characterised by the
impairment of corneal sensitivity associated with epithelial breakdown, a deficiency in the
healing process leading to corneal ulceration, and subsequent vision loss.
Neurotrophic corneal ulcers are classified into three stages (Mackie, 1995):
Stage 1 is characterised by rose bengal staining of the inferior palpebral conjunctival surface, a
decrease in tear breakup time, and punctate keratopathy. If these changes become chronic,
superficial vascularisation, stromal scarring, epithelial hyperplasia and irregularity may develop.
Stage 2 is characterised by epithelial breakdown where cells assume an oval or circular shape that
is often localised in the superior half of the cornea. The area of epithelial deficit is usually
30
Chapter 1….Literature Review
surrounded by loose epithelium that, in the absence of a healing process, becomes hazy,
oedematous, and poorly adherent to Bowman’s layer. The edges of the defect become smooth
and rolled as the defect ages without appreciable epithelial growth (Cavanagh et al., 1976).
Stage 3 is characterised by stromal involvement with a corneal ulcer that may progress to melting
and perforation. Secondary infection or topical treatment with corticosteroids increases the risk of
perforation (Groos, 1997).
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Chapter 1….Literature Review
Table 1.1 Causes of decreased corneal sensitivity
Category
Disease
Genetic
Riley-Day syndrome (familial dysautonomia)
Goldenhar-Gorlin syndrome
Mobius syndrome
Familial corneal hypoesthesia
Systemic
Diabetes
Leprosy
Vitamin A deficiency
Central nervous system
Neoplasm
Aneurysm
Ischaemia
Neurosurgical procedures for acoustic
neuroma or trigeminal neuralgia
Other surgical injury to the trigeminal nerve
Ocular
Herpes infection (herpes simplex or herpes
zoster)
Chemical and physical burns
Abuse of topical anaesthetics
Topical toxicity (timolol, betoxolol,
diclofenac sodium, sulfacetamide)
Surgical or laser treatment to cornea
Corneal dystrophies (lattice, granular)
Chronic ocular surface injury or inflammation
Contact lens wear
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Chapter 1….Literature Review
Over the past few years, there has been growing interest in exploring the mechanisms by which
various growth factors and therapeutic agents promote healing of corneal epithelial cells in order
to establish any significant correlations between topical administration and accelerated corneal
healing (Kaji, 2005; Micera et al., 2004; Schultz et al., 1981 ; Woo et al., 2005). Before
discussing the treatment options, different factors ameliorating altered corneal healing will be
mentioned.
1.3.4. Mechanisms of corneal maintenance and healing
The corneal epithelium is composed of a squamous epithelium of 5-7 layers of stratified cells
attached to a basement membrane and is separated from the stroma by Bowman’s layer. Trauma,
disease and corneal surgery, including the increasingly popular refractive laser surgery, all cause
the cornea to enter a phase of wound healing. Upon wounding, the cornea, which has less
immune protection than other tissues due to the lack of blood vessels, needs to re-epithelialise
urgently in order to restore its barrier function.
The healing of epithelial wounds can be divided into several distinct but continuous phases:
sliding of superficial cells to cover the denuded surface, cell proliferation, and stratification for
re-establishment of multicellular layers (Lu et al., 2001). Although several layers participate in
cell migration, the majority of the defect is initially covered by the sheet-like movement of a
monolayer of epithelial cells, followed by a landslide-like mass movement of the epithelium
(Suzuki et al., 2003).
Based on the XYZ hypothesis for corneal epithelial maintenance put forward by (Thoft & Friend,
1983), researchers have assumed that limbal stem cells and transient amplifying cells are induced
after acute wounding and that these cells always migrate centripetally to repair the wound.
However, it has been recently reported that after corneal wounding, the capacity for epithelial cell
proliferation and migration appears to be as active in the central cornea as in the peripheral/limbal
areas (Chang et al., 2008). Indeed, central and peripheral epithelial recovery remains equal even
after ablation of the limbus. Thus corneal epithelial recovery may be independent of limbal stem
cells (the mother cells of the Y component in the previous hypothesis) in a human corneal
organotypic model. Therefore, central human corneal epithelial cells are fully capable of corneal
epithelial regeneration, at least in the first 12 h after wounding. This observation may lead to new
insights into the pathogenesis and therapy of corneal epithelial diseases.
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Chapter 1….Literature Review
Corneal epithelial cell migration is modulated by various humoral and extracellular matrix
(ECM) proteins (Lu et al., 2001). Growth factors, such as insulin-like growth factor (IGF)-1
(Nagano et al., 2003), epidermal growth factor (EGF)(Maldonado & Furcht, 1995a), interleukin 6
(Nishida et al., 1992), fibroblast growth factor-2 (FGF-2) (David et al., 1995), transforming
growth factor-β (TGF-β) (Saika et al., 2004), keratinocyte growth factor (KGF), and hepatocyte
growth factor (HGF) (Sharma et al., 2003; Wilson et al., 1993) have been shown to stimulate
corneal epithelial cell migration both in vivo and in vitro. Glycoproteins of the ECM, such as
fibronectin (Fn), laminin (Ln), and collagen IV, also facilitate cell migration (Maldonado &
Furcht, 1995b; Ohji et al., 1993). However, the precise relationship between these cytokines and
ECM protein production in the corneal epithelium is not well understood.
Once the wound area is covered the cells are further induced to proliferate to restore the stratified
nature of the epithelium. Corneal wounding induces an increased release of several growth
modulating cytokines and many of these cytokines have been linked to the regulation of epithelial
cell proliferation (Lu et al., 2001). The presence of TGF-β in the later stages prevents over
proliferation of the epithelial cells (Joyce & Zieske, 1997).
Whilst the above data seems to indicate specific roles for specific cytokines in corneal wound
healing, the interaction between growth factors and cells, and growth factor to growth factor,
inevitably means that the right combination of factors needs to be expressed with spatial and
temporal coordination to ensure correct wound healing.
1.3.5. Treatment of diabetic keratopathy
1.3.5.1. Standard treatment
The standard treatment of neurotrophic corneal ulcers consists of a number of components
including: maximising preservative free topical lubricants, minimising evaporative tear loss, use
of topical antibiotics, protecting the corneal surface with a bandage contact lens, covering the
ocular surface via lid closure by patch, tarsorrhaphy or induced ptosis, and more permanent
solutions such as construction of a conjunctival flap. However, even in combination these
measures may be ineffective, and the outcome is often severe impairment or loss of vision
(Lambiase et al., 1998b).
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Chapter 1….Literature Review
1.3.5.2. Growth factors
A number of growth factors has been identified in the corneal epithelium and their gene
expression unravelled (Hongo et al., 1992; Kiritoshi et al., 1991; Schultz et al., 1990). The role of
growth factors in maintaining the normal structure and function of the cornea, and in corneal
epithelial healing has emerged over the past three decades (Schultz et al., 1990; Tripathi et al.,
1990). Autologous serum has been shown to be beneficial in epithelial wound healing both in
vitro and in clinical studies (Schulze et al., 2006). Although the exact mechanism for these effects
is not entirely clear, growth factors such as EGF and TGF may play a role.
A brief overview of some of the key growth factors involved in corneal epithelial healing
follows.
Insulin-like growth factor-1 (IGF-1)
IGF-1 is a multifunctional regulatory peptide that shares structural homology with proinsulin. It
has been shown to mediate proliferation, differentiation, and survival effects, depending on the
target cell and the presence of other hormones and growth factors (Gockerman et al., 1995). IGF1 is secreted by the liver. Like other growth factors, it is present in high concentrations in serum,
produced locally, and may act in an autocrine or paracrine manner (McAvoy & Chamberlain,
1990). Accumulated evidence indicates that IGF-1 promotes cell motility in a variety of normal
and malignant cell types. However, the effect of IGF-1 on corneal epithelial cell motility and
migration is controversial (Nishida et al., 1996). It has been reported that substance P (SP) and
IGF-1 synergistically stimulated corneal epithelial migration in an organ culture of the cornea.
Accordingly, it was found that addition of SP or IGF-1 separately did not affect epithelial cell
migration, yet co-treatment with SP and IGF-1 significantly stimulated epithelial cell migration
(Nakamura et al., 1997). Also, it was noted that IGF-1 significantly increased migration and
expression of laminin-5 in cultured human corneal epithelial cells (Lee et al., 2006).
In the clinical setting, the administration of eye drops containing both SP and IGF-1 has been
shown to be an effective treatment in the case of a child with neurotrophic and anhidrotic
keratopathy (Brown et al., 1997). Moreover, eye drops containing peptides based on SP and IGF1 have been reported to be effective in the prevention of superficial punctate keratopathy in
diabetic patients after cataract surgery and have also successfully induced rapid epithelial
resurfacing in patients with persistent corneal epithelial defects (Yamada et al., 2008).
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Chapter 1….Literature Review
Insulin
Insulin, an anabolic peptide hormone closely related to IGF, is another growth factor implicated
in wound repair, but its role is not well documented (McAvoy & Chamberlain, 1990). Insulin
stimulates hepatotactic migration of human epidermal keratinocytes (Benoliel et al., 1987) and
topical insulin therapy has been shown to aid the healing of ulcerations (Rosenthal, 1968; Van
Ort & Gerber, 1976) and burns (Pierre et al., 1998). More interestingly, it has been shown that
insulin is present in human tear fluid, and receptors to insulin have been detected on the human
ocular surface (Rocha et al., 2002), cornea (Naeser, 1997) and neuronal and vascular tissues of
the retina (Im et al., 1986; Thomopoulos & Pessac, 1979). The functions of insulin receptors
within structures of the eye are not yet known, but diabetes is the major cause of blindness in
people of working age and is often associated with disorders of the corneal epithelium
(Rosenberg et al., 2000; Zagon et al., 2002a).
Recently, intensive systemic therapy with insulin which establishes normoglycemia in rats with
diabetes was found to prevent the delay in wound healing of the ocular surface epithelium in
poorly controlled diabetic animals (Zagon et al., 2006c). Normoglycemia in diabetic animals,
induced by systemic treatment with insulin, restores decreased levels of DNA synthesis in the
ocular surface epithelium to normal values when examined three weeks after wounding (Zagon et
al., 2006c). Also, it has been reported that treatment with topical insulin significantly accelerates
wound healing in diabetic rats compared with the untreated diabetic group. However, topical
insulin had no effect on corneal re-epithelialisation of corneal wounds in healthy rats (nondiabetic) (Zagon et al., 2007a). These results were attributed to the ability of topical insulin to
restore the decreased levels of DNA synthesis in basal epithelial cells to normal values, seen 48 h
after wounding.
These observations present a new and promising therapeutic indication for insulin in the
treatment of corneal ulcers in diabetics. Topical application of insulin to the surface of the eye
will inevitably result in some systemic absorption. It has been shown previously monitoring
serum glucose levels up to 14 h of topical exposures to 1 U of insulin had no effect on plasma
glucose values in diabetic or healthy rats (Zagon et al., 2007a). However, the insulin used in this
study was compounded as a simple solution. This is different from the commercially available
insulin dosage forms. Commercial insulin contains pharmaceutical additives that are added to
36
Chapter 1….Literature Review
maintain and prolong the physical and microbial stability of the formulation. These may include
benzalkonium chloride (preservative) and sodium editate (antioxidant and chelating agent). These
additives have well-established penetration enhancing effects. This might increase systemic
insulin absorption and as such might have an effect on blood glucose levels. Therefore, the
aforementioned therapeutic benefits might be offset by possible systemic absorption of insulin
via the ocular route, hence potentiating the effect of co-administered hypoglycaemic drugs (Lee
et al., 2002). Table 2.1 presents the major attributes of insulin.
Table 1.2 Major attributes of insulin
Advantages
Successfully enhanced corneal healing
rate in diabetic rodent
Disadvantages
Limited clinical trials (unknown
mechanism)
Insulin (6 kDa protein) cannot easily
cross intact corneal barrier
Long term safety on ocular tissue is
controversial
Nerve growth factor (NGF)
NGF is a non-covalently linked dimer consisting of two 118-residue polypeptides, each of which
contains three intramolecular disulphide bridges. NGF is a prototypical member of the
neurotrophin family of growth factors that promote survival and growth of sympathetic and
sensory neurons and differentiation of neurons in the central nervous system (Levi-Montalcini,
1987). Its biological action is not restricted to cells of neuronal origin but extends to cells of the
immune system. NGF receptors (TrKA) were identified on the human corneal surface (Lambiase
et al., 1998a). NGF is expressed constitutively in normal human and rat corneas. Published data
supports the possibility that NGF modulates ocular inflammation and corneal epithelial
proliferation and differentiation through its receptors. Also, recent evidence has demonstrated
that NGF is identified in the cornea, conjunctiva, tear fluid, and lacrimal glands (Ghinelli et al.,
2003; Ríos et al., 2007; Touhami et al., 2002; You et al., 2000).
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Chapter 1….Literature Review
Many reports describe the relationship between NGF and the ocular surface health and wellbeing: one in vitro study showed that NGF induces proliferation and differentiation of rabbit
corneal epithelial cells (Kruse & Tseng, 1993). A clinical report showed an increase in NGF
plasma levels in vernal keratoconjunctivitis (VKC) with a direct correlation between mast cell
conjunctival infiltration and NGF levels (Lambiase et al., 1995).
Topical murine NGF eye drops was applied to 14 eyes with non-infectious corneal ulcers caused
by essential neurotrophic keratitis (5 eyes), chemical burns (3 eyes), abuse of topical anaesthetics
(2 eyes), orbital tumour surgery (1eye), surgery of acoustic neuroma (1 eye), penetrating
keratoplasty of unknown reason (1 eye) and a lamellar keratoplast for a herpatic vascularized scar
(1 eye) (Lambiase et al., 1998b). Remarkably, considering the diverse aetiology, all patients had
complete corneal healing after 10 days to 6 weeks of NGF treatment. However, all patients
experienced mild to moderate conjunctival hyperaemia accompanied by pain, photophobia, and
superficial or deep corneal neovascularisation occurred in 9 of the 14 treated eyes. All ocular
symptoms disappeared once the corneal ulcers had completely healed.
In another study, it has been reported that murine NGF (dosage: one drop of 200 µg/ml NGF
solution every 2 h for 2 days, followed by one drop six times daily until the ulcer showed signs of
healing, followed by one drop four times daily of 200 µg/ml NGF solution) improved corneal
sensitivity and promoted corneal epithelial healing in 45 eyes with both moderate and severe
neurotrophic keratitis within 12 to 42 days of NGF treatment (Bonini et al., 2000). Visual
functions such as corneal sensitivity and visual acuity were improved, and healing of the corneal
ulcers was promoted in NGF-treated eyes. Interestingly, except in a few cases, no relapse of the
disease occurred during the follow-up period. However, transient adverse effects such as
hyperaemia and pain in the eye were observed on instigation of NGF treatment. Supporting to
that, a report has suggested NGF as a future treatment in several pathologies of both the ocular
surface and the retina (Micera et al., 2004).
Therefore, NGF has attracted the attention of pharmaceutical scientists to develop specialised
drug delivery systems aimed at inhibiting NGF’s degradation, prolonging its biological half-life
and enhancing its biological activity (Pfister et al., 2008; Sakiyama-Elbert & Hubbell, 2000; Xie
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Chapter 1….Literature Review
et al., 2005). All of the aforementioned evidence augments the clinical interest in NGF as a
potential therapeutic agent to promote corneal wound healing. However, most of the
experimental evidence in relation to the clinical efficacy of NGF has been obtained with the 2.5S
murine NGF which has several limitations (Table 1.3).
Several attempts have been made to produce recombinant human nerve growth factor (rhNGF)
using different micro-organisms, including Saccharomyces cerevisiae (Kanaya et al., 1989;
Nishizawa et al., 1993) and Escherichia coli (Negro et al., 1992; Rattenholl et al., 2001) as well
as insect (Allen et al., 2001; Barnett et al., 1991) and mammalian cells (Iwane et al., 1990).
However, most of these studies have been carried out in vitro, and the available evidence in vivo
indicates that the action of rhNGFs in human peripheral neuropathies was not comparable with
the effect of murine NGF (Apfel, 2002). The technology of rhNGF preparation is not complex;
however the biological activity of β-NGF relies on the formation of three disulfide bonds and a
cysteine knot within two β-chains of 120 amino acids each after cleavage of pro-peptide
sequences from a larger precursor molecule (Edwards et al., 1988; Shooter, 2001; Ullrich et al.,
1983). Recently, it has been reported that the production (on a laboratory scale) of large amounts
of rhNGF was shown to be effective both in vitro and most importantly in vivo (Colangelo et al.,
2005).
Table 1.3 Major attributes of murine NGF
Advantages
Successfully used in many preclinical
and clinical trials
Disadvantages
High cost
Limited sources and insufficient quantity
(adult male mouse submandibular
glands, snake venom)
Allodynia (hyperalgesia induced upon
topical administration)
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Chapter 1….Literature Review
Hepatocyte growth factor (HGF), epidermal growth factor (EGF), keratinocyte growth factor
(KGF), transforming growth factor-β (TGF-β)
HGF, EGF, KGF, and TGF-β are expressed in keratinocytes, corneal endothelial cells and upregulated in rabbit keratocytes after wound healing (Li & Tseng, 1996; Wilson et al., 1993).
However, conflicting results have been reported in human trials, such as those described for EGF
(Daniele et al., 1992; Hosotani et al., 1995). Moreover, TGF-β has been implicated as a potent
stimulant of the scarring process in the eye. Corneal scarring is a major problem in influencing
results of photorefractive surgery, giving rise to symptoms of haze and resulting in a reduction of
the best corrected visual acuity (Jester et al., 1997; Mayers et al., 1997).
Opioid growth factor (OGF)
Endogenous opioids and their receptors were originally thought to be related to neural
modulation (Akil et al., 1984). Although there are numerous endogenous opioids and respective
receptors, only the native opioid peptide, [Met5]enkephalin, is classified as an opioid growth
factor (OGF). OGF is a pentapeptide with the sequence Tyr-Gly-Gly-Phe-Met. OGF has been
determined to serve a growth regulatory role and its binding receptor is not homologous to classic
opioid receptors. OGFs act as growth regulators in many normal and malignant tissues (Zagon &
McLaughlin, 1991b, 1993a). It has been identified in both eukaryotes and prokaryotes as a potent
inhibitor of cell replication (Zagon & McLaughlin, 1991a, 1991b, 1993a). In vertebrates, OGF is
also thought to be important in cellular maturation and survival (Meriney et al., 1991; Zagon &
McLaughlin, 1993a). OGF has been documented as being produced in an autocrine and/or
paracrine, manner (Zagon et al., 1994a). OGF activity is mediated by the zeta (ζ)-opioid receptor
(Zagon & McLaughlin, 1993a, 1993b).
Endogenous opioid systems influence proliferation in a wide variety of cells and tissues both in
vivo and in vitro. These include developing neurons and galia of the mammalian nervous system
(including the retina), glial cells in culture, myocardial and epicardial cells in neonatal heart,
neuronal and non-neuronal cancer cells, and cellular renewal in the epithelium of the adult tongue
(McLaughlin, 1994; Zagon & McLaughlin, 1991a, 1991b, 1993a; Zagon et al., 1994b). OGF has
been detected in the bovine corneal epithelium (Tinsley et al., 1990; Tinsley et al., 1989). OGF
and the ζ-receptor are also present in the normal rabbit corneal epithelium (Zagon et al., 1995).
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Chapter 1….Literature Review
A study was performed to show the physiological role of endogenous opioid systems (opioid
peptides and receptors) in the mammalian corneal epithelium in vitro using rabbit corneal
explants (Zagon et al., 1995). The following strategies were adopted; firstly, naltrexone (opioid
antagonist) was administered to explant cultures of rabbit corneal epithelium to examine whether
this approach modulates epithelial outgrowth; secondly, OGF was administered to cultures of
corneal explants to investigate the effects of this opioid peptide on the extent and organization of
epithelial outgrowth. Immunocytochemistry experiments using antibodies specific to OGF and ζreceptor were performed to confirm that OGF systems are present in normal corneal epithelium
in culture. The study demonstrated that OGF and ζ-receptor are present in the normal corneal
epithelium and, more surprisingly, OGF exerted an inhibitory effect on cellular proliferation and
growth in the corneal epithelium. This tonically active growth regulatory inhibitory pathway is
involved in cell replication. OGF targets cyclin-dependent kinase inhibitors (P16 and/or P21)
(McLaughlin et al., 2010). The regulation of cells proliferation by OGF-OGF receptor (OGFr), as
aforementioned, has been termed OGF-OGFr axis for cellular renewal and proliferation
(McLaughlin et al., 2010).
Fluorescence imaging of OGFr by green fluorescent protein-labelled OGFr revealed that OGFr is
assembled into outer nuclear envelope. This complex is shuttled to the nucleus. The OGF-OGFr
complex unregulated p16 and/or p21 leading to a marked delay in the G1-S phase of the cell
cycle. Naltrexone (a long acting potent opioid antagonist) administration blocks OGF-OGFr
interactions. Hence, this increases cell proliferation (Cheng et al., 2009). Blockade of opioid
receptors by naltrexone (a long acting potent opioid antagonist) for seven days significantly
increased the extent of proliferation and outgrowth of the corneal epithelium in vitro. This study
suggested, for the first time, an inhibitory role for an endogenous growth factor. Moreover, the
study demonstrated that blocking of the corresponding receptor by naltrexone significantly
increased DNA synthesis, cell migration and proliferation of corneal epithelium.
Other reports have demonstrated that topical or systemic application of naltrexone markedly
accelerates DNA synthesis and wound healing of corneas in humans (Zagon et al., 2000), rats
(Zagon et al., 1998b) and rabbits (Zagon et al., 1998a) as well as in diabetic rodents (Zagon et al.,
2002a). Indeed, topical application of naltrexone healed corneal epithelial wounds in uncontrolled
Type 1 diabetic rodents at rates equal to or surpassing that in normal controls (Klocek et al.,
41
Chapter 1….Literature Review
2007). However, concomitant application of topical naltrexone and topical insulin does not have
an additive or synergistic effect on corneal re-epithelialisation (Klocek et al., 2009).
In another study, the same group used a gene gun to introduce sense and antisense
complementary DNA (cDNA) for OGFr to establish that the effects of OGF on corneal
reepithelialization are specifically dependent on the OGFr. This study establishes, for the first
time, that the OGF-OGFr axis is an autocrine loop that serves as a crucial regulatory system for
corneal epithelial wound healing. The plasmid pcDNA3.1-OGFr, carrying the rat OGFrcomplementary DNA in both the sense and antisense orientations, and control, were delivered by
gene gun to the rat cornea. After 24 h, corneas were abraded and re-epithelialisation was
monitored by fluorescein photography. Corneal cells exposed to sense OGFr exhibited corneal
defects that were up to 52% larger than the control group. On the other hand, corneas subjected to
the antisense constructs of OGFr exhibited defects that were up to 56% smaller than the control
group (Zagon et al., 2006b).
Naltrexone has been found to enter human cells by passive diffusion. Fluorescent-tagged
naltrexone was found to accumulate in the cytoplasm of the human head and neck squamous cell
carcinoma cell line as little as 60 s after incubation. Neither fluorescein dye nor fluoresceintagged dextrans were found to enter the cells (Cheng et al., 2009). In the same study, untagged
naltrexone and fluorescent-tagged naltrexone were found with an increase of up to 70% in cell
proliferation markers compared with the vehicle-treated group (Cheng et al., 2009). Table 1.4
summarises the major features of naltrexone.
42
Chapter 1….Literature Review
Table 1.4 Major attributes of naltrexone
Advantages
Successfully reversed most signs of
diabetic keratopathy (corneal sensation,
delayed wound repair, dry eye)
Well-defined mode of action
Synthetic chemical entity (commercially
available)
Safe on ocular tissues
Disadvantages
Extremely bitter taste (nasolacrimal
drainage)
1.3.5.3. Substance P
Substance P (SP) (11-amino acid polypeptide) is a neurotransmitter of the trigeminal nerve and a
member of the tachykinin family. SP is present in physiologically relevant concentrations in the
normal cornea (Nishida et al., 1996). SP concentrations in tears are thought to reflect the
neuropeptide levels in ocular tissues. In patients with unilateral corneal hypesthesia, substance P
concentrations in tears from the affected eye were significantly lower than those in tears from the
unaffected eye (Yamada et al., 2000).
Topical application of SP alone has no detectable effect on the rate of corneal epithelial wound
closure in rabbits (Kingsley & Marfurt, 1997). However, SP in combination with IGF-1 has been
demonstrated to facilitate synergistically corneal epithelial migration in rats with trigeminal
denervation (Nagano et al., 2003) and in organ cultured corneas (Nishida et al., 1996). As
discussed above, this combination has been shown to be effective in healing corneal epithelium in
humans in the clinical setting. Unfortunately, SP is not available commercially.
1.3.5.4. Aldose reductase inhibitors
Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway in which glucose is
converted to sorbitol (Herse, 1988). AR activity is implicated in diabetic corneal epithelial
disease primarily because of the effect that AR inhibitors have on these documented diabetic
epithelial abnormalities (Herse, 1988). The AR inhibitor was applied topically to treat a non43
Chapter 1….Literature Review
healing corneal ulcer in a patient with diabetes. The corneal ulcer healing was dramatically
improved (Ohashi et al., 1988). Topical CT-112 (an AR inhibitor) was used to treat two patients
with diabetes with non-resolving corneal epithelial lesions (Ohashi et al., 1988). Not only did the
lesions resolve, but they returned when the CT112 therapy was discontinued and resolved again
when CT-112 therapy was reinstituted.
The patients remained lesion-free on long-term
maintenance therapy with CT112. Also, topical CT-112 was applied to a group of galactosaemic
rats, vehicle alone to a second group of galactosaemic rats and vehicle alone to a third group of
normal rats (Yokoi et al., 1997). After 3 weeks, fluorescein uptake (as an indicator of corneal
permeability) was measured by fluorophotometry in the three groups. The galactosaemic rats that
received vehicle alone displayed a significantly higher sodium fluorescein uptake than the normal
rats and the CT-112 treated galactosaemic rats showed no significant difference in the fluorescein
uptake between the vehicle-treated normal rats and the CT-112-treated galactosaemic rats.
Treatment with AR inhibitors has been shown to reduce corneal changes after they occurred, and
limit the development of these corneal changes when compared to untreated controls in both
animals and humans. The mechanism by which increased AR activity contributes to diabetic
corneal pathology remains unknown (Herse, 1988).
1.3.5.5. Miscellaneous
Nicergoline is an ergoline derivative known to cross the blood-brain barrier and is now widely
and safely used to treat cognitive impairment from stroke and degenerative dementia. In vivo
studies have shown that nicergoline treatment induces significant increase in the NGF levels
within the frontal region of the brain and supports cholinergic neurons, increasing the content of
NGF and brain-derived neurotrophic factor in the brain of aged rats (Giardino et al., 2002; Nishio
et al., 1998). Recently, it has been found that oral nicergoline (10 mg/kg per day) for 2 weeks
increased the rate of corneal wound healing in 50 rat eyes (Kim et al., 2009). The promoting
effect of nicergoline in corneal wound healing is attributed to increased NGF in corneas and/or
lacrimal glands. However, the exact ocular tissue responsible for increasing NGF levels as a
response to nicergoline has not yet been identified. Recently, oral treatment of nicergoline at least
for 4 weeks has been found to restore corneal sensation in rats after photorefractive keratectomy
(Kim et al., 2011).
44
Chapter 1….Literature Review
1.4. Thesis aims and structure
The cornea is the most densely innervated mammalian tissue and the integrity of corneal nerves is
of paramount importance for the refractive and protective functions of the cornea. With the
advent of modern refractive surgeries and the increasing number of diabetic patients worldwide,
the cornea is routinely subjected to injury and this damage may cause transient or chronic
neurotrophic deficits (corneal scarring). If this damage does not receive proper medical attention
loss of vision can ensue.
The corneas of diabetic patients show significant and characteristic signs known collectively as
diabetic keratopathy. These characteristic diabetic signs include: delayed wound repair, reduced
tear production, reduced corneal sensation and neurotrophic corneal ulcers. The prevalence of
diabetic keratopathy is approximately 47% to 64% during the course of diabetes mellitus (Schultz
et al., 1981 ).
The available treatment options are topical antibiotics, tear substitutes and eye patches. This
treatment is in most cases insufficient because it does not treat the underlying causes
(McLaughlin et al., 2010).
Naltrexone (a potent opioid antagonist) has recently been found to reverse the signs and
symptoms of diabetic keratopathy. Naltrexone successfully accelerates corneal wound healing,
both systemically and topically, in normal and diabetic rats, rabbits and humans (McLaughlin et
al., 2010; Zagon et al., 2009; Zagon et al., 1997; Zagon et al., 1998a, 1998b; Zagon et al., 2007b).
Recently, it has been found that naltrexone can restore impaired corneal sensation in diabetic rats
(McLaughlin et al., 2010; Zagon et al., 2009). These effects are attributed to blocking opioid
growth factor receptors in corneal tissues and consequently accelerate DNA synthesis in the
cornea and re-epithelialisation. Whilst there is substantial clinical evidence on the effectiveness
of naltrexone in diabetic keratopathy, there are hardly any reports on its formulation or
incorporation into an appropriate ocular delivery system.
Naltrexone has an extremely bitter taste. This bitterness can be tasted upon ocular administration
of the drug from simple solution due to rapid and extensive nasolacrimal drainage. There are also
limited reports on the chemical stability of naltrexone in aqueous solution. Parallel to that,
frequent instillation of a simple drug solution to the eye is associated with pulse-type entry and
may prompt patient incompliance.
45
Chapter 1….Literature Review
As previously outlined, niosomes lend themselves to potential ocular drug delivery systems. They
have the convenience of being delivered as eye drops, the ability to prolong precorneal drug
residence, a localised effect, complete biodegradability and minimal irritancy.
This work aims to formulate and characterise niosomal formulations for the ocular delivery of
naltrexone hydrochloride. To achieve this aim, this thesis will explore the following objectives:
•
Develop and validate an analytical method for accurate and selective determination of
naltrexone hydrochloride in the prepared formulations. In addition, forced degradation
studies will be conducted under different stress conditions to demonstrate that the
developed method is a stability-indicating method.
•
Study the chemical stability of naltrexone hydrochloride in aqueous solutions.
•
Study the fundamental physicochemical properties of naltrexone hydrochloride such as
melting behaviour, aqueous solubility and lipophilicity (octanol-buffer partition
coefficient determination).
•
Perform formulation studies on niosomes that includes:
o Investigating the effect of surfactant type (sorbitan esters and ployoxyethylene
alkyl ethers) and cholesterol (membrane stabiliser) on vesicle sizes, entrapment
efficiency
and
vesicle
membrane
fluidity
(gel/liquid
phase
transition
temperatures). Also, the effect of total lipid and initial drug concentration on the
entrapment efficiency of the prepared niosomes.
o Determining whether some selected membrane additives could alter the physical
properties such as drug entrapment efficiency, morphology, size and gel/liquid
transition temperatures of the prepared niosomes with emphasis on topical
application to the eye surface.
o Determining the physical and chemical properties of the prepared niosomes such
as contact angle, spreading coefficient, viscosity measurements and influence of
vesicular encapsulation on oxidative degradation of naltrexone hydrochloride.
•
Evaluate the prepared niosomes for ocular naltrexone hydrochloride delivery; ocular
toxicity using suitable in vitro models; in vitro drug release; ex vivo drug permeation
through excised bovine corneas; and physical stability of the selected formulations at
different temperatures.
46
Chapter 2….Preformulation of Naltrexone hydrochloride
2. Preformulation studies of naltrexone hydrochloride
2.1. Introduction
Preformulation characteristics are certain fundamental physical and chemical properties of a drug
that can eventually help in selecting the suitable formulation approach to develop an effective,
stable and economic dosage form (Wells & Aulton, 2007). The selection of particular
preformulation properties to study depends on the proposed dosage form. For example, powder
flow, compressibility, intrinsic solubility and dissolution studies are essential for developing solid
dosage forms. Generally, fundamental preformulation experiments include: development of a
suitable analytical method; aqueous solubility studies; melting point measurement; partition
coefficient and dissociation constant determination. Studying these preformulation characteristics
for the drug under investigation is essential especially at the early stages of designing and
developing a new dosage form.
Up to the early seventies, the available compounds for treatment of narcotic addiction were
cyclazocine and naloxone. These two drugs proved to be excellent opioid receptors antagonists
but both had drawbacks (Fink et al., 1968; Freedman et al., 1968; Renault, 1981). Cyclazocine
was limited because of dysphoric side effects due to weak agonistic activity (not pure antagonist).
Naloxone is a pure antagonist and thus free of side effects, but lack potency when administered
orally (Blumberg & Dayton, 1973; Freedman et al., 1968; Renault, 1981). Naltrexone has been
developed to fulfil the need for a long-acting, potent and pure antagonist with minimal unwanted
side effects. These requirements are met by naltrexone; it is about twice as active as naloxone as
an antagonist in mice. Furthermore, it is longer acting than naloxone in human (Blumberg &
Dayton, 1973). Currently, naltrexone is a widely used opioid antagonist for treatment of heroin
dependence (Renault, 1981) and alcoholism (Sweetman, 2007). Naltrexone is marketed
commercially as hydrochloride salt. Naltrexone hydrochloride is administered orally either in
liquid (extemporaneously prepared) or solid (tablets and capsules) form. The capsule form is
preferable, because naltrexone has an extremely bitter taste (Renault, 1981).
47
Chapter 2….Preformulation of Naltrexone hydrochloride
2.1.1. Physical properties of naltrexone hydrochloride
Naltrexone hydrochloride (NTX) is a hydrochloride salt of naltrexone. It is chemically named Ncyclopropylmethyl-14-hydroxydihydromorphinone. NTX is a white crystalline solid, soluble in
water. NTX has an empirical formula of C20H23NO4. HCl (mol wt 377.87) and a melting point of
275oC (crystals from methanol)(O’Neil et al., 2006). It has an extremely bitter taste (Crabtree,
1984); has a low partition coefficient (P), a log P (octanol/water) value of 1.9 (Galichet et al.,
2006); it is a weak basic drug, a pKa value of 8.38 at 20oC (Kaufman et al., 1975). Figure 2.1
shows a schematic of the chemical structure of NTX.
Figure 2.1 Chemical structure of NTX
2.1.2. Pharmacological uses and dosage for systemic administration of NTX
NTX is widely used in treatment of alcohol dependence and rescue medications to reverse the
side effects of opioid agonists (respiratory depression overdose) (Goodman et al., 2007; Kastrup
et al., 2009). Off-labelled uses of NTX include treatment of obesity, severe pruritus, psychosis
and Parkinson’s disease (Goodman et al., 2007). The usual dosage of NTX is 50 mg once daily or
350 mg per week in three divided doses (Crabtree, 1984; Goodman et al., 2007).
2.1.3. Pharmacokinetics of NTX
Absorption: NTX is a pure opioid receptor antagonist. Although it is absorbed well orally,
naltrexone is subject to significant first-pass metabolism. Its oral bioavailability ranges
from 5% to 40% (Kastrup et al., 2009).
48
Chapter 2….Preformulation of Naltrexone hydrochloride
Distribution: The volume of distribution for NTX following iv injection is estimated to be 1350
litres. In vitro tests of human plasma show NTX to be 21% bound to plasma proteins over
the therapeutic dose range (Kastrup et al., 2009).
Metabolism: The systemic clearance (after iv administration) of naltrexone is 3.5 ml/min. The
major metabolic site is the liver and the major metabolite is 6-β-naltrexol. The primary
metabolic pathway is glucuronide conjugation (Misra, 1981).
Excretion: Urinary excretion is the major excretion route of NTX and its metabolites (> 95%).
Less than 5% of an orally administered dose is found in faeces after 48 h (Crabtree, 1984;
Misra, 1981).
2.1.4. Adverse reactions of systemic administration of NTX
Combined reporting of adverse events from oral and injectable formulations includes:
Cardiovascular syncope (13%), headache (25%), insomnia (14%), anxiety (12%), nausea (33%),
vomiting (14%), abdominal pain and cramps (11%) (Fuller & Sajatovic, 2009; Kastrup et al.,
2009).
Contrary to expectation, injectable forms of NTX seem to induce more adverse reactions than
oral forms, leading to a higher rate of treatment discontinuation by patients. Amongst these
adverse reactions associated with injectable NTX administrations are pain, bruising, induration,
angioedema and nodules at the injection site (Roozen et al., 2007).
2.1.5. Warnings on the systemic administration of NTX
The following warnings are reported with oral administration of NTX (Kastrup et al., 2009):
•
NTX has the capacity to cause hepatocellular injury when given in excessive doses.
•
NTX is contraindicated in acute hepatitis or liver failure, and its use in patients with
active liver disease must be carefully considered in light of its hepatotoxic effects.
•
Patients should be warned of the risk of hepatic injury and advised to stop the use of NTX
and seek medical attention if they experience symptoms of acute hepatitis.
•
NTX and its primary metabolite are excreted primarily in urine and caution is
recommended in administering the drug to patients with renal impairment.
49
Chapter 2….Preformulation of Naltrexone hydrochloride
2.1.6. Naltrexone as a new ophthalmic pharmaceutical
Topical ocular application of NTX in doses of up to 0.4 mg/ml has been found to accelerate
markedly the wound healing of cornea in humans (Zagon et al., 2000), rats (Zagon et al., 1998b)
and rabbits (Zagon et al., 1998a), as well as diabetic rodents (Zagon et al., 2002a). Moreover,
topical application of NTX can enhance diabetic corneal epithelial healing without causing
morphologic abnormalities in the reassembly of adhesion structures (Zagon et al., 2007b).
Furthermore, topical treatment with NTX has been found to normalise tear production and
corneal sensitivity in type 1 diabetic rats (Zagon et al., 2009).
The mechanism of NTX for accelerating corneal wound healing is the blockade of opioid growth
factor (OGF) interaction with the OGF receptor (Zagon et al., 2002b). Consequently, it can
enhance DNA synthesis and corneal epithelialisation. However, the exact mechanism for
normalising tear production and restoring corneal sensation in diabetes mellitus is still unknown
(Zagon et al., 2009). It is worth mentioning that the corneal safety of topically applied NTX has
been studied (Zagon et al., 2006a). The results showed that concentrations of naltrexone ranging
from 10-3-10-7 M are not toxic when applied topically to the cornea.
2.1.7. Chemical stability of NTX
There are few reports on the chemical stability of NTX and its major degradation pathways in
aqueous solutions (Fawcett et al., 1997; Gupta, 2008). Table 2.1 summarises some of the reported
HPLC methods for NTX analysis.
The physicochemical stability of NTX oral liquid formulations prepared from commercially
available tablets and powder at two different concentrations (1 mg/ml and 5 mg/ml) was studied
(Fawcett et al., 1997). A vehicle of pH 3.5 was used and contained ascorbic acid (0.5%), sodium
benzoate (0.1%), glycerol (20%) and distilled water (to 100%).
The prepared formulations were stored in the dark at three temperatures 4, 25, and 70oC and
monitored for chemical and physical stability of NTX up to 90 days. The samples were assayed
for NTX concentration to assess decomposition over 90 days. The results showed that
decomposition of both NTX from drug powder and tablet stored at 4 and 25oC was not significant
over 90 days. However, the percentage of NTX remaining after storage for 90 days at 70oC was
51.1 % and 64.1% for NTX liquid formulations prepared from tablet and powder respectively. In
50
Chapter 2….Preformulation of Naltrexone hydrochloride
terms of physical stability, powder formulations stored at 4oC developed a pale yellow colour
after 90 days. Both solutions prepared from powder and tablets stored at 25 and 70oC developed a
pale yellow colour by day 30 and day 2 respectively.
In another study, the chemical stability of a NTX injection (extemporaneously prepared) was
studied. NTX was dissolved in sufficient sterile water for injection under aseptic conditions to
form a concentration of 1.4 mg/ml. The solution was stable for at least 42 days when stored in
clear glass vials at room temperature. Also, the physical appearance of the injection did not
change during the study period (Gupta, 2008).
Having a morphinan structure, oxidation could be the main culprit for the loss of potency and
degradation of NTX. Furthermore, oxidation is the main degradation pathway for phenolic
compounds such as opioids and catecholamines (Florence & Attwood, 1998a; Lachman et al.,
1986).
51
Chapter 2….Preformulation of Naltrexone hydrochloride
Table 2.1 Summary for HPLC conditions of NTX determination
Author
Chromatographic conditions
Column
(Fawcett et al., 1997)
(Iyer et al., 2007)
(Gupta, 2008)
C18: SupelcosilTM LC-18-DB
(250 mm × 4.6mm, 5µm)
C18: SupelcosilTM LC-18-DB
(150 mm × 4.6mm, 5µm)
C18: Beckman UltrasphereTM
LC-18-DB (150 mm × 4.6mm,
5µm)
Mobile phase
A : 0.06% triethylamine
solution in Phosphate buffer
(40mM) pH 5 (50%v/v) B:
Acetonitrile (50% v/v)
A : 0.1% of 85% phosphoric
acid and 0.1% glacial acetic
acid solution (90% v/v) pH 2.6
B: Acetonitrile (10% v/v)
Injection volume (µl)
Elution type
Temperature (oC)
Flow rate (ml/min)
Detection
Retention time (min)
Linearity range (µg/ml)
10
Isocratic
Ambient
1
UV at 214 nm
6
10-200
A : 0.06% triethylamine
solution in Phosphate buffer
(40mM) pH 4.75 (88 parts)B:
Acetonitrile (12 parts), A and
B were premixed together
5
Isocratic
50
1.25
UV at 204 nm
4.5
0.16-20
80
Isocratic
Ambient
1.5
UV at 285 nm
4
140-300
52
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.1.8. Oxidation
Oxidation is one of the most common causes of the chemical degradation of a considerable
number of pharmaceutical compounds (Florence & Attwood, 1998a; Lachman et al., 1986). For
instance, the degradation of steroids, vitamins, antibiotics and morphine is due to oxidation
reactions (Lachman et al., 1986). Oxidation involves the removal of electrons, radicals or
electropositive atoms (Florence & Attwood, 1998a).
The most common form of oxidation occurring in pharmaceutical dosage forms is autoxidation,
which is the reaction between molecular oxygen and organic compounds under mild conditions
(Johnson & Gu, 1988). Apart from the loss of drug potency due to autoxidation, other instability
problems can occur such as discolouration, changes in dissolution rate, precipitation, loss of
preservative efficacy, evolving of off-odours and sometimes generation of toxic compounds
(Johnson & Gu, 1988). It is well accepted that most liquid-phase autoxidations are free-radical
chain reactions. A free radical is an atom or molecule with one or more unpaired valence
electrons. Three well-distinct steps are identified in autoxidation reactions (Johnson & Gu, 1988):
Initiation:
equation 2.1
This stage is initiated once free radicals are generated. This inevitably takes place over a certain
period of time but the generation of free radicals can be accelerated by the action of heat, light
and traces of heavy metals (Florence & Attwood, 1998a; Johnson & Gu, 1988). The length of the
initiation is called the induction period (Stewart & Tucker, 1984).
Propagation:
equation 2.2
In the propagation stage, the generated free radicals in the initiation stage react with molecular
oxygen to yield peroxy radicals. Therefore, the oxygen concentration is of importance in the
autoxidation process, especially when determining the reaction rate of oxidation at different
temperatures due to the effect of temperature on the oxygen content (concentration of oxygen) of
the liquid (Lachman et al., 1986); the higher the temperature, the lower the oxygen concentration.
The hydroperoxide reacts with a new available free radical to propagate the reaction.
53
Chapter 2….Preformulation of Naltrexone Hydrochloride
Termination:
equation 2.3
The oxidation reaction proceeds until the free radicals are scavenged by inhibitors or halted by
side reactions which eventually break the chain reactions.
Figure 2.2 is a schematic showing a typical example of autoxidation reactions of morphine (M)
with atmospheric oxygen in aqueous solution to morphine N-oxide (MNO) (Yeh & Lach, 1961).
54
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.2 Schematic of morphine autoxidation in aqueous solutions
55
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.1.9. Arrhenius plot and predicting drug stability
Temperature is a key factor in determining the velocity of the degradation reaction of
pharmaceutical products. It is necessary to evaluate the temperature dependency of the reaction.
This allows prediction of the stability of the product (Lachman et al., 1986). The speed of many
degradation reactions doubles for each 10o rise in temperature (Lachman et al., 1986). The most
satisfactory method for quantitative determination of the influence of temperature on the reaction
velocity is that proposed by the Arrhenius equation which will be described later in the method
section in this chapter. The most important parameter which can be calculated from the Arrhenius
equation is the heat of activation (∆Ea), which represents the energy the reaction molecules must
acquire to undergo reaction. Plotting reaction rate constant values obtained at several elevated
temperatures versus the inverse of absolute temperature gives rise to what is known as the
Arrhenius plot. Since the plot is linear, the prediction of stability at shelf temperature is possible
by extrapolating the curve to the lower temperature (Lachman et al., 1986). The use of the
Arrhenius equation to predict drug stability at shelf temperature is most useful when the reaction
is too slow to be monitored conveniently and when∆Ea is relatively high. For example, for a
reaction with a ∆Ea value of 25 kcal/mol, a temperature increase from 25 to 45 oC brings about a
14-fold increase in the reaction rate constant (Guillory & Poust, 2002).
2.1.9.1. Limitations of the Arrenius plot for predicting oxidative degradation
There are some issues related to accelerated thermal degradation methods hindering the ability of
the Arrhenius equation to predict the shelf-life of dosage forms at lower temperatures (Lachman
et al., 1986; Waterman et al., 2002):
•
Higher temperatures may evaporate solvents, thus producing unequal moisture
concentration.
•
At higher temperatures, there is less oxygen solubility, thus hindering the predictability of
the room temperature stability of drugs sensitive to oxidation.
•
The permeability of oxygen through packaging is temperature dependent.
•
If the generation of radicals is rate limiting, the kinetics can show autocatalysis, the rate of
reaction increases as the radical concentration increases.
•
Instability may be related to the amount of peroxide impurities present in a particular lot
of excipient. This is related to age of the excipient and the conditions under which the
material was stored.
56
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.2.
Chapter aims
Designing a new ophthalmic liquid dosage form for NTX compels performing preformulation
studies for NTX. This chapter aims at carrying out preformulation studies of NTX which can
help select subsequent approaches during the formulation development. Specific objectives
include:
•
Development and validation of a sensitive and standard HPLC method for NTX analysis
in the prepared formulation.
•
Studying various stress conditions which can affect the chemical stability of NTX in
accordance to International Conference on Harmonization (ICH) guidelines.
•
Determining the following fundamental physicochemical properties of NTX:
o Melting behaviour.
o Spectrometric fingerprints (infrared, mass and 1H-NMR spectra).
o Aqueous solubility.
o Ionisation constants (pKa values) at different temperatures.
o
Lipid solubility (distribution and partition coefficients).
57
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.3. Materials and methods
2.3.1.
Materials
NTX was purchased from Mallinckrodt Inc., St. Louis, MO, USA; n-octanol was purchased from
Schalau Chemie, Sentmenat, Spain. All other solvents and buffer salts were of analytical grade
and used as received.
2.3.2.
Methods
2.3.2.1. Melting behaviour study of NTX using differential scanning calorimetry (DSC)
A small amount (typically 2 mg) of NTX powder was weighed in an aluminium pan and covered
with an aluminium lid and hermitically sealed using a pan press (Thermal Science, USA). The
temperature of the pan gradually increased from 50 to 300oC at a rate of 10oC/min using a
differential scanning calorimeter (Q 1000 series, Thermal Analysis, USA) precalibrated with
indium. The purging gas was nitrogen at a flow rate of 45 ml/min.
2.3.2.2. Spectrometric identification of NTX
Mass spectroscopy (MS)
A sample weight (approx. 1 mg) was dissolved in 1 ml methanol and injected directly into the
ionisation chamber. The atmospheric pressure chemical ionisation (APCI) technique was used to
ionise the sample and generate ions using a LC-M spectrometer (Surveyor MSQ,
ThermoFinnigan, New Jersey, USA).
Fourier transform infrared (FT-IR) spectroscopy
A FT-IR spectrometer (Bruker Miracle Micro ATR, Tensor 37, GmbH, Germany) was used to
record the FT-IR spectrum. A clean diamond window was used to measure the background
spectrum. A sufficient amount (approx. 1mg) of NTX was placed to form a thin film covering the
diamond window. The data was acquired and analysed using OPUS software (OPUS 6.5,
Germany).The FTIR spectrum was recorded at spectral resolution of 2 cm-1 with an average of
120 scans.
Proton nuclear magnetic resonance (1H-NMR) spectroscopy
1
H-NMR spectrum of NTX was determined in deuterated dimethyl sulfoxide (CD3)2SO on a 1H-
NMR spectrometer (Bruker Avance 400 Spectrometer, GmbH, Germany) at 400 MHz. Chemical
shifts are recorded in ppm.
58
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.3.2.3. Analytical methods
Ultraviolet (UV) spectrophotometry
An amount of NTX (10 mg) was accurately weighed and dissolved in 10 ml of phosphate buffer
saline (PBS) pH 7.4 to produce a concentration of 1mg/ml. Aliquots of 0.3-1ml of the stock
solution were transferred in triplicate to a series of 10-ml volumetric flasks and diluted to 10 ml
using PBS to obtain final concentrations of 30-100 µg/ml. Then, absorbance values of these
dilutions were measured at 281.5 nm using a UV-spectrophotometer (Thermospectronic, Helios
Gamma, England). A plot of absorbance vs. concentrations (µg/ml) was constructed.
High performance liquid chromatography (HPLC) method development
A modification of the method described by (Fawcett et al., 1997) was performed. In brief, an
HPLC system (Agilent 1200, Agilent Corporation, Germany) comprising a quaternary pump, an
automatic sampler and a photodiode array (PDA) detector was used with data acquisition by
ChemStation® software (Agilent Corporation, Germany). The chromatographic separation was
achieved using a Supelcosil C18 column (5 µm; 250 mm×4.6 mm, Supelco Corporation, PA,
USA) maintained at 25◦C. The mobile phase was prepared and premixed. Seventy parts (by
volume) of monobasic potassium dihydrogen phosphate solution (40 mM) pH 4 were mixed with
thirty parts (by volume) of acetonitrile; and the isocratic flow rate was 0.85 ml/min. A water:
acetonitrile (70:30 v/v) mixture was used as a rinse solution for the injector, and the injection
volume was fixed at 5 µl. Detection was carried out using a wavelength of 214 nm. The peak
purity for NTX was also determined.
Validation of the HPLC analytical method
The developed method was validated according to International Conference on Harmonisation
(ICH) guidelines (Shah et al., 1992).
Approximately 10 mg of NTX were accurately weighed, transferred into a 10 ml volumetric
flask and dissolved in triple-distilled water obtained in-house by reverse osmosis and
commercially known as Milli-Q water (MilliQ, Millipore, USA) to yield a stock solution of
1mg/ml. From the stock solution, a serial dilution was performed using the water:acetonitrile
(70:30 v/v) mixture to yield a standard calibration curve with a concentration range from 5 to 50
µg/ml.
59
Chapter 2….Preformulation of Naltrexone Hydrochloride
Precision and accuracy
Intra-day and inter-day variabilities were determined by repeated injections of quality control
(QC) samples. The QC samples were prepared at 8, 18 and 40 µg/ml representing low, middle
and high controls respectively. Accuracy was assessed by comparing the predicted concentrations
of the QC samples with the nominal 8, 18 and 40 µg/ml concentrations.
Limit of detection (LOD) and limit of quantitation (LOQ)
LOD is the lowest amount of analyte in a sample which can be detected but not necessarily
quantitated as an exact value, calculated using equation 2.1.
LOD =
3σ
S
equation 2.1
Where σ is the standard deviation of the intercept, and S is the slope of the calibration curve
LOQ is the lowest amount of analyte in a sample, which can be quantitatively determined in
suitable precision and accuracy calculated using equation 2.2.
LOQ =
10σ
S
equation 2.2
Specificity (peak purity determination)
The HPLC-PDA detector with the Agilent ChemStation software allowed on-line data acquisition
of UV-spectra between 190-400 nm during peak elution. The PDA detector provides more
information on sample composition than single-wavelength detection (Krull & Szulc, 1997;
Sinha et al., 2007). The UV spectra were obtained at four different points across the NTX peak;
two points before the peak apex (leading front), one point at the apex and one point after the apex
(tailing front). The peak purity was assessed by examining the similarity of the UV spectra
obtained at the five points. If an impurity or degradation product coelutes with the NTX peak, the
five UV spectra obtained across the peak are different. The peak purity analysis was carried out
for all NTX samples throughout the study.
2.3.2.4. Forced degradation studies
NTX was subjected to common stress conditions according to ICH guidelines to force NTX
degradation. The stress conditions were acid (0.1 M HCl) at 60oC, base (0.1 M NaOH) at 60oC,
oxidative (2% v/v H2O2) at room temperature, thermal (PBS pH 7.4) at 60oC and photolysis
(exposure to artificial daylight illumination of 10,000 lux) at 40oC for 24 h (Alsante et al., 2003;
60
Chapter 2….Preformulation of Naltrexone Hydrochloride
Reynolds et al., 2002). The samples were allowed to cool, neutralise (acid and base samples), be
filtered and be diluted before injection into the chromatographic system. All experiments were
performed in triplicate.
2.3.2.5. Degradation product identification using electron spray ionisation-mass spectroscopy
(ESI-MS)
To identify peaks of the resultant degradation products, samples were run on a Bruker
MicroOTOFQ mass spectrometer (Bruker Daltronics, Bremen, Germany) coupled with a KD
Scientific syringe pump (KD Scientific, Holliston, Massachusetts, USA). Samples were diluted to
1-10 µg/ml and were introduced by direct infusion at 180 µl/h into an ESI source in a positive
ionisation mode (capillary voltage -4500V) using 99.999% nitrogen gas as drying nebuliser gas
(180oC, 5 L/min and pressure of 0.5 bar). External mass calibration was performed using sodium
formate clusters and an enhanced cubic fit for the mass curve. Data was acquired for 3 min over a
50 to 1000 m/z range. Spectra were averaged over the 3 min and processed using Compass
software 1.3 (Bruker Daltonics, Bremen, Germany).
2.3.2.6. pH-degradation rate studies
NTX solutions were prepared at a concentration of 0.4 mg/ml in phosphate buffers of pH 4.5-9.5.
The buffer solutions were prepared by dissolving a suitable amount of NaH2PO4 or Na2HPO4 to
make up final concentrations of 40 mM with MilliQ water. The pH of the final solutions was
adjusted using H3PO4 (1M) or NaOH (1M) solutions to the desired pH values at room
temperature using a pH-meter (SevenEasy, Mettler-Toledo, Schwerzenbach, Switzerland)
precalibrated using standard buffers at pH 4 and pH 7 (BDH, England). The prepared NTX
solutions at pH 4.5-9.5 were incubated in a stability chamber (BINDER, Tuttlingen, Germany) at
60oC.
2.3.2.7. Accelerated stability and chemical kinetic studies
Screw-capped glass vials containing NTX solutions (0.4mg/ml) in PBS pH 7.4 were stored at 40,
50, 60 and 70oC to force degradation of NTX. Aliquots were withdrawn at suitable time intervals
depending on the decomposition rate, and analysed immediately using the HPLC method
described above. The observed first order degradation rate constant (kobs) was calculated from the
slope of log C versus time (t) plot according to equation 2.3 (Lachman et al., 1986).
61
Chapter 2….Preformulation of Naltrexone Hydrochloride
kobst
C
log  = −
2.303
 Co 
equation 2.3
Where C is the percentage of the drug remaining at time (t) and Co is percentage of the drug at t
equal to zero.
Plotting log kobs at different temperatures versus the reciprocal of absolute temperature (1/T) in
degrees absolute (oK) gives rise to what is known as the Arrhenius plot and the plots were then
fitted to the Arrhenius equation (equation 2.4) (Lachman et al., 1986).
log Kobs = log A −
∆Ea
2.303RT
equation 2.4
Where A the Arrhenius factor or the frequency factor (a measure of the frequency of collisions
between the reacting molecules),∆E
a
is the activation energy and R is the gas constant (1.987
cal.mol-1. degree-1). The kobs values at lower temperatures can be estimated using equation 2.5.
log
∆Ea  T 2 − T 1 
kobs 2
=


kobs1 2.303R  T 2T 1 
equation 2.5
2.3.2.8. Solubility studies of NTX
Equilibrium solubility studies of NTX were performed in both MilliQ water and PBS (NaCl 137
mM, Na2HPO4 10 mM, KH2PO4 1.47 mM, KCl 2.68 mM) pH 7.4 at 25oC ± 0.5oC. An excess
amount of NTX was dispersed in 5 ml of either PBS solution or water in 10 ml-screw-capped
glass vials and shaked at 200 strokes/min using a thermally-controlled shaking water bath (Heto
SBD 50, Jouan Nordic, Denmark) for 24 h and left for another 24 h to attain equilibrium. The
samples were withdrawn through 0.22 µm filter membranes, properly diluted and analysed
spectrophotometrically at 281.5 nm.
2.3.2.9. Determination of n- octanol/PBS distribution coefficient and partition coefficient of
NTX
The distribution of NTX between equal volumes of octanol (oil phase) and PBS at pH 7.4
(aqueous phase) was determined at three different temperatures 25, 35 and 37oC. Both n-octanol
and PBS were mutually saturated over overnight for at least 12 h at the corresponding
temperature before use. A specified amount of NTX was dissolved in presaturated PBS with the
oil phase to make a final concentration of 1 mM. Aliquots of 2 ml of the NTX solution were
62
Chapter 2….Preformulation of Naltrexone Hydrochloride
mixed with equal volumes of the oil phase in screw-capped glass vials and vortexed for 2 min.
The glass vials were left for a period of 12 h which was sufficient to allow the drug to reach
distribution equilibrium between the two phases (preliminary experiments showed that
equilibrium was reached at this time). After equilibration, a Pasteur pipette was used to separate
the two phases, followed by centrifugation to remove any of the remaining immiscible octanol
phase. The drug concentration in the oil phase was determined by subtracting the drug
concentration in the aqueous phase before and after distribution using the HPLC method
described above. The distribution coefficient (D) was calculated using equation 2.6 (Florence &
Attwood, 1998d; Wu et al., 2005):
D=
Co Cinitial − CE
=
Cw
CE
equation 2.6
Where Co is the concentration of NTX in n-octanol, Cw is the total concentration of NTX in PBS,
and Cinitial and CE are the concentration of NTX in PBS initially and at equilibrium respectively.
The true partition coefficient (P) was calculated from D using equation 2.7 (Schoenwald &
Huang, 1983):
P=

(CB )o = D1 +
1


(CB )w  anti log( pH − pKa) 
equation 2.7
Where (CB)O and (CB)W are the concentration of NTX free base in octanol and water respectively;
pH at 7.4. No activity corrections were used as the concentration of the drug was significantly
less than 0.01 M (Leo et al., 1971).
To calculate the pKa value at 25 and 35 oC, equation 2.8 was used (Albert & Serjeant, 1971):
− d ( pKa ) pKa − 0.9
=
dT
T
equation 2.8
Where the left side of the equation is the temperature coefficient which is defined as the rate of
decrease of pKa as the temperature is raised by 1o, pKa is the dissociation constant at T in
degrees absolute (oK).
63
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.3.3. Statistical analysis
A one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparisons at the 5%
significance level was used to test statistical significance differences between the test solutions
for forced degradation studies, distribution coefficient and partition coefficient at different
temperatures. These were performed using GraphPad Software Version 3.05, San Diego
California, USA.
64
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4. Results and discussion
2.4.1. DSC study
Figure 2.3 shows the DSC thermogram of NTX. A broad thermal event appeared at 60-110 oC.
This could be due to surface moisture evaporation. A second endothermic peak was observed at
220oC. This was due to NTX melting. These results were in a good agreement with those
reported elsewhere (Fuentes et al., 1997). According to the report, NTX showed endothermic
peaks at 102 and 217 oC. These peaks were attributed to dehydration and melting of NTX
respectively.
This result is in contrary to that of the reference melting point of NTX which is 275oC (Galichet
et al., 2006). The marked difference between the observed melting point and the reference one is
attributed to crystallisation from two different solvents, resulting in two different crystal forms.
The reference melting point was of the drug crystallised from methanol (O’Neil et al., 2006).
However, the NTX powder purchased from Mallinckrodt Inc, St. Louis, MO, USA is crystallised
from another solvent system (undisclosed by the company). Therefore, spectrometric studies
were carried out to confirm the identity of the NTX powder used.
Figure 2.3 DSC thermogram of NTX
65
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4.2. Spectrometric analyses of NTX powder
2.4.2.1. Mass spectroscopy
The mass spectra were determined on an LC-MS system, using atmospheric pressure chemical
ionisation (APCI) to ionise the sample (Figure 2.4). Under these detection conditions, the sample
ionises by interacting with the solvent (in this case methanol). The detector can detect positive
ions (lower trace) and negative ions (upper trace) at the same time.
To form positive ions the sample picks up a hydrogen ion: [M + H]+ so the mass is one atomic
mass unit (AMU) more than the molecular weight (the lower trace, Figure 2.4)
To form negative ions the sample loses a hydrogen ion: [M – H]- so the mass is one AMU less
than the molecular weight (the upper trace, Figure 2.4).
Figure 2.4 APCI mass spectra of NTX
2.4.2.2. FTIR-spectroscopy
Figure 2.5 shows the FT-IR spectrum of NTX powder. The spectrum shows the characteristic
peaks of NTX assigned at wave numbers 1717, 1660, 1504, 1314 and 1274 cm−1(Galichet et al.,
2006).
A high intensity peak at 1717 cm-1 is due to strong C=O of saturated cyclic aliphatic ketone
stretching absorption (Silverstein et al., 2005). The C=C stretching band of C=C-O-H occurs at
1660 cm-1. The C=C aromatic ring stretching appears at 1504 cm-1, O-H bending absorption at
66
Chapter 2….Preformulation of Naltrexone Hydrochloride
-1
1314 cm , and C-O-C asymmetric stretching peak observed at 1274 cm-1. A strong peak at 900
cm-1 is due to aromatic C-H bending vibration.
Wavenumber (cm-1)
Figure 2.5 FT-IR spectrum of NTX
2.4.2.3. 1H-NMR spectroscopy
Figure 2.6 and Table 2.2 show the 1H-NMR spectrum of NTX and chemical shifts (δ) in ppm for
the assigned hydrogens. The spectrum shows that all characteristic protons, which are chemically
attached to characteristic functional groups of NTX such as alcoholic, phenolic and ammonium
ion salt, were assigned. Conclusively, the data collected from the spectrometric analyses confirm
the integrity of the chemical structure of NTX.
67
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.6 1H-NMR spectrum of NTX in CD3SO, 400 MHz
68
Chapter 2….Preformulation of Naltrexone Hydrochloride
Table 2.2 Assignments of 1H-NMR spectra for NTX
δ (ppm)
0.3 - 0.7
1.05
1.4
2
2.15
2.45
2.7
3
3.3
4
5
6.55
6.7
7
9
9.45
Assignment
H19, H19’, H20, H20’
H18
H8’, H15’
H8
H7’
H16’
H15
H7, H10’, H16, H17’
H10, H17
H9
H5
H1
H2
H14 (OH Alcoholic)
H (+NH)
H3 (OH Phenolic)
Integral
4H
1H
2H
1H
1H
1H
1H
4H
2H
1H
1H
1H
1H
1H
1H
1H
69
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4.3. UV-spectrophotometric assay
The normal absorption spectrum of NTX was carried out in PBS pH 7.4 and showed maximum
absorption at 281.5 nm (λmax) (Figure 2.7). Under the experimental conditions described above,
the graph obtained by plotting absorbance (A) at 281.5 nm against concentration of NTX, in the
range of 30 to100 μg/ml, shows a linear relationship (Figure 2.8).
Figure 2.7 UV-Spectrum of NTX (100 µg/ml) in PBS pH 7.4
70
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.8 Standard UV-spectrophotometric calibration curve of NTX in PBS at pH 7.4
(Results expressed as mean, n = 5)
2.4.4. HPLC method validation
The system suitability parameters were found to be within acceptable limits. An analytical run
time of 8 min was optimised for each sample. The retention time of NTX under the experimental
conditions was 4.16 min and well resolved from the formulation excipients used (Figure 2.9 A
and B). The following validation parameters were estimated and evaluated according to the ICH
guidelines (ICH., 1996) (Shah et al., 1992):
2.4.4.1. Linearity
The standard calibration curve was linear in the range of 5–50 µg/ml (R2 > 0.999). Percentage
(%) recovery ± % relative standard deviation (RSD) ranged from 98.4 ± 3.84 to 103.53 ± 1.59
(Table 2.3).
71
Chapter 2….Preformulation of Naltrexone Hydrochloride
Table 2.3 Reverse predicted concentrations, % recovery and regression coefficient (R2)
Set number
Nominal concentration (µg/ml)
R2
Slope
Intercept
5
10
15
20
25
30
35
50
1
5.34
9.68
14.50
19.91
25.44
30.95
34.13
50.00
0.9984
13.481
9.5682
2
4.95
10.11
14.96
19.43
25.15
30.26
35.48
49.65
0.9995
13.58
5.127
3
5.07
10.31
15.35
19.77
25.14
29.55
33.93
50.86
0.9984
13.210
3.0877
4
5.29
10.02
14.48
19.71
25.67
29.14
35.88
49.80
0.9983
13.324
7.0479
5
5.21
9.77
14.51
20.22
26.46
29.22
36.00
50.32
0.9975
13.280
7.6631
Mean
5.17
9.98
14.76
19.80
25.57
29.83
34.75
50.13
0.9996
13.375
6.4988
% RSD
1.59
4.39
3.84
0.73
1.60
3.40
3.18
0.81
% Recovery
103.53
99.80
98.40
99.00
102.00
99.43
99.28
100.26
2.4.4.2. Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ were estimated to be 0.55 µg/ml and 1.85µg/ml respectively.
2.4.4.3. Precision and accuracy
The precision and accuracy of the method were reported as the relative standard deviation
(%RSD) and % recovery of the QC samples (Table 2.4). The inter-day and intra-day precision for
the three samples was found to be between 0.64 to 2.73%, and 0.14 to 1.16% respectively. These
values comply with the acceptance criteria of the ICH guidelines (Shah et al., 1992).
The purity of NTX peaks was checked all over the study using a PDA detector and Agilent
ChemStation software through examining the UV spectra acquired of four predetermined points
on the eluted drug peak from beginning to end by PDA detector. Figure 2.10 is a representative
example showing four superimposable NTX spectra acquired during NTX elution. The examined
points were 2 upslope points, a base point and a downslope point for purity determination. The
UV spectra obtained across the peak were superimposable indicating a single component and a
pure peak (Krull & Szulc, 1997; Wiberg et al., 2004).
72
Chapter 2….Preformulation of Naltrexone Hydrochloride
Table 2.4 Precision and accuracy data of the QC samples (Results were expressed as mean
values, n = 5)
Mean
% RSD
% Recovery
8
7.92
1.16
99.00
Nominal concentration (µg/ml)
Intra-day
Inter-day
18
40
8
18
40
18.20
38.36
7.95
18.60
40.10
0.14
0.71
2.73
1.40
0.64
101.00 96.00
99.40 103.00 100.24
Figure 2.9 Representative HPLC chromatograms of standard NTX (0.4 mg/ml) solution (A)
and NTX resolved from Span 60: cholesterol: DCP-niosomal formulation (B)
73
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.10 PDA- spectra of the NTX peak acquired from 4 different positions throughout
the eluted peak for peak purity assessment
2.4.5. Forced degradation studies
Forced degradation studies were performed in order to demonstrate the specificity of the
developed method, to ensure that the method is stability-indicating and to provide insights on the
stability of the drug under common stress conditions (Alsante et al., 2003; Reynolds et al., 2002).
The results of % recovery of NTX from the various stress conditions used after 24 h exposure are
shown in Figure 2.11. The chromatograms of NTX resolved after exposure to base-heat, acidheat, heat, oxidation and artificial daylight illumination at 40oC for 24 h are shown also in Figure
2.12 and Figures 2.14 -2.17 respectively.
74
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.11 Percentage (%) remaining of NTX after 24 h exposure to various
stress conditions (Results are expressed as mean values ± SD, n = 3)
Approximately 50% of the potency of NTX was lost after exposure to strong alkaline conditions
for 24 h at 60oC and discolouration of the solution to yellowish brown. Figure 2.12 and Figure
2.13 show the HPLC chromatograms and the different spectra of both NTX and the main
degradation products of NTX after exposure to a strong base and heat. Figure 2.12 shows the
chromatogram of NTX after base-catalysed degradation at 60oC for 24 h. The chromatogram
shows three peaks at 3.13, 3.39 and 3.8 min in addition to the parent drug at 4.15 min. The peak
resolved at 3.13 min was due to medium or salt-related impurities, as seen in the blank
chromatogram (Figure 2.12), whereas the peaks at 3.39 and 3.8 min were due to NTX
degradation. The chromatogram shows a resolution factor of 1.8 for NTX and its degradation
product at 3.8 min. Although the resolution of peaks of different heights should be ≥ 2 (Krull &
Szulc, 1997), this can be accepted because the smallest peak was eluted first (Gleditsch &
Waaler, 2001). The similarity of the parent compound and its degradation products makes them
difficult to separate (Gleditsch & Waaler, 2001; Krull & Szulc, 1997). For example, a resolution
factor of morphine and morphine-N-oxide is found to be 1.6. This is due to the similarity of
hydrophilicity of morphine and morphine-N-oxide (Gleditsch & Waaler, 2001).
75
Chapter 2….Preformulation of Naltrexone Hydrochloride
Exposure of NTX to a strong acid and heat showed moderate degradation (Figure 2.11). The
extent of NTX degradation at pH 1.2 was significantly less (P < 0.01) than that at pH 10. Also,
the extent of degradation (16%) at pH 1.2 was significantly lower (P < 0.01) than that (23.5%) at
pH 7.4. These results demonstrate that the chemical stability of NTX is pH-dependent. Assuming
that the main degradation reaction is oxidation, it is generally more difficult to remove an
electron from a drug when it is more positively charged. Therefore, drug stability against
oxidation is often greater under lower pH conditions. An acidic pH promotes protonation of the
basic drug. On the other hand, higher pH conditions deprotonate the basic drug. The basic
conditions make the basic drug more susceptible to oxidation than the acidic conditions
(Waterman et al., 2002). For example, NTX is completely ionised in pH 1.2 whereas NTX, with a
pKa value of 8.13, is predominantly unionised at pH 10. This explains why the degradation of
NTX decreased in the following pH order pH 10 > pH 7.4 > pH 1.2.
Furthermore, NTX underwent extensive degradation when subjected to the oxidising agent (H2O2)
(Figure 2.16). The percentage of NTX remaining was found to be 16%. The major
degradation product was eluted at 4.3 min (Figure 2.16), whereas it was eluted at 3.8 min in the
heat and base stress test. These findings suggest that the product of oxidation due to H2O2 is
different from that produced in the heat-base degradation. H2O2 is often non-predictive of
molecular oxygen reactions because it does not involve the radical chain reaction common with
the oxygen-based processes (Waterman et al., 2002). However, H2O2 is a common oxidising
agent and is recommended by ICH to induce drug oxidation (Alsante et al., 2003; Reynolds et al.,
2002).
The order of the degradation of NTX under the various stress conditions is as follows: oxidation
> base > heat > photolysis > acid.
76
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.12 HPLC chromatograms of the blank solution and aquous NTX solution (0.4
mg/ml) pH 10 after 24 h exposure to heat at 60oC
77
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.13 PDA spectra of NTX eluted at 4.16 min (A), the
degradation product resolved at 3.7 min (B) and the second
degradation product resolved at 3.3 min (C) after exposure to pH 10 at
60 oC
78
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.14 HPLC chromatograms of the blank solution and aquous NTX solution
(0.4 mg/ml) of pH 1.2 after 24 h exposure to heat at 60oC
Figure 2.15 HPLC chromatograms of the blank solution and aquous NTX solution
(0.4 mg/ml) of PBS pH 7.4 after 24 h exposure to heat at 60oC
79
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.16 HPLC chromatograms of the blank solution and aquous NTX solution (0.4
mg/ml) of PBS pH 7.4 after 24 h exposure to H2O2 (2% v/v)
Figure 2.17 HPLC chromatograms of the blank and aquous NTX solution (0.4 mg/ml) of
PBS pH 7.4 after 24 h exposure to artificial daylight illumination (10,000 lux) at 40oC
80
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4.6. Degradation products identification using ESI-MS
NTX degradation was indicated by appearance of additional peaks at 3.39 min and 3.8 min as
well as discolouration of NTX solutions. These peaks increased in area as a function of
temperature and time and the intensity of the discolouration obviously increased, as well. Heat
and base-catalysed degradation solutions were introduced to ESI-MS. The mass spectra were
generated to determine the molecular weight of the major degradation products (Figure 2.18).
The results showed that major ion peaks appeared at 342, 358 and 374 m/z. These peaks were
attributed to the positively charged molecular ion of naltrexone [M+1]; and other metabolites
(degradation products) at [M+16] and [M+32]. The metabolite at [M+16] was called product I
and attributed to 10-hydroxy naltrexone (Figures 2.18 and 2.19). According to the United States
pharmacopeia (USP), 10-hydroxy naltrexone is eluted at around 0.3 min earlier than the
naltrexone peak (USP32/NF27, 2009). The metabolite at [M+32] was attributed to N-oxide-10hydroxy naltrexone, as nitrogen has a lone pair of electrons. This makes it a nucleophilic centre
for oxidative reaction. An N-oxide product is a common oxidation product for morphinan-bearing
compounds (Gleditsch & Waaler, 2001; Yeh & Lach, 1961). Figure 2.19 shows the proposed
degradation pathway of NTX. The N-oxide position in product II was evidence based on a
published report on a similar compound where N-oxide morphine is the oxidation product of
morphine (Yeh & Lach, 1961). Other m/z ratio peaks at 364, 380 and 396 were observed and
related to the sodium and potassium adducts of naltrexone and sodium adduct of sodium 10hydroxy-naltrexone respectively.
Figure 2.18 ESI mass spectra of product I and Product II of NTX oxidation
81
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.19 Schematic of the proposed oxidative degradation pathway for NTX
82
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4.7. pH-degradation rate of NTX
Figure 2.20 shows semi-log degradation plots of NTX in phosphate buffers over pH 4.5-9.5 at
60oC. The semi-logarithmic plots of % NTX remaining versus time showed good linearity; and
the correlation coefficient (R2) ranged from 0.98 to 0.995. This indicates that the degradation of
NTX followed first-order kinetics. A pH-rate profile of a sigmoid or S- shape was obtained over a
pH range of 4.5 to 9.5 at 60oC (Figure 2.21). The S-shaped pH-rate profile is most likely to be
associated with autoxidation reactions (Johnson & Gu, 1988). Additionally, a yellowish-brown
colour was observed with the degraded samples and the intensity of colour increased as the
percentage of degradation increased. Discolouration of the NTX solution is another symptom
which supports the autoxidation process (Johnson & Gu, 1988). This indicates that autoxidation
reactions are involved in the decomposition of NTX. The degradation occurred more rapidly in
basic than acidic solutions (Figure 2.21). For example, the observed rate constants (kobs) were
0.02 and 0.172 day-1 at pH 4.5 and 8.5 respectively; and the estimated half-life (t1/2) values were
found to be 35 and 4 days at pH 4.5 and 8.5 respectively at 60oC. These results are attributed to
the pH-dependency of oxidation reactions in aqueous solutions.
Figure 2.20 First-order degradation plots for NTX at 60oC in phosphate buffers of
various pH values (Results are expressed as mean values ± SD, n=3)
83
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.21 pH-rate (kobs, day-1) profile for NTX at 60oC
The removal of electrons from a positively charged compound is more difficult than a neutral
one. This causes an increase of the oxidation potential with a concurrent increase in stability
against oxidation (Lachman et al., 1986). A basic compound undergoes accelerated
decomposition in alkaline conditions where the drug is dominantly unionised. Another factor
which can enhance the oxidation reaction is the hydroxyl ion concentration through a catalytic
effect (Florence & Attwood, 1998a; Johnson & Gu, 1988; Waterman et al., 2002). For instance,
the autoxidation rate for the fully protonated form of NTX at pH 4.5 is approximately 17 times
slower than that for the fully unprotonated form of NTX at pH 9.5 at 60oC. Two steeps were
observed at pH ranges of 6.5-7.5 and 8.5 and 9.5. The state of NTX ionisation apparently
influences the oxidation reaction rate. Below pH 6.5, NTX molecules are dominantly in an
ionised state, hence the reaction rate proceeds at a low rate. The steep at pH 6.5-7.5 is likely due
to availability of 50% unionised NTX species at pH 7.5, which is equal to pKa of NTX, at 60oC.
The second steep at pH higher than 8.5, NTX molecules are dominantly in an unionised state.
Further, NTX is degraded by two oxidation reactions (Figure 2.19); these could explain the two
steeps in the pH-oxidation profile of NTX.
84
Chapter 2….Preformulation of Naltrexone Hydrochloride
The pH at which NTX shows a maximum stability (pH 4.5), unfortunately, is not suitable for
ocular administration. This is because it is well below the physiological pH of the resident tears
(pH 7.4) leading to possible precorneal tear film breakdown, reflex lacrimation and blinking.
Consequently, the administered dose is rapidly diluted or removed from the surface of the eye
(Lang et al., 2002). Additionally, NTX is dominant in the ionised form (the least absorbable
form) at the acidic conditions. NTX penetration via the lipophilic epithelial corneal membrane is
expected to be minimal at the pH of maximal stability. Therefore, ophthalmic solution should be
ideally buffered to a pH of 7.4 which is the normal physiological pH of the tear fluid. The reason
for this is that the product would be comfortable and offer optimum therapeutic activity (Lang et
al., 2002).
2.4.8.
Accelerated stability and chemical kinetic studies
The effect of temperature on NTX stability was studied in PBS at pH 7.4. NTX was subjected to
forced degradation at different temperatures 40, 50, 60 and 70oC. Semi-log plots of the % NTX
remaining versus time showed linear relationships indicating first order kinetics (Figure 2.22).
Table 2.5 shows the calculated and predicted kobs at different temperatures. The results show the
higher the temperature, the greater the value of kobs (Table 2.5). There was also an inverse
relationship between the temperature and t1/2 and t90% (time of 10% degradation). The kobs values
at the assigned temperature were plotted against the reciprocal of the absolute temperature
according to the Arrhenius equation. The activation energy (Ea) was estimated to be 15.28
kcal/mol. The Ea value reported for morphine sulphate (a morphinone-based drug) is 23 kcal/mol
(Kennon, 1964).
From the literature, the Ea values of degradation reactions for most of the drugs in aqueous
solutions are found to be in the range of 10-30 kcal/mol (Lachman et al., 1986). The stability of
NTX in ambient and cold storage conditions was predicted by calculating kobs values at 25 and
4oC respectively (Table 2.5). These findings show that NTX is less chemically stable than
morphine sulphate. Hence, encapsulating NTX in bilayer membranes in lipid-based vesicles
could enhance its chemical stability, because this reduces the direct interaction of NTX with light
and other initiators of oxidative catalysis. The photo-degradability of tretinoin encapsulated by
niosomes was lower than the free tretinoin solution (Manconi et al., 2003). Therefore, this is an
incentive to study the effect of encapsulating NTX in niosomes on the chemical stability.
85
Chapter 2….Preformulation of Naltrexone Hydrochloride
Figure 2.22 First-order degradation plots for NTX in PBS pH 7.4 at various temperatures
(Results are expressed as mean values ± SD, n=3)
Table 2.5 First–order kinetic parameters and correlation coefficient of NTX degradation in
aqueous solution of PBS pH 7.4 at different temperatures
Temperature (oC)
4
25
40
50
60
70
R2
0.97
0.99
0.96
0.97
kobs (day-1)
0.001*
0.008*
0.02
0.086
0.14
0.188
t1/2 (days)
693
87.00
35.00
8.00
5.00
3.50
t90% (days)
104.00
13.00
5.20
1.21
0.74
0.55
* Average kobs values at 4oC and 25oC were predicted using the Arrhenius equation. The other Kobs values were
experimentally determined.
86
Chapter 2….Preformulation of Naltrexone Hydrochloride
2.4.9. Solubility studies of NTX
Thermal analysis using DSC showed that the NTX powder used had a melting peak at 220oC
suggesting that it is a different crystalline form for NTX. The object of this study was to
determine the aqueous solubility of the available NTX crystalline form. The results showed that
the respective solubility of NTX in water and PBS pH 7.4 was > 100 mg/ml and 60 mg/ml. These
results indicated that the available NTX form is freely soluble in water, and there is no noticeable
difference between the solubility of the NTX form used and the solubility of the reference NTX.
The lower solubility of NTX in PBS pH 7.4 compared to that in water is attributed to the lower
ionisation (PKa of NTX is 8.26 at 25oC) at pH 7.4 than water (pH 6) where ionisation percentage
of NTX in pH 7.4 and 6 is 87.87% and 99.45% respectively. Additionally, the ionic strength of
the buffer system might also contribute to lowering NTX solubility compared with pure water.
This negative effect of ionic strength on NTX solubility is due to the salting-out effect (Miyazaki
et al., 1981). Furthermore, the availability of chloride ions in the PBS might decrease the
solubility of the hydrochloride salt due to the common ion effect on the solubility product
(Florence & Attwood, 1998d).
2.4.10. Determination of n-octanol/PBS distribution coefficient (D) and partition coefficient
(P) of NTX
For ionisable drugs, the distribution coefficient (D) is the ratio between drug concentration
(unionised species) in oil phase and drug concentration in the aqueous phase (both free base and
acid salt). The true partition coefficient (P) for ionisable drugs is the ratio between concentration
of unionised species in the oil phase (octanol) and concentration of unionised species in the
aqueous phase. The aqueous phase used was a PBS solution with pH 7.4 and osmolality of 278 ±
5 mOSm/kg to mimic the pH and osmolality of the tear fluid (Lang et al., 2002).
The D values were determined at three different temperatures 25oC (ambient temperature), 35oC
(ocular surface temperature) (Schoenwald & Huang, 1983) and 37oC (body temperature). The D
values at the three different temperatures were converted through equation 2.4 to The P values to
determine lipohilicity of NTX. The reported NTX pKa values are 8.38 and 8.13 at 20oC and 37oC
respectively (Kaufman et al., 1975). Nitrogenous bases are highly temperature sensitive where
they become weaker as the temperature is raised. The pKa of NTX decreases when the
temperature increases. Equation 2.11 governs the temperature variation for the ionisation of
monoacidic bases. This equation has been well supported by experimental evidence (Albert &
Serjeant, 1971). The average temperature coefficient was calculated using equation 2.11 and was
87
Chapter 2….Preformulation of Naltrexone Hydrochloride
estimated to be 0.024. This value was multiplied by 5 and the product was subtracted from the
pKa value at 20oC to give the value of pKa at 25oC (Table 2.6). To calculate pKa at 35oC, the
average temperature coefficient was multiplied by 2 and the product was added to pKa at 37oC
(Table 2.6).
Table 2.6 n-octanol-buffer distribution coefficient (D) and partition coefficient (P) and
calculated pKa at different temperatures (Results are expressed as mean values ± SD, n =3)
Temperature (oC)
25
35
37
D
4.28 ± 0.25
6.05 ± 0.24
9.10 ± 0.07
pKa
8.26
8.18
8.13
P
35.30 ± 2.00
42.50 ± 1.70
58.00 ± 0.51
Log P
1.54 ± 0.02
1.61 ± 0.001
1.76 ± 0.002
In addition, Table 2.6 presents D and P values at the tested temperatures. The D values at 25, 35
and 37 oC were converted to P values to judge lipophilicity of NTX. The results showed that both
D and P values increased significantly (P < 0.001) as a function of the studied temperatures. For
example, 1.4- and 2-fold increases in the D values were obtained as the temperature is raised
from 25oC to 35oC and 37oC respectively. These results demonstrate the dependency of the D
value on the temperature. The temperature dependency of D value has been reported with most of
opioid agonists and antagonists (Kaufman et al., 1975). This is partly attributed to decreasing in
the ionisation constant as the temperature increases. Hence, the amount of unionized species of
NTX increases and diffuses to the oil phase, and the concentration of unionised species increases
in the octanol phase. The P value of NTX was found to be 42.5 at 35oC. Based on β-adrenergic
blockers classification on lipophilicity, NTX can be considered as a hydrophilic drug and hence,
the corneal epithelium is expected to be the rate-limiting barrier for ocular absoprtion
(Schoenwald & Huang, 1983). This is an additional incentive to consider niosomes
(surfactant/lipid-based system) for the ocular delivery of NTX.
88
Chapter 2….Preformulation of Naltrexone Hydrochloride
Conclusion
•
The available crystal form of NTX is freely soluble in water and soluble in PBS.
•
NTX undergoes chemical decomposition upon melting.
•
The validated HPLC method was rapid, precise, sensitive and stability indicating.
•
NTX is vulnerable to autoxidation as demonstrated by S-shaped pH-degradation rate
constant profile, discolouration of NTX solutions and ESI-mass spectroscopy.
•
NTX shows temperature-dependent lipid solubility as shown from the distribution
coefficient/partition coefficients at different temperatures.
•
According to P values, NTX is a hydrophilic agent and its permeation through the cornea
is likely to be dependent on the epithelium barrier.
From the preformulation study, there are incentives to consider surfactant-based vesicles
(niosomes) as a delivery system for NTX in order to enhance NTX uptake by the lipophilic
corneal epithelium and provide NTX with a shield of bilayer membranes to protect it from
oxidation. Therefore, the next chapter is concerned with preparing and characterising NTX
niosomes.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3. Preparation of niosomes using biocompatible surfactants:
optimisation of surfactants and cholesterol level
3.1. Introduction
3.1.1. Niosomes
Niosomes are non-ionic surfactant based vesicles. They were originally developed as an
alternative controlled drug delivery system to liposomes, in order to overcome the problems
associated with sterilisation, large-scale production and stability (Azmin et al., 1985, 1986; El
Maghraby & Williams, 2009).
The hydration of a film, comprising a mixture of a single or double-alkyl chain and cholesterol,
leads to the formation of vesicular dispersion. These dispersions were termed niosomes (Baillie et
al., 1985). Basically, these vesicles do not form spontaneously. Thermodynamically stable
vesicles form only in the presence of proper mixtures of surfactants and a membrane stabilising
agent (cholesterol), at a temperature above the gel/liquid transition of the main lipid forming
vesicles (Azmin et al., 1985, 1986; Nefise, 2007).
The first niosome formulations were developed and patented by L’Oreal in 1975 (Nefise, 2007).
Niosomes were first utilised as a drug delivery system for anticancer drugs (Azmin et al., 1985,
1986). The developed niosomes were capable of altering the pharmacokinetic profile, organ
distribution and metabolism of methotrexate in mice (Azmin et al., 1985, 1986). Niosomes are
versatile in structures, morphology and sizes; they can entrap hydrophilic drugs in aqueous
compartments or lipophilic drugs by partitioning of these molecules into bilayer domains (Figure
3.1).
90
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.1 Locations of entrapped hydrophilic, lipophilic and amphipathic
drugs in niosomes
Furthermore, they can be formulated to be unilamellar, oligolamellar or multilamellar (Figure
3.2). Niosomes also possess great stability, are cost-effective, and provide a simple methodology
for routine and large-scale production (Baillie et al., 1985; Uchegbu & Florence, 1995; Uchegbu
& Vyas, 1998).
Figure 3.2 Major types of niosomes, MLV (multilamellar vesicles), OLV (oligolamellar
vesicles) and ULV (unilamellar vesicles). Small circles (o) represent polar head group, sticks
(–) represents apolar tails of single-chain surfactant molecules and a bilayer membrane
represent a circular double surfactant molecules layer oriented in continuous tails-to-tails
and polar heads lining the inner and outer circle
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.1.1.1. Self-assembly of lipid molecules
The principle of opposing forces
Formerly, the self-assembly of amphiphilic molecules, composed of hydrophilic and lipophilic
portions, in an aqueous environment into well-defined structures, such as micelles, open bilayers
and vesicles, was solely ascribed to favourable hydrophobic and hydrophilic interactions in the
aggregated phase (Tanford, 1973). The major forces derive from the hydrophobic interactions
between the hydrocarbon tails, which induces the molecules to associate; and the hydrophilic
nature of the head group, which imposes the opposite requirement, that they remain in contact
with water (the theory of opposing forces) (Tanford, 1973). These two interactions compete
giving rise to the idea of two ‘opposing forces’ acting mainly in the interfacial region: one tends
to decrease and the other tends to increase the interfacial area per molecule (the head group area)
exposed to the aqueous phase. While this notion is true and can be accepted thermodynamically,
it does not explain all the possible structures into which lipids can aggregate. For instance, some
form micelles (e.g. lysolecithin), and some form bilayers (e.g. lecithin), while others form only
inverted micellar structures or precipitate out of solution (Israelachvili et al., 1980).
The principle of geometrical or packing properties
The role of molecular geometry or the ‘packing’ properties of amphiphiles (surfactants), as
shown in Figure 3.3, in determining these structures was recognised by (Israelachvili et al.,
1976). Accordingly, an optimal polar head group area ao, an optimal volume of the hydrocarbon
chains v and a maximum effective length that the hydrocarbon chains can assume (critical chain
length) lc will determine the self-assembly of the amphiphiles into organised lipid aggregates.
The value of the dimensionless ‘packing parameter’ v/ ao lc will determine whether a certain
amphiphile or lipid structure will form spherical micelles (v/ ao lc < 1/3); non-spherical micelles
(1/3 < v/ ao lc < 1/2); bilayers (1/2 < v/ ao lc <1); an inverted micellar structure (v/ ao lc >1); or
even precipitate out of solution (e.g. cholesterol), since cholesterol molecules possess a headgroup too small to pack into organised structures (Israelachvili et al., 1980; Israelachvili &
Mitchell, 1975; Israelachvili et al., 1976).
Apart from the selection of amphiphiles with proper geometrical properties, the formation of
niosomes involves some input of energy (physical agitation and heat), as closed vesicles do not
form spontaneously (Baillie et al., 1985; Sahin, 2006).
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.3 Schematic of a single-chain surfactant, ao = hydrophilic head group area, v =
hydrophobic chain volume, lc = hydrophobic chain length
In pharmaceutical research, the hydrophilic-lipophilic balance (HLB) system, which is a measure
of the relative contributions of the hydrophilic and lipophilic regions of the surfactant molecules,
is more commonly used as an indicator to niosomes formation. Surfactants possess HLB values
of 4 to 8 have been found to be optimal for the formation of niosomes (Girigoswami et al., 2006).
Surfactants of high HLB values (9.6-16.7) such as polysorbates (Tween®) tend to form micelles
or open lamellar structures (Manosroi et al., 2003). Other surfactants cannot assemble into
niosomes due to their geometry (packing) properties, such as Span 80 (HLB of 4.3). This is
because of the kink in the hydrocarbon chain due to double bond at C9 (Yoshioka et al., 1994).
3.1.1.2. Raw Materials used in manufacturing niosomes
Niosome forming lipids (Surfactants)
Unlike naturally occurring phospholipids (the principal liposome-forming lipids) which have
double alkyl chains, most widely used niosome forming surfactants are single chain, synthetic,
non-ionic surfactants (Florence & Baillie, 1989; Uchegbu & Florence, 1995). Alkyl ethers, alkyl
esters and alkyl amides, as well as fatty acid and amino acid compounds, can form vesicles
(Uchegbu & Florence, 1995). Recently, niosomes consist of Pluronic L64 and P105 (copolymers
of ethylene oxide and propylene oxides) have been prepared and used as a transdermal delivery
system for sulfadiazine sodium (a model hydrophilic drug) (Muzzalupo et al., 2011). However,
both alkyl ethers and alkyl esters have a wider application as niosomes drug delivery carriers, due
to their commercial availability and low toxicity.
93
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Alkyl ethers
Alkyl ethers can be divided broadly into two subclasses, based on their hydrophilic head group:
alkyl ethers whose heads consist of glycerol subunits or larger sugar molecules, and alkyl ethers
whose hydrophilic head groups consist of repeat ethylene oxide subunits (Uchegbu & Florence,
1995).
Alkyl glycerol ethers, most notably hexadecyl diglycerol ether (C16G2), have been reported as
niosome-forming surfactants and have been used in many drug delivery applications such as
experimental cancer chemotherapy (Baillie et al., 1985; Uchegbu & Florence, 1995). These
surfactants were used to alter the pharmacokinetics of methotrexate (Azmin et al., 1985) and
doxorubicin (Uchegbu et al., 1996a). Alkyl glycerol ethers were also used to encapsulate sodium
stibogluconate to control leishmaniasis (Baillie et al., 1986).
The second group of alkyl ether surfactnats, in which the hydrophilic region consists of repeat
oxyethylene units, was used to encapsulate insulin for oral delivery, in order to prevent the
inactivation of insulin by the gastric juice (Pardakhty et al., 2007).
Alkyl esters
Alkyl esters, such as sorbitan fatty acid esters (Span®) and polyoxyethylene sorbitan fatty acid
esters (Tween®), are widely used in cosmetics and foodstuffs, as well as oral, parenteral and
topical pharmaceutical formulations. They are regarded as non-toxic and non-irritant materials
(Lawrence, 2003). These surfactants have been utilised in manufacturing niosomes as a drug
delivery system, including oral (Varshosaz et al., 2003), trandermal (Balakrishnana et al., 2009;
Ibrahim et al., 2005) and ocular delivery (Abdelbary & El-gendy, 2008; Aggarwal et al., 2004;
Kaur et al., 2008). Cell culture toxicity studies have shown that niosomes composed of ester-type
surfactants are less toxic than ether-type niosomes. This could be attributed to the possible
enzymatic degradation of ester bonds (Hofland et al., 1992).
Cholesterol (bilayer membrane stabiliser)
Some water-soluble surfactants, such as Tween 20 and Tween 61, could not form vesicles under
normal conditions; these surfactants prefer to form micelles, in a manner identical to lyolecithin
(Florence & Baillie, 1989; Manosroi et al., 2003). These surfactants might form vesicles when
mixed with cholesterol. Cholesterol molecules operate cooperatively to form bilayer vesicles.
Cholesterol is considered as lipid, but it is a comparatively rigid molecule and lacks the
accommodating ability of an acyl-chained lipid. In particular, cholesterol does not form bilayers
and its shape can be modelled as an inverted cone (Figure 3.4) (Israelachvili et al., 1980).
94
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
However, cholesterol has been called the ‘mortar’ of bilayers because, by virtue of its molecular
shape and solubility properties, it fills in empty spaces among the amphiphiles, thereby anchoring
them more strongly into the bilayer structure.
Figure 3.4 Chemical structure of cholesterol
Cholesterol in niosomes is reported to increase the membrane stability, decrease the fluidity of
the membrane and alter membrane permeability (Uchegbu & Florence, 1995; Uchegbu & Vyas,
1998). Other excipients, such as dicetyl phosphate and stearyl amine, were used in niosomes
technology to impart negative and positive charges respectively (Yoshioka et al., 1994). In such
cases, the operational packing parameter is estimated for all the components.
3.1.1.3. Vesicle preparation
Vesicle formation requires an input of energy, in the form of either gentle agitation or probe
sonication. Usually, the surfactant/lipid mixtures are hydrated in the presence of the drug in an
aqueous solution at a temperature above the gel/liquid transition temperature of the surfactant.
The unentrapped drug can be separated by ultracentrifugation, low-pressure gel filtration
chromatography or exhaustive dialysis. Since vesicle size is an important determinant of drug
biodistribution, it may be reduced by extrusion via nucleopore filters, microfluidisation or highpressure homogenisation. The different techniques used in surfactant/lipid hydration and drug
loading are summarised as follows.
Hydration and drug loading techniques
Below are the commonly used laboratory methods of niosome preparation and drug loading
identified in the literature:
95
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
1. The injection of an organic solution of surfactant/lipid into a hot aqueous solution of the
drug to be encapsulated, which is heated above the boiling point of the organic solvent
(ether injection) (Baillie et al., 1985).
2.
The formation of a surfactant/lipid film by evaporation of an organic solution of
surfactant/lipid. This lipid film is then hydrated with a solution of the drug (Azmin et al.,
1985; Baillie et al., 1985). This method is commonly known as thin film hydration (TFH)
or the conventional method. The niosomes generated by THF are multilamellar vesicles
(MLV). The TFH method was previously described by (Bangham et al., 1965) for the
preparation of liposomes.
3. The formation of a W/O emulsion from an organic solution of surfactant/lipid and an
aqueous solution of the drug. The organic solvent is then slowly evaporated to leave
niosomes dispersed in the aqueous phase. In some cases, a gel results which must be
further hydrated to yield niosomes [reverse phase evaporation (REV)] (Kiwada et al.,
1985) (Szoka & Papahadjopoulos, 1978). The vesicles generated by the REV method are
called unilamellar or oligolamellar vesicles and are relatively more uniform in size
compared with the TFH method (Szoka & Papahadjopoulos, 1978).
4. The injection of melted surfactant/lipid into a highly agitated heated aqueous phase in
which presumably the drug is dissolved (Wallach & Philippot, 1993) or the addition of a
warmed aqueous phase dissolving the drug to a mixture of melted lipids and hydrophobic
drug (Niemac et al., 1995). This method does not require the use of organic solvents,
which are expensive, hazardous and difficult to completely remove.
5. The homogenisation of a surfactant/lipid mixture, followed by bubbling of nitrogen gas
through this mixture (Talsma et al., 1994). The bubbling unit consisted of a round bottom
flask with three necks, positioned in a water bath to control temperature. A water-cooled
reflux column was connected to the central neck of the flask to avoid loss of water during
the experiment. A thermometer for temperature monitoring was positioned in the second
neck. The third opening was used for the tubing. A continuous stream of gas bubbles was
generated at the bottom of the flask by forcing nitrogen gas through a sintered glass filter
with a surface area of 1 cm2 by a slight over pressure. Prior to the gas bubbling step, the
lipid materials are added to the aqueous solution and then mixed for 30 min with a
homogenizer. The prepared vesicle dispersion was in the size range of 0.2 µm to 0.5 µm.
96
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Omitting the homogenisation step has been found not to affect the particle size; however,
a longer ‘bubbling time’ was required.
6. The supercritical carbon dioxide (scCO2) technique has been used as a more
environmentally friendly alternative. The carbon dioxide gas can be in a fluid state
(scCO2) under a temperature of 31.1oC and pressure of 78.8 bars. The scCO2 has good
solvating properties (Manosroi et al., 2008a). Niosomes composed of different molar
ratios of Tween 61: cholesterol mixtures were successfully prepared using the scCO2
method and encapsulated glucose (a model water soluble agent). The scCO2 niosomes
demonstrated higher drug entrapment efficiency than the niosomes prepared using the
TFH method. However, The TFH niosomes showed superior stability and dispersibility
compared with the scCO2 niosomes (Manosroi et al., 2008a).
Niosome size
The particle size of niosomes can determine their behaviour in the body (Uchegbu & Vyas,
1998). It has been reported that particles smaller than 100 nm easily penetrate tumour tissues
(Mayer et al., 1989). This is because these nano-particles can penetrate the discontinuous
capillaries and fenestra of tumour tissues (Nakano et al., 2008). A size reduction step is
occasionally incorporated into the niosome production procedure, subsequent to the initial
hydration step. This is in order to improve the biodistribution of systemically injected niosomes
and to target the administered niosomes at the affected area (Azmin et al., 1986; Uchegbu et al.,
1995; Uchegbu & Vyas, 1998). Niosome sizes > 10 µm are suitable for ophthalmic drug delivery
(Sahin, 2006). This allows them to encapsulate a considerable amount of therapeutic drug
molecules, resist removal by lacrimation and prolong ocular residence time. Micro-size niosomes
are not easily washed out by lacrimal fluid compared with nano-size niosomes (Uchegbu & Vyas,
1998). For instance, large niosomes (discomes) sized 12 - 60 µm were prepared and used for
ocular delivery of timolol maleate (water soluble drug). These were found to entrap a relatively
larger amount of timolol maleate (Vyas et al., 1998). The prepared discomes produced a
sustained drug effect. They were found to lower significantly intraocular pressure (IOP),
compared to an aqueous solution. The systemic drug absorption measured from discomes was of
a negligible level (Vyas et al., 1998). Figure 3.5 demonstrates a schematic of suitable niosome
sizes for particular routes of administration as adopted from (Uchegbu & Vyas, 1998).
97
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.5 Suitable niosome sizes for particular routes of administration
Generally, size reduction (sonication or extrusion methods) adversely affects the entrapment
efficiency of the vesicles to the active agents. This could be ascribed to their small inner volume
and drug leakage from small and intermediate-sized unilamellar vesicles (up to 100 nm)
(Uchegbu & Florence, 1995; Uchegbu & Vyas, 1998; Zhigaltsev et al., 2001). A reduction in
vesicle size may be achieved by a number of methods:
•
Probe sonication can produce niosomal vesicle sizes in the range of 100 to 140 nm
(Azmin et al., 1985; Baillie et al., 1985). Although it is more effective than bath
sonication, probe sonication suffers from high energy input to the solution, leading to a
sharp local increase in the temperature of the solution and shedding of titanium particles
into the solution. This necessitates an additional centrifugation step to remove the metallic
particles from the niosomal dispersion.
•
Extrusion through polycarbonate (Nucleopore™) filters using liposome extruders.
•
A combination of sonication and filtration has been utilised to prepare doxorubicin
niosomes in the 200 nm size range (Uchegbu et al., 1995).
•
High pressure homogenisation also yields vesicles below 100 nm in diameter, although
drug loading is ultimately sacrificed to achieve this small size (Uchegbu & Vyas, 1998).
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.1.2. Non-conventional niosomes
3.1.2.1. Elastic niosomes
Over the past three decades, researchers in the field of liposomes have introduced a new class of
liposomes. These have significant elasticity and ultradeformability, compared with conventional
liposomes. These elastic or ultradeformable liposomes, Transferosomes®, demonstrated the
ability to penetrate intact skin while carrying therapeutic concentrations of drugs (Cevc & Blume,
1992). They consist of phospholipids and an edge activator; an edge activator is often a single
chain surfactant that can destabilise the lipid bilayers of the vesicles, and increase the
deformability of the bilayers (Cevc et al., 1996). Parallel to this development, other classes of
vesicles were produced; vesicles with ethanol (ethosomes) to provide flexibility were developed
(Touitou et al., 1997). Ethosomes embodying a high concentration (20–45%) of ethanol
demonstrated enhanced skin delivery and were reported to penetrate intact skin layers (Touitou et
al., 2000). For more detail about these two delivery systems, the reader can refer to references (El
Maghraby & Williams, 2009; Elsayed et al., 2007).
When it comes to the literature on niosomes, there is scarcely any detailing on non-conventional
forms of niosomes. To our knowledge, only one study has reported on elastic niosomes
(Manosroi et al., 2008b). Tween 61 and Span 60 niosomes entrapping diclofenac
diethyammonium and embodying 0-25% ethanol have been developed. The prepared niosomes
were called elastic niosomes. This is because the prepared niosomes registered deformability
index values of 13.76 and 3.44 times higher than conventional niosomes loaded and non-loaded
with the drug respectively (Manosroi et al., 2008b).
3.1.2.2. Proniosomes
One approach to minimise the water content in liposomes and niosomes dispersions is to develop
a vesicle proconcentrate, called proliposomes or proniosomes. These contain all the vesicular
components with ethanol and only traces of water. These systems are gel-like preparations that
are believed to generate vesicles used for application to the skin (El Maghraby & Williams,
2009). Proniosomes are mainly composed of non-ionic surfactants, phospholipids, alcohols and
water, with or without cholesterol (Mokhtar et al., 2008).
The effects of formulation composition, surfactant and alcohol type, and preparation method on
the transdermal permeation profile of various proniosomes have been studied (Fang et al., 2001;
99
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Vora et al., 1998). The effects of non-ionic surfactants on the levonorgestrel permeation profile
showed that the flux value was the highest for Span 80 and lowest for Span 60. No significant
difference was observed in the skin permeation profile of formulations containing Span 40 and
Span 60, due to their higher phase transition temperature, which is responsible for their lower
permeability. Conversely, opposite effects in the case of estradiol were reported (Fang et al.,
2001). Span 60 showed the highest flux on the estradiol permeation profile. Proniosomes from
estradiol and levonorgestrel formulations differed in their content of cholesterol, and type and
content of alcohol. The effect of different alcohols on the drug permeation profile was found to
be as follows: isopropanol > butanol > propanol > ethanol. Proniosomes could become a useful
dosage form for transdermal drug delivery. This is due to their simple and scaling-up
manufacturing and the ability of proniosomes to modulate drug delivery across the skin (El
Maghraby & Williams, 2009).
3.1.3. Niosomes as drug delivery systems
Over the past three decades, niosomes have been successfully utilised as a drug delivery system
to solve some major biopharmaceutical problems. Poor bioavailability of slightly soluble drugs
can be improved, as niosomes can accommodate hydrophobic molecules in their bilayer domains
(Nasr et al., 2008; Suwakul et al., 2006). Niosomes can also minimise the side effects of some
potent drugs by localising the pharmacological effects to the target tissues (Baillie et al., 1986;
Baillie et al., 1985; Guinedi et al., 2005; Vyas et al., 1998). Niosomes can serve as a non-toxic
penetration enhancer for transdermal drug delivery (Schreier & Bouwstra, 1994; Solanki et al.,
2010). Moreover, niosomes can improve the chemical stability of photosensitive drugs by
entrapping drug molecules in the aqueous internal milieu (Manconi et al., 2003). Below are
discussed the most common applications of niosomes as drug delivery systems.
3.1.3.1. Anticancer delivery
Methotrexate-loaded niosomes were first utilised as a more stable and economic alternative drug
delivery system to liposomes, in order to improve the therapeutic index of the anticancer drug by
localizing the cytotoxic effects to target cells (Baillie et al., 1985). The anti-tumour activity of
vincristine also increased in S-180 sarcoma and Ehrlich-ascites-bearing mice when encapsulated
in niosomes (Parthasarathi et al., 1994). Moreover, Span 60 bleomycin niosomes increased the
anti-tumour action of bleomycin in the abovementioned two tumour models (Naresh & Udupa,
1996). Cytotoxic side effects of doxorubicin decreased when encapsulated in C16G2 niosomes.
Doxorubicin copolymer-loaded niosomes were not hemolytic in vitro at the doses selected for in
100
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
vivo use. C16G2 niosomes also showed localised and sustained effects on liver and spleen
(Uchegbu & Duncan, 1997). Furthermore, Span 40- and Span 60-based niosomal delivery
systems for 5-flurouracil showed a sustained and higher local concentration of the drug in the
liver and kidney of the rats, compared with the injectable drug solution (Namdeo & Jain, 1999).
3.1.3.2. Anti-infective delivery
Sodium stibogluconate is a drug used in the treatment of visceral leshmniasis (a protozoan
infection of the reticuloendothelial system). The drug’s pharmacokinetics were equally altered by
encapsulation into liposomes and niosomes (Hunter et al., 1988). It was found that niosomal
sodium stibogluconate was significantly more active in reducing parasite burden than the free
drug, and the effect observed after multiple dosing suggesting that niosomal formulations act as a
depot within the liver.
Rifampicin, an anti-tuberculosis drug, encapsulated in Span 85 niosomes was found to
accumulate in the lungs of mice (Jain and Vyas, 1995b). Therefore, it can offer the possibility of
improving anti-tuberculosis therapy (Jain & Vyas, 1995). Moreover, niosomes composed of Span
60: cholesterol: dicetyl phosphate at 4:2:1molar ratio successfully encapsulated ribavirin (an
antiviral agent used for treatment of hepatitis C). The concentration of ribavirin in the rat liver
obtained from the ribavirin niosomes was 6-fold higher than that obtained from the ribavirin
solution after an intraperitoneal injection of a single dose equivalent to 30 mg/kg of the drug.
This demonstrates effective liver targeting properties for niosomes (Hashim et al., 2010).
3.1.3.3. Macromolecules and vaccine delivery
Span 60 niosomes loaded with tetanus toxoid (TT) were prepared by the REV method. The
prepared niosomes were coated with a modified polysaccharide O-palmitoyl mannan (OPM), in
order to protect the niosomes from enzymatic degradation in the gastrointestinal tract and to
enhance their affinity toward the antigen presenting cells of Peyer’s patches. The results were
compared with alum-adsorbed TT following oral and intramuscular administration, and it was
observed that OPM-coated niosomes produced higher IgG levels compared with plain uncoated
niosomes and alum-adsorbed TT upon oral administration. Oral niosomes also elicited a
significant mucosal immune response (sIgA levels in mucosal secretions). The developed
niosomes also elicited a combined serum IgG2a/IgG1 response suggesting that they were capable
of eliciting both humoral and cellular response. The proposed system was simple, stable, and
cost-effective and may be clinically acceptable. The study emphasised the potential of OPM101
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
coated niosomes as an oral vaccine delivery carrier (Jain & Vyas, 2006). Moreover, niosomes
loaded with insulin were prepared using the TFH method. The Span 60 niosomes protected
insulin against proteolytic enzymes, and showed good stability in the presence of sodium
desoxycholate (Varshosaz et al., 2003).
3.1.3.4. Transdermal delivery
Niosomes were studied as a transdermal drug delivery systems and their ability to enhance drug
permeation through the intact stratum corneum was also investigated (Reddy & Udupa, 1993;
Schreier & Bouwstra, 1994; Van Hal et al., 1996).
For instance, small niosomes (100 nm) have been visualised by freeze fracture electron
microscopy technique between the first and second layer of human corneocytes 48 h after
incubation with niosomes (Junginger et al., 1991). The penetration of this upper layer by
niosomes appeared feasible (Junginger et al., 1991). However, no transport of niosomes took
place across the whole skin layer (Junginger et al., 1991).
In vitro studies on the transdermal penetration of oestradiol using high-phase transition sucrose
ester niosomes or C18EO7 niosomes and low phase transition poly-7-oxyethylene alkyl ether
(C12EO7) niosomes revealed that the latter were better transdermal carriers (Van Hal et al., 1996).
C12EO7 micelles were ineffective as drug carriers in this study. The higher flexibility of these
bilayers is believed to be responsible for the improved transdermal penetration (Van Hal et al.,
1996). Also, it was found that reducing the cholesterol content in niosomes increased the
transdermal delivery of oestradiol. These can be attributed to increases in the flexability of the
vesicles (Van Hal et al., 1996).
Niosomes were developed for the transdermal drug delivery of nimesulide (non-steroidal antiinflammatory drug). The prepared niosomes were evaluated in vivo in rats using the carrageeninduced rat paw oedema method. This investigation conclusively demonstrated that the niosomes
successfully prolonged the drug release into the skin and across the skin (Shahiwala & Misra,
2002).
Furthermore, permeation of ketorolac (a potent nonsteroidal anti-inflammatory drug) across
excised rabbit skin from various proniosome gel formulations was investigated using Franz
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
diffusion cells. The prepared proniosomes significantly improved drug permeation and reduced
lag time (Ibrahim et al., 2005).
3.1.3.5. Ophthalmic delivery
For the ideal properties and application of niosomes as an ocular drug delivery system refer to
Niosmes at Section 1.2.4.7 in Chapter 1. Furthermore, the physical stability of niosomes will be
discussed in Chapter 5.
3.2. Chapter aims
The drug under investigation (NTX) is water-soluble, and as general rule of thumb, hydrophilic
drugs are less preferentially up taken by niosomes than lipophilic ones. The aim of this chapter is
to study the effect of the main constituting lipids (surfactants and cholesterol) on niosomes
formation and their physical properties. Two classes of surfactants are used; sorbitan esters
(Span®) and polyoxyethylene alkyl ethers (Brij®). These surfactants are commercially available
and widely used in the pharmaceutical, food and cosmetic industries (Lawrence, 2003; Yu, 2003).
The surfactants used are selected for their versatility in chemical structures and gel/liquid
transition temperatures. They have different polar head groups and hydrocarbon chains of various
lengths and degrees of saturation. The specific objectives of this chapter were to:
•
Prepare niosomes by the conventional thin film hydration method using two classes of
surfactants, which include sorbitan esters (Span 20, Span 40, Span 60 and Span 80) and
polyoxyethylene alkyl ethers (Brij 52 and Brij 72).
•
Image niosomes macroscopically and microscopically using different techniques such as
light/polarised light microscopy and cryogenic scanning electron microscopy.
•
Optimise cholesterol (a bilayer membrane stabiliser) levels in the prepared niosomes by
studying the effect of cholesterol concentrations on:
o
Vesicle size.
o Membrane fluidity (gel/liquid phase transition temperature).
o Drug entrapment efficiency and loading efficiency.
•
Study the effect of total lipid content (surfactant/cholesterol) and the initial amount of
NTX on entrapment efficiency and drug loading.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.3. Materials and methods
3.3.1. Materials
NTX was purchased from the same source as mentioned in Chapter 2. Span 20, Span 40, Span
60, Span 80, Brij 52 and Brij 72 were purchased from Sigma-Aldrich, St. Louis, USA. All other
solvents and buffer salts were of analytical grade and used as received.
3.3.2. Methods
3.3.2.1. Niosome preparation
Thin film hydration method (TFH)
Niosomes were prepared by adopting the TFH method (Azmin et al., 1985). Briefly, a specified
amount (300 µmol) of lipids (surfactant: cholesterol) at a molar ratio of 1:0, 9:1, 8:2, 7:3, 6:4, and
1:1 (Table 3.1) was dissolved in a chloroform: methanol (2:1 v/v) mixture and rotary evaporated
(Heidolph, Laborota 4000, GmbH, Germany) to form a thin lipid film. The dried lipid film was
purged with a stream of nitrogen for 5 min in order to remove any residual traces of the organic
solvent. The lipid film was then hydrated with either 5 ml of PBS pH 7.4 or NTX solution
(1mg/ml) in PBS at 200 rpm for 1 h. The selected hydration time at 1h was based on preliminary
studies. Longer hydration time did not show further improvement in terms of NTX loading. The
resultant niosomal suspension was set aside for at least 2 h at room temperature, to allow the
vesicle’s membrane to anneal. The formed niosomes were stored in a fridge for subsequent
analyses.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Table 3.1 Composition of the prepared niosomes using various surfactant: cholesterol
molar ratios
Molar ratio
Span 20
1:1
6:4
7:3
8:2
9:1
Span 40
1:1
6:4
7:3
8:2
9:1
Span 60
1:1
6:4
7:3
8:2
9:1
Span 80
1:1
6:4
7:3
8:2
9:1
Brij 52
1:1
6:4
7:3
8:2
9:1
Brij 72
1:1
6:4
7:3
8:2
9:1
Surfactant
(g/mol)
346.00
52.00
62.28
72.66
83.00
93.42
403.00
60.45
72.54
84.63
96.72
108.81
431.00
64.65
77.58
90.50
103.44
116.37
429.00
64.35
77.22
90.10
102.96
115.83
330.00
49.50
59.40
69.30
79.20
89.10
358.00
53.70
64.44
75.19
85.92
96.66
Cholesterol
(g/mol)
386.65
58.00
46.40
35.00
23.20
11.60
386.65
58.00
46.40
35.00
23.20
11.60
386.65
58.00
46.40
35.00
23.20
11.60
386.65
58.00
46.40
35.00
23.20
11.60
386.65
58.00
46.40
35.00
23.20
11.60
386.65
58.00
46.40
35.00
23.20
11.60
Total lipid weight
(mg)
Equivalent no. of
moles (µmol)
110.00
108.68
107.46
106.20
105.02
300
300
300
300
300
118.45
118.94
119.43
119.92
120.41
300
300
300
300
300
122.65
123.98
125.50
126.64
127.97
300
300
300
300
300
122.35
123.62
125.10
126.16
127.43
300
300
300
300
300
107.50
105.80
104.30
102.40
100.70
300
300
300
300
300
111.70
110.84
110.19
109.12
108.26
300
300
300
300
300
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.3.2.2. Physical properties
Niosome imaging
Plane and polarised light microscopy
To assure vesicle formation, all lipid dispersions were examined using a light microscope
equipped with a cross polariser. Briefly, a drop of the prepared lipid dispersion was placed on a
microscope glass slide, covered with a glass cover slip and examined using a polarised light
microscope (Leica DMR, GmbH, Germany). The samples were examined for vesicle formation.
Large multilamellar vesicles were recognised by their characteristic Maltese cross textures
(Manosroi et al., 2003; Weder & Zumbuehl, 1984).
Cryogenic scanning electron microscopy (Cryo-SEM)
The morphology and lamellarity of the prepared niosomes were studied using cryo-SEM. A drop
of the prepared niosomal suspension was sandwiched between two fracture rivets (internal
diameter = 1.58 mm). This assembly was held by a gold-plated copper sample holder. The whole
assembly was connected to a transfer rod, slushed in liquid nitrogen under a vacuum and then
transferred into a Cryo-unit (Gattan Alto 2500, England). A fracture was created by removing the
upper rivet using a scalpel. The fractured sample was heated up to -85oC for 30 min to sublime
the surface moisture. Subsequently, the sample was gold coated and transferred to SEM (Philips
XL30S FEG, Netherlands) for imaging at a temperature -120oC and voltage of 5 kV.
Entrapment efficiency percentage (EE %) and drug loading percentage (DL%)
The EE% and DL% determination methods used relied on destruction of the bilayer membranes
and subsequent quantification of the released material (Abdelbary & El-gendy, 2008; Nasr et al.,
2008). Ultracentrifugation was adopted to determine the encapsulated amount of NTX in
niosomes. In brief, NTX-loaded niosomes were separated from the non-entrapped NTX
molecules by diluting 2 ml of niosome dispersion to 30 ml with PBS, prior to centrifugation at
60,000 g (Sorvall Discovery 100S, USA) for 30 min. The formed niosomal pellets were washed
with an additional 30 ml of PBS and centrifuged for a further 30 min. The niosomal pellets were
re-suspended in 2 ml of PBS and 2ml of isopropanol were added (1:1 v/v), followed by 5 ml of
PBS. The dispersion was re-centrifuged and the supernatant was quantitatively assayed for NTX
content using high performance liquid chromatography (HPLC). The HPLC system used (Agilent
1200, Germany) was equipped with a C18-reverse phase column (Supelcosil LC-18DB 25 cm x
4.6 mm, 5 µm). The mobile phase comprised an acetonitrile: phosphate buffer pH 4 (40 mM)
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
30:70 system, and detection was achieved using a PDA detector at 214 nm. The drug entrapment
efficiency was expressed as a percentage (EE %) and calculated using equation 3.1(Nasr et al.,
2008):
A
X 100
Ao
EE % =
equation
3.1
Where A is the amount of NTX entrapped by niosomes and Ao is the initial amount of NTX used.
DL % was calculated using equation 3.2 (Wang et al., 2004).
DL% =
A
X 100
W
equation 3.2
Where A is the amount of NTX entrapped by niosomes and W is the total weight of lipid used
initially.
Niosome size measurements
The size of the prepared vesicles was determined by a laser diffraction technique (Mastersizer
2000, Malvern Instruments, UK). The samples were properly diluted with PBS and measured at
25 oC. Niosomes size was expressed in terms of volume diameter (D [4,3]) and the measurements
were done in triplicate and the average values were used.
Membrane fluidity (gel/liquid transition temperature) studies of niosomes using differential
scanning calorimetry (DSC)
Niosomal dispersions containing 100 mg/ml of the total surfactant/lipid mixtures were prepared
using the TFH method, as described in Section 3.3.2.1. The niosomal dispersions comprised 1:0,
9:1, 8:2, 7:3, 6:4, and 1:1 Span 60: cholesterol molar ratio. A small amount (approx. 5 mg) of the
prepared niosomes was accurately weighed in an aluminium pan, covered with an aluminium lid
and hermitically sealed using a pan press (Thermal Science, USA). Another pan containing an
equivalent amount of water was sealed and used as a reference cell. The temperature of the pans
was raised from 4 to 80oC at a rate of 5oC/min using a differential scanning calorimeter
connected to an automatic sampler (Q 1000, Tzero series, Thermal Analysis, USA). Nitrogen gas
was purged at a flow rate of 45 ml/min.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.3.3. Statistical analysis
Statistical significant differences between formulations were analysed, using one-way analysis of
variance (ANOVA) and student t test at the 5% significance level, by GraphPad Software
Version 3.05, San Diego California, USA.
3.4. Results and discussion
3.4.1. Niosome formation and imaging using plane /polarised microscopy
The following surfactants were investigated for their ability to form niosomes in combination
with different concentrations of cholesterol (0% to 50 % mol/mol) using the TFH method:
sorbitan esters (Span 20, Span 40, Span 60 and Span 80) and polyoxyethylene alkyl ethers (Brij
52 and Brij 72). Table 3.2 shows the chemical structures, gel-liquid transition temperatures (Tm)
and hydrophilic-lipophilic balance (HLB) values of the surfactants used, and summarises the
ability of each surfactant to form niosomes alone and the minimal level of cholesterol required to
form niosomes. The formed niosomes were visualised under a polarised light microscope, and
Figure 3.6 shows representative micrographs of niosomes. Different textures, such as uniform
spheres and Maltese crosses, are characteristic under the normal and polarised light microscope
(Mura et al., 2007).
Span 40, Span 60, Brij 52 and Brij 72 formed niosomes without cholesterol. On the contrary,
Span 20 and Span 80 did not self-assemble into niosomes unless 10 to 20% mol/mol of
cholesterol was incorporated. These findings can be attributed to the geometrical packing
parameters of the surfactants used. The geometrical packing parameters depend on the optimal
polar head group area ao, the volume of the hydrocarbon chains v and the maximum effective
length that the hydrocarbon chains can assume (critical chain length) lc. Accordingly, the value of
the dimensionless ‘packing parameter’ v/ ao lc will determine whether a certain amphiphile will
form niosomes (1/2 < v/ ao lc < 1), or other surfactant aggregates, such as spherical micelles ( v/
ao lc < 1/3), non-spherical micelles (1/3 < v/ ao lc < 1/2), or inverted micellar structures (v/ ao lc >
1), or even precipitate out of solution (Israelachvili et al., 1980; Israelachvili & Mitchell, 1975).
The niosomal dispersions formed and contained 0% cholesterol exhibited extensive gelling at the
room temperature. The formed gel was difficult to separate from the non-entrapped drug
molecules. Additionally, all niosomal dispersions prepared from Brij 52 showed phase separation
and creaming upon storage overnight (Figure 3.7A). Therefore, all Brij 52 niosomes were
excluded from any further investigation.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Table 3.2 Chemical structure, phase transition temperature, HLB and vesicle-forming ability of
the used surfactants
a Phase transition temperature (Tm) of the unhydrated samples.
b Hydrophilic- lipophilic balance (HLB) values were given by suppliers.
* Vesicle formation was determined by visualisation of Maltese cross textures under polarised light
109
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.6 Representative micrographs of Brij 72:cholesterol 7:3 mol/mol-based niosomes
under plane light (left) and polarised light microscope (right)
A
1:1
6:4
7:3
B
1:1
6:4
7:3
Figure 3.7 Niosomal dispersions containing various molar ratios of Brij 52:cholesterol (A) and Span
60:cholesterol (B); and showing phase separation of the Brij 52 niosomes (A) and poor lipid hydration
and non-vesicle aggregates at higher contents of cholesterol in Span 60 niosomes (B)
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.4.2. Niosome imaging using Cryo-SEM
The microstructure and lamellarity of the prepared niosomes were studied using Cryo-SEM
(Figure 3.8). The micrographs captured revealed spherical-shaped niosomes and typical
concentric multilayers, organised in an onion-like or rose-like morphology, indicating
multilamellarity of the prepared niosomes. Niosomes of rough surfaces were obvious due to the
sample preparation technique used which is based on snap freezing in liquid nitrogen and surface
fracture formation by a scalpel.
It is worth noting that Cryo-SEM has been reported to be more reliable than freeze-fracture
transmission electron microscopy (FF-TEM) (Egelhaaf et al., 2003; Perrie et al., 2007). The steps
involved in FF-TEM are likely to promote the formation of artefacts during sample preparation
and replica creation (Egelhaaf et al., 2003; Perrie et al., 2007).
Figure 3.8 Cryo-SEM micrographs of Span 60: cholesterol 7:3 mol/mol (left) and Brij 72:
cholesterol 7:3 mol/mol (right) niosomes
3.4.3. Niosome size measurements
Table 3.3 shows the D [4,3] of the prepared niosomes composed of various surfactants and
contained various cholesterol levels (30%-50% mol/mol). The mean D [4,3] values of the
prepared niosomes ranged from 7.0 ± 1.0 to 14.6 ± 0.8 µm (Table 3.3). The size measurements
were repeatable and reproducible from batch to batch (acceptable deviations from the average
values, Table 3.3).
111
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Table 3.3 Effect of surfactant type and cholesterol level on the D [4,3] and EE % of the
prepared niosomes (Results are expressed as mean values ± SD, n = 3)
Span 20
Span 40
Span 60
Span 80
Brij 72
Cholesterol level (% mol/mol)
30%
40%
50%
D [4,3] (µm)
14.2 ± 0.6
14.5 ± 0.65 14.6 ± 0.8
12.7± 1.0
13.0±1.50
13.4±1.20
7.3 ± 0.62
7.6 ± 0.75 7.9 ±0.90
7.0 ± 1.00
7.4 ± 0.88 7.5 ± 1.20
9.0 ± 0.82
9.5 ± 0.88
9.8 ± 1.0
Cholesterol level (% mol/mol)
30%
40%
50%
EE% (w/w)
3.5 ± 0.23 3.0 ±0.15
2.5 ±0.23
9.0 ± 0.67 7.0 ± 0.62
6.0 ±0.54
13.5 ± 0.56 9.0 ± 0.45
7.0 ±0.67
3.2 ± 0.22 4.0 ± 0.35 2.8 ± 0.25
7.0 ± 0.55 5.0 ± 0.47 4.5 ± 0.32
The increases in the D [4,3] values were insignificant (P > 0.05), when cholesterol level ranged
from 30% to 50% mol/mol. Similar results were reported elsewhere (Essa, 2010). Span 40:
cholesterol-based niosomes showed insignificant increase in sizes when cholesterol ratio
increased from 2:1 to 1:1 mol/mol. Cholesterol is a rigid molecule with an inverted cone shape. It
can be intercalated between the fluid hydrocarbon chains of the amphiphile when hydrated at a
temperature above the gel/liquid transition temperature. It can also produce larger vesicles (Essa,
2010; Uchegbu & Florence, 1995). Cholesterol is a membrane stabiliser and can be
accommodated and work cooperatively with the surfactant bilayer membranes. However, this
accommodating characteristic is limited to a certain cholesterol level which is apparently equal to
30% mol/mol. The ability of the investigated surfactants to accommodate cholesterol was
obviously decreased when cooled below the melting point, where the hydrocarbon chains are
fully extended (all-trans) and exist in a gel state. Figure 3.7B shows plain niosomal dispersions
containing different levels of cholesterol after storage at the room temperature overnight. Such
storage allows the vesicle’s membrane to anneal and correct any membrane defect. Niosomal
dispersions containing cholesterol > 30% mol/mol were poorly hydrated and excess cholesterol
precipitated out (Figure 3.7B). This may be attributed to the compromised ability of the
hydrocarbon chains of the surfactant used to accommodate cholesterol (Grant et al., 2001;
Israelachvili et al., 1980).
Table 3.3 also showed that the D [4,3] values of the prepared niosomes were significantly (P <
0.05) dependent on the HLB of the used surfactants. This effect was more clearly illustrated in
112
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.9. The D [4,3] values of Span 60-based niosomes were significantly smaller than the
Span 40-based ones. Lipid aggregation and self-assembly in an aqueous medium is mainly
controlled by two opposing electrostatic forces. These are the hydrophobic attraction forces
between the hydrocarbon tail chains and the electrostatic repulsion of the polar head groups
(Tanford, 1973). The surface free energy increases with any decrease in hydrophobicity
(Yoshioka et al., 1994). Hence, surfactants of higher hydrophilicity (higher HLB) should yield
larger vesicles.
Figure 3.9 Effect of HLB on Span-based niosomes composed of surfactant:cholesterol
at 7:3 molar ratio (Results are expressed as mean values ± SD, n = 3)
* Significant effect (P < 0.05)
** No significant effect (P > 0.05)
113
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.4.4. NTX EE%
3.4.4.1. Effect of surfactant type on NTX EE%
The EE% values for the prepared niosomes ranged from 2.5% ± 0.23 to 13.5% ± 0.56 (Table
3.3). Span 40 and Span 60 established the highest EE%, whereas the lowest EE% values were
related to Span 20 and Span 80. The main factors affecting the entrapment of small water-soluble
drug molecules in the bilayer vesicles (niosomes) are the permeability of the biomelcular
membranes and the structure continuity of the hydrocarbon chain of the bilayer-forming
surfactant (Grant et al., 2001). Span 40 and Span 60 have saturated acyl chains [palmityl (C-16)
and stearyl (C-18) chains respectively] which are in a gel state at ambient conditions (Grant et al.,
2001). Such features render these vesicles less likely to leak the encapsulated water-soluble drug
molecules (NTX). In contrast, Span 20 and Span 80 are fluid at room temperature and, in the
presence of cholesterol, are disorganised due to the trans-gauche conformations of their acyl
chains. The introduction of double bond at C9 position of the oleyl chain of Span 80 creates a
twist or imperfection of the niosomal bilayer membranes (kinks). These kinks create vacancies in
the hydrophobic chains and renders Span 80-based niosomes more leaky and permeable to NTX
than even Span 20 (C12)-based niosomes. These findings explain the lowest EE% achieved by
Span 80-based niosomes. The EE% values for the prepared niosomes can be summarised as
follows: Span 60 > Span 40 > Brij 72 > Span 20 > Span 80. These results support the hypothesis
that the higher the transition temperature of the surfactant is, the higher the corresponding EE%
will be for water-soluble solutes (Yoshioka et al., 1994). This effect is clearly illustrated in Figure
3.10.
114
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.10 Effect of gel/liquid transition temperature on EE% for
niosomes composed of surfactant:cholesterol at 7:3 molar ratio (Results
are expressed as mean values ± SD, n = 3)
* Significant effect (P < 0.05)
3.4.4.2. Effect of cholesterol on NTX EE%
The literature details controversial results concerning the effect of cholesterol on EE%. Some
studies have shown that cholesterol has no effect on EE% (Uchegbu & Florence, 1995). Other
studies demonstrated that EE% increases with increasing cholesterol concentrations from 0% to
50% mol/mol (Yoshioka et al., 1994). On the other hand, other reports have shown that
cholesterol had a limiting (up to 30% mol/mol) enhancement of EE% (Abdelbary & El-gendy,
2008; Moazeni et al., 2010). Our results were found to agree with the final opinion.
Figure 3.11 illustrates the effect of cholesterol levels on NTX EE%. The results demonstrated
that the cholesterol level significantly (P < 0.05) affected the ability of niosomes to entrap NTX.
Increasing the cholesterol concentration can improve the EE% of NTX, but only to a certain
extent. Any further increase of cholesterol beyond a certain concentration (30% mol/mol for Span
40, and Span 60 and 40% mol/mol for Span 80) markedly decreased EE%.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
The initial increase in NTX EE% is likely due to the initial intercalation of cholesterol molecules
between the hydrocarbon chains of the surfactant bilayers, hence, the permeability to NTX (a
water soluble molecule) decreases. The prepared niosomes established EE% peaked at 30%
mol/mol. Span 80 niosomes exhibited EE% peaking at 40% mol/mol due to structural
disorganisation and kinks in the oleyl chains of Span 80 niosomes. These vacancies in the bilayer
chains can accommodate more cholesterol molecules to reach the peak of EE%.
Further increases in cholesterol concentration could increase the rigidity of the bilayer
membranes, by virtue of cholesterol’s rigid structure and characteristic inverted cone shape
(Israelachvili et al., 1980). This results in disruption of the vesicles’ bilayer structure which
compromises their ability to entrap NTX (Manosroi et al., 2003). This could lead to a decrease in
the total amount of lipid available for encapsulating the drug. Consequently, there is a decrease in
the ability of the niosomes to entrap such water-soluble molecules. These findings are displayed
in Figure 3.7B; the glass vials show precipitates at a cholesterol level higher than 30 mol%.
These results were in a good agreement with a previous study. Uncharged niosomes composed of
Tween 60 and cholesterol at 1: 0.5 mol/mol had a higher EE% than those containing higher ratio
of Tween 60 and cholesterol at molar ratio of 1:1 (Abdelbary & El-gendy, 2008).
116
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.11 Effect of cholesterol concentration on EE% for Spanbased niosomes (Results are expressed as mean values ± SD, n = 3)
3.4.4.3. Effect of total lipid content on NTX EE% and DL%
The effect of total lipid content on the EE% and DL% of NTX was investigated (Figures 3.12 and
3.13). The results revealed a linear increase (R2 > 0.98) in the EE% of the drug with increasing
the total lipid content of the same composition (Span 60:cholesterol in 7:3 mol/mol). This is due
to the increasing the lipid/drug ratio available for hydration.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.12 Effect of the total surfactant/lipid content on EE % for Span 60:
cholesterol
(7:3 mol/mol) niosomes (Results are expressed as mean values ± SD, n = 3)
On the contrary, DL% non-linearly decreased with increasing surfactant/lipid content (Figure
3.13). Similar results have been reported with both liposomes (Kirby & Gregoriadis, 1984a) and
proniosomes (Mokhtar et al., 2008). The DL% values were found to decrease with increasing the
total surfactant/lipid amounts. This could be ascribed to the lower hydration efficiency associated
with increasing the lipid content. The anhydrous lipid film becomes hydrated and rounds off into
vesicles encapsulating a small portion of the aqueous phase. When the surfactant/lipid content
increases, an inevitable portion of the surfactant/lipid never comes in contact with the aqueous
phase. This unhydrated portion could increase with increasing the the surfactant/lipid content.
118
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.13 Effect of the total surfactant/lipid content on DL % for Span 60: cholesterol
(7:3 mol/mol) niosomes (Results are expressed as mean values ± SD, n = 3)
3.4.4.4. Effect of initial NTX concentration on NTX EE% and DL%
Figure 3.14 shows the effect of the initial amount of NTX on EE%. The results revealed that the
EE% markedly decreased when the initial amount of NTX used increased. Since it is a weakly
basic drug with a low octanol/buffer partition coefficient, NTX can show amphipatic properties,
especially at the elevated temperature encountered during niosomes formation where ionisation
of the nitrogenous basic drug (NTX) considerably decreased. The interaction of unionised species
with bilayer membranes could not be ruled out especially at higher NTX concentrations and the
elevated temperature during the self-assembly of the surfactant molecules. This interaction could
disrupt the bilayer formation of niosomes and limit the ability of niosomes to entrap NTX. This
was evidenced by precipitating out non-bilayer lipid aggregates when increasing the initial NTX
concentration.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.14 Effect of the initial amount of NTX on EE % for Span 60: cholesterol
(7:3 mol/mol) niosomes (Results are expressed as mean values ± SD, n = 3)
The effect of the initial amount of NTX on DL% is illustrated in Figure 3.15. The DL% slightly
increased at an initial NTX concentration of 2 mg/ml and then steeply declined at a higher NTX
concentration (3 mg/ml). The DL % results suggested that the initial NTX concentration of 2
mg/ml is optimal for NTX loading and it can be utilised for further NTX loading studies.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.15 Effect of the initial amount of NTX on DL % for Span 60:
cholesterol (7:3 mol/mol) niosomes (Results are expressed as mean values
± SD, n = 3)
3.4.5. DSC studies
3.4.5.1. Effect of cholesterol on gel/liquid phase transition of Span 60 niosomes
DSC was employed to understand the interaction of cholesterol with the single-chain non-ionic
surfactant Span 60, and to monitor its effect on the gel/liquid transition of the prepared niosomes.
The bilayer membrane is an ordered structure which may exist in a gel or liquid crystalline state.
Essentially, molecules are more mobile in a liquid crystalline state, enjoying the lateral diffusion
within the bilayer that they are denied in the gel state (Israelachvili et al., 1980). The liquid
crystalline state exists at a higher temperature than the gel state. An increase in temperature
favours the transition from the gel to the liquid state because of the entropy gain (ΔS) associated
with this transition. This ultimately leads to a lowering of the free energy (ΔG) of the system
(Israelachvili et al., 1980).
Figure 3.16 demonstrates the effect of cholesterol level on the gel-liquid transition of Span 60
niosomes. Table 3.4 presents two parameters: the gel/liquid transition temperature (Tm), which is
the temperature peak point at which transition (melting) from gel to solid is half-complete; and
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
the transition enthalpy, which is the amount of heat in calories/mol required for the entire
transition to take place. The thermogram of Span 60-based vesicles with 0% cholesterol shows a
thermal event at 63oC (Figure 3.16), which was markedly higher than that of the constituting
Span 60 surfactant (melting point 55oC) in the unhydrated form. This increase in the Tm of the
Span 60 might be attributed to the hydration and self-assembly of the surfactant molecules
forming closed bilayer structures in water. As such, greater energy was required to break the
strong hydrophobic interaction between surfactant moieties in the hydrated state. However,
unlike phospholipids, no pre-transition peak was seen with Span 60 (Figure 3.16). This might be
due to the well defined structure and composition of the non-ionic surfactant used.
Cholesterol exhibited a marked effect in decreasing both Tm and transition enthalpy. This was
clearly evident when gradually decreasing both the area under the peaks of DSC thermograms
and Tm values. The cholesterol contents in the membrane increased until the complete
disappearance of the gel-liquid transition of Span 60 at 50% mol/mol. This is due to intercalation
of cholesterol molecules between the lipid bilayer, which weakens Van der Waals forces between
the hydrocarbon chains of surfactant’s bilayer membranes. These findings were in accordance
with previous reports, where it was found that cholesterol could alter the fluidity of the bilayer
membranes of liposomes (Taylor & Craig, 2003). Finally, it is worth noting that the residual gelliquid transition recorded for niosomes containing 30% and 40% mol/mol of cholesterol might be
advantageous. Such transition can impart bilayer flexibility and thermo-responsiveness. As such
this renders them promising carriers for ocular drug delivery. On the contrary, complete
abolishment of the gel-liquid transition makes such niosomes rigid and as such less attractive for
ocular applications.
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Table 3.4 DSC parameters of Span 60-based niosome dispersions containing different
concentrations of cholesterol
Tm (oC)
∆H (Cal/mol)
0
63.44
89.00
10
55
45.10
20
51
2.05
30
47.22
0.82
40
43.5
0.43
50
No transition
-
Cholesterol concentration
(% mol/mol)
123
Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Figure 3.16 DSC thermograms of Span 60-based niosomes containing different
concentrations of cholesterol
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
3.4.5.2. Effect of NTX on gel-liquid transition of Span 60 niosomes
The effect of the encapsulated NTX on the Tm and enthalpy (heat content,
∆H) was
also
investigated (Figure 3.17). The results showed that the Tm and enthalpy values of NTX free and
loaded niosomes (7 Span 60: cholesterol: 3 mol/mol) were 47.22, 46.8oC and 0.82, 0.73 cal/mol
respectively. The results showed a slight difference in Tm and enthalpy of the NTX loaded
niosomes and NTX free niosomes. However, the differences were insignificant (P > 0.05, Student
t-test). Therefore, the entrapment of NTX molecules have a slight or even no effect on the fluidity
of the bilayer membranes of the prepared niosomes, suggesting that the drug was encapsulated
inside the aqueous core. These results are correlated well with the partition coefficient studies
which indicate that NTX is a hydrophilic drug.
Figure 3.17 Effect of NTX on gel/liquid transition temperature of (Span 60: cholesterol
7:3 mol/mol) niosomes
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Chapter 3….Effect of Surfactants and Cholesterol on Niosomes Physical properties
Conclusion
•
Span 60 is a promising surfactant, in terms of its ability to encapsulate NTX compared
with other surfactants.
•
NTX solutions of 2 mg/ml and 1mmol of lipid will be used for further studies, for better
NTX loading in niosomes.
•
Optimising cholesterol level can significantly alter the physical properties of the prepared
niosomes, most notably their thermo-responsiveness.
•
Such behaviour is likely to render these vesicles more amenable to the ocular temperature
and less likely to create a foreign body sensation (and subsequent reflex tear production
and blinking) when instilled topically to the eye surface.
Unfortunately, conventional niosomes (surfactant and cholesterol) have limited ability to
encapsulate a considerable amount of NTX, in order to attain the required therapeutic dose
(0.4 mg/ml). Therefore, the next chapter is concerned with studying the effect of some
selected excipients and other non-conventional methods of niosome preparation, in an attempt
to enhance NTX EE% and to obtain various niosomal formulations with versatile properties
for the effective ocular delivery of NTX.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4. Effect of membrane additives on the physical properties of
niosomes: size, morphology, spreading ability, rheological and
stabilising properties
4.1. Introduction
4.1.1. Bilayer membrane additives
Bilayer membrane additives can be defined as lipid excipients which are unable to form lipid
vesicles (liposomes and niosomes) by their own but they can be accommodated by the bilayer
domains. The membrane additives have been found in the lipid vesicles basically for two reasons;
either to alter the biodistribution, or to improve the physical properties. Non-ionic surfactants can
form niosomes in absence of other membrane additives under specified conditions. However,
such niosomes have been found to be highly permeable and leaky to the solutes. Cholesterol is
the most common membrane additive found in lipid vesicles. Cholesterol can significantly alter
membrane permeability, membrane fluidity, vesicle stability, entrapment efficiency and niosomes
size and it is widely known as a membrane stabiliser. Other membrane additives can be broadly
classified into charge inducers and water-soluble surfactants.
4.1.1.1. Charge inducers
Stearyl amine and dicetyl phosphate are positive and negative charge inducers respectively, and
are frequently incorporated in the bilayer membranes. These charge inducers have been mainly
utilised to improve the physical stability of the vesicular dispersions against aggregation
(Uchegbu & Vyas, 1998); to prolong the half life of circulating lipid vesicles in plasma (Maurer
et al., 2007); and to prolong the ocular residence time of topically instilled ophthalmic lipid
vesicles (Kaur et al., 2004).
For example, incorporation of positively charged lipids such as stearyl amine is essential for
successful delivery of negatively charged polynucleotides and efficient transfection into the cells
(Maurer et al., 2007). Also, incorporation of dicetyl phosphate in niosomes significantly
improved gentamicin sulfate entrapment efficiency in the prepared niosomes for ocular delivery
compared with the neutral niosomal formulations (Abdelbary & El-gendy, 2008). Negatively
charged niosomes have been also found most effective in prolonging gentamicin release rate
(Abdelbary & El-gendy, 2008).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
On the other hand, introduction of water soluble surfactants into vesicles’ bilayer membranes
produces liposomes and niosomes with numerous applications.
4.1.1.2. Water soluble surfactants
Liposomes
The addition of surfactant molecules to vesicular dispersions of phospholipids leads to
breakdown or solubilisation of the liposomes and formation of a mixed micellar phase. Such
vesicle to micelle transitions of phosphatidylcholine liposomes have been achieved by the
addition of non-ionic surfactants of the polyoxyethylene cetyl ether class (Kim & Kim, 1991). In
this system a region occurs where lamellar and mixed micelles coexist. In another study, sodium
cholate was found to induce a vesicle to micelle transition in cholate-phosphatidylcholine
systems (Walter et al., 1991b). On increasing the bile salt concentration, more multilamellar
vesicles were detected. In addition, “open” vesicles, large (twenty to several hundred nanometres
in diameter) bilayer sheets, and long (150-300 nm) flexible cylindrical vesicles were also
detected (Walter et al., 1991b).
Furthermore, new classes of liposomes, called Transferosomes®, have been developed upon
mixing phospholipids with some water-soluble surfactants, such as sodium cholate and Tween
80. Transferosomes® have the same morphology as liposomes, but they have ultradeformable
bilayer membranes. These non-conventional liposomes have been reported to penetrate intact
skin when applied non-occlusively. To prepare these vesicles, surfactants such as sodium cholate
and sodium deoxycholate have been incorporated in the vesicular membrane, in a concentration
range from 20%-50% mol/mol (Cevc et al., 1996; Planas et al., 1992).
Niosomes
Niosomes derived from non-ionic surfactants of low aqueous solubility are analogues of
liposomes (Florence & Baillie, 1989). Niosomes are similar in many respects to liposomes, being
prepared in the same way forming unilamellar or multilamellar structures which entrap lipidsoluble or water-soluble solutes (Uchegbu et al., 1992). The incorporation of poly-24oxyethylene cholesteryl ether (Solulan C24) into niosomes has been found to generate atypical
niosomal structures such as polyhedral niosomes and discomes (Uchegbu & Florence, 1995;
Uchegbu & Vyas, 1998).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Polyhedral niosomes
Polyhedral niosomes first obtained from surfactant systems were non-uniform spherical vesicles
and had between 4 to 12 straight edges of similar length (Uchegbu et al., 1997). These nonconventional structures are termed polyhedral niosomes. Typically, the reported polyhedral
niosomes are formed from mixtures of hexadecyl diglycerol ether (C16G2) and Solulan C24, and
could also be formed from C16G2, Solulan C24 and minimum amount of cholesterol. These
polyhedral vesicles have been found to be stable for at least 36 days and able to entrap and
release slowly water-soluble markers, such as carboxyfluorescein, and nucleotides (Uchegbu &
Florence, 1995; Uchegbu et al., 1997; Uchegbu & Vyas, 1998).
Luteinizing hormone releasing hormone (LHRH)-loaded polyhedral and spherical niosomes were
prepared as a slow release depot system for intramuscular administration. Spherical conventional
niosomes showed more stable membranes than polyhedral ones, suggesting that the conventional
niosomes are more effective intramuscular depots (Arunothayanun et al., 1999).
Giant niosomes (discomes)
Large disc-like niosomes (discomes) of 11-60 µm in size were formed upon incubation of the
preformed conventional (spherical) niosome dispersion with various proportions of Solulan C24
in a shaking water bath at a temperature of 74oC for 1 h (Uchegbu et al., 1992). Larger discomes
were formed with higher Solulan C24 levels and vice versa. A C16G2: cholesterol molar ratio of
7:3 favours the formation of discomes. Increasing the cholesterol level in the vesicular dispersion
above 30% mol/mol suppresses the production of discomes due to increasing the rigidity of
niosomes (Uchegbu et al., 1992).
Discomes are thermoresponsive vesicles, which become leakier as the temperature is increased
from room temperature to 37oC (Uchegbu & Vyas, 1998). Discomes have been proposed a
potential drug delivery system for ophthalmic administration (Uchegbu et al., 1992; Uchegbu &
Vyas, 1998). They are capable of entrapping water-soluble drugs and their large faceted structure
may prevent their drainage and absorption into systemic circulation via the nasolacrimal duct.
They have been investigated to control the ocular delivery of timolol maleate (Vyas et al., 1998).
The prepared discomes showed a sustained drug release pattern and systemic absorption was
minimised to a negligible level (Vyas et al., 1998). However, the formation of this system
requires the incubation of the niosomal dispersion with Solulan C24 at 75oC for at least 1 hr.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
These preparation conditions might affect the chemical stability of some thermolabile therapeutic
agents when incubated at such high temperature.
4.1.2.
Surfactant and ocular delivery
The use of surfactants in ophthalmic formulations and ocular drug delivery is occasionally
recommended. This is due to their potential ability to increase bioavailability by increasing drug
solubility, prolonging pre-corneal retention, and enhancing permeability(Jiao, 2008). Surfactants
are used as preservatives in multiple dose ophthalmic formulations to maintain sterility and
protect the formulations against microbial instability. For example, benzalkonium chloride and
chlorhexidine (cationic surfactants) are used as preservatives in ophthalmic formulations.
However, cationic surfactants are found to be highly irritant and cytotoxic (Furrer et al., 2000;
Saettone et al., 1996a). The order of surfactant toxicity is found to be anionic > cationic >> nonionic (Lang et al., 2002).
Many surfactants have been studied as drug ocular penetration enhancers. Non-ionic surfactants
including Span 20, Span 40, Span 85, Tween 20, Tween 40, Tween 81, Brij 35, Brij 58, Myrj 52
and Myrj 53 were tested for their ability to increase the corneal permeability of fluorescein in
human subjects (Marsh & Maurice, 1971). The study showed that an HLB range of 16-17 was
found to be effective as a corneal penetration enhancer in 1% solution. Only Brij 58 was harmful
at this concentration (Marsh & Maurice, 1971). In addition, some bile salts have been studied for
drug corneal penetration enhancing effect and ocular toxicity and irritancy; these include sodium
deoxycholate,
sodium
taurodeoxycholate,
sodium
ursodeoxycholate
and
sodium
tauroursodeoxycholate. The study pointed out that the aforementioned surfactants are safe and
effective as corneal penetration promoters (Saettone et al., 1996a).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.2. Chapter aims
The use of some membrane additives can alter the physical properties of niosomes, such as size,
morphology and EE% of the encapsulated drug. The aim of this chapter is to study stearyl amine,
dicetyl phosphate, sodium cholate and poly-24-oxyethylene cholesteryl ether (Solulan C24) as
potential membrane additives. The excipients used can be broadly categorised into charged lipids
(stearyl amine and dicetyl phosphate) and water-soluble surfactants (sodium cholate and Solulan
C24). The following objectives are considered:
•
Studying the effect of different membrane additives and preparation methods on EE% of
NTX.
•
Investigating the effect of the membrane additives on the physical properties of the
prepared niosomes such as vesicle size, morphology and gel/liquid phase transition
temperature.
•
Exploring the spreading ability and rheological properties of the prepared niosomes.
•
Studying the effect of NTX encapsulation by niosomes on NTX chemical stability under
stress factors such as artificial daylight illumination and oxidising agents.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.3. Materials and methods
4.3.1.
Materials
Stearyl amine (STA) and dicetyl phosphate (DCP) were purchased from Sigma-Aldrich, St.
Louis, USA; poly-24-oxyethylene cholesteryl ether (C24) was a generous gift from Lubrizol Inc.,
Cleveland, USA; sodium cholate (CH) was a generous gift from New Zealand Pharmaceuticals,
Palmerton North, New Zealand. All other chemicals were purchased or obtained from the same
source mentioned in Chapter 2 and Chapter 3.
4.3.2.
Methods
4.3.2.1. Niosome preparation
TFH method
Niosomes were prepared using the TFH method as described in Section 3.3.2.1. An amount
equivalent to 1 mmol of total lipid was used. The following lipid ratios were studied: Span 60:
cholesterol:additive (DCP, STA, C24 or CH) 7:3:0, 6.9:2.9:0.2, 6.75:2.75:0.5, 6.5:2.5:1
mol:mol:mol and the final percentages of the membrane additive used were 0%, 2%, 5% and
10% mol/mol respectively.
Three formulations as well as control niosomes (F-S60) were selected based on the highest EE%.
Their compositions are presented in Table 4.1.
Table 4.1 Codes and composition of the prepared niosomal formulations
Formulation code
F-S60
F-DCP
F-C24
F-CH
Span 60
7
6.75
6.9
6.75
Molar ratio
Cholesterol
3
2.75
2.9
2.75
DCP
0
0.5
0
0
C24
0
0
0.2
0
CH
0
0
0
0.5
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Freeze and thaw method
Frozen and thawed multilamellar vesicles (FAT-MLV) were generated by subjecting 8 ml of
MLV suspensions prepared using the TFH method to 5 alternate cycles of freezing in liquid
nitrogen for 60 s and thawing in a water bath at 60°C for another 60 s (Mayer et al., 1985).
Reverse phase evaporation (REV) method
The REV method used was a modification of the method described by (Kirby & Gregoriadis,
1984b). A thin film of the lipid was formed, as previously described in the TFH method. The thin
film was then dissolved in 12 ml of a mixture of ether:chloroform (1:1 v/v). Four ml of the
aqueous NTX solution (4 mg/ml) of PBS was added to the organic phase, such that the organic:
aqueous phase ratio was 3:1. This mixture was then sonicated for 3 min in a bath sonicator
(Bandelin Snorex, Berlin, Germany), until an opalescent w/o emulsion was formed. The final
dispersion was rotary evaporated at 60oC until a semi-solid gel-like mass or aqueous lipid
dispersion (depending on the lipid contents) was obtained. The resulting system was purged with
a stream of nitrogen gas for 3 min to remove any traces of the organic solvent. The final
dispersion was diluted with 4 ml PBS solution and rotary evaporated at a speed of 200 rpm for at
least 30 min. This method was previously used to generate large unilamellar vesicles (LUV)
(Kirby & Gregoriadis, 1984b).
Dehydration-rehydration method
Dehydration-rehydration vesicles (DRV) were prepared by a modified version of the method
described by (Hope et al., 1986). Eight ml of MLV dispersion prepared by the TFH method was
frozen in liquid nitrogen, followed by lyophilisation in a freeze dryer (Labconco, Missouri, USA)
overnight at a temperature of -20°C and vacuum≤ 0.133 mbar . The freeze-dried mass was
hydrated with 4 ml (4 mg/ml) NTX solution of PBS pH 7.4 at 60°C. After complete lipid
hydration, the final volume of the preformed niosomes was diluted to 8 ml using a PBS solution.
4.3.2.2. Physical properties
Entrapment efficiency (EE %) of NTX
EE% was determined as mentioned in method section Chapter 3.
Niosome size and size distribution measurements
The size of the prepared niosomes was determined by the laser diffraction technique (Mastersizer
2000, Malvern Instruments, UK). The samples were properly diluted with PBS and measured at
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
25 oC. The size was expressed in terms of volume diameter (D [4,3]) and the experiments were
done in triplicate. Span was calculated using equation 4.1:
equation 4.1
Where d(0.9) is the particle diameter at 90% cumulative size, d(0.1) is the particle diameter at
10% cumulative size and d(0.5) is the particle diameter at 50% cumulative size
Morphology
Cryo-SEM
The ultrastructure of the prepared niosomes was studied using Cryo-SEM as mentioned in
method section Chapter 3.
Confocal laser scanning microscopy (CLSM)
Niosomes preparation
Niosomes were prepared using the REV method, as described in Section 4.3.2.1. The NTX
solution was replaced with a 1.5 mM carboxyfluorescein (CF) solution of PBS pH 7.4 as a
fluorescent probe. The free CF was separated from the niosomes by exhaustive dialysis. A
cellulose bag containing 2 ml of the prepared niosomes was dialysed against a 500 ml PBS
solution at 4oC. The PBS solution was replaced at least 3 times over 48 h.
Niosomes imaging
Three wells were drilled into a microscopic plastic slide by a laser cutter. Each well was 1 cm in
diameter and 1.5 mm in depth. The wells were created to hold niosome samples, in order to avoid
squashing of niosomes between the slide and the cover slip. Measurements were conducted on a
Leica CLSM (Leica DMRXA-2 microscope fitted with a TCS-SP2 scan head, Leica
Microsystems, Heidelberg, Germany) using a 40x water immersion lens; a zoom of 1; a pinhole
with an Airy disk diameter of 2; and a combination of lasers and emission band pass filters (510521 nm) to visualise the prepared niosomes. The images were visualised and analysed with Leica
Confocal Software® (Leica Microsystems, Heidelberg, Germany).
DSC studies
DSC studies were conducted to study the effect of different membrane additives on the
membrane fluidity of the prepared niosomes (gel/liquid transition temperatures). Special batches
134
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
of the four niosomal formulations (10 mmol of total surfactant/lipid) were prepared by the TFH
method. The DSC study was performed as mentioned in method section Chapter 3.
Surface tension measurements
The surface tension (γ) of the prepared niosomes was determined at the ambient conditions using
an interfacial tensiometer (Torsion Balance, Malvern Wells, UK). All measurements were
performed in triplicate.
Contact angle and spreading coefficient measurements
Contact angle measurements were performed using a drop shape analyser (goniometer) (KSVCAM 101, Helsinki, Finland). Goniometry is the analysis of the shape of a drop of test liquid
placed on a solid surface. The basic elements of the goniometer include a light source, sample
stage, lens and image capture. The contact angle can be assessed directly by measuring the angle
formed between the solid surface (a glass slide) and the tangent to the drop surface (Figure 4.1).
A Hamilton syringe was filled with each of the tested niosomal formulations. Approximately 20
µl of each formulation were dropped onto a glass slide at ambient conditions (Figure 4.2). The
image of the drop was captured and measured by CAM 101 software. The measurements were
performed in triplicate and compared with that of an NTX (0.4 mg/ml) aqueous solution in PBS.
Figure 4.1 Equilibrium between forces acting on a drop of liquid on a
solid surface
135
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Poor wetting (θ > 90o)
Good wetting (90o > θ > 0o)
Figure 4.2 KSV-CAM 101 goniometer setup for measuring contact angle, (A)
poor wetting, and (B) good wetting of a liquid drop on a glass slide
136
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
The contact angle (θ) is defined as the angle formed by a liquid at the three phase boundary
where a liquid (L), air (A) and solid (S) intersect (Figure 4.1). A representation of the several
forces acting on a drop of liquid placed on a flat solid surface is outlined in Figure 4.1. The
surface tension of the solid (γS/A) will favour spreading of the liquid, but this is opposed by the
solid-liquid interfacial tension (γS/L) and the horizontal component of the surface tension of the
liquid (γL/A) in the plane of the solid surface (γL/ACOS θ). Equating these forces gives equation
4.2, which is generally referred to as Young’s equation (equation 4.2) (Florence & Attwood,
1998c).
γS/A = γS/L + γL/A COS θ
equation 4.2
The condition required for complete wetting of a solid surface is that the contact angle should be
zero. This condition is fulfilled when the forces of attraction between the liquid and the solid are
equal to or greater than those between liquid and liquid (Florence & Attwood, 1998c). The type
of wetting in which a liquid spreads over the surface of the solid is referred to as spreading
wetting. The tendency for spreading can be quantified in terms of the spreading coefficient (S)
using equation 4.3 (Florence & Attwood, 1998c).
S=
If
θ
γL/A (COS θ – 1)
is larger than 0o, the term (COS
equation 4.3
θ – 1) will be negative, and the value of S as well. The
condition for complete or spontaneous wetting is thus a zero value for the contact angle.
Rheological properties measurements
Viscosity measurements were performed using a Brookfield DV-III programmable cone and plate
rheometer (Brookfield Engineering Laboratories Inc., Stoughton, Massachusetts, USA). A CP-40
cone spindle was used and a rotation speed was 100 rpm. A water-jacketed sample cup was
thermostatically controlled by circulating water pumped using a water pump (Biolab Scientific
137
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Limited, Victoria, Australia) immersed in a water bath maintained at the required temperature
(25oC ± 0.5oC and 35oC ± 0.5oC). Brookfield Rheocalc operating software was employed for data
acquisition and analysis. The system was calibrated and found to be accurate within ± 2% of the
working range. Prior measurements, the tested niosomes were diluted with PBS to a
concentration equivalent to the required therapeutic NTX concentration (0.4 mg/ml). A sample
volume of 0.5 ml was used and the measurements were performed in triplicate. Viscosity was
calculated using equation 4.4 (Martin et al., 1993).
η=
F
G
equation 4.4
Where G is shear rate (s-1) which is the difference of velocity between two planes of liquid
separated by an infinitesimal distance, F is shear stress (dynes/cm2) which is the force per unit
area required to bring about flow and η is the viscosity (Martin et al., 1993).
4.3.2.3. Effect of niosomal encapsulation on NTX oxidation
An aqueous NTX solution of 0.4 mg/ml in PBS pH 7.4 or an equivalent concentration of NTX
encapsulated in niosomes were exposed to 2% v/v hydrogen peroxide (H2O2) for 2 h at ambient
conditions. Another group of samples were stored in a BINDER KBF 240 series stability
chamber (BINDER, Tuttlingen, Germany) and subjected to artificial daylight illumination of
10,000 lux at 40oC. Samples were withdrawn at a suitable time interval and analysed for NTX
content using the HPLC method.
4.3.3. Statistical analysis
A one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparisons at the 5%
significance level was used to test statistical significance differences between the prepared
formulations and control for size measurements, contact angle, spreading ability, rheological and
protective effects of niosomes. These were performed using GraphPad Software Version 3.05,
San Diego California, USA.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.4.
Results and discussion
The structures, molecular weights and critical micelle concentrations of the used additives are
presented in Table 4.2.
Table 4.2 Chemical structure, molecular weight, CMC and phase transition temperature of
the investigated additives
* The phase transition temperature (Tm) of the unhydrated samples of Solulan C24 and dicetyl phosphate
were measured using DSC. Sodium cholate was obtained from a data sheet by the supplier.
** Critical micelle concentrations (CMC) of the two water-soluble surfactants (Solulan C24 and sodium
cholate) were determined by surface tension measurements using a torsion balance.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.4.1.
Effect of selected membrane additives on NTX EE%
The effect of different concentrations (0%, 2%, 5% and 10% mol/mol) of the selected membrane
additives on NTX EE% was studied (Figure 4.3). The EE% values calculated for the prepared
niosomes were highly dependent on the type and concentration of the additives used. F-S60 (0%
additive) achieved EE% of 11.5% w/w whereas the EE% declined to 7% w/w in niosomes
composed of Span 60:cholesterol:STA at molar ratio of 6.9:2.9:0.2. Incorporation of STA in a
concentration above 2% mol/mol was unsuccessful. This was due to the formation of large lipid
aggregates which precipitated out from the dispersion. In contrast, the addition of DCP
significantly (P < 0.05) increased NTX EE%. For instance, there were 2.7-fold and 3.5-fold
increases in the NTX EE% when 2% and 5% mol/mol of DCP were incorporated in the niosomes
respectively (Figure 4.3).
Figure 4.3 Effect of selected membrane additives concentration on NTX EE% for niosomes
prepared using the TFH method (Results are expressed as mean values ± SD, n = 3)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
STA and DCP are positively and negatively charged lipids respectively. Being a basic drug (pKa
8.380), NTX molecules carried positive charges at the selected buffer pH (7.40). Electrostatic
interactions between charged drug molecules (NTX molecules) and STA or DCP were likely to
be the main factor controlling the EE% for niosomes (Abdelbary & El-gendy, 2008; Junyaprasert
et al., 2008). Further, as the concentrations of the ionic additives increased, niosomes’ charge
densities increased. These observations could explain the marked reduction and significant
increase of the EE% of NTX in STA- and DCP-containing niosomes respectively. Figure 4.3 also
shows levelling off the DCP curve at a concentration of > 5% mol/mol. This could be ascribed to
the saturation of the bilayer membranes of the niosomes with DCP molecules.
The effect of different concentrations of C24 and CH on the EE% was also illustrated in Figure
4.3. The results revealed that the incorporation of C24 or CH in niosomes significantly (P < 0.05)
increased NTX EE% compared with F-S60. The EE% values calculated for niosomes containing
5% mol/mol CH (F-CH) and 2% mol/mol C24 (F-C24) were 1.74-fold and 2.4-fold higher
respectively than that for F-S60. These increases in the EE% were limited to critical
concentrations (Table 4.2). For example, any further increase in C24 concentration beyond 2%
mol/mol was accompanied by a significant (P < 0.05) decrease in EE% of NTX, whereas 5%
mol/mol of CH was found to be optimal EE% of NTX. These critical concentrations were
dependent on the type of the additive used. This could be due to the difference in the
accommodating abilities of the bilayer membrane to the additives used. For instance, the structure
of C24 is inherently bulky (mol wt 1443) compared with Span 60 (mol wt 431) and cholesterol
(mol wt 386.7). The presence of 24 units of polyoxethylene chains, which are highly hydrophilic
and hydrated with numerous water molecules, might account for such behaviour. Furthermore,
different additives could show different affinities to the bilayer membrane and water (El
Maghraby et al., 2004).
The positive effect of C24 and CH on the EE% of NTX could be ascribed to the improved
hydration of the thin lipid film and the enhanced quality of the formed lipid dispersion due to
minimisation of vesicle aggregation. This is due to the steric effect of the bulky hydrophilic head
group of C24 and the possible electrostatic repulsion between negatively charged CH moieties.
On the other hand, the use of additives at concentrations higher than the critical concentrations
markedly decreased NTX EE%. For example, NTX EE% declined to 14% and 20% in niosomes
containing 10% mol/mol CH and 10% mol/mol C24 respectively. Such behaviour could be due to
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
the increase in the concentration of the water-soluble surfactant additives used (CH and C24),
which in turn could lead to an increase in the permeability of the formed niosomes to the
entrapped solutes. Additionally, increasing the concentration of the water-soluble surfactants
could lead to self-assembling of the lipids into other aggregates, such as open lamellar structures
or even mixed micelles. It has been reported that intermediate aggregates between closed bilayer
vesicles and mixed micelles were identified with cholate-phosphatidylcholine systems using
turbidity measurement (Ollivon et al., 1988; Walter et al., 1991a). Intermediate structures
between niosomes and mixed micelles were also identified with hexadecyl diglycerol ether:
cholesterol: C24: DCP 49:19.5:29:2 (Uchegbu et al., 1992).
The additives used achieved a significant enhancement of EE% and Table 4.2 shows formulation
codes and compositions of the selected niosomal formulations. Thanks to these additives, F-DCP,
F-C24 and F-CH formulations were of clinical significance, as they were able to carry the
required therapeutic concentration (0.4 mg/ml), whereas F-S60 (0% additive) niosomes carried a
sub-therapeutic dose of NTX. Studying the effect of different preparation methods could be other
solution to enhance EE% for F-S60 control niosomes.
4.4.2. Effect of preparation methods on NTX EE%
The four selected niosomal formulations (F-S60, F-DCP, F-C24 and F-CH) were prepared using
three different methods (FAT-MLV, REV and DRV) and were compared with the conventional
TFH method. Figure 4.4 outlines the effect of the method of preparation on NTX EE%. The
results showed that, for the same niosomal formulation, NTX EE% values were significantly (P <
0.05) different and dependent on the method of preparation. For instance, the NTX EE% values
for F-DCP prepared by TFH, FAT-MLV, DRV and REV were 40%, 29%, 43% and 61.5%
respectively.
Niosomes prepared using the REV method showed the highest ability to encapsulate NTX. On
the contrary, those prepared using the FAT-MLV method showed the lowest EE%. There was a
1.4-fold decrease in the EE% obtained from FAT-MLV F-DCP compared with that obtained
from TFH F-DCP. These findings could be ascribed to the different preparation methods.
Subjecting the bilayer vesicles to drastic conditions such as 5 alternate cycles of freezing and
thawing was thought to rupture the bilayer membrane and consequently increase their
permeability to the entrapped solutes. These results were in a good agreement with those reported
on ethosomes (liposomes containing ethanol) (Maestrelli et al., 2009). According to this report,
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
the effect of preparation methods was studied for ethosomal formulations for topical delivery of
the local anaesthetic agent, benzocaine. The prepared ethosomes were prepared using TFH, FATMLV, REV and small unilamellar vesicles (SUV) by sonication or extrusion through 200 nm
polycarbonate filters. Both SUV and FAT-MLV ethosomes showed the lowest EE%.
Figure 4.4 Effect of the preparation method on NTX EE% (Results are expressed as
mean values ± SD, n =3)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
On the other hand, it was found that a 1.5-fold increase in the EE% for REV F-DCP compared
with TFH F-DCP. This could be attributed to the ability of niosomes to encapsulate large aqueous
cores during the slow evaporation of the organic phase and conversion from inverted micelles to
bilayer vesicles.
Niosomes prepared by the DRV method showed higher EE% than TFH niosomes. This could be
due to the increase in the lipid/drug ratio which was achieved when rehydrating the freeze-dried
niosomes in half of the original solution volume (Hope et al., 1986; Kirby & Gregoriadis, 1984a).
This result was in a good agreement with that mentioned for the effect of total lipid content on
EE% (Figure 3.12) in Chapter 3.
4.4.3. Niosome size and distribution measurements
The effects of the membrane additives used on the volume diameter (D [4,3]) and span values for
REV niosomes were studied (Table 4.3 and Figure 4.5).
The results showed that the average D [4,3] value for F-DCP was 7.5 ± 0.59 µm, whereas that for
F-S60 was 8.30 ± 1.14 µm. Also, the span index for F-DCP (1.68) was less than that for F-S60
(2) indicating a narrow distribution of size and polydispersity (unimodal). Similar span values
(1.3 to 2.75) were reported for DCP liposomes and prepared by the REV method (Mehanna et al.,
2009). Incorporation of DCP imparted negative charges to niosomes; and the resultant
electrostatic repulsion is likely to account for the reduction in the tendency for niosomes to
aggregate.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Table 4.3 Effect of selected membrane additives on D [4,3] and span values for niosomes
prepared by the REV method (Results are expressed as mean values ± SD, n = 3)
REV
Formulation
F-S60
F-DCP
F-C24
F-CH
D [4,3] µm
8.30 ± 1.14
7.50 ± 0.59
22.41 ± 1.40
5.37 ± 0.81
Span
2.0 ± 0.15
1.68 ± 0.13
5.55 ± 1.40
1.72 ± 0.05
The D [4,3] calculated for F-CH was 5.37± 0.81 µm, compared with 8.3 ± 1.14 µm for F-S60.
The results showed that incorporation of CH in the prepared niosomes significantly decreased (P
< 0.01) D [4,3] for F-CH. CH is a highly hydrophilic molecule (HLB 16.70) (El Maghraby et al.,
2004). The introduction of such a highly polar molecule should allow niosomes to self-assemble
into smaller closed bilayer structures. Similar results have been observed with liposomes where
the introduction of CH to the phospholipid bilayer markedly decreased the size of the liposomes
formed. Hence, CH is well-known as an edge activator (Cevc et al., 1996; El Maghraby et al.,
2004). More interestingly, the D [4,3] for F-C24 was found to be 22.41 ± 1.40 µm and span index
was found to be 5.55. This reflects a 2.7-fold increase in F-C24 niosome size compared with FS60 niosomes. C24 has a bulky structure (mol wt 1443) and highly hydrophilic long poly-24oxyethylene chains. The bulky structure and the water molecules associated with the
polyoxyehtylene chains could lead to an increase in the cross-sectional area of the head groups
and produce giant niosomes and a relatively broad size distribution (bimodal). Similar findings
were reported upon incorporation of C24 in hexadecyl diglycerol ether (C16 G2) and Span 40based niosomes (Uchegbu et al., 1992; Vyas et al., 1998). Giant vesicles or discomes, with sizes
ranging from 11 to 60 µm, were detected upon incubating the preformed C16 G2- or Span 40based niosomes with C24 solution at 75oC (Uchegbu et al., 1992; Vyas et al., 1998). These giant
niosomes are called discomes and coexist with smaller spherical niosomes (2-10 µm) and
suggested as a preferable vehicle over conventional spherical niosomes (Uchegbu et al., 1996b).
Discomes are more likely to resist nasolacrimal drainage by virtue of larger sizes. Hence, they are
thought to offer improved ocular bioavailability than conventional niosomes (Uchegbu &
Florence, 1995; Uchegbu et al., 1997; Vyas et al., 1998).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
F-S60
F-DCP
F-C24
F-CH
Figure 4.5 Size-frequency distribution curves of the prepared
niosomes
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.4.4. Morphology and lamellarity
Microstructure and lamellarity were studied using Cryo-SEM, and the results are shown in Figure
4.6 and Figure 4.7. The micrographs revealed spherical and concentric bilayers, arranged in an
onion-like organisation, indicating the multilamellarity of F-S60 and F-CH niosomes. On the
other hand, F-DCP vesicles were large, spherical and unilamellar (Figure 4.6). These structures
are characteristic of vesicles generated by the REV method. Large unilamellar vesicles (LUVs)
are likely to self-assemble during the transition from inverted micelles to bilayer vesicles upon
the slow evaporation of the organic phase (Kirby & Gregoriadis, 1984b). Incorporating CH and
DCP in the bilayer membrane produced spherical niosomes similar to those of F-S60, suggesting
that CH and DCP had no obvious effect on the morphology. Incorporation of C24 in the bilayer
membrane yielded large or giant vesicles (approx. 22 µm) with oval shapes. These giant vesicles
coexisted with smaller spherical vesicles (Figure 4.7). Large elliptical vesicular structures have
been produced when incubating the performed Span 40-based niosomes in a C24 solution for 1 h
at 75oC. The size of these giant structures ranged from 11 to 60 µm and they had a disc-like
appearance; hence the term discomes (Uchegbu et al., 1992). These discomes were reported to be
an enhanced and controlled ocular delivery system (Mainardes et al., 2005; Uchegbu & Vyas,
1998). Their inherent large size was thought to prevent discomes from being rapidly washed out
by tear dynamics. Also, their non-uniform spherical structure could provide a better fit on the
ocular surface (Mainardes et al., 2005; Vyas et al., 1998). For instance, discomes have been
found to achieve higher entrapment efficiency and improved the ocular bioavailability of timolol
maleate (Vyas et al., 1998).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
F-S60
F-DCP
F-S60
F-DCP
Figure 4.6 Cryo-SEM micrographs of F-S60 and F-DCP prepared using the REV method
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
F-C24
F-CH
F-C24
F-CH
Figure 4.7 Cryo-SEM micrographs of F-C24 and F-CH prepared using the REVmethod
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figures 4.8-4.11 show CLS micrographs of the prepared niosomes loaded with a fluorescent
probe (CF). Typical uniform spherical-shaped niosomes were imaged from conventional
niosomes (F-S60, Figure 4.8). F-DCP and F-CH were found to be spherical in shape, as shown in
Figures 4.9 and 4.11 respectively. These results indicate that the incorporation of DCP and CH
into bilayer membranes has no observable effect on niosomes morphology. On the other hand,
oval giant vesicles (approx. 20 µm) were imaged for F-C24 niosomes (Figure 4.10). These results
were in a good agreement with those obtained from Cryo-SEM micrographs and confirmed
discomes formation. As previously mentioned, incorporating such a bulky surfactant (mol wt
1443) with a long ployoxyethylene chain of C24 could influence niosomes geometry, especially
at a relatively low level of cholesterol typically ≤ 30% mol/mol (Uchegbu et al., 1992; Uchegbu
et al., 1996b).
In this study, the discomes were prepared in a single step and under relatively mild conditions.
These were formed at 60oC, whereas all the literature on discomes necessitates incubation of the
preformed spherical niosomes in a solution of C24 for 1 h at 75oC (Uchegbu et al., 1992; Vyas et
al., 1998). This method of preparation could be of value for encapsulating thermolabile watersoluble drugs.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.8 CLS micrographs of F-S60 niosomal formulation loaded with CF produced
by the REV method
151
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.9 CLS micrographs of F-DCP niosomal formulation loaded with CF produced by the
REVmethod
152
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.10 CLS micrographs of F-C24 discomes loaded with CF produced by the REV
method
153
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.11 CLS micrographs of F-CH niosomal formulation loaded with CF produced by
the REV method
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.4.5. DSC studies
The DSC studies were exploited in the previous chapter to study the interaction of cholesterol
with the bilayer membranes, and to find out the location of entrapped NTX molecules in Span 60
niosomes. Cholesterol exhibited a marked effect in decreasing both Tm and transition enthalpy.
This was clearly evident when gradually decreasing both the area under the peaks of DSC
thermograms and Tm values. The cholesterol contents in the membrane increased until the
complete disappearance of the gel/liquid transition of Span 60 at 50% mol/mol. Additionally, the
DSC studies pointed out that NTX molecules are located in the aqueous domains due to the fact
that NTX is a hydrophilic.
Figure 4.12 Effects of different membrane additives on gel/liquid transition
temperatures of the prepared niosomes
In this study, DSC was employed to understand further the interaction of the selected membrane
additives with the bilayer domains of Span 60 niosomes, and to monitor their effects on the
gel/liquid transition temperatures of the prepared niosomes. The residual phase transition of FS60 niosomes (7:3 mol/mol Span 60: cholesterol) was completely abolished upon incorporating
the selected membrane additives (Figure 4.12). Such thermal behaviours suggest the inclusion of
the additives used into the bilayer membranes of the prepared niosomes.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figures 4.13-4.15 show hypothetical positions of the membrane additives based on DSC data. It
is believed that cholesterol is a common membrane stabilizer and can be accommodated by the
bilayer membranes. Its steroidal nucleus aligns itself parallel to the hydrocarbon chains of the
surfactants and the hydroxyl groups project to the adjacent ester linkages of surfactants’ polar
head groups. Cholesterol has a rigid structure, poor water solubility and extreme hydrophobicity.
Cholesterol can offer membrane stabilising properties to vesicles; alter vesicle sizes; abolish their
phase transition; decrease their membrane permeability; and alter membrane fluidity
(Israelachvili et al., 1980; Israelachvili & Mitchell, 1975; Uchegbu & Florence, 1995).
The fully extended (all trans) double hydrocarbon chains of DCP align themselves parallel to the
hydrocarbon chains of Span 60, and the polar phosphate groups of DCP are parallel to the polar
heads the of the sorbitan ester groups. This type of interaction could impart more packing by
filling any irregularities running through the bilayer membranes of niosomes. Such improvement
in the packing properties could reduce the permeability to the entrapped water-soluble solutes
(Figure 4.13).
The extremely hydrophobic steroidal nucleus of C24 is more likely to align itself parallel to the
accommodating hydrocarbon chains of Span 60 in a manner similar to that of cholesterol
molecules in niosomes. The long poly-24-oxyethylene chains align themselves parallel to the
sorbitan ester groups (Figure 4.14).
Although sodium cholate (CH) has a steroidal nucleus like cholesterol, it is energetically
favoured to flip vertically, compared to the orientation of cholesterol in the bilayer membranes.
This is due to presence of the carboxylate functional group at the hydrocarbon terminal, and
hydroxyl groups attached to the steroidal nucleus. This means the carboxylate group can be
accommodated by the sorbitan ester groups and the steroidal nucleus aligns itself parallel to the
hydrocarbon chains of the surfactant. Further, due to the high polarity of carboxylate groups and
the presence of hydroxyl groups attached to the steroidal nucleus, CH molecules more
preferentially protrude into the aqueous environment and consequently, some vacancies in the
bilayer membrane could be created (Figure 4.15).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.13 Hypothetical position occupied by DCP in the bilayer membrane of F-DCP
based on DSC
157
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.14 Hypothetical position occupied by C24 in the bilayer membrane of F-C24
based on DSC
158
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.15 Hypothetical position occupied by CH in the bilayer membrane of F-CH based
on DSC
159
Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
4.4.6. Surface tension measurements
Figure 4.16 Surface tension measurements for the prepared niosomes
compared with NTX aqueous solution (0.4 mg/ml) (Results are
expressed as mean values ± SD, n =3)
* Significant difference (P < 0.05), ** insignificant difference (P > 0.05)
Figure 4.16 shows the surface tension (γ) measurements for the prepared niosomes compared
with NTX aqueous vehicle (0.4 mg/ml in PBS). The γ values measured for the prepared niosomes
ranged from 38 to 41 dynes/cm. These results revealed that the γ values for the prepared
niosomes were significantly (P < 0.001) lower than that for the aqueous solution (72 dynes/cm).
However, there was no significant (P > 0.05) difference amongst the γ values measured for the
prepared niosomes.
It is well accepted that the lower the surface tension is, the easier it is for the formulations to wet
the hydrophobic surface of the corneal epithelium (Pawar & Majumdar, 2006; Rathore &
Majumdar, 2006). For example, two gatifloxacin eye drops were prepared with two distinctly
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
different surface tension properties and their transcorneal permeation was evaluated. Control eye
drops (3 mg/ml gatifloxacin isotonic solution) and optimised formulations composed of 3 mg/ml
gatifloxacin isotonic solution containing benzalkonium chloride (BAC) (0.01%) and disodium
edetate (EDTA) (0.01%). The amounts of gatifloxacin permeated through excised goat cornea
from the control and optimised formulations were 29 and 83 µg respectively. The optimised
formulation showed 2.9-fold increase in the amount of the drug permeated. This was attributed to
the lower surface tension of the optimised formulations (41.2 dynes/cm) than that (69.5
dynes/cm) of the control. Hence, a better spreading ability and a permeation-enhancing effect
were observed with the optimised eye drops (Rathore & Majumdar, 2006). Both BAC (cationic
surfactant) and EDTA (chelating agent) are penetration enhancers. Nevertheless, BAC was found
to have a more pronounced penetration enhancing effect than EDTA (Rathore & Majumdar,
2006). This in part due to its ability to lower the surface tension compared with EDTA and also
due to disruption of corneal epithelium (Rathore & Majumdar, 2006). This report can highlight
the role of surface tension of the vehicle in enhancing the corneal penetration via the exclusive
corneal epithelium tight junctions.
4.4.7. Contact angle and spreading coefficient measurements
The contact angle is a measure of the spreading or wetting of a solid surface by a liquid, adhesion
and biocompatibility (Florence & Attwood, 1998c; Lerk et al., 1977). Low values indicate that
the liquid spreads or wets well while high values indicate poor wetting. If the angle is less than
90°, the liquid is said to wet the solid. If it is greater than 90° it is said to be non-wetting. A zero
contact angle represents complete wetting (Florence & Attwood, 1998c; Lerk et al., 1977).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Table 4.4 Contact angle and spreading coefficient measurements for the prepared niosomes
(Results are expressed as mean values ± SD, n=3)
Formulation
PBS solution
F-S60
F-DCP
F-C24
F-CH
Contact angle
(θo)
57.00 ± 1.80*
42.00 ± 1.00
40.00 ± 2.20
43.00 ± 1.81
44.00 ± 2.60
Spreading
coefficient
( dynes/cm)
-32.79 ± 1.90*
-9.79 ± 0.81
-9.00 ± 0.94
-11.20 ± 0.90
-11.65 ±1.26
* The PBS solution was significantly (P < 0.01) different to the prepared formulation, while all the niosomes
were insignificantly different from each other (P > 0.05)
Table 4.4 shows the contact angle (θ) and the spreading coefficient (S) measurements of the
prepared formulations compared with the plain vehicle (PBS). The θ and S values of the prepared
formulations ranged from 40o ± 2.20o to 44o ± 1.81o, and -9.00 ± 0.94 to -11.40 ± 0.90 dynes/cm
respectively, compared with those for PBS which were 57o ± 1.80o and -32.79 ± 1.90 dynes/cm.
The θ values of all tested niosomes were significantly less (P < 0.05) than that of the aqueous
vehicle. Additionally, the S values of the prepared formulations were up to 5 times higher than
that of the aqueous vehicle. These results suggest that spreading of niosomes on the solid surface
is more energetically favoured than rounding itself. These findings suggest that the prepared
formulations have better wetting properties than the aqueous vehicle. Hence, the prepared
formulations could more promptly and easily wet, spread and adhere to the hydrophobic surface
of the cornea than the aqueous vehicle.
4.4.8. Rheological properties measurements
Many reports have shown that increasing the viscosity of ophthalmic solutions increases the
ocular residence time and the pharmacological effect (Burgalassi et al., 2000; Lang et al., 2002;
Ludwig, 2005). However, applying an extensive gel into the eye can blur the vision and make the
adjustment of the dose very difficult (Winfield et al., 1990).
Increasing the ocular bioavailability due to increasing the viscosity is limited to a plateau, where
further increases in the viscosity produces a slight or no increase in the ocular bioavailability.
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
This plateau effect has been found to be dependent on drug and formulation type (Lang et al.,
2002).
It has been reported that an increase in the viscosity range over 1 to 12.5 mPa.s using
methylcellulose as a viscosity imparting agent showed a 3-fold decrease in the drainage rate. This
decrease in the drainage rate increased the concentration of drug in the precorneal tear film (Chrai
& Robinson, 1974). In addition, Isopto® and Liquifilm® are widely used commercial viscous
vehicles and consist of 0.5% hydroxypropyl methylcellulose and 1.4% polyvinyl alcohol. The
viscosity ranges for these products are 10-30 cps and 4-6 cps respectively, and both have the
convenience of being an eye drop (Lang et al., 2002).
In this study, the rheological properties of the prepared niosomes were studied at two different
temperatures 25oC (ambient temperature) and 35oC (ocular temperature). Data was collected by
Reholac software. A direct relationship between F and G at a constant viscosity (η) was obtained
for all tested niosomes and PBS which indicates Newtonian flow properties (Figure 4.17).
Figure 4.18 shows the η values of PBS solution and the prepared niosomes at 25oC and 35oC.
Generally, the η values were higher at 25oC than at 35oC. The effect of temperature on the η
values was significant (P < 0.05) in some instances (Table 4.5). For example, the η values for FS60, F-C24 and F-CH were significantly lower (P < 0.05) at 35oC. These results reveal the
dependency of viscosity on temperature. Hydrogen bonds, responsible for solvent-solvent or
solute-solvent interactions, can be broken by thermal movement at higher temperatures (Martin et
al., 1993).
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.17 Representative rheograms for the aqueous vehicle (PBS) and the prepared
niosomes 35oC
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.18 Viscosity values for the aqueous solution (PBS) compared with the
prepared niosomes at two different temperatures 25oC and 35oC (Results are
expressed as mean values ± SD, n=3)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Table 4.5 Tukey’s pair wise comparison of viscosity values for the prepared niosomes at
25oC and 35oC
PBS solution F-S60
F-DCP
F-C24
F-CH
25; 35 25; 35
Temperature(oC)
25; 35
25; 35
25; 35
PBS solution
NS
-
-
-
-
F-S60
S;S
S
S;S
-
-
F-DCP
S;S
S;S
NS
-
-
F-C24
S;S
S;S
S;S
S
-
F-CH
S;S
S;S
NS ; NS
S;S
S
S = significant difference (P < 0.05)
NS = non-significant (P > 0.05)
One S or NS indicates the statistical significance difference for one formulation at 25oC and 35oC
S;S or NS;NS indicates the statistical significance differences between two formulations at 25oC and 35oC
Irrespective of the temperature, all η values for the prepared niosomes were significantly higher
(P < 0.01) than the aqueous vehicle. The viscosity range for the prepared niosomes was 1.7 to 8.2
times higher than that for the aqueous vehicle at 25oC. Similarly, the viscosity for the prepared
niosomes was found to be 1.7 to 6.3 higher than that for the aqueous vehicle at 35oC.
The tested niosomes consisted of different sizes, compositions and lipid contents. The order of
the total surfactant/lipid contents of the tested niosomes was as follows F-S60 > F-CH > F-C24 >
F-DCP.
The highest η values (9.7 and 5.5 cps) were recorded for F-S60 niosomes at both 25oC and 35oC,
whereas the lowest η values recorded for the prepared niosomes were 2 and 1.5 cps for F-DCP
and F-CH niosomes respectively. F-C24 discomes came in the middle and demonstrated a 2.8fold increase in the η value compared with that for the aqueous vehicle at 25oC.
The highest surfactant/lipid content estimated for F-S60 showed the highest η value. Conversely,
F-DCP showed the lowest η value, which is mainly due to having the lowest surfactant/lipid
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
concentration. These results suggest that the higher the surfactant/lipid concentration, the higher
the vesicle-solvent interactions are more likely to occur and consequently the greater the viscosity
of the final dispersion. Other factors such as size and composition of niosomes could affect the
viscosity of niosomal dispersions. For example, the total surfactant/lipid content for F-CH was
approximately double that for F-DCP and the η values for F-DCP and F-CH showed nonsignificant difference (P > 0.05) (Figure 4.18). F-DCP niosomes had a lower concentration but a
larger size compared with F-CH niosomes which proves the surfactant/lipid content is not the
sole factor determining the viscosity of the niosomal dispersion.
Similarly, the η value for F-C24 discomes was significantly higher (P < 0.05) than that for F-CH,
although F-CH had a higher content of surfactant/lipid than F-C24.
The average diameter for F-C24 discomes was approximately 4 times larger than that for F-CH
niosomes. Also, inclusion of C24, with a head group consists of 24 units of highly hydrophilic
polyoxyethylene units, in F-C24 discomes increases vesicles-solvent interactions. These findings
suggest that many factors such as the lipid content, size and composition of niosomes control the
viscosity of the niosomal dispersions.
Conclusively, the prepared niosomes showed η values significantly higher than the aqueous
vehicle at the same time they have the convenience of being eye drops. It is well accepted that
more viscous vehicles have longer precorneal residence times than simple eye drop solutions.
This is another potential advantage for the prepared niosomes as ocular delivery vehicles for
NTX rather than their spreading and wetting ability.
4.4.9. Effect of niosomal encapsulation on NTX oxidation
From the preformulation studies performed in Chapter 2, autoxidation was found to be the main
degradation pathway of NTX as indicated from the S-shaped degradation profile, discolouration
of NTX solutions stored for forced degradation studies and MS studies. It is well accepted that
most liquid-phase autoxidations are free-radical reactions. Free radicals are atoms and molecules
that have an unpaired valence electron. Metal ions, light, heat and peroxides are involved as
catalysts (Johnson & Gu, 1988; Lachman et al., 1986).
Light energy, similar to heat, provides the activation energy necessary for oxidation reactions.
Daylight is a primary source for creating free radicals and an initiator of the autoxidation
propagation (Florence & Attwood, 1998a). One of the genuine properties a certain drug delivery
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
system should have is the ability to offer a chemical protective effect to the drug. Niosomes have
been proposed as systems capable of improving the chemical stability of photosensitive drugs
such as doxorubicin (Uchegbu & Florence, 1995) and tretinoin (Manconi et al., 2003). The
prepared formulations were tested for their ability to protect the encapsulated NTX molecules
from the light-induced degradation and the oxidizing agent such as H2O2.
Figure 4.19 shows the percentages remaining of NTX for the free PBS solution of NTX and NTX
encapsulated niosomes after exposure to daylight and H2O2 (2% v/v). The results showed that
84% and 20% of NTX solution potency was lost after exposure of the free NTX solution to H2O2
(2% v/v) and daylight illumination (10,000 lux) respectively; and the potency of NTX loaded
niosomes was also adversely affected by exposure to H2O2 but was significantly (P < 0.05) lower
than that of the free drug solution. In terms of protective effects against light-induced degradation
(photo-degradation) and apart from F-S60, NTX molecules encapsulated in the prepared
niosomes exhibited 24 h protection against the photo-degradation.
Figure 4.19 Effect of niosomes encapsulation on the chemical stability of NTX against
oxidation and daylight illumination (Results are expressed as mean values ± SD, n=3)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Table 4.6 Tukey’s pair wise comparison of oxidation and photolysis for the prepared
niosomes
NTX solution
F-S60
F-DCP
F-C24
Oxid; Phot
Oxid; Phot
Oxid; Phot
Oxid; Phot
F-S60
S; NS
-
-
-
F-DCP
S;S
NS; S
-
-
F-C24
S;S
NS ; S
NS ; NS
-
F-CH
S;S
S;S
NS ; NS
NS ; NS
S = significant difference (P < 0.05)
NS = non-significant (P > 0.05)
Oxid = Oxidation due to H2O2
Phot = Photo-oxidation
Longer term (10 days) photo-oxidation profiles of NTX from the PBS solution of NTX and NTX
encapsulated niosomes were studied. Figure 4.20 shows semi-logarithmic plots of % NTX
remaining versus time. Linear correlations were obtained (R2 > 0.99) for both the PBS solution of
NTX and NTX encapsulated niosomes indicating first-order photo-degradation kinetics.
Exposure of NTX aqueous solution to the artificial daylight obviously accelerated degradation of
NTX. The observed degradation rate constants (kobs) for the aqueous NTX solution stored in the
dark and exposed to the daylight illumination were found to be 0.02 and 0.22 day-1 respectively.
The calculated kobs value for the aqueous NTX solution exposed to the daylight illumination was
11-fold faster than that of the dark stored sample (Figure 4.21). This indicates the significant
impact of the daylight illumination as a potential initiator for autoxidation reactions.
The calculated kobs values for the photo-oxidised NTX niosomes were significantly smaller (P <
0.001) than the photo-degraded the PBS solution of NTX. A 1.4-3-fold decrease in kobs was
estimated for NTX encapsulated niosomes compared with the free NTX solution. These results
could be attributed in part to the ability of niosomes to protect niosomes-encapsulated NTX from
peroxide radicals formed by the daylight illumination by virtue of lipid bilayer membranes. Also,
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
the bilayer membranes could scavenge the free radicals and prevent them from propagation and
slowing autoxidation kinetics (Roda et al., 1998). These findings show the protective effect of the
prepared niosomes against photolytic and oxidative degradation of NTX. However, the calculated
kobs value for NTX F-S60 was significantly higher (P < 0.001) than all other NTX encapsulated
niosomes. The lower protective effect for F-S60 could be ascribed to leakage of NTX molecules
from F-S60 due to the presence of a residual gel/liquid transition and its thermo-responsiveness.
Abolishment of the gel/liquid transition of the bilayer membranes due to incorporation of the
membrane additives would improve the stability of niosomes especially at higher temperatures
than the gel/liquid transition temperature (Manconi et al., 2003). These results also suggest that
the composition of the bilayer membranes has an influence on the photostability.
Figure 4.20 First-order degradation kinetics for PBS solution of NTX and NTX encapsulated
niosomes under artificial daylight illumination at 40oC in PBS pH 7.4 (Results are expressed as
mean values ± SD, n=3)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Figure 4.21 First-order degradation rate constants for PBS solution of NTX and NTX
encapsulated niosomes under artificial daylight illumination at 40oC in PBS pH 7.4 (Results are
expressed a mean values ± SD, n = 3)
* Significant difference (P<0.05) and no significant difference ** P > 0.05
* Significant difference (P < 0.05), ** non-significant difference (P > 0.05)
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Chapter 4….Effect of Membrane Additives on the Physical properties of Niosomes
Conclusion
•
Four different niosomal formulations were successfully prepared, encapsulating a
considerable amount of NTX.
•
Both the composition and method of preparation were found to have a significant effect
on NTX EE%.
•
Inclusion of membrane additives (DCP, C24 and CH) to the bilayer membrane generated
niosomes with versatile morphology and sizes as confirmed by Cryo-SEM and CLSM. As
such discomes were detected with F-C24.
•
The contact angle and spreading coefficient measurements of niosomes exhibited better
wetting and spreading abilities than the aqueous vehicle.
•
The prepared niosomes were significantly more viscous than the PBS solution. The lipid
content, size and composition of niosomes are the main factors affecting the viscosity.
•
Exposure of NTX solution to artificial daylight illumination can produce extensive
degradation of NTX. The prepared formulations were able to significantly protect
encapsulated NTX from the photo-degradation compared with free NTX solutions.
From the abovementioned key findings, the next and final experimental chapter in the thesis is
concerned with evaluating the prepared niosomes for ocular delivery.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5. Evaluation of niosomal formulations for ocular delivery of
naltrexone hydrochloride
5.1. Introduction
In recent decades, researchers testing ocular dosage forms have recorded toxicological signs of
ocular tissues exposed to topically applied drugs. Ocular tissues, such as the cornea and
conjunctiva, are susceptible to injuries and adverse ocular effects, either from the administered
drug or from excipients used in formulating pharmaceutical products (Basu, 1984; Li et al.,
2008).
For instance, amphotericin B and ketoconazole (antifungal agents) can cause corneal oedema and
corneal abnormalities when administered topically (Foster et al., 1981). Excessive use of topical
anaesthetics can produce corneal lesions and ulcers (Basu, 1984). Further, anti-inflammatory
corticosteroids are found to retard epithelial corneal wound healing and induce glaucoma (Basu,
1984; Basu et al., 1981; Li et al., 2008).
Ocular side effects due to excipients in the pharmaceutical products have also been reported. For
instance, benzalkonium chloride (BAC), a quaternary ammonium cationic surfactant, is a
commonly used preservative in ophthalmic products. BAC has been reported to cause corneal
opacity, a decrease in corneal epithelial microvili, conjunctival hyperaemia (red eye) and delayed
wound healing (Li et al., 2008; Pfister & Burstein, 1976). Assessment of the toxicity of
ophthalmic formulations and the potential for ocular irritation represents an essential step in the
development of new ocular delivery systems (Basu, 1984; Lang et al., 2002).
On the regulatory viewpoints, there is relatively little guidance from ICH regulatory expectations
for non-clinical toxicity needed for ocular drugs, including those with novel delivery approaches
(Avalos et al., 1997; Short, 2008).
European regulatory authorities recommend an ocular tolerance study (CPMMP/SWP/21/00),
they propose that these studies consist of a single-dose study in a small number of rabbits (1-3),
with a drop size of 20 to 30 µl in a single dose administration, along with observation and scoring
for any ocular abnormalities (Short, 2008). There are many tests and assays which can be utilised
to evaluate the ocular potential of ocular pharmaceuticals.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
A brief review of the different tests used for assessing the ocular toxicity of ophthalmic dosage
forms is included below.
5.1.1. Eye irritation tests
5.1.1.1. In vivo ocular irritation test (Draize test)
Standard procedures for assessing the eye irritancy potential of a chemical substance are based on
the Draize eye irritation test or rabbit eye test (Draize et al., 1944). According to the Draize test,
100 µl of liquid or 100 mg of a solid test material are placed into the lower conjunctival sac of the
albino rabbit eye and the eyelids are held shut for a fixed period. Any ocular response is assessed
by an observer at various time intervals for a period of up to three weeks after treatment. The
contralateral eye is used as an untreated control. The scoring procedures evaluate the irritation
response of three ocular tissues, namely the cornea, iris, and conjunctiva. A numerical score was
given to different aspects of the irritation observed (Table 5.1). The maximum score is 110, with
80 points possible from the cornea, 10 from the iris and 20 from the conjunctiva. Since damage to
the cornea is so critical, its contribution to the scoring is the greatest.
Table 5.1 Ocular tissue and score for assessing ocular irritation using the Draize test,
adopted from (Draize et al., 1944)
Ocular tissue
Maximum
Cornea score = opacity (0-4) X area (0-4) X 5
80
Iris score = grading value (0-2) X 5
10
Conjunctiva score = [redness (0-3) + oedema (0-4) +
discharge (0-3)] X 2
20
Total score
110
Over the years, the Draize test has received both technical and ethical criticism (Anderson &
Russell, 1995; Bruner, 1992). Technically, the reproducibility of the test is poor, as scoring and
interpretation are subjective, depending on the visual observation of the observer. Also, it tends to
over-predict the human response, because it uses a high dose of the test materials and the site of
application is at the deep conjunctival sac of the rabbit eye. Ethically, the use of large numbers of
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
live animals, the application of large doses of painful and stressful test materials and the length of
recovery time are criticised by animal welfare groups (Anderson & Russell, 1995; Bruner, 1992).
5.1.1.2. Modification of Draize test (Low volume eye test)
One of drawbacks of the Draize test is the over-prediction of human risk resulting from using a
large amount of the test material (100 µl for liquid or 100 mg for solid). A modification of the
test is carried out; this uses only 10 µl of the test materials, which is applied directly to the cornea
instead of the lower conjunctival sac, and the eyelids are not held shut. This procedure gives rise
to a procedure called the low volume eye test (LVET) (Griffith et al., 1980). The applied volume
and the site of application closely mimic accidental human exposures (Avalos et al., 1997;
Bruner, 1992).
5.1.1.3. In vitro ocular toxicity models
Over the past three decades, there has been growing need in developing an in vitro alternative to
in vivo animal studies for ocular toxicity by ethics committees and animal welfare organisations,
to provide more objective and reproducible results, avoid the use of animals and end animal
suffering (Bruner, 1992). Potential in vitro alternatives include (Anderson & Russell, 1995;
Bruton et al., 1981; Curren & Harbell, 1998):
•
Cytotoxicity tests
•
Chorioallantoic membrane of the chicken eggs
•
Isolated enucleation eyes
•
Combination of chorioallantoic membrane and isolated enucleation eyes
These in vitro tests can offer many advantages over the conventional in vivo tests. These include
reducing the number of animals involved, and using more quantifiable and objective end-point
measurements. These tests are also more convenient and less time-consuming (Avalos et al.,
1997).
Cytotoxicity tests
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test is a colorimetric
reaction that relies on the ability of viable cells to reduce yellow tetrazoliums to purple
formazans, a reaction catalysed by the mitochondrial succinate dehydrogenase of the viable cells.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
The MTT assay is also subject to some limitations, including the chemical reduction of
tetrazolium by the test agents. Also, chemical interference occasionally occurs due to the cellular
reduction of tetrazolium. There is also a variety of metabolic conditions (e.g. depletion of glucose
and variations in pH) which might affect the production of formazan from tetrazolium
(Rubinstein et al., 1990; Skehan et al., 1990).
Another rapid and sensitive cytotoxicity test relies on the interaction of sulforhodamine B (SRB)
with the viable cellular protein content. The SRB assay shows good linearity with the number of
cells (Rubinstein et al., 1990). The SRB assay offers major practical advantages over the MTT
assay. Notably, the MTT assay involves a timed step, in which cells are incubated with
tetrazolium and the number of viable cells is estimated on the basis of the amount of formazan
produced. When many hundreds of 96-well microtiter plates are handled daily in the laboratory,
scheduling this step and subsequently reading the plates becomes difficult. In the SRB assay,
cells in the culture wells are chemically fixed at the end of the assay, allowing simultaneous
staining and further processing of extremely large batches of plates without time-critical steps
(Rubinstein et al., 1990).
There are some limitations to the use of cytotoxicity testing of water-insoluble solids or oilybased formulations (e.g. w/o emulsions), since such test materials do not come into direct contact
with the aqueous cell culture media. Also, cell culture media lack the protective shield of the
precorneal tear film, and as a result the test materials are directly exposed to the test tissue. Other
drawbacks include a possible interaction of the test materials (drug or formulation additives) with
the components of the growth medium for the cell line, producing false results.
The chorioallantoic membrane (CAM) test
The CAM is the vascularised respiratory membrane that surrounds an embryonic bird developing
in the egg. It is a complete tissue including veins, arteries and capillaries. This test was initially
developed as a possible model for predicting the irritant effect of the chemical on the conjunctiva
(Leighton et al., 1985). The test material is applied directly to the membrane surface, and then
incubated for a certain period, depending on the protocol used. The membrane is inspected
visually and changes in morphology (size of the lesion and presence of necrosis or capillary
haemorrhage) are scored (Bruner, 1992). The CAM test has been used to test the toxicity and
irritancy of some surfactants commonly used in pharmaceutical industries. The test can
successfully discriminate between the test materials according to their irritancy potential (Bagley
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
et al., 1991a, 1991b). It has also been found to correlate well with rankings of the same
preparation from the in vivo rabbit eye test (Draize test) (Leighton et al., 1985).
A modification of the CAM test, called the Hen’s egg test-CAM (HET-CAM), was developed by
(Luepke, 1985). The HET-CAM is a rapid, sensitive and inexpensive test, whereby fresh
fertilised viable eggs are incubated at 37oC ± 0.5 and relative humidity of 62.5 ± 7.5%. On day 10
of incubation, the egg’s shell is scratched around the air cell and the inner egg membrane is
removed. The test substance is instilled onto the membrane, and after 20 seconds the membrane
is irrigated with warm water. The membrane is then scored for irritant effects including
hyperaemia, haemorrhage, and coagulation at 0.5, 2 and 5 min. A variety of household materials
were tested including pyrithiones, phenols and isothiozolinones. The HET-CAM showed a good
correlation with the ranking of the same preparation tested using the Draize test (Luepke, 1985).
In addition, the HET-CAM has achieved good discrimination and sensitivity to the irritation
potential for pharmaceutical excipients and agricultural pesticides (Alany et al., 2006; Budai et
al., 2010). For instance, several formulation additives have been tested for ocular irritancy using
the HET-CAM test, including ethyl oleate, sorbitan mono-laurate, polyoxyethylene 20 sorbitan
mono-oleate, 1-propanol, 1-butanol, 1-hexanol, 1,2 propanediol, and 1,2 hexandiol. The test
successfully discriminated between the individual test materials in a homologous series. For
example, ocular irritation of n-alcohols showed that there was a stronger irritation potential above
a critical hydrocarbon chain length (2–3 carbon atoms). Moreover, the introduction of a second
hydroxyl group caused a reduction of irritation only for 1,2-propanediol compared with 1propanol (Alany et al., 2006).
The HET-CAM offers greater advantages than the Draize test. These advantages include reduced
animal suffering, less subjective scoring and more reproducible scoring systems. The HET-CAM
is also more rapid, less expensive (no animals or animal housing) and uses simpler methods
(Alany et al., 2006; Bagley et al., 1991a, 1991b; Budai et al., 2010; Leighton et al., 1985).
Isolated enucleated eyes
Isolated enucleated eyes (IEEs) from bovines (Muir, 1985), pigs (Bruton et al., 1981), chickens
(Bruton et al., 1981) and rabbits (Scaife, 1985) were fortunately used as potential alternatives
tests. The results obtained from the IEE test correlated well with in vivo data. IEEs do not require
the use of live animals but they use animal by-products and ready to use animal tissues. More
advantageously, IEE tests do not necessitate animal and human ethics’ approval and as such they
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
save the time required for that. The main argument against the acceptance of the use of isolated
rabbit eyes is that there are no rabbit slaughterhouses to supply rabbit meat for human
consumption. Therefore, laboratory rabbits are still needed as eye donors (Prinsen & Koeter,
1993).
The bovine corneal opacity and permeability (BCOP) assay is a good example for IEE assay. The
BCOP assay involves an assessment of corneal opacity and disruption of the corneal barrier (as
assessed by the passage of a fluorescent dye) after exposure to the test material. This test has
been widely utilised as an in vitro alternative to the in vivo test (Draize test). The BCOP assay is
currently applied across cosmetic and pharmaceutical industries in order to evaluate the ocular
irritation potential of surfactants, pharmaceutical intermediates and final products (Cooper et al.,
2001; Gautheron et al., 1992; Muir, 1985; Vanparys et al., 1993).
Combined in vitro assay
There is general agreement that the HET-CAM test serves as a possible model for conjunctival
irritation testing, as it responds to irritant substances with an inflammatory reaction similar to that
produced by conjunctival tissue (Alany et al., 2006; Anderson & Russell, 1995; Bruner, 1992).
However, good eyesight relies on the cornea as a refractive component. The cornea serves as the
gateway to the eye for external images. The transparency and smoothness of the cornea is
essential to maintaining its refractive and protective functions (Nishida, 2005). Consideration
must be given to the safety of the corneal tissue when using the developed ocular formulation. It
is not surprising that both corneal and conjunctival damage together constitute 100 out of 110
possible points when scoring the Draize test. Therefore, it has been found more advantageous to
develop an in vitro alternative model to investigate the safety of the test material on both the
cornea and the conjunctiva (Weterings & Vanerp, 1987). Combined isolated enucleated eyes and
HET-CAM models for the cornea and conjunctiva respectively were developed. These combined
tests were evaluated against the effects observed in the rabbit test. A good correlation was
obtained with a broad range of chemical substances (Weterings & Vanerp, 1987). Moreover, this
method is easy to perform, inexpensive, reproducible and uses less subjective scores than the in
vivo rabbit test (Avalos et al., 1997; Budai et al., 2010; Weterings & Vanerp, 1987).
However, predicting ocular irritation using opacity and permeability endpoints is challenging
when the test substances produce a delayed reaction by interacting with nucleic acids and
mitochondrial proteins, rather than causing an immediate loss of epithelial integrity. Therefore,
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
histological examination of the cornea after treatment with the test substances can provide
comprehensive assessment of the depth of injury and cellular damage of the three principle layers
of the cornea (Curren et al., 2000; Curren & Harbell, 1998).
In this thesis, combined HET-CAM and bovine eye tests are conducted to study the irritation
potential of the test substances used and the prepared niosomes, followed by histological
evaluation of the excised bovine cornea.
5.1.2. Toxicity of niosomes
There are limited research reports on the toxicity of niosomes. Different niosomal formulations
were evaluated for inhibiting the cell proliferation of SV-40 human keratinocytes (Hofland et al.,
1991). The effects of molecular structure of the surfactant and the incorporation of cholesterol on
the cell proliferation were investigated. The surfactants were composed of different hydrocarbon
chain lengths (12, 14, 17 and 18) and polyoxethylene chain lengths (3 and 7). Also, ether- and
ester-type surfactants were examined. It was found that both the hydrocarbon chain length and
the polyoxyethylene chain length had minor effects on cell proliferation. However, the bond (an
ether or ester bond) by which the alkyl chain was linked to the polyoxyethylene head group had a
strong effect on the cell proliferation. The concentration of the ether type-surfactants that
inhibited cell proliferation by 50% was 10 times lower than for the ester-type surfactants. The
cholesterol content of the bilayers has not been found to have an effect on cell proliferation. The
low toxicity of ester-type surfactants compared with ether-type surfactants could be attributed to
enzymatic degradation of ester bonds (Hofland et al., 1992).
In some instances, encapsulation of some drugs in niosomes reduces the toxicity. For example,
niosomes encapsulating vincristine decreased the neurological symptoms, diarrhea and alopecia
following intravenous administration of vincristine (Parthasarathi et al., 1994).
On the other hand, evaluating the physical stability of niosomes with respect to changes in
niosome size and drug retention ability should be carried out at an early stage of formulation
development. Therefore, the tendency of niosomes to aggregate and leakage of entrapped
therapeutic molecules over time have been investigated (Guinedi et al., 2005; Manosroi et al.,
2003; Uchegbu & Vyas, 1998).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.1.3.
Physical stability of niosomes
One of the most important features of niosomes compared with liposomes is their chemical
stability. Niosomes are less vulnerable to chemical degradation or oxidation than phospholipids
(Baillie et al., 1985; Zuidam et al., 2003). However, both liposomes and niosomes can equally
show physical stability problems. Vesicle aggregation, bilayer fusion and drug leakage are the
most common physical changes exhibited. Physically stable niosomal dispersions must exhibit a
constant particle size and a constant level of entrapped drug over time.
It has been shown that niosomes are also more physically stable than liposomes (Mukherjee et al.,
2007; Nasr et al., 2008). For example, aceclofenac Span 60 niosomes showed higher physical
stability (in terms of particle aggregation and drug leakage) than liposomes when stored for three
months at 4oC (Nasr et al., 2008). Also, both reconstituted and lyophilised Span 20-based
niosomes were more physically stable than liposomes when stored at 4 and 20oC for 3 months
(Mukherjee et al., 2007). Additionally, Tween 61 niosomes with 25% ethanol and Span 60
niosomes with 20% ethanol (elastic niosomes) exhibited no sedimentation, no layer separation
and an unchanged particle size at 4, 27 and 45oC within three months (Manosroi et al., 2008b).
It is worth mentioning that the main factors determining the stability of niosomes include
surfactant/lipid type, original size and temperature of storage.
First, the choice of surfactant determines the fluidity of the bilayer membrane. It has been found
the higher the gel-liquid transition temperature, the higher the drug-retentive properties of
niosomes. This is because the bilayer membranes are always in a gel state at the storage
conditions. The likelihood of carboxyfluorescein-loaded Span niosomes leaking was found to be
in the following order: Span 60 > Span 40 > Span 20 > Span 80 (Yoshioka et al., 1994). Also, the
incorporation of cholesterol (membrane stabiliser) in niosomes improves their physical stability
(Uchegbu & Vyas, 1998).
Secondly, niosomes sized between 1 and 10 µm have been found to be more stable than those in
the sub-micron range. Thermodynamically, smaller niosomes have higher surface free energy and
tend to aggregate more than larger ones in order to lower the excess free energy (Moazeni et al.,
2010). For example, niosomes composed of hexadecyl diglycerol: cholesterol: Solulan C24 at a
40:40:10 molar ratio with an original size of 70 nm were monitored for changes in size. The size
of the niosomes increased 250-fold. This result demonstrated that the original size has a
significant effect on the stability of niosomes (Uchegbu & Vyas, 1998).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Thirdly, the storage temperature for niosomes must be controlled, as a change in temperature
often leads to a change in the nature of the surfactant bilayer membranes (Tanaka, 1990;
Uchegbu & Vyas, 1998). In this context, Span 40 niosomes have been found to be more stable
than liposomes at higher storage temperatures such as 25 and 37oC since Span 40 has a higher
transition temperature than phospholipids (Ning et al., 2006).
5.2. Chapter aims
This chapter aims to evaluate the prepared niosomes for ocular delivery of NTX. The following
specific objectives are considered:
•
Investigating the ocular irritation potential of the surfactants and additives used, and the
prepared niosomes at different concentrations (1-10% w/v of total lipid). This involves the
use of the HET-CAM and bovine eye tests to study conjunctival and corneal irritation
potential respectively.
•
Examining the extent of corneal tissue damage from the prepared niosomes via
histological sectioning and examination of the bovine corneas after exposure to the test
substances at different time points.
•
Exploring the in vitro release profiles and release kinetics of NTX from the prepared
niosomes.
•
Studying the ex-vivo permeation of NTX from a simple NTX solution and from the
prepared niosomal formulations using excised cow corneas.
•
Investigating the physical stability of the prepared niosomes through monitoring changes
in particle size and NTX EE% over 90 days at different temperatures.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.3.
Materials and methods
5.3.1.
Materials
NTX, solvents and buffer salts were purchased from the same sources mentioned in Chapter 3
and Chapter 4. Fertilised Shaver Brown hen’s eggs were purchased from Bromley Park
Hatcheries Ltd, Tuakau, New Zealand; whole cow eyes were collected from Auckland Meat
Processors, Auckland, New Zealand and were used on the same day; cellulose membrane,
molecular weight cut-off 12,000-14,000, was purchased from Sigma-Aldrich, St. Louis, USA.
5.3.2.
Methods
5.3.2.1. Ocular irritation studies
Conjunctival (modified HET-CAM) test
Preparing and growing the CAMs
Freshly collected fertilised hen’s eggs (Shaver brown) were incubated at 37.5oC ± 0.5oC and 66 ±
5% relative humidity (RH) for 3 days according to the HET-CAM method (Luepke, 1985). The
eggs were incubated horizontally in growing trays and gently rotated every day to ensure that the
embryo was properly positioned. On days 3 and 4, the eggshells were sterilised by spraying them
with a rectified spirit and opened by cracking the underside of the egg against the edge of a
plastic Petri dish. The egg was then poured into a growing chamber, according to the modified
HET-CAM method (Alany et al., 2006).
The growing chamber was made of a cylindrical plastic holder, to which a piece of cellophane
membrane (commercial Glad Wrap™) was attached to make a pouch. The cellophane pouch was
fixed using a plastic circular sleeve. Once in the growing chamber, the egg was examined to test
the viability of the embryo (intact CAM and yolk) (Figure 5.1). Only viable embryos with an
intact CAM and yolk sac were further incubated at 37.5oC ± 0.5oC and 66% ± 5% of relative
humidity (RH). These were the optimum conditions for CAM growing (Luepke, 1985). The
temperature and RH were recorded every hour using a RH/Temp data logger (EL-USB-2, UK)
operated by Easylog software. Figure 5.2 shows a 10-day recording of the temperature and RH%
inside the in-house modified egg incubator.
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Three day old dead embryo with intact yolk sac Three day old embryo with broken yolk and CAM
CAM
Three day old embryo with intact yolk and CAM Ten day old embryo with viable CAM covering the
entire surface
Figure 5.1 Development stages of the growing embryos
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Figure 5.2 Temperature, percentage relative humidity (% RH) and dew point inside the
egg incubator over the 10-day incubation period
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Test substances and noisomal formulations
The ocular irritation potential of the following test substances was investigated:
•
NTX solution (40 mg/ml) in PBS pH 7.4.
•
Span 60 and cholesterol, DCP, C24 and CH in their pure (powder) forms.
•
Surfactant solutions of 0.3% w/v CH and 0.4% C24 solutions in PBS representing the
actual concentrations of the surfactant additives in the proposed niosomal formulations.
•
The plain niosomal formulations (F-S60, F-DCP, F-C24 and F-CH) at three different total
surfactant/lipid concentrations (1, 5 and 10 % w/v).
•
NTX loaded F-S60, F-DCP, F-C24 and F-CH niosomal formulations.
Irritation testing, scoring and classification
On day 10, 0.2 ml (for liquid test materials) or 0.1 g (for solid test materials) of the test
substances were dropped onto the membrane. For each test substance three eggs were used.
NaOH (0.5 M) was used as a positive control for a strong irritant, acetone as a moderate irritant,
propylene glycol as a slight irritant, and normal saline as a negative control (Alany et al., 2006).
After the application of the test substance, the blood vessels and capillaries were examined for
irritant effects. The irritant effects were hyperaemia, haemorrhage and clotting at different time
points post-application for 5 min (Figure 5.3). A time-dependent numerical score was allocated to
each test substance or formulation (Table 5.2).
The sum of the time-dependent numerical scores for all three irritant responses gave a single
numerical value. This value interpreted the irritation potential of the test substance (Table 5.3).
The mean score value of the test allowed the assessment by a classification scheme analogous to
the well-known Draize test (Luepke, 1985).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.2 HET-CAM scoring system
Score
Effect
time (min)
0.5
2
5
Hyperemia
5
3
1
Haemorrhage
7
5
3
Clotting/coagulation
9
7
5
Table 5.3 Classification of cumulative scores in HET-CAM
Cumulative score
Interpretation
0-0.9
None
1.0-4.9
Slight
5.0-8.9
Moderate
9.0-21.0
Severe
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
No response
Hyperaemia
Haemorrhage
Clotting
Figure 5.3 Vascular responses used to score the test substances
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Corneal (Bovine eye) test
Biological materials
Cow eyes were obtained from a local slaughterhouse (Auckland Meat Processors, Auckland,
New Zealand). The eyes were excised by standard procedures within 10 min of the animal being
killed. The collected eyes were transported to the laboratory in cold saline (8-10oC). The eyes
were examined for epithelium detachment, corneal opacity and corneal vascularisation. Eyes with
corneal damage or abnormalities were discarded. A summary of the average dimensions of the
cow corneas, compared with human corneas is presented in Table 5.4.
Table 5.4 Average dimensions of human and cow corneas
Species
Cornea
Cornea
Cornea
diameter
surface area
thickness
mm
mm2
mm
Human
11
95
0.52
Cow
20
314
1
Data collected from (Worakul & Robinson, 1997) and own measurements
Test substances and niosomal formulations
Four different controls were used for validation purposes; 0.5 M NaOH was used as a strong
irritant control, acetone as a moderate irritant, propylene glycol as a slight irritant, and normal
saline as a negative control. The same components and formulations described in the HET-CAM
test were also investigated for their corneal irritation potential.
Irritation testing, scoring and classification
Small plastic cups were used to hold the eyes (cornea upwards), and the whole kept in the humid
atmosphere of a closed water bath at 37oC ± 0.5oC for 10 min (Weterings & Vanerp, 1987). A
silicon O-ring (thickness 1.78 mm, an internal diameter 7.6 mm) was carefully placed on the
central part of the cornea, to localise the application site and for easy and reproducible test
material application (Figure 5.4). One drop of saline was applied inside the ring and the eyes
were equilibrated in a closed water bath for 5 min. The test substance was applied to the cornea
inside the ring at a volume of 0.1 ml or 0.1 g of the solid test substance. After 30 s, the eyes were
rinsed with saline (approximately 10 ml), followed by further incubation in the closed water bath
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
for 10 min. Then, the extent of corneal injury was assessed by evaluating the opacity, followed by
application of sodium fluorescein solution (2% w/v pH 7.4) to examine the integrity of the
corneal epithelium, using an examination lamp and cobalt blue filter (Leica, GmbH, Germany).
The observations were graded according to individual numerical scores for opacity, epithelial
integrity (degree of staining) and epithelial detachment (Weterings & Vanerp, 1987). Corneal
injuries and scores are demonstrated in Figure 5.5 and Figure 5.6 and Table 5.5. The sum score
was calculated and the mean scores for each of the 3 exposed eyes was used to interpret the
corneal irritation potential (Table 5.6).
Figure 5.4 Excised cow eyes immersed in normal saline with silicone O-rings centered on top
of the cornea and incubated a water bath thermostatically equilibrated at 37oC ± 0.5oC
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.5 Bovine eye scoring system
Opacity
Score
Epithelial integrity
Score
Epithelial detachment
Score
None
0
None
0
No gross abnormalities
0
Slight
1
Diffuse and weak
0.5
Wrinkling of corneal surface
2
Marked
2
Confluent and weak
1
Loosening of epithelium
3
Severe
3
Confluent and intense
1.5
Epithelium absent
4
Opaque
4
-
-
-
-
Table 5.6 Classification of cumulative scores in bovine eye test
Cumulative
score
Interpretation
≤0.5
None
0.6-1.9
Slight
2.0-4.0
Moderate
>4
Severe
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Transparent cornea (none)
Opaque cornea
No staining (none)
Complete staining and loss
of lustre (no epithelium)
Figure 5.5 Degree of corneal opacity (left) and fluorescein permeability (right) used to score
the test substances [non-irritant (upper) and strong irritant (lower) models]
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Marked opacity
Diffuse and weak opacity
Confluent and intense staining
Confluent and weak staining
Figure 5.6 Degree of corneal opacity (left) and fluorescein permeability (right) used to
score the test substances [moderately irritant (upper) and slightly irritant (lower) models]
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Histopathological evaluation of the bovine corneas
The treated areas of the cornea in the previous bovine eye test and bovine eyes treated with the
prepared niosomes for longer periods of time (1, 3 and 8 h) were histologically studied. The
samples were dissected and fixed in 10% v/v neutral buffered formalin (NBF) for at least 24 h,
and then transferred to 70% v/v ethanol for another 24 h. The fixed samples were transferred into
labelled cassettes and placed in 70% v/v ethanol (Zagon et al., 2006a). They were then sent to the
histology laboratory for tissue processing, embedding, sectioning and staining. Each section was
paraffin-embedded, bisected into two equal halves and finally mounted in a paraffin block so that
a section of each half cut and placed on a single microscopic slide. The slides were stained with
haematoxylin and eosin (H&E). The stained corneal sections were imaged using a light
microscope (Leica DMRE, GmbH, Germany) and the thickness of stroma was measured using
Leica DMRE software.
5.3.2.2. In vitro release studies
In vitro release studies were performed using standard Franz diffusion cells. The Franz–diffusion
cells had a 15 mm diameter orifice (providing a diffusion area of 1.7 cm2) and were thermostated
by means of a water jacket connected to a VTC-220 heat circulator (Logan Instrument
Corporation, Somerset, NJ, USA). Receptor chambers (12 ml volume) were filled with PBS pH
7.4 (osmolality 297 mOSm/kg) and stirred constantly using small magnetic bars. Donor and
receptor chambers were separated by means of a 12,000-14,000 mol wt cut-off dialysis
membrane, presoaked in the receptor medium overnight prior to the experiment. The temperature
was set at 35oC ± 0.5ºC. Two ml of each formulation were loaded into the donor compartment
before occluding the chamber with Parafilm. Care was taken to avoid air bubbles at the
membrane/liquid interface. Samples (0.4 ml each) were withdrawn at predetermined time points
for up to 12 h, and replaced with an equal volume of the receptor medium. The experiments were
done in triplicate and the samples were determined using the HPLC method as described in
Section 2.3.2.
5.3.2.3. In vitro release data analysis
Three in vitro release parameters were determined for comparing NTX release profiles from the
prepared niosomes.
•
Q2h: the percentage drug release per unit surface area at 2 h.
•
Q6h: the percentage drug release per unit surface area at 6 h.
•
DE %-12: the percentage dissolution efficiency at 12 h (equation 5.1).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
12
DE % − 12 =
∫
y.dt
0
1200
.100%
equation 5.1
12
The DE%-12 was defined as the area under the % release-time curve ( ∫ y.dt ) at 12 h, expressed
0
as a percentage of the area of the rectangle described by 100% release at the same time which is
12
1200 (Khan, 1975). The
∫
y.dt was estimated using the trapezoidal method.
0
5.3.2.4. In vitro release kinetic studies
To elucidate the mechanism of drug release from the prepared niosomes, the release data was put
through equation 5.2 which is widely known as the Korsmeyer-Peppas equation (Korsmeyer et
al., 1983).
Mt
= kt n
M∞
equation 5.2
Mt/M∞, t, k and n are the fraction of drug release at time t, the kinetic constant, release exponent
respectively (Peppas, 1985). The release exponent (n) indicates the general operating release
mechanism; an n value of 0.43 indicates Fickian diffusion-mediated release; 0.43 < n < 0.85
indicates non-Fickian release or coupled diffusion and erosion; and n = 0.85 indicates erosionmediated release (zero-order kinetics) (Peppas, 1985; Ritger & Peppas, 1987). The release
exponent n is the slope of the log fraction of the drug release (Mt/M∞) versus log time plot.
5.3.2.5. Ex-vivo permeation studies
Enucleated bovine eyes were carefully examined for any corneal damage such as epithelium
detachment, corneal opacity and corneal vascularisation, before the corneas were dissected. The
dissection was performed with extreme care to avoid touching the surface of the cornea.
Therefore, a small ring of sclera tissue was left around the cornea (Figure 5.7). The scleral tissue
served as a gasket and permitted the cornea to be mounted where the endothelium was facing the
receptor compartment and the epithelium was facing the donor compartment.
Franz-diffusion cells were used for ex-vivo study. A modification was made in the donor and
receptor compartments to hold cow corneas and maintain normal corneal curvature without
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
wrinkling (Figure 5.7). The receptor compartment was concave and the donor component was
convex providing a diffusion area of 1.7 cm2. The temperature was kept at 35oC ± 0.5oC.
The receptor chambers were filled with a modified ringer’s solution to preserve the integrity of
the excised cornea over 8 h (Schoenwald & Huang, 1983). The permeation medium comprised
sodium chloride (122 mM), sodium bicarbonate (25 mM), magnesium sulfate (1.2 mM), dibasic
sodium phosphate (0.5 mM), calcium chloride (1.4 mM), glucose (10 mM) and 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mM). The medium’s pH and
osmolality were 7.4 and 299 mOSm/kg respectively. The medium was constantly stirred using
small magnetic bars. A two ml-sample of each formulation was introduced into the donor
compartment. Samples of 0.4 ml were withdrawn at predetermined time points for up to 12 h and
replaced with fresh receptor medium. The experiments were performed under non-occlusive
condition, to allow air permeation through the corneal tissues (n = 5). The samples were analysed
as previously mentioned in Section 2.3.2.
Figure 5.7 Excised bovine cornea, modified parts and top view of the final assembly of the
Franz- diffusion cell (from left to right)
5.3.2.6. Ex-vivo data analysis
The apparent permeability coefficient (Papp, cm/s) was calculated using equation 5.3 (Schoenwald
& Huang, 1983).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Papp =
∆Q
∆t (3600 )ACo
equation 5.3
∆Q/∆t is the permeability rate of NTX across the excised cow corneas. It was calculated from the
slope of the straight line obtained from the plot of the amount of NTX permeated versus time
curve. Co is the initial drug concentration (µg/cm3), A is the corneal surface area (cm2) and 3600
allows for the conversion of hours to seconds. The lag time (tL) was determined by extrapolating
the steady-state line to the time axis.
5.3.2.7. Physical stability of the prepared niosomes
Four different batches of F-S60, F-DCP, F-C24 and F-CH were prepared, purged with nitrogen
gas and stored in screw-capped 20-ml glass vials at three different temperatures (4oC, 25oC and
37oC) for three months. Samples were withdrawn at specified time intervals and evaluated for:
•
Changes in size by measuring the average D [3,4] as previously mentioned in size
measurements at Section 4.3.2.2.
•
The amount of NTX retained in the niosomes, by measuring the residual EE% as
previously described in EE% measurements at Section 3.3.2.2.
5.3.3. Statistical analysis
A one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparisons at the 5%
significance level was used to test statistical significance differences between the prepared
formulations for in vitro release studies, kinetic studies, ex vivo studies and physical stability
studies. These were performed using GraphPad Software Version 3.05, San Diego California,
USA.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.
Results and discussion
5.4.1. Conjunctival (HET-CAM) test
During the cracking stage of the CAM preparation process, about 30% were excluded for several
reasons, some of which were beyond experimental control like eggs had dead embryos (Figure
5.1) and infertile eggs. In addition, the technique of cracking the shell against the side of the
growing chamber did not allow adequate control of breaking and in some occasions resulted in
damaging of the young CAM and/or the yolk membrane (Figure 5.1).
In some instances, the damage of yolk membrane was in the form of small leaks that were not
detected initially and showed up after 24-72 h. These findings suggest the need for an alternative
technique for the eggshell breaking with better control of the breaking process. This would
reduce the chances of damaging the embryo and extra-embryonic membranes and thus increasing
the number of embryos incubated. There are many methods of opening the eggs reported in the
literature (Alany et al., 2006; Auerbach et al., 1974; Luepke, 1985).
According to the original method, the CAMs were fully grown in the eggshells until test day. On
day 10 of incubation, a window was created in the eggshell using a dentist rotary saw-blade and
the inner egg membrane was removed to expose the CAM for applying test substances (Luepke,
1985). However, exposing the CAM by this method was found to damage the CAM occasionally
when eggshell particles fell onto the CAM surface during the drilling procedure. Moreover, the
window created in the eggshell is relatively small and hence gives limited access to and visibility
of the CAM. This impedes the evaluation of vascular responses and minimises the total area of
CAM surface available for testing (Lawrence et al., 1986).
A modified method was reported in the literature, where the chick embryo was grown in a Petri
dish from day 3 onwards to allow ready access to the entire CAM surface for better visibility and
convenience (Auerbach et al., 1974). This method was slightly modified for the studies
performed in this thesis using in-house made growing chambers to replace the Petri dishes.
Accordingly, the growing chambers can offer the embryo a more natural environment to grow.
The curved, elastic nature of the cellophane membrane mimics the inner membrane of the
eggshell compared with the flat, rigid surface of the Petri dish. This could explain the higher
percentage of embryos surviving (60%) compared with that obtained when using a Petri dish to
grow the embryos (50%). These findings suggest that the cracking procedure and growing
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
conditions are critical to the survival of the embryos and therefore the number of CAMs available
for test substances.
Figure 5.8 shows the cumulative HET-CAM scores for the controls, NTX solution (40 mg/ml) in
PBS, the test substances used in fabricating the niosomal formulations and the prepared
niosomes.
Strong irritant
Moderate irritant
Slight irritant
Non irritant
Figure 5.8 Cumulative HET-CAM scores for the controls and representative examples of
the test substances (Results expressed as mean values ± SD, n = 3)
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
The average cumulative scores calculated for NTX solution, Span 60 powder, cholesterol powder
and DCP powder were found to be < 0.9. These results reveal that these test substances are
practically non-irritant when applied to the surface of the CAM. Span 60 and cholesterol are
regarded as non-irritant and are widely used in the cosmetic and food industry (Buehler, 2003;
Lawrence, 2003). In contrast, CH powder was found to be strongly irritant with a cumulative
score of 14 ± 1.4. This result is in accordance with the in vivo rectal irritation in rats. A solution
of 25 mM (equivalent to 1.1 % w/v) CH was found to be strongly irritant to rectal mucosa of rats.
Immediate strong contractions were observed after application into the rectum. Lumen
congestion, oedema and haemorrhage were also detected after 20 min (Kinouchi et al., 1996).
All the prepared niosomal formulations, total lipid concentrations up to 10 % w/v, were assessed
for the irritation potential and the results did not show any signs of inflammatory reactions at the
nominated time points. Similar results have been reported on niosomes assessed using the in vivo
rabbit eye test (Draize test) (Abdelbary & El-gendy, 2008). Niosome formulations composed of
Tween 60:cholesterol:DCP at 1:1:0.1 molar ratio, Tween 80: cholesterol at 1:1 molar ratio and
Brij 35:cholesterol: DCP at 1:1:0.1 molar ratio did not show any signs of redness, inflammation
or increased tear production when applied onto the eyes of albino rabbits (Abdelbary & El-gendy,
2008).
C24 powder developed minimal irritation potential such as hyperaemia after 3 min. This indicates
that C24 is slightly irritant when applied on the surface of the CAM. Further, no signs of vascular
toxicity were detected after application of niosomes containing sodium cholate (F-CH). The
concentrations of CH in the tested F-CH niosomes were 0.13% to 0.53% w/v. It is likely that the
amount of CH in these formulations was insufficient to produce an irritant effect. Rabbit corneas
demonstrated good ocular tolerability and no signs of ocular damage after the application a 0.5 %
w/v CH solution onto rabbit eyes (Furrer et al., 2002b). Another possible explanation is that CH
molecules were included in the bilayer membrane of the prepared niosomes, which is likely to
reduce the direct contact of free CH molecules with the ocular surface.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.2. Cornea (bovine eye) test
Figure 5.9 shows the cumulative bovine eye test scores for the controls, NTX solution (40 mg/ml)
in PBS, the test substances used in manufacturing the prepared niosomal formulations and the
prepared niosomes.
The cornea has both refractive and protective functions. The normal cornea is transparent,
lustrous and completely impermeable to fluorescein dye, due to the exclusive tight junctions of
the corneal epithelium. Figure 5.5 shows photographs for normal cornea before and after staining.
Apart from CH powder, all the test substances and the prepared formulations did not show any
signs of corneal injuries (corneal opacity, corneal permeability (stained green) or epithelial
damage), and the cumulative corneal scores were < 0.5. Additionally, all the prepared niosomal
formulations in the three concentrations tested showed no signs of corneal damage when applied
to the corneas. Moreover, the lustre of the niosome-tested corneas was as normal as the negative
control corneas. Similar results were reported on the in vivo rabbit eye test for niosomes (Guinedi
et al., 2005). Niosomes composed of Span 60: cholesterol 7:4 mol/mol were applied topically on
rabbit eye test daily for 40 days. Rabbits’ corneas were excised and assessed histologically for
corneal irritation. The tested corneas showed slight stromal oedema and no major harmful corneal
signs, and as such the tested niosomes were regarded as safe for short and long term treatment
(Guinedi et al., 2005).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Strong irritant
Moderate irritant
Slight irritant
Non irritant
Figure 5.9 Cumulative bovine eye scores for the controls and representative examples of the
test substances (Results expressed as mean values ± SD, n = 3)
Table 5.7 shows the interpretations of the cumulative test scores for the HET-CAM and bovine
eye tests. The irritation potential for all the prepared niosomes and the excipients used was
equally interpreted by the HET-CAM (the conjunctival model) and bovine eye assay (the corneal
model). These findings indicate that a good correlation was achieved between the two toxicity
models. The only exception to these results was recorded for acetone. Based on the HET-CAM
test, acetone was regarded as a strong irritant, whereas it was found a moderate irritant when
applied on the bovine cornea. The scoring system of bovine cornea is based on assessing the
disruption of the test substances to the epithelium barriers and consequently, corneal opacity and
permeability induced by the test substances are estimated. Acetone, an organic solvent, could
induce damage to the corneal tissues other than the immediate effects recorded for the surfactants
used. Therefore, histological examination to the corneal layers could be helpful as discriminatory
tool and confirmatory to the HET-CAM and bovine corneal opacity and permeability (BCOP)
tests.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.7 Summary of HET-CAM and bovine eye test interpretations
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.3. Histopathological evaluation of the bovine corneas
Histological sectioning, fixing and H&E staining of the corneal samples were carried out to help
understand and evaluate the degree of corneal injuries for the test substances.
Negative (Figure 5.10A) and positive (Figure 5.11 and Figure 5.12) controls were included to
facilitate the interpretation of the results. The negative controls provide the baseline against
which histological changes are compared. Furthermore, the histology of the negative control
corneas was used in this study to evaluate the quality/acceptability of the sections from batch to
batch, to minimise or rule out batch processing and cattle age effects. Older cattle have thicker
corneal sections where Descemet’s membrane increases in thickness with the age of the donor.
Also, poorly-prepared sections (over-trimmed or poorly-embedded) show thicker corneal layers
(Cuellar et al., 2002). In this study, a negative control was used as a baseline for each processed
batch to evaluate the irritation potential for the test substances (Table 5.8).
5.4.3.1. Evaluating the negative control- treated corneas
Figure 5.10 A and Figure 5.10 B show photomicrographs for the whole corneal layers at 10x
magnification; and the epithelial layers at 40x magnification for the saline-treated cornea (the
negative control).
The cornea is an avascular tissue (no circulatory system), hence no leukocyte infiltration is
evident. Since the test materials were applied topically onto a silicon O-ring on the surface of the
cornea, the evaluation was conducted top-down, starting with the upper epithelium and
proceeding down through the epithelial layers, via the stroma and down to the endothelium. The
depth of corneal injury basically depends on the penetration of the test substance through the
three principles corneal layers: the epithelium, stroma and endothelium.
Figure 5.10 B shows magnified corneal epithelium which is composed of three distinct layers (the
basal cell layer, wing cell layer and squamous cell layer). The basal cell layer is a columnar-cell
layer directly attached to the basement membrane above Bowman’s layer. Several cell-layers of
wing cells are on top of the basal layer. The superficial layer or squamous layer is composed of
flattened with limited cytoplasm and highly condensed nuclei. The second principal layer is the
stroma which accounts for 90% of the total thickness of the cornea. It is composed of wellorganised collagen matrix fibres with dispersed keratocytes. The third layer is endothelium,
which is a single layer of flattened cells attached to the basal surface of Descemet’s membrane.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) Corneal layers (10x magnification)
(B) Epithelial layer (40x magnification)
Figure 5.10 Photomicrographs (A 10x; B 40x) of H&E stained corneal sections of
negative control treated with saline for 30 s
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.3.2. Evaluating the positive control-treated corneas
Epithelial defects and stromal oedema are two common causes of ocular irritation after topical
application of test substances (Harbell et al., 2006; Harbell et al., 1999). Cell loss, vacuolisation
(presence of vacuoles or holes), pyknosis (nuclei coagulation), saponification (due to the effect of
alkali) and separation of cells from the Bowman’s membrane are characteristic lesions observed
in the epithelium (Figure 5.11 and Figure 5.12).
The lesions of stroma are predominantly presented in form of stromal oedema, keratocytes
pyknosis and vacuolisation. The stromal swelling or oedema is associated with the loss of the
normal ordered array of extracellular collagen matrix fibres. Stromal swelling is directly related
to corneal opacity. The goal of scoring the corneal lesions is not to report separately but rather to
correlate histological scores with the opacity and permeability values. It is accepted that the
greater the stromal oedema, the greater the corneal opacity found (Nishida, 2005). A potential
utilisation of histological scoring of the corneal lesions was to quantify the degree of opacity by
measuring stromal thickness using employed Leica software.
Stromal swelling could be manifested in the form of vacuoles in the organised collagen matrix.
As the degree of vacuolisation increases, the overall thickness of the stroma is expected to
increase. Lastly, the damage to endothelium would be expected to result from mechanical
damage rather than the topical application of test substances.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) 5x magnification
(B) 40x magnification
Figure 5.11 Photomicrographs (A at 10x magnification; B at 40x magnification) of H&E
stained corneal sections of acetone-treated cornea for 30 s
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Figure 5.11 A & B shows the corneal layers treated with acetone and a magnified
photomicrograph for the epithelium layer. Squamous layer vacuolisation, squamous layer
sloughing and wing layer coagulation were observed (Figure 5.11 B). The stroma showed a
significant increase (P < 0.001) in thickness compared with the stroma of the saline-treated
cornea. The stromal thickness was 2 times thicker than that for the negative control-treated.
Further, marked stromal collagen matrix condensation and keratocytes condensation were
observed. The lower opacity and permeability scores calculated for acetone-treated eyes from the
bovine eye test are attributed to coagulation of the epithelium and consequently this could
decrease the permeability of the epithelium to the fluorescent dye. Hence, additional scores
should be counted for the marked coagulation and condensation of acetone-treated cornea as
shown by histological examination.
Figure 5.12 A & B shows the corneal layers treated with NaOH (0.5M) and a magnified
photomicrograph for the remnants of the epithelium and stroma. Extensive corneal damage was
observed for NaOH treated control. For example, epithelial rupture, saponification and epithelial
loss were registered. Furthermore, the stroma was exhibited extensive vacuolisation and swelling.
These results were correlated well with opacity and permeability scores from the bovine eyes test.
The marked opacity and loss of lustre induced by NaOH can be attributed to complete loss of
epithelial barriers.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) 5x magnification
(B) 40x magnification
Figure 5.12 Photomicrographs (A at 5x magnifications; B at 40x magnification)
of H&E stained corneal sections of NaOH (0.5 M)-treated cornea for 30 s
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.3.3. Evaluating the test substances
Test substances applied for 30 s
Figure 5.13 A-F shows photomicrographs of H&E stained histological sections of corneas treated
with CH powder, C24 powder, F-S60, F-DCP, F-C24 and F-CH for 30 s according to the bovine
eye assay.
Figure 5.13 A demonstrates an extensive epithelial loss and swelling or oedema induced by CH
powder. Further, epithelial detachment, vacuolisation and stromal collagen matrix vacuolisation
were observed (Table 5.8). This indicates that CH is a strongly irritant substance. In contrast,
intact epithelial layer and mild stromal oedema were recorded for C24 powder indicating that
C24 is a slightly irritant. These results are in accordance with those obtained from the opacity and
permeability scores.
Additionally, there were no obvious signs of epithelial loss or stromal oedema recorded for the
prepared niosomes. The slight superficial sloughing of squamous layer exhibited by F-DCP and
F-CH (Figure 5.13 D&F) indicates good corneal tolerability. These results are also well
correlated with opacity and permeability scores.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) CH powder (5x magnification)
(B) C24 powder (10x magnification)
(C) F-S60 (10x magnification)
(D) F-DCP (10x magnification)
(E) F-C24 (10x magnification)
(F) F-CH (10x magnification)
Figure 5.13 Photomicrographs (A at 5x magnification; B, C, D, E and F at 10x magnification) of H&E
stained corneal sections treated with CH Powder, C24 powder and the prepared niosomes for 30 s
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Test substances applied for 1, 3 and 8 h
The duration of exposure was also studied by applying the prepared niosomes for a longer time 1,
3 and 8 h. The treated corneas were dissected, fixed, sectioned, stained and photographed.
Figures 14-16 show photomicrographs of H&E stained corneas treated with the prepared
niosomes for 1, 3 and 8 h respectively. Table 5.8 summarises the histological toxicity and stromal
thickness for all test substances at the different time points.
After 1 h, all the prepared niosomes showed minimal to no histopathological signs. Intact
epithelium and non-significant differences between corneal stromal thicknesses were observed
after 1 h compared with 30 s-treated samples.
Similarly, the prepared niosomes did not demonstrate notable differences after 3 h. However,
epithelial vacoulisation and slight epithelial swelling induced only by F-CH (Figure 5.15 D).
After 8 h, moderate stromal collagen matrix vacoulisation exhibited by the prepared niosomes
(Figure 5.16 A-D) and marked epithelial cell loss and erosion induced only by F-CH (Figure 5.16
D). The stromal oedema induced by the prepared niosomes is likely to be reversible and the
cornea regains its normal configuration and normal structure after stopping treatment (Guinedi et
al., 2005; Hughes et al., 2004).
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) F-S60 (10x magnification)
(C) F-C24 (10x magnification)
(B) F-DCP (10x magnification)
(D) F-CH (10x magnification)
Figure 5.14 Photomicrographs (A, B, C and D at 10x magnification) of H&E stained corneal
sections treated with the prepared niosomes for 1 h
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) F-S60 (10x magnification)
(C) F-C24 (10x magnification)
(B) F-DCP (10x magnification)
(D) F-CH (10x magnification)
Figure 5.15 Photomicrographs (A, B, C and D at 10x magnification) of H&E stained corneal sections
treated with the prepared niosomes for 3 h
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
(A) F-S60 (10x magnification)
(C) F-C24 (10x magnification)
(B) F-DCP (10x magnification)
(D) F-CH (10x magnification)
Figure 5.16 Photomicrographs (A, B, C and D at 10x magnification) of H&E stained corneal sections
treated with the prepared niosomes for 8 h
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.8 Histological corneal lesions scores for controls and the prepared niosomes at
different time points (Thickness values are expressed as mean values ± SD, n = 3)
Test substance
Epithelium
Cell loss
Stroma
Vacuolisation/coagulation
Thickness
Collagen matrix
(µm)
Vacuolisation
Control (30 s)
-
-/-
657 ± 60
-
NaOH (1M) (30 s)
+++
+++/+++
1180 ± 267
+++
Acetone (30 s)
+
+/+++
1278 ± 110
+
CH powder (30 s)
+++
+++/+++
1280 ± 258
+++
C24 Powder (30 s)
-
+/-
756 ±211
+
F-S60 (30 s)
-
-/-
653 ± 64
-
F-DCP (30 s)
-
-/-
649 ± 21
-
F-C24 (30 s)
-
-/-
663 ± 15
-
F-CH (30 s)
-
-/-
745 ± 22
-
Control (1 h)
-
-/-
740 ± 39
-
F-S60 (1 h)
-
-/-
621 ± 70
-
F-DCP (1 h)
-
-/-
672 ± 58
-
F-C24 (1 h)
-
-/-
691 ± 12
-
F-CH (1 h)
-
-/-
664 ± 17
-
Control (3 h)
-
-/-
674 ± 91
-
F-S60 (3 h)
-
-/-
720 ± 15
+
F-DCP (3 h)
-
-/-
733 ± 49
+
F-C24 (3 h)
-
-/-
632 ± 14
+
F-CH (3 h)
-
-/-
677 ± 25
+
Control (8 h)
-
+/-
687 ± 73
+
F-S60 (8 h)
-
+/-
702 ± 16
+
F-DCP (8 h)
-
+/-
635 ± 31
+
F-C24 (8 h)
-
+/-
679 ± 28
+
F-CH (8 h)
++
++/-
781 ± 28
+
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
5.4.4.
In vitro release studies
In vitro dissolution or release testing is a regulatory requirement in drug development and quality
control to warrant that a dosage form will release the active pharmaceutical ingredient at
appropriate rate and extent (Burgess, 2010). Development of an in vitro release test should
consider physiological conditions such as the temperature, the pH of the fluid, ionic composition,
buffer capacity, surface tension and hydrodynamic stirring rate, in order to closely represent the
in vivo performance.
In this study, many physiological factors were considered, such as the ocular temperature (35oC),
pH (7.4) and osmolality (isotonic with the tear fluid) in order to simulate ocular surface
conditions.
The in vitro release performances of NTX from the prepared niosomes were studied on Franzdiffusion cells. An aqueous solution of PBS was used as a release medium which is composed of
electrolytes (NaCl 137 mM, Na2HPO4 10 mM, KH2PO4 1.47 mM, KCl 2.68 mM), a pH value of
7.4, osmolality of 278 ± 5 mOSm/kg, stirred and equilibrated at 35oC ± 0.5. The tested niosomes
in the donor compartment were separated from the receptor compartment, which was filled with
12 ml of the release medium, by a cellulose membrane.
Figure 5.17 shows various release profiles of NTX from the aqueous solution and the prepared
niosomes. Levelling off the NTX release from NTX solution formulation observed at the 6 h
point is shown in Figure 5.18. This can be attributed to attaining equilibrium between the donor
and receptor compartments. The sample size used (0.4 ml) was insufficient to maintain a
concentration gradient between the donor and receptor compartments. Unperturbed NTX release
profiles were obtained when relatively larger sample sizes (1 ml and 2 ml) were used (Figure
5.18). Such flattening was not observed with the prepared niosomal formulations over the 12 hrelease period. The partitioning of NTX molecules through the bilayer membranes of the
niosomes was the rate-controlling step in the release process. Hence, an appreciable
concentration gradient was maintained.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Figure 5.17 In vitro release profiles of NTX from the NTX solution and the prepared
niosomes (Results are expressed as mean values ± SD, n = 3)
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Figure 5.18 Effect of sample volume on the diffusion of NTX from the aqueous solution
(Results represent mean values, n = 3)
Table 5.9 In vitro release parameters for NTX from the prepared niosomes compared with
NTX aqueous solution (Results are expressed as mean values ± SD, n = 3)
Formulation
Q2h (%.cm-2)
Q6h (%.cm-2)
DE% -12.cm-2
NTX- solution
17.06 ± 1.70
35.29 ± 3.05
26.34 ± 0.34
F-S60
4.53 ± 0.26
11.00 ± 0.29
8.86 ± 0.47
F-DCP
2.29 ± 0.11
6.00 ± 0.41
5.03 ± 0.38
F-C24
5.38 ± 0.94
13.88 ± 1.77
11.60 ± 1.4
F-CH
8.15 ± 0.21
20.11 ± 0.16
17.01 ± 0.29
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.10 Tukey’s pair wise comparison of Q2h, Q6h and DE%-12 for NTX from the
aqueous PBS solution and the prepared niosomal formulations
NTX - solution
F-S60
F-DCP
F-C24
Parameters
Q2h; Q6h;DE% -12
Q2h; Q6h;DE%-12
Q2h; Q6h;DE%-12
Q2h; Q6h;DE%-12
F-S60
S;S;S
-
-
-
F-DCP
S;S;S
S;S;S
-
-
F-C24
S;S;S
NS ; S ; S
S;S;S
-
F-CH
S;S;S
S;S;S
S;S;S
S;S;S
S = significant difference (P < 0.05)
NS = non-significant (P > 0.05)
The release parameters (Q2h, Q6h and DE %-12) calculated for the NTX solution were 17.06,
35.29 and 26.34 respectively (Table 5.9). These parameters were significantly higher (P < 0.01)
than those calculated for the prepared niosomal formulations. For example, the Q6h value
calculated for NTX solution was 5.88 and 2.54 times greater than that for F-DCP and F-C24
respectively. These results demonstrate the NTX release retarding efficiency for the prepared
niosomes. Similar results were obtained from acyclovir encapsulated liposomes. The in vitro
release rate for acyclovir from liposomes was markedly lower than that for an aqueous solution of
acyclovir indicating that the prepared liposomes established controlled acyclovir release (Law et
al., 2000).
The drug release rate and extent were significantly different (P < 0.5) amongst the prepared
niosomal formulations (Table 5.9 and Table 5.10). F-DCP showed the highest NTX retarding
efficiency, whereas NTX release rate was highest for F-CH. The drug release rates for F-S60 and
F-C24 came in the middle. For instance, the Q6h values for F-S60, F-DCP and F-CH were 11, 6
and 20 %.cm2 respectively. These results revealed good discrimination amongst the drug release
profiles for the prepared niosomes. This is partly due the ability of the developed in vitro release
test to show perfect sink conditions. In addition, this can be attributed to the effect of lipid
composition and membrane additives used to fabricate the tested niosomes.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
The highest NTX release retarding efficiency ascribed for F-DCP niosomes could be due to the
incorporation of DCP in the bilayer membranes. This could lead to two possible effects: firstly,
the electrostatic interaction between the positively charged NTX molecules and the negatively
charged DCP was likely to slow the NTX release from the vesicles. Secondly, the double
hydrocarbon chains of DCP could impart more order and improve the packing of the bilayer
membranes, as previously outlined in Figure 4.13. Similar results have been reported with
charged liposomes. The lowest release rate was obtained from positively charged liposomes
entrapping acetazolamide (weak acid). This has been ascribed to the electrostatic interaction
between positively charged liposomes and the negatively charged drug (Hathout et al., 2007).
Incorporating bulky structures with long and highly hydrophilic poly-24-oxyethylene chains (C24
molecules) in the bilayer layer of F-C24 could increase the interstitial spaces between the bilayer
membranes. This could increase the permeability of the membrane to water-soluble solutes and
hence the drug release rate.
F-CH showed 1.18- and 3.33-fold increases in Q6h compared with those calculated for F-S60 and
F-DCP respectively. CH molecules could interact with the hydrocarbon region such that its
carboxylate group interacts with the head group of Span 60 and the steroidal nucleus aligns itself
parallel to the acyl chains. Such orientation is likely to create some vacancies in the hydrophobic
domains of the bilayer membranes (Figure 4.15). This could provide increase NTX permeability
through the bilayer membranes and increase release rate for NTX compared with F-DCP and FS60.
5.4.5. In vitro release kinetic studies
The Higuchi model has been used to explain the drug release kinetics for many niosomal
formulations. The Higuchi model was also used to interpret the mechanism of water-soluble and insoluble drugs from uni- and multi-lamellar niosomes (Bayindir & Yuksel, 2010; Guinedi et al.,
2005).
In this study, the Korsmeyer-Peppas equation was utilised to elucidate the release kinetics of
NTX from the prepared niosomes. This equation does not force the time data points to a
particular exponent (t1/2) like Higuchi equation does. In Addition, it can give more insights on
other drug release mechanisms, such as Fickian (diffusion), non-Fickian and erosion-mediated
(zero-order) release. Further, this equation has been successfully used to explain release
220
Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
mechanisms from thin film, cylindrical, disc and spherical controlled release devices (Ritger &
Peppas, 1987).
Table 5.11 summarises the release exponent (n) values and regression coefficients (R2) calculated
for the tested formulations. The n values for F-S60, F-DCP and F-C24 were found to be close to
0.85. These results suggested that the general operating release mechanism was zero-order
(erosion-mediated release). For F-CH, the n value was found to be 0.77 suggesting that the NTX
released by coupled diffusion and erosion mechanism, however, the dominant release mechanism
was by erosion. Zero-order kinetics prevails if n > 0.66 (Mockel & Lippold, 1993).
Table 5.11 In vitro release kinetic parameters for NTX from the prepared niosomes (Results
are expressed as mean values ± SD, n=3)
Formulations
Release exponent
(n)
Regression
coefficient
(R2)
F-S60
0.80 ± 0.015
0.996
F-DCP
0.83 ± 0.071
0.998
F-C24
0.80 ± 0.061
0.999
F-CH
0.77 ± 0.032
0.999
5.4.6. Ex-vivo corneal permeation studies
Figure 5.19 shows transcorneal permeation profiles of NTX from the investigated formulations
using excised bovine corneas. The following transcorneal permeation parameters were calculated:
steady-state flux, apparent permeability coefficient (Papp) and lag time (tL) (Table 5.12). Table
5.13 shows statistical significance differences among the transcorneal permeation parameters for
NTX from the aqueous solution and the prepared niosomes. The prepared niosomes achieved
controlled NTX permeation through the excised bovine cornea compared with NTX solution. The
steady-state flux and Papp calculated for F-DCP were 2.4 times lower than those for the NTX
solution. In addition, the permeation parameters obtained from the NTX solution were 1.6 times
higher than those from F-CH. These results correlated well with the in-vitro release data
suggesting that the corneal permeation process of NTX is dependent on its release characteristics.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Similar results were reported for lipid-based delivery systems, such as liposomes and solid lipid
nanoparticles (SLNs), using excised rabbit corneas and bioengineered human corneas (Attama et
al., 2009; Law et al., 2000). The permeation rate and apparent permeability coefficient for
acyclovir-encapsulated liposomes and timolol maleate loaded SLNs were significantly controlled
compared with that of the corresponding drug solutions. When the prepared acyclovir liposomes,
which demonstrated lower permeability, were applied on rabbit eyes, higher ocular
bioavailability for acyclovir was obtained, compared with that for the aqueous drug solution
(Law et al., 2000). This is attributed to the ability of liposomes to provide longer precorneal
residence time and less precorneal drug loss due to tear dilution and spillage than the acyclovir
aqueous solution.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Figure 5.19 Transcorneal permeation profiles of NTX from the NTX solution and the
prepared niosomes using excised cow corneas (Results are expressed as mean values ± SD, n
= 5)
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.12 Steady state flux, apparent permeability coefficient (Papp) and tL for NTX from
the aqueous PBS solution and the prepared niosomes though excised cow corneas (Results
are expressed as mean values ± SD, n = 5)
Steady state flux
Papp x 106
tL
(µg/h)
(cm/s)
(h)
NTX solution
18.74 ± 2.00
7.65 ± 0.83
1.27± 0.26
F-S60
8.00 ± 0.37
3.26 ±0.36
0.68 ± 0.35
F-DCP
7. 89 ± 1.23
3.22 ± 0.73
0.5 ± 0.30
F-C24
9.30 ± 2.25
3.79 ± 0.76
0.90 ± 0.44
F-CH
11.88 ± 1.45
4.85 ± 1.00
1.00 ± 0.23
Formulation
Table 5.13 Tukey’s pair wise comparison of steady state flux, Papp and tL for NTX from the
aqueous PBS solution and the prepared niosomal formulations
NTX - solution
F-S60
F-DCP
F-C24
Parameters
Flux; Papp; tL
Flux; Papp; tL
Flux; Papp; tL
Flux; Papp; tL
F-S60
S;S;S
-
-
-
F-DCP
S;S;S
NS ; NS ; NS
-
-
F-C24
S ; S ; NS
NS ; NS ; NS
NS ; NS ; NS
-
F-CH
S ; S ; NS
S ; S ; NS
S;S;S
NS ; NS ; NS
S = significant difference (P < 0.05)
NS = non-significant (P > 0.05)
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
To demonstrate the degree of correlation between in vitro and ex vivo studies, a plot of Papp
values versus in vitro release parameters (Q2h, Q6h and DE%-12h) was constructed (Figure 5.20).
A direct relationship was established between Papp and in vitro release parameters for NTX
solution and the prepared formulations. These findings show the good in vitro/ex vivo correlation
and confirm the ability of the prepared niosomes to retard NTX release on the excised corneal
surface.
Figure 5.20 Relationship between NTX release rate (Q2h, Q6h and DE%-12h values) and
apparent permeability coefficient (Papp) for NTX from the NTX solution and the prepared
niosomes
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
With regard to the lag time (tL), the NTX solution produced the longest tL value, yet produced the
highest steady-state flux, whereas F-DCP achieved the shortest. The tL values for F-DCP and FS60 demonstrated 2.54 and 1.86 times shorter than that for the aqueous NTX solution. The tL
values for F-C24 and F-CH came in the middle (Table 5.12). These results could be attributed to
the fact that the formulated niosomes possess better wetting and spreading abilities on the
lipophilic corneal surface than the aqueous solution. The calculated tL values were in accordance
with the contact angle and spreading coefficient measurements for the prepared niosomes.
Superior wetting and spreading abilities in niosomes can promote intimate contact with the
lipophilic corneal epithelium, increase the available corneal surface area in contact with the
applied formulations and promote better corneal absorption. Further, by virtue of their
amphiphilic nature, niosomes are reported to impart a corneal permeation-enhancing effect
(Saettone et al., 1996b). These were thought to enhance the penetration of NTX, and
consequently shorten the lag time. To support the previous assumption, a θ versus tL plot was
drawn (Figure 5.21). A direct relationship between the θ and tL values was obtained.
Figure 5.21 Relationship between contact angle and lag time from the NTX solution and
four niosomal formulations
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
It is worth noting that negatively charged niosomes (F-DCP) produced a shorter tL than neutral
ones (F-S60). This suggests that negatively charged niosomes could be more effective than
neutral ones for improving the permeation of NTX through the cornea, despite the negatively
charged nature of the epithelial corneal surface. Similar results were observed elsewhere.
Negatively charged multilamellar liposomes showed enhanced corneal permeability of penicillin
G (water soluble drug), compared with both neutral and negatively charged unilamellar
liposomes (Schaeffer & Krohn, 1982). Further, these findings support the hypothesis that flux
rate is independent on the lag time. Similar results have been reported with other colloidal drug
delivery systems for transdermal delivery of indomethacin (El Maghraby, 2010). Longer tL values
were reported for Self–microemulsifying drug delivery systems (SMEDDS), although these
formulations exhibited the greatest flux values. In the same report, application of indomethacin in
microemulsion forms produced a transdermal flux value relatively smaller than that obtained
from SMEDDS but with a shorter tL value.
5.4.7. Physical stability of the prepared niosomes
The physical stability of the prepared niosomes against aggregation and leakage of the payload
was studied over three months at three different temperatures; 4, 25 and 35oC. More specifically,
the effects of ageing (storage time) and storage temperature on the physical stability were
evaluated.
5.4.7.1. Effect of ageing on volume diameter D[4,3]
The prepared niosomes were stored for three months at three different temperatures (4, 25 and
35oC). The three different temperatures were used to represent fridge, room and physiological
ocular temperatures. Over the 90-day storage period, samples were withdrawn and evaluated for
size by measuring D [4,3].
Table 5.14 shows the D [4,3] values recorded for the prepared niosomes over 3 months’ storage
at the three different temperatures. Apart from F-C24, the prepared niosomal formulations
showed slight (< 2%) to significant (40%) increases in the D [4, 3] values compared with the
original D [4, 3] values over the storage period at the three different temperatures.
After 90 days, the average D [4, 3] values measured for F-S60 were 8.48, 9.57 and 9.13 µm at 4,
25 and 35oC respectively. There was no significant increase (P > 0.1) in the D [4,3] at 4oC,
whereas the D [4,3] values significantly increased (P < 0.05) after 3 months at 25 and 35oC.
Similar trends were recorded for F-DCP and F-CH.
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Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
These results showed that the ambient storage temperature is more likely to have a noticeable
effect on the stability of the prepared niosomes against aggregation. At higher temperatures, the
niosomes were relatively unstable compared with the cold storage temperature. The thermal
energy imparted to vesicles increases when the temperature increases. Hence, the rate and force
of collision between vesicles increases. Niosomes tend to aggregate and coalesce to become more
energetically stable and, consequently, niosome size increases. These findings suggest that the
storage temperature of choice is 4oC where the prepared niosomes showed minimal changes in
sizes over three months.
Conversely, the prepared discomes (F-C24) exhibited a notable decrease in the D [3,4] during
storage. This decrease was significant (P< 0.05) when the temperature increased from 4 to 25oC.
For example, the D [4,3] values for F-C24 were 17.2 and 12.54 µm at 4 and 25oC respectively
after 3 months. The respective decreases in the D [4,3] value compared to that for the freshly
prepared discomes were 23% and 44%.
F-C24 is considered to be a giant discoid-shaped niosome, but the results showed that the
prepared discomes were not able to retain their characteristic size for a period of 2 months at
higher storage temperatures. These results were in accordance with the fact that discomes lose
their unique discoidal morphology at higher temperatures (> 35oC) and turn into small spherical
vesicles due to partitioning C24 molecules throughtout of the bilayer membranes (Uchegbu et al.,
1996b; Uchegbu & Vyas, 1998). These results suggest that cold storage is also the storage
temperature of choice for discomes in order to maintain their morphological stability.
228
Chapter 5….Evaluation of Niosomal Formulations for Ocular Delivery
Table 5.14 Effect of ageing on the volume diameter (D [4,3]) for the prepared niosomes over three months at three different
temperatures (Results are expressed as mean values ± SD, n = 3)
Control (0 time)
Formulation
4oC
25oC
35oC
1 month
4oC
D [4,3] µm
25oC
2 month
35oC
4oC
D [4,3] µm
25oC
3 month
35oC
4oC
D [4,3] µm
25oC
35oC
D [4,3] µm
F-S60
8.30 ±
1.14
8.30 ±
1.14
8.30 ±
1.14
8.12 ±
0.42
8.39 ±
0.33
9.05 ±
0.75
8.49 ±
0.21
8.67 ±
0.43
9.75 ±
1.12
8.48 ±
0.21
9.57 ±
1.11
9.13 ±
1.33
F-DCP
7.50 ±
0.59
7.50 ±
0.59
7.50 ±
0.59
7.56 ±
0.66
7.72 ±
0.33
9.39 ±
1.59
7.89 ±
1.12
8.34 ±
0.76
11.18 ±
1.10
8.10 ±
1.34
9.17 ±
1.85
10.71 ±
0.98
F-C24
22.41 ±
1.40
22.41 ±
1.40
22.41 ±
1.40
22.3 ±
4.30
13.00 ±
0.77
16.79 ±
1.60
20.16 ±
2.74
13.60 ±
1.53
13.00 ±
1.22
17.20 ±
4.2
12.54 ±
1.73
10.50 ±
2.5
F-CH
5.37 ±
0.81
5.37 ±
0.81
5.37 ±
0.81
4.99 ±
0.08
5.67 ±
1.33
6.60 ±
0.96
5.28 ±
0.39
6.84 ±
1.51
9.96 ±
2.12
5.09 ±
1.02
8.77 ±
1.65
14.22 ±
3.80
229
Chapter 5….Evaluation of the Prepared Niosomes for Ocular Delivery
5.4.7.2. Effect of ageing on NTX retention
Another physical stability parameter to determine is the drug leakage from the prepared niosomes
over the 90-day storage period. The percentages of NTX retained in niosomes were measured for
three months at the three different temperatures; 4, 25 and 35oC.
A blanket of nitrogen gas was used to replace air in the head space of the glass vials in order to
minimise or slow down autoxidation of NTX. This was carried out to determine the amount of
NTX retained in the niosomes without interference from the drug loss due to oxidative
degradation over the period of study. It has been reported that the degradation rate of an
oxidisable drug can be retarded by replacing air in ampoules with inert nitrogen gas (Johnson &
Gu, 1988).
Firstly, the effects of storage temperatures on percentages of NTX retained in the prepared
niosomes over the 90-day period are presented in Figure 5.22. The results showed that the
prepared niosomes were relatively more stable to NTX leakage at 4oC than at higher
temperatures. After 90 days, the percentages of NTX retained in F-S60 were 85, 64.5 and 53% at
4, 25 and 35oC respectively. Similarly, the respective percentages of NTX retained in F-DCP at
the three temperatures were 87, 73 and 66%. The effect of temperature on NTX leakage from all
the prepared niosomes was significant (P < 0.001). This effect is more pronounced with ageing
(storage time). For example, the drug leakage after a month was not significant (P > 0.05) at 4oC
but a statistically significant (P < 0.05) difference was obtained after three months.
These results could be attributed to defects in the niosomes’ bilayer integrity, as a consequence of
ageing and the effect of temperature on the fluidity of the bilayer membranes leading to
partitioning of NTX molecules throughout the bilayer membranes. At 4oC, the bilayer
membranes are extensively in a gel state, hence NTX leakage is relatively minimal compared
with the state of bilayer membranes at higher temperatures.
Secondly, the effect of bilayer membrane composition on the NTX leakage over three months is
outlined in Figure 5.23. The percentages of NTX retained in F-S60, F-DCP, F-C24 and F-CH
after three months at 4oC were 85%, 87%, 83% and 80.5% respectively. The NTX leakage from
F-CH was significantly higher (P < 0.05) than that calculated for both F-S60 and F-DCP. Further,
the percentages of NTX retained in F-S60, F-DCP, F-C24 and F-CH after three months at 25oC
were 64.5%, 73%, 58 and 52% respectively. In addition to the temperature, incorporation of the
230
Chapter 5….Evaluation of the Prepared Niosomes for Ocular Delivery
membrane additives was found to have an additional effect on niosomes stability. The
percentages of NTX retained for the prepared niosomes were significantly different (P < 0.05)
due to the incorporation of the membrane additives used. F-DCP was found to be the most stable
formulation, whereas F-CH showed the highest NTX leakage. The electrostatic interaction
between the positively charged NTX and negatively charged DCP partly decreases the
partitioning of NTX throughout the bilayer membrane. The packing effect of DCP on the bilayer
membrane can also decrease its permeability to NTX compared with the conventional niosomes
(F-S60). In contrast, the incorporation of CH can alter the nature of the bilayer membrane by
disturbing its packing properties in a manner opposite to cholesterol which is attributed to its
highly hydrophilic nature and the introduction of two hydroxyl groups in the steroidal nucleus.
CH can create vacancies in the bilayer membrane as shown in Figure 4.15. Hence, the
partitioning of NTX throughout F-CH was faster than F-S60. However, the higher NTX leakage
calculated for F-C24 rather than F-S60 is mainly ascribed to the discomes breaking down into
relatively smaller sizes.
It is worth mentioning that over the three-month study period, no degradation peaks were
detected for NTX by the HPLC chromatograms. These could be attributed to two possible
reasons: firstly, replacing the oxygen gas in the heads of the glass vials with inert nitrogen gas
can minimise the oxygen concentration available to initiate autoxidation (Johnson & Gu, 1988);
secondly, the influence of vesicular encapsulation and its stabilising effect against NTX oxidative
degradation, as shown and discussed earlier in Chapter 4, Section 4.4.9.
231
Chapter 5….Evaluation of the Prepared Niosomes for Ocular Delivery
Figure 5.22 Effect of storage temperature on NTX retention in the prepared niosomes over three months
(Results are expressed as mean values ± SD, n = 3)
232
Chapter 5….Evaluation of the Prepared Niosomes for Ocular Delivery
Figure 5.23 Effect of niosome composition on NTX retention over three
months (Results are expressed as mean values ± SD, n = 3)
233
Chapter 5….Evaluation of the Prepared Niosomes for Ocular Delivery
Conclusion
•
The prepared niosomes were found to be non-irritant, when applied onto the CAM
surfaces (Conjunctival model) and bovine corneas.
•
The HET-CAM test, BCOP assay and histological examination were well correlated and
successfully used to study the ocular irritation potential of niosomes and can be
considered as an alternative in vitro toxicity tests for the in vivo rabbit eye test.
•
The in vitro release rate (Q2h and Q6h) and extent (DE-12%) were significantly dependent
on surfactant/lipid composition.
•
The prepared niosomes demonstrated significant controlled release and enhanced
penetration of NTX on the excised bovine corneas, compared with the aqueous NTX
solution.
•
The prepared niosomes were found to be more physically stable at 4oC than ambient
conditions over 90 days.
The four niosomal formulations were demonstrated versatile sizes, morphological features, in
vitro NTX release and transcorneal permeation profiles. These key findings point out the utility
of the prepared niosomes as a safe, effective and new ophthalmic pharmaceutical for the opioid
growth factor antagonist-NTX- for treatment of diabetic complications on the surface of the eye.
234
General Discussion, Conclusion and Future Directions
6. General discussion, conclusion and direction for future
research
6.1.
General discussion and conclusion
Diabetic keratopathy is the term given to diabetic complications in the cornea and affects up to
60% of diabetics (Schultz et al., 1981 ). Diabetic keratopathy or diabetic corneas demonstrate
corneal nerve damage, reduced corneal sensation and delayed wound repair. The current
treatment consists of topical lubricants and antibiotics. However, these treatments, even in
combination, are not effective in most cases (McLaughlin et al., 2010; Schultz et al., 1981 ).
Recently, a group of macromolecules (insulin and nerve growth factor) and small molecules
(naltrexone and nicergoline) have shown effects which accelerate corneal wound healing, but
only NTX has both desirable pharmacological properties and a high safety profile with ocular
tissues. However, there has been no data yet on formulating NTX for ocular use.
Eye drops are the most popular ophthalmic dosage form. This is because of simplicity and patient
acceptability. However, these advantages are offset by low bioavailability, frequent instillations,
and the typical pulse-type entry of the drug, which might promote both patient incompliance and
systemic side effects. Hence, topical ocular delivery is still a challenging endeavour; the two
available strategies to improve both patient compliance and ocular bioavailability are to increase
corneal permeability and prolong precorneal residence time.
Niosomes (non-ionic surfactant vesicles) were proposed in this study as a potential ocular
delivery system for NTX. Niosomes are economic and more chemically stable alternatives to
liposomes; they have the convenience of being eye drops; they can be fabricated from
biodegradable and biocompatible materials; can provide controlled drug-release and penetrationenhancing effects; and they can protect the encapsulated drug molecules from enzymatic
degradation.
For the effective ocular drug delivery, the following properties should be considered during
niosomes fabrication:
•
The size of the niosomes should be > 10 µm to resist drainage by reflex tearing.
235
General Discussion, Conclusion and Future Directions
•
The shape of the prepared niosomes should show some irregularities to fit properly into
the cul-de-sac of the eye and lodge on the eye surface.
•
Ocular niosomes should ideally be thermo-responsive in order to release the drug content
in a controlled yet timely manner, before being flushed by blinking and nasolacrimal
drainage.
This thesis was concerned with designing and characterising the major attributes of niosomes as
ocular delivery systems for NTX. Prior to the formulation stage, the fundamental physical
properties of NTX, such as aqueous solubility, lipid solubility and ionisation constant as well as
developing and validating an HPLC method, were determined (Chapter 2). The results showed
that NTX is freely water-soluble and is a hydrophilic moiety. The HPLC method was rapid and
sensitive. The short run time of approximately 4 min, with isocratic flow, makes it ideal for
routine analysis. LOD and LOQ were 0.55 µg/ml and 1.85µg/ml respectively. Additionally, the
method developed was stability-indicating. There was no interference with the excipient used and
the assay can be applied to quantify NTX in niosomal formulations.
The forced degradation studies showed that autoxidation is the major pathway of the degradation
of NTX. The results of NTX preformulation studies recommended using a surfactant-based
delivery system to enhance corneal penetration through the lipophilic epithelium of the cornea,
and to encapsulate NTX in bilayer vesicles in an attempt to protect NTX against oxidative
stresses.
In Chapter 3, two different classes of non-ionic surfactants (Span® and Brij®) were used for
forming niosomes. The surfactants used for niosomes preparation were Span 20, Span 40, Span
60, Span 80, Brij 52 and Brij 72. These surfactants are widely used in food, cosmetic and
pharmaceutical industries. They are also regarded as non-toxic and non-irritant materials
(Lawrence, 2003; Yu, 2003). The surfactants used are composed of different hydrocarbon chain
lengths, degree of saturation and various HLB values. Cholesterol was used as a membrane
stabiliser at different concentrations. The effects of both surfactants and cholesterol on the
niosomes’ physical properties, such as EE %, D [4,3] and gel/liquid phase transition temperature,
were studied. Surfactant/lipid mixtures composed of different surfactant: cholesterol molar ratios
(1:0, 9:1, 8:2, 7:3, 6:4 and 1:1 mol/mol) were studied. The surfactant:cholesterol ratio 7:3 was
found to show the highest NTX EE%. Generally, Spans-based niosomes were found to be more
236
General Discussion, Conclusion and Future Directions
physically stable than Brij-based niosomes. The Brij-based niosomes showed phase separation
and creaming upon storage, irrespective of the cholesterol level.
Span 60: cholesterol 7:3 mol/mol niosomes showed a residual phase transition, with the onset of
the gel/liquid transition at the ocular temperature (approximately 35oC) indicating its thermoresponsiveness. In addition, The Span 60: cholesterol (7:3) niosomes exhibited the highest NTX
EE% compared with other niosomes but the amount of NTX entrapped by the niosomes was still
sub-therapeutic (< 0.4 mg/ml). In fact, these findings support the hypothesis that hydrophilic
drugs are less preferentially entrapped by niosomes than lipophilic drugs (Essa, 2010). Hence,
other attempts were sought elsewhere to improve EE% of NTX. The effect of membrane
additives and various methods for niosome preparation were studied.
In Chapter 4, both charged lipids (dicetyl phosphate [DCP] and stearyl amine [STA]) and watersoluble surfactants (poly-24-oxyethylene cholesteryl ether [C24] and sodium cholate [CH]) were
investigated as bilayer membrane additives. The selected membrane additives were studied for
their effects on the physical properties of niosomes. Different concentrations (0-10% mol/mol) of
the selected additives were used. Niosomal formulations containing 2% to 5% mol/mol of C24,
DCP and CH encapsulated a considerable amount of NTX. For example, F-DCP showed a 3-fold
increase in NTX EE%, compared with the conventional niosomes (F-S60), using the TFH
method. Additionally, the REV method was superior to other methods (TFH, MLV-FAT and
DRV methods). REV niosomes showed roughly a 1.5-fold increase in EE% compared with TFH
niosomes. The REV method has more lipid hydration efficiency than the TFH method. Also, the
REV method generates niosomes with larger aqueous core to accommodate a considerable
amount of water-soluble solutes compared with the TFH method.
Incorporation of CH and DCP produced spherical niosomes similar to the conventional (F-S60)
niosomes in shape, as shown by Cryo-SEM and CLSM. CH containing niosomes (F-CH)
achieved smaller D [4,3] (5 µm) than that (8 µm) for F-S60, whereas introduction of C24 into
the bilayer membrane produced niosomes significantly larger sizes (22 µm) with an oval-to-disc
shape, indicating the formation of atypical giant niosomes, better known as discomes.
It is worth mentioning that ocular drug bioavailability can be affected by the size of instilled
ophthalmic suspensions. It is likely that as the particle size increases, eye discomfort could be
created. As a consequence, tear production and blinking rate are likely to increase, leading to a
237
General Discussion, Conclusion and Future Directions
decrease in residence time. It has been reported that an ophthalmic suspension made up of solid
particles should have a diameter of 10 µm or less. This is to ensure uniform ocular absorption and
prevent eye irritation (Lang et al., 2002). However, the upper limit of particles mentioned in the
literature is based on a study on ophthalmic suspensions of water-insoluble anti-inflammatory
steroids (Schoenwald & Stewart, 1980).
According to this study, dexamethasone suspensions of different particle sizes were prepared.
Suspensions A, B and C were formulated with particle sizes of 5.57, 11.5 and 22 µm
respectively. The area under aqueous humour drug concentration versus time curve (AUC) was
determined for each suspension formulation. The results showed that as the particles decrease in
size, the time taken to reach peak concentration (Cmax) decreased and AUC increased. The ocular
bioavailability obtained from a dexamethasone suspension, measured in terms of the AUC of
suspensions A-C, can be expressed in the following order: A > B > C. The AUC was markedly
dependent on the particle size of the suspension. These results were attributed to the fact that as
the particles increase in size, the in vivo dissolution rate decreases, such that the particles are
removed from the conjunctival sac before dissolution is completed.
Therefore, both the rate and extent of penetration into aqueous humour decreased. Based on
observation of the animals during the study, there was no reason to believe that dexamethasone
induced abnormal tearing or blinking at an average size of 5.75-22 µm. There is no firm
conclusion to support the need for particle size being less than 10 µm to minimise irritation in the
eye (Schoenwald & Stewart, 1980). Nevertheless, shape, concentration and density are additional
factors that make it difficult to select a specific particle size above which irritation or discomfort
might result. The authors came to the conclusion as particle sizes increase, the surface area
available for drug dissolution decreases. Hence, the drug dissolution rate decreases providing
dissolution rate-limited absorption.
For niosomes ophthalmic dispersion, the entrapped drug molecules are in the soluble molecular
forms and they are either entrapped in the bilayer domains or encapsulated in the aqueous
domains, depending on the drug physical properties. Partitioning of the entrapped/encapsulated
drug molecules throughout the bilayer membranes at the eye temperature could be the ratecontrolling step for ocular drug absorption from niosomes. Controlled and constant drug
partitioning is likely to be important for consistent ocular bioavailability. To design niosomes
capable of responding to ocular temperature and release the entrapped drug molecules at
238
General Discussion, Conclusion and Future Directions
controlled and constant rate (thermo-responsiveness) is more favourable than rigid niosomes
(without gel/liquid transition temperature close to ocular temperature). The vesicles could spread
promptly and in an even layer on the precorneal surface. Hence, niosomes should ideally be
thermo-responsiveness for optimum ocular bioavailability. Other factors could affect the ocular
bioavailability of the drug molecules entrapped by niosomes are; firstly, the ability of niosomes
to carry the therapeutic molecules (EE %); secondly, the shape of niosomes could be an
additional factor. Oval shaped niosomes (discomes) are thought to be of value for ocular delivery,
due to better fitting on the conjunctival sac (retained by inner canthus) than spherical niosmes
(Mainardes et al., 2005; Uchegbu & Vyas, 1998). Niosomes > 10 µm are suitable for ophthalmic
drug delivery (Sahin, 2006). For instance, large niosomes (discomes) sized between 12 and 60
µm were found to entrap a relatively larger amount of timolol maleate (Vyas et al., 1998). The
prepared discomes produced a controlled drug release and high bioavailability compared with the
aqueous drug solution. The systemic drug absorption measured from discomes was at a negligible
level (Vyas et al., 1998).
Returning to Chapter 4, DSC thermograms revealed that the additives used completely abolished
the residual gel/liquid transition of F-S60 niosomes indicating that the tested additives were
accommodated by the bilayer membranes of the prepared niosomes. Niosomes achieved marked
improvement in spreading ability compared with the aqueous vehicle. Viscosity measurements
also demonstrated a 1.7- to 8.2-fold increase in the viscosity compared with that for the aqueous
solution. These results demonstrate the ability of the tested niosomes to spread and wet easily the
lipophilic corneal surface as well as prolong precorneal residence time. More importantly, the
prepared niosomes were able to protect chemically the encapsulated NTX from photolytic
degradation compared with dark storage.
In Chapter 5, the HET-CAM test, bovine eye assay and histological examination of the bovine
corneas were utilised for evaluating the ocular irritation and toxicity of NTX, the raw materials
used to fabricate niosomes and the prepared niosomes. Further, in vitro release studies, release
kinetics and the transcorneal permeation of NTX from the prepared niosomes were studied.
Finally, investigations of the physical stability of the prepared niosomes were performed at three
different temperatures 4, 25 and 35oC to evaluate their ability to maintain their size and retain
their NTX payload.
239
General Discussion, Conclusion and Future Directions
The results obtained from the HET-CAM (conjunctival) model were correlated well with those
obtained from the corneal model. The HET-CAM and bovine eye tests were thought to be simple,
less expensive, rapid and discriminating in vitro ocular toxicity models for niosomes as a
potential ocular drug delivery. Histological examination of the corneas treated with the prepared
niosomes at different time points 30 s, 1 h, 3 h and 8 h showed slight stromal oedema and no
harmful signs. Such effects are reversible upon discontinuation of treatment or can be easily
reversed by ocular administration of a simple hyperosmotic salt solution (Dawson & Edelhauser,
2010).
In vitro drug release parameters (Q2h, Q6h and DE-12h) calculated for the tested niosomes
indicate significantly prolonged (P < 0.01) NTX release rate. The calculated Q6h values for the
prepared niosomes were approximately 1.6 to 6 times lower than those for the NTX solution. The
four different niosomes generated four well-discriminated controlled-release profiles for NTX
and the general operating release mechanism was zero-order release.
Parallel to the in vitro release data, ex vivo transcorneal permeation profiles showed that
niosomes potentially controlled NTX permeation though the excised bovine corneas. The steady
flux rate values calculated for the prepared niosomes were 1.6 to 2.4 times slower than those for
the NTX solution. This controlled flux rate is likely to be desirable for prolonging precorneal
residence time and reducing the typical pulse type entry of eye drop solutions. Furthermore,
controlling NTX release and permeation is specifically advantageous for taste-masking NTX,
since the NTX solution is extremely bitter taste.
The lag time (tL) values calculated for the prepared niosomes were shorter than those for the
aqueous vehicle, suggesting that the prepared niosomes additionally have permeation-enhancing
effects. A linear correlation between tL values and contact angles (θ) was discovered.
In addition, Chapter 5 focused on studying the physical stability of the prepared niosomes stored
at three different temperatures; 4, 25 and 35oC for 90 days. The effect of storage time (ageing) on
size and the amount of NTX retained in niosomes was evaluated. Ageing provoked various
changes in niosomes’ D [4,3] and in the percentages of NTX retained. These changes were
dependent on the storage temperature and the composition of niosomes. Minimum changes in D
[4,3] and the highest percentage of NTX retained were recorded at 4oC.
240
General Discussion, Conclusion and Future Directions
In conclusion, this thesis has presented NTX as a new and potential treatment for diabetic
keratopathy. It has also studied the physicochemical properties of NTX necessary for developing
a new liquid dosage form. The main attributes of niosomes as an ocular delivery system is to
prolong and enhance corneal uptake of NTX. These delivery devices can provide the
pharmaceutical formulator with insights into using a surfactant-based delivery system as a
potential ophthalmic pharmaceutical dosage form for NTX.
6.2. Limitations and future direction
This project is an attempt to develop ocular niosomes for an ambitious therapeutic entity (NTX).
Four different niosomal formulations have been designed and tailored to fit the anatomical and
physiological barrier of the eye surface in terms of suitable size, morphology and thermoresponsiveness.
Although the formulation approach was adopted in this thesis in an attempt to protect NTX
molecules from oxidative stresses, it has apparently seemed to be insufficient. Other strategies, to
overcome NTX oxidative degradation, should be considered for any future work, most notably,
the use of stabilisers.
Stabilisers are pharmacologically inactive ingredients added to the formulation to decrease the
rate of decomposition of the active ingredient (Lang et al., 2002). A typical example for the
stabilisers is antioxidants. Antioxidants can reduce or prolong the autoxidation process by
different mechanisms.
•
Chain terminators are molecules with weak bonds to hydrogen atoms, such as thiol or
phenol groups, which attack and scavenge free radicals and hence do not propagate
oxidation chains reactions.
•
Sacrificial reductants (e.g. ascorbic acid) are compounds that can be oxidised more
readily than the drug. They effectively scavenge oxygen while they are themselves
consumed (Waterman et al., 2002).
Other stabilisers to protect oxidisable drugs are chelating agents, such as editic acid and citrate,
which can inhibit oxidative degradation by chelating any traces of heavy metals (Waterman et al.,
2002). The pH also has an important effect on NTX chemical stability in aqueous solution, as it
has been demonstrated that oxidation reaction is pH-dependent. The tear film pH 7.4 was selected
as vehicle’s pH, although it is not the optimum pH to offer maximum chemical stability to NTX.
This is because:
241
General Discussion, Conclusion and Future Directions
•
The physiological pH can offer greater eye comfort and better drug permeability.
•
Being a basic drug, NTX molecules exist predominantly in their ionised (the least
absorbable) form below physiological pH.
•
Drug loss from the cul-de-sac of the eye on cheek and via nasolacrimal drainage can be
exacerbated following instillation of drug solutions buffered below physiological pH due
to increased drainage and induced lacrimation (Ahmed & Patton, 1984; Lang et al., 2002).
•
Prolonged tear film pH depression can reduce corneal permeability (Francoeur et al.,
1983; Rathore & Majumdar, 2006).
Therefore, the propective research will study the influence of various stabilisers on the chemical
stability of NTX.
With respect to the physical stability of the prepared niosomes, there are two approaches to
improve the physical stability of lipid vesicles at ambient conditions. Dispersing liposomes in a
viscous gel has been used either to reduce the rapid leakage of the encapsulated drug from
liposomes (Meisnera & Mezeib, 1995) or to minimise the burst release effect observed with
liposomes (Mehanna et al., 2009, 2010). This approach may be extrapolated to improve the
physical stability of niosomes. However, the drug release from such a system is likely to be
complex, as the drug molecules have to release from the bilayer membranes and diffuse through
the viscous gel within the short ocular residence time. Hence, the improvement in the physical
stability of the niosomes can be offset by reducing the ocular bioavailability of the administered
drug in such vehicles. Additionally, a topical application of a viscolised gel is less convenient
than eye drops in terms of ocular administration and the adjustment of the dose.
In addition to the previous approach, converting the final liposomal/niosomal liquid dispersion to
a powder form by lyophilisation (freeze-drying) or spray-drying not only enhances the physical
stability of the vesicles (Ingvarsson et al., 2011), but can also dramatically reduce the oxidative
instability of oxidisable drug molecules, by minimising the formation of hydroxyl free radicals
(Uchegbu & Vyas, 1998; Waterman et al., 2002).
These advantages can be offset by high production costs; long production time; and ending up
with more complicated formulations containing cryoprotectants. The ready-to-use eye drops are
the easiest and most convenient for producing and administering a dosage form. Accordingly, the
242
General Discussion, Conclusion and Future Directions
direction of our future research on niosomes will be concerned with studying different strategies
for improving the physical stability of niosomes.
An ophthalmic dosage form must be sterile; however, the niosomes used in this thesis were
prepared under non-sterile conditions because the study is still in the early stages of formulation
development. Sterilisation of the final lipid-based (niosomes/liposomes) products is a challenging
endeavour. Heat sterilisation is not recommended for lipid-based delivery systems (Zuidam et al.,
1993). Dry heat and steam sterilisation are unsuitable and destructive for lipid-based formulations
with a gel/liquid transition temperature which is relatively lower than temperature involved in
heat sterilisation, since this may cause extensive drug leakage from the bilayer vesicles.
Membrane filtration is also unsuitable for particulate delivery systems larger than the pore size
(0.22 µm) of the bacterial filters. On the other hand, preparation under aseptic conditions, gas
(ethylene oxide) and gamma irradiation could be useful, since minimal heat is generated during
the sterilisation process. Liposomes and niosomes are prepared in a sterile form by passing all
organic lipid, buffer and drug solutions through bacterial filters; and then the final product is
prepared under aseptic conditions (Abdelbary & El-gendy, 2008; Hathout et al., 2007). Gamma
irradiation has high penetration power, so it can be applied to a packaged product and is
applicable to heat-sensitive drugs. Many marketed pharmaceuticals are gamma radiationsterilised, such as ophthalmic ointments, drops and injectables (Waterman et al., 2002). These
sterilisation methods could be of a potential value for producing sterile niosomes, and hence the
effect of gamma irradiation on the physical stability of niosomes could be a potential research
line for future work.
Future work will also look at studying precorneal retention time in vivo (in rabbit models) by
evaluation of the nasolacrimal drainage of encapsulated radioisotopes in niosomes and discomes,
and compared with an aqueous solution of the radioactive material using a gamma camera. This
is in order to evaluate the ability of niosomes and discomes to prolong the precorneal residence
time, compared with aqueous solution.
In conclusion, the future of NTX in diabetic keratopathy has been documented (Abdelkader et al.,
2011; McLaughlin et al., 2010). NTX competitively blocks the interaction between OGF and
OGFr (ζ-opioid receptors) on the basal corneal epithelial cells. This leads to a marked increase in
DNA synthesis, cell division and cell migration as well as regeneration of corneal epithelium and
243
General Discussion, Conclusion and Future Directions
improvement of diabetic keratopathy (McLaughlin et al., 2010; Zagon et al., 1997; Zagon et al.,
2000). Other diabetic complications are often experienced by diabetic patients are
keratoconjunctivitis sicca or commonly known as dry eye symptoms and delayed corneal
sensation. Recently, NTX has demonstrated an ability to reverse dry eye symptoms and improve
delayed diabetic corneal sensation in diabetic rat models (Zagon et al., 2009). However, the exact
mechanism for these effects is still unknown (McLaughlin et al., 2010).
It is worth mentioning that there is an ongoing collaboration between the Department of
Ophthalmology and our research group in the School of Pharmacy to test the hypothesis that
topical ocular application of insulin or naltrexone promotes corneal nerve regeneration. Diabetic
rats will be used to model neurotrophic keratopathy and non-invasive in vivo confocal
microscopy will be utilised to image and quantitatively analyse the corneal nerves non-invasively
over time.
244
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Appendix-Publications
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