Download interactions of mucins with biopolymers and drug delivery

INTERACTIONS OF MUCINS
WITH BIOPOLYMERS AND
DRUG DELIVERY PARTICLES
Malmö University
Health and Society Doctoral Dissertations 2008:2
© Olof Svensson 2008
ISBN 978-91-7104-212-5
ISSN 1653-5383
Holmbergs, Malmö 2008
OLOF SVENSSON
INTERACTIONS OF MUCINS
WITH BIOPOLYMERS AND
DRUG DELIVERY PARTICLES
Malmö University, 2008
The Faculty of Health and Society
To my family
CONTENTS
ABSTRACT .................................................................................
LIST OF PAPERS ..........................................................................
INTRODUCTION..........................................................................
Background and aim .............................................................................
The mucous gel and mucins ....................................................................
Polyelectrolyte multilayers .......................................................................
MATERIALS AND METHODS . ........................................................
Proteins and polymers.............................................................................
Surfaces ...............................................................................................
Ellipsometry ..........................................................................................
Particle electrophoresis............................................................................
Atomic force microscopy.........................................................................
Electrochemistry ....................................................................................
RESULTS AND DISCUSSION ..........................................................
Layer-by-layer film formation with mucin....................................................
Interactions between drug delivery particles and mucin...............................
SUMMARY AND CONCLUDING REMARKS ......................................
POPULÄRVETENSKAPLIG SAMMANFATTNING .................................
ACKNOWLEDGEMENT.................................................................
REFERENCES...............................................................................
APPENDIX . ................................................................................
10
12
14
14
16
22
27
27
30
31
40
41
42
44
44
58
65
67
70
71
81
ABSTRACT
The main components in the mucous gels apart from water are mucins, which
are proteins with high molecular weights and an abundance of negatively
charged oligosaccharide side chains. The aim of the investigations was to characterize interactions between mucins and other proteins that are present in the
mucous gel, and also between mucins and components used in pharmaceutical
formulations. More specifically, the main objectives were (I) to investigate the
possibility to assemble multilayer films with mucins and oppositely charged
polymers or proteins on solid substrates; (II) to evaluate mucoadhesive properties of drug delivery particles by examination of their interactions with mucins.
The construction of multilayer films was performed on silica and hydrophobized silica surfaces by alternate adsorption, and the adsorbed amount and
thickness of the films were measured in situ by time resolved ellipsometry. It
was demonstrated that films could be assembled using mucins in combination
with both chitosan and lactoperoxidase. The build-up was characterized by adsorption and redissolution processes, and the extent of redissolution could be
explained by taking the charge densities and concentrations of the components
into account. It was also demonstrated that the nature of the substrate can be
crucial for the possibilities to assemble multilayer films, and from the results it
may be concluded that a high amount of mucin in the first step is important for
successful layer-by-layer assembly. Furthermore, it was demonstrated that lactoperoxidase is catalytically active when adsorbed to mucin layers, and it may
thereby exert its antimicrobial action.
10
The evaluation of mucoadhesive properties of drug delivery particles was performed with lipid nanoparticles stabilized by a poly(ethylene oxide) based
polymer and with particles modified by chitosan. Both types of model particles
(unmodified and chitosan modified) were investigated by measuring their adsorption to mucin-coated silica surfaces by ellipsometry. It was shown that the
binding of unmodified particles to mucin-coated silica surfaces was weak and
pH-dependent. Based on the pH and electrolyte dependence of the adsorption,
it was proposed that the interaction is mediated by hydrogen bonding between
protonated carboxyl groups in the mucin molecule and oxygen atoms in
poly(ethylene oxide). Chitosan modified particles, on the other hand, showed a
substantial and strong binding to mucin-coated surfaces, which can probably be
attributed to interactions between amino groups in chitosan and negatively
charged groups in the mucin layer. The findings from the present investigations
are in agreement with previous reports on the interaction of mucins with
poly(ethylene oxide) and chitosan. It can therefore be concluded that the methodology applied is useful for evaluating mucoadhesive properties of nanoparticles.
11
LIST OF PAPERS
I. Layer-by-layer assembly of mucin and chitosan - Influence of surface
properties, concentration and type of mucin. Olof Svensson, Liselott
Lindh, Marité Cárdenas and Thomas Arnebrant, Journal of Colloid and
Interface Science 2006, 299(2), 608-16.
II. The salivary mucin MUC5B and lactoperoxidase can be used for layerby-layer film formation. Liselott Lindh, Ida Svendsen, Olof Svensson,
Marité Cárdenas and Thomas Arnebrant, Journal of Colloid and Interface Science 2007, 310(1), 74-82.
III. Activity of lactoperoxidase when adsorbed on protein layers. Karolina
Haberska, Olof Svensson, Sergey Shleev, Liselott Lindh, Thomas Arnebrant and Tautgirdas Ruzgas, Manuscript
IV. Interactions between drug delivery particles and mucin in solution and at
interfaces. Olof Svensson, Krister Thuresson and Thomas Arnebrant, Ac-
cepted for publication in Langmuir
V. Interactions between chitosan-modified particles and mucin-coated surfaces. Olof Svensson, Krister Thuresson and Thomas Arnebrant, Manu-
script
Reprint permission of papers I and II has been granted by Elsevier Inc. and a
blanket permission is granted by the American Chemical Society for reprinting
of paper IV.
12
Contributions to the publications
I performed most of the planning and essentially all experimental work in papers I, IV and V. In addition I did the writing of the manuscripts with support
from the co-authors. My contribution to paper II was to perform data analysis,
take part in discussions of the results and write parts of the manuscript. I also
made minor contributions to the experimental work. In paper III, I was contributing to the planning of the experimental work as well as performing most
of the ellipsometric measurements.
13
INTRODUCTION
Background and aim
The mucous gel layer is a highly hydrated protein gel that covers the mucosal
surfaces of our body and its general function is to protect the underlying mucosal tissues from dehydration, mechanical stress and bacterial infections. In humans the average thickness of the mucous gel is estimated to be a few hundred
micrometers, and the main component apart from water is a group of glycoproteins referred to as mucins. This class of high molecular weight glycoproteins is
important in many aspects and is for example considered to form the backbone
of the gel.
As all nutrients and most pharmaceuticals on the market enter our body
through the mucous gel, the composition and structure of this gel is of obvious
scientific interest. From this perspective my research at Malmö University has
been focused on the interactions of mucins with other types of proteins that are
naturally present in the mucous gel as well as molecules and assemblies of
molecules used in pharmaceutical formulations. The general aim has been to
gain a deeper understanding of how molecules present in the native mucous gel
can combine to form a three-dimensional network and how mucins interact
with pharmaceutical constituents.
The main objectives have been:
I. To investigate the possibility to form multilayer films with mucins and oppositely charged polymers or proteins on solid substrates. The possibility to measure enzymatic activity of proteins in these structures was also addressed. This
work was done with the ambition to create artificial gels that could act as mu-
14
cous models to study the interactions with for example pharmaceutical formulations. In addition the assembled films could have interesting lubricating and
antiadhesive properties that would be of interest for coatings of contact lenses
and dental implants.
II. To study the interactions between drug delivery particles and mucin in order
to evaluate their mucoadhesive properties and also to understand interactions
between mucin and pharmaceutical constituents. Such knowledge is of interest
in the area of mucosal drug delivery and the development of novel mucosal
drug delivery systems.
The Introduction of the thesis consists of a description of the general properties
of the mucous gel with emphasis on mucins and their interactions with other
mucus components and adsorption to solid surfaces. Also the layer-by-layer assembly of oppositely charged polymers or proteins is described focusing on the
assembly process and the use of proteins in these structures. In the Materials
and Methods part, the experimental techniques are described and information
about the key proteins and polymers is provided. The emphasis of this section is
on ellipsometry, which was the main experimental technique used, and mucins,
which were the key proteins in my investigations.
The Result and Discussion section is divided into two parts, where the first part
presents layer-by-layer assembly of mucin and oppositely charged biopolymers
(papers I, II and III). The second part is devoted to the interactions between
particles aimed for drug delivery and mucin-coated surfaces (papers IV and V).
I have presented what I consider to be the most important and interesting observations and the results obtained in the individual papers are discussed in relation to each other.
15
The mucous gel and mucins
The mucous gel
The mucous gel layer (mucus) is a highly hydrated protein gel that covers the
mucosal surfaces in for example the gastrointestinal, pulmonary, oral, nasal
and genital tracts. Its function and composition differs at different locations of
our body, but a general function of the mucus is to protect mucosal tissues
from dehydration, mechanical stress, harmful microorganisms and toxic substances.
Mucus proteins originate from mucous producing goblet cells that are localized
in the epithelial cell layer or in mucous producing glands. The secreted mucous
forms a viscoelastic gel on the epithelial surfaces, and the thickness of the gel
depends on its location. In for example the gastrointestinal tract of rats, the
thickness has been reported to vary between 100 µm in the jejunum to 800 µm
1
in the colon. The water content of mucus is high and reported values suggest
2, 3
that the water content of native mucus is approximately 90%.
The compositions of various mucous gels have been investigated in several studies, with the conclusion that a group of glycoproteins identified as mucins is the
main component apart from water in terms of mass of the gel, with an ap3
proximate concentration of 50 mg/mL. In addition to mucins, other proteins,
4, 5
lipids and nucleic acids have been identified in the mucous gel. Many proteins
that are specifically secreted in the body have an active role in the protection
against bacterial infections. For example IgA, lysozyme, lactoferrin and lactoperoxidase, which all have protective functions, have been identified in mucous
4, 6
secretions.
Mucins
Mucins are structurally similar and have many properties in common, although
a high degree of diversity exists within this group. The molecular weight is generally high, ranging between 0.2 and 10 million Dalton, and all mucins contain
one or more domains which are highly glycosylated. The glycosylated domains
are enriched in serine and threonine residues which serve as anchoring points
16
for oligosaccharide side chains. These O-linked oligosaccharide side chains are
complex both in terms of composition and length, and apart from differences in
glycosylation between different mucins, different “glycoforms” have been iden7
tified. The carbohydrate weight fraction is substantial and values between 68
8, 9
and 81% by weight have been reported. Apart from glycosylated domains,
mucins also contain “naked domains” with little or no glycosylation and these
domains are typically found in the N-terminal and C-terminal part of the protein and are enriched in cysteine residues. The cysteine residues can form intermolecular bonds, and in the native state mucins are often found as oligomers
composed of several end-to-end linked mucin subunits. Figure 1 presents a
10
model of mucin according to Carlstedt and co-workers. This particular mucin
has on average four subunits per mucin molecule, and each subunit contains on
average four to five glycosylated domains.
S-S
S-S
S-S
Figure 1. A proposed architecture of cervical mucin adopted from Carlstedt and coworkers.10 Black thick lines represent glycosylated domains, thin lines represent nonglycosylated domain and sulphate bonds between subunits are shown as S-S.
17
A common feature of mucins, apart from a high molecular weight and a high
carbohydrate content, is the abundance of negatively charged groups. The nega11
tive charges arise mainly from sialic acid residues (pKa ≈ 2.6 ) and in some
12
cases from sulphated sugars (pKa ≈ 1 ). These acidic groups account for the low
13-15
isoelectric point of mucins that is estimated to be between 2 and 3.
The glycosylated regions of mucins interact favourably with water and force the
molecule to an extended random coil conformation, and the high molecular
weight enables individual mucin molecules to overlap and entangle at relatively
low concentrations. These characteristics are ideal with respect to the formation
of hydrogels and investigations have shown that reconstructed mucous gels
from mucins have similar rheological properties as native mucous gels at
16
physiological concentrations. Although the ability of mucins to form the structural backbone of the mucous gel is one of its most important functions, other
physiological functions have been reported and a more comprehensive review
3, 17
on mucins and their biological functions can be found elsewhere.
Mucin association in solution
Entanglement is a general feature of polymer solutions and depends on both the
molecular weight of the polymer and the concentration of the polymer solution.
The concentration at which the individual polymer coils starts to overlap and
entangle is referred to as overlap concentration (C*) and above this concentration the viscosity increases rapidly with increasing concentration. This general
type of polymer interaction is also the most important type of interaction that
accounts for the viscoelastic properties of concentrated mucin solutions. As
mucins are high molecular weight molecules, the overlap concentration is low
18
(2-4 mg/mL ) and thus the mucin molecules in a native mucous gel (approx. 50
3
mg/mL ) are expected to be highly entangled. Since entanglement is dependent
on the molecular size of the molecules, reduction of molecular weight should
have a strong influence on the viscoelastic properties of mucin solutions. This
has also been demonstrated by showing that a reduction of disulphide links be16
tween mucin subunits causes the gel to collapse and form a viscous solution.
18
Hydrophobic interactions between the non glycosylated parts of mucin molecules may also be important for the gel properties of mucin solutions. For example, it has been proposed that pig gastric mucin self-assembles through hy19, 20
drophobic interactions at low pH.
Mucin interactions with other mucous gel components
Raynal and co-workers have investigated the gel forming properties of mucin
purified from human saliva (MUC5B) and no evidence was found of any spe21
cific interactions besides entanglement in aqueous solution. Furthermore, since
the investigation showed that mucin solutions did not replicate the gel forming
properties of saliva, a subsequent investigation was performed to examine the
22
influence of calcium. It was evident that calcium had the ability to crosslink
mucin into larger aggregates and it was also suggested that the binding was
mediated by a protein site.
Trefoil factors is a group of peptides that are co-secreted with mucins in most
mucus producing cells in the gastrointestinal tract and their importance for the
23
rheological properties of pig gastric mucin solutions have been investigated. It
was found that the addition of trefoil peptides could result in a tenfold increase
in viscosity of mucin solution. This result demonstrates that these peptides interact with mucin and it is likely that they are important for the rheological
properties of the native mucous gel.
The association between the separated gel phase of human saliva and IgA, lactoferrin and lysozyme has been reported, indicating that these proteins bind to
24
salivary mucins. The complex formation between human salivary mucin and
other salivary proteins has also been investigated, with the conclusion that amylase, proline-rich proteins, statherins and histatins could form complexes with
25
mucins.
19
Mucin adsorption to solid surfaces
Proteins usually readily adsorb to solid surfaces from aqueous solutions to form
a protein film, which is usually mixed with water. Many types of interactions
can mediate the adsorption, and among these hydrophobic and electrostatic interactions have been identified as central factors determining protein adsorp26
tion. In addition, structural rearrangements of proteins as well as hydrogen
27
bonding are suggested to influence the adsorption. The predictions made for
protein adsorption can be applied for mucins as well, but some features of
mucins require some special attention. First, the mucin molecule has an amphiphilic character with hydrophilic glycosylated regions as well as regions with no
or little glycosylation. It could therefore be advisable to consider the adsorption
behaviour of both regions separately. Second, the molecular weights of mucins
are generally high, which in turn requires adsorption studies to be performed
over long time periods. Low diffusion coefficients also put demands on purity
of mucin preparations since low molecular weight impurities may preferentially
be adsorbed, at least in the initial phase.
Numerous studies have been devoted to the adsorption of mucins to solid surfaces, and it is evident that mucins adsorb to most types of surfaces independently of mucin preparation or solution properties. The characteristics of the adsorbed layer has been investigated by surface force measurements for different
types of mucins adsorbed to different types of surfaces with the conclusion that
13, 28, 29
a long range steric repulsion exists between mucin-coated surfaces.
Steric
forces could be detected at a distance between surfaces of 100 nm or more, indicating that mucin segments protrude far into the ambient solution. The morphology of adsorbed mucin layers has been examined by transmission electron
microscopy and atomic force microscopy with the general conclusion that the
adsorbed mucin can be found as fibres with average contour lengths of a few
5, 30
hundred nanometers.
Thus the morphology of adsorbed mucin seems to reflect their extended conformation in solution. As indicated from surface force
measurements, segments of the mucin molecule extend from the surface, and by
considering the amphiphilic character of the mucin molecules it is likely that the
non-glycosylated parts of the mucin molecule interact with the surface while
glycosylated regions are oriented towards the ambient solution. Thus mucin adsorbs in a fashion similar to synthetic poly(ethylene oxide) based block co
polymers (PEO-PPO-PEO) and provides steric repulsion with promising anti-
20
adhesive properties. Their anti-adhesive properties has, for example, been util31-33
ized to suppress cell adhesion to polymeric surfaces.
The influence of surface properties has been investigated with respect to the
28, 31, 34, 35
amount of mucin adsorbed.
The conclusion is that the adsorbed amount
is very dependent on the substrate although no correlation could be found between the hydrophobicity of the surface, as determined by contact angle meas31
urements, and the adsorbed amount. However, firm attachment of adsorbed
mucin on hydrophobic surfaces is indicated as only a very small fraction was
29, 36
removed when rinsing with a mucin free solution.
The electrolyte concentration and pH of the ambient solution have also been investigated with respect to
adsorption. For electrolyte concentration below 0.1 M a general trend is that
14, 15, 35
the amount increases with increasing electrolyte concentration.
Also, effects of solution pH has been examined, showing a trend of increasing adsorbed
14, 15
amount with decreasing pH at low ionic strength.
The dependence of electrolyte concentration and pH can be understood by considering electrostatic interactions between the surface and the mucin molecules as well as the electrostatic interactions between mucin molecules. Accordingly, an increase in electrolyte concentration is screening the electrostatic repulsion between the surface
and the mucin molecules as well as the electrostatic repulsion between mucin
molecules. Similarly, by lowering the pH, the net charge of the mucin molecule
decreases.
A final remark is that mucins constitute a diverse group of molecules with differences in molecular weight, charge density and structure which has also been
14, 35, 37
reflected in the adsorbed amount.
Furthermore, it should be noted that the
preparation procedure may affect the quality of the mucin sample as well as the
amount of impurities, which in turn may affect the adsorption behaviour.
21
Polyelectrolyte multilayers
General
The alternate adsorption of oppositely charged polyelectrolytes was demonstrated by Decher and co-workers who showed that polyelectrolyte multilayers
(PEM) with arbitrary thickness can be obtained by simply controlling the num38
ber of adsorption cycles. It was proposed that the surface charge is reversed
during the adsorption and this has later been confirmed by zeta potential meas39
urements. Figure 2 illustrates the basic principle of how to construct a bilayer
on a solid substrate. The surface can either be consecutively dipped in the solutions or the ambient solution can be exchanged while keeping the surface fixed.
A variation of these assembly procedures is alternate deposition by spraying,
40
which enables a more rapid build-up.
Polymer 1
Rinse
Polymer 2
Rinse
1 adsorption cycle / 2 layers
Figure 2. Illustration of how to construct polyelectrolyte multilayers.
As electrostatic interactions are important for the interaction electrolyte concentration, polyelectrolyte charge density and solution pH should influence the
41-43
build-up and this has also been demonstrated.
Also for overcompensation to
occur the combined effect of polyelectrolyte concentration and adsorption time
41
has to be considered in the experimental set-up. Few studies have so far re39, 44
ported on how substrate properties affect the subsequent build-up.
In some
investigations the solid substrate is used without modification and in other
cases the substrate is modified to provide a high surface charge density to facilitate build-up. For example, chemical modification with amino groups or preadsorption of poly(ethylene imine) is frequently reported.
22
Build-up mechanisms
In initial investigations it was reported that the film thickness increases linearly
38, 41, 45
with the number of adsorption cycles for highly charged polymers.
However, it was later discovered that many systems that include polypeptides and
polysaccharides show an exponential growth with the number of adsorption
46-49
cycles.
To explain this exponential increase, a growth mechanism was presented in which the polyelectrolytes are able to diffuse into and out from the
50
film during build-up. The adsorbing polymer diffuses into the polymer film,
which acts as a “reservoir” for the polymer. When the oppositely charged
polyelectrolyte adsorbs, the polymer that had diffused into the film diffuses out
from the film and form complexes. The existence of such a mechanism has later
been verified, showing that one of the polyelectrolytes indeed diffused into and
51
out from the film during build-up. Figure 3 presents build-up mechanisms for
a system with no diffusion of polyelectrolytes within the film, resulting in a linear increase with the number of layers and a system in which one of the polyelectrolytes is able to diffuse into and out from the film during build-up, resulting in an exponential growth.
a)
-
-
-
+
-
+
+
+
+
+
+
+
+
-
+
b)
-
-
+
-
-
-
-
+
Figure 3. A linearly growing system (a) and a system where one of the polyelectrolytes
is able to diffuse into and out from the film, leading to an exponential growth (b).
Recently a different build-up pattern was reported which is characterized by an
alternating increase and decrease during build-up using hyaluronic acid and chi49
tosan. At a low salt concentration the mass of the film decreased after chitosan addition and increased after hyaluronic acid addition. However, the net
23
growth with the number of bilayers proved to be linear. To explain this complex behaviour, a build-up mechanism similar to the one depicted in figure 3b
was suggested. According to this mechanism, chitosan is able to diffuse into the
film and when hyaluronic acid is added it interacts with chitosan on the surface
of the film and form complexes. In addition, chitosan diffuses out from the film
and form other complexes with hyaluronic acid. However, these complexes
formed from chitosan diffusing out from the film are considered to be of different nature and dissolve upon the second addition of chitosan. Even if no explanation is given to why complexes formed from chitosan that diffuses out from
the film are of a different nature and in what way they differ, this mechanism
explains the redissolution as well as the linear growth.
To illustrate different build-up mechanisms, figure 4 presents three theoretical
systems, where the film mass versus the number of adsorption cycles is shown.
Figure 4a presents a linearly growing system in which the polymers are not able
to diffuse during build-up. The increase in adsorbed amount for a system in
which one of the polymers is able to diffuse into and out from the film and
form complexes is shown in figure 4b. Finally, figure 4c shows the build-up
pattern for a system in which one of the polymers diffuses into and out from
the film and forms complexes, which are subsequently dissolved.
Proteins in PEM
The incorporation of proteins in multilayered structures is of significant interest
in the areas of biotechnology and bioengineering, and multilayers containing
proteins have potential applications in for example catalytic processes, the con52
struction of biosensors and coating of implants. The protein could be embedded in a sandwich structure composed of polyelectrolytes, or the net charge of
the protein itself could be utilized to build structures in combination with oppositely charged polymers.
24
12
a)
Adsorbed amount
10
8
6
4
2
0
120
b)
Adsorbed amount
100
80
60
40
20
0
12
c)
Adsorbed amount
10
8
6
4
2
0
0
1
2
3
4
5
6
7
8
Number of adsorption cycles
Figure 4. Schematic illustration of the build-up of polyelectrolyte multilayers for a nondiffusing system (a), a system in which one of the polyelectrolytes is able to diffuse into
and out from the film and form stable complexes at the surface (b) and a system in
which one of the polyelectrolytes diffuses into and out from the film, forming complexes
that are subsequently dissolved (c).
25
The stability of proteins in polyelectrolyte multilayers has been examined in
several investigations concluding that proteins preserve their structure and that
enzymes are active. For instance, it was shown that embedded fibrinogen retained its secondary structure and that incorporation protected the protein from
53
aggregation and improved heat stability. In addition, heat stability with respect to enzyme activity has been investigated using glucose oxidase, and the
enzyme was found to have a thermostability higher than that of free enzyme in
54
solution.
When constructing multilayers containing proteins, the surface is often precoated with a few polyelectrolyte layers. The motivation for using these precur55
sor layers is to facilitate the subsequent build-up with proteins. Also, direct
contact between the protein and the solid surface can be avoided, which could
reduce structural changes and denaturation of the native protein upon adsorption. The majority of the investigations of proteins in multilayered structures
involve proteins in combination with oppositely charged synthetic polymers or
polysaccharides, and the reported cases of layer-by-layer build-up using oppositely charged proteins have so far been few, indicating that assembly of pure
56
protein structures is a difficult task.
Mucins in PEM
In the present investigations we have focused on the multilayer constructions
with mucins in combination with cationic polymers or proteins. Bovine submaxillary mucin was used in combination with chitosan (paper I) and human
MUCB5 mucin was used in combination with cationic proteins present in the
native mucous gel (paper II). Although the interfacial properties of mucins have
been investigated in numerous studies, only one investigation has so far re57
ported on multilayer formation with mucin.
26
MATERIALS AND METHODS
Proteins and polymers
Bovine submaxillary mucin from Sigma-Aldrich Co. (M3895, Type I-S) was the
most used mucin in the present investigations. The preparation method is de58
scribed elsewhere and the molecular weight is approximately 0.4 MDa. This
preparation has been demonstrated to include other protein components, and
purification to remove these impurities resulted in a (mucin) fraction with a
30
molecular weight of 1.6 MDa. Bovine serum albumin (BSA) was later identified in the preparation, and fractionation was shown to generate two main
59
mucin fractions with different molecular weights. The mucin preparation also
60
contain aggregates and a hydrodynamic radius of above 500 nm has been de61
termined by dynamic light scattering. In paper IV the amount of these aggregates in the preparation was estimated to be less than 10 wt % in accordance
59
with a previous report.
A human mucin purified from saliva, identified as MUC5B, was also used in
the investigations (papers I-III), and the preparation of this mucin was done ac7
cording to Wickström and co-workers. Before use the mucin was dialyzed using a membrane with a molecular weight cut-off of 6-8000 Da as described by
35
Lindh and co-workers. Some physiochemical parameters of the mucins are
listed in table 1. It is evident that the molecular weights of the mucin preparations are very different and this difference is also reflected by the hydrodynamic
radius. The difference could be explained by different molecular weights of the
mucins in their native states, and this may also be a result of differences in
preparation of the samples. Both mucins appear to have the same content of
sialic acid, with values in the range of 9–17 wt %. The aliphatic index is a
measure of the hydrophobic character of a protein and is defined as the relative
27
62
volume occupied by aliphatic side chains. This parameter, which could be of
interest for predicting the interactions with other molecules or surfaces, was estimated for both bovine submaxillary mucin and MUC5B mucin. The terminal
non-glycosylated regions were identified in the protein sequence (The Swiss Institute of Bioinformatics, Swiss-Prot) and the aliphatic index of these regions
was found to vary between 50 and 65, depending on the mucin and the nonglycosylated part considered.
Table 1. Molecular weight, hydrodynamic radius and sialic acid (N-Acetylneuraminic
acid) content of Bovine Submaxillary Mucin from Sigma-Aldrich Co. and human
MUC5B mucin.
Molecular weight
(MDa)
BSM
MUC5B
1.6, 2.9
21
13.5
a
Hydrodynamic
radius (nm)
b
44
21
86
Sialic acid
content
(wt %)
9–17 wt %
9
14 wt %
c
a) Purified fractions30, 59
b) Dissolved mucin aggregates by sodium dodecyl sulphate61
c) Specified content
The other proteins used in the investigations were lysozyme (L6876, from
chicken egg white, 95% pure), lactoferrin (L0520, from human milk, 98%
pure), lactoperoxidase (L8257, from bovine milk, 86% pure), α-amylase
(10092, from human saliva ≥95% pure) and albumin (A8531, from bovine serum). These proteins were obtained from Sigma-Aldrich Co. and some relevant
physiochemical properties of these proteins are summarized in table 2. Note
that isoelectric points, net charges and aliphatic indexes are theoretical values
calculated on the basis of the protein backbone and thus they do not always
agree with the properties of the native proteins.
28
Table 2. Selected physiochemical parameters of lysozyme, lactoferrin, lactoperoxidase,
α-amylase and albumin. Molecular weights are approximate values obtained from
Sigma-Aldrich Co. or taken from literature. Values of the isoelectric points, net charges
and aliphatic indexes are theoretical values calculated from the amino acid sequence,
obtained from the Swiss-Prot database provided by The Swiss institute of Bioinformatics.
Lysozyme
Lactoferrin
Lactoperoxidase
α-Amylase
Albumin
Molecular
weight
(kDa)
Theoretical
isoelectric
point
Net charge at
b
pH 7
Aliphatic
a
index
14
90
78
56
66
9.3
8.6
8.3
6.3
5.6
+8
+12
+4
-4
-17
65
75
81
66
76
a) Aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains.
62
b) Calculated as the difference between the number of positively charged amino acid residues (lysine and arginine) and the number of negatively charged amino acid residues (aspartic acid and
glutamic acid)
Chitosan used in paper I was obtained from Fluka Production (22741) with a
specified molecular weight of 150 kDa and a degree of acetylation of 15.5%. In
paper V chitosan from Fluka BioChemika (50494, low-viscous) was used. The
degree of acetylation of this product was determined to 19% from titrimetric
63
analysis of the amino groups as described elsewhere.
29
Surfaces
Silica surfaces with an oxide layer thickness of approximately 30 nm were prepared by heating silicon slides (Okmetic OY, Espoo, Finland) in an oxygen atmosphere as described in reference 64. These surfaces were cleaned at low pressure in a glow discharge plasma cleaner for 5 minutes (PDC -32 G, Harrick Scientific Corporation, New York, USA). Plasma cleaning was followed by gentle
boiling in an alkaline solution for 5 minutes, rinsing three times in water, and
gentle boiling in an acidic solution for 5 minutes. The components of the first
solution were NH3 (25%), H2O2 (30%) and water (1:1:5 by volume), and the
second solution was composed of HCl (37%), H2O2 (30%) and water (1:1:5 by
volume). Finally, the surfaces were rinsed in water three times and then in ethanol (96%) twice. The cleaned surfaces were stored in ethanol. Before ellipsometric measurements the cleaned silica surface was rinsed in water, dried in
nitrogen, and subjected to plasma cleaning for 5 minutes and immediately
transferred to the cuvette for ellipsometric measurements. Hydrophobized silica
surfaces were prepared from surfaces cleaned as described above by rinsing in
trichloroethylene twice followed by immersion in a solution of trichloroethylene containing 0.05 vol. % dichlorodimethylsilane for one hour. The surfaces
were subsequently rinsed three times in trichloroethylene and three times in
ethanol and stored in ethanol. The hydrophobized surfaces were rinsed in ethanol, followed by rinsing in water and then dried in nitrogen directly before ellipsometric measurements.
Silica surfaces and hydrophobized silica surfaces have been characterized by
65
contact angle measurements and electroosmosis. The contact angle for silica
was reported to be less than 10º and the advancing and receding angles for hydrophobized silica surfaces was reported to be 95º and 88º respectively. The
zeta potential was reported to -45 mV for both silica and hydrophobized silica
determined by electroosmosis in 1 mM NaCl at pH 7.0.
Gold surfaces were manufactured at the Laboratory of Applied Physics,
Linköping University, Sweden using a Balzers UMS 500 P system by electronbeam deposition of 200 nm of gold onto silicon wafers, precoated with a 2.5
nm-thick titanium adhesion layer. Prior to each experiment, the gold surface
was cleaned electrochemically in 0.5 M H2SO4 by means of cyclic voltammetry.
By this procedure it was possible to determine the electrode surface area for
subsequent electrochemical measurements.
30
Ellipsometry
Introduction
Ellipsometry is an optical technique based on the detection of changes in polarization of light upon reflection at the interface between media with different
refractive indices. Light with its electric vector parallel to the plane of incidence
(p-direction) is reflected with a change in amplitude and phase that is different
from light with its electric vector parallel to the surface (s-direction). The differences in reflection will usually result in a change in the ellipticity of the light,
which gives the technique its name. The measurements are non-destructive and
suitable for characterization of optical properties of surfaces and thin films,
with thicknesses ranging from a few ångströms up to about one micrometer.
Figure 5 illustrates reflection and refraction for incident light at an interface,
with the p and s-directions indicated.
p
p
s
s
Figure 5. Reflection and refraction at an interface. Electric waves polarized in the plane
of incidence (p) and waves polarized in the plane of the surface (s) are indicated with
arrows in the figure.
31
In biology and surface chemistry, ellipsometry is commonly used to characterize
films formed by adsorbing proteins, polymers or surfactants from aqueous solutions. A film is usually then formed containing both the adsorbed molecules
and the solvent. Such a film can be characterized in terms of thickness and refractive index, where the refractive index depends on the concentration of the
adsorbed molecules. If the solid substrate and the wavelength are chosen with
care, both the thickness and the refractive index can be determined from ellipsometric measurements, and from these two parameters the mass of the film
can be calculated with high accuracy and precision.
Theory
To understand how the polarization of light changes upon reflection, it is essential to decompose the incident light into light oscillating parallel to the plane of
incidence (p-direction) and light oscillating parallel to the surface (s-direction,
perpendicular to the plane of incidence). From an optical model of the surface,
complex reflection coefficients are calculated in both directions (rp and rs), and
the complex ratio of these coefficients contains information about changes in
amplitude and phase shift upon reflection (Equation 1). The absolute value (tan
Ψ) gives the changes in amplitude ratio and the argument (∆) gives the phase
shift between the p and s components upon reflection.
rp
rs
= ρ = tan(Ψ )ei∆
( Eq. 1)
The calculation of the complex reflection coefficients (Fresnel coefficients) of
light oscillating in the plane of incidence, and in the plane of the surface as well
as combined reflection coefficients for an optical model that contains more than
66
one interface, is given in appendix. A useful way to present how the phase
shift and the amplitude ratio vary with changes in an optical model, is to construct Ψ-∆ plots. These plots are usually constructed by calculating Ψ and ∆
values for films with different thicknesses and refractive indices.
32
(a)
45
Distance between marks (x) = 10 nm
40
x
x
x
35
n = 1.50
x
x
x
x
x
x
x
30
Psi
x
15
x
x
x
x
x
x
x
x
x
x
x
n = 1.40
x
x
x
x
x
140
x
x
180
Delta
x
x
x
x
x
x
160
x
x
x
o
x
x
x
x
x
x
x
x
x
x
x
(b)
x
x
x
x
10
120
x
x
x
x
x x
x
xx
xx
x
x
x
x
x x
20
n = 1.45
x
x
x
25
x
x
200
220
240
16
n = 1.50
Distance between marks (x) = 1 nm
15.9
15.8
x
x
15.7
n = 1.45
15.6
x
Psi
x
15.5
x
n = 1.40
x
15.4
x
x
15.3
x
15.2
x
x
15.1
15
135
o
135.5
136
136.5
137
Delta
137.5
138
138.5
139
Figure 6. Ψ-∆ plot for films on silicon with an oxide layer. The wavelength is 442.9 nm,
the refractive index of the ambient is 1.341 (water) and the angle of incidence is 67.83º.
The refractive index of silicon is 4.753 – 0.16i and the thickness and refractive index of
the silicon oxide layer are 31 nm and 1.466 respectively. O indicates the starting point
(zero thickness). Figure a shows Ψ and ∆ values for films with thicknesses below 250
nm and plot b gives a more detailed view of Ψ and ∆ values for thin films. Values of
refractive indices are taken from the literature.67
33
Figure 6 presents Ψ-∆ plots for films on an oxidized silicon surface, which is the
most used type of surface in the present investigations. Parameters in the optical
model are chosen to match the experimental conditions used in our investigations, and Ψ and ∆ values are given for films with different refractive indices
(1.40, 1.45 and 1.50) with increasing thicknesses. It can be concluded from the
figure that thickness and refractive index can be resolved independently since Ψ
and ∆ values for films with different refractive indices do not overlap. The periodicity of the system is the film thickness which gives Ψ and ∆ values equal to
that of a film with zero thickness (bare surface). These values were calculated to
vary between 260 nm (n=1.50) and 340 nm (n=1.40).
Experimental set-up
Figure 7 illustrates a typical experimental set-up for null ellipsometry. The light
beam passes from the light source through a polarizer and a compensator before it is reflected on a sample surface. If the measurement is performed in a
liquid environment the light beam also has to pass the walls of the cuvette and
the liquid surrounding the sample surface. After reflection, the light passes
through a second polarizer (analyzer) and the light intensity is finally detected
by a photo detector. This order of optical components is referred to as a PCSA
arrangement.
The polarizer is transmitting light in one direction and the compensator induces
a relative phase shift of a quarter of a wavelength between light travelling parallel and perpendicular to its fast axis. In null ellipsometry the orientation of
the fast axis is set to +/- 45 degrees relative to the plane of incidence. This will
generally result in elliptically polarized light. The plane of transmission of the
polarizer is set so that the elliptically polarized light becomes plane polarized
after reflection at the sample surface. The plane polarized light can now be extinguished by setting the plane of transmission of the analyzer perpendicular to
the plane polarized light. This results in a minimum light intensity and the
method is thus referred to as null ellipsometry. The output parameters from
null ellipsometry are the angular settings of the polarizer and analyzer resulting
in extinction, which are used to determine the change in phase (∆) and amplitude ratio (tan Ψ) upon reflection.
34
Light source
Light detector
Polarizer
Compensator
Analyzer
(second polarizer)
α0
“null”
Sample cell and surface
Figure 7. Null ellipsometry set-up (PCSA arrangement)
Determination of Ψ and ∆ values from the settings of the polarizer and
analyzer
In null ellipsometry the optical properties (Ψ and ∆ values) of a substrate can be
obtained from the nulling settings of the polarizer and analyzer. The phase shift
(∆) is determined from the setting of the polarizer while changes in amplitude
ratio (Ψ) can be determined from the setting of the analyzer. If the fast axis of
the compensator is located at -45°, two sets of polarizer and analyzer settings
can be found that gives a minimum in light intensity, and similarly two different pairs of polarizer and analyzer values can be found with the fast axis located at +45°. These settings represent different “zones” and the calculation of
Ψ and ∆ values in the different zones are presented in table 3. Even though
measurements and characterization of the substrate can be done in only one
zone in theory, measurements are often performed in two or four zones to reduce errors that originate from instrumental imperfections.
35
Table 3. Calculation of Ψ and ∆ values from the analyzer (A) and polarizer (P) settings
in different zones.68
Zone
Compensator
setting (fast axis)
ψ
∆
1
2
3
4
-45°
+45°
-45°
+45°
A
A
180°-A
180°-A
2⋅P+90°
-90°-2⋅P
2⋅P-90°
90°-2⋅P
Calculating surface properties from Ψ and ∆ values
In a two phase model the substrate and the ambient constitutes the two phases,
and from the ellipsometric parameters (Ψ and ∆ ) the complex refractive index
can be calculated analytically. The Fresnel coefficients are inserted in equation
1, and equation 2 gives the resulting analytical solution for the complex refrac69
tive index of the substrate (n1). The input parameters are the refractive index
of the ambient medium (n0), angle of incidence (α0) and the experimentally obi
tained Ψ and ∆ values (ρ = tan(Ψ)e ∆).
⎛
⎞
4ρ
n1 = n0 tan(α 0 ) ⎜⎜ 1 −
sin 2 α 0 ⎟⎟
2
⎝ (1 + ρ )
⎠
( Eq. 2)
In a three phase model a plane parallel and homogenous film with a certain
thickness and refractive index is present between the ambient and the substrate.
In this case, no analytical expression can be derived, and an iterative procedure
has to be performed to obtain the thickness and refractive index of the film
69
from the Ψ and ∆ values. In this procedure refractive indices of the transparent film are assumed and the correct values are obtained when a real value of
the thickness is found.
36
Mass calculations and water content
The refractive index is frequently related to the concentration by the LorentzLorenz equation, where the refractive index depends on the molar refraction
70
and concentration of all components. A more simple method, based on the
empirical observation of how the refractive index varies with concentration, is
71
to assume that the refractive index increases linearly with the concentration.
For a system containing two components (solvent and dissolved molecules) the
refractive index can be calculated from the refractive index of the solvent (ns),
the refractive index increment (dn/dc) and the concentration of the dissolved
molecules (c) according to equation 3. The amount of molecules in an adsorbed
layer (Γ) can subsequently be calculated by multiplying the expression for the
concentration with the ellipsometric thickness as shown in equation 4. To facilitate the calculations of the adsorbed amount, the refractive index of the solvent (ns) can normally be approximated by the refractive index of the ambient
solution (n0).
⎛ dn ⎞
n = ns + ⎜ ⎟ ⋅ c ( Eq. 3)
⎝ dc ⎠
Γ=
n − ns
⋅d
⎛ dn ⎞
⎜ ⎟
⎝ dc ⎠
( Eq. 4 )
In ellipsometric measurements the adsorbed amount can be determined more
accurately than the thickness and refractive index since an overestimation of the
thickness will result in an underestimation of the refractive index and vice
versa. It can be seen in equation 4 that reverse co-variations in thickness and
refractive index will in part be cancelled out in the calculation of the adsorbed
amount. The limited accuracy and precision in thickness is more pronounced at
2
a low surface coverage, and below a surface coverage of 0.5 mg/m the thickness data is generally unreliable at the experimental conditions used in the pre72
sent investigations.
37
The mean water content of the film is a useful parameter, which can be calculated directly from the ellipsometric thickness and the adsorbed amount according to equation 5.
Water content ( wt %) =
(d − Γ ⋅ V )ρ
Γ + (d − Γ ⋅ V )ρ
sp
w
sp
( Eq. 5)
w
Vsp = specific volume of the adsorbed molecules
ρ w = density of water
Instrument set-up
The instrument used throughout the investigations was a Rudolph thin-film ellipsometer (type 43603-200E, Rudolph Research, Fairfield NJ, USA) and the
experimental set-up was based on null ellipsometry as illustrated in figure 7.
66
The automatization was done according to the concept of Cuypers , improved
73
by Landgren and Jönsson, which enables a time resolution of a few seconds. A
xenon arc lamp was used as a light source, and light was detected at 442.9 nm
using an interference filter with UV and infrared blocking (Melles Griot, Netherlands). The 5 mL trapezoid cuvettes made of optical glass (Hellma, Germany)
was thermostated and equipped with a magnetic stirrer.
Evaluation
When oxidized silicon surfaces were used as substrate, an optical model composed of two layers had to be assumed in the evaluation of its properties. The
unknown parameters in this optical model were the complex refractive index of
the silicon and the refractive index and thickness of the oxide layer (the silicon
oxide layer is assumed to be transparent). In order to determine these optical
constants, both air and aqueous phase were used as ambient media in the char73
acterization. For the gold surfaces a one layer model was assumed and the
bare surface was characterized in the relevant liquid media.
38
After determination of the optical properties of the bare surface, the properties
of the adsorbed film were monitored in situ with the assumption that the molecules formed a homogenous layer. The Ψ and ∆ values were determined from
the readings of the polarizer and analyzer, and the thickness and refractive index of the film were calculated as well as the adsorbed amount. To reduce systematic errors, two zone measurements were conducted in the characterization
of the substrates, and the derived correction factors for Ψ and ∆ were used in
the determination of the properties of the adsorbed layers.
39
Particle electrophoresis
Charged particles in a solution will migrate if an electric field is applied across
the dispersion, and from their electrophoretic mobility, the zeta potential (ζ)
can be calculated. By definition the zeta potential is the potential in the slip
plane between the stationary solution and the moving particle with adherent
liquid. In the general case, the exact position of the slip plane is not known, but
it is expected to be in the order of a few molecular diameters for particles with
74
a sharp boundary towards the liquid.
In particle electrophoretic measurements, the electrophoretic mobility (u) is determined by dividing the velocity of the particles by the electric field and the
zeta potential can then be calculated from the Hückel or Smoluchowski equa74
tion (equations 6 and 7). Apart from the electrophoretic mobility (u) the viscosity of the solution (η), the permittivity of vacuum (ε0) and the relative dielectric permittivity (εr) are used in the calculations. The choice of equation depends
on the radius of the particles (R) and the screening length of the ambient solu-1
tion (κ ).
3 ηu
2 ε oε r
ζ = ⋅
ζ =
ηu
ε oε r
κR << 1 Hückel equation ( Eq. 6 )
κR >> 1 Smoluchowski equation ( Eq. 7 )
As it has been shown that the zeta potential depends on the electrolyte concentration, type of counter ion, pH and temperature, it is important to work with
75
well defined systems for comparative studies. Complications also arise when
non-spherical particles are studied and thus comparisons of absolute values of
27
zeta potentials in the literature are often problematic. However, zeta potential
measurements are well suited to follow relative changes in electrophoretic mobility when, for example, polymers or proteins adsorb at the surface of particles.
40
Atomic force microscopy
Atomic force microscopy (AFM) is a useful technique that is used for characterization of surface morphology and colloidal forces between particles and sur76
faces. In imaging mode AFM, topographic images are usually obtained and
useful parameters such as surface roughness and size distributions of, for example, adsorbed particles can be determined. The resolution is dependent on
the nature of the sample, and as a general rule softer material gives a lower
resolution. For adsorbed proteins and polymers the resolution is a few nanometers at ideal conditions, and this enables individual proteins to be visualized. In
addition, information can sometimes be obtained about the tertiary structure of
globular proteins and contour lengths of random coil proteins.
Topography images are obtained by using a cantilever with a very sharp tip to
scan the surface. The force between the tip and the sample causes the cantilever
to bend, and the bending is monitored by a laser beam reflected on the surface
of the cantilever. In contact mode the force/bending of the cantilever is usually
kept constant while scanning the surface. The constant force between the sample and the tip of the cantilever is achieved by shifting the vertical position of
the sample, and the resulting topographic image is obtained from the height
signal. The main advantages using AFM is that high resolution images can be
obtained and that pre-treatment of the sample surface is not required. Also
measurement can be conducted in situ in air or liquid at ambient temperatures.
Disadvantages using this technique are that soft structures are not easily visualized and that scanning may distort the structures on the surface so that the topographic images do not reflect true conformations. However, direct contact
between the sample and the tip can in some cases be avoided by taking advan77
tage of electrostatic repulsions.
In the present investigations we determined the topography by contact mode
AFM in liquid, and we strived to use a minimum force between the tip and the
sample surface in order to minimize distortion of the loosely bound soft protein
structures during scanning. Cantilevers with spring constants of less than 0.3
N/m and cantilever tips made of silicon nitride were used. The instrument employed was a scanning probe microscope from Veeco (Picoforce multimode
SPM with a Nanoscope IV control unit).
41
Electrochemistry
The enzymatic activity of surface bound lactoperoxidase (LPO) was evaluated
by measuring the current obtained by electrochemical reduction of catechol,
which was used as a mediator in the enzymatic process. Activity measurements
were generally carried out through the following sequence of experimental procedures: Lactoperoxidase was adsorbed on a gold surface or on a gold surface
with preadsorbed mucin or albumin while monitoring the adsorption process
by ellipsometry. After adsorption and rinsing, the surface was transferred to an
electrochemical cell and connected as a working electrode to a potentiostat
(ZPta Elektronik, Höör, Sweden). A silver wire served as a combined reference
and counter electrode. The cell was filled with a buffered solution and after that
a -50 mV potential was applied to the working electrode. 100 µM of catechol
and up to 50 µM of hydrogen peroxide were added to the cell to start the enzymatic reaction, and the process was followed by measuring the resulting electrode current. In both ellipsometric and electrochemical measurements a 10
mM phosphate buffer was used (pH 7.0), containing 100 mM NaCl and 1 mM
CaCl2.
A simplified reaction sequence of the enzymatic and electrochemical process is
summarized below.
3+
LPO(Fe ) + H2O2
5+
LPO(Fe ) + catechol
o-quinone + 2e-(Au) + 2H+
-
+
H2O2 + 2e (Au) + 2H
3+
→
→
→
→
5+
LPO(Fe ) + H2O
3+
LPO(Fe ) + o-quinone + H2O
catechol
2H2O
5+
LPO(Fe ) and LPO(Fe ) represent native and 2-electron oxidized lactoperoxidase respectively. 2e (Au) represents two electrons at the gold electrode. From
the reaction scheme above, it is clear that the rate of the catalytic process can be
determined from the electrochemical reduction rate of o-quinone at the gold
surface, measured as a current.
Enzyme activity is by definition equal to the amount of substrate converted per
unit time. One international unit (U) equals 1 µmol of substrate converted per
minute. If the activity unit (U) is related to the amount of the enzyme, the specific activity is obtained expressed as U/mg. According to the reaction scheme it
42
can be concluded that the conversion of one mole of hydrogen peroxide requires 2 moles of electrons. Thus, the rate of enzymatic reduction of hydrogen
peroxide can easily be related to the current of the lactoperoxidase modified
electrode expressed in the equation 8.
Reduction rate of H 2 O2 ( µmol / s ) =
imax ( µA)
2⋅F
( Eq. 8 )
(imax) is the electric current and F is the Faraday constant (C/mol). The amount
2
of enzyme per unit area (Γ in mg/m ) from ellipsometric measurements can be
2
multiplied by the real surface area (A in m ) of the electrode to obtain the total
amount of enzyme, which is needed to calculate the specific activity (U/mg) as
exemplified in equation 9.
Specific LPO activity (U / mg ) =
imax ( µA) ⋅ 60
2⋅ F ⋅Γ⋅ A
( Eq. 9 )
It should be pointed out that we must assume that the maximum current (imax)
at these electrodes is not limited by the diffusion of reaction substrates (H2O2,
catechol, and o-quinone). This assumption is valid since the experimentally
measured current was at least 10 times lower than what could be expected from
diffusion limited processes involving H2O2, catechol, or o-quinone.
43
RESULTS AND DISCUSSION
Layer-by-layer film formation with mucin
Papers I and II describe the build-up of multilayers containing mucin and oppositely charged polymers or proteins. The first paper includes layer-by-layer assembly of bovine submaxillary mucin (BSM) and chitosan, and the second paper includes a human mucin purified from saliva (MUC5B) in combination
with cationic proteins naturally present in the mucous gel. In addition to this αamylase was used as a control protein. Also included is a part that describes activity of lactoperoxidase adsorbed on gold surfaces precoated with mucin and
albumin (paper III).
Mucin (BSM) and chitosan (paper I)
In paper I, bovine submaxillary mucin (BSM) was used in combination with
chitosan. Assembly was done in an aqueous 0.1 vol % acetic acid solution to
ensure that chitosan was in its protonated and soluble form. The build-up was
investigated on silica and hydrophobized silica as model surfaces and figure 8
illustrates the results. On silica, the amount of mucin after rinsing was low in
35, 57
comparison with the values found in other studies.
However, this difference
can be explained by the absence of added salt resulting in essentially unscreened
electrostatic repulsions between the silica surface and mucin, as discussed in the
introduction. Upon the first addition of chitosan the adsorbed amount increased, but the build-up with the number of adsorption cycles was limited and
2
after 8 adsorption cycles the amount was found to be less than 0.4 mg/m . On
hydrophobized silica, the amount of mucin after rinsing was found to be much
28, 35
higher than on silica and in agreement with other investigations.
The subse-
44
quent addition of chitosan and rinsing did not significantly change the total adsorbed amount and in fact a small decrease was detected. The following adsorption was characterized by an alternating increase upon mucin addition and
a decrease upon chitosan addition. However, the net result showed that the adsorbed amount increased approximately linearly with the number of adsorption
cycles, and after 8 adsorption cycles the amount was found to be about 6
2
mg/m . The increase in thickness with the number of adsorption cycles is illustrated in figure 9, and as for the adsorbed amount the increase in thickness was
found to be approximately linear.
a)
0.5
b)
10
Silica
Hydrophobized silica
2
Adsorbed amount (mg/m )
8
2
Adsorbed amount (mg/m )
0.4
0.3
0.2
0.1
6
4
2
0
0
0
1
2
3
4
5
6
Number of adsorption cycles
7
8
0
1
2
3
4
5
6
7
8
Number of adsorption cycles
Figure 8. Adsorbed amount on silica (a) and hydrophobized silica (b) versus the number
of adsorption cycles, (mucin (BSM) - chitosan) x 8. The build-up was monitored in 0.1
vol % acetic acid and the mucin and chitosan concentration was 0.1 mg/mL. Values after mucin addition and rinsing ({) and chitosan addition and rinsing (†) are presented.
Note the different scales on the y-axes.
The results clearly show that while the build-up was limited on silica a linear
build-up with the number of adsorption cycles was possible on hydrophobized
silica. From this result it may be concluded that the amount of mucin in the
first step is crucial for the subsequent build-up. Hydrophobization is thus an
attractive approach to facilitate the subsequent build-up when working with
mucins or other amphiphilic molecules that show limited adsorption to hydrophilic substrates.
45
To illustrate the build-up kinetics, figure 9 shows adsorbed amount, thickness
and refractive index versus time. From this figure it can be seen that the first
addition of mucin led to a relatively slow increase in the adsorbed amount. The
subsequent addition of chitosan caused a small decrease in the adsorbed
amount whereas the thickness increased. After the second addition of mucin, a
rapid increase was detected in adsorbed amount, accompanied by an increase in
thickness. The second addition of chitosan led to gradual decrease in the adsorbed amount, while the thickness remained essentially constant.
The subsequent adsorption cycles were similar to the second adsorption cycle in
the way that we detect a rapid increase in adsorbed amount upon the addition
of mucin, and a gradual decrease upon the addition of chitosan. The thickness
increases rapidly upon all additions of mucin, whereas from the third addition
of chitosan and on a gradual and pronounced decrease in thickness was detected. It can also be noted the refractive index of the film decreases after all
additions of chitosan.
A more detailed investigation of the kinetic curves reveals that, from the fourth
addition of mucin and on, the initial rapid increase was followed by a slow decrease in adsorbed amount. Also, from the fifth addition of chitosan and on, a
small increase could be detected before the decrease in adsorbed amount and
thickness. An observed initial increase followed by a decrease in adsorbed
49, 78
amount has been reported previously.
This phenomena, often referred to as
overshoot, can be explained by the fact that the polymer fist adsorbs to the surface of the film, but complexes formed between the two polymers in the film
are in a later stage dissolved and diffuse out from the film.
The complex build-up pattern of adsorption followed by redissolution can be
explained by the build-up mechanism proposed by Richert and co-workers de49
scribed in indtroduction. Accordingly, chitosan is assumed to be able to diffuse into and out from the film and form loosely bound complexes that are subsequently dissolved. The proposed mechanism will result in a linear growth
with the number of adsorption cycles indicating that this mechanism may be
valid for the build-up in the present investigation. By comparing our results
(figure 8b) with the theoretical behaviour of such a system (figure 4c) it is evident that they indeed have similar features.
46
Number of adsorption cycles
0
1
2
3
4
5
6
7
8
15
2
Adsorbed amount (mg/m )
20
C
C
C
10
C
C
C
C
C
5
0
100
M
M
M
M
M
M
M
C
M
C
80
C
Thickness (nm)
C
C
60
C
C
M
40
C
M
M
M
20
M
M
M
1.39
0
M
C
Refractive index
1.38
1.37
C
C
C
C
C
C
C
1.36
1.35
M
M
M
M
80
160
240
M
M
M
M
320
400
480
560
1.34
0
640
Time (min)
Figure 9. Adsorbed amount, thickness and refractive index versus time for layer-by-layer
build-up on hydrophobized silica, (mucin (BSM) - chitosan) x 8. The mucin and chitosan concentration was 0.1 mg/mL. M indicates mucin addition and C indicates chitosan
addition.
47
Ionic strength →
However, for a deeper understanding of the redissolution process, it is of interest to consider the stability of polyelectrolyte coacervates (polyelectrolyte complexes) formed in aqueous solution, from a thermodynamic point of view. Figure 10 presents a stability diagram proposed for polyelectrolytes in solution,
where the ratio between the polyelectrolytes and the ionic strength is taken into
78
account. From the figure it is evident that the most stable coacervates (shaded
area) are formed at equal amounts of polyelectrolytes at low ionic strength, and
that the coacervates dissolve in excess of one of the polyelectrolytes. During the
construction of polyelectrolyte multilayers, the overall composition is normally
alternating between a high mole fraction of the cationic polymer and a high
mole fraction of the anionic polymer. This is also the case for our system and
thus the desorption seen in figure 9 can be explained by the stability diagram. It
should be kept in mind, however, that the stability diagram is used to explain
the adsorption behaviour of polyelectrolytes in solution, and thus it may not be
valid for the part of the film that is directly associated with the surface. In addition, the presented stability diagram, with its symmetric shape of the stability
region, is an idealized description that is most appropriate to describe systems
that includes similar polyelectrolytes with equal (and opposite) charge densities.
Stable
coacervates
0
Polyelectrolyte ratio →
1
Figure 10. Schematic representation of the stability of coacervates redrawn from reference 78. The horizontal axis represents the mole fraction of the cationic (or anionic)
polyelectrolyte and the vertical axis represents the ionic strength. The shaded grey region symbolises the existence of stable coacervates. Arrows represent changes in the system during layer-by-layer build-up with oppositely charged polyelectrolytes.
48
Nevertheless, some questions that cannot be easily explained from the stability
diagram remain. First, it is evident that only part of the film is dissolved when
chitosan is added, in spite of the fact that the stability diagram suggests that the
film would be completely dissolved except for the part of the film that is directly associated with the solid surface. By looking at the adsorption kinetics it
is evident that equilibrium was not reached when the cuvette was rinsed after
chitosan addition. Therefore the absence of a total redissolution of the film
when chitosan is added can be explained by the slow kinetics of polymer systems, especially involving high molecular weight molecules.
It is also evident that redissolution when mucin is added is very limited compared to chitosan additions. This difference may not be a surprise considering
that these molecules are very different and in relation to the layer-by-layer
build-up of polyelectrolytes it is of interest to consider the charge balance. Accordingly, the charge density of chitosan was calculated from the number of
amino groups in the molecule and the charge density of mucin was calculated
on the basis of the amount of bound sialic acid. The charge density in 0.1 vol.
% acetic acid (pH 3.4) was calculated to be 5 mmol/g for chitosan and 0.3
mmol/g for mucin. As the coacervates are dissolved when the charge balance is
moving towards the extremes (in figure 10), it is understandable that the highly
charged chitosan will cause a rapid redissolution upon addition. In comparison
with chitosan the charge density of mucin is much lower and this could be the
reason to why redissolution is limited or absent after mucin addition.
To minimize redissolution, we decided to decrease the concentration of chitosan from 0.1 mg/mL to 0.01 mg/mL while keeping the mucin concentration
constant. The effect of a lower chitosan concentration on the layer-by-layer
build-up with mucin is presented in figure 11, and it was found that redissolution was reduced significantly, increasing the mass of the final film threefold.
49
Number of adsorption cycles
0
1
2
3
4
5
6
7
C
8
20
C
C
2
Adsorbed amount (mg/m )
15
M
C
C
10
M
C
M
C
5
C
M
M
M
0
0
M
M
80
160
240
320
400
480
560
640
Time (min)
Figure 11. Adsorbed amount versus time for layer-by-layer build-up on hydrophobized
silica, (mucin (BSM) – chitosan) x 8. The mucin and chitosan concentration were 0.1
mg/mL and 0.01 mg/mL respectively. M indicates mucin addition and C indicates chitosan addition.
50
Mucin (MUC5B) and cationic proteins (paper II)
A human mucin purified from saliva (MUC5B) was used in combination with
cationic proteins that are known to be present in the mucous gel and the work
is described in paper II. Conditions such as temperature, pH and ionic strength
were chosen to resemble in vivo conditions, and as the surface properties was
found to have a profound influence on the build-up with mucin (BSM) and chitosan (paper I), we investigated the build-up on both silica and hydrophobized
silica. Lactoferrin, lactoperoxidase and lysozyme, which are all present in the
4, 6
native mucous gel, as well as α-amylase as control, were used in combination
with mucin. Two adsorption cycles were performed with these systems in order
to study the possibilities to build layer-by-layer structures and the results are
presented in figure 12.
a)
10
b)
10
Mucin - Lactoferrin
Mucin - Lactoferrin
Mucin - Lactoperoxidase
Mucin - Lactoperoxidase
8
Mucin - Lysozyme
2
Adsorbed amount (mg/m )
2
Adsorbed amount (mg/m )
8
Mucin - Lysozyme
6
4
2
6
4
2
0
0
0
0.5
1
1.5
Number of adsorption cycles
2
0
0.5
1
1.5
2
Number of adsorption cycles
Figure 12. Adsorbed amount on silica (a) and hydrophobized silica (b) versus the number of adsorption cycles, (mucin (MUC5B) - cationic proteins) x 2. The mucin and cationic protein concentration were 0.05 mg/mL and 0.01mg/mL respectively. Values after
protein addition and rinsing are presented.
For comparison it is of interest to determine the net increase of the second adsorption cycle for the different protein systems. On silica this increase was similar for the systems that included lactoperoxidase and lysozyme, while a very
modest increase was detected for the system that included lactoferrin. On hydrophobized silica a substantial increase was found for the system that included
lactoperoxidase while almost no increase could be detected for the systems that
included lactoferrin or lysozyme. For the system that included α-amylase, a lim-
51
ited increase was detected for the second bilayer on silica whereas no increase
was detected on hydrophobized silica (see paper II, figure 2). This was expected
from the net negative charge of α-amylase with an isoelectric point of 6.3. From
these result it can be concluded that lactoperoxidase is the best candidate to
build layer-by-layer structures with mucin, at our experimental conditions.
The reason to why lactoperoxidase was the best candidate among the investigated proteins to assemble multilayers with mucin is still open to speculations.
In paper II, we proposed that matching charge densities between the glycosylated domains of mucin and lactoperoxidase could offer one explanation. This
79
value was calculated based on the amount of negatively charged sugars. However, in another reference the sialic acid content of this mucin is reported to 14
9
wt %, which gives a higher value of the charge density. This means that other
contributions to the interactions have to be the discriminating ones. In spite of
the nature of the interaction, it can be speculated that lactoperoxidase may
have the ability to cross-link mucin molecules in the native mucous gel.
As a significant net increase for the second bilayer was detected for the system
that included lactoperoxidase, we performed a deeper investigation of this system, and figure 13 shows the build-up on silica and hydrophobized silica with 4
adsorption cycles. On silica, the amount of mucin after the first addition was
2
found to be 1 mg/m and the subsequent increase in adsorbed amount was indicating either a linear increase or an increasing net increase with the number of
adsorption cycles. Thus, it could be concluded that the amount of mucin in the
first step was sufficient for successful build-up. On hydrophobized silica the
2
amount of mucin after the first addition was 4 mg/m and the subsequent buildup was found to be linear with respect to adsorbed amount. The build-up on
both silica and hydrophobized silica was characterized by an alternating decrease upon mucin addition and increase upon lactoperoxidase addition after
the first bilayer and this alternation was more pronounced on silica.
52
a)
12
b)
12
Hydrophobized silica
Silica
2
Adsorbed amount (mg/m )
10
2
Adsorbed amount (mg/m )
10
8
6
4
2
8
6
4
2
0
0
0
1
2
Number of adsorption cycles
3
4
0
1
2
3
4
Number of adsorption cycles
Figure 13. Adsorbed amount on silica (a) and hydrophobized silica (b) versus the number of adsorption cycles, (mucin (MUC5B) - lactoperoxidase) x 4. The mucin and lactoperoxidase concentrations were 0.05 mg/mL and 0.01 mg/mL respectively. Values after mucin addition and rinsing ({) and lactoperoxidase addition and rinsing (†) are
presented.
From the kinetic curves on silica (paper II, figure 3) it can be seen that the additions of mucin led to a slow and gradual decrease in the adsorbed amount,
while the adsorbed amount increased rapidly to plateau values upon the additions of lactoperoxidase. Adsorption and redissolution processes were discussed
in the previous section (Mucin (BSM) and Chitosan) and the reader is referred
to this section for a more detailed discussion of the complex build-up behaviour.
That redissolution only occur upon the addition of mucin can be explained by a
higher charge density of this protein, originating from sialic acids and sulphate
groups. Another factor that can be taken into account is that the mucin concentration is five times higher than the lactoperoxidase concentration in this work.
Mucins are also more hydrophilic than lactoperoxidase due to the high carbohydrate content and therefore the complexes formed in excess of mucin could
be expected to be more water soluble than complexes formed in excess of lactoperoxidase.
53
Activity of lactoperoxidase when adsorbed on protein layers
80
Despite the well known low stability of lactoperoxidase, the enzyme is interesting for its involvement in the mammalian defence system. The basic antimicrobial principal of action is due to the lactoperoxidase assisted generation of
oxidised halogens (I2) and pseudohalogens ( (SCN)2), which react and inactivate
80
proteins of bacterial cells. From the understanding of how lactoperoxidase
acts as antimicrobial agent, a number of applications or products have been
81
proposed that include this enzyme.
In paper II we have shown that it is possible to build multilayer structures with
lactoperoxidase in combination with mucin by alternate adsorption. In this
context it is of interest to be able to measure the activity of lactoperoxidase in
these structures. We therefore performed investigations of lactoperoxidase activity on gold surfaces and on mucin-coated gold surfaces by means of electrochemistry. The activity of lactoperoxidase was also measured on a preadsorbed
layer of albumin as a control experiment.
Adsorption of lactoperoxidase
Gold
Ellipsometric measurements showed that lactoperoxidase adsorbs on gold surfaces and that the adsorbed layer is irreversibly bound with respect to rinsing
(paper III, figure 2). After rinsing, the thickness and adsorbed amount were 33
2
Å and 2.9 mg/m respectively. A thickness of 41 Å has previously been reported
82
for adsorption on gold, which is in fair agreement with our measurements,
2
and the amount of 2.9 mg/m is in between values reported for silica and hydrophobized silica (paper II, table 1). The adsorbed amount is close to the
theoretical value of an end-on monolayer (55 Å x 81 Å) of lactoperoxidase, indicating a high surface coverage. When gold surfaces were modified by preadsorption of other proteins, the adsorption of lactoperoxidase was found to be
different and below you will find a description of the sequential adsorption of
lactoperoxidase on mucin (BSM and MUC5B) and albumin (BSA).
Mucin (BSM)
2
BSM adsorbed on gold resulted in an approximate amount of 1.6 mg/m (paper
III, figure 3). Subsequent addition of lactoperoxidase resulted in an increase in
54
2
the total amount to 2.6 mg/m . However, lactoperoxidase adsorption simultaneously led to a substantial decrease in the thickness of the adsorbed layer. A
simultaneous increase in the adsorbed amount and a decrease in thickness were
also observed previously (paper II, figure 3). These observations can be explained by complexation between mucin and lactoperoxidase leading to a more
compact film structure, or it could be attributed to an exchange of mucin by
lactoperoxidase from the surface. Similar changes in film properties have re59
cently been reported for the sequential adsorption of mucin and albumin.
Mucin (MUC5B)
MUC5B was adsorbed at different concentrations (50 µg/mL and 0.5 µg/mL)
and the results are presented in paper III (figure 5). For the higher MUC5B con2
centration a surface coverage of more than 3 mg/m was obtained and the subsequent adsorption of lactoperoxidase did not significantly change the ellipsometric parameters of the film. This result was unexpected since a significant
binding of lactoperoxidase was detected to this mucin when adsorbed to silica
(paper II), and at present we do not understand the reason for this difference.
At 100 times lower concentration of MUC5B, the adsorbed amount on the gold
2
surface was low (0.2 mg/m ), and the subsequent adsorption resulted in an increase in adsorbed amount that was comparable to the amount of adsorbed lactoperoxidase on a clean gold surface.
Albumin (BSA)
The adsorption profile of albumin on a gold surface and further addition of lactoperoxidase is presented in paper III (figure 4). The amount of albumin was
2
2
1.8 mg/m and the total amount increased to 2.7 mg/m after addition of lactoperoxidase, whereas the thickness remained almost constant.
Activity measurements
After ellipsometric studies of the adsorption of lactoperoxidase to gold and protein-coated gold surfaces, the enzymatic activity of surface bound lactoperoxidase was evaluated. This was done by monitoring the current resulting from the
electrochemical reduction of the enzymatically oxidised electron donor, oquinone.
55
Figure 14 shows that significant differences in current responses can be observed at different electrodes. The current response varied between 0.2 and 5.5
2
µA/cm , with the highest value from lactoperoxidase adsorbed on gold modified
with a low amount of MUC5B. In figure 15 the estimates for the specific activities of lactoperoxidase are presented, and it is evident that the specific activity
varied from 2.7 up to 7.9 U/mg. The first conclusion that can be made is that
the specific activity of the enzyme (to some extent) is preserved on all surfaces.
The highest estimated specific lactoperoxidase activity was found on gold surfaces precoated with BSM or a low amount of MUC5B.
10
Au-LPO
BSM-LPO
BSA-LPO
MUC5B (50 µg/mL)-LPO
MUC5B (0.5 µg/mL)-LPO
100
Activity (%)
2
Current density (µA/cm )
8
6
75
50
25
4
0
1
2
2
3
4
Number of measurements
L)–
Au
Au
(0.
5µ
g/m
uc
5B
-M
LP
O
O
-M
uc
5B
(50
µ
g/m
L)–
Au
LP
(50
µg
/m
L)
–
SA
-B
LP
O
LP
O
-B
SM
LP
O
(50
µg
/m
L)
–
–A
Au
u
0
Figure 14. Current density of lactoperoxidase on gold electrodes and on protein-coated
gold electrodes. Insert: Operation stability of lactoperoxidase on gold electrodes and on
protein-coated gold electrodes. Mean values are presented and error bars represent the
range of the individual measurements.
56
Specific activity (U/mg)
10
8
6
4
2
Au
50
0.5
µg
/m
µg
/m
L)–
L)–
Au
u
–A
µg
/m
L)
LP
O
-M
uc
5
B(
B(
-M
uc
5
LP
O
-B
SA
LP
O
LP
O
-B
SM
(50
(50
µg
/m
LP
O
L)
–
–A
Au
u
0
Figure 15. Specific activity of lactoperoxidase on gold electrodes and on protein-coated
gold electrodes. Mean values are presented and error bars represent the range of the individual measurements.
The amounts of adsorbed lactoperoxidase used in the estimation of the specific
activity are subject to some uncertainty. Some of the initially adsorbed protein
might for example be exchanged by lactoperoxidase. If this is the case, the
amount of lactoperoxidase will be underestimated, leading to an overestimation
of the specific enzymatic activity. However, in the case of MUC5B adsorption
at the low concentration (0.5 µg/mL) the small amount of adsorbed MUC5B
will reduce the possible error resulting from exchange of protein. If, for example, lactoperoxidase is assumed to exchange all adsorbed mucin upon adsorption the calculated specific activity is calculated to 6.9 U/mg, which is still
higher than the value found for lactoperoxidase on bare gold.
The stability of adsorbed lactoperoxidase was also investigated and it was
found to be almost independent on surface modification and relatively poor
(figure 14, insert). On average, about 25% of the activity is lost after each electrochemical enzyme activity assay.
57
Interactions between drug delivery particles and mucin
The general aim of papers IV and V was to evaluate the mucoadhesive properties of drug delivery particles. Lipid nanoparticles with an inner cubic structure
stabilised by a poly(ethylene oxide) (PEO) based polymer were used as model
®
particles in the investigations (Cubosome particles). A second type of particles
was also prepared by modifying the lipid nanoparticles by adsorption of chitosan. A description of the lipid nanoparticles can be found in paper IV, and the
modification procedure is described in paper V.
In both studies (papers IV and V), silica surfaces were coated with bovine submaxillary mucin (BSM) and adsorption of particles was monitored by ellipsometry. Therefore, the focus of this section is to compare differences between
the lipid nanoparticles and the chitosan modified lipid nanoparticles with respect to their interaction with mucin. For simplicity, the lipid nanoparticles and
the chitosan modified lipid nanoparticles are referred to as unmodified and
modified particles respectively. All measurements in this section were conducted
in aqueous solutions containing 50 mM NaCl at 37 ºC if nothing else is specified.
Particle characterization
The mean particle size of the unmodified particles was determined to 311 nm
(standard deviation 58 nm) in water and the size of the modified particles was
334 nm (standard deviation 88 nm), indicating that the particle size increases
somewhat when modified. A detailed description of the method for size deter83
mination can be found elsewhere. The particles were furthermore characterized by particle electrophoresis, and figure 16 shows the zeta potential versus
the pH. For the unmodified particles, the zeta potential was found to be negative and relatively low in magnitude. To investigate the origin of the negative
charge, we performed electrophoretic measurements on particle dispersions of
the stabilizing polymer and found that the potential was close to zero, with an
absolute value of less than 2 mV (pH 5.4). The essentially neutral charge of this
polymer determined from the electrophoretic measurements is in agreement
with other studies, and thus the negative charge should be associated with the
84, 85
lipid phase.
The negative charge could originate from free oleic acid or ad84
sorption of hydroxyl ions at the oil-water interface as proposed by others.
58
Zeta potential (mV)
For the modified particles, the zeta potential was found to be positive below pH
7, and the pH dependence followed the general behaviour of chitosan in solu86
tion. The isoelectric point was determined to pH 7, although at this pH approximately 20% of the amino groups are protonated. However, this could be
explained by the intrinsic negative charge of the particles compensating for the
positively charged amino groups.
20
20
15
15
10
10
5
5
0
0
-5
-5
-10
-10
-15
-15
2
3
4
5
6
7
8
9
pH
Figure 16. Zeta potential versus pH for unmodified particles ({) and modified particles
(†). Connecting lines have been added to guide the eye. Error bars represent 95% confidence intervals.
Characterisation of mucin-coated silica surfaces
In the ellipsometric investigations we coated the surface by mucin adsorption
before studying the interactions with particles, and these measurements revealed that the irreversibly bound fraction of mucin varied between 0.6 and 1.0
2
mg/m , and the ellipsometric thickness varied between 20 and 45 nm. No correlation was found between electrolyte concentration (50 and 150 mM NaCl)
and the thickness and adsorbed amount of the adsorbed mucin layer. Therefore
50 mM NaCl appears to be sufficient to screen electrostatic repulsions that are
expected to decrease the adsorbed amount on silica. The low adsorbed amount
59
on silica at low electrolyte concentration was for example shown in paper I. As
for the electrolyte concentration no difference could be detected in the properties of the mucin layer at the different pH (4 and 6).
In addition to ellipsometry, the mucin-coated silica surfaces were characterised
by atomic force microscopy and particle electrophoresis as described in paper
IV. It was found that the mucin adsorbed as closely packed disc-shaped aggregates approximately 20 nm high and 150 nm in lateral dimension. The existence of surface aggregates after adsorption to silica has previously been re87
ported for this mucin preparation and they probably originate from aggregates
in solution. When mucin was adsorbed to silica nanospheres (490 nm in diameter), a small increase in electrophoretic mobility was detected, corresponding to
an increase in the absolute value of the zeta potential from 11mV to 13 mV
(pH 6). This increase is probably a result of the high amount of negatively
charged sugar residues (e.g. sialic acid) oriented towards the ambient solution
after mucin adsorption.
Interaction between mucin and unmodified particles
The binding of unmodified particles to adsorbed mucin is illustrated in figure
17, which shows the adsorbed amount and thickness versus time. It is evident
that a small increase in adsorbed amount could be detected at pH 4 and that
the adsorption was reversible with respect to rinsing. However, no significant
changes could be detected upon addition of particles at pH 6. Thus we could
conclude that the interaction between adsorbed mucin and particle was weak
and pH-dependent. These findings are in good agreement with other investigations on the interactions between PEO chains and bovine submaxillary mucin.
For example, the interactions between a lipid bilayer with grafted PEO chains
and mucin in solution were examined by surface plasmon resonance measurements, with the conclusion that the mucin adsorbs reversibly to PEO chains at
60
neutral pH. Also, the interactions between a block copolymer of PEO and
88
PPO and mucin were examined by rheological measurements. Only a small
increase in viscosity was observed when mixing a mucin solution with PEO,
and it was concluded that the interactions between PEO chains and mucin are
weak.
60
200
150
2
100
1
0
20
40
Part.
60
80
Time (min)
100
Rinse
120
Thickness (nm)
3
0
250
Adsorbed amount
Adsorbed amount (mg/m2)
Thickness
4
5
pH 4
2
Adsorbed amount (mg/m )
Adsorbed amount
pH 6
Thickness
4
200
3
150
2
100
50
1
50
0
0
0
0
20
40
Part.
60
80
Time (min)
100
120
Rinse
Figure 17. Adsorbed amount and thickness versus time upon the addition of unmodified particles to a silica surface precoated with mucin (BSM) at pH 4 (left figure) and
pH 6 (right figure). Arrows indicate the addition of particles (Part.) and rinsing (Rinse).
At pH 6 both the mucin and the particles have a higher negative charge than at
pH 4, and we speculated that electrostatic repulsion could prevent the adsorption at pH 6 and explain the pH-dependence. We therefore performed adsorption experiments at a higher electrolyte concentration (150 mM NaCl) at both
pH 4 and pH 6. As we did not observe any effect of increasing electrolyte concentration on the adsorbed amount, we could conclude that electrostatic repulsion was not the factor that prevented the adsorption. Instead, the difference
could be explained by assuming that carboxyl groups (e.g. sialic acids) are important for the interactions. A higher fraction of the carboxyl groups in the
mucin molecule are protonated at pH 4 than at pH 6, and more hydrogen
bonds can form with oxygens in PEO. These hydrogen bonds are expected to
be strong as the hydroxyl groups of carboxyl acids are more polarized than for
example hydroxyl groups of alcohols due to the presence of adjacent carbonyl
89
groups.
Hydrogen bonding between mucin and PEO was also suggested by Efremova
60
and co-workers to explain a stronger interaction at lower pH. Furthermore,
the pH dependent interaction between PEO and carboxylic groups has been
90
demonstrated recently. In this study polymer films were assembled by alternate adsorption of PEO and poly(acrylic acid) at low pH, and these films could
be disintegrated by changing the ambient solution to neutral pH.
61
Thickness (nm)
250
5
To investigate how the surface coverage of mucin affected the adsorption of
particles, a final set of experiments were performed with the unmodified particles at pH 6. These results are shown in figure 18 and it could be concluded
2
that only 0.3 mg/m of mucin is needed to completely prevent the adsorption of
particles. In our experiments the adsorbed amount of mucin varied between 0.6
2
and 1.0 mg/m , and thus we could be confident that the interaction was investigated above the critical value of surface coverage (at pH 6). A second interesting observation that can be made from figure 18 is that, at a mucin surface cov2
erage of 0.25 mg/m , there is a modest increase in adsorbed amount of ap2
proximately 1 mg/m whereas the ellipsometric thickness increases to 230 nm
after addition of particles. The calculated amount of a closed packed monolayer
2
of particles was estimated to be above 100 mg/m and adsorbed amount ac91
cording to the random sequential adsorption (RSA) model was estimated to
2
70 mg/m . These calculations clearly demonstrate that a very low surface coverage results in an ellipsometric thickness that is close to the dimensions of the
adsorbed particles.
250
Mass increase
20
200
Total thickness
15
150
10
100
5
50
Total thickness (nm)
Increase in adsorbed amount (mg/m2)
25
0
0
0
0.2
0.4
0.6
0.8
1
2
Adsorbed mucin (mg/m )
Figure 18. Changes in adsorbed amount and the total thickness of the adsorbed layer
after addition of unmodified particles versus the amount of preadsorbed mucin (BSM)
on silica at pH 6. Lines have been added to guide the eye.
62
Interaction between mucin and modified particles
The interactions with adsorbed mucin were also investigated for modified particles, and figure 19 illustrates the results. Upon addition of particles a significant increase in adsorbed amount and thickness was detected at both pH 4 and
pH 6. In these experiments, the decrease in adsorbed amount and thickness
upon rinsing was low, indicating strong interactions. The obtained result is in
agreement with other studies showing that chitosan particles have good muco92, 93
adhesive properties.
The pH was found to influence the adsorption, and at
pH 4 the adsorbed amount was higher, whereas the thickness was found to be
lower. We explain this difference by a higher amount of protonated amino
groups present at pH 4 that can form electrostatic bonds with negatively
charged groups in the mucin molecule. A lower thickness may be a consequence
of the stronger interaction resulting in pronounced deformation of particles.
25
250
250
Adsorbed amount
pH 6
Thickness
20
200
150
10
100
Rinse
5
50
15
150
10
100
5
50
Rinse
0
0
0
20
40
Mod. Part.
60
80
Time (min)
100
120
0
0
0
20
40
Mod. Part.
60
80
100
120
Time (min)
Figure 19. Adsorbed amount and thickness versus time upon the addition of modified
particles to a silica surface precoated with mucin (BSM) at pH 4 (left figure) and pH 6
(right figure). Arrows indicate the addition of modified particles (Mod. Part.) and rinsing (Rinse).
63
Thickness (nm)
15
Thickness (nm)
2
200
Adsorbed amount (mg/m )
pH 4
Thickness
20
25
2
Adsorbed amount (mg/m )
Adsorbed amount
Drug delivery aspects
The use of nanoparticles in mucosal drug delivery is an expanding field in
pharmaceutical formulations since the interior of the particles may offer protection of the active drug substance and the particles may be designed to provide
94
sustained release properties.
The relation between mucoadhesive properties of nanoparticles and the therapeutic effect of a pharmaceutical formulation may be very complex. However,
it has been shown that chitosan-coating of liposomes enhanced their mucoad92
hesive properties and improved systemic delivery after oral administration.
The improved delivery was attributed to a prolonged retention in the gastrointestinal tract and penetration into the mucus layer. Pulmonary delivery of chitosan-coated nanospheres has also been shown to improve and prolong pharma95
cological action. This result was correlated to the slow clearance of the chitosan modified nanospheres in comparison with the unmodified nanospheres. It
was also suggested that chitosan can enhance drug absorption by opening of
intercellular tight junctions in the epithelium.
To evaluate mucoadhesive properties of particles, interactions with mucins have
been investigated in solution by following changes in electrophoretic mobility
96, 97
98, 99
and size,
or by assessing the amount adsorbed by depletion techniques.
Also, the mucoadhesive properties of polymer-coated liposomes have been
evaluated by counting the remaining liposomes in a dispersion after exposure to
92
mucosal surfaces. However, few other in vitro techniques have been reported
100
that address the mucoadhesive properties of nanoparticles , and from this perspective ellipsometry is an alternative and promising tool to examine the interaction.
Although this investigation has been devoted to the interfacial properties of
nanoparticles, it is important to keep in mind that particle size is crucial for the
diffusion within the mucous gel layer and subsequent uptake through the mu101, 102
cosa.
For instance, it has been shown that the systemic levels of a model
drug after mucosal delivery of particles increased with decreasing size of the
103
particles.
64
SUMMARY AND CONCLUDING REMARKS
Layer-by-layer film formation with mucin (papers I-III)
We have demonstrated that mucins can be used to assemble layer-by-layer films
with oppositely charged polysaccharides or proteins (papers I and II). Construction was performed on solid substrates by alternate adsorption, and the adsorbed amount and thickness of the films were measured in situ by time resolved ellipsometry. The build-up was characterized by adsorption and redissolution processes, but the net increase with the number of adsorption cycles was
approximately linear. Redissolution of polymer complexes from the film was
explained by considering the stability of polyelectrolyte complexes and the extent of redissolution was accounted for by taking the charge density and concentration into account. In addition, it was shown that the substrate properties
influence the amount of mucin adsorbed in the first step and the possibilities for
subsequent layer-by-layer assembly (paper I).
The assembled films are interesting as mucous models, because thick films with
high amounts of mucin can be obtained, which implies that the influence of the
underlying surface will be minimized. Especially interesting are the assembled
films that include human MUC5B mucin and lactoperoxidase, since they only
contain proteins that are naturally present in the mucous gel. It would, for example, be interesting to study how molecules and assemblies of molecules used
in pharmaceutical formulations interact with these films. Despite the fact that
multilayer films are interesting as mucous models, a single layer of adsorbed
mucin may provide a sufficient surface coverage to estimate mucoadhesive
properties as demonstrated in papers IV and V.
65
104
Adsorbed mucin layers may function as lubricating films and antiadhesive
31-33
coatings to prevent cell attachment.
It would therefore be interesting to investigate whether multilayer films with mucin will further improve these functions, as a result of a more complete surface coverage. In addition, the multilayer structures including lactoperoxidase could have additional antimicrobial
properties. It was shown in paper III that lactoperoxidase displays enzymatic
activity when adsorbed to surfaces. The results also indicate that beneficial interactions between mucin and lactoperoxidase occur in terms of lactoperoxidase activity and stability.
Interactions between drug delivery particles and mucin (papers IV-V)
The interactions between lipid nanoparticles stabilized by a poly(ethylene oxide) based polymer and mucin-coated silica surfaces were studied by ellipsometry (paper IV). It was found that the particles adsorbed reversibly at pH 4 while
no adsorption was detected at pH 6. This weak and pH-dependent interaction
is in agreement with other investigations on the interaction between
poly(ethylene oxide) chains and mucin. From the pH and electrolyte dependence on the adsorption we propose that hydrogen bonds between carboxyl
groups (e.g. sialic acids) in mucin and poly(ethylene oxide) is important for the
interaction. In paper V the lipid nanoparticles were surface modified by chitosan. These modified particles showed substantial and strong binding to mucincoated silica surfaces, in agreement with previous reports on this interaction.
The binding of modified particles to the adsorbed mucin can be attributed to
electrostatic interactions between protonated amino groups in chitosan and
negatively charged groups in mucin.
Based on the result it is evident that ellipsometry is a useful tool to evaluate
mucoadhesive properties of nanoparticles. However, additional techniques
could provide a better understanding of the interactions between mucin-coated
surfaces and particles. For example, AFM could be used to confirm deformation of particles suggested from ellipsometric measurements and neutron reflection can be used to obtain density profiles of the adsorbed layers.
66
POPULÄRVETENSKAPLIG
SAMMANFATTNING
Den yttersta delen av kroppens slemhinnor utgörs av en viskös proteinfilm med
hög vattenhalt som benämns mucus. Dess biologiska funktion är att skydda den
underliggande vävnaden från uttorkning och mekaniska påfrestningar. Denna
barriär fungerar även som ett skydd mot bakterieangrepp och skadliga substanser. Den största gruppen proteiner i mucus utgörs av muciner, som är negativt
laddade vattenlösliga molekyler med hög molekylvikt. Dessa egenskaper gör att
muciner kan bilda en gel vid låga koncentrationer och binda mycket vatten. Eftersom muciner är den största och viktigaste gruppen av molekyler i mucus, har
forskningen varit inriktad på att undersöka hur muciner binder in till andra
molekyler som exempelvis proteiner och ämnen som återfinns i läkemedel.
Den första undersökningen genomfördes för att studera möjligheterna att återskapa den proteinfilm som utgör den yttersta delen av slemhinnan genom att
binda samman muciner med motsatt laddade molekyler. Undersökningen visar
att det är möjligt att bygga upp filmer med muciner i kombination med kitosan
(en positivt laddad polysackarid) och med laktoperoxidas (ett positivt laddat
protein som återfinns naturligt i mucus). Uppbyggnaden av dessa filmer har visat sig vara komplex och mycket arbete har ägnats åt att förklara och optimera
uppbyggnadssprocessen. Det har bland annat visat sig att egenskaperna hos
ytan som filmerna byggs upp på kan vara avgörande för om uppbyggnaden är
möjlig, och att koncentrationerna av de molekyler som bygger upp filmen påverkar tillväxten. Molekylernas laddningstäthet är en annan faktor som har beräknats för att bättre kunna förklara hur uppbyggnaden fungerar.
Slutsatsen från dessa undersökningar är att det går att bygga upp filmer med
muciner och motsatt laddade molekyler samt att bindningar mellan muciner
67
och motsatt laddade molekyler kan vara viktiga för den sammanhållande proteinstrukturen i mucus. Studierna har även bidragit till en ökad generell förståelse
för hur filmer kan byggas upp med motsatt laddade proteiner och polysackarider.
De filmer som konstruerats innehåller mycket vatten och kan därför ha en
smörjande effekt, som kan utnyttjas för ytbeläggning av exempelvis kontaktlinser och tandproteser. I detta sammanhang är filmer med polysackarider särskilt
intressanta eftersom de har en bra vattenbindande förmåga. Vidare kan de filmer som byggs upp ha antiadhesiva egenskaper som gör att bakterier har svårt
att binda in till filmerna. Dessutom kan filmer byggas upp med enzymer (proteiner), som har en hämmande effekt bakteriernas tillväxt. Ett exempel på denna
typ av antimikrobiella enzymer är laktoperoxidas som vi använde i uppbyggnaden med muciner. Ett annat tillämpningsområde för filmer som byggts upp med
proteiner är utvecklandet av biosensorer. Principen för biosensorer bygger ofta
på att enzymer används för att detektera ett visst ämne. I den typ av filmer som
konstruerats kan en stor mängd enzymer bindas och vidare kan enzymerna stabiliseras i filmerna. Detta kan leda till utveckling av sensorer med bättre detektionsförmåga och ökad livslängd.
Som en andra del i forskningsarbetet studerades partiklar som kan fungera som
bärare av läkemedel. Läkemedelsbärare i form av partiklar är intressanta eftersom de kan innesluta de aktiva substanserna och därmed skydda dem från nedbrytning innan de tas upp genom slemhinnan. Partiklarna kan även ge en depåeffekt genom att den aktiva substansen frisätts långsamt från partiklarna till
följd av att partiklarna bryts ner eller att de aktiva substanserna diffunderar
(sipprar ut) från partiklarna. Dessutom kan partiklar som är mindre än cirka en
mikrometer (en tusendels millimeter) tas upp intakta genom slemhinnan. Hur
pass effektiva partiklarna är på att förmedla de aktiva substanserna i kroppen
beror till stor del på deras storlek, men man har även visat att partiklarnas ytegenskaper har betydelse. Tidigare studier visar bland annat att de partiklar
som binder in bättre till slemhinnan också ökar upptaget i kroppen.
Partiklar eller andra substansers förmåga att binda in till slemhinnan går under
benämningen mucoadhesion och syftet med studien var att utveckla en metod
för att kunna studera partiklars mucoadhesiva egenskaper. Kortfattat så skapades ett mucinlager på en fast yta genom att låta muciner spontant binda in från
en omgivande lösning. I ett andra steg tillsattes en lösning med partiklar och
68
genom att mäta partiklarnas inbindning till muciner på ytan erhölls ett mått på
partiklarnas mucoadhesiva egenskaper.
De partiklar som studerades är cirka en halv mikrometer i diameter och består
av en inre lipidstuktur, som syftar till att bära de aktiva substanserna. För att
stabilisera partiklarna i en vattenlösning innehåller partiklarna även ytaktiva
molekyler som har en fettlöslig och en vattenlöslig del. Den vattenlösliga delen
av dessa molekyler består av polyetylenoxid (polyetylenglykol) och utgör partiklarnas yttersta delar. Genom att ytmodifiera dessa partiklar med kitosan
skapades även en annan typ av partiklar. I undersökningen jämfördes de omodifierade partiklarna med de modifierade partiklarna med avseende på inbindningen till mucinfilmer.
Resultaten visade att en mycket liten mängd av de omodifierade partiklarna
band in till mucinfilmen. Inbindningen av de modifierade partiklarna var å
andra sidan stor, och resultaten tyder även på att bindningen är stark. Dessa
resultat stämmer väl överens med undersökningar som andra genomfört beträffande muciners interaktioner med polyetylenoxid och kitosan. I studien har
även pH-värde och salthalt varierats för att bättre förstå hur molekylerna på
ytan av partiklarna interagerar med mucin. Våra resultat tyder på att vätebindningar mellan polyetylenoxid och mucin är den viktigaste typen av växelverkan
mellan dessa molekyler. För modifierade partiklarna pekar resultaten på att positivt laddade grupper i kitosan är viktiga för bindningen till muciner.
Slutsatsen är att den metod som vi utvecklat är användbar för att uppskatta
mucoadhesiva egenskaper hos partiklar och deras förmåga att binda in till
slemhinnan. Studierna har även visat att ytan på partiklarna enkelt kan modifieras med kitosan och denna typ av partiklar kan vara intressanta som läkemedelsbärare, genom att förbättra upptaget av aktiva substanser.
69
ACKNOWLEDGEMENT
Först och främst vill jag ge ett stort tack till min handledare Thomas Arnebrant,
som har ställt upp i alla lägen och som har visat vägen till hur man presenterar
sin forskning med vederbörlig veteskaplig stil och klass. Min andra handledare,
Tautgirdas Ruzgas har kommit med många reflektioner och nya infallsvinklar
och hjälpt mig i under min senare tid som doktorand. Samarbetet med forskarkolleger och medförfattare vid Malmö högskola har varit utmärkt och jag vill i
detta sammanhang nämna Ida Svendsen, Liselott Lindh, Marité Cárdenas, Karolina Haberska och Sergey Shleev. Vitaly Kocherbitov och Tobias Halthur
med flera har givit mig många och intressanta vetenskapliga samtal under min
tid här. Som extern samarbetspartner vill jag framhålla Krister Thuresson, som
har gett mig mycket stöd och uppmuntran.
Ekonomiskt stöd har erhållits från KK-stiftelsen (Biofilms – Research center for
Biointerfaces), Stiftelsen Gustav Th Ohlssons fond och Malmö Högskola. Mikael Nilsson har granskat språket på ett utmärkt sätt och Ulla Gertsson har
svar på de flesta av de jordiska frågor som rör tryckandet av en avhandling.
Enheten för biomedicinsk laboratorievetenskap är en gemytlig och hemtrevlig
arbetsmiljö, och vi har haft många trevliga stunder. Peter och Linda har hängt
med som trogna följeslagare på den fyra år långa berg och dalbanan, tillsammans med många andra doktorander här.
Sist men inte minst vill jag tacka mina föräldrar Aki och Torsten. Ni har varit
ett stort stöd under alla år, och de sista dagarna med manushets - tack för alla
kloka ord och korrekturläsning. Tillsammans med mina bröder Linus och Harald utgör ni den bästa familj som man kan önska sig. Och vad vore livet utan
min kära Pamela från Santiago.
70
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APPENDIX
Fresnel coefficients for light polarized in the plane of incidence (rp) and perpendicular to the plane of incidence (rs):
rp =
n1 ⋅ cosα 0 - n0 ⋅ cosα 1
n1 ⋅ cosα 0 + n0 ⋅ cosα 1
rs =
n0 ⋅ cosα 0 - n1 ⋅ cosα 1
n0 ⋅ cosα 0 + n1 ⋅ cosα 1
α0
n0
n1
α1
α0 = angle of incidence
α1 = angle of refraction
n0 = refractive index of ambient medium
n1 = refractive index of surface
n0 sin α0 = n1 sin α1
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If the optical model is composed of more than one interface the reflection coefficients are calculated for each interface and combined pair wise to obtain the
overall reflection coefficients.
rp (comb.) =
rs (comb.) =
d=
rp01 + rp 12 ⋅ e − id
1 + rp 01 ⋅ rp12 ⋅ e −id
rs 01 + rs 12 ⋅ e − id
1 + rs 01 ⋅ rs 12 ⋅ e −id
4 ⋅ π ⋅ d1 ⋅ n1 ⋅ cos α 1
λ
α0
n0
n1
α1
α2
αx = angle of incidence/angle of refraction
λ = wavelength of light in vacuum
nx = refractive index
d1 = thickness of the intermediate phase
82
n2
d1
Malmö University Health and Society Doctoral Dissertations
Ross, M. W. Typing, doing and being. A study of men who have sex with men and
sexuality on the Internet. 2006:1
Stoltz, P. Searching for meaning of support in nursing. A study on support in family care
of frail aged persons with examples from palliative care at home. 2006:2
Gudmundsson, P. Detection of myocardial ischemia using real-time myocardial contrasts
echocardiograpy. 2006:3
Holmberg, L. Communication in palliative home care, grief and bereavement. A
mother’s experiences. 2007:1
Ny, P. Swedish maternal health care in a multiethnic society – including the fathers.
2007:2
Schölin, T. Etnisk mångfald som organisationsidé. Chefs- och personalpraktiker i äldreomsorgen. 2008:1
Svensson, O. Interactions of mucins with biopolomers and drug delivery particles.
2008:2
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See www.mah.se/muep
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