Download Shenyang Pharmaceutical University LAB 10: LIPOSOMES

Shenyang Pharmaceutical University
LAB 10: LIPOSOMES
PHARMACEUTICS III
LABORATORY 10: Preparation of liposomes and
determination of encapsulation efficiency
1. LABORATORY OBJECTIVES
a) To learn how to prepare liposomes using the thin-film dispersion method.
b) To learn how to determine encapsulation efficiency of liposomes using a cation exchange
resin.
c) To know the principles for the formation of liposomes and the methods of characterization.
d) To understand the concept of “active” and “passive” drug loading into liposomes.
2. INTRODUCTION
Liposomes are microscopic vesicles consisting of an aqueous core which is enclosed
by a bilayer membrane of lipid molecules (usually phospholipids). These lipid molecules
possess a hydrophilic and a lipophilic part. Liposomes form spontaneously when theses
lipids are dispersed in an aqueous medium such as water or a buffer solution. In the bilayer
membrane, the lipophilic part of a lipid molecule is associated with the lipophilic part of
another lipid molecule. The hydrophilic part of one lipid molecule encloses the inner
aqueous core and the hydrophilic part of another lipid molecule forms the outer layer of the
bilayer membrane, orienting towards the external aqueous phase.
Phospholipids capable of forming liposomes include natural and synthetic
phospholipids such as natural egg phosphatidylcholine, soybean phosphatidylcholine,
lecithin and synthetic dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine and
so on. Cholesterol, a zwitterionic or a neutral lipid, is used as the basic lipid for the
preparation of liposomes. The incorporation of cholesterol into the lipid bilayer membrane
generally enhances the stability of liposomes by reducing the permeability of the
membrane to water soluble molecules and decreasing the fluidity or increasing the
microviscosity of the bilayer. The net surface charge of liposomes can be modified by the
incorporation of either positively charged lipids such as stearylamine or negatively charged
lipids such as diacetylphosphate, phosphatidyl glycerol and phosphatidyl serine.
According to their microstructures, liposomes are classified as small unilamellar
vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs).
The size of SUVs can range from approximately twenty to fifty nanometers in diameter.
They are consisted of a single lipid bilayer surrounding an aqueous compartment and can
be obtained by applying ultrasonic treatment to MLVs. LUVs are named because of their
large size with a diameter ranging from about 200 nanometers to 1 micron and are often
prepared by the injection method. MLVs have the size ranging from 400 nanometers to 3.5
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microns in diameter and show a multi-compartmental structure (under a microscope)
which is similar to the cross-sectional appearance of an onion or a human fingerprint.
There are a variety of methods applicable for preparing liposomes. (a) The thin-film
dispersion method. This is the most commonly used method for the preparation of MLVs
and SUVs can be obtained by subjecting MLVs to ultrasonification. Liposomes with the
characteristic bilayer structures can be readily prepared by using this method, but the
encapsulation efficiency is usually low. (b) The injection method (ether and ethanol
injection methods). The ether injection method involves the injection of an immiscible
ether solution of the lipid very slowly into an aqueous phase maintained at 55-65℃ with
additional stirring for 1-2h. At such a high processing temperature, ether will be removed
by evaporation leading to the formation of liposomes. (c) The reversed-phase evaporation
method. An emulsion is formed with the aqueous solution containing the drug and the
organic solvent containing the lipids. The organic solvent is later removed by evaporation
under reduced pressure resulting in the formation of liposomes. This method is suitable for
the encapsulation of water soluble drugs and biomacromolecules such as insulin due to its
high encapsulation efficiency of aqueous phase. (d) The freeze drying method. This
method is suitable for preparing liposomes containing drugs unstable in an aqueous
solution. (e) The fusion-method. The vesicles formed by using this method are multiphasic
liposomes which exhibit good physical stability and can be sterilized by the exposure to
high temperature.
According to the different drug loading principles, the preparation methods of
liposomes can be divided into “active loading” and “passive loading” method. In the
“active loading” method, the drugs can be loaded by creating a diffusion gradients for the
ions or drugs across the external and internal aqueous phases such as the K+-Na+ gradient
and H+ gradient. The most commonly used method is the “passive loading” technique by
which the drug is encapsulated by introducing an aqueous phase of a water-soluble drug or
an organic phase of a lipid-soluble drug before or at some stage during the manufacture of
the liposomes. In this process, owing to the same drug concentration across the bilayer of
the liposome, the percentage encapsulation depends on the affinity of drug to the lipid
membrane, the lipid composition of membrane, the volume of internal aqueous phase, the
concentration of liposomes formed, and the drug-to-lipid ratio. For lipid-soluble drugs with
a high affinity to the lipid membrane, the “passive loading” method should be used and
high drug encapsulation efficiency can be achieved. When the microencapsulation of an
amphoteric drug is considered, because their oil/water partition coefficient can be
significantly affected by pH and ionic strength of the medium, a slight change in the
entrapment conditions can show great impact on the encapsulation efficiency.
Key testing parameters for evaluating the quality of liposomes include the particle size,
size distribution, and encapsulation efficiency. Among these parameters, encapsulation
efficiency is an important indicator for the quality of liposomes. The methods commonly
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used to determine encapsulation efficiency include gel filtration column chromatography,
ultracentrifugation, and ultrafiltration. In the cation exchange resin method, the free drug
with a positive charge (not being entrapped into liposomes) can be separated from the
negatively charged liposomes by being bound to the anionic group in the resin.
3. METHODS
3.1 Preparation of blank liposomes
3.1.1 Formulation
Soybean phosphatidylcholine
0.9 g
Cholesterol
0.3 g
Dehydrated alcohol
1-2 mL
Phosphate buffer
q.s.
Total volume of liposome suspension
30 mL
3.1.2 Procedures
a) Preparation of a phosphate buffer solution (PBS): Weigh 0.37 g of sodium phosphate
dibasic (Na2HPO4·12H2O) and 2.0 g of sodium phosphate monobasic (NaH2PO4·2H2O)
and dissolve both ingredients in 1000 mL of distilled water. The pH of the solution
should be around 5.7.
b) Weigh 0.9 g of Soybean phosphatidylcholine and 0.3 g of cholesterol in a 50 mL beaker,
and add 1-2 mL of dehydrated alcohol, followed by stirring the mixture at the
temperature of 65~70℃ until the lipid is dissolved. Dried lipid film can be prepared
from this solution by using a Rotary Evaporator. (Alternatives: the dried lipid film can
also be formed in a small beaker by spreading the solution over the wall of beaker and
evaporate the alcohol by an air stream produced using a rubber pipette bulb).
c) Place 30 mL of PBS into another beaker and keep it in a thermostatic water bath with a
temperature of 65-70℃.
d) Add 30 mL of the PBS obtained from (c) into the beaker with the dried lipid film,
followed by hydrating the lipid film in a thermostatic water bath at a temperature of
65-70℃ for 10 min under stirring with a stirring bar on a magnetic stirring plate. After
hydration, stir the suspension for another 30~60 min at room temperature. Adjust the
total volume to 30 mL with distilled water and the blank liposomes are obtained.
e) Examine the morphology of the liposomes under an optical microscope using an oil
immersion lens and record the structure and particle size of the liposomes (including
the maximum and average size).
3.1.3 Notes
a) Flame is not allowed during this experiment.
b) The alcohol solution containing lipids should be clear and should not be kept in the
water bath for a long time.
c) The dried film formed by phospholipids and cholesterol should be as thin as possible.
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PHARMACEUTICS III
d) During the first 10 min of the hydration process, make sure that all the lipid film on the
wall of the beaker has been hydrated completely to form a homogeneous suspension
without any visible pieces of lipids.
3.2 Preparation of liposomes containing berberine hydrochloride by the “passive
loading” method
3.2.1 Formulation
Soybean phosphatidylcholine
0.6g
Cholesterol
0.2g
Anhydrous alcohol
1-2 mL
Berberine hydrochloride solution (1mg/mL)
30 mL
Total volume of liposome suspension
30 mL
3.2.2 Procedures
a) Preparation of the berberine hydrochloride solution: Dissolve the predetermined
amount of berberine hydrochloride in PBS to give a solutions with the concentrations
of 1mg/mL and 3mg/mL respectively.
b) Preparation of liposomes containing berberine hydrochloride: Weigh the prescribed
amounts (in the formulation above) of soybean phosphatidylcholine and cholesterol in
a small beaker, add about 1-2 mL of anhydrous alcohol, and follow the same
procedures as described in “3.1.2” except substituting the PBS buffer with the
berberine hydrochloride solution.
3.2.3 Notes
Pay attention to the same precautions as those described in “3.1.3”.
3.3 Preparation of liposomes containing berberine hydrochloride by the “active
loading” method
3.3.1 Procedures
a) Preparation of a citrate buffer solution: Weigh 10.5 g of citric acid and 7.0 g of sodium
citrate, transfer to a 1000mL volumetric flask, add distilled water to 1000mL and mix
until complete dissolution.
b) Preparation of a sodium bicarbonate (NaHCO3) solution: Weigh 50 g of NaHCO3,
transfer to a 1000mL of volumetric flask, add distilled water to 1000mL and mix until
complete dissolution.
c) Preparation of blank (empty) liposomes: The procedures are the same as those
described in “3.1”, except that PBS is replaced with citrate buffer. The blank liposome
suspension is passed through a 0.8 μm microporous filter membrane twice to achieve
a uniform size distribution.
d) Active loading drug: Introduce 2mL of the blank liposomes into a 10mL of vial, add
1mL of 3mg/mL of berberine hydrochloride solution and 0.5mL of NaHCO3 solution
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(50mg/mL) while shaking the vial. After mixing well, keep the vial for 20min in a
thermostatic water bath at a temperature of 70℃ and then cool it down immediately
with cold water.
3.3.2 Notes
a) The order of addition of the formulation ingredients is important and the order should
not be altered during the “active loading” process. When adding the ingredients,
continuous shaking is required to ensure uniform mixing.
b) When incubating the vials in a water bath, a moderate degree of shaking should be
used but vigorous shaking should be avoided.
c) During the cooling process, the vial should also be shaken gently.
3.4 Determination of encapsulation efficiency of berberine hydrochloride in liposomes
3.4.1 Packing of cation exchange resin column
Remove the plunger from a 5-mL disposable plastic syringe, plug the barrel with a
glass cotton pad, and fill the barrel to the top with well-conditioned cation exchange resins.
The top of the bed should be level at the 4mL mark and the column is equilibrated with
PBS.
3.4.2 Investigation on the separation efficiency of the column
a) Preparation of a liposome mixture: Mix 0.1mL of 3mg/mL of berberine hydrochloride
solution with 0.2mL of blank liposomes obtained from 3.1.
b) Preparation of reference solution: Add 0.1mL of the mixture obtained from step (a)
into a 10mL of volumetric flask, add 6mL of 95% alcohol, dissolve the mixture in the
alcohol by shaking, make to the final volume with PBS, mix again, and then filter this
solution with a filter paper. After filtration, transfer 4mL of the filtrate to a 10mL of
volumetric flask and make to volume with PBS followed by mixing.
c) Preparation of sample solution: Add exactly 0.1mL of the mixture obtained from step
(a) drop wise to the top of the cation exchange resin bed and allow to stand for 5min.
Elute with 2~3mL of PBS (pay attention not allowing the resins on the top of the
column to be disturbed), collect the eluant in a 10mL volumetric flask, add 6mL of
95% alcohol, shake the flask to ensure complete dissolution, and then make to the
final volume of 10mL with PBS. Mix again and filter this solution with filter paper.
Use the filtrate as the sample solution.
d) Preparation of the blank solvent: Transfer 30mL of 95% alcohol in a 50mL of
volumetric flask, add PBS to the volume and mix until uniform.
e) Absorbance determination: Measure the absorbance of the sample and reference
solution respectively at 345nm against the blank solution. Calculate the separation
efficiency of the column as described in Equ.1, which should be larger than 0.95.
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resolution  1 
LAB 10: LIPOSOMES
A sample
A reference 25
PHARMACEUTICS III
Equ.1
where, Asample and Areference are the absorbance of the sample solution and the
reference solution, respectively. 2.5 is the dilution factor for the reference solution.
3.4.3 Determination of encapsulation efficiency
Take two 0.1mL samples of the berberine hydrochloride liposome mixture. Add one
sample to a 10mL of volumetric flask and treated by the same procedures as those
described in 3.4.2-b. Add the other sample drop wise to the top of the cation exchange
resin column and follow the same procedures as those described in 3.4.2-c. After
processing, measure the absorbance of the samples at 345nm against the blank solution.
Calculate the encapsulation efficiency (EE) according to Equ.2.
A
Equ.2
EE%  L  100%
AT
where, AL is the absorbance of berberine hydrochloride encapsulated in liposomes after
column separation. AT is the absorbance of the total drug in the liposome suspension.
4. RESULTS AND DISCUSSION
4.1 Draw the morphology of liposomes as observed under a microscope and discuss the
differences in the shape of liposomes, emulsions, and microcapsules.
4.2 Record the particle size of liposomes measured under a microscope including the
maximum and average size.
4.3 Calculate the column separation efficiency and drug encapsulation efficiency.
4.4 Based on the encapsulation efficiency of berberine hydrochloride in liposomes,
evaluate the advantages and disadvantages of liposome preparation by the “active
loading” and the “passive loading” methods with respect to berberine.
5. QUESTIONS
a) Discuss the characteristics of liposomes used as the carrier of drugs and the factors
affecting the formation of liposomes.
b) How to increase the encapsulation efficiency of liposomes?
c) How to select the analytical methods for determining the encapsulation efficiency of
liposomes? What are the advantages and disadvantages of the cation exchange
resins method as compared with the gel column chromatograph method and the
ultracentrifugation method?
d) Design a new experiment for the preparation of liposomes and describe how the
results can be improved by using this new experiment?
(Li YANG)
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