CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 9, (2004) pp 603 – 615

http://www.cmbl.org.pl

Received 13 February 2004

Accepted 9 July 2004

A SINGLE-STEP METHOD OF LIPOSOME PREPARATION

ZYGMUNT H. ZAWADA
Department of Physical Pharmacy, Medical University of Silesia, Jagiellońska 4,
41-200 Sosnowiec, Poland

Abstract: All the liposome preparation protocols, which involve drug

encapsulation are multi-step processes, i.e. they consist of one or several steps of
preparation and homogenization. The conditions of converting all lipids into
vesicles smaller than 200 nm were determined by replacing ultrasonication with
mechanical stirring of the buffer and solution of lipids in a low-boiling point
organic solvent or solvents in a simple preparator. Preferably, the process should
be carried out at a temperature higher than the temperature of the gel/fluid phase
transition (Tm), and higher than the boiling point of the organic solvent(s) used
to obtain the lipid solution. For many lipid membrane compositions, the products
of preparation are as follows: a dominant fraction of unilamellar vesicles (vesicle
of diameter smaller than 200 nm) and a fraction of much larger multivesicular or
multilamellar vesicles, easily separated by simple centrifugation at 15000×g.
If PEG-phosphatidylethanolamine or cholesteryl palmitate are additional
membrane components, multivescular or multilamellar vescicles are virtually
absent in the final product, of a single-step process and all the used lipids were
quantitatively converted into vesicles smaller than 200 nm in diameter.

Key Words: Reverse Phase Evaporation, Double Bilayer Vesicles, Cholesteryl

Palmitate, PEG-PE, NBD-PE

Abbreviations used: SUV - small unilamellar vesicles; LUV - large unilamellar vesicles;
MLV - multilamellar vesicles; MVV - multivesicular vesicles; EPC - egg
phosphatidylcholine; DPPC - 1;2-dihexadecanoyl-sn-glycero-3-phosphocholine; DPPE -
1;2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; NBD-PE - N-(7;nitrobenz-2-
oxa-1,3-diazol-4-yl)-1;2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethyl-
ammonium salt; Chol - cholesterol; pChol - cholesteryl palmitate; cChol - cholesteryl
phosphoryl choline; LPC - 3-sn-lysophosphatidylcholine; PEG (2000) - polyethylene
glycol; SPC - 3-sn-phosphatidylcholine from soybean; SA - octadecylamine; PB - Patent
Blue V; LNBD - vesicles labeled with NBD-PE; TRIZMA - tris(hydroxymethyl)-
aminomethan; PEG-PE - 1,2-dihexadecanoyl-sn-glycero-3-phosphoethano-lamine with
covalently attached poly(ethylene glycol); FAT MLV - freeze thawed multilamellar
vesicles; REV - reverse phase evaporated vesicles; LUV ET - extruded large unilamellar
vesicles.
604 CELL. MOL. BIOL. LETT. Vol. 9. No. 4A. 2004

INTRODUCTION

Liposomes are prepared by many methods and the obtained vesicles may vary
significantly in terms of diameter and a number of bilayers. Liposomes are
classified as small or large unilamellar vesicles (SUV, LUV), multilamellar
vesicles (MLV) and multivesicular vesicles (MVV) containing several vesicles
and, in consequence, several separate aqueous phases [1]. The bilayers of all
MVVs and MLVs have identical lipid composition, which is their characteristic
feature. The majority of the liposome preparation methods are based on either
dry lipid hydration or the evaporation of an organic solvent in which the lipids
are dissolved directly into an aqueous phase. The methods based on dry lipid
hydration are originally multi-step processes (organic solvent evaporation from
lipid solution, lipid drying, hydration, calibration, and other). The methods based
on the injection of ethanol or ether lipid solution into the buffer results in small
vesicles, useful only as model membranes [2, 3]. There are several multi-step
methods based on the aqueous phase dispergation in organic, lipid solubilizing
solvents, and on the organic solvent evaporation directly during vesicle
formation. The reverse-phase evaporation method is the oldest of these [4]. In
accordance with the original procedure, an aqueous phase is dispergated in an
organic solvent (ethyl ether, halothane, chloroform, methylene chloride or other)
to form a water-in-oil emulsion by sonicating the mixture of both of these
phases. Next, the emulsion is transfered to a rotary evaporator, and the solvent is
removed under reduced pressure. At this stage, some of the aqueous phase
droplets combine and form the environment where buffer droplets, enveloped in
lipid membrane, are suspended. Then, the aggregates are centrifuged and the
supernatant is filtered to obtain LUVs, smaller than the defined sizes of filter’s
pores. Another method using double emulsion characteristics is based on
aqueous emulsion formation in chloroform and ethyl ether solutions of
liposomal lipids by mechanical agitation with a shaker. Constant bubbling of
nitrogen agitates the combined emulsions and unilamellar [5] or multivesicullar
vesicles are formed [6]. During the preparation of multi-component model
membranes, a primary consideration is to maintain the compositional
homogeneity of the suspension. This may be obtained when the substitution of
the hydrophobic solvents into the buffer is quick [7]. When liposomes are
introduced into bloodstream, they must be smaller than 200 nm in diameter. In
accordance with this and other requirements, it is beneficial to form vesicles
smaller than 200 nm in a quick (compositional homogeneity) single-step
process.
In this paper, two devices and the conditions of converting (by using of these
devices) all the used lipids into LUVs (smaller than 200 nm) in a single-step
process are described. This preparation method is a substantial modification of
reverse-phase evaporation method.
CELLULAR & MOLECULAR BIOLOGY LETTERS 605

MATERIALS AND METHODS

Components of the vesicleses and other chemicals

Egg lecithin was purified according to the Singleton method [9], and kept in
sealed glass vials under nitrogen at -20ºC until needed. DPPC, DPPE and NBD-
PE were obtained from Molecular Probes (Eugene). Cholesterol was obtained
from Mallinckrodt Inc. (St. Louis). Cholesteryl palmitate, cholesteryl phosphoryl
choline, 3-sn-lysophosphatidylcholine, PEG-2000, SPC, octadecylamine, and
Patent Blue V and other reagents were purchased from Fluka (Buchs).
Phosphatidic acid was purchased from Sigma Chemical Co. (St. Louis).
Sephacryl-S1000 was obtained from Pharmacia-LKB (Uppsala). Solvents were
obtained from POCH (Gliwice). All the organic solvents were freshly distilled
before use. PEG-PE was synthesized by the activation of PEG-2000 with
disuccimidyl carbonate, followed by condensation with DPPE in chloroform in
the presence of triethylamine [10]. The PEG-PE derivative with a chain ending
with a methoxy group was used for the liposome preparation.

Devices for vesicle preparation

A single-step protocol of liposome preparation was achieved using the device,
which is schematically depicted in Fig. 1A. A 30-cm long, jacketed
chromatographic column (35 mm in diameter) was modified by inserting teflon
stoppers, custom-engineered to guide a multi-paddle mixer, and by adding
openings equipped with a funnel (top) and a drain-cock (bottom). This device
facilitates the preparation of 10-30 ml of liposome solution. At the top and
bottom ends of the column, there are teflon guides (1) of a multi-paddle mixer
(2) with a paddle of 3.4 cm in diameter. In the top guide, there is an additional
opening for a funnel through which the preparator is inserted. In the bottom
guide, there is a drain-cock (3) through which the vesicle suspension is drained
off after the preparation procedure. The preparator has N2 or Ar 10-20 (ml/min.)
blown through it to remove O2 and organic solvents from the “dead” space of the
preparator during the preparation.

Standard procedure of liposome preparation

DPPC (250 µM) and 50 µM Chol were diluted into 100 ml of the mixture of
methylene chloride/chloroform (10:1 v/v). The obtained solution was the organic
phase. The large system (Fig. 1A) was filled with 30 ml of saline buffered with
0.01M TRIS-HCl, pH=7.4. The level of this solution was marked on a wall of
the thermostating jacket. Next, described above the organic phase was added.
Through the opened drain-cock (3), nitrogen gas was supplied via a rubber
tubing at a flow rate of 10-20 (ml/min). The mixer (driven by a 60 W motor of a
standard laboratory mixer with a flexible shaft ML-2, from Mechanika
Precyzyjna, Kraków, Poland) was turned on and set at 1800 rpm. A two-phase
system, water-in-oil, was formed during stirring at 47ºC. After 10-20 minutes,
low-boiling point organic solvents evaporated, and the level of the vesicle
suspension decreased to the mark on the jacket wall (the level when only buffer
606 CELL. MOL. BIOL. LETT. Vol. 9. No. 4A. 2004

is present in the preparator). The resulting vesicle suspension contained 1%

(w/v) lipids. When, during the preparation process, the concentration of
phospholipids in aqueous phase was in the range of 1-4% (w/v), a “creamy”
phase was initially formed, which subsequently broken into the vesicle
suspension.

Fig. 1. The scheme of vesicle preparation systems for the modified reverse-phase
evaporation method: A – a large device for the preparation of 10-30 ml of vesicle
suspension, B – a small device for the preparation of about 3 ml of vesicle suspension.

Next, during the evaporation of the organic solvents, the deposition of

a phospholipid film (which later disappeared) could be observed on the glass
wall. The complete clearance of the inner glass wall of the film was used as
a signal for terminating the whole preparation process (the stirring should be
stopped to observe this). At this time, the drain-cock (3) must be closed and the
nitrogen flow should be stopped. When the lipid concentration was lower than
1% (w/v), the “creamy” phase was not formed and the deposition of
a phospholipid film on the glass wall could not be observed. In this way, the
vesicles from pure: EPC, SPC and DPPC and those one enriched with Chol
and/or SA, PA, cChol, pChol, PEG-PE or NBD-PE were obtained, all in saline
solution buffered with 10 mM TRIS-HCl.
Fig, 1B shows a small, glass device with a capacity of 20 ml and an inner
diameter of 1.5 cm. A wire step mixer, 1.4 cm in diameter, was found to be best
for mixing the solutions (a laboratory micro-stirrer with a flexible shaft and
a rotation speed of 11.000 rpm was used). This small system was filled with 3.2
ml of saline solution containing PB (0.2 mg/ml) buffered with 10 mM TRIS-
HCl, and a lipid solution of 25µM DPPC and 5 µM of Chol in 10 ml of low-
boiling point organic solvents (methylene chloride/diethyl ether/chloroform
5:5:1 (v/v)). The preparation process was carried out in the same way as in the
large system at 47ºC. The size and heterogeneity patterns of vesicles were
determined by dynamic light scattering with a Malvern Zeta Sizer 5000
(Malvern Instruments Ltd, Malvern, UK).
CELLULAR & MOLECULAR BIOLOGY LETTERS 607

Vesicle encapsulation efficiency

The aim of the assay was to compare the efficiency of the aqueous phase
encapsulation within the following liposomes: those which underwent size
calibration performed by extrusion through 0.45 and 0.2 µm pore filters, and the
centrifuged ones, which were larger than 0.2 µm. The suspension (3 ml) of
DPPC/Chol[PB] (100:20 mol%) PB trapping vesicles was divided into three
equal parts. The first was calibrated by 5-fold extrusion through 0.45 and 0.2 µm
pore polycarbonate filters from Nucleopore Inc. (Pleasanton, CA) [11]. The
second was passed through a 0.45 µm pore filter twice. After diluting to 6 ml,
the third was centrifuged at 15000 × g for 30 minutes by using a MPW 375
laboratory centrifuge from Med-Instrument (Warszawa). The lipid phosphorus
content in the pellets was assayed according to Hellen [12]. Untrapped PB from
filtered samples and supernatants were removed on a Sephacryl-S1000 column
(50 × 1.5 cm). According to the suggestions of Reynolds et al. [13] and the gel’s
supplier, the column was pre-saturated with adequate lipids and allowed to
stabilize for at least 2 days at the experimental flow rate (1.2 ml/min). The
sample loading and fraction volumes were 2.0 ml. Untrapped PB was
determined spectrophotometrically (λ=634 nm). The trapped volume was
expressed as ml of PB solution per gram of lipids (as determined by phosphorus
assay). In control experiment the same volume of PB, diluted in a suspension of
empty DPPC/Chol vesicles, was separated on the same column.

Exterior surface fraction of vesicles

Exterior surface fraction here means the part of the outer surface of the
outermost liposome bilayer(s) in relation to the total surface of the all liposome
bilayers. The assay allows the differentiation of LUVs from MLVs or MVVs,
and the determination of the degree of vesicle perforation after ultrafiltration.
Half of the LNBD vesicles were in turn passed five times through 0.45 and 0.2
µm pore polycarbonate filters and diluted to 6 ml. The remaining vesicles were
centrifuged at 15000 × g for 30 minutes. The pellets and supernatants were
diluted to 6 ml. The extruded vesicles, re-suspended pellets and supernatants
were used for the analysis of the exterior surface fraction of vesicles. Before the
analysis, in order to change the environment to basic, each of them was dialyzed
once against 200 ml of 0.1 M TRIZMA buffer, pH=10.5, for 12 hours, at 4ºC.
Next, 200 µl of dialyzed vesicles were dropped into a cuvette containing 3 ml of
TRIZMA buffer and then 30 µl of Na2S2O4 (100 mg of sodium dithionite in 2 ml
of 0.1 M TRIZMA buffer) was added [14-16]. The changes in fluorescence
emission intensity (before and after the addition of sodium dithionite) were
measured at λem=535 nm (exc=463 nm). Both the excitation and emission
bandwidths were 5 nm. In order to avoid possible sedimentation of the vesicles,
the content was gently stirred during all the assays. Fluorescence intensity was
recorded continuously before (2-4 minutes) and after (15 minutes) the addition
of sodium dithionite. After the addition of the dithionite, fluorescence first
decreased rapidly (2-3 minutes) and then was nearly constant. The fluorescence
608 CELL. MOL. BIOL. LETT. Vol. 9. No. 4A. 2004

intensity after a fast reaction was normalized to the ratio of fluorescence before
dithionite addition, to obtain the percentage of fluorescence quenched by
dithionite, i.e. the external surface fraction, in accordance with the protocol
described by Lentz et al. [16].

RESULTS AND DISCUSSION

Standard preparation of liposomes

In both small and large device, approximately 250 vesicle preparations have
been obtained so far, and the relevant vesicles have had similar sizes and
homogeneity patterns, independently of the system used. Liposomes of various
lipid compositions, enclosing various aqueous phases, were obtained. Fig. 2
shows a typical distribution of DPPC/Chol (100:20 mol%) vesicle sizes, as
obtained from the results of dynamic light scattering measurements, prior to
centrifugation. Two fractions of vesicles were most often observed. Tab. 1
shows the fraction participations within the liposomes obtained in the described
preparations for the various lipid compositions used. The distributions of
liposomal sizes reveal the presence of a quantitatively dominant fraction of
vesicles of 80-120 nm in diameter, irrespective of the quantity of lipids used for
the preparation. For the largest quantity of lipids used for the preparation, the
main fraction A (70-130 nm in diameter) is accompanied by relatively few
vesicles (fraction B) of 310-550 nm in diameter or, occasionally, slightly larger
ones. In both the large and small systems, regularities occurred when the
distance between the mixer paddles and the inside wall of the preparator was

Fig. 2. The diameter and heterogeneity pattern of DPPC/Chol (100:20 mol%) vesicles
prepared in the large device before and after centrifugation at 15000 × g for 30 minutes.
The total lipid concentration per aqueous phase (saline buffered with 0.01M TRIS-HCl,
pH=7.4) was 0.6% (w/v), organic solvents: methylene chloride/diethyl ether/chloroform
5:5:1 (v/v), temperature 47ºC. A - fraction of the small vesicles before and after
centrifugation (grey and black, respectively) B - fraction of the large vesicles before and
after centrifugation (grey and black, respectively). Data was obtained from Malvern Zeta
Sizer 5000.
CELLULAR & MOLECULAR BIOLOGY LETTERS 609

about 0.5 mm. Fraction B rapidly increased with increasing distance between the
mixer paddles and the inside wall. Thus, it may be suggested that the
homogenization of the vesicles takes place in the space between the inside wall
of the preparator and the mixer paddles. Liposomes containing stearylamine
were always large, i.e. approximately 2500 nm in diameter. This could be caused
by the binding of positively charged membranes to the negatively charged glass
of the preparator and therefore problems in their disruption.

Tab. 1. The size distribution and heterogeneity pattern of the vesicles obtained in the
small (*) and large preparator. Sizes were determined via the laser light scattering method
on a ZETA SIZER 5000 (MALVERN)**, for details see text.
Fraction A Fraction B
Composition of liposome Entrapped (LUV) (LUV+MLV+MVV)
bilayer volume Diameter Volume Diameter Volume
[mol %] [ml/g] [nm] [%] [nm] [%]
98 ± 7*** 33.9 420 ± 40 42.6
EPC 8.8 ± 2.1 82 ± 9 54.5 420 ± 50 32.4
126 ± 16 56.3 440 ± 80 31.1
78 ± 8*** 54.6 420 ± 35 21.9
EPC(*) 9.1 ± 1.4 88 ± 6 57.7 420 - 700 25.0
69 ± 6 63.5 400 ± 90 26.4
EPC/Chol[100:20] 10.8 ± 1.2 110 ± 19 42.7 450 ± 110 38.5
EPC/Chol/SA[100:10:5] 4.3 ± 1.1 - - 1520 ÷ 2240 100
EPC/Chol/PA[100:10:5] 5.2 ± 2.2 78 ± 18 48.5 430 ± 95 40.6
**-Values of entrapped volume and diameter of liposomes represent mean values ± S.D.
determined from 2-5 preparations. ***Data from preparations with different
concentrations of lipids: 0.25% (top), 0.6% (middle), 3% (bottom) [w/v], respectively.
For the other examples, the concentration of lipids was 0.6% [w/v].

Centrifugation at 15000 × g leads a to nearly complete separation of these two

fractions, i.e. fraction A to the supernatant and fraction B to the pellets. It is
important since a simple centrifugation gives a supernatant containing almost
only liposomes smaller than 200 nm and trace quantities of larger ones. Multiple
filtration of DPPC/Chol vesicles through a 200 nm pore filter, converts them into
vesicles not larger than 200 nm. The data from the Tab. 2 show that the
ultrafiltration of liposomes damages their structures, resulting in the leakage of
the encapsulated solute. Similar results were obtained by Berger et al. [11].
As mentioned above, 0.5 mol% of NBD-PE was added to the vesicle membranes
during preparation to determine their permeability after calibration and to define
the exterior surface fraction of the vesicles, which do not encapsulate PB. NBD-
PE is equally distributed on both sides of bilayer [18]. The NBD-PE fraction
spread in the outer vesicle bilayer was assayed in the dithionite reaction. The
product of the NBD and dithionite reaction does not exhibit fluorescence [14,
15]. When, after the reaction with dithionite, normalized fluorescence decreased
to approximately 50%, LUVs had been obtained. Fluorescence higher than 50%
610 CELL. MOL. BIOL. LETT. Vol. 9. No. 4A. 2004

meant MLVs or MVVs, but significantly lower one than 50% suggested that the
liposomes were leaky. When the liposomes were prepared from EPC, DPPC or
SPC only, fluorescence decreased to 48-51% of the initial value. When the
unfiltered liposomes also contained cholesterol, the fluorescence decreased to
65-78% of its initial value. It can be concluded that, when liposomes were
prepared from only EPC or SPC, the pellets (fraction B after centrifugation) are
mainly large LUVs. When the vesicles were prepared with EPC/Chol,
DPPC/Chol or SPC/Chol, a large MLVs or MVVs were also obtained. For all
supernatants, fluorescence decreased to 48-51% of its initial value, which means
they were LUVs, irrespective of the lipid composition.

Tab. 2. The encapsulation efficiency and exterior surface fraction of vesicles before and
after filtration or centrifugation.
Total vesicles
Filtered Supernatant
Lipid composition of Unfiltered Filtered 5 × 450 nm
vesicles 2 × 450 nm and 5 × 200 only
[mol%] nm
PB encapsulation efficiency [%]
DPPC/Chol[PB] 13.0 ± 1.3* 13.1 ± 1.2 5.1 ± 1.0 6.1 ± 1.0
100:20
EPC/Chol[PB] 13.6 ± 1.4 13.9 ± 1.1 5.3 ± 1.0 5.5 ± 1.4
100:20
Percentage of surface inaccessible to dithionite [%]
DPPC/Chol/NBD-PE 67 ± 3 63 ± 4 29 ± 4 51 ± 1
100:20:0.5
EPC/Chol/NBD-PE 71 ± 4 59 ± 3 26 ± 3 48 ± 3
100:20:0.5
*- mean values ± S.D. determined from 3-5 preparations.

Unfiltered LNBD: DPPC/Chol/NBD-PE (100:20:0.5 mol%) vesicles in reaction

with sodium dithionite exhibited 67 ± 3% of NBD inaccessible to dithionite
(Tab. 2). They exhibited 63 ± 4 % of NBD inaccessible to dithionite after 2-fold
extrusion through a 450 nm pore filter. Having undergone further 5-fold
extrusion through a 200-nm pore filter, they exhibited 29 ± 4% of NBD
inaccessible to dithionite several hours after filtration. This is a sign of
a decrease in vesicle sizes, albeit with simultaneous and significant perforation.
The results are in quantitative agreement with the data on PB encapsulate vesicle
filtration, where an over two-fold decrease of encapsulated PB in vesicles was
observed after multiple extrusion.
The influence of temperature (55, 50, 45 and 40ºC) on the size and heterogeneity
patterns of the prepared vesicles was only studied for EPC/Chol (100:20 mol%)
vesicles. A slight decrease in the average liposomal size of fraction A and
a slight relative increase in fraction B vesicles quantity were observed. Lowering
the preparation temperature necessitates prolonging the preparation, which is
CELLULAR & MOLECULAR BIOLOGY LETTERS 611

connected with an organic solvent evaporation. Hence, it is hard to decide

whether the lower temperature of the preparation or the longer mixing time
caused the changes in liposomal sizes. When the preparation was carried out at
50-55ºC, the process lasted 5-10 minutes in both systems. There was no point in
prolonging the mixing because it had been found that a further 60 minutes of
mixing caused insignificant liposomal size decrease.
The main purpose of the REV method modification was to alter it so as to
facilitate the conversion of all the used lipids into smaller than 200 nm liposomal
vesicles, which could be used as carriers of hydrophilic drugs in a single step
process. The calibration filtering in particular was to be avoided since it leads to
a significant decrease in the degree of drug encapsulation. Sufficient
impermeability of a membrane requires cholesterol presence in the membrane
[17, 18]. The described lipid compositions enable vesicles smaller than 200 nm
to be obtained, but after two stages, i.e. preparation and simple sedimentation.
The alterations of both the preparation temperature and mixing time do not
significantly affect vesicle sizes and heterogeneity pattern. The next stage of
modification included the study of the chosen lipids, i.e. PEG-PE, pChol, cChol
and 1-PC, and their influence on the increase in the membrane curvature, with
preservation of 10 mol% of Chol to ensure membrane impermeability.

PEG-PE or pChol containing liposomes

When a relatively small PEG-PE or pChol quantity was the component of the
vesicles’ lipid composition, unilamellar vesicles with a diameter of 40-80 nm
were formed during the preparation. The total concentration of lipids per
aqueous phase volume used for the preparation should be smaller than 1% (m/v).
The content of PEG-PE (up to 4 mol%) or pChol (up to 5 mol%) used for the
preparation virtually eliminates the possibility of forming multilamellar or
multivesicular vesicles smaller than 200 nm (Tab. 3). A similar effect can be
observed when a small amount of detergent is present in an aqueous phase
(Tween, Brij; data not shown). Apart from liposomes, stacked disc-like
structures on a border of dark bodies (looking like a dark spot [19]) were
observed in the DPPC/Chol/PEG-PE (100:10:6 mol%) vesicles (data not
shown). Dark bodies are amorphous lipid structures that occur in the PEG-
derivatives containing lipid structures [20]. I believe that dark bodies present in
the preparation containing 6 mol% of PEG-PE result from intensive mechanical
mixing.
Depending on the corresponding quantities of PEG-PE and bilayer-forming
lipids, these components may coexist in the solution as vesicles, mixed micelles,
bilayer discs, threadlike micelles, bilayer sheets and dark bodies [20-23]. Betsito
et al. [24], while studying MLVs, estimated that for DPPC/PEG-PE(2000)
vesicles at 7.5 mol% of PEG-PE, the fraction of micelles in the total population
was 10%, approximately. At lower PEG-PE content, the relative optical density
of the suspension significantly increased, which was, according to the authors,
related to MLV conversion into smaller, unilamellar vesicles. Similar results
were obtained for EPC/PEG-PE(2000) vesicles [25]. At high concentrations of
612 CELL. MOL. BIOL. LETT. Vol. 9. No. 4A. 2004

PEG-lipids, the interaction between different PEG-chains gives rise to

a considerable lateral pressure in the liposome membrane [26]. To prevent the
membrane from rupturing, this lateral pressure must be counter-balanced by
bilayer cohesion, which in turn can be increased by the inclusion of cholesterol
[17].

Tab. 3. PEG-PE, pCHol, cChol and 1-PC influence on the diameter and heterogeneity
patterns of vesicles, when a constant concentration of lipids (0.6% w/v) was used for
their preparation.
Lipid composition of Fraction A (LUV) Fraction B
vesicles (LUV+MLV+MVL)
[mol %] Diameter* Volume Diameter* Volume
[nm] [%] [nm] [%]
PC/Chol/PEG-PE[100:10:2] 62±5 95.8 290±19 4.2
PC/Chol/PEG-PE[100:10:4] 49±5 95.4 160±28 4.6
PC/Chol/PEG-PE[100:10:6] 38±3 97.8 165±35 2.2
PC/Chol/pChol[100:5:5] 58±3 95.4 275±32 4.6
PC/Chol/cChol[100:5:5] 79±6 69 405±55 31.0
PC/CHOL/l-PC[100:5:5] 80±6 88 380±40 11.8
*-mean values ± standard errors determined from 3-4 preparations.

Tab. 4. The encapsulation efficiency, external surfaces fraction and entrapped volume of
various liposomal preparations.
External Entrapped Encapsulation
Preparative method surface volume efficiency Reference
fraction [ml/g] [%]
[%]
SUV n.d. 0.5 0.5-1 [4]
MLV n.d. 0.5-6.7 5-15 [4]
FAT MLV 13.4 ± 2.5* 5.02-1.77 31.3-88.6 [32]
LUV n.d. 9.1 5-15 [4]
LUV ET 17-47 n.d. n.d. [11]
Ethanol injection n.d. 0.72 n.d. [2]
Crossflow injection n.d. n.d. 26 ± 1 [31]
Rapid solvent exchange 33.1 ± 1.6* 4.5 ± 0.09* n.d. [7]
REV n.d. 8.1-13.7 30-57 [4]
Modified REV 50 ± 2 4.3-33 13-28 (described
here)**
*-mean value ± S.D. **-vesicles smaller than 200 nm

The conformation of the cholesteryl palmitate is the horseshoe conformation,

where the ester bond is close to the aqueous interface, and the cholesteryl and
palmitate chain extends into the center of the bilayer [27]. Increased amounts of
pChol have been incorporated into sonicated EPC multilayers, but the extent of
the incorporation is still relatively low, i.e. up to 5 mol% [28]. When 20 mol% of
CELLULAR & MOLECULAR BIOLOGY LETTERS 613

Chol is present in the SUV EPC bilayer, the maximum solubility is decreased
from about 5 mol% to about 2-3 mol% [29]. To conclude, about 4 mol% of
PEG-PE and up to 5 mol% of pChol in the EPC/Chol or DPPC/Chol membrane
enable vesicles smaller than 200 nm to be obtained in a single step preparation.
Cholesteryl-phosphoryl-choline increases membrane fluidity [30], but it is
mainly a cholesterol substitute and, as can be observed, does not significantly
facilitate small vesicle formation. Lysophosphatidylcholine, as a weak surfactant
incorporated into a liposomal membrane, facilitates smaller vesicle formation
but a fraction of vesicles larger than 200 nm is still present.
When different protocols of vesicle preparation are compared (Tab. 4), it appears
that none of them is much better than the others. On the one hand, freeze-thawed
multilamellar vesicles (FAT MLV) encapsulate a relatively large amount of the
aqueous phase, but the concentration of ballast lipids from the vesicles damaged
by filtration is the highest one [32]. Though REV vesicles encapsulate a
relatively large quantity of the aqueous phase, they are 200 to 1000 nm in
diameter [4]. To sum up, the described modification of the REV method of
vesicle preparation differs from all the other methods, since the obtained vesicles
are grouped into two fractions instead of a single broad one. The two fractions
are composed of small vesicles of ~100 nm in diameter and large vesicles of 400
nm in diameter, respectively. They can be separated quantitatively by simple
sedimentation. If PEG-PE or pChol are additional components of the membrane,
without involving any additional procedure, almost exclusively vesicles smaller
than 200 nm are obtained, which is important for certain medical uses. After the
separation of the fraction B liposomes, only impermeable unilamellar vesicles
with no ballast lipid structures are present in the supernatant. This can be
particularly useful for the preparation of stealth liposomes, i.e. PEG-vesicles,
smaller than 200 nm, in a single-step process.

Acknowledgements. The Medical University of Silesia (Katowice, Poland)

supported this study.

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