Method of preparing lipid structures

A method of forming lipid structures, such as liposomes is disclosed. Lipids are dissolved in a solvent system containing a water-miscible solvent, such as ethanol, and an aqueous medium. The water:solvent ratio is raised by removing water and solvent in a reverse osmosis system, and replacing lost solvent with added aqueous medium, preferably at a rate which just balances volume loss by reverse osmosis. In one embodiment, the method is used to produce uniform-size liposomes.

1. FIELD OF THE INVENTION 
The present invention relates to a method of preparing lipid structures 
such as liposomes. 
2. REFERENCES 
Gregoriadis, G., in Liposomes, Vol. III. 
Poznansky, M. L., et al, Pharm Revs, 36(4):277 (1984). 
Szoka, F., et al, Proc Nat Acad Sci (USA) 75:4194 (1978). 
Szoka, F., et al, Ann Rev Biophys Bioeng, 9:467 (1980). 
3. BACKGROUND OF THE INVENTION 
The use of liposomes and other lipid structures, such as micro-emulsions, 
micelles, and drug/lipid complexes, for drug delivery has been widely 
proposed. Such lipid structures, and particularly liposomes, have the 
potential for providing controlled "depot" release of an administered drug 
over an extended time period, and of reducing the side effects of the 
drug, by limiting the concentration of free drug in the bloodstream. These 
advantages apply to a variety of routes of administration, including 
intravenous, intramuscular, and subcutaneous, application to mucosal 
tissue, or delivery by inhalation. Where liposomes are administered by 
intravenous delivery, liposomes provide a further advantage of altering 
the tissue distribution of the drug. Liposome drug delivery systems have 
been reviewed (Poznansky, Gregoriadis). 
In the case of liposomes, optimal size for use in parenteral administration 
is generally between about 100 nm and 300 nm. Liposomes in this size range 
can be sterilized by passage through conventional filters having a 
particle size discrimination of about 200 nm. This size range of liposomes 
also favors biodistribution in certain target organs, such as liver, 
spleen, and bone marrow (Gabizon), and gives more uniform and predictable 
drug-release rates and stability in the bloodstream. Liposomes whose sizes 
are less than about 300 nm also show less tendency to agglutinate on 
storage, and are thus generally safer and less toxic in parenteral use 
than larger-size liposomes. 
It may also be desirable to prepare uniformsize liposomes in a selected 
size range less than about 100 nm. For example, small unilamellar vesicles 
(SUVs) having sizes between about 30-80 nm are useful in targeting to 
tumor tissue or to hepatocyte cells, because of their ability to penetrate 
the endothelial lining of capillaries. SUVs are also advantageous in 
ophthalmic liposome formulations, because of the greater optical clarity 
of the smaller liposomes. 
Many techniques for preparing liposomes and other lipid structures have 
been proposed (e.g., Szoka 1983). Typically, prior art liposome 
preparation methods yield liposomes which are heterodisperse, and 
predominantly greater than about 1 micron (1,000 nm) in size. These 
initial heterodisperse suspensions can be reduced in size and size 
distribution by a number of known methods. One size-processing method 
which is suitable for large-scale production is homogenization. Here an 
initial heterodisperse liposome preparation is pumped under high pressure 
through a small orifice or reaction chamber. The suspension is usually 
cycled through the reaction chamber until a desired average size of 
liposome particles is achieved. A limitation of this method is that the 
liposome size distribution is typically quite broad and variable, 
depending on a number of process variables, such as pressure, number of 
homogenization cycles, and internal temperature. Also, the processed fluid 
tends to pick up metal and oil contaminants from the homogenizer pump, and 
may be further contaminated by residual chemical agents used to sterilize 
the pump seals. 
Sonication, or ultrasonic irradiation, is another method that is used for 
reducing liposome sizes by shearing, and especially for preparing SUVs. 
The processing capacity of this method is quite limited, since long-term 
sonication of relatively small volumes is required. Also, localized heat 
build-up during sonication can lead to peroxidative damage to the lipids, 
and sonic probes shed titanium particles which are potentially quite toxic 
in vivo. 
A third general size-processing method known in the prior art is based on 
liposome extrusion through uniform pore-size polycarbonate membranes 
(Szoka 1978). This procedure has advantages over homogenization and 
sonication methods in that several membrane pore sizes are available for 
producing liposomes in different selected size ranges. In addition, the 
size distribution of the liposomes can be made quite narrow, particularly 
by cycling the material through the selected-size filter several times. 
Nonetheless, the membrane extrusion method has limitations in large-scale 
processing, including problems of membrane clogging, membrane fragility, 
and relatively slow throughput 
Co-owned U.S. Pat. No. 4,737,323 for "Liposome Extrusion Method" describes 
a liposome sizing method in which heterogeneous-size liposomes are sized 
by extrusion through an asymmetric ceramic filter. This method allows 
greater throughput rates, and avoids problems of clogging since high 
extrusion pressure and reverse-direction flow can be employed. However, 
like the membrane extrusion method, the filter-extrusion method requires 
post-liposome formation sizing. Further, the method may be limited where 
uniform-size SUVs are desired. 
One limitation of all of the above-mentioned methods is the loss of 
encapsulated material as large liposomes are broken and reformed as 
smaller vesicles. 
In none of the liposome-preparation methods mentioned above are liposomes 
with a narrow, substantially symmetrical size distribution produced. 
4. SUMMARY OF THE INVENTION 
It is a general object to provide a novel method of preparing a variety of 
lipid structures, including liposomes, micelles, and emulsion particles. 
It is a more specific object of the invention to provide a liposome 
preparation method which solves or substantially overcomes above-mentioned 
problems associated with the prior art. 
Another object is to provide such a method which can be used to produce 
uniform-size liposomes, without any requirement for a shearing and/or 
extrusion processing steps after initial liposome formation. 
Still another object of the invention is to provide such a method which can 
be practiced to achieve uniform liposome sizes, and relatively high 
encapsulation rates, and in which loss of non-encapsulated material is 
minimized. 
It is another object of the invention to provide a novel method for 
preparing uniform-size small unilamellar vesicles (SUVs) without requiring 
sonication or other post-liposome formation shearing procedures. 
In practicing the method of the invention, there is first formed a mixture 
of lipids in a single phase solvent system containing a water-miscible 
lipid solvent and water. The water:solvent ratio of the mixture is raised 
by removing solvent and water from the mixture by reverse osmosis, and, as 
the solvent and water are removed, by adding aqueous medium to the 
mixture, until lipid-structure formation occurs. 
In a preferred embodiment of the invention, the addition of aqueous medium 
to the mixture is controlled to balance the removal of solvent and water 
by reverse osmosis, to maintain the volume of the mixture substantially 
constant during lipid structure formation. In the case of liposome 
formation, the resulting liposomes have a narrow, substantially 
symmetrical size distribution. 
According to another aspect of the invention, the average size of the 
liposomes formed can be varied selectively, from about 30-300 nm, 
according to lipid composition and ionic strength of the mixture. By way 
of example, SUVs are formed with neutral lipid components, and with lipid 
components containing 5-10 mole percent negatively charged phospholipid at 
low ionic strength during liposome formation. Liposomes with average sizes 
about 250 nm are formed with lipid components containing 5-10 mole percent 
negatively charged phospholipid at higher ionic strength. 
The invention also includes a unique method for producing SUVs without 
post-liposome formation shear processing. 
These and other objects and features of the invention will become more 
fully appreciated when the following detailed description of the invention 
is read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
A. Lipid/Solvent/Water Mixture 
The lipid/solvent/water mixture formed in practicing the invention is 
composed of lipids, a water-miscible lipid solvent, and an aqueous medium, 
which preferably includes charged solute components. 
Where the method is used in forming liposomes, the lipids are 
vesicle-forming lipids which generally include neutral and negatively 
charged phospholipids, such as phosphatidylcholine (PC) and 
phosphatidylglycerol (PG). The vesicle-forming lipids may also include 
sterol lipids, such as cholesterol, and/or glycolipids, such 
phosphatidylinositol, gangliosides, and the like. A variety of lipids 
having selected acyl chain compositions are commercially available or may 
be obtained by standard lipid isolation procedures. 
One factor in the choice of lipid components in the mixture is the 
size-effect of charged lipid species. For example, egg PC (with no charged 
lipid) produces uniform-size liposomes having average sizes of about 30-60 
nm. By contrast, a lipid composition containing 95% egg PC and 5% egg PG 
yields an average liposome size which is dependent on ionic strength 
(Example 1 and 2). 
Where the method is used to produce other lipid structures, suitable lipid 
components, such as triglycerides for producing emulsions, and a variety 
of amphipathic lipids, such as fatty acids, for producing micelles, are 
employed. 
The water-miscible lipid solvent in the mixture is defined herein as one 
which can be mixed with an aqueous medium in substantially any proportion 
without forming a two-phase system. Preferred solvents are small alcohols, 
such as ethanol, methanol, propanol, and isopropanol, dimethylsulfoxide 
(DMSO). The solvent may contain additional solvent components, such as 
chloroform, acetone, or chlorofluorocarbon solvents, which are typically 
added to increase lipid solubility, and are present at a sufficiently low 
concentrations that solvent phase separation does not occur. For example, 
one suitable solvent for preparing liposomes containing phospholipid and 
cholesterol components contains ethanol as primary solvent component and 
between about 10%-20% chlorofluorocarbon solvent. 
As indicated above, the ionic strength of the mixture during liposome 
formation may determine liposome mean diameter, when charged lipid 
components are present. As seen in Examples 1 and 2, a relatively low 
ionic strength results in liposome sizes less than about 50 nm, whereas at 
a tenfold higher ionic strength, liposome sizes in the 200-300 nm range 
are produced. The ionic component of the mixture is typically provided by 
a salt, such as sodium or potassium chloride or phosphate salts, which is 
preferably added as an aqueous salt or buffer solution to the lipid 
solvent. Alternatively, the ionic component may include a charged compound 
which is to be encapsulated or entrapped in liposomes. For forming SUVs 
with an average size of less than about 50 nm, and with charged lipid 
components, the ionic strength of the medium during liposome formation is 
typically less than about 30 mM, and preferably between about 5-20 mM. 
Alternatively, where neutral lipid components alone are used, the size of 
the liposomes is relatively independent of ionic strength. 
In preparing the lipid/solvent/water mixture, the selected lipids are 
preferably first dissolved in the lipid solvent. The amount of lipid added 
is calculated to give a final lipid concentration in the mixture of less 
than about 250-300 umole/ml, depending on desired final lipid 
concentration and encapsulation efficiency. The lipid solution may also 
include lipophilic drug components which are to be entrapped in the lipid 
structures. 
The aqueous medium used in forming the final lipid/solvent/water mixture 
may include salts or other ionic species and water-soluble drug compounds 
which are to be encapsulated in the liposomes. Typically, a relatively 
small portion of the aqueous medium is added to the solvent mixture by 
dilution, prior to reverse osmosis (RO). The solvent is then substantially 
exchanged with aqueous medium by reverse osmosis, as described below in 
Section B. The procedure described in Examples 1 and 2 is illustrative. 
Here an initial lipid in ethanol solution is diluted with distilled water 
to a final water:solvent volume ratio of about 1:9. Additional aqueous 
medium is then added as solvent and water are removed by RO, at a rate 
which maintains the volume of the mixture substantially constant, by 
addition of aqueous medium containing the desired salt concentration. 
As the water:solvent volume ratio of the mixture is increased, during 
reverse osmosis, a mixture containing vesicle-forming lipids typically 
become translucent. With continued replacement of solvent by aqueous 
medium, lipid aggregates or assemblies which appear to include large 
vesicular structures as well as amorphous lipid bodies (as observed by 
light microscopy) begin to form. In the case of ethanol as solvent, the 
solvent ratio at which lipid structure first form is typically between 
about 40%-55%, depending on lipid concentration and percent charged lipid 
components. Experiments conducted in support of the present invention show 
that the lipid structure assembly occurs at about 56% ethanol for 
uncharged lipid components, and falls to about 40% ethanol as the percent 
negatively charged lipid is increased to about 30 mole percent. 
B. Solvent Exchange by Reverse Osmosis 
FIG. 1 shows a reverse osmosis (RO) system 10 designed for solvent exchange 
in a lipid-structure preparation method. The system includes a holding 
tank 12, and a vessel 14 which feeds the aqueous medium to the tank, at a 
controlled rate, via a pump 16. In a specific embodiment of the invention, 
where the volume of the mixture in the tank is maintained at about 1 
liter, the pump is operated to supply medium to the tank at between about 
5-200 ml/min and preferably about 8-50 ml/min, and the pumping rate is 
preferably controlled to match the rate of removal of liquid from the tank 
by RO filtration. 
A crossflow RO filter 18 is connected to the tank through a high pressure 
pump 20 which circulates the fluid in the tank in the direction shown. The 
speed of the pump is adjustable to regulate the rate of fluid flow through 
the filter. The pump speed is typically set to a flow rate of about 80% of 
the maximum. A valve 24 is adjustable to control the pressure within the 
filtration device. Typically the filtration pressure is between about 
400-600 psi. 
Filter 18 is selected on the basis of ability to pass the solvent(s) in the 
lipid/solvent/water mixture. RO filters designed for passage of a variety 
of small, water-miscible solvents are commercially available, such as from 
Millipore Corporation (Bedford, Mass.). 
In operation, a given volume of lipid/solvent or lipid/solvent/water 
mixture is added to the tank. In a preferred embodiment, the water:solvent 
ratio in the initial mixture is substantially less than that at which 
lipid assembly first occurs. A first aqueous medium stored in vessel 14 
preferably contains the solute molecules to be encapsulated or entrapped 
in the lipid structures, and salt or other ionic species added to control 
particle size, in the case of liposomes. If the compound to be 
encapsulated is contained in the first wash, the total volume of the wash 
preferably should be no greater than that required to bring the mixture in 
the tank to the lipid assembly point. In the method illustrated in 
Examples 1 and 2, where the first aqueous-medium volume contains the ionic 
species in the mixture, but not drug solute molecules, the aqueousmedium 
volume and the initial mixture in the tank both have the same 1 liter 
volume. Four additional volumes of distilled water are added to remove all 
but residual amounts of the solvent from the liposome preparation. 
FIG. 2 is a flow diagram of the processing steps in the RO method, as 
exemplified for liposome formation. The reverse osmosis step in the 
diagram involves circulating the tank mixture under pressure across an RO 
membrane, with addition of aqueous medium from vessel 14 to balance 
solvent/water RO filtrate loss. As the water:solvent ratio in the system 
is raised, the mixture will reach a water:solvent ratio at which lipid 
assembly first occurs. With continued solvent exchange, the mixture passes 
through the stage where large lipid aggregates reform, with the final 
reformation producing uniform-size liposomes having the selected average 
size. After final liposome formation, the solvent is conveniently removed 
by continued RO, with addition of several volumes of distilled water. 
FIG. 3 shows the course of solvent exchange in the liposome-preparation 
method described in Example 1. Here filtrate weight versus time was 
measured to determine flow rate during the course of the process. The 
filtrate rate, expressed in g/min (left ordinate) is shown in open circles 
in the figure. Samples were collected from the processing tank every 
minute. Between tubes 6 and 15, the appearance of the mixture changed from 
clear to translucent, but with no indication of liposome formation. The 
flow rate curve shows a sharp drop at about 16-17 minutes, indicating 
initial formation of lipid structures, and tubes 16 and 17 showed large 
lipid aggregates (FIG. 4A). From sample 16 to sample 19, flow rate was 
substantially constant, as the large lipid structures showed a gradual 
reduction in size (FIG. 4B). At tube 24, the lipid structures included a 
mixture of smaller and medium-size vesicles (4C), and at tube 30, the 
lipid structures had the appearance of the final uniform-size liposomes 
seen at the end of the solvent exchange (FIG. 4D). 
The dotted line in FIG. 3 shows the ethanol concentration at that given 
time point, calculated on the basis of theoretical dilution in a 
constant-volume RO system. The first liter of wash volume was supplied by 
a 158 mM (0.9 M) NaCl solution. At sample 16, when lipid assembly first 
occurred, about 600 ml of the wash fluid had been added to the tank, 
producing a salt concentration of about 95 mM, and an ethanol 
concentration of about 50%. After the entire 1 liter aqueous medium was 
added (sample 31) an additional 4 liters of distilled water were added, 
producing a final ethanol concentration of less than 1%. FIG. 5 shows the 
size distribution of the liposomes formed in the Example 1 method. The 
size range is from about 110-340 nm and the average size, about 258 nm. As 
seen, the size distribution of the vesicles is substantially symmetrical 
about the mean size, in contrast to the size distribution of liposomes 
prepared by liposome sizing methods which involve shear forces on 
pre-formed liposomes. 
As indicated above, one aspect of the invention is the ability to control 
average liposome size, by varying the proportion of charged lipid 
components and/or ionic strength. The effect of ionic strength can be seen 
from the RO method described in Example 2. This example is identical to 
the one in Example 1, except that the ionic strength of the first aqueous 
medium is only one-tenth as great, i.e., about 15 mM. FIG. 6 shows the 
change in flow rate (left ordinate) and calculated ethanol concentration 
(right ordinate). Flow rate was measured by change in filtrate weight over 
time. The curves are similar to those shown in FIG. 3. The histogram of 
liposome sizes, seen in FIG. 7, shows a narrow substantially symmetrical 
distribution of sizes between about 30-50 nm, with a mean diameter of 
about 33 nm. 
The lipid mixtures in Examples 1 and 2 both contained 95% egg PC and 5% egg 
PG, with buffers at varying ionic strengths. The RO method has also been 
applied to uncharged lipid compositions, e.g., 100% egg PC. The liposomes 
formed were uniformly sized SUVs, with a mean diameter of 30-50 nm. Little 
effect of ionic strength was observed with uncharged lipids. 
C. Filter Sterilization and Free-Drug Removal 
Lipid structures prepared as above may be readily sterilized by passage 
through a sterilizing membrane having a particle discrimination size of 
about 200 nm, such as conventional 220 nm depth or membrane filter. 
Where the lipid structures, e.g., liposomes, are formulated to contain an 
entrapped drug, for use in parenteral drug administration, it is usually 
desirable to further process the structures to remove free drug, i.e., 
drug present in the bulk aqueous phase of the suspension. Several methods 
are available for removing free drug from a liposome suspension. The sized 
liposome suspension can be concentrated by ultrafiltration, then 
resuspending the concentrated liposomes in a drug-free replacement medium. 
Alternatively, gel filtration can be used to separate larger liposome 
particles from solute (free drug) molecules. Ion-exchange chromatography 
may provide an efficient method of free drug removal, in instances where a 
suitable drug-binding resin can be identified. One preferred method of 
free drug removal is by diafiltration, using a conventional hollow fiber 
or stacked filter device, which preferably has a molecular weight cutoff 
of between about 10,000-100,000 daltons. 
II. Utility 
Liposome suspensions prepared according to the invention are useful in 
therapeutic applications requiring liposomes with encapsulated or 
entrapped drug compounds. Uniform-size liposomes having selected sizes 
less than about 300 nm are useful in particular for parenteral drug 
administration. As noted above, liposomes in this size range are readily 
sterilized, give favorable biodistribution, and show less tendency to 
aggregate on storage. 
SUVs formed in accordance with the invention are useful in therapeutic 
applications which involve tumor targeting or infiltration into hepatic 
cell liver sites. An advantage of SUVs in ophthalmic liposome applications 
is greater optical clarity. SUVs are also used for producing larger, 
high-encapsulation liposomes by freeze-thaw methods. 
Other lipid structures prepared according to the method of the invention 
are also useful in therapeutic or cosmetic formulations. For example, 
lipid emulsions can be employed for parenteral nutrition, and in 
concentrated form, can be used in cosmetic cream or paste formulation. 
Drug lipid complexes particularly involving amphipathic drugs which by 
themselves are poorly soluble in the blood, are also advantageous for 
parenteral drug delivery. 
From the above, it can be appreciated that the present invention offers a 
number of advantages over prior art methods of preparing lipid structures, 
particularly liposomes. Where the liposomes are formed under conditions of 
substantially constant-volume water/solvent exchange by reverse osmosis, 
the method produces substantially uniform liposomes sizes, without the 
requirement for extrusion or other additional liposome sizing steps. This 
feature is due, at least in part, to the relatively uniform environment of 
the lipid during liposome formation, i.e., as the lipid structures are 
gradually converted to small lipid vesicles. In particular, the constant 
volume ensures that localized lipid dilution effects are avoided. This in 
contrast with earlier-proposed methods in which the water:solvent ratio in 
a lipid/solvent/water mixture is raised by dilution with water, e.g., as 
disclosed in EP patent application No. 0,158,441 for "Liposome-Forming 
Composition", where formation of relatively heterogeneous-size liposomes 
is observed. 
The present method also provides for immediate solvent removal, thus 
lessening encapsulation loss. In addition, liposomes formed by the present 
method have a significantly more symmetrical size distribution about the 
mean diameter particle size than has been observed in liposomes produced 
by prior art shear methods. 
Inherent in the reverse osmosis system is a uniform environment solute 
concentration during liposome formation, since localized solute dilution 
effects are minimized. Solute retention also minimizes problems associated 
with solvent recovery and loss of non-encapsulated water-soluble drugs. 
Since the solute compound is neither lost nor diluted during liposome 
formation, higher encapsulation efficiencies/added compound are possible. 
The ability to selectively vary the mean diameter of liposomes, according 
to lipid composition and/or ionic strength, is another useful feature of 
the invention. One selected size range, between about 100-300 nm, is 
advantageous for a variety of parenteral uses, as discussed. 
Finally, the invention provides a unique method for forming SUVs, having 
selected sizes between about 30-80 nm, without shearing-energy input, such 
as by prolonged sonication or homogenization. Problems of sample 
contamination, scale-up, oxidative degradation, and extended processing 
time associated with sonication and homogenization are avoided or 
minimized. 
The following examples illustrate both use and results achievable with the 
method of the invention, but are in no way intended to limit the scope of 
the invention. 
Materials 
Egg phosphatidylcholine (egg PC) was obtained from Lipid KG (Federal 
Republic of Germany), and egg phosphatidylglycerol (egg PG) was obtained 
from Avanti Lipids (Birmingham, Ala.). Reverse osmosis membranes, 
#SK2P473E5, were obtained from Millipore Corp (Bedford, Mass.). A Prolab 
reverse osmosis (RO) filtration apparatus was obtained from Millipore, 
Model #MSDPROLAB. 
EXAMPLE 1 
Preparation of 250 nm Liposomes 
Egg PC (38 g) and egg PG (2 g) were dissolved in 473 ml 100% ethanol in a 1 
liter flask. After addition of 100 ml distilled water, with stirring, the 
lipid/ethanol/water mixture was brought to 1 liter with ethanol. The 
resulting mixture was approximately 50 umole/ml lipid in 90% ethanol. 
The mixture was placed in the processing tank of a Prolab filtration 
apparatus for RO filtration. The RO filter was flushed with a 90% ethanol 
solution prior to use. The system was run at 80% crossflow with 500 psi 
back pressure. 
NaCl (9 g) was dissolved in 1 liter of distilled water, and this solution 
was used as the first wash volume, for replacement of ethanol and water 
lost by RO filtration. Subsequent washes were with 4 liters of distilled 
water. Samples from the process vessel were collected every minute. Volume 
replacement with the five aqueous-media volumes was complete after about 
110 minutes, and the filtration rate varied from about 30 to 60 ml/min 
during the five-volume replacement. The final concentration of ethanol in 
the filtered mixture was less than 1%. 
The filtrate weight was measured vs. time to determine flow rate during the 
filtration period. FIG. 4 (left ordinate) is a plot of flow rate vs time 
(solid circles). The percentage of ethanol in the filtrate shown in dotted 
lines in the graph was calculated from theoretical value. At an ethanol 
concentration corresponding to about 50% ethanol, a sharp decrease in flow 
rate occurred. This flow gradually increases as the ethanol in the mixture 
is replaced by water. 
To examine the relationship between the formation of lipid structures in 
the mixture, and the sudden reduction in flow rate observed, samples taken 
from the processing tank every minute were consecutively numbered by their 
collection time. Those spanning this portion of the curve were examined by 
light microscopy. The samples examined were identified as 6, 12, 15, 16, 
17, 18, 19, 20, 24, 30, and 36, where sample 16 corresponds to the peak in 
filtrate rate. The appearance of samples 16 (FIG. 4A), 19 (FIG. 4B), 24 
(FIG. 4C), and 30 (FIG. 4D) are discussed above. No change in liposome 
appearance was observed after sample 30. 
The size distribution of the final liposomes was examined with a dynamic 
light scattering instrument with Nicomp and Brookhaven correlators. FIG. 5 
shows a histogram of liposome sizes. The particles have a mean diameter of 
258.5 nm, with a standard deviation of 88.9 and a chi square value of 7.1. 
No evidence of size change in the liposomes was observed during storage. 
EXAMPLE 2 
Preparation of 33 nm Liposomes 
A lipid/solvent/water mixture containing 95% egg PC, 5% egg PG in 90% 
ethanol was prepared as in Example 1. The ethanol in the mixture was 
replaced by RO filtration, as above, except that the initial 1 liter wash 
volume was 0.09% NaCl, rather than 0.9% as used in Example 1. The initial 
1 liter aqueous-medium volume was followed by 4 liters of distilled water. 
One minute samples of the process solution were taken. 
FIG. 6 (right ordinate) is a plot of flow rate vs time (solid circles) in 
the RO procedure, where the calculated percent ethanol is also shown here 
in dashed line. As above, the RO flow rate peaks at about tube 16 or 17, 
corresponding to about 50% ethanol, declines sharply, then slowly 
increases as progressively more of the ethanol in the mixture is replaced. 
The size distribution of the final liposomes was examined as above, and 
FIG. 7 shows a histogram of liposome sizes. The particles have a mean 
diameter of 33 nm, with a standard deviation of 7.1 nm and a chi square 
value of 6.8. 
Although the invention has been described with respect to particular 
embodiments and uses, it will be appreciated that the scope of the 
invention includes a variety of modifications and applications not 
specifically mentioned.