WO1996040061A1

WO1996040061A1 - Method for encapsulating pharmaceutical materials

Info

Publication number: WO1996040061A1
Application number: PCT/US1996/007892
Authority: WO
Inventors: Eric A. Forssen; Mitsuo Oka
Original assignee: Nexstar Pharmaceuticals, Inc.
Priority date: 1995-06-07
Filing date: 1996-05-29
Publication date: 1996-12-19
Also published as: AU5938996A

Abstract

A method for encapsulating pharmaceutical materials is described. The invention comprises the use of immiscible perhalocarbon cosolvents in the manufacturing process and results in high entrapment (encapsulation efficiencies exceeding 50 %) of aqueous soluble materials in liposomes.

Description

METHOD FOR ENCAPSULATING PHARMACEUTICAL

MATERIALS

FIELD OF THE INVENTION The present invention relates generally to the field of pharmaceuticals and more particularly to a method for encapsulating aqueous soluble materials with high entrapment efficiency, by using an immiscible perhalocarbon cosolvent during fabrication.

BACKGROUND

Liposomes are closed, nearly spherical, vesicles containing one or more concentric bilayer membranes which enclose an equal number of aqueous compartments. Bilayer membrane vesicles can be formed from a variety of synthetic and/or naturally occurring substances, either alone or in mixtures. A common physico-chemical property shared by molecules that form bilayer membranes is that they are amphiphilic. Amphiphilic molecules contain both a polar (hydrophilic) region or head group and a non-polar (lipophilic) region.

The hydrophilic portion comprises phosphato, glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline and other polar groups. Examples of non-polar groups are saturated or unsaturated hydrocarbons such as alkyl, acyl, alkenyl or other lipid groups, which form chains or tails extending from the head group. The amphiphiles most often included in liposomes are the phospholipids, of which phosphatidylcholine is the most widely used. In phosphatidylcholine, the head group is zwitterionic and consists of a negatively charged phosphate and a positively charged choline.

The lipophilic portion typically comprises two fatty acid chains

which can range in length from two carbon atoms each (diacetylphosphatidylcholine) to over twenty-two carbon atoms each (dibehenoylphophatidylcholine). The hydrocarbon chains are typically long; fatty acid hydrocarbon chains of less than fourteen carbon atoms are found in only small amounts.

Another important amphiphile is cholesterol which is composed principally of pure, lipophilic hydrocarbon in the form of a rigid steroid ring and a small polar region consisting of a single hydroxy group. Examples of synthetic compounds used in the manufacture of liposomes include dicetyl phosphate and N,N-dimethyl-N,N-didodecylamine. Naturally occurring compounds include the phosphatidylcholines, phosphatidylserines, and sphingomyelins. Other pharmaceutically acceptable adjuvants, including anti-oxidants such as alpha-tocopherol, are sometimes included to improve vesicle stability or to confer other desirable characteristics to the liposome. Upon exposure to water, phospholipids containing two fatty acid tails, when present at sufficient concentration, spontaneously align to exclude water and associate to form bilayer membranes with the lipophilic ends of the molecules in each layer associated in the center of the membrane and the opposing polar ends forming the respective inner and outer surface of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium. These membranes are initially arranged in a system of concentric multilamellar closed membranes, in a manner not dissimilar to the layers of an onion, around an internal aqueous space. With the application of a shearing force, these multilamellar vesicles (MLVs) can be converted into unilamellar vesicles (UVs).

Amphiphiles with head groups sufficiently polar to be dispersed readily into aqueous media, and which possess only a single hydrocarbon chain (e.g., sodium stearate or /yso-phosphatidylcholine), form self- aggregates in water known as micelles. Micelles are relatively small structures with diameters on the order of 5 nm and are composed of approximately 100 amphiphilic molecules. In water, micellar amphiphiles are arrayed with the lipophilic chains directed inward, forming a hydrocarbon core with the polar head groups located at the surface, in order to maintain aqueous solubility. Micelles, unlike liposomes, do not contain an aqueous core, and consequently, cannot encapsulate water-soluble materials.

The arrangement of amphiphiles within a micelle or liposome is driven by the internal "structure" of water. Water molecules exert a strong attractive force between one another (hydrogen bonds) that must be disrupted or distorted in order for solute molecules to be dispersed. Any attractive forces between amphiphilic hydrocarbons and water are quite weak relative to that between the water molecules themselves. The organization of the micelle or liposome bilayer minimizes the surface areas of the hydrocarbon chains exposed to water and this, in turn, minimizes the disruption of the strong attractive force between the water molecules.

Attraction between the nonpolar hydrocarbon chains plays only a minor role in the organizational arrangement of the micelle. As mentioned above, amphiphiles with two hydrocarbon chains also attempt to minimize interactions between the lipophilic groups and water when dispersed in aqueous media, and curve back on themselves to form closed bilayer vesicles (liposomes). The bilayer structure is preferred over that of the micelle since it has a larger radius of curvature and can thus accommodate the added bulk of the second hydrocarbon chain in each amphiphile.

The behavior of the lipids in the bilayer is strongly influenced by the length and homogeneity of the hydrocarbon chains. At sufficiently low temperatures, amphiphiles show little lateral motion in the plane of the membrane bilayer and behave like solid hydrocarbon crystals. As the temperature is increased, the bilayer can demonstrate abrupt changes in its properties at one or more transition temperatures. Above a transition temperature the bilayer behaves more like a fluid system, and the lipid molecules can display rapid translational motion within the bilayer. The bilayer can also show an increased permeability or leakiness. The temperature(s) at which the phase transition(s) occurs is (are) a function of the precise lipid composition of the vesicles. Lipid bilayers that are composed primarily of unsaturated phospholipids (hydrocarbon chains with one or more double bonds), or include phospholipids with shorter hydrocarbon chains and/or limited amounts of single chain amphiphiles, have lower transition temperatures. For example, liposomes composed of the unsaturated phospholipid, dioleoylphosphatidylcholine, have a phase

transition temperature of about -22°C. On the other hand, liposomes

composed of fully saturated distearoylphosphatidylcholme have a phase

transition temperature of about 58°C. The addition of cholesterol to

phosphatidylcholine vesicles causes the phase transition to occur over a wide temperature range that broadens as the proportion of cholesterol

increases. At a 2:1 phospholipid:cholesterol mole ratio, the phase transition nearly disappears and the liposomes demonstrate a particularly high stability and resistance to leakage of entrapped materials. The presence of cholesterol in the membrane bilayer also helps to stabilize the vesicles in the presence of serum by controlling the loss of phospholipids to high density lipoproteins. The attractive van der Waals forces between the nonpolar hydrocarbon chains are relatively weak; however, for fully saturated acyl moieties, additive effects can be important, leading to significantly increased liposome stability.

Methods for producing liposomes are well known in the art, and there are numerous liposome preparation techniques that may be employed to produce various types of liposomes. Liposomes are generally classified

as multilamellar vesicles (MLVs), which are composed of a series of concentric bilayer membranes with intervening aqueous spaces or as unilamellar vesicles (UVs) which contain a single bilayer surrounding an aqueous center and generally having a size between about 30 and 200 nm. There are several techniques known to those skilled in the art that may be used to produce UVs, including homogenization, sonication or extrusion (through filters) of MLVs. In addition, UVs can be formed by detergent removal techniques. Liposomes that have been referred to as large unilamellar vesicles (LUVs) can be prepared using the solvent infusion or reverse-phase evaporation (REV) methods. In this technique, lipids are dissolved in an organic solvent, to which is added an aqueous buffer; formation of liposomes proceeds via the formation of inverted micelles which collapse into a gel-state upon evaporation of the organic solvent. See F. Szoka, Jr. and D. Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75, 4194-4198 (1978), and U.S. Patent No. 4,235,871 to Papahadjopoulos.

It is well known in the art that liposomes are advantageous for encapsulating or incorporating a wide variety of therapeutic and diagnostic agents. Drugs or other biologically active compounds may be entrapped in the liposomes interior aqueous space, in the case of aqueous soluble materials, or in the membrane bilayer, in the case of lipid-soluble materials. Lipophilic agents may be incorporated directly into the lipid bilayer by inclusion in the lipid formulation. Typically, this technique involves mixing the lipid-soluble agent with the liposomal lipid components in an organic solvent, evaporating the solvent to form a dried film and dispersing the dried mixture in an aqueous medium to form MLVs.

Aqueous soluble agents may be incorporated into liposomes by one of two methods: active loading or passive entrapment. Active loading of certain ionizable materials (i.e., charged at certain pH's) into preformed liposomes is possible where an ionic or pH gradient has been generated by entrapment of a counterion of opposite charge within the vesicle. This method is described in U.S. Patent No. 4,946,683 to Forssen, whose disclosure is incorporated herein by reference. If sufficiently lipid soluble, the ionizable agent will then pass through the membrane bilayer, neutralizing the counterion. With this method, encapsulation efficiencies approaching 100% may be obtained. However, this active loading approach is limited generally to agents that are ionizable and small enough or sufficiently lipid-soluble to be able to pass through a lipid membrane, or where there is included a membrane-associated carrier. This technique is not appropriate for the majority of aqueous soluble materials, especially those with high molecular weights. In addition, a particular drawback of using pH gradients for loading agents into liposomes, especially in large-scale manufacturing, is that significant amounts of lipid hydrolysis can occur as a result of exposing the lipids to extreme pH changes during loading, unless process parameters are carefully controlled. Unstable lipid domains may also form within membrane bilayers resulting from changes in the electrical charge and/or hydration states of the lipid head groups due to protonation-deprotonation of titratable moieties in the lipids. High molecular weight, aqueous soluble molecules that cannot

be entrapped using an active loading process must be entrapped passively. Passive entrapment was historically the first method developed for encapsulating materials within liposomes and relies on the natural ability of amphiphilic molecules to form liquid-filled spheres when dispersed in aqueous media. The extent of encapsulation using passive techniques is limited simply by the aqueous volume that can be entrapped during the

liposome formation process. As liposomes are formed, aqueous soluble molecules dissolved in the solution in which the phospholipids are dispersed will be incorporated into the aqueous interior and thus be

passively entrapped in the liposomes. Typically, only about 0.5 - 10% of the agent is entrapped using passive techniques. Since entrapment percentages are proportional to the aqueous lipid concentration, one strategy for maximizing entrapment efficiency has involved minimizing, for a given amount of lipid, the total volume of aqueous media used in the preparation. Thus, greater efficiencies can be achieved by using higher lipid concentrations. However, the maximum aqueous concentration of lipids which is practical for making liposomes is limited to about 15 to 20% (150 to 200 mg/ml), due largely to processing difficulties associated with the high viscosity encountered at the increased lipid concentrations. Unfortunately, even at these lipid concentrations, it is typically possible to entrap only about 5 - 10% of aqueous soluble agents. Thus, passive entrapment of such agents is impractical, especially where the agent is expensive, since unentrapped material must either be discarded or, if possible, recycled. Previous attempts to increase encapsulation efficiency have failed to provide liposomes suitable for administration in vivo. For example, S. Kim and G.M. Martin, Biochim. Biophys. Ada 646, 1-9 (1981), reported achieving up to 89% encapsulation of water soluble compounds in cell-size LUVs. See also, S. Kim et al., Biochim. Biophys. Ada 728, 339-348 (1983). However, the size of the liposomes (median diameters of 10 - 30 μm) made them unsuitable for

many in vivo applications because of possible emboli formation. A review of liposome preparation methods is provided in R.R.C. New, ed., Liposomes: A Practical Approach (IRL Press, Oxford, 1992).

Perfluorocarbons have previously been used as solvents for the preparation of phospholipid emulsions. Such a preparation is described in Japanese Patent No. 51-26213, which discloses a method for preparing an aqueous emulsion containing water-soluble drugs, phospholipids, vegetable oils, and perhalocarbon. Lyophilization of the dispersion produced micro-

particles with a size distribution of 0.5 - 2.0 μm (500 - 2000 nm). In these

formulations, a vegetable oil (triglyceride) was used as a formulation component along with the phospholipids. It is well known that aqueous suspensions prepared from phospholipids and triglycerides are emulsions rather than liposomal suspensions. Accordingly, it is a desideratum to provide a method for passively encapsulating aqueous soluble agents in liposomes at high entrapment efficiencies; the need for such a method is particularly acute with respect to higher molecular weight compounds which due to their size cannot be actively loaded.

SUMMARY OF THE INVENTION

The invention relates to the formation of liposomes using immiscible perhalocarbon solvents. This method results in encapsulation of aqueous soluble agents with entrapment efficiencies exceeding 50%, substantially greater than typical passive encapsulation values of 1 - 10%. High entrapment efficiencies may be obtained by preparing a liposome suspension consisting of phospholipids, aqueous media and one or more

perhalocarbon solvents, followed by lyophilization of the suspension to

remove water and perhalocarbon solvent. Rehydration of the lyophilized cake yields liposomes suitable for in vivo administration.

The perhalocarbon solvent is not only immiscible with the aqueous phase, it is immiscible with the lipid components as well. Accordingly, a wide variety of lipids and other amphiphilic molecules may be used in the practice of the present invention. In addition, due to their inertness and low vaporization temperature, perhalocarbon solvents are

easily and quickly removed from phospholipid membranes, unlike organic solvents like ethanol. Any of a number of perhalocarbon solvents which have a high vapor pressure and are a liquid at room temperature may be used in the invention described here. Preferably, the perhalocarbon solvent is also pharmacologically acceptable and non-toxic in trace amounts. Examples of perhalocarbons suitable for use in the present invention include perfluorocarbons, especially perfluorodecalin, perfluoromethyldecalin and perfluorotributylamine. Perfluorodecalin is particularly preferred. A preferred embodiment of the present invention involves using a mixture of a neutral phospholipid and cholesterol in a 2:1 mole ratio. Examples of neutral phospholipids suitable for use in the present invention include phospholipids having lipid tails from 14 to 22 carbons, such as egg phosphatidylcholine (egg-PC) and hydrogenated egg phosphatidylcholine (HEPC). Preferably, the lipids contain carbon chain lengths between sixteen and eighteen carbons long. Distearoylphosphatidylcholine (DSPC) is particularly preferred as a neutral phospholipid component of the liposomes. In a most preferred embodiment, the present invention does not include cholesterol esters, triglycerides, or other molecules which prevent liposome formation.

In the practice of the present invention, the liposomes may be formed according to a variety of techniques known to those skilled in the art and include sonication, homogenization and extrusion. It is also readily apparent to one skilled in the art that the particular liposome preparation method will depend on the intended use, the agent to be entrapped and the type of lipids used to form the bilayer membrane. Any aqueous soluble agent is suitable for encapsulation according to the method described herein. Multiple aqueous soluble agents may be encapsulated simultaneously; lipophilic compounds may also be incorporated subsequent to liposome formation using, for example, active loading techniques. Therapeutic and diagnostic agents are contemplated within the scope of the present invention.

One aspect of the present invention is the removal of the perhalocarbon solvent following liposome formation by lyophilization (freeze- drying) or evaporation. The low vaporization temperature of perhalocarbons ensures that sensitive lipids are not oxidatively degraded and that maximum membrane stability is preserved. In practicing the present invention, it is preferred that the perhalocarbon solvent is removed by lyophilization and that a lyoprotectant agent such as a carbohydrate, particularly sucrose, is present during lyophilization. According to the present invention, the lyophilized cake may be stored for an extended period of time until ready for use, at which time rehydration with aqueous media will reconstitute the liposome suspension with entrapped agent.

The high encapsulation efficiency achieved by the present invention is presently believed to be attributable to exclusion of aqueous components by the immiscible perhalocarbon solvent. In this manner, aqueous components, including solubilized drugs or agents, are "forced" into the aqueous volume of the liposomes during their formation. However, the invention is not limited to this particular proposed mode of encapsulation, and other mechanisms may be applicable.

The method provides the ability to dilute the lipids and agent using a solvent which is immiscible both with these components and a small amount of water which is used to hydrate the lipids. Ideally, the immiscible cosolvent is removed from the liposome suspension following this processing step. Perhalocarbons, especially perfluorocarbons such as perfluorodecalin, are biologically inert, removable by evaporation or lyophilization and are immiscible with water, lipids, and most agents that are candidates for liposome entrapment. Because of the immiscibility of perhalocarbons, lipids are not dissolved in the solvent and liposome formation does not proceed via a micelle or inverted micelle intermediate.

Other features and advantages of the present invention will be apparent upon review of the detailed description of the preferred embodiment, the examples and the claims. DETAILED DESCRIPTION

According to the present invention, a method for encapsulating aqueous soluble agents in liposomes with high efficiency is described. Specifically, a suspension of amphiphilic molecules (e.g., lipids), an aqueous medium (containing one or more agents to be entrapped), and one or more perhalocarbon solvents is prepared. The suspension is exposed to a shearing force (e.g., sonication or homogenization) in order to form small, uniformly sized liposomes. Following formation of the liposomes, additional agents may optionally be incorporated into the liposomes by active loading techniques.

Lyophilization of the suspension removes water and perhalocarbon solvent from the suspension and yields a dried cake which is stable upon long-term storage. Rehydration of the lyophilized cake with an aqueous medium reconstitutes the liposomes with the aqueous soluble agent still encapsulated. Rehydration may be accomplished using any pharmaceutically acceptable aqueous medium, including water-for-injection (WFI), buffer solutions, or solutions which contain a carbohydrate such as sucrose.

Unentrapped material may be separated by any of a variety of techniques known in the art, including chromatography, dialysis and ultrafiltration. The removal of unentrapped material may be performed either following initial liposome formation or following rehydration.

The rehydrated liposomes may be used for a variety of applications, including administration to a patient for therapeutic or diagnostic purposes. Optionally, the rehydrated liposome suspension may be filtered immediately prior to use in order to remove larger liposomes.

The present invention may be practiced with a wide variety of agents, but is particularly advantageous for aqueous soluble materials which are poorly entrapped using conventional passive entrapment techniques. Aqueous soluble agents are substances which partition predominantly within the interior aqueous space of liposomes, rather than in the bilayer membrane. The aqueous soluble agent to be entrapped is preferably a biologically active substance, i.e., one which is a naturally occurring substance in vivo or one which elicits a physiological response. This method is particularly suitable for encapsulating nucleic acid (DNA and RNA) oligonucleotides in liposomes intended for use in gene therapy. The inertness of the perhalocarbon solvent and the gentle processing steps of the present invention prevent damage to or modification of sensitive biomolecules.

Example 1 Encapsulation of a Nucleic Acid Oligonucleotide

A liposome encapsulated oligonucleotide (20-bp) sample was prepared as follows. A spray-dried powder of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 molar ratio; 200 mg total weight) were mixed with perfluorodecalin (0.80 ml) and 1.20 ml of an aqueous 9% sucrose solution (pH 6.7) containing the oligonucleotide (1.0 mg). The heterogeneous system was sonicated for approximately 15 min. using a Sonics and Materials Vibra Cell Sonicator at 65°C to produce a

homogeneous dispersion of liposomes. The size distribution of the resultant

liposomes was bimodal with median diameters of 0.288 μm (67% relative

population by volume) and 0.054 μm (33% by volume).

Lyophilization of the liposome suspension produced a white lyophilized cake. Rehydration of the lyophilized cake with 9% sucrose yielded

a size distribution of 1.78 μm (50% by volume), 0.25 μm (42%), and 0.067 μm

(8%). Similar results were obtained using water-for-injection (WFI) as the aqueous medium for rehydration. HPLC chromatography using Sephacryl S-300 and an eluting solvent of 9% sucrose with TRIS (10 mM) and EDTA (1 mM) at pH 7.4 separated the liposomes from free oligonucleotide. The concentration of the oligonucleotide in the two fractions (1.2 ml each) was measured by UV absorption at 254 nm. The liposome fraction (1 ml) was dried under nitrogen gas and 1:1 (v/v) THF: water (1.5 ml total volume) was used to lyse the liposomes. The free oligonucleotide fraction (1 ml) was mixed with 1 :1 (v/v) THF: water (0.5 ml total volume) and UV absorption was measured.

From the UV absorption values, the entrapment efficiency of the oligonucleotide was calculated to be 58%.

Example 2 Encapsulation of Carboxyfluorescein A liposome encapsulated 4-(and -5)-carboxyfluorescein sample was prepared in a manner similar to Example 1. A spray-dried powder of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 molar ratio; 200 mg total weight) were mixed with perfluorodecalin (0.80 ml) and a 4-(and -5)-

carboxyfluorescein solution (1.20 ml total volume; 263 μg/ml concentration).

The heterogeneous system was sonicated for approximately 15 min using a Sonics and Materials Vibra Cell Sonicator at room temperature to produce a homogeneous dispersion of liposomes. The size distribution was bimodal

with median diameters of 0.490 μm (47% by volume) and 0.094 μm (53%).

In order to check qualitatively the successful encapsulation of 4- (and -5)- carboxvfluorescein, a small portion of the sample was passed through a PD-10 column (Sephadex G-25 M by Pharmacia; 1:18 liposome:gel volume ratio) eluted with water-for injection (WFI). The liposome fraction contained most of the yellow 4-(and -5)-carboxyfluorescein, and the unentrapped 4-(and -5)-carboxyfluorescein was only a very minor fraction. Lyophilization of the liposome fraction produced a light peach color lyophilized cake, and rehydration in WFI yielded a yellow colored liposome

sample with a size distribution of 5.7 μm (20% by volume), 1.4 μm (77%), and

0.27 μm (3%).

All publications and patent documents cited in this specification are incorporated herein by reference as if each individual document were specifically and individually indicated to be incorporated by reference.

While the foregoing examples and descriptions set forth the preferred embodiments and various ways of accomplishing the present invention, they are not intended to be limiting as to the scope of the invention, which is as set forth in the following claims. Moreover, it will be recognized in view of the foregoing disclosure that the invention embraces alternative embodiments and structures that are the lawful equivalents of those described herein.

Claims

I CLAIM:

1. A method for encapsulating an aqueous soluble agent in the interior

aqueous space of liposomes comprising forming the liposomes in a suspension comprising one or more bilayer membrane-forming amphiphilic compounds; an aqueous medium; a perhalocarbon solvent; and the aqueous soluble agent to be entrapped.

2. The method of claim 1 wherein the liposomes have a median diameter between about 30 nm and 10 :m.

3. The method of claim 1 wherein the liposomes comprise unilamellar vesicles.

4. The method of claim 3 wherein the unilamellar vesicles have a median diameter between about 30 nm and 200 nm.

5. The method of claim 1 wherein the one or more amphiphiles comprises a phospholipid.

6. The method of claim 5 wherein the phospholipid is neutral.

7. The method of claim 5 wherein the phospholipid is a phosphatidylcholine.

8. The method of claim 7 wherein the phosphatidylcholine is distearoylphosphatidylcholine.

9. The method of claim 1 wherein the aqueous medium comprises a buffer solution.

10. The method of claim 1 wherein the aqueous medium comprises a carbohydrate.

11. The method of claim 10 wherein the carbohydrate is sucrose.

12. The method of claim 1 wherein the perhalocarbon solvent comprises a perfluorocarbon.

13. The method of claim 12 wherein the perfluorocarbon is selected from the group consisting of perfluorodecalin, perfluoromethyldecalin, and perfluorotributylamine.

14. The method of claim 1 wherein the aqueous soluble agent is an

oligonucleotide.

15. The method of claim 1 wherein the one or more amphiphiles is present at a total concentration between about 10 and 250 mg per ml of total solvent volume (aqueous and perhalocarbon).

16. The method of claim 1 wherein the volume ratio of the aqueous medium and the perhalocarbon solvent is between 1:100 and 10:1 [aqueous:perhalocarbon, v:v].

17. A method for preparing dehydrated liposomes that encapsulate an aqueous soluble agent, comprising the steps of:

(a) forming the liposomes in a suspension comprising one or more bilayer membrane-forming amphiphilic compounds, a perhalocarbon solvent, an aqueous medium comprising water and the aqueous soluble agent to be entrapped; and

(b) evaporating the water and solvent from the liposome suspension.

18. The method of claim 17 wherein the evaporation is accomplished by lyophilization.

19. A method for preparing rehydrated liposomes which encapsulate an aqueous soluble agent, comprising the steps of:

(a) forming initial liposomes in a suspension comprising one or more bilayer membrane-forming amphiphilic compounds, a first aqueous medium, a perhalocarbon solvent, and the aqueous soluble agent to be entrapped;

(b) lyophilizing the liposome suspension; and

(c) rehydrating the lyophilized liposomes with a second aqueous medium.

20. The method of claim 19 wherein the rehydrated liposomes have a

median diameter between about 30 nm and 10 μm.

21. The method of claim 19 wherein the rehydrated liposomes comprise unilamellar vesicles.

22. The method of claim 20 wherein the unilamellar vesicles have a median diameter between about 30 nm and 200 nm.

23. The method of claim 19 wherein the one or more amphiphiles comprises a phospholipid.

24. The method of claim 23 wherein the phospholipid is neutral.

25. The method of claim 23 wherein the phospholipid is a phosphatidylcholine.

26. The method of claim 25 wherein the phosphatidylcholine is distearoylphosphatidylcholine.

27. The method of claim 19 wherein the first aqueous medium comprises a buffer solution.

28. The method of claim 19 wherein the first aqueous medium comprises

a carbohydrate.

29. The method of claim 19 wherein the second aqueous medium comprises a buffer solution.

30. The method of claim 19 wherein the second aqueous medium comprises a carbohydrate.

31. The method of claim 19 wherein the perhalocarbon solvent comprises a perfluorocarbon.

32. The method of claim 31 wherein the perfluorocarbon is selected from the group consisting of perfluorodecalin, perfluoromethyldecalin, and perfluorotributylamine.

33. The method of claim 19 wherein the aqueous soluble agent is a biologically active substance.

34. The method of claim 33 wherein the biologically active substance is an oligonucleotide.

35. The method of claim 19 wherein the one or more amphiphiles is present at a total concentration between about 10 and 250 mg per ml of total solvent volume (first aqueous medium and perhalocarbon solvent).

36. The method of claim 19 wherein the volume ratio of the first aqueous medium and the perhalocarbon solvent is between 1 :100 and 10:1 [aqueous:perhalocarbon, v:v].

37. Liposomes formed according to the method of claim 1.

38. Dehydrated liposomes prepared according to the method of claim 17.

39. Rehydrated liposomes prepared according to the method of claim 19.