METHOD FOR PRESERVING LIPOSOMES
Field of the Invention
The present invention relates generally to liposomes, and more particularly relates to a method of preserving liposomes containing biologically active mol¬ ecules. This process is useful in applications such as in vivo drug delivery and preservation of diagnostic agents.
This invention was made with Government support under Grant No. PCM 82-17538 with the National Science Foundation and the University of California. The Govern¬ ment has certain rights in this invention.
Background of the Invention
Liposomes are unilamellar or multilaπiellar lipid vesicles which enclose a fluid space. The walls of the vesicles are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In an aqueous (or polar) solution, the polar heads of one layer orient outwardly to extend into the surrounding medium, and the non-polar tail portions of the lipids associate with each other, thus providing a polar surface and a non-polar core in the wall of the vesicle. Unilamellar liposomes have one such bimolecu¬ lar layer, whereas multilamellar liposomes generally have a plurality of substantially concentric bimolecular layers.
Liposomes are well recognized as useful for encapsulation of drugs and other therapeutic agents and for carrying these agents to in vivo sites. For example.
U.S. Patent 3,993,754, inventors Rahman et al., issued November 23, 1976, discloses an improved chemotherapy method in which an anti-tumor drug is encapsulated within liposomes and then injected. U.S. Patent 4,263,428, inventors Apple et al., issued April 21, 1981, discloses an antitumor drug which may be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drug within uniformly sized liposomes. Drug administration via liposomes can have reduced toxicity, altered tissue distribution, increased drug effectiveness, and an improved therapeutic index.
Liposomes have also been used successfully for introducing various chemicals, biochemicals, genetic material and the like into viable cells _irι vitro, and as carriers for diagnostic agents.
A variety" of methods for' preparing liposomes are known, many of which have been described by Szoka and Papahadjopoulos, Ann-. Rev. Biophysics Bioeng. 9: 467-508 (1980). Also, several liposome encapsulation methods are disclosed in the patent literature, notably in U.S. patent 4,235,871, to Papahadjopoulos et al., issued November 25, 1980, and in U.S. patent 4,U16,100 to Suzuki et al., issued April 5, 1977.
Although encapsulation of therapeutic agents and biologically active compounds in liposomes has sig¬ nificant commercial potential, a major difficulty that has been encountered in the commercial use of liposome encapsulates is with their long term stability. Although liposome structures may be maintained intact under certain storage conditions, such conditions are often inconvenient or unavailable. It is as a solution to this problem that the method of this invention is presented.
Summary of the Invention
Accordingly, it is an object of the present invention to provide a commercially feasible method of preserving liposomes.
It is another object of the present invention to provide a commercially feasible method of preserving liposomes by means of freeze-drying, wherein upon rehy- dration, resultant liposomes can retain as much as 100% of their original encapsulated material.
It is still another object of the present invention to provide a method of preserving liposomes by means of a carbohydrate compound capable of preserving structure and function in biological membranes.
It is a further object of the present invention to provide a method of preserving liposomes by means of a preserving agent such as trehalose, present either as encapsulated material inside the'liposome or externally in solution during freeze-drying, or both.
It is yet another object of the present invention to provide a lyophilized composition which upon rehydration retains up to 100% of original encapsulated material.
Further objects and advantages of this inven¬ tion will become apparent from the study of the following portion of the specification, the claims and the attached drawings.
In one aspect of the present invention, a method for preserving liposomes includes freeze-drying liposomes in the presence"of a preserving agent capable of preserving structure and function in biological mem¬ branes. Preferred preserving agents include carbohy¬ drates having at least two monosaccharide units, and especially preferred compounds include the disaccharides sucrose, maltose, and trehalose.
In another aspect of the present invention, the method comprises freeze-drying liposomes which in addi¬ tion to containing biologically active molecules or therapeutic agents contain a preserving agent such as trehalose internally. In a preferred embodiment of the inventive method, an appropriate compound such as trehal¬ ose is present both inside and outside the lipid membrane; preferred weight ratios of total preserving agent to lipid range from about 0.1:1 to 3.0:1. An especially preferred weight ratio is about 1.0:1.0.
The invention also embodies a lyophilized composition such that when reconstituted by rehydration, resultant liposomes retain substantially all of their originally encapsulated material. Such a lyophilized composition may be prepared by the method as outlined above.
Detailed Description of the Invention
The invention comprises a method for preserving liposomes containing biologically active molecules using a preserving agent. The method involves either freeze- drying liposomes in the presence of a preserving agent, or freeze-drying liposomes which contain a preserving agent internally in addition to encapsulated medicaments, or both. Preferred preserving agents are carbohydrates having at least two monosaccharide units joined in glycosidic linkage, and particularly preferred pre¬ serving agents include sucrose, maltose and trehalose. Of these, trehalose has been found to be the most effective preserving agent for use with the inventive method.
Trehalose is a naturally occurring sugar found at high concentrations in organisms capable of surviving dehydration. Trehalose is especially effective in pre¬ serving structure and function in dry biological mem¬ branes. Liposomes which are freeze-dried in the presence of trehalose and which additionally contain encapsulated trehalose, exhibit particularly good retention of encap¬ sulates. That is, when liposomes are exposed to tre¬ halose both internally and externally during freeze- drying, they can retain as much as 100% of their original encapsulated contents upon rehydration. This is in sharp contrast to liposomes which are freeze-dried without any preserving agent, which show extensive fusion between liposomes and loss of contents to the surrounding medium.
Representative phospholipids used in forming liposomes which may be used in this process include phos- phatidylcholine, phosphatidylserine, phosphatidic acid and mixtures thereof. Both natural and synthetic phos¬ pholipids may be successfully used.
The biologically active or therapeutic encap¬ sulated material is preferably water soluble. Examples of suitable therapeutic agents with which this preserva¬ tion method can successfully be carried out include sympathomimetic drugs such as amphetamine sulfate, epi- nephrine hyάrochloride, or ephedrine hydrochloride; an- tispasmodics such as atropine or scopalamine; broncho- dilators such as isoproternol; vasodilators such as dilthiazen; hormones such as insulin; and antineoplastic drugs such as adria ycin. Suitable biologically active molecules include, for example, RNA, DNA, enzymes and immunoglobulins.
Small unilamellar vesicles (SUV's) are pre¬ pared as starting materials prior to encapsulation of trehalose, and may be prepared by any of the available techniques. Suitable techniques include injection of the lipid in an organic solvent into water, extrusion from a French pressure cell, and sonication. The material to be trapped may be added at any stage during preparation of the small unilamellar vesicles, but in practice it is most convenient to mix the small unilamellar vesicles with an aqueous solution of the material to be trapped immedi¬ ately before preparation of large unilamellar vesicles. Preferred weight ratios of encapsulate to lipid are about 1.0:1.0.
Large unilamellar vesicles (LUV's) with in¬ creased trapping efficiency may then be prepared by either freeze-thawing or rotary evaporation. An exemplary rotary evaporation method and one which is especially effective in conjunction with the method disclosed herein is illustrated in Deamer, D. ., "A Novel Method for Encapsulation of Macromolecules in Liposomes" in Gregori- adis, G. (ed.) Liposome Technology (1984). The method comprises providing a polar solution having initial liposomes and a quantity of material to be encapsulated. Substantially all of the solution is removed, and the resultant liposomes are then recovered by hydration of the concentrated admixture. This method is also the subject of pending U.S. Patent Application Serial No. 493,952, inventor Deamer, et al., filed May 12, 1983. The resulting vesicles may then be made more uniform by filtration, centrifugation or gel permeation chromato- graphy.
Trehalose may be added at any stage during preparation of the large unilamellar vesicles, but greatly improved preservation is attained with trehalose present on both sides of the phospholipid bilayer. Therefore, trehalose is preferably added before the large
unilamellar vesicles are prepared, so that trehalose is trapped inside. The preferred weight ratio of total trehalose to lipid ranges from about 0.1:1 to about 3.0:1; a particularly preferred weight ratio is approximately 1.0:1.0. The large unilamellar vesicles are then frozen in liquid nitrogen and lyophilized. Under some circum¬ stances, as when lipids are used which are susceptible to damage due to the presence of oxygen, it may be desirable to seal the dry preparations under vacuum. Rehydration is accomplished simply by adding water to the dry mixture.
Although in a preferred embodiment of the invention, the liposomes are exposed to trehalose, it should be understood that a variety of preserving agents may be substituted for trehalose, including carbohydrate compounds which are composed of at least two monosac¬ charide units. In particular, sucrose and maltose are suitable alternatives.
The following examples illustrate certain as¬ pects and embodiments of the present invention, and are not intended to limit the scope of the invention as defined in the appended claims.
Example 1 A phopholipid mixture consisting of approximately 40 mg. dipalmitoyl phosphatidylcholine and phosphatidic acid in a molar ratio of 95:5 was sonicated to optical clarity in a bath sonicator. Large unilamellar vesicles were prepared by freeze-thawing in a 50 mM solution of isocitric acid in water as the compound to be encap¬ sulated. Excess isocitric acid was removed by dialysis. Trehalose (2.0:1.0 trehalose:phospholipid weight ratio) was added either after freeze-thawing or beforehand, thus providing some large unilamellar vesicles with external trehalose only and some vesicles with trehalose both externally and internally. Isocitric acid was assayed by adding isocitrate dehydrogenase and NADP to the outside of the vesicles according to the method of Plaut, et al. (Eds.), Methods in Enzy ology, Volume 5 (New York: Aca¬ demic Press). Isocitrate external to the vesicles was oxidized by the isocitrate dehydrogenase, resulting in reduction of NADP to NADPH, the rate and amount of which may be recorded fluorometrically. Total isocitric acid in the vesicles was assayed following addition of Triton X-100 (octylphenoxy polyethoxyethanol, a detergent and emulsifier manufactured by Rohm & Haas Co., Philadelphia, PA; "TRITON" is a registered trademark of Rohm & Haas Co.), which releases the trapped isocitric acid into the surrounding medium. Isocitric acid trapped in the vesi¬ cles was assayed before and after both lyophilization and rehydration, thus providing an estimate of the efficiency with which the trapped isocitrate was retained. As may be seen in Table 1, the results show that over sixty percent (60%) of the trapped isocitrate was retained when the vesicles were lyophilized with trehalose both inside and outside the vesicles. When trehalose was present externally only, there was still a significant increase in the efficiency of retention, but to a lesser degree than in the case where trehalose was present on both sides of the lipid membrane. Examination of lipid concen¬ tration at time of freezing showed that such had no significant effect on retention of trapped material following lyophilization. Table 1
Method of
Preparing Concentration g Trehalose Trehalose %Reten- LUV ' s of Lipid /g Lipid External Internal tion
(mg/ml)
FT* 10.8 0 0
FT 11.1 0.08 0
FT 10.8 1.78 + 42
FT 11.1 1.78 + 61
rFT = freeze-thaw
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Example 2 Small unilamellar vesicles of were made by sonication of 43 mg egg phosphatidylcholine in 4 ml of water. Large unilamellar vesicles were prepared by rotary drying the phospholipid in the presence of 32 mg of trehalose and 13 mg of isocitric acid. The weight ratios of phospholipid:trehalose:isocitric acid were approxi¬ mately 4:3:1. Excess isocitric acid and trehalose were removed by dialysis against distilled water, and the amount of isocitric acid trapped in the vesicles was determined by the enzyme assay described in Example 1. Trehalose was added to the dialyzed liposomes to give a final weight ratio of phospholipid:trehalose of 1.0:1.4, and the sample was lyophilized. The sample was then rehydrated with distilled water, and the amount of isocitric acid remaining in the liposomes was determined by enzyme assay. The lyophilized vesicles retained 75% of their original contents.
Example 3 A phospholipid mixture of palmitoyloleoyl phosphatidylcholine (90%) and phosphatidylserine (10%) was hydrated to 10 mg./ l., and small unilamellar vesicles were then prepared by sonication. Large uni¬ lamellar vesicles were prepared by rotary drying in the presence of isocitric acid, which served as the encap¬ sulated molecule. Essentially the same techniques as previously described in Examples 1 and 2 were used. Efficiency of retention of isocitric acid following lyophilization and rehydration was recorded as before, with large unilamellar vesicles lyophilized first in the presence and then in the absence of trehalose. As may be seen in Table 2, the results show that 100% of the trapped
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isocitric acid is retained when the large unilamellar vesicles are lyophilized and rehydrated under the stated conditions. As the previous examples demonstrated, tre¬ halose is preferably present both externally and inter¬ nally to optimize retention of the encapsulate.
Example 4 One of the damaging events presumed to be occurring during lyophilization is close approach of the large unilamellar vesicles to each other, leading to fusion and leakage of the vesicular contents. Fusion has been assayed by resonance energy transfer, a fluorescence method which depends upon energy transfer from an excited probe (the "donor probe") to a second probe (the "acceptor probe"). The acceptor probe fluoresces when the energy transfer occurs. In order for the transfer to occur the two probes must be in close proximity. Thus probe intermixing can be used as an assay for fusion between vesicles during lyophilization. Large unilamellar vesi¬ cles were prepared with donor probe in one preparation and acceptor probe in another, and the two preparations were mixed before lyophilization. Following lyophilization and rehydration, probe intermixing was measured, with the results listed in Table 3. The results show that with increasing trehalose concentration there is a decrease in probe intermixing. Furthermore, the presence of tre¬ halose inside the liposomes alone significantly reduce probe mixing. Thus, use of trehalose tends to reduce fusion of the vesicles.
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Table 2
Method of
Preparing g Trehalose Trehalose %Reten-
LUV's /q Lipid External Internal tion
RD* 0.06 + 0
RD 3.2 + + 100
RD 0 - - 0
RD 3.9 + - 26
RD 0.11 + + 22
RD 0.19 + + 49
RD 0.33 + + 69
RD 0.63 + + 76
RD 0.91* + + 86
RD 1.76 + + 99
*RD = rotary drying
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Table 3
Method of
Preparing g Trehalose Trehalose % Probe
LUV's JS Lipid External Internal Mixing
RD* 0.05 + 72
RD 0.15 + + 39
RD 0.25 + + 29
RD 0.50 + + 12
RD 0.95 + + 8
FT** 0. - - 93.0
FT 0.4 + + 79.0
FT 0.8 + + 59.0
FT 1.2 . + + 54.0
FT 1.6 + + 38.0
FT 2.0 + + 15.0
*RD = rotary drying **FT = freeze-thaw
FUSION BETWEEN PALMITOYLOLEOYL PHOSPHATIDYLCHOLINE: PHOSPHATIDYLSERINE (90:10) LARGE UNILAMELLAR VESICLES, AS ASSAYED BY RESONANCE ENERGY TRANSFER BETWEEN FLUORES¬ CENT PROBES 14
Example 5 A further experiment was carried out identical to that set forth in Example 3, with first maltose and then sucrose as the preserving agent. Results are set forth in Tables 4 and 5. As may be concluded from those tables, both maltose and sucrose provide good retention of encapsulated material following lyophilization.
Table 4
Method of
Preparing g Trehalose Trehalose %Reten-
LUV's JSL Lipid External Internal tion
RD 0.05 + 3
RD 0.15 + + 41
RD 0.25 + + 88
RD 0.49 + + 95
RD 0.64 + + 100
Table 5
Method of
Preparing g Trehalose Trehalose %Reten-
LUV's /g Lipid External Internal tion
RD 0.07 + 20
RD 0.35 + + 57
RD 0.49 + + 89
RD 0.83 + + 86
RD 1.15 + + 91