Method of encapsulating biologically active materials in lipid vesicles

• US 4,235,871 A
• Filed: 02/24/1978
• Issued: 11/25/1980
• Est. Priority Date: 02/24/1978
• Status: Expired due to Term

• Current Assignee: Liposome Technology, Inc.
• Original Assignee: Demetrios P. Papahadjopoulos, Francis C. Szoka Jr
• Inventors: Papahadjopoulos, Demetrios P., Szoka, Francis C. Jr.
• Primary Examiner(s): Daus, Donald G.
• Assistant Examiner(s): Eakin, M. C.
• Application Number:
  US05/881,116
• Time in Patent Office: 1,005 Days
• Field of Search: 424/38, 424/19, 424/177, 424/178, 252/316, 260/112.7
• US Class Current: 424/450
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: C12N 1/04 Preserving or maintaining viable microorganisms immobilised microorganisms C12N11/00
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 9/98 Preparation of granular or free-flowing enzyme compositions C12N9/96 takes precedence
• CPC Class Codes: Y10S 424/812 Liposome comprising an antibody, antibody fragment, antigen, or other specific or nonspecific immunoeffector
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

The disclosure is of a method for encapsulating biologically active materials in synthetic, oligolamellar lipid vesicles (liposomes). The method comprises providing a mixture of lipid in organic solvent and an aqueous mixture of the material for encapsulation, emulsifying the provided mixture, removing the organic solvent and suspending the resultant gel in water. The method of the invention is advantageous over prior art methods of encapsulating biologically active materials in that it provides a means for a relatively high capture efficiency of the material for encapsulation. The disclosure is also of intermediate compositions in the encapsulation method, the product vesicles, compositions including the product vesicles as an active ingredient and their use.

Claims

1. A method of encapsulating biologically active materials in synthetic, oligolamellar, lipid vesicles, which comprises:

providing a mixture of a vesicle wall forming compound in organic solvent and an aqueous mixture of the biologically active material to be encapsulated, the ratio of organic phase to aqueous phase being that which will produce an emulsion of the water-in-oil type;

forming a homogeneous water-in-oil type of emulsion of said mixture;

evaporating organic solvent from the emulsion, until a mixture is obtained having a gel-like character; and

converting the gel-like mixture to a suspension of synthetic, oligolamellar vesicles encapsulating the biologically active material by one of the steps of (a) agitating said gel-like mixture and (b) dispersing said gel-like mixture in an aqueous media.

2. The method of claim 1 wherein the wall forming compound is a phospholipid.

3. The method of claim 2 wherein the phospholipid comprises phosphatidylcholine in admixture with a second phospholipid and cholesterol.

4. The method of claim 3 wherein the proportion of phosphatidylcholine is in a ratio of 4:

1 to the second phospholipid.

5. The method of claim 1 wherein the wall forming compound is initially provided in an inert solvent and is then deposited on the side wall of a reaction vessel by evaporation of the inert solvent.

6. The method of claim 1 wherein the organic solvent is selected from the group consisting of diethyl ether, chloroform, tetrahydrofuran and isopropyl ether.

7. The method of claim 1 wherein the aqueous mixture is buffered to a pH suitable to maintain stability of the biologically active material.

8. The method of claim 7 wherein the buffer is an ionic compound, at a strength of less than 0.3 units.

9. The method of claim 1 wherein the proportion of wall forming compound is within the range of from about 0.5 mg to about 50 mg per milliliter of the organic solvent.

10. The method of claim 1 wherein emulsification is carried out with ultrasonic radiation at a temperature of from about -10°

to about 50°

C.

11. The method of claim 1 carried out under an inert atmosphere.

12. The method of claim 1 wherein the aqueous mixture comprises water buffered to a pH suitable to maintain stability of the biologically active material.

13. The method of claim 1 wherein the oligolamellar vesicles are then separated from unencapsulated biologically active materials.

14. The method of claim 1 wherein the biologically active material is an enzyme.

15. The method of claim 1 wherein the biologically active material is an antibiotic.

16. The method of claim 1 wherein the biologically active material is a nucleic acid.

17. The method of claim 16 wherein the nucleic acid is a deoxyribonucleic acid.

18. The method of claim 1 wherein the biologically active material is a pesticide.

19. The method of claim 1 wherein the biologically active material is a polypeptide.

20. The method of claim 19 wherein the polypeptide is insulin.

21. The method of claim 1 wherein the biologically active material is a bacterial organism.

22. The method of claim 1 wherein the biologically active material is a virus.

23. The method of claim 1 wherein the wall forming compound is a mixture of cholesterol, phosphatidyglycerol and phosphatidylcholine in a ratio of 5:

1;

4 by weight.

24. The product of the process of claim 1.

25. The method of claim 1 wherein the ratio of organic phase to aqueous phase is within the range of from about 2:

1 to about 20;

1 v/v.

26. The method of claim 25 wherein said ratio is 4:

1.