Fused liposome and acid induced method for liposome fusion

• US 5,283,122 A
• Filed: 02/14/1992
• Issued: 02/01/1994
• Est. Priority Date: 04/19/1984
• Status: Expired due to Fees

• Current Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Original Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Inventors: Huang, Leaf, Connor, Jerome
• Primary Examiner(s): Lovering, Richard D.
• Application Number:
  US07/836,125
• Time in Patent Office: 718 Days
• Field of Search: 264/4.1, 264/4.3, 264/4.6, 428/402.2, 424/450, 436/829
• US Class Current: 428/402.2
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Liposomes containing phosphatidylethanolamine, palmitoyl homocysteine or oleic acid or palmitic acid, fuse rapidly when the pH of the medium is reduced below 7. Liposome fusion was measured by (a) mixing of the liposomal lipids as shown by resonance energy transfer, (b) gel filtration and (c) electron microscopy. The presence of phosphatidylethanolamine or acid addition esters thereof in the liposomes greatly enhances fusion; whereas the presence of phosphatidylcholine inhibits fusion. During fusion of liposomes containing phosphatidylethanolamine:palmitoyl homocysteine (8:2), almost all of the encapsulated calcein is released. Inclusion of cholesterol (40%) in the liposomes substantially decreases leakage without impairing fusion. Those pH sensitive liposomes are fused to deliver biologically active molecules such as DNA, into living cells.

Claims

1. A method of fusing liposomes which comprises:

(a) preparing a suspension of liposomes containing at least one lipid which has a tendency to form the inverted hexagonal phase and at least 20 mol percent of one or more amphipathic molecules containing one or more weakly acidic functional groups selected from the group consisting of C₁₂ to C₃₀ fatty acids and palmitoyl homocysteine; and

(b) in the absence of externally added divalent cations, proteins or other macromolecules, acidifying the liposome suspension to reduce the pH of these liposomes to below pH 7, whereby at least about 20% of said liposomes fuse to one another.

2. The method of claim 1 wherein said weakly acidic functional group of said amphipathic molecule is one or more carboxylic groups.

3. The method of claim 2, wherein said fusion occurs within the pH range of from about 4.8 to 6.8 and said amphipathic molecule is a C₁₆ to C₂₄ fatty acid.

4. The method of claim 3, wherein said long chain fatty acid is selected from the group consisting of palmitic acid and oleic acid.

5. The method of claim 2, from wherein said fusion occurs within the pH range of about 5.75 to 6.0 and said amphipathic molecule is palmitoylhomocysteine.

6. The method of claim 1 wherein the pH is reduced in step (b) to below 6.0.

7. The method of claim 1 wherein the pH is reduced in step (b) to below 5.0.

8. The method of claim 1 wherein step (b) is carried out using an aqueous mineral acid.

9. The method of claim 8, wherein the aqueous mineral acid is hydrochloric acid.

10. The method of claim 1 which further comprises forming the liposomes of step (a) with an amphipathic molecule which has a tendency to form hexagonal phase.

11. The method of claim 1, wherein said inverted hexagonal phase forming lipid of step (a) is selected from the group consisting of the phosphatidylethanolamines.

12. The method of claim 11, wherein the phosphatidylethanolamine is dioleoylophosphatidylethanolamine.

13. The method of claim 12 wherein the mol ratio of dioleoylphosphatidylethanolamine to said amphipathic molecule containing one or more weakly acidic functional groups is about 8:

2.

14. The method of claim 13, wherein said amphipathic molecule containing one or more weakly acidic functional groups is selected from the group consisting of palmitoyl homocysteine, palmitic acid and oleic acid.

15. Fused nonagglomerated liposomes comprised of at least one lipid which has a tendency to form the inverted hexagonal phase and at least 20 mol percent of an amphipathic molecule containing one or more acidic functional groups an amphipathic molecule selected from palmitoylhomocysteine, palmitic acid and oleic acid. PG,30

16. The fused liposomes of claim 15, consisting of from 1:

9 mol ratio of phosphatidylethanolamine and palmitoylhomocysteine.

17. The fused liposomes of claim 16 wherein the mol ratio of PE to PHC is from 5:

1 to 1;

5.

18. The fused liposomes of claim 16 wherein the mol ratio of PE to PHC is 8:

2.

19. Fused liposomes containing phosphatidylethanolamine and oleic acid in a mol ratio of 8:

2.

20. Fused liposomes containing phosphatidylethanolamine and palmitic acid in a mol ratio of 8:

2.