Fused liposome and acid induced method for liposome fusion

• US 4,789,633 A
• Filed: 04/19/1984
• Issued: 12/06/1988
• Est. Priority Date: 04/19/1984
• Status: Expired due to Fees

• Current Assignee: BioWhittaker, Inc. (Lonza Group AG)
• Original Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Inventors: Huang, Leaf, Connor, Jerome
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/602,177
• Time in Patent Office: 1,692 Days
• Field of Search: 424/19, 424/38, 424/85, 424/88, 424/89, 424/92, 424/94, 424/131, 424/177, 424/180, 424/248.57, 424/250, 424/33, 424/417, 424/420, 424/450, 264/4.6, 264/7.1, 428/402.2, 435/240.1, 435/240.2, 435/243, 435/253, 435/254, 435/257
• US Class Current: 435/458
• CPC Class Codes: A61K 9/127 Liposomes
• 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: 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 encapsulate 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 palmitoylhomocysteine; and

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

2. The process of claim 1, wherein the inverted hexagonal phase forming lipid is a phosphatidylethanolamine.

3. The process of claim 1 or 2, wherein the liposomes further comprise cholesterol.

4. A method of inserting foreign material into a living cell which comprises:

(a) encapsulating the foreign material in non-leaky, pH sensitive liposomes prepared from cholesterol, at least one lipid which has a tendency to form the inverted hexagonal phase and at least about 20 mol percent of palmitoyl homocysteine; and

(b) in the absence of externally added divalent cations, proteins, or macromolecules, containing said living cell, at its physiologicla pH, with said encapsulated, non-leaky, pH sensitive liposomes, whereby insertion occurs.

5. The method of claim 4, wherein the inverted hexagonal phase forming lipid is a phosphatidylethanolamine.

6. The process of claim 4 or 5, wherein the liposomes further comprise at least about 40 mole percent cholesterol.

7. Liposomes, fused to one another at a pH of less than about 6.5, at a fusion level of at least about 50%, said lipsomes containing at least one lipid which has a tendency to form the inverted hexagonal phase and at least about 20 mol percent of palmitoyl homocysteine.

8. The liposomes of claim 7, wherein the inverted hexagonal phase forming lipid is a phosphatidylethanolamine.

9. The liposomes of claim 7 or 8, wherein the liposomes further comprise cholesterol.