Liposome for entrapping gene, liposomal preparation and process for the manufacture of the preparation

• US 5,552,157 A
• Filed: 03/28/1995
• Issued: 09/03/1996
• Est. Priority Date: 08/27/1990
• Status: Expired due to Term

• Current Assignee: Kabushiki Kaisha Vitamin Kenkyuso
• Original Assignee: Kabushiki Kaisha Vitamin Kenkyuso
• Inventors: Ohishi, Nobuko, Kurono, Masayasu, Yagi, Kunio, Noda, Hitoshi
• Primary Examiner(s): Rollins, John W.
• Assistant Examiner(s): Ware, Deborah K.
• Application Number:
  US08/412,107
• Time in Patent Office: 525 Days
• Field of Search: 424/450, 435/174, 435/177
• US Class Current: 424/450
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Abstract

For increasing gene entrapping efficiency, it is preferable that liposome membrane to be formed is positively charged. For this purpose, a lipid with quaternary amine has been employed as one of constitutional lipids, but the such lipid may show toxicity to cells to be transformed with the gene for expression. The invention selects, as constitutional lipids for liposomes, N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylethanolamine (DOPE) with molar ratio of 1:2:2, and vortex treatment for preparing the liposomes with multi-layers to entrap the gene therein.

Claims

1. A process for producing a deoxyribonucleic acid entrapping multilamellar liposome, comprising the following steps:

dissolving lipids of N-(α

-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroylphosphatdylcholine and dioleophosphatidylethanolamine in a molar ratio of 1;

2;

2, in a solvent suitable for forming a lipid film,

removing the solvent with an evaporator to form a lipid film,

adding a deoxyribonucleic acid solution to said lipid film to form a mixture, and

shaking said mixture with a vortex mixer to form a deoxyribonucleic acid entrapping multilamellar liposome.

2. The method according to claim 1, wherein said evaporator is a rotary evaporator.

3. The method according to claim 1, wherein said solvent is chloroform.

4. The method according to claim 1, further comprising separating any deoxyribonucleic acid not entrapped in the liposome using a density gradient centrifugal method with Ficoll-Paque.