METHODS FOR ENCAPSULATING PLASMIDS IN LIPID BILAYERS

• US 20100041152A1
• Filed: 03/16/2009
• Published: 02/18/2010
• Est. Priority Date: 06/07/1995
• Status: Abandoned Application

• Current Assignee: Tekmira Pharmaceuticals Corporation, University of British Columbia
• Original Assignee: Tekmira Pharmaceuticals Corporation, University of British Columbia
• Inventors: Wheeler, Jeffery J., Bally, Marcel B., Cullis, Pieter R., Hope, Michael J.
• Application Number:
  US12/404,837
• Publication Number:
  US 20100041152A1
• Time in Patent Office: Days
• US Class Current: 435/458
• 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: Y10S 977/80 Nucleic acid, e.g. DNA or RNA
• CPC Class Codes: Y10S 977/907 Liposome

Abstract

Plasmid-lipid particles which are useful for transfection of cells in vitro or in vivo are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. The particles are typically 65-85 nm, fully encapsulate the plasmid and are serum-stable.

Claims

1. A method for the preparation of serum-stable plasmid-lipid particles, comprising:

(a) combining a plasmid with cationic lipids in a detergent solution to provide a coated plasmid-lipid complex;

(b) contacting non-cationic lipids with said coated plasmid-lipid complex to provide a solution comprising detergent, a plasmid-lipid complex and non-cationic lipids; and

(c) removing said detergent from said solution of step (b) to provide a solution of serum-stable plasmid-lipid particles, wherein said plasmid is encapsulated in a lipid bilayer and said particles are serum-stable and have a size of from about 50 to about 150 nm.

2. A method in accordance with claim 1, wherein said removing is by dialysis.

3. A method in accordance with claim 1, wherein step (b) further comprises adding a polyethylene glycol-lipid conjugate.

4. A method in accordance with claim 2, 3, wherein said polyethylene glycol-lipid conjugate is a PEG-ceramide conjugate.

5. A method in accordance with claim 1, further comprising;

(d) sizing said particles to achieve a uniform particle size.

6. A method in accordance with claim 1, wherein said cationic lipids are selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.

7. A method in accordance with claim 1, wherein said non-cationic lipids are selected from the group consisting of DOPE, POPC, EPC and combinations thereof.

8. A method in accordance with claim 1, wherein said detergent solution comprises a detergent having a critical micelle concentration of between about 20 mM and 50 mM.

9. A method in accordance with claim 8, wherein said detergent is n-octyl-β

-D-glucopyranoside.

16. A plasmid-lipid particle prepared according to claim 1.

17. A method for introducing a plasmid into a cell, comprising;

(a) preparing a plasmid-lipid particle according to the method of claim 1; and

(b) contacting said cell with said plasmid-lipid particle for a period of time sufficient to introduce said plasmid into said cell.

18. A method in accordance with claim 17, wherein said plasmid-lipid particle comprises a plasmid, DODAC, POPC and a PEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.

19. A method in accordance with claim 17, wherein said plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a PEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.

10. A method for the preparation of serum-stable plasmid-lipid particles, comprising;

(a) preparing a mixture comprising cationic lipids and non-cationic lipids in an organic solvent;

(b) contacting an aqueous solution of plasmid with said mixture prepared in step (a) to provide a clear single phase; and

(c) removing said organic solvent to provide a suspension of plasmid-lipid particles, wherein said plasmid is encapsulated in a lipid bilayer, and said particles are stable in serum and have a size of from about 50 to about 150 nm.

11. A method in accordance with claim 10, wherein said non-cationic lipids comprise a polyethylene glycol-lipid conjugate.

12. A method in accordance with claim 11, wherein said polyethylene glycol-lipid conjugate is a PEG-ceramide conjugate.

13. A method in accordance with claim 10, further comprising;

(d) sizing said plasmid-lipid particles to achieve a uniform particle size.

14. A method in accordance with claim 10, wherein said cationic lipids are selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.

15. A method in accordance with claim 10, wherein said non-cationic lipids are selected from the group consisting of DOPE, POPC, EPC and combinations thereof.

20. A plasmid-lipid particle prepared according to claim 10.

21. A method for introducing a plasmid into a cell, comprising;

(a) preparing a plasmid-lipid particle according to the method of claim 10; and

(b) contacting said cell with said plasmid-lipid particle for a period of time sufficient to introduce said plasmid into said cell.

22. A method in accordance with claim 21, wherein said plasmid-lipid particle comprises a plasmid, DODAC, POPC and a PEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.

23. A method in accordance with claim 21, wherein said plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a PEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.