Methods for encapsulating plasmids in lipid bilayers

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.

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.  
     
     
         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.  
     
     
         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-C 20  and PEG-Cer-C 14 .  
     
     
         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-C 20 , and PEG-Cer-C 14 .  
     
     
         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-C 20  and PEG-Cer-C 14 .  
     
     
         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-C 20  and PEG-Cer-C 14 .