High efficiency encapsulation of charged therapeutic agents in lipid vesicles

Abstract

Methods for the preparation of a lipid-nucleic acid composition are provided. According to the methods, a mixture of lipids containing a protonatable or deprotonatable lipid, for example an amino lipid and a lipid such as a PEG- or Polyamide oligomer-modified lipid is combined with a buffered aqueous solution of a charged therapeutic agent, for example polyanionic nucleic acids, to produce particles in which the therapeutic agent is encapsulated in a lipid vesicle. Surface charges on the lipid particles are at least partially neutralized to provide surface-neutralized lipid-encapsulated compositions of the therapeutic agents. The method permits the preparation of compositions with high ratios of therapeutic agent to lipid and with encapsulation efficiencies in excess of 50%.

1 . A method for preparation of a composition comprising lipid-encapsulated therapeutic agent particles, said method comprising the steps of:
 (a) combining a mixture of lipids comprising at least a first lipid component and a second lipid component with a buffered aqueous solution of a charged therapeutic agent to form an intermediate mixture containing lipid-encapsulated therapeutic agent particles, said first lipid component being selected from among lipids containing a protonatable or deprotonatable group that has a pKa such that the lipid is in a charged form at a first pH and a neutral form at a second pH, said buffered solution having a pH such that the first lipid component is in its charged form when in the buffered solution, said first lipid component being further selected such that the charged form is cationic when the charged therapeutic agent is anionic in the buffered solution, and anionic when the charged therapeutic agent is cationic in the buffered solution, and said second lipid component being selected from among lipids that prevent particle aggregation during lipid-therapeutic agent particle formation. and   (b) changing the pH of the intermediate mixture to neutralize at least some exterior surface charges on said lipid-encapsulated therapeutic agent particles to provide at least partially-surface neutralized lipid-encapsulated therapeutic agent particles.   
     
     
         2 . The method of  claim 1 , wherein the therapeutic agent is a polyanionic nucleic acid. 
     
     
         3 . The method of  claim 2 , wherein said composition consists essentially of lipid-nucleic acid particles, said particles having a size of from 70 nm to about 200 nm. 
     
     
         4 . The method of  claim 2 , wherein said mixture of lipids in step (a) is a mixture of lipids in alcohol. 
     
     
         5 . The method of  claim 2 , wherein the first lipid component is an amino lipid. 
     
     
         6 . The method of  claim 2 , wherein the second lipid component is a polyethylene glycol-modified or polyamide oligomer-modified lipid. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 6 , wherein the first lipid component is an amino lipid. 
     
     
         9 . The method of  claim 2 , wherein said lipids present in said lipid mixture comprises an amino lipid having a pKa of from about 5 to about 11, a neutral lipid, Chol and a PEG-modified or polyamide oligomer-modified lipid. 
     
     
         10 . The method of  claim 9 , wherein said lipids are present in molar percents of about 25-45% neutral lipid, 35-55% Chol, 10-40% amino lipid and 0.5-15% PEG-modified or polyamide oligomer-modified lipid. 
     
     
         11 . The method of  claim 2 , wherein said mixture of lipids comprises DODAP, DSPC, Chol and PEG-CerC14. 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 2 , wherein said mixture of lipids comprises DODAP, POPC, Chol and PEG-CerC14. 
     
     
         14 . The method of  claim 2 , wherein said mixture of lipids comprises DODAP, SM, Chol and PEG-CerC14. 
     
     
         15 .- 18 . (canceled) 
     
     
         19 . The method of claim  17 , wherein the buffered solution comprises 10 to 50 mM citrate or phosphate buffer. 
     
     
         20 . The method of  claim 2 , wherein the nucleic acid contains at least some phosphorothioate or phosphorodithioate linkages. 
     
     
         21 . The method of  claim 20 , wherein the buffered solution comprises 10 to 300 mM citrate or phosphate buffer. 
     
     
         22 .- 24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein the first lipid component is an amino lipid. 
     
     
         26 . The method of  claim 1 , wherein the second lipid component is a polyethylene glycol-modified or polyamide oligomer-modified lipid. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . The method of  claim 1 , wherein said lipids present in said lipid mixture comprises an amino lipid having a pKa of from about 5 to about 11, a neutral lipid, Chol and a PEG-modified or Polyamide oligomer-modified lipid. 
     
     
         30 . The method of  claim 29 , wherein said lipids are present in molar percents of about 25-45% neutral lipid, 35-55% Chol, 10-40% amino lipid and 0.5-15% PEG-Ceramide. 
     
     
         31 .- 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the pH is changed in step (b) to physiological pH. 
     
     
         36 . The method of  claim 1 , wherein the step of changing the pH is performed using tangential flow dialysis. 
     
     
         37 .- 83 . (canceled)