Method of producing lipid nanoparticles for drug delivery
What is described is a method for preparing a liposome that efficiently encapsulates a negatively charged therapeutic polymer, e.g., siRNA. The process involves preparing a lipid mixture comprising a cationic lipid in a water miscible organic solvent, such as ethanol, at a concentration of 2.3 mg/ml, and adding this solution to the polymer dissolved in water to a final concentration of 35% ethanol in water. The final charge ratio of drug:lipid is 1:2.5. The resulting nanoparticles have a mean size of 50 to 150 nm.
1. A method for preparing a lipid nanoparticle encapsulating a RNA molecule, comprising the steps of
(a) transferring to a mixing container a first aqueous buffer, pH 3.5-6.5, comprising the RNA molecule at 0.08 to 0.8 mg/ml;
(b) injecting a lipid solution comprising a cationic lipid, a helper lipid, a sterol, and a PEG lipid dissolved in a water-miscible organic solvent into the aqueous solution in the mixing container at a constant rate for 1-100 minutes while stirring the aqueous solution until a mixture comprising 25-45% (v:v) organic solvent is reached;
(c) diluting the organic solvent in the mixture to less than 10% (v:v); and
(d) removing the organic solvent from the diluted mixture by diafiltration against a second aqueous buffer, pH 6.5-8;
wherein step (b) is performed batchwise, and wherein the mixture comprises lipid nanoparticles having a RNA:lipid ratio 0.06 to 0.16 (w:w), and a RNA:lipid charge ratio of 1:2.5 to 1:1.
2. The method of claim of 1 , wherein the second aqueous buffer further comprises a polysaccharide.
3. The method of claim 2 , wherein the polysaccharide consists of sucrose, trehalose, mannitol, sorbitol, xylitol, lactose, maltose, or inulin.
4. The method of claim of 1 , further comprising the step of lyophilizing the lipid nanoparticle encapsulating the RNA molecule.
5. The method of claim 1 , wherein the organic solvent is ethanol.
6. The method of claim 1 , wherein the cationic lipid is 40 to 60 mole percent of the lipids.
7. The method of claim 1 , wherein the cationic lipid is selected from the group consisting of
8. The method of claim 1 , wherein the cationic lipid is selected from an ionizable cationic lipid or permanently charged cationic lipid.
9. The method of claim 1 , wherein the lipids further comprise a targeting lipid.
10. The method of claim 1 , wherein the first aqueous solution and the second aqueous buffer are at 25-55° C.
11. The method of claim 1 , wherein the first aqueous solution comprises citrate.
12. The method of claim 5 , wherein the ethanol solution is added to the aqueous solution by injection to an air-water interface.
13. The method of claim 5 , wherein the ethanol solution is added to the aqueous solution by a submerged injection.
14. A pharmaceutical formulation comprising a lipid nanoparticle encapsulating a RNA molecule produced by a process of claim 1 wherein the nanoparticle does not require mechanical processing to obtain a polydispersity index of less than 0.2.
15. The pharmaceutical formulation of claim 14 , wherein the cationic lipid is 40 to 60 mole percent of the lipids.
16. The pharmaceutical formulation of claim 14 , wherein the cationic lipid is selected from the group consisting of
17. The pharmaceutical formulation of claim 14 , wherein the cationic lipid is selected from an ionizable cationic lipid or permanently charged cationic lipid.
18. The pharmaceutical formulation of claim 14 , wherein the lipids further comprise a targeting lipid.
19. The pharmaceutical formulation of claim 14 , wherein the process further comprises lyophilization of the liposome-encapsulated RNA molecule.
20. The pharmaceutical formulation of claim 14 , further comprising a polysaccharide.
21. The pharmaceutical formulation of claim 14 , wherein the polysaccharide consists of sucrose, trehalose, mannitol, sorbitol, xylitol, lactose, maltose, or inulin.
22. The pharmaceutical formulation of claim 14 , wherein the mean particle diameter of the lipid nanoparticle encapsulating the RNA molecule is 50-100 nm in size.
23. The pharmaceutical formulation of claim 14 , wherein the lipid nanoparticle encapsulating the RNA molecule has a polydispersity index less than 0.2.