Liposomal apparatus and manufacturing methods
The present invention provides apparatus and processes for producing liposomes. By providing a buffer solution in a first reservoir, and a lipid solution in a second reservoir, continuously diluting the lipid solution with the buffer solution in a mixing chamber produces a liposome. The lipid solution preferably comprises an organic solvent, such as a lower alkanol.
1. An apparatus for producing a lipid vesicle encapsulating a nucleic acid within the lipid vesicle, the apparatus comprising:
a first reservoir containing an aqueous solution including a nucleic acid;
a second reservoir containing an organic lipid solution, wherein the lipids present in the organic lipid solution are solubilized in a lower alkanol at a concentration of about 75% v/v to 100% v/v; and
a pump mechanism configured to pump the aqueous solution and the organic lipid solution into a mixing chamber at different flow rates relative to each other;
wherein the mixing chamber is configured such that the aqueous solution and the organic lipid solution are introduced into the mixing chamber as opposing flows at about 180° relative to each other and mixed within the mixing chamber to instantaneously produce a lipid vesicle encapsulating the nucleic acid within the lipid vesicle by diluting the concentration of the lower alkanol in the organic lipid solution.
2. The apparatus of claim 1 , wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
3. The apparatus of claim 2 , wherein a plurality of lipid vesicles are produced and said plurality of lipid vesicles have a mean particle diameter of less than 150 nm.
4. The apparatus of claim 2 , wherein the aqueous solution includes an acidic buffer.
5. The apparatus of claim 2 , wherein the aqueous solution includes a buffer having a pH lower than the pKa of the cationic lipid component.
6. The apparatus of claim 2 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 6.
7. The apparatus of claim 2 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 8.
8. The apparatus of claim 1 , wherein the nucleic acid comprises an RNA.
9. The apparatus of claim 8 , wherein the lower alkanol comprises ethanol and wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
10. The apparatus of claim 9 , wherein the aqueous solution includes an acidic buffer.
11. The apparatus of claim 9 , wherein the aqueous solution includes a buffer having a pH lower than the pKa of the cationic lipid component.
12. The apparatus of claim 9 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 6 and said plurality of lipid vesicles have a mean particle diameter of less than 150 nm.
13. The apparatus of claim 8 , wherein the RNA comprises an mRNA.
14. The apparatus of claim 13 , wherein the lower alkanol comprises ethanol and wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
15. The apparatus of claim 14 , wherein the aqueous solution includes an acidic buffer.
16. The apparatus of claim 14 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 6 and said plurality of lipid vesicles have a mean particle diameter of less than 150 nm.
17. The apparatus of claim 14 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 8.
18. An apparatus for producing a lipid vesicle encapsulating a nucleic add within the lipid vesicle, the apparatus comprising:
a first reservoir containing an aqueous solution including a nucleic acid;
a second reservoir containing an organic lipid solution, wherein the lipids present in the organic lipid solution are solubilized in a lower alkanol at a concentration of about 75% v/v to 100% v/v; and
a pump mechanism configured to pump the aqueous solution and the organic lipid solution into a mixing chamber at different flow rates relative to each other;
wherein the mixing chamber is configured such that the aqueous solution and the organic lipid solution are introduced into the mixing chamber at an angle of between 90° and 180° relative to each other and mixed within the mixing chamber to instantaneously produce a lipid vesicle encapsulating the nucleic acid within the lipid vesicle by diluting the concentration of the lower alkanol in the organic lipid solution.
19. The apparatus of claim 18 , wherein the lower alkanol comprises ethanol and wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
20. The apparatus of claim 19 , wherein the aqueous solution includes an acidic buffer having a pH lower than the pKa of the cationic lipid component.
21. The apparatus of claim 20 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 6 and said plurality of lipid vesicles have a mean particle diameter of less than 150 nm.
22. The apparatus of claim 20 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 8.
23. The apparatus of claim 18 , wherein the nucleic acid comprises an RNA.
24. The apparatus of claim 23 , wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
25. The apparatus of claim 24 , wherein the aqueous solution includes an acidic buffer.
26. The apparatus of claim 24 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 8.
27. The apparatus of claim 23 , wherein the RNA comprises an mRNA.
28. The apparatus of claim 27 , wherein the lipids present in the organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
29. The apparatus of claim 28 , wherein the aqueous solution includes a buffer having a pH lower than the pKa of the cationic lipid component.
30. The apparatus of claim 28 , wherein the aqueous solution and the organic lipid solution are mixed within the mixing chamber to produce a plurality of lipid vesicles at a pH range of about 3.5 to about 6 and said plurality of lipid vesicles have a mean particle diameter of less than 150 nm.