Liposomal apparatus and manufacturing methods
View Patent ↗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. A process for producing a lipid vesicle encapsulating a nucleic acid within the lipid vesicle, said process comprising:
providing an aqueous solution including a nucleic acid in a first reservoir;
providing an organic lipid solution in a second reservoir, wherein the lipids present in said organic lipid solution are solubilized in a lower alkanol at a concentration of about 75% v/v to 100% v/v; and
mixing said organic lipid solution with said aqueous solution by introducing said organic lipid solution and said aqueous solution into a mixing environment at about equal flow rates;
wherein said mixing instantaneously produces a lipid vesicle encapsulating the nucleic acid within the lipid vesicle by diluting said lower alkanol to a concentration of between 45% v/v to about 60% v/v; and
wherein the mixing environment includes a T-connector, wherein the aqueous solution and the organic lipid solution are introduced into the T-connector as opposing flows at about 180° relative to each other and mixed within the T-connector.
2. The process of claim 1 , further comprising diluting said lipid vesicle with a buffer solution wherein said lipid vesicle undergoes a continuous stepwise dilution to further stabilize the lipid vesicle.
3. The process of claim 1 , wherein said lipid vesicle is in a solution having a pH of about 3.5 to about 8.0.
4. The process of claim 1 , wherein the lipid vesicle is in a solution having a pH of about 6 or lower, and wherein said lipid vesicle has a nucleic acid encapsulation efficiency of greater than 50%.
5. The process of claim 1 , wherein the lipid vesicle is in a solution having a pH of about 5 or lower, and wherein said lipid vesicle has a nucleic acid encapsulation efficiency of between about 80% and about 90%.
6. The process of claim 1 , wherein said lipid vesicle has a diameter of about 150 nm or less.
7. The process of claim 1 , wherein said lipid vesicle is in a solution having a salt concentration of about 100 mM to about 200 mM.
8. The process of claim 1 , wherein said nucleic acid is selected from the group consisting of a plasmid, an antisense polynucleotide, a ribozyme, tRNA, snRNA, siRNA, and pre-condensed DNA.
9. The process of claim 1 , wherein said nucleic acid comprises an siRNA.
10. The process of claim 1 , wherein said lower alkanol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, isomers thereof, and combinations thereof.
11. The process of claim 1 , wherein said lower alkanol comprises ethanol.
12. The process of claim 1 , wherein the lipids present in said organic lipid solution are solubilized in said lower alkanol at a concentration of between about 80% v/v to about 90% v/v.
13. The process of claim 1 , wherein the lipid vesicle is a liposome.
14. The process of claim 2 , wherein said lipid vesicle is in a solution having a concentration of less than 25% or about 25% v/v of said lower alkanol after dilution.
15. The process of claim 1 , wherein said lipid vesicle is produced at a flow rate of between about 0.075 L/min and about 0.3 L/min.
16. The process of claim 1 , wherein said lipid vesicle is produced at a shear rate of between about 500/s and about 3300/s.
17. The process of claim 1 , wherein one or both of the first and the second reservoirs are temperature controlled.
18. The process of claim 1 , wherein one or both of the first and the second reservoirs include a jacketed stainless steel vessel with an overhead mixer.
19. The process of claim 1 , wherein the T-connector is polypropylene or stainless steel.
20. The process of claim 1 , wherein the lipids present in said organic lipid solution comprise a phospholipid, cholesterol, a PEG-lipid, and a cationic lipid.
21. The process of claim 2 , further comprising concentrating said lipid vesicle by tangential flow ultrafiltration.
22. The process of claim 1 , wherein the lipids present in said organic lipid solution are solubilized in said lower alkanol at a concentration of about 100% v/v.