Method of encapsulating a nucleic acid in a lipid nanoparticle host
Encapsulated nucleic acid nanoparticles of uniformly small particle size are produced by intersecting one or more nucleic acid streams with one or more lipid streams. The encapsulated nucleic acid nanoparticles include a nucleic acid encapsulated within a lipid nanoparticle host. Uniformly small particle sizes are obtained by intersecting an aqueous nucleic acid stream and a stream of lipids in organic solvent at high linear velocities and with total organic solvent concentrations less than 33%.
1. A method of encapsulating a nucleic acid in a lipid nanoparticle host to provide an encapsulated nucleic acid nanoparticle, the method comprising:
providing two nucleic acid streams, each of the streams comprising a mixture of a nucleic acid in a first aqueous solution, and together having a combined linear velocity of greater than or equal to about 3 to about 14 meters/second;
providing one lipid stream, the lipid stream comprising a mixture of one or more lipids in an organic solvent, and having a linear velocity of about 1.5 to about 4.5 meters/second;
joining the lipid stream with the two nucleic acid streams at a first intersection point configured as a cross connector, wherein the two nucleic acid streams enter the cross connector from opposing directions at about 180° relative to each other, and the lipid stream enters the cross connector at about 90° relative to the two nucleic acid streams to provide a first joined stream;
flowing the first joined stream in a first direction out of the cross connector in substantially the same direction as the lipid stream and about 90° relative to the two nucleic acid streams; and
mixing the components thereof to provide a first outlet solution comprising the encapsulated nucleic acid nanoparticle;
wherein the first aqueous solution and the organic solvent are miscible, one of the first aqueous solution or first organic solution contains a buffer, and the concentration of the organic solvent in the first outlet solution is in an amount to minimize aggregation.
2. The method of claim 1 , wherein:
the first aqueous solution is a first buffer solution comprising an aqueous solution of an alkali metal salt; and
the organic solvent comprises an alcoholic solvent.
3. The method of claim 1 , further comprising:
diluting the first joined stream with a dilution solvent selected from a second buffer solution, which second buffer solution contains at least one alkali metal salt, and water, to provide the first outlet stream;
the alkali metal salt is selected from one or more of NaCl and sodium citrate, and
the concentration of the organic solvent in the first outlet solution is less than about 33%.
4. The method of claim 1 , wherein:
the combined linear velocity of the one or more lipid streams is between about 3 to about 4 meters/second; and
the combined linear velocity of the one or more nucleic acid streams is between about 9 to about 14 meters/second.
5. The method of claim 2 , wherein diluting the first joined stream with a diluting solvent comprises:
providing a diluting stream, the diluting stream comprising an aqueous solvent selected from a second buffer solution and water; and
joining the first joined stream with the diluting stream to provide the first outlet stream;
wherein the combined linear velocity of the one or more lipid streams is between about 3 to about 4 meters/second; and the combined linear velocity of the one or more nucleic acid streams is between about 6 to about 8 meters/second.
6. The method of claim 1 , further comprising incubating the first outlet solution;
diluting the incubated first outlet solution with a second dilution solvent selected from a third buffer solution and water, to provide a second outlet solution; and
concentrating and dialyzing the second outlet solution, to provide a concentrated dialyzed solution.
7. The method of claim 6 , wherein the concentrating comprises concentrating by tangential flow filtration.
8. The method of claim 7 further comprising sterile filtering the concentrated dialyzed solution.
9. The method of claim 1 , wherein the nucleic acid is a siRNA.
10. The method of claim 1 , wherein the nucleic acid is an mRNA.
11. The method of claim 1 , wherein the lipid nanoparticle host comprises one or more of the lipids selected from Table 2.
12. The method of claim 1 , wherein the encapsulated nucleic acid nanoparticle has an average particle size diameter of about 30 nm to about 80 nm.
13. The method of claim 1 , wherein the encapsulated nucleic acid nanoparticle has an average particle size diameter of about 40 nm to about 70 nm and a polydispersity index of less than about 0.1 as determined by dynamic light scattering.
14. The method of claim 1 , wherein the ratio by mass of lipids:nucleic acid is about 15-20:1; the concentration of nucleic acid in the one or more nucleic acid streams is about 0.1 to about 1.5 mg/mL and the concentration of lipids in the one or more lipid streams is about 10 to about 25 mg/mL; and the concentration of ethanol in the second outlet solution is about 10-15%.
15. The method of claim 5 , wherein joining the first joined stream with the diluting stream comprises intersecting the first joined stream with the diluting stream from about 90° relative to the direction of the first joined stream.
16. The method of claim 1 , wherein greater than about 90% of the nucleic acid from the nucleic acid stream is encapsulated in the first outlet solution.
17. The method of claim 1 , wherein the concentration of the organic solvent in the first outlet solution in an amount to minimize aggregation is less than about 33%.