Nucleic acid-containing lipid particles and related methods

Abstract

Lipid particles containing a nucleic acid, devices and methods for making the lipid particles, and methods for using the lipid particles.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
         1 . A lipid particle, comprising:
 (a) a core consisting of (i) nucleic acid and cationic lipid; and   (b) second lipids surrounding the core;   wherein the 31P nuclear magnetic resonance spectrum of the lipid particle measured in solution after treatment with 150 mM ammonium acetate does not exhibit a resonance due to the nucleic acid.   
     
     
         2 . The particle of  claim 1 , comprising from 30 to 95 mole percent cationic lipid. 
     
     
         3 . The particle of  claim 1 , wherein the second lipids are selected from neutral lipids, anionic lipids and PEG-lipids. 
     
     
         4 . The particle of  claim 3 , wherein the second lipid is selected from the group consisting of a PEG-lipid, a zwitterionic lipid, and a sterol; preferably wherein the second lipid is selected from a PEG-lipid and wherein said PEG-lipid is present in said lipid particle in are amount from 1.0 to 10 mole percent. 
     
     
         5 . The particle of  claim 1  having a diameter from 15 nm to 300 nm. 
     
     
         6 . The particle of  claim 1 , which comprises nucleic acid, cationic lipid, neutral lipid, PEG-lipid, and sterol. 
     
     
         7 . An in vitro method for introducing a nucleic acid into a cell, comprising contacting a cell with the lipid particle of  claim 1 . 
     
     
         8 . A method for making lipid particles containing a nucleic acid, comprising:
 (a) introducing a first stream comprising a nucleic acid in a first solvent into a microfluidic device; wherein the device has a first region adapted for flowing one or more streams introduced into the device and a second region for mixing the contents of the one or more streams with a microfluidic mixer;   (b) introducing a second stream comprising lipid particle-forming materials in a second solvent into the device to provide first and second streams flowing under laminar flow conditions, wherein the lipid particle-forming materials comprise a cationic lipid, and wherein the first and second solvents are not the same;   (c) flowing the one or more first streams and the one or more second streams from the first region of the device into the second region of the device; and   (d) mixing the one or more first streams and the one or more second streams in the second region of the device to provide a third stream comprising the lipid particles of  claim 1 .   
     
     
         9 . The method of  claim 8 , wherein the nucleic acid is encapsulated in the lipid particle with an efficiency of from 90 to 100%. 
     
     
         10 . The particle of  claim 1 , wherein the cationic lipid is an ionizable lipid. 
     
     
         11 . The particle of  claim 1 , wherein the cationic lipid is an amino lipid. 
     
     
         12 . The particle of  claim 3 , wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. 
     
     
         13 . The particle of  claim 1 , wherein the nucleic acid is a DNA, an RNA, a locked nucleic acid, a nucleic acid analog, or a plasmid capable of expressing a DNA or an RNA. 
     
     
         14 . The particle of  claim 1 , wherein the nucleic acid is mRNA, siRNA, or microRNA. 
     
     
         15 . The method of  claim 8 , wherein the first solvent is an aqueous buffer and the second solvent is an aqueous alcohol. 
     
     
         16 . The method of  claim 8 , wherein the second stream further comprises a second lipid. 
     
     
         17 . The method of  claim 8 , wherein the first solvent is an aqueous solvent and the second solvent is an organic solvent, and wherein the volume ratio between the first and second solvent exceeds 1.0 (aqueous:organic). 
     
     
         18 . The method of  claim 17 , wherein the volume ratio is 3:1.