US 12,215,342 B2
Method for preparing PEI-lipid nanoparticles used for delivering a mRNA vaccine and transfecting stem cells
Bin Zheng, Tianjin (CN); Yulin Cao, Beijing (CN); Tingting Hua, Tianjin (CN); and Shixiang Cheng, Tianjin (CN)
Assigned to Healthina Stem Cell Industry Platform (Tianjin) Limited, (CN); and Tangyi Holdings(Shenzhen) Limited, (CN)
Filed by Healthina Stem Cell Industry Platform (Tianjin) Limited, Tianjin (CN); and Tangyi Holdings(Shenzhen) Limited, Shenzhen (CN)
Filed on Jul. 12, 2022, as Appl. No. 17/862,421.
Claims priority of application No. 202210189584.6 (CN), filed on Feb. 28, 2022.
Prior Publication US 2023/0272426 A1, Aug. 31, 2023
Int. Cl. C12N 15/88 (2006.01); A61K 9/127 (2006.01); A61K 9/51 (2006.01)
CPC C12N 15/88 (2013.01) [A61K 9/1272 (2013.01); A61K 9/5123 (2013.01)] 6 Claims
 
1. A method of preparing polyethyleneimine-lipid nanoparticles (PEI-LNPs) for delivering an mRNA vaccine and transfecting stem cells, comprising the following specific steps:
1) adding anhydride to a PEI solution to produce polycarboxylic acid and PEI-LNPs, wherein the anhydride is C8-C18 alkyl anhydride;
2) dissolving the PEI-LNPs obtained in step 1, together with DPPC, DPPG, DPPE-PEG-COOH and cholesterol in chloroform to form a chloroform solution, dissolving cGAMP in a sterile water to form a cGAMP solution and then adding the cGAMP solution to the chloroform solution, and performing vacuum ultrasonication for 30 min and then vacuum evaporation to remove the chloroform and obtain a PEI-LNPs mixture;
3) adding PBS to the PEI-LNPs mixture obtained in step 2 for hydration, and then performing ultrasonication for 20 min to obtain a liposome solution; and
4) adding EDC/NHS to the liposome solution obtained in step 3 to perform a reaction for 15 min, then adding an mRNA to form a mixed solution, and stirring the mixed solution at a room temperature for 1 h to obtain a bionic virus nanovaccine.