Patent
Method for preparing PEI-lipid nanoparticles used for delivering a mRNA vaccine and transfecting stem cells
| العنوان: | Method for preparing PEI-lipid nanoparticles used for delivering a mRNA vaccine and transfecting stem cells |
|---|---|
| Patent Number: | 12215,342 |
| تاريخ النشر: | February 04, 2025 |
| Appl. No: | 17/862421 |
| Application Filed: | July 12, 2022 |
| مستخلص: | The method of preparing polyethyleneimine-lipid nanoparticles (PEI-LNPs) for transfecting stem cells includes the following steps: S1, adding anhydride to a PEI solution to produce polycarboxylic acid; S2, dissolving the PEI-LNPs from S1, together with DPPC, DPPG, DPPE-PEG-COOH and cholesterol in chloroform, dissolving cGAMP in a water and then adding to such chloroform solution, and performing vacuum evaporation to remove the chloroform; S3, adding PBS for hydration, and performing ultrasonication to obtain liposomes; and S4, adding EDC/NHS to the liposome solution obtained for reaction for 15 min, then adding mRNA, and stirring the mixed solution to obtain a bionic virus nanovaccine. The prepared PEI-LNPs are used for delivering an mRNA vaccine and transfecting stem cells, wherein through a lipid PEI derivative library synthesized based on short-branched PEI with a variable length and hydrophobic tail substitution ratio, short-branched PEI can form a stable complex with the mRNA vaccine. |
| Inventors: | Healthina Stem Cell Industry Platform (Tianjin) Limited (Tianjin, CN); Tangyi Holdings(Shenzhen) Limited (Shenzhen, CN) |
| Assignees: | Healthina Stem Cell Industry Platform (Tianjin) Limited (CN), Tangyi Holdings(Shenzhen) Limited (CN) |
| Claim: | 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. |
| Claim: | 2. The method according to claim 1 , wherein step 2 comprises: dissolving the PEI-LNPs obtained in step 1, together with 13 μg of DPPC, 2 μg of DPPG, 2 μg of DPPE-PEG-COOH and 1 μg of cholesterol in 3 mL of chloroform to form the chloroform solution, and dissolving cGAMP in 1 mL of DNase/RNase-free water to form the cGAMP solution, and then adding the cGAMP solution to the chloroform solution, and then performing the vacuum ultrasonication for 30 min and then the vacuum evaporation to remove the chloroform and obtain the PEI-LNPs mixture; wherein step 3 comprises adding 2 mL of PBS to the PEI-LNPs mixture for hydration, and then performing the ultrasonication for 20 min to obtain the liposome solution; and wherein in step 4, EDC and NHS are each added to the liposome solution obtained in step 3 in an amount of 10 mg per mL of the liposome solution; when the mRNA is added to the liposome solution containing EDC and NHS, a volume ratio of mRNA:liposomes is 2:1. |
| Claim: | 3. The method according to claim 1 , wherein in step 1, a mass ratio of PEI-LNPs:anhydride is 1:0.5. |
| Claim: | 4. The method according to claim 1 , wherein in step 1, the anhydride is C12 alkyl anhydride. |
| Claim: | 5. The method according to claim 1 , wherein in step 4, the mRNA vaccine has the following synthetic process: (1) adding an mRNA aqueous solution to such mixed solution, and performing ultrasonication for 15 min and vacuum evaporation; and (2) adding PBS for hydration to obtain the mRNA vaccine. |
| Claim: | 6. The method according to claim 5 , wherein in step 2, the PEI-LNPs are composed of 50% of ionogenic cationic lipid DLin-MC3-DMA, 10% of DSPC, 35% of cholesterol and 5% of DPPE-PEG-COOH. |
| Patent References Cited: | 2018/0161272 June 2018 Obaid 2020/0206134 July 2020 Kalinichenko 2021/0330600 October 2021 Talukder WO-2009129354 October 2009 |
| Other References: | BroadPharm: A Worldwide Leading PEG Supplier. What is a helper lipid? (Year: 2022). cited by examiner G-Biosciences: Water, Endotoxin Free (Year: 2019). cited by examiner Warriner et al. Biomacromolecules 2018 19 (11), 4348-4357 (published: Oct. 24, 2018) and (Year: 2018). cited by examiner Mali et al. J Appl Polym Sci 2021, 138(45), e51323 (published: Jun. 2, 2021). (Year: 2021). cited by examiner |
| Primary Examiner: | Alley, Genevieve S |
| رقم الانضمام: | edspgr.12215342 |
| قاعدة البيانات: | USPTO Patent Grants |
| الوصف غير متاح. |