# US 12,215,342 B2 · Method for preparing PEI-lipid nanoparticles used for delivering a mRNA vaccine and transfecting stem cells

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- Number: US 12,215,342 B2
- Document ID: US12215342B2
- Status: Granted
- Kind: B2
- Application number: 17/862,421
- Filed: 2022-07-12
- Published: 2023-08-31
- Granted: 2025-02-04
- Earliest claimed: 2022-02-28
- Assignees: Healthina Stem Cell Industry Platform (Tianjin) Limited; Tangyi Holdings(Shenzhen) Limited
- Applicants: Healthina Stem Cell Industry Platform (Tianjin) Limited; Tangyi Holdings(Shenzhen) Limited
- Inventors: Bin Zheng; Yulin Cao; Tingting Hua; Shixiang Cheng
- Web page: https://patentgrep.com/patent/US12215342B2

## CPC

- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- A61K 9/5123: {Organic compounds, e.g. fats, sugars}
- A61K 9/5146: {obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides}
- A61K 9/5192: {Processes}
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61K 39/0011: {Cancer antigens}
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

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.

## Claims

### 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 (depends on 1)

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 (depends on 1)

The method according to claim 1, wherein in step 1, a mass ratio of PEI-LNPs:anhydride is 1:0.5.

### Claim 4 (depends on 1)

The method according to claim 1, wherein in step 1, the anhydride is C12 alkyl anhydride.

### Claim 5 (depends on 1)

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 (depends on 5)

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.