SiRNA delivery system compound as well as preparation method and application thereof

Patent metadata

Field	Value
Publication number	US-12194145-B2
Application number	US-202117227408-A
Country	US
Kind code	B2
Filing date	Apr 12, 2021
Priority date	Jun 30, 2020
Publication date	Jan 14, 2025
Grant date	Jan 14, 2025

Abstract

The invention relates to the technical field of gene treatment and nano targeted delivery, in particular to a siRNA delivery system compound as well as a preparation method and application thereof. The preparation method comprises the following steps of reacting Boc-protected histidine with cholesterol to generate Boc-protected histidine cholesteryl ester, and removing BOC-released protected amino in trifluoroacetic acid to generate histidine cholesteryl ester; then, enabling amino to react with carboxyl of carboxymethyl chitosan to modify long-chain carboxymethyl chitosan to generate carboxymethyl chitosan modified by histidine cholesteryl ester, and performing targeted labeling on carboxymethyl chitosan modified by histidine cholesteryl ester to generate a carboxymethyl chitosan high-molecular compound modified by histidine cholesteryl ester and a targeted marker together, then forming a stable spherical delivery vector through self-assembly above critical micelle concentration, and mixing the stable spherical delivery vector with siRNA (small interfering ribonucleic acid) to obtain the siRNA delivery system compound.

First claim

The invention claimed is:

1. A preparation method of a siRNA delivery system compound, comprising the following steps:
S1: adding cholesterol and Boc-protected histidine to dichloromethane, continuing to add dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the dichloromethane, reacting at room temperature for 14-16 h, successively filtering off a white precipitate, removing a solvent and purifying under decompression to obtain a Boc-protected histidine cholesteryl ester intermediate;
S2: dissolving the Boc-protected histidine cholesteryl ester intermediate prepared in S1 in dichloromethane, continuing to add trifluoroacetic acid, stirring for 1-1.5 h, then neutralizing with an aqueous ammonia solution, evaporating a solvent under decompression, diluting a residue in ethyl acetate, washing and drying successively, and evaporating the solvent under decompression again to obtain a white powdery histidine cholesteryl ester intermediate;
S3: dissolving the histidine cholesteryl ester prepared in S2 in tetrahydrofuran to prepare a solution A, dissolving carboxymethyl chitosan, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and N-hydroxysuccinimide in a 1% acetic acid solution, activating at room temperature, adding the solution A, reacting at room temperature for 70-72 h, evaporating tetrahydrofuran under decompression, then adding absolute ethanol to accumulate a precipitate, retaining the precipitate, and washing the precipitate sequentially with tetrahydrofuran, acetone and ether to obtain a white powdery histidine cholesteryl ester modified carboxymethyl chitosan polymer compound;
S4: dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in a targeted marker solution with a concentration of 0.1 mmol, activating the carboxyl group for 1-1.5 h to obtain an activated targeted marker solution, dissolving the histidine cholesteryl ester modified carboxymethyl chitosan polymer compound prepared in S3 in a 0.8-1% hydrochloric acid solution, dripping the activated targeted marker solution under stirring, reacting for 22-24 h at 4° C., then freeze-drying to obtain a carboxymethyl chitosan polymer compound modified by the histidine cholesteryl ester and the targeted marker;
S5: dissolving the carboxymethyl chitosan polymer compound modified by the histidine cholesteryl ester and the targeted marker in a HEPES buffer solution with a concentration of 20 mM and a pH value of 7.4, treating with ultrasonic, then purifying to obtain a nano spherical carrier; and
S6: dissolving siRNA in a nano spherical carrier solution with a concentration of 0.56 mg/mL, and the ratio of the siRNA to the nano spherical carrier in the solution is 1 pmol: 0.01-0.2 μg, and storing in a dark place for more than 30 min to obtain the siRNA delivery system compound.

2. The preparation method according to claim 1 , wherein in S1, the ratio of the cholesterol, the Boc-protected histidine, the dichloromethane, the dicyclohexylcarbodiimide and the 4-dimethylaminopyridine is 200-220 mg: 125-135 mg: 10-12 mL: 1.8-2 mg;
in S2, the mass volume ratio of the Boc-protected histidine cholesteryl ester intermediate, the dichloromethane, the trifluoroacetic acid and the aqueous ammonia solution is 200-220 mg: 6-8 mL: 0.8-1 mL: 1.8-2 mL;
in S3, the mass volume ratio of the histidine cholesteryl ester to the tetrahydrofuran is 500-520 mg: 100 mL, and the mass volume ratio of the carboxymethyl chitosan, the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the N-hydroxysuccinimide, the solution A and the absolute ethanol is 480-500 mg: 240-260 mg: 150-160 mg: 48-50 mL: 198-200 mL;
in S4, the mass volume ratio of the carbodiimide hydrochloride, the N-hydroxysuccinimide, the targeted marker solution, the cholesteryl histidine ester modified carboxymethyl chitosan polymer compound and the hydrochloric acid solution is 38-40 mg: 22-23 mg: 1.8-2 mL: 40-42 mg: 10-12 mL;
in S5, the mass-volume ratio of the carboxymethyl chitosan polymer compound modified by the histidine cholesteryl ester and the targeted marker and the HEPES buffer solution is 10-12 mg: 5-6 mL.

3. The preparation method according to claim 2 , wherein in S4, the targeted marker is one selected from the group consisting of EGFR monoclonal antibody, HER2 monoclonal antibody, PSMA monoclonal antibody, or RGD short peptide containing arginine, glycine and aspartic acid sequences.

4. The preparation method according to claim 3 , wherein the siRNA silences genes related to tumorigenesis, proliferation, and metastasis.

Assignees

• Univ Zhengzhou

Inventors

• Wang Zhimin
• Zhu Chengshen
• Liu Wentao
• Zhang Xiangyang
• He Suqin
• Wang Yaohe
• Liu Hao
• Huang Miaoming
• Zhang Chenglin
• Qin Bin
• Huang Lei

Classifications

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C08B37/003

  Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof · CPC title

• A61K31/713 Primary

  Double-stranded nucleic acids or oligonucleotides · CPC title

• A61K9/5161

  Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin · CPC title

• A61P35/04

  specific for metastasis · CPC title