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CN-122005828-A - Nucleic acid delivery system and preparation method and application thereof

CN122005828ACN 122005828 ACN122005828 ACN 122005828ACN-122005828-A

Abstract

The invention discloses a nucleic acid delivery system, a preparation method and application thereof, and belongs to the technical field of biological materials. According to the invention, a novel benzenesulfonamide functional gemini surfactant (NGS) is constructed by introducing benzenesulfonamide functional groups into GSs molecular skeletons. Wherein, the benzenesulfonamide group is a specific endoplasmic reticulum targeting group, and endoplasmic reticulum targeting enrichment can be realized by specific binding with sulfonylurea receptors on the endoplasmic reticulum membrane, so that the NGS gene delivery vector is distributed to the endoplasmic reticulum. After NGS and pDNA form a complex, they enter the cell through the cellular protein-mediated endocytosis, then target to the endoplasmic reticulum and release the pDNA, and the pDNA is delivered into the nucleus by using the close relationship between the endoplasmic reticulum and the nuclear membrane, so that the gene transfection efficiency is finally improved, and meanwhile, the negatively charged gamma-polyglutamic acid (gamma-PGA) is introduced to further improve the biocompatibility, so that the application prospect is wide.

Inventors

  • CHEN HUALI
  • QIAN PENG
  • CHEN YUXIN

Assignees

  • 重庆医科大学

Dates

Publication Date
20260512
Application Date
20260318

Claims (7)

  1. 1. A method of preparing a nucleic acid delivery system, comprising the steps of: S1, mixing 2- (4-sulfamoyl phenyl) acetic acid and N, N '-carbonyldiimidazole for dissolution, stirring and reacting for 20-40min, adding 3,3' -iminobis (N, N-dimethylpropylamine), and carrying out reflux reaction for 1-3h at 60-80 ℃ after uniform mixing to obtain an intermediate product, wherein the molar ratio of 2- (4-sulfamoyl phenyl) acetic acid to N, N '-carbonyldiimidazole to 3,3' -iminobis (N, N-dimethylpropylamine) is 1-2:1-2; s2, mixing and dissolving the intermediate product and bromooctadecane in a molar ratio of 0.5-1.5:2-3, and then carrying out reflux reaction for 70-74h at 90-110 ℃ to obtain the gemini surfactant; s3, mixing the gemini surfactant and a substance to be delivered in a mass ratio of 6-10:0.5-1.5, repeatedly blowing for 5-8 times, then swirling for 5-10S, and standing at room temperature for incubation for 0.5-1.5h to obtain the NGS nano-particles, wherein the substance to be delivered is plasmid DNA, messenger RNA, small interfering RNA or antisense oligonucleotide.
  2. 2. The method for preparing a nucleic acid delivery system according to claim 1, wherein the solvent used for the dissolution in S1 and S2 is tetrahydrofuran or acetonitrile.
  3. 3. The method of claim 1, wherein the molar ratio of 2- (4-sulfamylphenyl) acetic acid, N '-carbonyldiimidazole, and 3,3' -iminobis (N, N-dimethylpropylamine) is 1:1:1.
  4. 4. The method of claim 1, further comprising mixing the gamma-polyglutamic acid solution and the NGS nanoparticles at a ratio of 1.9-9.7. Mu.L/15-20. Mu.g, repeatedly blowing for 3-5 times, and incubating at room temperature for 30-60min to obtain the NGS-PGA nanoparticles.
  5. 5. The method for preparing a nucleic acid delivery system according to claim 4, wherein the concentration of the gamma-polyglutamic acid solution is 90-110. Mu.g/mL.
  6. 6. A nucleic acid delivery system, characterized in that it is produced by the production method according to any one of claims 1 to 5.
  7. 7. Use of the nucleic acid delivery system of claim 6 for the preparation of a nucleic acid delivery medicament.

Description

Nucleic acid delivery system and preparation method and application thereof Technical Field The invention belongs to the technical field of biological materials, and particularly relates to a nucleic acid delivery system, a preparation method and application thereof. Background In recent years, gene therapy has shown a broad application prospect in the fields of cancer, infectious disease, genetic disease, rare disease and the like, and is focused on the realization of accurate treatment of diseases by delivering exogenous nucleic acids such as plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA) and the like to regulate or repair abnormal gene expression. However, nucleic acids themselves have large molecular weight, negative charge, poor stability, and the like, and in vivo delivery needs to overcome multiple extracellular and intracellular barriers, including nuclease degradation in blood, reticuloendothelial system removal, cell membrane electrostatic repulsion, intracellular lysosome retention, and limited nuclear entry. Thus, the construction of efficient, safe nucleic acid delivery systems is critical to achieving successful gene therapy. Compared with a viral vector, the non-viral vector system is widely researched and focused due to the advantages of low immunogenicity, strong structure controllability, low preparation cost and the like. The cationic gemini surfactant (Gemini surfactants, GSs) is formed by connecting two monomer surfactants through a spacer, has higher charge density and excellent nucleic acid compression capacity, can form a nano-composite with smaller particle size and positively charged surface with nucleic acid through electrostatic action, and is one of important research directions in the current non-viral delivery system. However, in the existing GSs gene delivery system, the problem of low transfection efficiency is still faced due to the challenges of lysosomal retention or nuclear membrane barrier restriction in the intracellular transport process, so that the clinical transformation is limited, and meanwhile, the biomimetic modification based on the cell membrane may have problems in terms of sources, mass production and long-term storage, so that the transformation of GSs delivery vectors into clinical application is severely limited. Therefore, there is a need for rational design of next generation GSs-based nucleic acid delivery vectors to achieve integration of high transfection efficiency with low cytotoxicity. Disclosure of Invention The invention aims to solve the technical problems of low transfection efficiency and poor delivery effect of the existing GSs gene delivery system by providing a nucleic acid delivery system and a preparation method and application thereof. In order to achieve the above purpose, the technical scheme adopted by the invention is that a preparation method of a nucleic acid delivery system is provided, which comprises the following steps: S1, mixing 2- (4-sulfamoyl phenyl) acetic acid and N, N ' -carbonyldiimidazole for dissolution, stirring and reacting for 20-40min, wherein 2- (4-sulfamoyl phenyl) acetic acid provides carboxyl (-COOH) as a reactive site, N, N ' -carbonyldiimidazole is used as a condensing agent and reacts with the carboxyl of 2- (4-sulfamoyl phenyl) acetic acid to generate an acylimidazole intermediate, then nucleophilic amine reactants, namely 3,3' -iminobis (N, N-dimethylpropylamine), are added, and after uniform mixing, the nucleophilic amine reactants and the activated acylimidazole intermediate are subjected to reflux reaction for 1-3h at 60-80 ℃ to generate amidation reaction, so that an intermediate product containing benzenesulfonamide groups is obtained, and the molar ratio of 2- (4-sulfamoyl phenyl) acetic acid, N, N ' -carbonyldiimidazole and 3,3' -iminobis (N, N-dimethylpropylamine) is 1-2:1-2; S2, mixing and dissolving an intermediate product and bromooctadecane in a molar ratio of 0.5-1.5:2-3, and then carrying out reflux reaction for 70-74h at 90-110 ℃, wherein the bromooctadecane is used as a long-chain alkylating agent, and a typical nucleophilic substitution reaction (Menshutkin reaction) is carried out on the bromooctadecane and tertiary amine groups in the intermediate product to generate a gemini surfactant (NGS) with a quaternary ammonium salt structure, the electrostatic interaction capability and compression capability between the NGS and negatively charged nucleic acid molecules are obviously enhanced by the gemini ammonium salt structure, and meanwhile, the stability of the nano structure is maintained by the two introduced long-chain alkyl hydrophobic tail chains; S3, mixing a gemini surfactant (NGS) and a substance to be delivered in a mass ratio of 6-10:0.5-1.5, repeatedly blowing for 5-8 times, then swirling for 5-10S, and standing at room temperature for incubation for 0.5-1.5h to obtain NGS nanoparticles, wherein the substance to be delivered is plasmid DNA (pDNA), mess