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Reduction-responsive hyperbranched poly-beta-amino ester with efficient gene delivery capability and preparation method and application thereof

A technology of hyperbranched polyaminoester, which can be used in medical preparations without active ingredients, medical preparations containing active ingredients, gene therapy, etc., can solve problems such as high cytotoxicity, improve endocytosis efficiency, positive charge The effect of high density and rapid degradation

Inactive Publication Date: 2020-09-29
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing gene carrier is limited by its structure, and often requires a high mass ratio to effectively condense nucleic acid molecules, which also brings high cytotoxicity

Method used

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  • Reduction-responsive hyperbranched poly-beta-amino ester with efficient gene delivery capability and preparation method and application thereof
  • Reduction-responsive hyperbranched poly-beta-amino ester with efficient gene delivery capability and preparation method and application thereof
  • Reduction-responsive hyperbranched poly-beta-amino ester with efficient gene delivery capability and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The method for preparing reduction-responsive hyperbranched poly-β-amino esters is as follows:

[0065] Bis(2-hydroxyethyl)disulfide (7.7 g, 50 mmol) and triethylamine (18.75 mL, 150 mmol) were dissolved in 200 mL of anhydrous tetrahydrofuran (THF). Under nitrogen protection, a solution of acryloyl chloride (12.2 mL, 150 mmol) in anhydrous THF (50 mL) was added dropwise within 2 h, and reacted at room temperature for 24 h. Na 2 CO 3 Solution (0.2 M, 5 × 100 mL) and distilled water (3 × 100 mL) were washed separately, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The crude product was subjected to silica gel column chromatography (developing solvent: n-hexane / ethyl acetate = 16 / 1, v / v) to give 2,2-dithiodiethanol diacrylate (SSDA) (8.1 g, 31 mmol, yield 62%) as a pale yellow oil. Deuterated chloroform for NMR, with figure 1 its NMR spectrum.

Embodiment 2

[0067] 2,2-Dithiodiethanol diacrylate (217 mg, 0.83 mmol), trimethylolpropane triacrylate (TMPTA, 74 mg, 0.25 mmol) and 4-amino-1-butanol (89 mg, 1 mmol), reacted at 50°C for 16 hours under solvent-free conditions. Add 1-(3-aminopropyl)-4-methylpiperazine (MPZ, 157 mg, 1 mmol) in dichloromethane solution (1 mL), and react at room temperature for 12 h. Then it was settled three times with diethyl ether, and the solvent was removed under vacuum to obtain a yellow viscous oil, namely BPAE-SS, with a yield of 90%. Deuterated chloroform for NMR, with figure 2 its NMR spectrum. The chemical structural formula of BPAE-SS (x is 9-11, y is 8-10, z is 7-9) is as follows:

[0068]

[0069] The above reaction at 50°C for 16 hours was replaced with reaction at 60°C for 8 hours, and the rest remained unchanged to obtain BPAE-SS with a yield of 75%.

Embodiment 3

[0077] Example 3 Preparation, Characterization and Application of Nanomedicine Packing siRNA

[0078] Prepare respectively the poly-β-amino ester (BPAE-SS) concentration of 1 mg / mL acetate buffer solution (pH = 5.2) and the DEPC aqueous solution of ICAM-1 siRNA (siICAM-1) concentration of 0.1 mg / mL in Example 2 ; then poly-β-amino ester and siRNA were mixed in different weight ratios (10 / 1, 20 / 10, 30 / 1, 40 / 1, 50 / 1, 80 / 1 and 100 / 1), and the mixture was vortexed for 10 seconds, and then incubated at 37 °C for 30 min to form a poly-β-amino ester / siRNA complex (BPAE-SS / siRNA). Then add 2% agarose gel electrophoresis to the sample well, run at 90 V for 20 min, and image with the gel imaging system to measure the loading efficiency of siRNA.

[0079] The particle size and potential of the polyβ-amino ester / siRNA complex under different weight ratio conditions were determined by dynamic light scattering (DLS).

[0080] Poly-β-amino ester was pretreated with glutathione (GSH, 5 mM, ...

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Abstract

The invention provides reduction-responsive hyperbranched poly-beta-amino ester with efficient gene delivery capability and a preparation method and application thereof. The polymer is polymerized byan A2 + B3 + C2 Michael addition method so that the polymer has a hyperbranched structure. Compared with a linear structure, the branched structure can enhance the interaction between the polymer andnucleic acid molecules, significantly improve the gene condensation ability of the polymer, and increase cellular uptake by enhancing the interaction with cell membranes. The main chain of the polymerhas a reduction-responsive group (disulfide bond), and under the action of GSH in damaged vascular endothelial cells, poly-beta-amino ester can be quickly degraded in cytoplasm to release an entrapped gene drug (ICAM-1 siRNA) so that efficient transfection of genes is realized, and meanwhile, the toxicity of the material is reduced. Due to the properties of the poly-beta-amino ester, the poly-beta-amino ester has a huge development prospect in biomedical materials, especially in the field of gene delivery.

Description

technical field [0001] The invention relates to the field of gene loading and delivery, in particular to a reduction-responsive hyperbranched poly-β-amino ester with high-efficiency gene delivery capability, its preparation method and application, and used for siRNA transfection. Background technique [0002] Gene vectors are important tools for loading nucleic acid molecules, delivering them into target cells and successfully expressing them. Gene carrier materials can be divided into two categories: viral vectors and non-viral vectors. Viral vectors have the advantages of high transfection efficiency, but their own shortcomings such as high immunogenicity, high risk of carcinogenesis, and low gene loading seriously restrict their application and development in the field of medicine. Based on this, non-viral gene vectors have gradually gained attention and development. Commonly used non-viral gene carriers mainly include liposomes, cationic polymers and polysaccharides. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G83/00C08G73/02A61K9/51A61K47/69B82Y5/00A61P9/10A61K31/713A61K48/00
CPCA61K9/5146A61K31/713A61P9/10B82Y5/00C08G73/0253C08G83/005
Inventor 殷黎晨王笑梁秋君
Owner SUZHOU UNIV
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