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Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof

A cationic polymer and gene carrier technology, applied in the fields of biomedicine and polymer medical materials, can solve the problems of high cytotoxicity and non-degradability, and achieve the effects of reducing cytotoxicity, good biocompatibility, and improving transfection efficiency

Inactive Publication Date: 2013-07-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, polyacetylimide PEI has good DNA loading capacity, and can promote the escape of DNA from endosomes to the "proton sponge" characteristics in the cytoplasm, and has high transfection efficiency. It is currently the cationic polymer gene carrier being studied. Studies have shown that increasing the degree of branching of PEI can enhance its ability to wrap DNA, but it is not degradable, the higher the transfection efficiency, the greater the cytotoxicity

Method used

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  • Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof
  • Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof
  • Sustained-and-controlled-release acid-sensitive cationic polymer gene vector with branched structure, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 has the preparation method of the acid-sensitive cationic polymer gene carrier of branched structure

[0021] Take 0.283g of ethylene glycol diglycidyl ether and place it in a double-necked bottle, add 1mL of DMF, stir and dissolve, then add 0.5g of diamine type acid-sensitive monomer (ie, ethylene glycol diglycidyl ether: binary Amine-type acid-sensitive monomer molar ratio = 1:1) Stir evenly, react for 48 hours under anhydrous and anaerobic room temperature conditions; put the reaction product into a dialysis bag with a cut-off molecular weight of 3000-4000, distilled water (in which 0.01% triethyl ether is added) amine) for 24-48 hours of dialysis; accurately measure 1 mL of the dialysis product to freeze-dry, weigh the mass after freeze-drying, and obtain the concentration of the polymer in 1 mL of the dialysis solution. The remainder is stored frozen in liquid form. The polymerization product is Polymer 1.

[0022] Take 0.424 g of ethylene glycol dig...

Embodiment 2

[0025] Embodiment 2 measures the cytotoxicity of polymkeric substance with MTT method

[0026] NIH / 3T3 cells were seeded into 96-well plates (1×10 4 each well), add 200 μL of DMEM medium containing 10% fetal bovine serum to each well at 37°C, 5% CO 2 Conditioned for 24h. Discard the medium, add 180 μL of fresh DMEM medium to each well and 20 μL of 20 mM HEPES solution to prepare a series of cationic polymer solutions, so that the final concentration of the polymer is: 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL mL, 100mg / mL, 500mg / mL, 1000mg / mL, 5000mg / mL. 37°C, 5% CO 2 Conditions for another 24 h. Add 20 μL of MTT solution (5 mg / mL) to each well, and continue to incubate at 37° C. for 4 h. Terminate the culture, discard the culture supernatant in the well, add 100 μL dimethyl sulfoxide (DMSO), and shake gently for 10 min. Measure the absorbance value of each well at 570nm, and calculate the cell activity (see Figure 2).

[0027]As shown in the figure, the cytotoxicity of poly...

Embodiment 3

[0028] Example 3 Complex Formation

[0029] The green fluorescent protein particle pEG-N1 was used as the research object to investigate the combination of DNA and cationic polymer. The pEG-N1 with an extraction purity >95% was dissolved in 20mM HEPES solution and configured as a 0.1 μg / μL DNA stock solution; polymers 1, 2, and 3 were diluted to an appropriate concentration gradient with 20 mM HEPES solution. Take 50 μL of the DNA stock solution (i.e., the amount of DNA is 5 μg), add 50 μL of the gradient concentration solution of the complex to it, so that the final volume is 100 μL, and the DNA:polymer (mass ratio) is 8:1, 4:1, 2, respectively. :1, 1:1, 1:2, 1:4, 1:8, 1:16. The complex was vortexed for 30 s and left at room temperature for 20 min.

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Abstract

The invention discloses a polymer prepared by polymerization with a framework of ethylene glycol diglycidyl ether and a diamines orthoester monomer (4'-methyleneoxy-di-(2-aminoethoxy-1,3-dioxolane) according to a molar ratio of 1:1-2. With different ratios, polymers with different branched degrees can be obtained by polymerization. The polymer has a branched structure, and has the characteristics of acid sensitivity, controllable degradation, low cytotoxicity, and high transfection efficiency. As a high-efficiency gene vector, the material has a wide application prospect in gene treatment. Through the comparison on the properties (such as DNA coating capacity and transfection effect) of polymers with different branched degrees, valuable experiences are provided for future gene vector researches.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and polymer medical materials, and specifically relates to a kind of acid with branched structure prepared from ethylene glycol diglycidyl ether and a new orthoester monomer containing amino groups at both ends. Synthesis and application of sensitive cationic polymers. Background technique [0002] Gene therapy is a new mode of disease treatment. It is to introduce normal genes or genes with therapeutic effects into human target cells for proper expression in a certain way, so as to correct or improve the defects caused by disease genes and achieve the purpose of treating diseases. With the deepening of gene therapy research, the lack of a safe and effective gene delivery system is a major bottleneck restricting the implementation of gene therapy. Therefore, the key to the success of gene therapy is to develop a suitable carrier, especially an acid-sensitive gene carrier with intracellular sl...

Claims

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Application Information

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IPC IPC(8): C08G59/50C08G59/22C12N15/87C12N15/85A61K47/34A61K48/00
Inventor 王睿唐汝培巩凯
Owner JIANGNAN UNIV
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