Preparation method and application of CBX3-containing L-arginine amination polyamine polymer gene vector

A gene carrier and polymer technology, applied in the fields of new formulations of biological preparations, gene carriers, and new excipients for pharmaceutical preparations, can solve the problems of non-degradability, high toxicity, inhibition, etc., and achieve the effect of good nuclear localization effect.

Active Publication Date: 2021-05-28
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyethylenimine is a gene carrier material with high transfection efficiency and is widely used, but its application in vivo is inhibited due to its non-degradable and high toxicity

Method used

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  • Preparation method and application of CBX3-containing L-arginine amination polyamine polymer gene vector
  • Preparation method and application of CBX3-containing L-arginine amination polyamine polymer gene vector
  • Preparation method and application of CBX3-containing L-arginine amination polyamine polymer gene vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0036] Embodiment 1 The preparation method of ARG-CBA polymer.

[0037] Take 0.11g of L-arginine guanidinium reagent, 0.105g of CBA, put it in a 50ml round bottom flask, add 2.5ml of water and 6ml of DMF as solvent, 45-60℃, avoid light, and react under nitrogen protection and magnetic stirring conditions for 3 1 day; TLC monitors the reaction progress, adds about 10% excess L-arginine guanidinium reagent (0.013g) after 72 hours, continues to react for 24 hours; Stops the reaction, adjusts the pH of the system to be 2-5 with 4mol / L hydrochloric acid , collect the whole reaction system solution, move it into a dialysis bag, dialyze and purify the reaction product, the dialysis medium is distilled water, the molecular weight cut off (MWCO) of the dialysis bag is 1000, dialyze for three days, and change the dialysis medium at least once a day; collect the solution in the dialysis bag, put In a dry petri dish, the polymer product was collected by lyophilization, and the structure w...

Embodiment 2

[0038] Example 2 Preparation of CBX3 / target gene / ARG-CBA gene carrier complex.

[0039] (1) Preparation of CBX3 / target gene complex.

[0040] pcDNA3.1-EGFP-cDNA plasmid, pSliencer TM 4.1 CMV-FANCF-shRNA plasmid, FANCF-shRNA, 328-microRNA were diluted to 0.016mg / ml with 50mM HEPES buffer solution with pH 7.4, and CBX3 powder was prepared with deionized water to a concentration of 5×10 -3 For the stock solution of μg / μl, the concentration will be 5×10 -3 The μg / μl CBX3 stock solution was diluted to different concentrations with HEPES, and then mixed according to the mass ratio of CBX3 and plasmid gene at 1:50-1:10000, and the mixture was vortexed in the EP tube for 5 seconds, and kept at room temperature. Incubate for 10-30min to obtain different ratios of CBX3 / pDNA complexes.

[0041] (2) Prepare CBX3 / target gene / ARG-CBA gene carrier complex.

[0042] The polymer gene carrier as described in Example 1 was dissolved in HEPES buffer at 0.9 mg / ml, and diluted with HEPES buffe...

Embodiment 3

[0046] Example 3 Evaluation of in vitro transfection efficiency of CBX3 / target gene / ARG-CBA gene vector.

[0047] (1) Study on in vitro transfection efficiency of pcDNA3.1-EGFP-cDNA plasmid carrying CBX3.

[0048] After preparing complexes with different N / P values ​​(1 / 12, 1 / 24, 1 / 48) according to the method described in Example 2, the three complexes were added to six wells of normally cultured MCF10A cells at a concentration of 500 μl per well In the plate, the medium contained serum (BSA); 48 hours after transfection, the expression of green fluorescent protein was detected by flow cytometry, and the transfection efficiency was determined by flow cytometry software. Select commercial transfection reagent Lipofectamine2000 and PEI as controls, the results are as follows figure 2 shown.

[0049] (2) Study on in vitro transfection efficiency of pSliencer TM 4.1CMV-FANCF-shRNA plasmid carrying CBX3.

[0050] After preparing complexes with different N / P values ​​(1 / 12, 1 / 2...

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Abstract

The invention belongs to the technical field of new auxiliary materials of pharmaceutical preparations, new dosage forms of biological preparations and gene vectors, and particularly relates to a polyaminoamine polymer compound gene vector containing target head protein CBX3 as well as a preparation method and application of the polyaminoamine polymer compound gene vector. The CBX3 target head protein is introduced into the compound gene vector, so that the compound gene vector has good membrane permeability and intracellular environment reduction sensitivity, and the CBX3 target head protein has a targeting effect of being specifically positioned in common chromatin. A disulfide bond and guanidyl of a main chain of the vector have reduction sensitivity in an intracellular environment and good membrane permeability; meanwhile, the CBX3 target head protein is introduced and can be specifically positioned in an ordinary chromatin region, gene expression is up-regulated, the CBX3 target head protein is combined with a target gene, so the compound vector has good intracellular reduction sensitivity and a targeting effect of exclusively positioning the compound vector to common chromatin, gene expression is promoted, and a new direction is opened up for gene therapy.

Description

technical field [0001] The invention belongs to the technical field of new auxiliary materials for pharmaceutical preparations, new dosage forms of biological preparations and gene carriers, and specifically relates to a polyamine polymer complex gene carrier containing target head protein CBX3 and its preparation method and application. Background technique [0002] As an emerging tumor treatment method, gene therapy has been widely favored by experts in the field of medicine in the past 20 years and has become an important part of the research and development of anti-tumor drugs. Gene therapy technology has entered the growth stage from the introduction stage, and has the following obvious advantages: (1) It can treat the causes of disease occurrence and development, and is expected to achieve a fundamental cure for the disease; (2) Based on the principle of complementary pairing of nucleotides, it has Certain natural targeting; (3) Compared with traditional chemical drugs...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K47/59A61K48/00A61K31/713A61P35/00
CPCA61K47/64A61K47/59A61K48/0041A61K48/0033A61K48/005A61K31/713A61P35/00
Inventor 余涧坤秦孟孟
Owner 中国医科大学
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