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Gene vector system, its preparation and application

A gene carrier and system technology, applied in the field of biomedical materials, can solve the problems of reducing gene transfection efficiency and being difficult to play a role, and achieve the effects of improving transfection efficiency, improving specific recognition and transmission, and increasing serum stability.

Active Publication Date: 2014-05-07
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because these masking materials are firmly combined with the gene carrier, they are not easy to fall off after entering the cell, and still tightly wrap the carried gene, making it difficult to function, thus greatly reducing the efficiency of gene transfection

Method used

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  • Gene vector system, its preparation and application
  • Gene vector system, its preparation and application
  • Gene vector system, its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of reduction sensitive masking system with targeting function

[0031] According to the feeding ratio described in Table 1, dissolve hyaluronic acid in phosphate buffer saline (PBS) with pH 6.8, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 1-hydroxybenzotriazole (HOBT), stirred, and reacted at room temperature for 2 hours to activate the carboxyl group. Add cystamine dihydrochloride (Cys), stir, and react the above reaction solution overnight at room temperature. After the reaction, the reaction product is dialyzed for 48 hours with a dialysis bag with a cut-off of 3500, and then freeze-dried to obtain cystamine-grafted transparent Hyaluronic acid (HA-Cys). The grafting ratio of cystamine in the product (HA-Cys) was calculated by nuclear magnetic spectrum, and the results are shown in Table 1.

[0032] Hyaluronic acid (HA-Cys) grafted with cystamine in different proportions was dissolved in phosphate buffere...

Embodiment 2

[0036] Example 2: Preparation of Gene Carrier System with Reduction Sensitive Masking System with Targeting Function

[0037] Dissolve plasmid DNA in sterilized water to prepare DNA solution A with a concentration of 0.1 mg / mL; dissolve cationic polymer gene carrier polyethyleneimine (PEI) in sterile HBG buffer (4-hydroxyethylpiperazine ethanesulfonic acid 20 mmol, pH 7.4, 5% glucose), to prepare PEI solution B with a concentration of 0.01-1 mg / mL; dissolve the reduction-sensitive masking system (HA-SS-COOH) with targeting function In sterile HBG buffer, prepare HA-SS-COOH solution C with a concentration of 0.01-1 mg / mL.

[0038] The cationic polymer PEI solution of different concentrations and the plasmid DNA aqueous solution were mixed at a mass ratio of 1.2:1, and the mixed aqueous solution was incubated at room temperature for 20 minutes to obtain a PEI / DNA complex. Add different concentrations of HA-SS-COOH solutions, and incubate the aqueous solution at room temperature...

Embodiment 3

[0041] Example 3: Using gel electrophoresis to identify the stability of complex particles

[0042] Mix 5 μL of 0.1 mg / mL DNA solution with 3 μL of 0.2 mg / mL PEI solution, incubate at room temperature for 20 minutes, then add 5 μL of HA-SS-COOH solution of different concentrations to make HA-SS-COOH / DNA The mass ratios were 10, 6, 3, 2, 1 and 0.5, and after incubation at room temperature for 10 minutes, the gel electrophoresis retardation experiment was used to detect the complex particles after adding different amounts of HA-SS-COOH shielding system. stability.

[0043] figure 2 The results of electrophoresis show that the complex formed between the cationic polymer and DNA will not be destroyed when the amount of the HA-SS-COOH hyaluronic acid masking system added reaches 10 times the amount of DNA.

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Abstract

The invention discloses a gene vector system of a redox sensitive shielding system having targeting function, its preparation and application in the field of gene therapy. The gene vector system disclosed herein comprises a redox sensitive shielding system having targeting function, a cationic high-molecular material and plasmid DNA; wherein the cationic high-molecular material and plasmid DNA form compound particles, the redox sensitive shielding system having targeting function shields the compound surface by electrostatic action, thus the toxicity of the vector can be reduced, the loaded genetic material is transferred into the cell successfully, the expression of the genetic material is realized, the transfection process is completed, the targeting of gene transfection can be raised, and simultaneously the gene transfection efficiency is raised.

Description

technical field [0001] The invention belongs to the field of biomedical materials, and relates to a gene carrier with targeting function and reduction sensitive response characteristic. Background technique [0002] Gene therapy is a promising therapeutic approach in the treatment of genetic diseases as well as tumors, which has been greatly advanced in the past decade. However, one of the bottlenecks of gene therapy is gene delivery. Nucleic acids are negatively charged biological macromolecules, so stable and efficient synthetic vectors are usually positively charged. A number of cationic lipid and polymer carrier systems have been developed to electrostatically bind DNA and be used to facilitate gene transfection in vitro. [0003] Polyethyleneimine (PEI) is a cationic polymer that has received the most attention among non-viral vectors. It has been widely used as a gold benchmark polymer carrier, but the inherent cytotoxicity and high surface positive charge of PEI lim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/79C12N15/85A61K48/00A61P35/00A61P11/06A61P9/00
Inventor 顾忠伟聂宇何一燕程刚谢丽
Owner SICHUAN UNIV
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