Method for preparing non-virus cation type gene vector

A cationic, gene carrier technology, which is applied in the field of preparation of new non-viral cationic gene carriers, can solve the problems of low transfection efficiency of naked DNA injection, lack of targeting mechanism of viral vectors, and reduced transfection efficiency, etc. control, narrow molecular weight distribution, and simple synthesis steps

Inactive Publication Date: 2008-08-06
SUZHOU UNIV
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Problems solved by technology

[0003] At present, there are mainly three different gene delivery systems in the field of gene therapy: (1) physical transfection technology systems, such as electroporation and particle bombardment technology, etc. relatively low transfection efficiency
(2) Viral vectors, including retroviruses, adenoviruses, and adeno-associated viruses, have shown high transduction efficiency in vitro; however, most viral vectors need to retain the outer shell and infection mechanism of wild viruses , there are some hidden dangers in terms of safety, such as the immune response to the carrier shell, the carcinogenicity of random gene integration, and potential endogenous virus recombination; at the same time, the viral vector lacks a targeting mechanism in the body, and its controllability is relatively poor. For example, in tumor gene therapy, the viral vector tumor gene therapy program is mainly aimed at some superficial and accessible tumors, such as skin cancer, head and neck tumors, etc., by intratumoral or peritumoral injection
This embedding method is simple and solves the compatibility problem of organisms, but at the same time, the in vitro cell transfection shows that its transfection efficiency is reduced

Method used

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  • Method for preparing non-virus cation type gene vector
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  • Method for preparing non-virus cation type gene vector

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Embodiment 1 4

[0043] In conjunction with embodiments one to four, describe the method for synthesizing polycationic electrolyte

Embodiment 1

[0044] Embodiment one: Chol (cholesterol)-PDMAEMA 30 preparation method

[0045] (1) Preparation of KH: Put the stirring rotor into a dry reaction bottle in advance, and plug it tightly with a reversible rubber stopper. Then use a needle and a latex tube to connect with a vacuum pump, and fill it with high-purity argon gas while vacuuming, and repeat the operation three times. After moving a certain amount of KH into the reaction bottle, inject 5ml of dry tetrahydrofuran (THF) with a dry syringe, stir and wash, and suck out the THF containing mineral oil with a syringe after standing still, repeat this three times, and finally dry the residual with high-purity argon THF solution. Accurately weigh the amount of KH in the reaction bottle (0.2-0.3 g, about 5.0-7.5 mmol) by the subtraction method.

[0046] (2) Preparation of initiator: inject a certain amount of THF into a polymerization bottle filled with KH. Put the reaction bottle in an oil bath (about 45°C), stir it magnet...

Embodiment 2

[0048] Embodiment two: Vitamin A (vitamin A 1 )-PDMAEMA 60 preparation method

[0049] (1) Preparation of KH: same as Example 1.

[0050] (2) Preparation of initiator: inject a certain amount of THF into a polymerization bottle filled with KH. Place the reaction bottle in an ice-water bath, stir it magnetically, and inject vitamin A (dissolved in anhydrous THF in advance, protected by argon) with KH and other substances at the same time, and react for 2 to 4 hours to fully react vitamin A and KH to form Oxygen anion.

[0051] (3) Polymerization reaction: then move the reaction bottle into a constant temperature oil bath at 25°C, add monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) with a dry syringe, and the molar ratio to the initiator is 60:1, react for 1~1.5h, and finally terminate the reaction with dry methanol. The reacted polymer is subjected to rotary evaporation at 30-40°C to remove the solvent, and the precipitation purification is continued with cold n-hexan...

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Abstract

The invention relates to a novel non-virus cationic gene vector and the process for preparation. The process for preparation comprises the following steps: firstly, utilizing an oxygen anion polymerization method to synthesize polycation type electrolyte and segmented copolymer which contains polycation type electrolyte, and then leading polycation type electrolyte and DNA to mutually compound in salting liquid whose PH is 7.4 and buffer solution, achieving the packing of polycation type electrolyte to DNA, and the surface of DNA / polycation compound which is formed is with positive charge, adding segmented copolymer MePEO-b-PMAA which contains polycation type electrolyte in a system, leading PMAA segment with negative charge to be absorbed on the surface of the DNA / polycation compound through electrostatic force action and hydrogen bond effect, achieving the packing to compounds. The novel non-virus cationic gene vector has the advantages of mild polymerization reaction condition, controllable molecular weight, narrow molecular weight distribution, simple process for preparing gene vector, high stability and bioavailability, low toxicity and the like.

Description

technical field [0001] The invention relates to the technical field of biomedical polymer materials, in particular to a preparation method of a novel non-viral cationic gene carrier. Background technique [0002] Gene therapy is a new treatment method that has important potential therapeutic effects on various gene-related diseases that threaten human life. Gene therapy has made many amazing achievements and encountered many difficulties in the development path of more than 40 years. In order for gene therapy to truly enter clinical application, there are still some fundamental problems that have not been resolved, the most important of which is the construction of a safe, efficient and controllable gene carrier. [0003] At present, there are mainly three different gene delivery systems in the field of gene therapy: (1) physical transfection technology systems, such as electroporation and particle bombardment technology, etc. relatively low trans...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/64C08F120/34C08F220/18
Inventor 顾子旭倪沛红袁媛何金林
Owner SUZHOU UNIV
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