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Multifunctional cationic polymer gene vector, and preparation method and application thereof

A cationic polymer and multi-functional technology, applied in gene therapy, active ingredients of heterocyclic compounds, drug combination, etc., can solve the problems of poor cell targeting, achieve low cytotoxicity, improve low transfection efficiency, and strong cell adhesion Adjunctive effect

Inactive Publication Date: 2013-11-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some researchers have linked chitosan with low molecular weight polyethyleneimine to form a graft copolymer. Although the carrier has low cytotoxicity and high gene transfection efficiency, the cell targeting of the carrier is poor. Therefore, it should be modified to improve its specificity and transfection efficiency.

Method used

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  • Multifunctional cationic polymer gene vector, and preparation method and application thereof
  • Multifunctional cationic polymer gene vector, and preparation method and application thereof
  • Multifunctional cationic polymer gene vector, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment one: the synthesis of cationic polymer chitosan-g-polyethyleneimine-candesartan (CS-g-PEI-CD, CPC)

[0046] 1) Activation of Candesartan (CD)

[0047] Weigh 0.0264g candesartan and dissolve it in 2ml N,N-dimethylformamide (DMF), and weigh 2 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiethylene Dissolve amine hydrochloride (EDCI) and 3 times the molar amount of N-hydroxysuccinimide (NHS) in 4ml of DMF, add it dropwise to the candesartan solution, and react with magnetic stirring for 1 hour at room temperature to obtain activated of candesartan.

[0048] 2) Synthesis of candesartan-polyethyleneimine conjugate (CD-PEI)

[0049] Weigh 0.0568g of polyethyleneimine and dissolve it in 6ml of water-N,N-dimethylformamide (1:2) mixed solvent, add the activation solution of above-mentioned candesartan to the polyethyleneimine solution dropwise, at room temperature The reaction was carried out under magnetic stirring for 24 hours, acetone was adde...

Embodiment 2

[0057] Embodiment two: the mensuration of cationic polymer buffer capacity

[0058] 6mg of the cationic polymer CPC prepared in Example 1 was dissolved in 30ml of 150mM NaCl solution, then the pH of the solution was adjusted to 10.0 with 0.1M NaOH, and then titrated with 0.1M HCl until the pH dropped to 3.0. Record the total volume of hydrochloric acid added and the corresponding pH of the solution at this time, and draw the hydrochloric acid volume-pH curve. 150mM NaCl solution, CS-PEI (see comparative example) and PEI25k were used as blank control, negative control and positive control respectively, the results are shown in the attached image 3 . The buffering capacity of the polymer is calculated according to the following formula:

[0059] (ΔH 上 样品 -ΔH 上 NaCl ) / N mol ×100%

[0060] In the formula, ΔH 上 It is the number of moles of hydrochloric acid consumed by the sample solution from pH 7.4 to 5.0 during titration. N mol is the number of moles of all protonata...

Embodiment 3

[0062] Example 3: Preparation of CPC / DNA complex and gel retardation experiment

[0063] The polymer CPC prepared in Example 1 was dissolved in PBS buffer (pH 7.2-7.4), and the plasmid DNA was dissolved in deionized water. The mass of fixed plasmid DNA (2 μg DNA) was 0, 0.25, 0.5, 0.75, 1, 1.5, and 2 according to the mass ratio of carrier and plasmid DNA. After mixing the polymer solution and plasmid DNA solution, vortex for 15 seconds, and let it stand at room temperature. Set it aside for 30 minutes to obtain the CPC / DNA complex solution. Take 20 μl of the complex solution for agarose gel electrophoresis to observe the DNA retardation. Electrophoresis conditions: 0.8% agarose (with GoldViewer TM ), 0.5×TBE buffer, voltage 90V, electrophoresis time 60min, the results are shown in the attached Figure 4 .

[0064] attached Figure 4 Middle lane 1 is the plasmid DNA control, and lanes 2 to 7 correspond to the mass ratios of vector and plasmid DNA being 0.25, 0.5, 0.75, 1, ...

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Abstract

The invention relates to a multifunctional cationic polymer gene vector and a preparation method and application thereof. A multifunctional cationic polymer is synthesized by oxidizing low molecular weight chitosan used as a framework material with potassium periodate and subjecting the oxidized low molecular weight chitosan and polyethyleneimine coupled with a sartan-class drug to reduction and ammonification. To overcome technical problems, the invention provides the novel cationic polymer with targeting performance of the acceptor of the tumor cell AT1, multiple proton buffering capability and adjuvant therapy effects on tumors and provides a preparation method with the advantages of mild conditions, low cost, applicability and reliability for the cationic polymer. A compound formed by the cationic polymer provided by the invention and plasmid DNA has the advantages of low cytotoxicity, good targeting performance, high transfection efficiency, substantial in-vitro antineoplastic effects, etc.; the polymer can be used for preparation of a non-virus gene transfer system capable of simultaneously transferring a gene drug and a chemical drug for cooperated treatment of a tumor.

Description

technical field [0001] The invention relates to the field of cationic polymer gene carrier and gene therapy, in particular to a multifunctional cationic polymer gene carrier and its preparation method and application. Background technique [0002] Gene therapy refers to the introduction of human normal genes or genes with therapeutic effects into target cells in a certain way to correct gene defects or play a therapeutic role to kill diseased cells or enhance the body's ability to eliminate diseased cells, so as to achieve the purpose of curing diseases . [0003] There are two types of gene vectors commonly used at present, including viral vectors and non-viral gene vectors. Viral vectors often have high transfection efficiency, but because of their high immunogenicity, poor targeting, limited gene loading capacity, inability to be reused in vivo, complex preparation, potential danger of causing malignant transformation of cells and infection, clinical application are gre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G81/00C08B37/08C08G73/04A61K48/00A61K47/48A61K31/41A61K31/4184A61P35/00
Inventor 周建平王伟鲍秀丽王玉丁学芳丁杨
Owner CHINA PHARM UNIV
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