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Preparation process of ammonia-ester-bond cross-linked poly(ethylene imine) polycation carrier

A technology of polyethyleneimine and urethane bonds, applied in the preparation and purification of small molecular weight PEI cross-linked derivatives, can solve the problems of high transfection activity, high toxicity, toxicity, etc., and achieve significant cell survival rate and low toxicity Effect

Inactive Publication Date: 2012-05-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Xu Songlin of our research group published the article "Novel poly(ethylene imine) biscarbamate conjugate as an efficient and nontoxic gene delivery system" in Journal of controlled release (Journal of controlled release, 2008, 130, 64-68). The synthetic urethane derivative PEIC achieves the maximum transfection activity when the mass ratio of the reporter gene complex is 40 / 1 (compared to commercial polyethyleneimine 25kDa), and the mass ratio of 40 / 1 will increase the load capacity of the carrier resulting in greater toxicity
At the same time, in the synthetic operation of PEIC, the purification is not considered, which will also bring greater toxicity

Method used

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  • Preparation process of ammonia-ester-bond cross-linked poly(ethylene imine) polycation carrier
  • Preparation process of ammonia-ester-bond cross-linked poly(ethylene imine) polycation carrier
  • Preparation process of ammonia-ester-bond cross-linked poly(ethylene imine) polycation carrier

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] PEI with a molecular weight of 800Da reacted with 1,4-butanediol dichloroformate at a molar ratio of 3:2. Calcium hydride was added to chloroform and triethylamine respectively, and then heated to reflux for 2 hours under the protection of high-purity nitrogen, and fresh fractions were collected for later use. Dissolve PEI800 and 1,4-butanediol dichloroformate in chloroform, respectively, and make the two into 3mL and 20mL solutions under anhydrous and oxygen-free conditions, and the reaction is carried out under ice bath conditions. The chloroform solution of PEI800 was added, and then 2.5 times excess triethylamine was added, and the chloroform solution of 1,4-butanediol dichloroformate was added dropwise to the reaction system under anhydrous and oxygen-free conditions to react for 24 hours. After the reaction was stopped, most of the organic solvent was removed with a rotary evaporator under reduced pressure and temperature, and then the obtained crude product was d...

Embodiment 2

[0036] Determination of Polymer Molecular Weight by Gel Permeation Chromatography (GPC)

[0037] Polyethylene glycol (PEG) standard and PEI-Bu as samples were dissolved in pure water to obtain a 10 mg / ml solution, shake well and stand still, then filter with a 0.45 μm microporous membrane, take the filtrate, and inject 20 μl , record the chromatogram. The logarithm value lgMw of the weight-average molecular weight of PEI standard substance and corresponding retention time (tR) are carried out linear regression, obtain regression equation. The molecular weight and distribution of PEI-Sc samples are calculated by the following formula:

[0038] Mn=∑RIi / ∑(RIi / Mi);

[0039] Mw=∑(RIi Mi) / ∑RIi;

[0040] D=Mw / Mn;

[0041] In the above formula, Mn and Mw are respectively the number average molecular weight and the weight average molecular weight; D refers to the distribution coefficient; RIi is the peak height of the test sample at the retention time i; Mi is the molecular weight ...

Embodiment 3

[0043] Preparation of Polymer (PEI-Bu) and Plasmid Complex (Polyplex)

[0044] Weigh a certain amount of polymer, add ultrapure water to make a 2mg / mL solution, then filter it with a 0.22μm sterile filter head, dilute the plasmid concentration to 1mg / mL, and prepare complex solutions with different mass ratios. The concentration of the plasmid solution remains the same, then dilute the concentration of the polymer solution according to different mass ratios, pay attention to keep the volumes of the diluted polymer solution and the plasmid solution equal, and finally add the polymer solution to the plasmid solution quickly and mix, at room temperature After incubation for 20 min, a series of complexes with mass ratios are obtained, which can be used for further determination of physical and chemical properties.

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Abstract

The invention relates to a preparation process of an ammonia-ester-bond cross-linked poly(ethylene imine) polycation carrier. The preparation process comprises the steps of: adding small-molecular-weight poly(ethylene imine) (PEI), 1,4-butanediol bischloroformate and triethylamine to a reaction solvent, stirring, shaking or vibrating the reaction system to carry out condensation reaction, then dialyzing with a 3500Da reactivated dialysis bag for 48 hours, finally filtering with a 0.22 mu m microporous filtering membrane, and freeze-drying to obtain the product. As compared with the prior art, the preparation process provided by the invention has the advantages that: a purified ammonia-ester poly(ethylene imine) derivative PEI-Bu is prepared; and as compared with poly(ethylene imine) biscarbamate conjugate (PEIC), PEI-Bu displays the highest transfection activity and lower cytotoxicity at a lower mass ratio of PEI-Bu to DNA (deoxyribonucleic acid), is an efficient, low-toxicity gene substance carrier, and can be used for conveying gene substances.

Description

technical field [0001] The invention relates to a high-efficiency and low-toxic polycation carrier, in particular to a preparation and purification method of a small molecular weight PEI cross-linked derivative that has been proved to have gene delivery function in cells in vitro tests. Background technique [0002] Gene therapy is a powerful tool in the treatment of many diseases, both congenital and acquired, because it can prevent, treat, and even cure diseases by regulating the expression of biologically active proteins in cells. Gene therapy has encountered a series of technical bottlenecks during its development, one of the most important bottlenecks is the safe and effective delivery of genetic material in vivo. [0003] Currently commonly used gene delivery vectors can be divided into recombinant virus vectors and artificially synthesized vectors (ie, non-viral vectors). Although viral vectors show high transfection efficiency, the mutation of the virus will cause p...

Claims

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Application Information

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IPC IPC(8): C08G73/04C12N15/87C12N15/85
Inventor 金拓苏靖徐松琳吴飞袁伟恩何沐
Owner SHANGHAI JIAO TONG UNIV
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