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Polyethylenimine derivative, preparation method thereof and application thereof

A polyethyleneimine and derivative technology, applied in the field of medicine, can solve the problems of reduced affinity, low transfection efficiency, inaccessibility of target genes, etc., and achieve the effects of low cytotoxicity and high transfection efficiency

Inactive Publication Date: 2013-07-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyethylenimine (PEI) is a type of non-viral gene carrier widely used at present, and has high transfection efficiency in vivo and in vitro. The transfection efficiency of PEI is related to its molecular weight, and its molecular weight is greater than 22000. A satisfactory transfection effect is obtained, but it also has high cytotoxicity, while PEI with a small molecular weight (Mw=2000) has little cytotoxicity, but its transfection efficiency is low, and the purpose of treatment cannot be achieved
Moreover, the complex formed by PEI and DNA is very easy to aggregate in vivo (such as being deposited in the pulmonary capillary bed, causing severe pulmonary embolism), and the particle size and dispersion stability of the carrier system are very sensitive to the buffer salt system and serum. Lead to high toxicity when applied in vivo, low transfection efficiency, poor pharmacokinetics and in vivo distribution
In order to overcome its shortcomings, biocompatible hydrophilic polymers such as polyethylene glycol (PEG) are generally used to modify PEI to improve the stability of PEI / DNA complexes and reduce toxicity, but because PEI is polymerized by hydrophilic After modification, the affinity with the cell is reduced, so that the target gene cannot enter the cell, and the transfection efficiency decreases

Method used

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  • Polyethylenimine derivative, preparation method thereof and application thereof
  • Polyethylenimine derivative, preparation method thereof and application thereof
  • Polyethylenimine derivative, preparation method thereof and application thereof

Examples

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preparation example Construction

[0028] The preparation method of polyethyleneimine derivative of the present invention is as follows (see figure 1 ):

[0029] (A) Activation of hydroxyl groups at both ends of poloxamers

[0030] Weigh an appropriate amount of poloxamer after water removal, add twice the molar amount of triphosgene, dissolve in a mixed solution of anhydrous toluene and anhydrous dichloromethane, and react overnight with magnetic stirring at room temperature. Remove the solvent by rotary evaporation in vacuo, then dissolve with an appropriate amount of anhydrous toluene and anhydrous methylene chloride, then add an appropriate amount of N-hydroxysuccinimide, and under magnetic stirring, anhydrous triethylamine (dissolved in 4ml anhydrous methylene chloride ) was added dropwise to the reaction solution, and the stirring reaction was continued for about 4h. After the reaction is complete, filter the reaction solution and remove the solvent by vacuum rotary evaporation again, dissolve the obtai...

Embodiment 1

[0034] Example 1 Preparation of Polyethyleneimine Modified by Poloxamer

[0035] Weigh 1.2mmol of poloxamer (F68=10.31g, P105=7.86g, P123=6.94g, L61=2.43g, BASF, Germany) after water removal, dissolve in anhydrous toluene and anhydrous dichloromethane To the mixed solution, 0.712 g of triphosgene (2.4 mmol) was added, and the reaction was carried out under magnetic stirring overnight at room temperature. The solvent was removed by rotary evaporation in vacuo, then dissolved with an appropriate amount of anhydrous toluene and anhydrous dichloromethane, then 0.274 g (2.4 mmol) of N-hydroxysuccinimide was added, and anhydrous triethylamine (0.4 ml dissolved in 4ml of anhydrous dichloromethane) was added dropwise to the reaction solution, and the stirring reaction was continued for about 4h. After the reaction is complete, the reaction solution is filtered and the solvent is removed by vacuum rotary evaporation again, the obtained residue is dissolved in 50ml of ethyl acetate, th...

Embodiment 2

[0037] Example 2 Cytotoxicity Evaluation Experiment of Polyethyleneimine Modified by Poloxamer

[0038] Hela cells were seeded on a 96-well plate, about 10,000 cells per well, and after 24 hours of culture, a series of concentrations (0, 4, 6, 8, 16, 24, 32 μg / ml) of PEI 25kd and modified PEI 2000, continue to culture for 24h, MTT assay cytotoxicity. from image 3 It can be seen that compared with PEI 25kd, the modified PEI 2000 showed stronger cell activity at each concentration, especially at high concentrations, the Hela cells treated with the modified PEI 2000 still showed very strong activity. The high biological activity provides an effective way to solve the problem of excessive cytotoxicity of PEI as a gene carrier. The main reason is that the polymerization product of low molecular weight PEI and poloxamer can be degraded into small molecules in vivo Fragments, which can be metabolized and excreted by somatic cells, reduce aggregation.

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Abstract

The invention discloses a polyethylenimine derivative. The polyethylenimine derivative is ploxamer modified polyethyleneimine. The invention also discloses a preparation method and an application of the polyethyleneimine derivative. The polyethyleneimine derivative of the invention, which has the advantages of small cytotoxicity and high transfection efficiency, can be used as a genetic vector.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a polyethylenimine derivative modified by Ploxamer (trade name Pluronic) and its preparation method and application. Background technique [0002] Gene therapy refers to the introduction of human normal genes or therapeutic genes into human target cells in a certain way to correct gene defects or exert therapeutic effects. Because there are many enzymes that can degrade DNA or RNA and different types of biological barriers in the body, in order to effectively reach the target site, the therapeutic gene must be transported by a certain carrier. Generally, plasmid DNA can be effectively expressed after entering the nucleus, while RNA (siRNA or miRNA) exerts its regulatory function in the cytoplasm. Therefore, how to construct different types of vectors so that they can either accumulate in the cytoplasm or penetrate the nucleus Membrane enrichment in the nucleus is the focus and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G73/04C08G81/00C12N15/87
Inventor 高申范伟尹东锋丁雪鹰武鑫高志祥高静刘继勇朱全刚
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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