Application of hydroxyl-containing crosslinked polymer guanidinated product in gene transfer

A cross-linked polymer, guanidine-based technology, used in gene therapy, medical preparations with non-active ingredients, genetic material components, etc., to achieve the effects of flexible spatial structure, high reactivity, and broad application prospects

Inactive Publication Date: 2013-07-10
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have directly converted amine groups into guanidinium groups on PEI or dendrimers with larger molecular weight (above 20kDa). Although this can improve transfection efficiency, it still shows obvious cytotoxicity.

Method used

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  • Application of hydroxyl-containing crosslinked polymer guanidinated product in gene transfer
  • Application of hydroxyl-containing crosslinked polymer guanidinated product in gene transfer
  • Application of hydroxyl-containing crosslinked polymer guanidinated product in gene transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the synthesis of the PEI derivative containing hydroxyl

[0050] 0.5 grams of diethylenetriamine (which can be regarded as PEI with a molecular weight of 103) is dissolved in 5ml of dichloromethane and placed in a 50ml reaction bottle. A certain amount of crosslinking agent L1, namely N',N'-tetracyclic Oxypropyl-4,4'-diaminodiphenylmethane (can also be one or two of L2-L5, quantified according to the molar ratio of the reaction) dissolved in 5ml of dichloromethane at 30-35 ° C, Add it dropwise into the above reaction bottle under magnetic stirring, and then continue to react for 22 hours. The volume of the reaction solution is reduced by more than half by rotary evaporation, and then dialyzed for more than 72 hours, and the hydroxyl-containing PEI derivative product is obtained after freeze-drying.

Embodiment 2

[0051] Embodiment 2: the synthesis of the PEI derivative containing hydroxyl

[0052] 0.5 grams of PEI (can be linear or branched, molecular weight 600) dissolved in 5ml of dichloromethane and placed in a 50ml reaction bottle, a certain amount of cross-linking agent L5 (can also be one of L1-L4 or Two kinds, quantified according to the molar ratio of the reaction) dissolved in 5ml of dichloromethane at 20-25°C, added dropwise to the above reaction flask under magnetic stirring, and then continued to react for 18 hours, and the volume of the reaction solution was reduced by rotary evaporation More than half of it was then dialyzed for more than 72 hours, and the hydroxyl-containing PEI derivative product was obtained after freeze-drying. Various hydroxyl-containing PEI derivatives (raw materials are shown in Table 1; Table 2), various reaction combinations and the resulting The polymer structure is schematically shown in Figure 1.

Embodiment 3

[0053] Embodiment 3: the synthesis of the PEI derivative containing hydroxyl

[0054] 0.5 g of PEI (can be linear or branched, molecular weight 2000) is dissolved in 5 ml of methylene chloride and placed in a 50 ml reaction bottle, a certain amount of cross-linking agents L2 and L5 (or one of L1-L4) One or two kinds, quantified according to the molar ratio of the reaction) dissolved in 5ml of dichloromethane at 20-25°C, added dropwise to the above-mentioned reaction flask under magnetic stirring, and then continued to react for 6 hours, and the reaction solution was evaporated by rotary evaporation to make it The volume is reduced by more than half, and then dialyzed for more than 72 hours, and the hydroxyl-containing PEI derivative product is obtained after freeze-drying. Various hydroxyl-containing PEI derivatives (raw materials are shown in Table 1; Table 2), various reaction combinations and the resulting The polymer structure is schematically shown in Figure 1.

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Abstract

The invention discloses a preparation method and an application of a hydroxyl-containing crosslinked polymer and guanidinated product thereof used for delivering nucleic acid and other bioactive agents. The method comprises the specific steps that: linear or branched polyethyleneimine with a molecular weight of 100-30000Da or various multi-amine compounds are subjected to a compounding reaction with one or more crosslinking agents with a molecular weight of 100-5000Da and containing two or more epoxy groups, such that the hydroxyl-containing crosslinked polymer is prepared. The invention also provides a synthesizing method of a novel guanidination reagent 3-guanidino propanol acrylate or 5-guanidino amyl acrylate. With the guanidination reagent, the hydroxyl-containing crosslinked polymer or other polymers can be guanidinated, such that gene transfection efficiency thereof can be substantially improved, and cytotoxicity can be reduced. The excellent-performance polymers screened by the invention have gene transfection efficiencies in various cell lines such as A549, B16F10, 3T3, and U87 substantially higher than conventional commercialized gene transfection reagents. The polymers are low-toxic high-efficiency non-virus gene transfection vectors. With the vectors provided by the invention, in-vitro high-efficiency and low-toxicity transfection of various cells can be realized, and in-vivo high-efficiency and low-toxicity local administration can be realized. The application has wide application prospect.

Description

technical field [0001] The invention relates to a method for crosslinking polyethyleneimine and its analogues with two or more epoxy group crosslinking agents to form hydroxyl-containing crosslinked polymers. Simultaneously also relate to the guanidinating reagent with certain chain length, i.e. 3-guanidinopropanol acrylate, the synthetic method of 5-guanidinopentyl alcohol acrylate etc. A method for the preparation of guanidinated polymers by chemical reaction. technical background [0002] It was discovered earlier that the human immunodeficiency virus (HIV) type 1 transactivator (TAT) has the ability to penetrate various types of cell membranes, which can greatly increase the transcription and replication levels of the HIV genome. According to the function of TAT, a class of small molecular polypeptides with the ability to penetrate cell membranes has been discovered, called cell penetrating peptides (Cell Penetrating Peptides, CPPs), whose length generally does not exce...

Claims

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Application Information

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IPC IPC(8): C08G73/04C08J3/24C08G59/50C08G59/32C08G59/24C08G59/30C08G59/16C12N15/87C12N15/85A61K47/34A61K48/00
Inventor 董伟王伯良丰连东刘洋洋赵玉瑜谢阿明
Owner NANJING UNIV OF SCI & TECH
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