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Preparation for transgenic carrier of temperature-sensitive poly isopropyl acrylamide and poly arginine conjugate

A technology of propylacrylamide and polyarginine, which is applied in gene therapy, medical preparations with inactive ingredients, and introduction of foreign genetic material using vectors, can solve the problems of low protein expression level and the like, and achieve a simple preparation method, Improved transfection efficiency and mild synthesis conditions

Inactive Publication Date: 2008-01-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Twaites et al. synthesized PEI-NIPAAm and NIPAAm-co-DMAEMA-co-HA (hexyl acrylic acid) polymers. The results of preliminary transfection experiments showed that the vectors in the experiments all had the ability to deliver plasmid DNA to the nucleus, but Low protein expression in mouse myocytes (C2C12)

Method used

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  • Preparation for transgenic carrier of temperature-sensitive poly isopropyl acrylamide and poly arginine conjugate
  • Preparation for transgenic carrier of temperature-sensitive poly isopropyl acrylamide and poly arginine conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Dissolve 2 grams of isopropylacrylamide in 25 milliliters of tetrahydrofuran, stir it magnetically for 20 minutes to dissolve it completely, add it to a 100-ml three-necked flask, add the initiator 4,4'-azobis(4-cyano valeric acid) 0.05 g, nitrogen 30min to get rid of the air in the reaction system, heated to 60°C in a water bath, reacted for 7h under nitrogen protection, precipitated and washed the product three times with ether, dissolved the product in distilled water, dialyzed for 5 days (molecular weight cut-off was 3500), and frozen Dry.

[0027] Take 100 mg of the sample obtained in the previous step and dissolve it in hydrochloric acid / N, N, N', N' tetramethylethylenediamine buffer solution (pH=4.5), weigh 10 mg of polyarginine (purchased from SIGMA-ALDRICH, USA CO company, batch number: P-4663) was added to the above solution, and 20 mg of carbodiimide was added, and the reaction was performed under magnetic stirring at 10° C. for 72 hours. The product was cen...

Embodiment 2

[0030] Dissolve 2 grams of isopropylacrylamide in 25 milliliters of tetrahydrofuran, stir it magnetically for 20 minutes to dissolve it completely, add it to a 100-ml three-necked flask, add the initiator 4,4'-azobis(4-cyano pentanoic acid) 0.02 g, 30 min of nitrogen flow to get rid of the air in the reaction system, heated to 50 ° C in a water bath, reacted for 6 h under nitrogen protection, precipitated and washed the product with ether three times, dissolved the product in distilled water, dialyzed for 5 days (molecular weight cut-off was 3500), and frozen Dry.

[0031] Take 100 mg of the sample obtained in the previous step and dissolve it in hydrochloric acid / N, N, N', N' tetramethylethylenediamine buffer solution (pH=5.0), weigh 20 mg of polyarginine and add it to the above solution, and add 10mg of carbodiimide was reacted with magnetic stirring at 15°C for 48 hours. The product was centrifuged at 32°C to remove PNIPAAm, the supernatant was collected, and then centrifu...

Embodiment 3

[0034] Dissolve 2 grams of isopropylacrylamide in 25 milliliters of tetrahydrofuran, stir it magnetically for 20 minutes to dissolve it completely, add it to a 100-ml three-necked flask, add the initiator 4,4'-azobis(4-cyano pentanoic acid) 0.10 g, nitrogen gas 30min to get rid of the air in the reaction system, heated to 45°C in a water bath, reacted for 8h under nitrogen protection, precipitated and washed the product three times with ether, dissolved the product in distilled water, dialyzed for 5 days (molecular weight cut-off was 3500), and frozen Dry.

[0035] Take 100mg of the sample obtained from the previous step and dissolve it in hydrochloric acid / N,N,N',N'tetramethylethylenediamine buffer solution (pH=5.5), weigh 10mg of polyarginine and add it to the above solution, and add 15mg of carbodiimide was reacted with magnetic stirring at 20°C for 60 hours. The product was centrifuged at 32°C to remove PNIPAAm, the supernatant was collected, and then centrifuged at 37°C ...

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Abstract

This invention is the preparation method of temperature-sensitive polyisopropyl acrylamide-polyarginine. It also belongs to temperature sensitive transgenetic vector preparation technology. The procedure is as followings: dissolve polyisopropyl acrylamide in tetrahydrofuran, add the initiating agent 4, 4-tetrahydrofuran (cyano valerianic acid), heat in water bath, wash the product within nitrogen production, dissolve with deionized water, get polyisopropyl acrylamide with N-end terminated by carboxyl after dialysis and freezing drying.then dissolve it in HCl / N,N,N',N'tetramethylethylenediamine buffer. Add polyarginine and carbodiimide, agitate while reacting, centrifuge at different temperatures. Get polyisopropyl acrylamide-polyarginine after freezing-drying. This invention is advantageous in simplified-manipulation and mild reaction temperature. The product could be used as transgenetic vector to regulate the transportation and expression of DNA.

Description

technical field [0001] The invention relates to a preparation method of a temperature-sensitive polyisopropylacrylamide-polyarginine conjugate, which belongs to the preparation technology of a temperature-sensitive transgene carrier. Background technique [0002] Gene therapy is the product of the combination of molecular biology and modern medicine. It is a method of correcting or replacing disease-causing genes by introducing normal or wild type genes using the most advanced life science and clinical medicine technologies. a treatment method. Introducing genes into target cells requires long-term effective vectors. [0003] So far, scientists have developed many transgenic vectors, which can be divided into two categories: viral vectors and non-viral vectors. Viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia virus vectors, and herpes simplex virus vectors; non-viral vectors include naked DNA, liposomes, polymer vectors, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F120/56C08F301/00C12N15/63A61K47/32A61K48/00
Inventor 刘文广程男姚康德梁东春郭刚张镜宇
Owner TIANJIN UNIV
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