Modified polyethyleneimine derivative as well as synthesis method and application thereof

A technology of polyethyleneimine and derivatives, which is applied in the field of polyethyleneimine derivatives and its application, and can solve the problems of cumbersome operation process and other problems

Inactive Publication Date: 2020-12-29
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some cells with thicker cell membranes, difficult to digest, and difficult to transfect, when the efficiency of electroporation or virus method is not high and the operation process is cumbersome, it is necessary to modify the PEI vector to enhance the resistance of difficult-to-transfect cells to nucleic acid drugs. uptake efficiency

Method used

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  • Modified polyethyleneimine derivative as well as synthesis method and application thereof
  • Modified polyethyleneimine derivative as well as synthesis method and application thereof
  • Modified polyethyleneimine derivative as well as synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Synthesis and characterization of PEI derivative PEI-Triton-N

[0040] Since the hydroxyl groups on Triton X-100 cannot undergo conjugation reactions with PEI, in order to realize the Triton X-100 modification of PEI, an active ester group is first introduced into the structure of Triton X-100, and the activated ester can be further reacted with the amino group of PEI to obtain Triton X-100 modified PEI derivatives, different numbers of Triton X-100 modified PEI derivatives are obtained by adjusting the ratio of raw materials in the synthesis process, the specific synthetic route is as follows figure 1 As shown in A.

[0041] 1. Synthesis and characterization of PEI derivative PEI1.8-Triton-N

[0042] Triton X-100 (6.56g, 1.0eq), N,N'-disuccinimidyl carbonate (3.84g, 1.5eq) and triethylamine (4.16mL, 3.0eq), the reaction was stirred overnight at room temperature. The next day, the mixture was amplified with dichloromethane, extracted three times with 100 m...

Embodiment 2

[0056] Example 2 Preparation, characterization and performance determination of PEI-Triton-N / pDNA complex

[0057] 1. Experimental method

[0058] 1.1 Preparation of PEI-Triton-N / pDNA complex

[0059]The ratio of PEI-Triton-N / pDNA complex is fed according to the mass ratio (w / wratio) of PEI-Triton-N:pDNA, and finally converted to nitrogen / phosphorus ratio (N / P ratio), and an appropriate amount of PEI-Triton -N was added dropwise to a certain amount of pDNA solution, vortexed for 10 seconds to mix, and incubated at room temperature for 15 minutes to obtain the mixture solution. Similarly, 1.8 kDa and 25 kDa PEI / pDNA complexes were prepared as controls.

[0060] 1.2 PEI-Triton-N / pDNA complex entrapment, release ability and stability evaluation

[0061] In order to study the binding ability of PEI-Triton-N to DNA, PEI-Triton-N / pDNA complexes with different N / P ratios (containing 200ng pDNA) were prepared in this experiment, with a final volume of 10 μL. Configure 2% agarose g...

Embodiment 3

[0076] Example 3 PEI-Triton-N carrier to cell activity, cell uptake experiment

[0077] 1. Experimental method

[0078] 1.1 Cell culture

[0079] Human normal skin keratinocytes HaCaT cells were donated by Lin Zhimiao's research group at Peking University First Hospital, HEK-293T, Hela, and MCF-7 cells were from the inventor's laboratory, and the above cells were all prepared using 10% fetal bovine serum, 100IU penicillin and DMEM cell culture medium with 100 μg / mL streptomycin was cultured in a constant 5% CO 2 , cultured in a carbon dioxide incubator at 37°C. When the cells grow to about 80%-90%, aspirate the medium, wash with PBS 2-3 times, add an appropriate amount of 0.25% trypsin to digest at 37°C for an appropriate time, add serum-containing medium to stop the digestion, carefully pipette and mix, transfer to 1.5 Centrifuge the mLEP tube at 1000rpm for 5min, discard the supernatant, and resuspend to make a single cell suspension for subculture.

[0080] 1.2 MTT meth...

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Abstract

The invention discloses a modified polyethyleneimine derivative as well as a synthesis method and application thereof. According to the invention, Triton X-100 is conjugated to polyethyleneimine, suchthat a PEI-Triton-N carrier is obtained. In-vitro cell evaluation shows that the carrier shows good safety and stability; cell transfection effect experiments show that the carrier shows efficient and low-toxicity gene delivery capacity, can significantly improve the gene transfection efficiency of various cells including skin immortalized cells HaCaT difficult to transfect, and is an excellent in-vitro transfection reagent; an in-vivo toxicity experiment shows that the carrier has no obvious systemic toxicity; and a gene delivery experiment result of mouse skin local administration shows that the carrier has good local administration gene delivery capability and is a good local administration gene delivery vector.

Description

technical field [0001] The present invention relates to polyethyleneimine derivatives, in particular to Triton X-100 modified polyethyleneimine derivatives and a synthesis method thereof, and the present invention also relates to the modified polyethyleneimine derivatives as transfection reagents in vitro or nucleic acids in vivo The application of the delivery carrier belongs to the field of polyethyleneimine derivatives and their applications. Background technique [0002] Nucleic acid therapy is a method of delivering a gene of interest to target cells through a carrier for the treatment of various diseases such as cancer, AIDS, cardiovascular disease, infectious diseases, and rare diseases with inherited gene mutations. Nucleic acid therapy has several advantages over other therapeutic methods, but in the actual process there are also many problems to be solved: (1) efficient and safe delivery system; (2) controllability of exogenous gene expression; (3) effective thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G73/02A61K48/00A61K47/59
CPCA61K48/005A61K47/59C08G73/0206C08G73/0213
Inventor 汤新景范馨莉
Owner PEKING UNIV
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