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MRNA transfection material, mRNA transfection system and application

A transfection system and reaction technology, applied in the biological field, can solve the problem that the system cannot be too stable

Pending Publication Date: 2022-05-27
深圳近邻生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem existing in the prior art is that in order to ensure the stability of nucleic acid outside the cell, the colloidal stability of the nucleic acid delivery system needs to be very high; however, in order to ensure that the nucleic acid can be released in the cell, the nucleic acid delivery system cannot be too stable, therefore, there is an urgent need to provide A nucleic acid delivery system with strong extracellular stability and intracellular destabilization to improve the transfection efficiency of the contained nucleic acid

Method used

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  • MRNA transfection material, mRNA transfection system and application
  • MRNA transfection material, mRNA transfection system and application
  • MRNA transfection material, mRNA transfection system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Synthesis of mRNA transfection material P-ss-DP of the present invention

[0046] (1) Synthesis of P-COOH: succinylated pullulan (P-COOH) was synthesized by esterification of succinic anhydride with pullulan (P). The specific synthesis steps are:

[0047] Dissolve 1.62 g of pullulan in DMSO, add 0.2 g of DMAP and 0.8 g of succinic anhydride, and continue stirring for 24 h on a magnetic stirrer at 45°C. After the reaction, the reaction solution was added to 500 mL of anhydrous ethanol and vigorously stirred, and a white precipitate appeared and centrifuged to remove the anhydrous ethanol. After collecting the reaction product, it was dispersed and dissolved in 10 mL of deionized water. After 3 days of dialysis in deionized water using a dialysis bag (MWCO, 10 kDa), the dialyzed solution was freeze-dried to obtain a white flocculent solid P-COOH.

[0048] (2) Synthesis of PS: Dissolve 1.42 g of P-COOH in 100 mL of PBS (0.2 M, pH 7.8), add 381.3 mg of NHS and 6...

Embodiment 2

[0051] Example 2 Structural analysis of the mRNA transfection material of the present invention

[0052] The structures of pullulan and its derivatives were analyzed by 1HNMR. like image 3 As shown, relative to the hydrogen spectrum of Pullulan, the carboxylated pullulan P-COOH appears between 2.4-2.7 ppm corresponding to the methylene group (C-CH) on succinic anhydride. 2 -CH 2 -C) of the new proton absorption peak. The degree of substitution of succinic anhydride on the sugar chain of pullulan (average number of substituents per 100 sugar units) was about 29 detected by acid-base titration. Cystamine methylene (NH-CH 2 -CH 2 -S) The chemical shift of the proton peak is between 2.8 and 2.9 ppm. The degree of substitution of cystamine is estimated to be about 18.5 by the integrated area of ​​the proton peak, and the degree of substitution of cystamine is estimated to be about 17.6 by elemental analysis. In the P-ss-D hydrogen spectrum, new proton peaks appear at 0.5-1.4...

Embodiment 3

[0053] Example 3 Preparation of nucleic acid delivery system (P-ss-DP / mRNA nanoparticle complex)

[0054] Weigh an appropriate amount of the P-ss-DP polymer powder of Example 1 and dissolve it in Tris-HCl buffer (10 mM, pH 7.4). Phosphorus ratio, the ratio of the amount of the positive charge provided by the amino group in the carrier molecule to the amount of the negatively charged substance provided by the phosphate group in the nucleic acid molecule) was 1, 5, 10, 20, 30, added to the mRNA solution, and vortexed. Homogenize, and adjust the mRNA concentration in the polymer & mRNA complex formed by the final self-assembly to 33.3 ng / μL. The mixed P-ss-DP / mRNA nanoparticles were incubated overnight at 4°C before use.

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Abstract

The invention relates to the technical field of biological medicine, in particular to an mRNA transfection material, an mRNA transfection system and application. The mRNA transfection material is prepared by grafting a cationic polymer on a pulullan framework, and the pulullan framework comprises pulullan with good hydrophilicity and biocompatibility, hydrophobic choline capable of keeping colloidal stability and disulfide bonds capable of responding to intracellular glucose and responding to intracellular glutathione responsive breakage. The cationic polymer fragment can realize the function of selectively releasing mRNA in cells. Experiments show that the stability of the mRNA transfection material provided by the invention is obviously improved, the mRNA transfection material can resist the competition of polyanions and inhibit the degradation of mRNA outside cells, and the colloid stability is obviously reduced in a reducing environment, so that the mRNA can be quickly released in reducing cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an mRNA transfection material, an mRNA transfection system and applications. Background technique [0002] Transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of exogenous DNA / RNA. The development of DNA transfection technology has had a huge impact on modern molecular biology. Gene transfection technology is not only an important tool for studying transgenes and gene expression, but also a key step in current gene therapy. An ideal gene transfection reagent should have the following characteristics: efficient transfection; safety; low cytotoxicity; simple method; time-saving and economical. [0003] The electric shock method is a short-term temporary perforation on the cell to allow the exogenous plasmid to enter; the calcium phosphate method and the liposome method use different carrier substances to carry the plasmid ...

Claims

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Application Information

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IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 林佳奇陈麒先鞠英辰
Owner 深圳近邻生物科技有限公司
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