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Preparation method of tumor pH response charge reversal loaded miRNA nanocomposite

A nanocomposite and charge reversal technology, applied in the field of polycationic gene carrier materials, can solve the problems of reduced blood circulation time, poor blood stability, poor cell uptake, etc., to enhance targeting and cell uptake, reduce side effects, The effect of the simple preparation process

Active Publication Date: 2022-03-11
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, miRNA therapy alone has the limitations of relatively short half-life, poor cellular uptake, rapid nuclease degradation, and poor blood stability
Under physiological conditions, cationic polymer polyethyleneimine (PEI) can self-assemble with negatively charged RNA through electrostatic interaction into a nanometer-sized positively charged nanocomplex (polyplex), which can effectively cover nucleic acid to avoid premature degradation. The gold standard of nucleic acid-coated nanoparticles, but the high positive charge will be adsorbed by proteins in serum, resulting in reduced blood circulation time and severe cytotoxicity

Method used

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  • Preparation method of tumor pH response charge reversal loaded miRNA nanocomposite
  • Preparation method of tumor pH response charge reversal loaded miRNA nanocomposite
  • Preparation method of tumor pH response charge reversal loaded miRNA nanocomposite

Examples

Experimental program
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Embodiment 1

[0034] A method for preparing a pH-responsive charge-reversal-loaded miRNA nanocomplex is provided, comprising the steps of:

[0035] (1) 250mg polyethyleneimine (PEI, molecular weight 25K) was dissolved in 4mL dimethylformamide (DMF); 514.2mg p-toluenesulfonyl arginine (Boc-Arg(Tos)-OH), 460.1mg 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 324.3 mg 1-hydroxybenzotriazole (HOBT) were dissolved in 8 mL dimethylformamide (DMF) , Stored at room temperature for 1h, a mixed solution was obtained. The DMF solution of polyethyleneimine (PEI) and diisopropylethylamine (DIPEA) (248.14mg, 1.92mmol) were added to the above mixed solution, stored at room temperature for 5 days, and the mixture was placed in deionized water (dialysis bag cut: 3500Da ) under dialysis for 72h, freeze-dried white solid. The obtained solid was added to 4ml of trifluoroacetic acid (TFA) and stirred at room temperature for 4h. Precipitate with anhydrous ether, filter and dry under vacuum, dialyze ...

Embodiment 2

[0041] A method for preparing a pH-responsive charge-reversal-loaded miRNA nanocomplex is provided, comprising the steps of:

[0042] (1) 250mg polyethyleneimine (PEI, molecular weight 25K) was dissolved in 4mL dimethylformamide (DMF); 514.2mg p-toluenesulfonyl arginine (Boc-Arg(Tos)-OH), 460.1mg 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 324.3 mg 1-hydroxybenzotriazole (HOBT) were dissolved in 8 mL dimethylformamide (DMF) , Stored at room temperature for 1h, a mixed solution was obtained. The DMF solution of polyethyleneimine (PEI) and diisopropylethylamine (DIPEA) (248.14mg, 1.92mmol) were added to the above mixed solution, stored at room temperature for 5 days, and the mixture was placed in deionized water (dialysis bag cut: 3500Da ) under dialysis for 72h, freeze-dried white solid. The obtained solid was added to 4ml of trifluoroacetic acid (TFA) and stirred at room temperature for 4h. Precipitate with anhydrous ether, filter and dry under vacuum, dialyze ...

Embodiment 3

[0046] A method for preparing a pH-responsive charge-reversal-loaded miRNA nanocomplex, comprising the steps of:

[0047] (1) 250mg polyethyleneimine (PEI, molecular weight 25K) was dissolved in 4mL dimethylformamide (DMF); 514.2mg p-toluenesulfonyl arginine (Boc-Arg(Tos)-OH), 460.1mg 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 324.3 mg 1-hydroxybenzotriazole (HOBT) were dissolved in 8 mL dimethylformamide (DMF) , Stored at room temperature for 1h, a mixed solution was obtained. The DMF solution of polyethyleneimine (PEI) and diisopropylethylamine (DIPEA) (248.14mg, 1.92mmol) were added to the above mixed solution, stored at room temperature for 5 days, and the mixture was placed in deionized water (dialysis bag cut: 3500Da ) under dialysis for 72h, freeze-dried white solid. The obtained solid was added to 4ml of trifluoroacetic acid (TFA) and stirred at room temperature for 4h. Precipitate with anhydrous ether, filter and dry under vacuum, dialyze with deioniz...

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Abstract

The invention provides a preparation method of a tumor pH (potential of hydrogen) response charge reversal miRNA (micro Ribonucleic Acid) loaded nano compound. The preparation method comprises the following steps: 1) grafting p-toluenesulfonyl arginine on a polyethyleneimine side group to obtain PER; (2) modifying the polyethyleneimine by using dimethyl maleic anhydride to obtain PEI-DMA; (3) carrying out self-assembly on the PER and the miRNA to obtain a PER / miRNA binary compound; and 4) carrying out self-assembly on the binary compound of the PEI-DMA and the PER / miRNA to prepare the nano compound. The preparation process is simple, the prepared charge reversal nanocomposite has the characteristic of pH response charge reversal, specific charge reversal occurs in the pH environment of a tumor site, targeting and cellular uptake are enhanced, miRNA is effectively controlled to be released to a target site to achieve gene therapy, side effects on other sites are reduced, and the application prospect is wide. Effective miRNA transfer and high transfection effects are achieved, and wide application prospects are achieved in the aspect of gene therapy.

Description

technical field [0001] The invention belongs to the field of polycationic gene carrier materials, and in particular relates to a preparation method of a tumor pH-responsive charge reversal-loaded miRNA nanocomposite. Background technique [0002] MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that can regulate the expression of most human genes and regulate cell differentiation, proliferation, and apoptosis through various mechanisms. Therefore, many miRNAs are now recognized as effective specific gene drugs for the treatment of cancer. However, miRNA therapy alone has the limitations of relatively short half-life, poor cellular uptake, rapid nuclease degradation, and poor blood stability. Under physiological conditions, cationic polymer polyethyleneimine (PEI) can self-assemble with negatively charged RNA through electrostatic interaction into a nanometer-sized positively charged nanocomplex (polyplex), which can effectively cover nucleic...

Claims

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Application Information

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IPC IPC(8): A61K47/58A61K31/7105A61K9/14A61K47/32A61P35/00C08G73/04C08G81/00
CPCA61K47/58A61K31/7105A61K9/146A61P35/00C08G73/02C08G81/00Y02P20/55
Inventor 殷义霞余丹
Owner WUHAN UNIV OF TECH