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Reductive polyglutamic acid/polyethyleneimine/siRNA composite nanoparticles as well as preparation and application thereof

A technology of polyethyleneimine and branched polyethyleneimine, which is applied in the application field of preparing tumor-targeted gene drugs, can solve the problems of high cytotoxicity and low gene transfection efficiency, and achieve the promotion of gene uptake and increase Active targeting, achieving the effect of passive tumor targeting delivery

Active Publication Date: 2020-05-08
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Macromolecular polyethylenimine has high gene transfection efficiency but high cytotoxicity, and low molecular weight polyethyleneimine has low toxicity but low gene transfection efficiency

Method used

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  • Reductive polyglutamic acid/polyethyleneimine/siRNA composite nanoparticles as well as preparation and application thereof
  • Reductive polyglutamic acid/polyethyleneimine/siRNA composite nanoparticles as well as preparation and application thereof
  • Reductive polyglutamic acid/polyethyleneimine/siRNA composite nanoparticles as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Weigh 10 g of branched polyethyleneimine with a molecular weight of 1200, and dissolve it in 300 mL of deionized water to obtain a BPEI 1.2k aqueous solution. Adjust the pH value of the BPEI1.2k aqueous solution to neutral by adding 0.1mol / L hydrochloric acid solution dropwise, and freeze-dry for 3 days;

[0031] (2) Dissolve the solid obtained in step (1) with 300 mL of anhydrous methanol in a three-necked flask, blow nitrogen gas for 20 minutes to remove the air in the system, add 3.3 mL of propylene sulfide with a 1 mL syringe, and Stirring reaction under the protection of nitrogen for 18 hours;

[0032] (3) The product obtained in step (2) was dried under reduced pressure to remove the solvent, then dissolved in 200 mL of anhydrous methanol, precipitated twice through cold ether, and dried under reduced pressure to remove the solvent to obtain BPEI-SH;

[0033] (4) The obtained BPEI-SH was dissolved in 30 mL of dimethyl sulfoxide solution, and stirred and reacted...

Embodiment 2

[0041] (1) Sample configuration: Add 1 μL of different concentrations of BPEI25k, SSBPEI, and PBS solution (PH=7.4) of SSBPEI and mPEG-γ-PGA polymer to 3.6 μL of siRNA solution with a concentration of 20 μM. Incubate for 30 minutes to prepare BPEI25k@siRNA, SSBPEI@siRNA N / P ratios of 1:1, 5:1, 10:1, 20:1 nanoparticles, and the mass ratio of mPEG-γ-PGA to SSBPEI@siRNA 0.5:1, 1:1, 2:1 SSBPEI@siRNA / mPEG-γ-PGA respectively. Add 2 μL of 6×RNA sample buffer to each solution with a pipette gun, pipette evenly and set aside.

[0042] (2) Agarose gel configuration: Weigh 0.3g of agarose and place it in a conical flask, add 30mL of 1×Tris-acetate-EDTA buffer solution and shake well, cover with a small beaker, then put the conical flask into the microwave Heat until the agarose is completely dissolved. When the heated agarose is cooled to about 60°C at room temperature, add 3uL of ethidium bromide (EB), shake well, then transfer the solution to the gel plate with the comb inserted, sha...

Embodiment 3

[0053] (1) Cytotoxicity: Take the A549 cells to be treated out of the incubator and place them in an ultra-clean workbench, absorb the original culture medium with a pipette gun, add PBS solution to wash twice, and then add 400 μL of trypsin digestion solution to carry out For digestion, place the culture bottle in a 37°C incubator for digestion for about 3 minutes, and then use a microscope to observe the conditions of the cells in the culture bottle. When the cells become round and the intercellular space becomes larger, the trypsin digestion solution is removed, and a certain amount of medium containing 10% FBS is added to terminate the digestion. Gently blow with a pipette to make a single-cell suspension, count it with a hemocytometer, and dilute it to a concentration of 5×10 4 cells / mL single cell suspension. Take out the 96-well plate, add 100 μL of the above cell suspension to each well, place it in the operating table for 10 minutes, and then put it in a 37°C incubat...

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Abstract

The invention provides a preparation method of reductive polyglutamic acid / polyethyleneimine / siRNA composite nanoparticles. The preparation method comprises the following steps: reacting micromolecular branched polyethyleneimine with propylene sulfide to obtain macromolecular disulfide bond modified branched polyethylenimine (SSBPEI); and preparing SSBPEI@ siRNA from SSBPEI and siRNA through electrostatic binding, and modifying the surface of the SSBPEI@ siRNA as an inner core with a polyethylene glycol modified polyglutamic acid (mPEG-gamma-PGA) shell through electrostatic binding, thereby obtaining the nanoparticles. The beneficial effects provided by the invention are mainly embodied in that the introduction of polyethylene glycol improves the biocompatibility and blood long-circulationcapability of a carrier; the polyglutamic acid can be specifically combined with tumor-related gamma-glutamyl transpeptidase (GGT) on tumor cells, so that a gene drug is actively delivered to a tumorpart in a targeted manner; SSBPEI can be degraded into low-toxicity small-molecule polyethyleneimine under the reduction of high-concentration glutathione in tumor cells, so that the toxicity of macromolecular SSBPE is reduced, and meanwhile, siRNA can be released at a tumor part in an accelerated manner to realize gene treatment.

Description

[0001] (1) Technical field [0002] The invention relates to a reducing polyglutamic acid / polyethyleneimine / siRNA composite nanoparticle and its preparation method, as well as its application in the preparation of tumor-targeted gene medicine. [0003] (2) Background technology [0004] Gene therapy refers to a biological treatment method that introduces exogenous normal genes into target cells through gene transfer technology, corrects or compensates diseases caused by gene defects and abnormalities, and achieves therapeutic purposes. In recent years, gene therapy has become one of the important means to treat various diseases, especially in the treatment of tumors. Tumor gene therapy can inhibit the proliferation and growth of tumor cells by changing the expression of genes in tumor cells. However, due to the disadvantages of poor targeting and easy degradation by serum in the process of tumor therapy, gene carriers have become an important part of gene therapy research. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K9/51A61K47/34A61K31/7105A61P35/00
CPCA61K9/5146A61K31/7105A61P35/00
Inventor 程翠王思远陈立刘沁颖
Owner FUZHOU UNIV
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