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Ternary gene delivery system based on cell-penetrating peptide and applications of ternary gene delivery system

A gene delivery and membrane-penetrating peptide technology, which is applied in gene therapy, medical preparations of non-active ingredients, non-active ingredients of polymer compounds, etc., can solve unfavorable multifunctional gene delivery system design and regulation, and poor escape ability of endosomes , lack of specificity and other issues, to achieve high-efficiency nuclear localization ability, good endosome escape ability, and effective gene delivery

Inactive Publication Date: 2018-08-17
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The gene delivery system based on penetrating peptide has high biocompatibility and special membrane penetrating effect, and has great development potential in the design of gene delivery system, but it is unstable, lacks specificity and endosome escape ability The difference makes its application also limited
In addition, current gene delivery systems are generally binary gene delivery systems of cationic polymers and genes, which is not conducive to the design and regulation of multifunctional gene delivery systems.
Therefore, the design and optimization of safe and efficient specific gene delivery systems still face great challenges.

Method used

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  • Ternary gene delivery system based on cell-penetrating peptide and applications of ternary gene delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A method for preparing a three-component gene delivery system based on penetrating peptides, comprising the steps of:

[0045] (1) According to the molar ratio of 1:8, octaamino POSS and NHS-PEG 2000 - OPSS was dissolved in PBS buffer solution with pH = 7.4, and reacted at room temperature for 2 hours;

[0046] (2) Add the REDV-G-TAT-G-NLS-C polypeptide to the solution obtained in step (1), react for 4 hours, use a dialysis bag with a molecular weight cut-off of 3500 for dialysis purification, and freeze-dry to obtain POSS-(PEG- NLS-G-TAT-G-REDV) 8 polymer;

[0047] The molar ratio of octaamino POSS and REDV-G-TAT-G-NLS-C is 1:8;

[0048] (3) Using PBS buffer solution with pH=7.4 as solvent, prepare POSS-(PEG-NLS-G-TAT-G-REDV) with a concentration of 0.5 mg / ml 8 Polymer liquid; by POSS-(PEG-NLS-G-TAT-G-REDV) 8The mass ratio of the polymer to the Cy5-labeled oligonucleotide is 1:0.6, and the Cy5-labeled oligonucleotide aqueous solution with a concentration of 0.05 m...

Embodiment 2

[0053] A method for preparing a three-component gene delivery system based on penetrating peptides, comprising the steps of:

[0054] (1) According to the molar ratio of 1:16, octaamino POSS and NHS-PEG 3000 - OPSS was dissolved in PBS buffer solution with pH = 7.4, and reacted at room temperature for 3 hours;

[0055] (2) Add the REDV-G-TAT-G-NLS-C polypeptide to the solution obtained in step (1), react for 8 hours, use a dialysis bag with a molecular weight cut-off of 3500 for dialysis purification, and freeze-dry to obtain POSS-(PEG- NLS-G-TAT-G-REDV) 8 polymer;

[0056] The molar ratio of octaamino POSS and REDV-G-TAT-G-NLS-C is 1:16;

[0057] (3) Using PBS buffer solution with pH=7.4 as solvent, prepare POSS-(PEG-NLS-G-TAT-G-REDV) with a concentration of 2 mg / ml 8 Polymer liquid; by POSS-(PEG-NLS-G-TAT-G-REDV) 8 The mass ratio of the polymer to the Cy5-labeled oligonucleotide is 1:0.8, and the aqueous solution of the Cy5-labeled oligonucleotide with a concentration o...

Embodiment 3

[0062] Control:

[0063] The binary gene delivery system was obtained from steps (1), (2) and (3) of Example 2.

[0064] The cytotoxicity of the binary gene delivery system and the ternary gene delivery system (ZNF580 gene) prepared in Example 1 and Example 2 was determined by MTT (3-(4,5-dimethylthiazole-2)-2,5-di Phenyl tetrazolium bromide) colorimetric method for evaluation.

[0065] The steps are as follows: Human umbilical vein endothelial cells (EA.hy926 cells) were inoculated into 96-well plates (1×10 4 cells / well) in the cell culture medium, after the cells grew to 90%, the cell culture medium was replaced with a serum-free medium, and the starvation treatment was performed for 12 hours. The medium was then changed to fresh growth medium (10% FBS DMEM). Add different concentrations of binary gene delivery system and ternary gene delivery system solutions into the growth medium, mix well, discard the supernatant after 24 hours, and add 5 mg / ml of MTT (solvent: pH = ...

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Abstract

The invention discloses a ternary gene delivery system based on cell-penetrating peptide and applications of the ternary gene delivery system. The ternary gene delivery system based on the cell-penetrating peptide is prepared by adopting the following method: multifunctional polypeptide REDV-G-TAT-G-NLG-C is connected to eight active sites of octa ammonium POSS (polyhedral oligomeric silsesquioxane) through orthopyridyl disulfide-PEG-NHS ester, and thus a star polymer is formed; the star polymer with positive charges and a gene with negative electricity are bonded through electrostatic interaction, and thus a binary gene delivery system with negative electricity on the surface is formed; a polypeptide sequence rich in histidine and the binary gene delivery system are bonded through electrostatic interaction, and thus the ternary gene delivery system is formed. The ternary gene delivery system based on cell-penetrating peptide provided by the invention has targeting ability for endothelial cells, and has the functions of cell-penetrating peptide, histidine and nuclear localization signals, so that carried genes can efficiently enter cells, the escape from endosome is effectively carried out, the entering of the genes into the cell nucleus is realized, and finally, the gene delivery effect is greatly improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a ternary gene delivery system based on a penetrating peptide and its application. Background technique [0002] In recent years, with the development of gene therapy, the treatment of various diseases by gene has attracted more and more attention. Among them, the main bottleneck restricting the wide application of this technology is the lack of efficient and low-toxic gene delivery system. Viral delivery systems have been controversial due to their safety concerns. The non-viral gene delivery system avoids the problem of viral vectors well, but the non-viral gene delivery system has high cytotoxicity, poor selectivity, low cell uptake efficiency, difficulty in endosome escape, difficulty in entering the nucleus, poor transfection efficiency, The low level of gene expression is the main problem that the gene delivery system carries the therapeutic gene and cannot treat the diseas...

Claims

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Application Information

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IPC IPC(8): A61K47/42A61K47/34A61K47/64A61K47/60A61K47/59A61K48/00
CPCA61K47/34A61K47/42A61K47/59A61K47/60A61K47/64A61K48/0025A61K48/005
Inventor 冯亚凯郝雪芳李茜郭锦棠任相魁
Owner TIANJIN UNIV
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