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Multi-targeted modified gene-loaded compound, preparation method and application

A technology of targeted modification and compound, applied in other methods of inserting foreign genetic materials, gene therapy, medical preparations of non-active ingredients, etc., can solve the problem of less research

Inactive Publication Date: 2016-11-09
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are few studies on the promotion of endothelial cell transfection by this multiple targeting modified gene complex

Method used

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  • Multi-targeted modified gene-loaded compound, preparation method and application
  • Multi-targeted modified gene-loaded compound, preparation method and application
  • Multi-targeted modified gene-loaded compound, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: the preparation method of the polycationic gene carrier (I) of membrane-targeting peptide modification, comprises the following steps:

[0058] Under nitrogen protection, polyethyleneimine-poly(glycolide-co-caprolactone)-polyethyleneimine copolymer with a weight average molecular weight of 30000, said polyethyleneimine-poly(glycolide- (co-caprolactone)-polyethyleneimine copolymer has the same weight-average molecular weight of branched polyethyleneimine, and is 10000, and the two ends with weight-average molecular weight of 2000 are respectively adjacent dithiopyridyl and succinimide-modified polyethylene glycol (OPSS-PEG-NHS), dissolved in a mixed solvent, reacted for 3 hours at room temperature and protected from light, added membrane targeting peptide, reacted for 6 hours, and the product was dissolved in distilled water Dialyze for 48h, change the distilled water every 2h, and freeze-dry to obtain the polycationic gene carrier (I) modified by the membr...

Embodiment 2

[0075] Embodiment 2: Preparation of REDV-NP, pGFP / REDV-NP complex and TAT-NLS / pGFP / REDV-NP complex:

[0076] (1) Dissolve the abbreviated TAT-NLS polypeptide in an appropriate amount of water at room temperature (the amount of water can dissolve the TAT-NLS polypeptide);

[0077] Nucleic acid (the nucleic acid used in this embodiment is a plasmid containing green fluorescent protein) is dissolved in an appropriate amount of water, (the amount of water can dissolve the plasmid of green fluorescent protein);

[0078] According to the mass ratio of TAT-NLS polypeptide and nucleic acid in abbreviation as 1:1, mix the TAT-NLS polypeptide aqueous solution and nucleic acid aqueous solution evenly, and let it stand for 30 minutes; obtain the mixed solution;

[0079] (2) Prepare the polycationic gene carrier (I) (REDV-NP) nanoparticle suspension modified by the membrane-targeting peptide by dialysis:

[0080] Weigh 5 mg of membrane-targeting peptide-modified polycationic gene carrier ...

Embodiment 3

[0089] Embodiment 3: the agarose gel electrophoresis analysis of TAT-NLS / pGFP / REDV-NP and pGFP / REDV-NP:

[0090] Add the prepared TAT-NLS / pGFP / REDV-NP, pGFP / REDV-NP and pure pGFP genes with different N / P ratios to the wells of 0.8% agarose gel respectively, and place them in 1×TAE buffer at 100V Electrophoresis in liquid for 30min. Observe the position of pGFP under ultraviolet irradiation and take pictures to analyze the binding ability of nanoparticles and pGFP. from Figure 4 As can be seen in , REDV-NP can fully compress loaded pGFP at N / P ratio ≥ 20. After adding TAT-NLS, TAT-NLS / pGFP / REDV-NP can completely block the migration of plasmids when the N / P ratio is ≥10, which indicates that TAT-NLS helps REDV-NP nanoparticles to bind better and Compress pGFP.

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Abstract

The invention discloses a multi-targeted modified gene-loaded compound, a preparation method and application. The method comprises the following steps: preparing a membrane targeted peptide modified polycation gene vector (I); evenly mixing a TAT-NLS polypeptide aqueous solution and a nucleic acid aqueous solution to obtain mixed liquor, and preparing the gene vector (I) into suspension liquid; and (4) evenly mixing the suspension liquid and the mixed liquor, and standing to obtain the multi-targeted modified gene-loaded compound. The multi-targeted modified gene-loaded compound is specifically recognized by REDV peptide in membrane targeted peptide and an integrin receptor on the surface of an endothelial cell, and uptake of the cell to the compound is improved. The compound which enters the cell is positioned in a lysosome / endosome structure, polyethyleneimine and TAT jointly act, the escaping capacity of the endosome of the compound is improved, and a target gene is promoted to enter cytoplasm. Through interaction of a nuclear localization signal NLS and a nuclear membrane, nuclear internalization of the target gene is promoted. Transfection efficiency of the target gene in the endothelial cells is high.

Description

technical field [0001] The invention relates to a multi-target modified gene-carrying complex, a preparation method and an application thereof, and belongs to the technical field of gene carriers with biological target recognition functions. Background technique [0002] At present, cardiovascular diseases are becoming more and more prevalent. Cardiovascular disease is one of the leading causes of death worldwide due to poor clinical treatment effects and huge treatment costs. Gene therapy is a promising approach for the treatment of congenital and acquired cardiovascular diseases. With the identification of pathophysiological molecular pathways of heart failure and related cardiovascular diseases, pre-clinical trials of gene therapy have been launched in small and large animal models, and gradually moved to the clinic. However, the initial clinical effects did not meet the expected goals . Viral vectors are still controversial due to their inherent lethal immunogenicity,...

Claims

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Application Information

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IPC IPC(8): C12N15/87A61K48/00A61K47/48A61P9/00A61P9/14
CPCA61K48/0041C12N15/87
Inventor 冯亚凯杨静李茜郭锦棠
Owner TIANJIN UNIV
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