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Cationization antheraea pernyi silk fibroin /adenovirus compound, and preparation method and application thereof

A technology of tussah silk fibroin and cationization is applied in the field of gene transfer carrier materials, which can solve the problems of poor degradability and affect the expression of target genes, and achieve the effects of reducing the effective use titer, improving the expression level and low immunogenicity.

Pending Publication Date: 2020-08-25
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the degradation of polyamide-amine dendrimers is poor, which affects the expression of target genes in target cells

Method used

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  • Cationization antheraea pernyi silk fibroin /adenovirus compound, and preparation method and application thereof
  • Cationization antheraea pernyi silk fibroin /adenovirus compound, and preparation method and application thereof
  • Cationization antheraea pernyi silk fibroin /adenovirus compound, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1. Polyethyleneimine modified tussah silk fibroin to prepare cationized tussah silk fibroin. The specific steps are as follows:

[0032] (1) 100 g of tussah silk in 5 L of Na with a mass concentration of 2.5‰ 2 CO 3Boil three times in the aqueous solution, the time is 30min, 30min, 45min respectively. After each cooking, wash three times with deionized water. The washed tussah silk was dried in an oven at 60°C to obtain refined tussah silk. Calcium nitrate tetrahydrate was heated and melted, filtered, and the temperature was kept at 105 °C, and the loose tussah silk was added in stages, and 10 g of tussah silk was dissolved for every 100 mL of calcium nitrate. Dissolve for about 5 hours until the tussah silk is completely dissolved without floc. The cooled tussah silk fibroin solution was transferred to a dialysis bag (molecular weight cut-off 12 kD), dialyzed in deionized water at a temperature of 0–4 °C to obtain a regenerated tussah silk fibroin protein solution,...

Embodiment 2

[0044] Cationic tussah silk fibroin / adenovirus complex infects target cells in vitro, including the following steps:

[0045] (1) The potency is 1×10 7 pfu / mL naked adenovirus and cationized tussah silk fibroin protein with a concentration of 100 μg / mL were mixed evenly at a volume ratio of 1:1, vortexed for 1 minute, and left at room temperature for 30-45 minutes to prepare silk fibroin / adenovirus Complex; titer 1 × 10 7 Pfu / mL naked adenovirus was mixed evenly with serum-free medium at a volume ratio of 1:1 to obtain naked adenovirus with the same titer as silk fibroin / adenovirus complex.

[0046] (2) Lung cancer cells H460 were used at 1×10 5 Cells / well density were seeded in six-well plates, and cultured in 1640 medium containing 10% fetal bovine serum for 24 h. Aspirate the medium, and add the cationized tussah silk fibroin / adenovirus complex prepared in step (1) and naked adenovirus respectively. The adenovirus encodes the ING4-IL-24 double gene. Eight hours after ...

Embodiment 3

[0051] Cytotoxicity of cationized tussah silk fibroin / adenovirus complex, the specific steps are as follows:

[0052] (1) The potency is 1×10 7 pfu / mL naked adenovirus and cationic tussah silk fibroin protein with a concentration of 100 μg / mL were mixed evenly at a volume ratio of 1:1, vortexed for 1 minute, and left at room temperature for 30-45 minutes to prepare silk fibroin / adenovirus Complex; titer 1 × 10 7 Pfu / mL naked adenovirus and serum-free medium were mixed evenly at a volume ratio of 1:1 to obtain naked adenovirus with the same titer as the silk fibroin / adenovirus complex. The adenovirus encodes the ING4-IL-24 double gene.

[0053] (2) HUVEC of umbilical vein endothelial cells in 1×10 4 The density of cells / well was inoculated in 96-well plate with 5 duplicate wells, and cultured in DMEM medium containing 10% fetal bovine serum for 24 h. Aspirate the medium, add 100 μL of the cationic tussah silk fibroin / adenovirus complex and naked adenovirus prepared in ste...

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Abstract

The invention discloses a cationization antheraea pernyi silk fibroin / adenovirus compound, and a preparation method and application thereof, and belongs to the technical field of biologic medical high molecular materials. The cationization antheraea pernyi silk fibroin is obtained through coupling of antheraea pernyi silk fibroin activated by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and polyethyleneimine, the antheraea pernyi silk fibroin having positive charges on the surface, is coated to the surface of an adenovirus through electrostatic adsorption action, and the cationization antheraea pernyi silk fibroin / adenovirus compound is obtained. The invention constructs a novel cationization antheraea pernyi silk fibroin / adenovirus compound genetic carrier high in infection efficiency, low in cytotoxicity and low in cell targeting properties, in accordance with defects of a naked adenovirus carrier and defects of an existing adenovirus compound carrier, and the cationization antheraea pernyi silk fibroin / adenovirus compound has favorable application prospects in in vivo and vitro infection of target cells.

Description

technical field [0001] The invention relates to a gene transfer carrier material, a preparation method and an application thereof, and belongs to the technical field of biomedical polymer materials. Background technique [0002] Tumor gene therapy refers to the use of gene manipulation methods to introduce foreign genes to correct the structural or functional defects of tumor cell genes, or to treat tumors by enhancing the host's ability to kill tumors and the body's defense functions. Gene therapy has the advantages of highly targeted, less toxic and side effects, and good curative effect. The key lies in the construction of safe and efficient gene delivery vectors. There are mainly two ways to deliver target genes to tumor cells, mediated by viral vectors and transfected by non-viral vectors. Non-viral vectors mainly include cationic liposomes and cationic polymers. Liposomes are expensive, and are prone to adsorption with plasma proteins, resulting in the disintegration...

Claims

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Application Information

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IPC IPC(8): C12N15/87C07K14/435C12N7/00
CPCC12N15/87C07K14/43586C12N7/00C12N2710/10021
Inventor 瞿静李明忠冯艳飞牛陇星
Owner SUZHOU UNIV
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