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Gene vector as well as preparation method and application thereof

A gene carrier and metal nanoparticle technology, applied in the field of biomedicine, can solve problems such as high cytotoxicity, achieve the effects of improving transfection efficiency, good biocompatibility, and reducing toxicity

Inactive Publication Date: 2014-10-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the carrier synthesized with polyethyleneimine can significantly improve the transfection efficiency, it has high cytotoxicity because it is easily decomposed by intracellular enzymes to produce toxic substances.

Method used

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  • Gene vector as well as preparation method and application thereof

Examples

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preparation example Construction

[0035] Preparation of epidermal stem cells

[0036] 1) Remove the back and head skin of the rat, remove fat and blood streaks, and cut into rat strips with a length of 1.5 cm and a width of 2 mm;

[0037] 2) Wash 3 times with ordinary D-Hanks (phosphate buffered saline solution without calcium and magnesium ions, Beijing Hongbokang Pharmaceutical Technology Co., Ltd.), 20 mL / time, 3 minutes each time;

[0038] 3) Then use high anti-D-Hanks (high anti-D-Hanks is obtained by mixing ordinary D-Hanks solution and double antibody solution, wherein, when mixing, the volume ratio of ordinary D-Hanks solution and double antibody solution is 100:4, The double-antibody is penicillin and streptomycin, and the double-antibody solution is purchased from Gibico, Brl, USA) and soaked for 150 minutes;

[0039] 4) put into the neutral protease aqueous solution that contains dispaseII neutral protease (purchased from Gibico, Brl, USA) weight percentage content is 0.25%, the volume of neutral p...

Embodiment 1

[0047] Embodiment 1: Gene carrier containing gold nanoparticles and polylysine (Au-PLL)

[0048] 1. Preparation of Au-PLL gene carrier

[0049] Add PLL to 10 mL of 1 mM (410 μg / mL) HAuCl 4 In the solution, make the concentration of PLL reach 5mg / mL, stir at room temperature for 20min, and make it fully mixed. With vigorous stirring, add 40 μL of NaBH 4 (0.1M4mg / mL) until the solution turns red immediately, and then continue to stir for 30min. The obtained colloidal solution was centrifuged at 3000 rpm for 30 min using an ultrafiltration centrifuge tube with a molecular weight of 100,000 to obtain the Au-PLL gene carrier. The particle diameter of the prepared Au-PLL gene carrier is 12.25±0.3nm, and the surface charge is 23±1.5mV.

[0050] 2. Preparation of complex (Au-PLL-DNA) combined with Au-PLL gene carrier and plasmid DNA

[0051] Mix the Au-PLL gene carrier and DNA at different mass ratios (see Table 1 for details) in equal volumes (carrier solution and DNA solution, ...

Embodiment 2

[0064] Embodiment 2: Gene carrier containing silver nanoparticles and polylysine (Ag-PLL)

[0065] 1. Preparation of Ag-PLL gene carrier

[0066] Add PLL to 10 mL of 1 mM (170 μg / mL) AgNO 3 In the solution, make the concentration of PLL reach 5 mg / mL, stir at room temperature for 20 min, and allow it to mix well. With vigorous stirring, add 40 μL of NaBH 4 (0.1M4mg / mL) until the solution turns bright yellow immediately, and then continue to stir for 30min. The obtained colloid solution was centrifuged at 3000 rpm for 30 min by using an ultrafiltration centrifuge tube with a molecular weight of 100,000 to obtain the Ag-PLL gene carrier. The particle size of the prepared Ag-PLL gene carrier is 35±1.2nm, and the surface charge is 29±1.7mV.

[0067] Two, the preparation of the complex (Ag-PLL-DNA) of Ag-PLL gene carrier and plasmid DNA combination

[0068] According to the preparation method of the complex of the gene carrier and the plasmid DNA in Example 1, the particle siz...

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Abstract

The invention discloses a gene vector as well as a preparation method and application thereof. The gene vector comprises metal nanoparticles as a core and a positive polymer connected to the surfaces of the metal nanoparticles, wherein the positive polymer is polylysine. The invention further discloses a preparation method of the gene vector and application of the gene vector to epidermal stem cell transfection. The gene vector is connected with the metal nanoparticles through polylysine and cell-penetrating peptide, so that the transfection efficiency of the gene vector is improved, the influence on cell toxicity is lowered, the low-toxicity characteristic of polylysine is effectively utilized, and the low transfection efficiency characteristic of polylysine is successfully optimized.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a gene carrier containing metal nanomaterials and cationic polymers, a preparation method and application thereof. Background technique [0002] In recent years, with the progress of the Human Genome Project and the continuous exploration of functional genes, the study of gene therapy has attracted more and more attention. Since gene therapy entered clinical trials in the 1980s, its application has evolved from genetic The treatment of diseases extends to the prevention and treatment of acquired diseases, such as tissue engineering and regenerative medicine. For gene recombination and gene transfection, the key to constructing vectors is a vector with good safety and high efficiency and targeted expression of genes. [0003] There are two types of gene transfection vectors commonly used at present: viral and non-viral. Viral vectors have the ability to efficiently deliver a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/64
Inventor 彭丽华高建青
Owner ZHEJIANG UNIV
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