A cationic silk fibroin/gene complex, preparation method and application thereof

A silk fibroin and cationization technology, applied in other methods of inserting foreign genetic materials, using vectors to introduce foreign genetic materials, recombinant DNA technology, etc., can solve the problem of affecting the binding efficiency of the carrier and the target cell, low transfection efficiency, Reduce adsorption and other problems, achieve good application prospects, improve transfection efficiency, and simple methods

Active Publication Date: 2017-01-25
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cationic polymers such as hyperbranched dendrimers, polyethyleneimine, etc., as non-viral gene carriers, have obvious advantages in biological safety and high transfection efficiency, but their disadvantages are cytotoxicity and non-degradable
However, since silk fibroin has a large amount of negative charge in a neutral environment, the isoelectric point pI is about 4.0-4.5, which greatly reduces the adsorption of the negatively charged cell surface to the carrier and affects the binding efficiency of the carrier and the target cell. Transfection still inefficient

Method used

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  • A cationic silk fibroin/gene complex, preparation method and application thereof
  • A cationic silk fibroin/gene complex, preparation method and application thereof
  • A cationic silk fibroin/gene complex, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of protamine cationized silk fibroin, concrete steps are as follows:

[0031] (1) Put 100 g tussah raw silk into 5 L Na with a mass concentration of 0.25 % 2 CO 3 In aqueous solution, treatment at 98-100°C for 45 min, repeated three times to degumming silk, washing and drying fully to obtain tussah silk fibroin fiber. Add the tussah silk fiber into the molten calcium nitrate tetrahydrate, stir and dissolve at 105 ℃. The obtained mixed solution was put into a dialysis bag (the molecular weight cut-off was 9-12 KDa), and dialyzed in deionized water for 4 days to remove impurities to obtain a pure tussah silk fibroin protein solution. Adjust the concentration of tussah silk solution to 20 mg / ml, and filter with a 0.22 μm microporous membrane;

[0032] (2) Dissolve sulfo-SMCC in PBS solution (phosphate buffered saline, pH=7.4) and prepare a solution with a concentration of 1mg / ml. Take 1 ml of sulfo-SMCC solution and slowly add it dropwise to 20 ml of th...

Embodiment 2

[0039] 1. Use boiling Na with a concentration of 3.5 ‰ 2 CO 3 The silkworm cocoon was treated with solution for 3 times, 30 min each time, to degumming the silk, and after washing and drying thoroughly, the silk fibroin fiber was obtained. Add celestial silk fibroin fiber into molten calcium nitrate tetrahydrate, stir and dissolve at 90 °C. The obtained mixed solution was put into a dialysis bag (the molecular weight cut-off was 9-12 KDa), and dialyzed with deionized water for 4 days to remove impurities to obtain a pure silk fibroin solution. Adjust the concentration of cecropin solution to 20 mg / ml, and filter it with a 0.22 μm microporous membrane;

[0040] 2. Dissolve sulfo-SMCC in PBS solution (pH=7.4), and prepare a solution with a concentration of 1 mg / ml. Take 1 ml of sulfo-SMCC solution and slowly add it dropwise to 20 ml of the silk fibroin solution obtained in step 1, stir magnetically for 2 h at 4 °C, and centrifuge the solution with an ultrafiltration centrifug...

Embodiment 3

[0048] Preparation of cationized silk fibroin / gene carrier, the specific steps are as follows: adjust the concentration of the cationized tussah silk fibroin solution with sample number 1 obtained in Example 1 of the present invention to 0.01 mg / ml, and filter it with a 0.22 μm microporous membrane , adjust the DNA concentration to 0.01 mg / ml with sterile water. Slowly stir the silk fibroin solution containing 4 μg cationized silk fibroin at 2-8°C with an electric stirrer at a speed of 60 rpm. After applying a shear force for 15 min, add 2 μg DNA solution, vortexed for 30 s, warmed up to 25°C, let the complex solution stand for 45 min, and self-assembled into a cationic silk fibroin / DNA complex by electrostatic interaction, which was recorded as sample 19, the mass of cationic silk fibroin and DNA The ratio is 2:1.

[0049] With reference to the above method, the mass ratios of cationized silk fibroin to DNA were respectively 3:1, 4:1, 5:1, 6:1, 7:1 for the samples whose samp...

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Abstract

The invention discloses a cationic silk fibroin / gene complex, a preparation method and an application thereof, and belongs to the technical field of biomedical polymer materials. The present invention constructs a novel biodegradable cationic silk fibroin gene transfer carrier with cell targeting and cell nucleus localization functions, which is mediated by water-soluble 2-iminothiolane hydrochloride protamine sulfate Produced by coupling silk fibroin activated with sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate and forms cations with genetic material by electrostatic interaction Fibroin / gene complex has good biocompatibility and degradability, controllable surface charge density, ability to effectively compress and protect DNA, high transfection efficiency, unique cell targeting and nuclear localization functions.

Description

technical field [0001] The invention belongs to the technical field of biomedical polymer materials, and in particular relates to a targeted gene carrier material, preparation method and application. Background technique [0002] In recent years, the development of gene therapy technology has made it possible to cure diseases or cancers caused by gene defects, and has also provided a new way for the repair and functional recovery of tissue defects caused by trauma, surgery and other reasons. Successful gene therapy requires efficient delivery of the target gene into cells. An ideal gene transfer vector should have cell targeting, high stability, low cytotoxicity and high transfection efficiency; at the same time, the vector should be simple and easy to manufacture, mass-produced and easy to use. Although viral vectors have high transfection efficiency, there are potential safety hazards such as immunogenicity and mutation, and the preparation methods are complicated, which ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N15/85
Inventor 刘雨李明忠卢神州王建南瞿静
Owner SUZHOU UNIV
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