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A kind of cationic silk fibroin, preparation method and application thereof

A silk fibroin, cationized technology, applied in the field of biomedical materials, can solve the problem of PEI being non-biodegradable, and achieve the effects of avoiding toxic side effects, improving targeting, and mild reaction conditions

Active Publication Date: 2016-08-17
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The literature "Antheraepernyi Silk Fibroin-Coated PEI / DNA Complexes for Targeted Gene Delivery in HEK 293 and HCT 116 Cells" [International journal of molecular sciences, 2014,15(5): 7049-7063] pointed out that in PEI-DNA gene binary A layer of tussah fibroin protein was electrostatically adsorbed on the outside of the complex, which was used to transfect human embryonic kidney and human colorectal cancer cells. The results showed that the introduction of tussah silk fibroin could reduce the cytotoxicity, and at the same time, the presence of the RGD sequence increased the gene-to-cell Transfection efficiency, but its disadvantage is that high molecular weight PEI is not biodegradable

Method used

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  • A kind of cationic silk fibroin, preparation method and application thereof
  • A kind of cationic silk fibroin, preparation method and application thereof
  • A kind of cationic silk fibroin, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Put 150 g of silkworm silk into 6 L of 0.05% Na 2 CO 3 In aqueous solution, at 98~100 o C treatment for 30 min, repeated 3 times to degumming silk, fully washed and dried to obtain pure silk fiber. Add pure silk fiber to the ternary solution (calcium chloride: water: ethanol molar ratio is 1:8:2), at 72 o C stirring and dissolving into a mixed solution of silk fibroin. Put the obtained silk fibroin mixed solution into a dialysis bag, and dialyze with deionized water for 4 days to obtain a purified silk fibroin solution.

[0029]Take 10 mL of silk fibroin solution with a concentration of 1 mg / mL, weigh PEI (800 Da) accounting for 64% of the mass of silk fibroin, dissolve it in 10 mL of phosphate buffer solution (100 mM, pH=6.2), and use 2 M Adjust the pH value of the HCl solution to 6.5, and then fully mix with the silk fibroin solution; dissolve tyrosinase in phosphate buffer solution (50mM, pH=6.0), and prepare tyrosinase with a concentration of 0.1 mg / mL solution...

Embodiment 2

[0033] The degummed silkworm silk fibers were added to 9.3 M LiBr solution at 60 o C stirring and dissolving into a mixed solution of silk fibroin. Put the obtained silk fibroin mixed solution into a dialysis bag, and dialyze with deionized water for 4 days to obtain a purified silk fibroin solution.

[0034] Take 10 mL of silk fibroin solution with a concentration of 10 mg / mL, weigh 1% PEI (800 Da) of the mass of silk fibroin, dissolve it in 10 mL of phosphate buffer solution (50 mM, pH=6.5), and use 2 M Adjust the pH value of the HCl solution to 6.9, and then fully mix it with the silk fibroin solution; dissolve tyrosinase in phosphate buffer solution (50mM, pH=6.8), and prepare tyrosinase with a concentration of 2 mg / mL solution, slowly drop 100 uL of tyrosinase solution to the above mixed solution, and react in a water bath at 25°C for 12 h. The reaction solution was removed by Sephadex (G-15) column chromatography to remove unreacted PEI, and then the solution was centr...

Embodiment 3

[0038] Add the degummed silkworm silk fiber into the ternary solution (the molar ratio of calcium chloride: water: ethanol is 1:8:2), at 72 o C stirring and dissolving into a mixed solution of silk fibroin. Put the obtained silk fibroin mixed solution into a dialysis bag, and dialyze with deionized water for 4 days to obtain a purified silk fibroin solution.

[0039] Take 10 mL of silk fibroin solution with a concentration of 20 mg / mL, weigh 32% PEI (1800 Da) of silk fibroin mass, dissolve it in 10 mL of phosphate buffer solution (100 mM, pH=6.8), and use 2 M HCl solution to adjust its pH value to 6.0, then fully mix with silk fibroin solution, dissolve tyrosinase in phosphate buffer solution (50mM, pH=6.5), and prepare tyrosinase with a concentration of 1 mg / mL Acidase solution, slowly drop 100 uL tyrosinase solution to the above mixed solution, and react in a water bath at 25°C for 12h. The reaction solution was removed by Sephadex (G-50) column chromatography to remove un...

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Abstract

The invention discloses a cationic silk fibroin as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: modifying silk fibroin by using low molecular weight polyethyleneimine through a tyrosinase catalysis oxidation method, grafting PEI into tyrosine residue side chain of the silk fibroin, wherein the modified silk fibroin is positively charged under the physiological environment and is capable of producing electrostatic bonding with charged DNA to form cationic silk fibroin-DNA binary compound which can be used as a non-virus transgene delivery for gene transfection. The cationic silk fibroin provided by the invention is low in cell toxicity, the reaction condition of the preparation method is mild, the problem that the high molecular weight PEI is non-degradable, and the application prospect of the cationic silk fibroin in the gene therapeutic field is developed.

Description

technical field [0001] The present invention relates to a modified silk fibroin, a preparation method and its application, in particular to a cationic silk fibroin, a preparation method, and its use as a non-viral gene transfer carrier for gene transfection, which belongs to biomedical material technology field. Background technique [0002] Gene therapy is a treatment method that introduces foreign genes into target cells and expresses them effectively, so as to achieve the purpose of curing diseases. The exposed exogenous gene is negatively charged, it is difficult to penetrate the negatively charged cell membrane to reach the nucleus, and it is easily metabolized and inactivated in the body, making it difficult to express. Therefore, in the gene therapy system, the gene delivery carrier plays a decisive role in the transfection efficiency. The vectors currently used for gene delivery include viral vectors and non-viral vectors. Viral vectors have high transfection effi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08H1/00C12N15/63
Inventor 李秀芳李明忠尤仁传瞿静
Owner SUZHOU UNIV
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