Preparing process and application of nano chitosan particle

A technology of chitosan nanoparticles and chitosan, which is applied in the field of preparation of chitosan nanoparticles, can solve the problems of complex preparation, low packaging efficiency, and small carrier capacity, and achieve good biocompatibility and high transfer efficiency , the effect of easy operation

Inactive Publication Date: 2007-06-27
WUHAN UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Its main disadvantage is that foreign gene expression is transient and multiple inputs can easily cause immune response
[0008] Adeno-associated virus is a defective provirus, which is non-pathogenic to humans. One of the B19 viruses can specifically integrate into human chromosome 19, thereby improving the safety of the gene; but its carrier capacity is small and cannot exceed 5kb, and the packaging efficiency is low, and the preparation is complicated, so its application is limited
[0009] Virus systems, including RNA viruses and DNA virus vectors, have high transfer efficiency, but they are limited in human applications due to infection, immune abnormalities, and possible carcinogenesis

Method used

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  • Preparing process and application of nano chitosan particle
  • Preparing process and application of nano chitosan particle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 uses chitosan nanoparticle to prepare non-viral vector to mediate the expression of human insulin gene

[0074] main reagent

[0075] E.Coli TOP10F' strain was purchased from Invitrogen Company of the United States; ultrapure plasmid miniprep kit and ultrapure plasmid midiprep kit were purchased from Hangzhou V-Gene Company; NIH3T3 cells were purchased from Wuhan University Typical Plant Collection Center; 6 Well culture plates were purchased from Dunk Company of Denmark; chitosan was purchased from Shanghai Boao Biotechnology Co., Ltd.; kanamycin and ampicillin were purchased from Promega Company; G418 was purchased from ALEXIS Company in the United States; From Shanghai Boda Bioengineering Company; calf serum, DMEM, and trypsin were purchased from Gibco Company in the United States; mouse anti-human insulin antibody was purchased from Maixin Company in Fuzhou City, Fujian Province; tryptone and yeast extract were purchased from Difco Company in the United...

Embodiment 2

[0192] Example 2 Using Chitosan Nanoparticles to Prepare Non-Viral Vectors to Mediate the Expression of Human Insulin Gene

[0193] 1. Construction and proliferation of expression plasmids

[0194] The cDNA fragment encoding human insulin was excised from PBAT16, hlnsG1.M2 with Bgl II / NotI, inserted into expression vector pCMV with cohesive ends, transformed into TOPIOF / bacterial strain (Invitrogen company product), screened for recombinants, and the recombinant plasmid was marked as pCMV.Ins . The pCMV and pCMV.Ins plasmids were respectively transformed into competent bacteria Top10F' and multiplied.

[0195] 2. Plasmid extraction and purification

[0196] The pCMV and pCMV.Ins plasmids were prepared in large quantities by alkaline lysis and purified by polyethylene glycol precipitation.

[0197] 3. Preparation of Chitosan Nanoparticle Carrier

[0198] Chitosan (Shanghai Bo'ao Biotechnology Co., Ltd. purification) is slightly heated and dissolved in 1% acetic acid, and t...

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Abstract

The present invention discloses preparation process and application of nanometer chitosan particle as one kind of non-viral gene transferring carrier. The preparation process is the following steps: 1. treating chitosan into chitosan solution through dissolving in acetic acid, regulating pH value, filtering membrane sterilizing and diluting with abacterial pure water; 2. treating DNA solution through dissolving 50-150 mg/l concentration DNA solution in 100 microliter in sodium sulfate solution; and 3. mixing the DNA solution and the chitosan solution in water bath through eddy flow vibration, letting stand at room temperature for 2 hr, packing and freeze drying. The nanometer chitosan particle may be used to carry several kinds of target gene for genetic treatment of diabetes. The present invention has high biocompatibility, non-toxicity, low cost and other advantages.

Description

technical field [0001] The invention relates to a non-viral gene transfer carrier—chitosan nanoparticle, in particular to a preparation method and application of the chitosan nanoparticle. The nanoparticle is safe and non-toxic, has economical sources, has the advantages of high transfection efficiency, easy operation and the like, and can carry multiple target genes. Background technique [0002] A very important reason restricting the development of gene therapy is the lack of a safe and effective gene delivery system, that is, gene transfer vectors. [0003] At present, there are more than ten kinds of gene transfer technologies that can transfer foreign genes into mammalian cells, which are generally divided into three categories: physical methods, chemical methods and biological (virus) methods. [0004] Physical and chemical methods have developed rapidly in recent years. The commonly used methods are electric shock, particle bombardment, calcium phosphate transfer an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J3/12C08L5/08A61K47/36
Inventor 徐焱成朱宜莲孙家忠牛力
Owner WUHAN UNIV
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