Chitosan-DNA nanometer granule complex and preparation method thereof

A nanoparticle and chitosan technology, which can be used in pharmaceutical formulations, genetic material components, gene therapy, etc., can solve problems such as low encapsulation efficiency, poor storage stability, and application limitations, and achieve endocytosis promotion and low cytotoxicity Effect

Inactive Publication Date: 2011-10-05
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-viral vectors include liposomes and cationic complexes. Although liposomes are currently widely used in cell transfection, their encapsulation efficiency is low, storage stability is poor, and they are easily removed by blood components, making them useful in the whole organism. App is restricted

Method used

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  • Chitosan-DNA nanometer granule complex and preparation method thereof
  • Chitosan-DNA nanometer granule complex and preparation method thereof
  • Chitosan-DNA nanometer granule complex and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment one: the preparation of chitosan nanoparticle DNA complex CNPs-DNA

[0026] (1) Dissolve 20mg of purified chitosan in 10% acetic acid and heat it slightly (do not exceed 42°C). After the chitosan particles are completely dissolved, roughly adjust the pH to 5.5 with 10M NaOH solution to obtain 0.2 % (w / v) chitosan stock solution, sterilized by filtration through a 0.22 μm filter membrane.

[0027] (2) Take an appropriate amount of chitosan stock solution and dilute it into a chitosan solution with a concentration of 0.08% (w / v), adjust the pH to 6.5-7.0 with 10M NaOH solution, and filter through a 0.22μm filter membrane for future use .

[0028] (3) Dilute the plasmid DNA (pEGFP-C1) with distilled water to a DNA solution of 400 μg / ml, add 1 / 10 volume of 500mM Na 2 SO 4 solution (to a final concentration of 50mM).

[0029] (4) Chitosan solution and plasmid solution were placed in a constant temperature incubator at 55°C for 30 min. After mixing equal volum...

Embodiment 2

[0030] Embodiment 2: Determination of the physical properties of CNPs-DNA

[0031] 1. Gel electrophoresis retardation and DNase I digestion experiment: please specify the specific steps of gel electrophoresis retardation digestion experiment,

[0032] (1) Gel electrophoresis retardation experiment: CNPs-DNA was subjected to 1.0% agarose gel electrophoresis at 60V for 40min. Since the negative charge of the DNA itself was covered by the chitosan-wrapped DNA, it could not move from the negative pole to the negative pole in the electrophoresis. The positive electrode moves and is blocked in the loading hole.

[0033] (2) DNaseⅠdigestion experiment: add 0.5U DNaseⅠ (1U / ul) to CNPs-DNA, incubate at 37°C for 1h, electrophoresis on 1.0% agarose gel at 60V, 40min, the wrapped DNA stays in the sample well , the unencapsulated DNA was digested and degraded by DNase I. In this way, the protective ability of chitosan nanoparticles to DNA was judged. see figure 1 and figure 2 , indi...

Embodiment 3

[0037] Example 3: Effect of CNPs-DNA with different N / P on transfection efficiency:

[0038] Apply CNPs-DNA to mediate the transfection of pEGFP-C1 to HEK293T cells, the specific steps are:

[0039] After recovery, the cells were subcultured, and the logarithmic growth phase was reached in about three generations (at this time, the cells grew vigorously and divided at the fastest speed), and the cells with good growth status were selected for testing. Count the cells after digesting them according to the previous cell passage steps, transfer them to a 24-well plate for culture, and the number of cells per well is about 5'105. After culturing for 16-24 hours, observe that the cells are uniformly attached to the wall and the density reaches 80%-90%, and then the transfection experiment can be performed.

[0040] (1) Remove the medium, carefully add PBS to wash the cells twice (do not damage or blow up the cells);

[0041] (2) Set up a blank control well, and only add complete ...

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Abstract

The invention relates to a chitosan-deoxyribonucleic acid (DNA) nanometer granule complex and a preparation method thereof. The complex is formed by combining NH3<+> converted from amino protons on chitosan with phosphate groups on sliverlight protein granule DNA under the static action, wherein the molar ratio of N to P is 4 to 8. The complex can protect DNA from being degraded by nuclease and generate a static adsorption effect together with electronegative cell membranes to promote the endocytosis of cells and realize the transfection of target genes. Due to low cytotoxicity, the complex can be used as an excellent gene vector for gene transfection. Therefore, the transfection of the target genes is realized, and a forceful condition is provided for the application of gene therapy.

Description

technical field [0001] The invention relates to a chitosan-DNA nano particle complex and a preparation method thereof. Background technique [0002] Gene therapy has been one of the hot spots of people's attention. It can treat genetic and non-genetic diseases by filling in missing genes, replacing defective genes or silencing unwanted genes. However, the naked gene is easily degraded by nucleases in vivo, and the transfection efficiency is very low. Therefore, the development of safe and efficient gene carriers has become a prerequisite for the success of gene therapy. In recent years, the main gene carrier systems are divided into two types: viral and non-viral vectors. Although viral vectors have high transfection efficiency in vivo, there are possible risks of toxicity, immune response and inflammation. Non-viral vectors include liposomes and cationic complexes. Although liposomes are currently widely used in cell transfection, their encapsulation efficiency is low, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/36
Inventor 文铁桥霍一楠刘芳
Owner SHANGHAI UNIV
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