Application of Pegylated Chitosan as Nucleic Acid Carrier
A technology of polyethylene glycol and chitosan, applied in the direction of introducing foreign genetic material using carriers, other methods of inserting foreign genetic material, gene therapy, etc., can solve the problem of insufficient transfection efficiency of transfection reagents, affecting application, Cytotoxic side effects and other issues, to achieve enhanced biocompatibility, low toxicity, high transfection efficiency
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Embodiment 1
[0044] Embodiment 1, preparation and identification of pegylated chitosan
[0045] 1. Preparation of PEGylated Chitosan
[0046]
[0047] Take 1.5g of chitosan, dissolve it in 150ml (pH=2) acetic acid aqueous solution, stir until completely dissolved, transfer the chitosan acetic acid aqueous solution to a three-necked flask, add 0.12g of sodium perborate, control the temperature at 60°C, and stir at a high speed. After reacting for 5 hours, add sodium hydroxide solution to adjust the pH value of the reaction system to 11, control the temperature at 4-5°C, add 10ml of ethylene oxide to the reaction system several times, continue to control the temperature at 60°C, and stir at 1400r / min for 70h. 20-40nm highly water-soluble O-hydroxyethyl chitosan self-assembled nanoparticles were obtained. The highly water-soluble O-hydroxyethyl chitosan self-assembled nanoparticle solution was placed in a dialysis bag, stirred and dialyzed for 12 hours; after freeze-drying, the O-hydroxye...
Embodiment 2
[0054] Embodiment 2, the preparation of pegylated chitosan / plasmid DNA complex particle
[0055] The preparation process of PEGylated chitosan / plasmid DNA complex particles is as follows:
[0056] (1) Take the PEGylated chitosan nucleic acid carrier (O-hydroxyethyl chitosan self-assembled nanoparticles) prepared in Example 1, dissolve it in water, and prepare an aqueous solution with a concentration of 2 mg / mL. A 0.22 μm microporous membrane was filtered to obtain a homogeneous and sterile aqueous solution of PEGylated chitosan, ready for use.
[0057] (2) Take the plasmid pEGFP-C1 and prepare it with water to make an aqueous solution with a concentration of 0.2 mg / mL. The PEGylated chitosan aqueous solution prepared in step (1) and the plasmid DNA aqueous solution were mixed in equal volumes to obtain a transfection complex carrier solution with a mass ratio of 10:1.
[0058] (3) Place the transfection complex carrier solution obtained in step (2) at room temperature (25°C)...
Embodiment 3
[0061] The cytotoxicity detection of the pegylated chitosan prepared by embodiment 3, embodiment 1
[0062] The MTT method was used to detect the cytotoxicity of the PEGylated chitosan carrier prepared in Example 1. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT (yellow dye) to water-insoluble Violet-blue formazan crystallizes and deposits in cells, whereas dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 490nm with an enzyme-linked immunosorbent detector, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. The specific operation is as follows:
[0063] set the density to 1.0x10 5 cells / ml HeLa cell suspension was inoculated in 96-well culture plate, 100 μl per well, after 12 hou...
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