Application of cationic magnetic nano-material as nucleic acid delivery vector, and application method
A magnetic nanometer and delivery carrier technology, applied in the field of nanomaterials and molecular biology, can solve the problems of high cytotoxicity and restrict the wide application of materials, and achieve the effects of low cytotoxicity, easy operation and simple preparation method
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Embodiment 1
[0043] This embodiment provides a preparation method of cationic magnetic nanomaterial G3-S-MNPs, comprising the following steps:
[0044] (1) Using ethylenediamine and methyl acrylate as raw materials, methanol as a solvent, adding reactants with a molar ratio of ethylenediamine and methyl acrylate of 1:4, and performing Michael addition reaction. After the reaction is complete, the product is purified by distillation under reduced pressure to remove the solvent and unreacted reactants, add distilled water as a solvent, and repeat the Michael addition reaction and amidation reaction three times to obtain the third-generation polyamide-amine (PAMAM) Dendrimers, referred to as PAMAM-G3;
[0045] (2) Disperse the ferroferric oxide magnetic nanomaterial in the aqueous solution, the wet weight is about 10%, that is, the mass volume ratio of the ferroferric oxide magnetic nanomaterial to water is 10%, add sodium hydrogen tartrate solution, and ultrasonicate for 4 hours , so that s...
Embodiment 2
[0051] This embodiment is to analyze the cytotoxicity of G3-S-MNPs in Example 1 as a nucleic acid delivery carrier, the steps are as follows: collect human lung cancer A549 cells or human breast cancer MCF-7 cells in the logarithmic growth phase, according to 5000 cells per well Cells were inoculated into a 96-well plate at a density of three cells, and three replicate holes were set in each group; they were placed in a cell incubator and cultivated for about 24 hours until the cells grew to about 70% to 80%; according to the concentration gradient (0,5,10, 20, 30, 40, 50, 75, 100 μg / mL) were added to the prepared material samples, and then continued to incubate for 46 hours; 10 μL of MTT (5mg / mL) solution was added to each well, and continued to incubate in the incubator for 2 hours; then aspirate Add 150 μL of dimethyl sulfoxide (DMSO) to each well to fill up the culture medium in the well, shake at a low speed for 10 minutes, and read the absorbance value of each well at 490...
Embodiment 3
[0055] This example is to test the in vitro nucleic acid binding ability of the G3-S-MNPs in Example 1 as a nucleic acid delivery carrier.
[0056] Flag-CHIP plasmid (expressing Hsp / Hsc70 interacting protein CHIP) DNA and CIP2A gene (cancer suppressor of protein phosphatase 2A) siRNA were used as target nucleic acids to illustrate G3-S-MNPs magnetic nanomaterials as nucleic acid delivery Carrier efficiency.
[0057] In vitro DNA binding assay steps are as follows:
[0058] The amount of immobilized plasmid is 1 μg, according to the gradient of 0.5:1, 1:1, 5:1, 10:1, 20:1 according to the mass ratio of material to plasmid, the binding system is 100 μL pure DMEM medium, at room temperature Incubate for 30 minutes. Then, 10 μL of the sample was taken for agarose gel electrophoresis, and a pure plasmid sample was set as a control, and the in vitro binding effect was judged according to the migration stagnation of the bands.
[0059] The steps of the in vitro siRNA binding exper...
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