Small interfering RNA loading N-succinyl chitosan nanoparticle drug delivery system, preparation method and applications thereof
A technology of succinyl chitosan and delivery system, which is applied in the field of biomedicine, can solve the problems of low efficiency of transfecting cells and high dose, and achieve the effects of strong anti-tumor effect, high drug encapsulation rate and good dispersibility
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Embodiment 1
[0041] (1) Weigh 5g of raw material chitosan into a 250mL round bottom flask, add 50mL of 10M NaOH, stir evenly, and intermittently hydrolyze at 110°C for 3 hours (after 1 hour of hydrolysis, cool to room temperature, wash the product with distilled water until neutral , the Buchner funnel was connected to a vacuum pump to dry it, and then transferred to a round bottom flask, and reacted under the same conditions as before, and reciprocated twice), the resulting white precipitate was washed with distilled water until neutral, and then washed with absolute ethanol and anhydrous acetic acid respectively. Suction filtration three times, vacuum drying at 60°C to obtain high deacetylated chitosan.
[0042] (2) Dissolve 2 g of high chitosan in 100 mL of 2% acetic acid solution, and slowly add 6 mL of hydrogen peroxide after the temperature reaches 60° C., and stir for 20 min. When neutralized with dilute sodium oxide solution to weak alkalinity, there will be a large amount of white...
Embodiment 2
[0048] VEGF-siRNA is a chemically synthesized cholesterol-modified symmetric siRNA targeting vascular endothelial growth factor (VEGF) (sequence: sense strand 'chol-AUGUGAAUGCAGACCAAAGAA dTdT 3', antisense strand 3'dTdTUACACUUACGUCUGGUUUCUU 5') , the synthesized VEGF-siRNA powder was dissolved in DEPC water to make 20 μM; VEGF-aiRNA was a chemically synthesized cholesterol-modified asymmetric aiRNA targeting vascular endothelial growth factor (sequence: sense strand 'chol-GUGAAUGCAGACCAAAGAAdTdT 3', The antisense strand is 3'dTdTUACACUUACGUCUGGUUUCUU 5'), and the synthesized VEGF-aiRNA powder was dissolved in DEPC water to make 20 μM. (wherein, chol- indicates cholesterol modification); Bcl2-siRNA (sequence: sense strand 'chol-CGGAGGCUGGGAUGCCUUUdTdT 3', antisense strand 3'dTdTGCCUCCGACCCUACGGAAA 5') The synthesized Bcl2-siRNA powder was dissolved in DEPC water to prepare 20 μM .
Embodiment 3
[0050] Weigh 250 mg of N-succinyl chitosan and dissolve it in 50 mL of 2% acetic acid solution to prepare a 5 mg / mL N-succinyl chitosan solution, and filter through a #3 sand core funnel to obtain a clear solution without impurities. Take 500 μL of 0.22 μm filtered N-succinyl chitosan solution and 50 μL of 20 μM small interfering RNA solution, mix them in a 55°C constant temperature water bath, keep warm for 10 minutes, and the volume after mixing is about 550 μL. The mixture was stored in a refrigerator at 4°C for later use. 50 μL small interfering RNA solution and 500 μL N-succinyl chitosan solution were used as the preparation ratio of 1:1 N-succinyl chitosan nanoparticles loaded with small interfering RNA. Then prepare nanoparticles with ratios of small interfering RNA solution and N-succinyl chitosan of 4:1, 3:1, 2:1, 1:2, 1:3, 1:4 respectively. The condition of N-succinyl chitosan loaded with aiRNA was judged by agarose gel electrophoresis test. The result is as imag...
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