Nano-composite vector for transfection, nucleic acid nano-drug as well as preparation method and application of nano-composite vector and nucleic acid nano-drug
A nanocomposite and nucleic acid nanotechnology, applied in the field of biomedicine, can solve the problems of limited application of siRNA delivery siRNA drugs, instability of lipid nanocarriers, etc., to improve transfection performance, avoid toxic side effects, and high treatment efficiency Effect
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Embodiment 1
[0044] Example 1: siRNA / tetrapiperazine epoxy C 60 Preparation of Derivative / Polythiophene Polymer Nucleic Acid Nanomedicine
[0045] (a) Take 1 mL of 1 mM tetrapiperazine epoxy C 60 Derivative (TPFE) aqueous solutions were added with different moles of 1mL polythiophene (PT), and the concentrations of thiophene monomers were 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM and 8mM PT aqueous solutions, and were fully mixed by a shaker , After standing for 3 minutes, use 0.22μm water-soluble filter membrane to filter, and finally obtain the molar ratio of TPFE and PT monomer as 1:1, 1:2, 1:3, 1:4, 1:5, 1: 6. 1:7, 1:8 nanocomposite carrier aqueous solution. Take 1mL of each solution and use the Zetasizer Nano ZSP instrument to measure the zeta potential of each nanocomposite carrier, see figure 1 . From the measurement results of the zeta potential, it can be seen that with the increase of the molar weight of PT, the ζ of the nanocomposite carrier gradually changes from positive to negat...
Embodiment 2
[0048] Embodiment 2: Nano-medicine and the hydration particle size detection of individual components
[0049] Take 1 mL of 0.01mM TPFE aqueous solution, PT aqueous solution with a polythiophene monomer molar concentration of 0.02mM, the nanocomposite carrier prepared in Example 1 of the present invention containing 0.01mM TPFE, and the nanocomposite carrier prepared in Example 1 of the present invention containing 0.01mM TPFE. Nucleic acid nano drug aqueous solution, adopt dynamic light scattering method (DLS) to measure the hydrated particle size of each material in ultrapure water, see figure 2 . Due to the electrostatic interaction and π-π interaction among the ternary components, the nucleic acid nanomedicine will self-assemble in water to form nanoparticles. The polydispersity of TPFE alone in water is high, and it fails to form stable and uniform nanoclusters. The average hydrated particle size of PT alone in water is 362.8nm, and the average particle size of nanocomp...
Embodiment 3
[0050] Embodiment 3: CCK-8 method detects the biosafety of nanocomposite carrier
[0051] The cytotoxicity of TPFE, PT and the nanocomposite carrier (T&P) compounded with TPFE: PT monomer molar ratio of 1:2 on mouse melanoma cells (B16F10) was detected by CCK-8 method.
[0052] 96-well plate, according to each well 1x10 4 When the confluence reached 40%-50%, add 200 μL of 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM of TPFE and 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM of PT and containing 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM TPFE nanocomposite carrier, the control group was added with 200 μL of PBS, and incubated under normal cell culture conditions for 24 hours after adding the drug. After 24 hours, the medium containing the drug was sucked away, and 100 μL of CCK-8 working solution diluted 10 times with colorless DMEM was added to each well. After incubation for 1 hour under normal cell culture conditions, a multifunctional microplate reader was used to measure t...
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