Biological nano drug delivery system for precisely targeting lung tumor cells and preparation method and application thereof
A bio-nano and drug delivery system technology, applied in the field of biomedicine, can solve the problems of poor biocompatibility and low toxicity of biocompatibility, achieve low controlled release, good water solubility, and improve the effect of treatment
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Embodiment 1
[0038] (1) Extraction of exosomes from lung tumor A549 cells:
[0039] (1) A549 cells were taken out and revived in liquid nitrogen, and strictly aseptically operated;
[0040] (2) After the cells have been passed on for 4-5 generations, culture the cells on a flat plate and medium without serum exosomes. After the cells grow to 80% (the cell growth surface area accounts for about 80% of the bottle), the supernatant is collected;
[0041] (3) Use differential centrifugation to remove large molecular weight substances from the supernatant. The parameters of differential centrifugation are set to 4°C, 300*g, 10min; 4°C, 2000*g, 10min; 1000*g, 30min;
[0042] (4) Sterilize the centrifuged supernatant with a filter membrane with a pore size of 0.22 μm; use an ultrafiltration tube with an interception capacity of 100 KDa to concentrate the supernatant; use an ultracentrifuge to separate all exosomes, and ultracentrifuge The parameters are set to 4°C, 100000*g, 70min;
[0043] (5...
Embodiment 2
[0056] (1) The extraction of exosomes from lung tumor A549 cells was the same as in Example 1;
[0057] (2) Importing drugs into exosomes
[0058] (1) The anti-tumor drug gefitinib was dissolved in DMSO, and the drug and exosomes were mixed in the electroporation buffer at a mass ratio of 1:10;
[0059] Steps (2)-(4) are the same as in Example 1;
[0060] (3) Detection of Exosome Drug Loading Example 1
[0061] The exosome nano drug delivery system obtained in Example 2 has a drug loading capacity of 0.0556 μg / μg, and the exosome drug delivery system has a good sustained release time of the drug, which can reach 72 hours, and has a good controlled release and sustained release effect. The detection of the marker protein by Western blot showed that no damage was caused to the marker protein.
Embodiment 3
[0063] (1) The extraction of exosomes from lung tumor A549 cells was the same as in Example 1;
[0064] (2) Importing drugs into exosomes:
[0065] (1) The anti-tumor drug gefitinib was dissolved in DMSO, and the drug and exosomes were mixed in the electroporation buffer at a mass ratio of 1:5; the anti-tumor drug was dissolved in a solvent.
[0066] Steps (2)-(4) are the same as in Example 1;
[0067] (3) Detection of drug loading in exosomes according to Example 1;
[0068] The exosome nano drug delivery system obtained in Example 3 has a drug loading capacity of 0.0553 μg / μg. The exosome drug delivery system has a good sustained release time of the drug, which can reach 72 hours, and has a good controlled release and sustained release effect. The detection of marker protein by Western blot showed that no damage was caused to the marker protein.
[0069] From Figure 1-4 It can be seen that the morphology of exosomes before and after drug loading did not change greatly, ...
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Abstract
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