Construction Of Luteolin And MicroRNA-199a-3p Co-transport Nanosystem And Estimation Of Its Anti-breast Cancer Effect

Objective: To construct a co-transport nanosystem of luteolin(Lut)and microRNA-199a-3p(miR-199a-3p)using cationic liposome CDO14 as a vector and investigate the effect and mechanism of co-transport nanosystem on human breast cancer cells MCF-7and MDA-MB-231 in vitro and in vivo.Methods:(1)Drug-loaded liposome CDO14/Lut was prepared by loading Lut with cationic liposome CDO14.Drug and gene co-transport nanosystem CDO14/Lut/miR-199a-3p was constructed by complexing miR-199a-3p with CDO14/Lut.The particle size,zeta potential and stability in the PBS buffer of liposomes and co-transport nanosystem were detected by laser particle size analyzer,and their morphology was observed by transmission electron microscopy(TEM).The encapsulation efficiency and drug loading of liposomes were detected by UV-Vis spectrophotometer.The cytotoxicity of drugloaded liposomes to human breast cancer cells MCF-7 and MDA-MB-231 were detected by CCK-8 assay to determine the optimal ratio of drug to lipid in CDO14/Lut.(2)CCK-8 assay,DAPI staining,AO/EB staining and cell scratch experiments were respectively used to investigate the effects of co-transport nanosystem on the proliferation,morphological change,apoptosis and migration of MCF-7 and MDA-MB-231 cells.Cell uptake and RT-qPCR experiments were used to investigate the ability of the liposomes to carry genes.The effect of co-transport nanosystem on the expression of miR-199a-3p targeting protein mTOR and apoptosis-related protein BAX was detected by Western blot.(3)BALB/c-nude mice were inoculated with MDA-MB-231 cells in the armpits to construct a xenograft tumor model.Liposomes and co-transport nanosystem were injected via tail veins every other day for seven times.Mice were sacrificed two days after the last administration.The anti-tumor effect and safety of the co-transport nanosystem were evaluated by tumor volume,weight,blood biochemical indexes,organ indexes and histopathological sections of mice.Results:(1)The particle size of the prepared blank liposome,drug-loaded liposomes and co-transport nanosystem were all between 90～210 nm,and TEM showed they presented good sphericity.The zeta potential of blank liposome,and drug-loaded liposomes were between 25～40 m V which decreased greatly after loading genes.The particle size of liposomes and co-transport nanosystem in PBS did not change significantly within 14 days,indicating good stability.When the molar ratio of CDO14:DOPC:Lut was 35:30:35,the encapsulation efficiency and drug loading of CDO14/Lut were higher and the anti-proliferation effect on tumor cells was the best.So liposomes were prepard at this ratio for follow-up experiments.(2)The results of CCK-8showed that Lut loaded with liposome CDO14 could enhance the anti-proliferation effect on tumor cells and reduce its dosage,and CDO14 had no significant effect on cell toxicity at the administration dose.The anti-proliferation effect was further enhanced by using cotransport nanosystem.The results of DAPI staining and AO/EB staining showed that the co-transport nanosystem promoted apoptosis of the tumor cells more strongly than drugloaded liposome.Cell scratch experiments showed that co-transport nanosystem had stronger effect on inhibiting tumor migration than drug-loaded liposome.Cell uptake experiments and RT-qPCR results confirmed that liposome had good ability of genes delivery,leading to high expression of miR-199a-3p in breast cancer cells.Western blot results showed that miR-199a-3p in the co-transport nanosystem could downregulate the expression of mTOR protein in cells,and inhibit cell proliferation and migration.Lut and miR-199a-3p could synergistically promote BAX expression and promote apoptosis in cells.(3)Tumor suppression experiments in mice showed that the co-transport nanosystem had the strongest tumor inhibition effect,and it did not affect the blood biochemical indexes and main organs of the mice,indicating it had high safety.Conclusion: The co-transport nanosystem CDO14/Lut/miR-199a-3p was successfully constructed.The co-transport nanosystem could inhibit tumor proliferation,migration and promote apoptosis in vitro and inhibit the growth of xenograft tumor in mice with high safety.