Preparation And Antitumor Effect Of Her2-targeted Delivery Of Shikonin Monomer Drug Nanomicelles

ObjectiveClinical studies have proved that many traditional Chinese medicines(TCMs)have obvious anti-tumor effects.In recent years,many researchers have reported that Shikonin,a source of Chinese herbal medicine,has a good anti-tumor effect.However,our previous studies have found that shikonin has low water solubility and poor targeting in vivo,which makes its clinical application difficult.In order to improve the specific targeting of shikonin to tumors,enhance intratumoral enrichment,and increase the degree of utilization,In this study,a temperature-sensitive block copolymer(Block Copolymer Micelles)was synthesized,which was self-assembled to form a shikonin-containing and temperature-sensitive nanomicelle(STN).In addition,studies have shown that tumor tissue is warmer than normal tissue(3-5?),and the delivery system is temperature sensitive and can passively target tumor tissue;Meanwhile,human epidermal growth factor receptor 2(Her-2)is a surface overexpressing antigen of human breast cancer Skbr-3 cells.Therefore,the anti-Her2 antibody is attached to the surface of STN to make it active for tumor tissue.The active and temperature-sensitive passive dual-target nano-medicine delivery system(Shikonin-loaded Dual-targeting Nanomicelle,SDN)is prepared by two techniques;and further evaluated the properties of the dual-targeted nanomicelles coated with shikonin and the anti-tumor mechanisms in vitro.Method:First,a standard curve of shikonin absorbance was prepared,which was used to calculate the content of shikonin in the polymer preparation containing shikonin.Then,the morphology of PNIPAN140-PDPA70-PDEA70 in water is adjusted by dialysis technology to form a high molecular weight polymer coated with shikonin.At the same time,the Her-2 antibody was ligated on the surface of the nanomicelle,and the double-targeted nanomicelle SDN loaded with shikonin was prepared to make it sensitive to temperature and passive targeting of the tumor tissue.The particle size of the prepared SDN was measured by a Dynamic/Static Light Scattering Instrument(DLS),and the morphology of the SDN was measured by a Transmission Electron Microscope(TEM).In an in vitro experiment,the serum stability of SDN was determined by measuring the particle size of the mixed SDN and BSA.Secondly,the laser scanning confocal microscopy(LSCM)was used to observe the endocytosis and enrichment of FITC-coated double-targeted nanomicelles(FDN)by breast cancer Skbr-3 cells.In addition,the effect of SDN on the killing of Skbr-3 and MCF-7 cells was evaluated by CCK-8 assay,and the temperature sensitivity and antibody targeting of SDN were verified.Finally,the effect of SDN on programmed cell death of Skbr-3 cells was examined by flow cytometry.Result:This subject successfully synthesized SDN.The results showed that SDN was spherical and had a complete core-shell structure with good serum stability,a potential of-6 mv and a particle size of 127 nm.LSCM shows that SDN can enrich tumor cells and promote endocytosis of cells.The CCK-8 toxicity experiment showed that the blank nanomicelle had a low killing effect on Skbr-3 cells,and SDN had a more killing effect on Her-2 highly expressed cells.At the same time,at 42 °C,SDN has better killing effect on cells,which proves that SDN has temperature sensitivity.Next,the effect of SDN on programmed cell death of Skbr-3 cells was further examined.The results showed that the programmed mortality of the antibody group was the highest,significantly higher than that of the BSA group and the single drug group.Conclusion:The dual-targeted nano-micelle SDN synthesized by this subject has dual targeting functions of temperature passive targeting and active antibody targeting.It has a complete spherical core-shell structure,stable potential,high drug loading,good serum stability,sustained release of drugs,killing Skbr-3 cells in vitro,and promoting endocytosis and programmed cell death.