Construction Of Salinomycin And IR780-loaded Nanoparticle Drug Delivery System And Preliminary Anti-Liver Cancer Study

Liver cancer is a malignant cancer with high morbidity and mortality.Traditional treatments usually fail to eliminate liver cancer completely,and the tumor is easy to relapse and metastasize after treatments.Liver cancer stem cells(LCSCs)are a subpopulation of liver cancer cells with ability of self-renewal and differentiation.Studies have shown that LCSCs have resistance to radiotherapy and chemotherapy and are closely related to the recurrence and metastasis of liver cancer.The existence of LCSCs may be an important reason for incomplete elimination of liver cancer after treatment.Therefore,the treatment strategy based on LCSCs may have important significance for preventing recurrence and metastasis of liver cancer.Salinomycin(SAL)is a kind of drug that can selectively inhibit the growth of many kinds of cancer stem cells,including LCSCs,and cancer cells with resistance to traditional chemotherapy drugs.IR780 iodide(IR780)is a new photosensitive reagent with excellent near-infrared(NIR)light absorption and photothermal conversion ability and is safe and controllable to be used in photothermal therapy(PTT)of cancers in recent years.However,salinomycin and IR780 are hydrophobic reagents,and the poor water solubility limits their clinical application.Therefore,it’s eager to develop an appropriate drug delivery system to improve the treatment efficacy of salinomycin and IR780 in clinic.This thesis aims to construct a nanoparticle drug delivery system based on upconversion nanoparticles(UCNPs),using distearoyl phosphorethanolamine-polyethylene glycol(DSPE-PEG)to encapsule salinomycin and IR780 to combine chemotherapy and PTT.The preliminary antitumor effect of the nanoparticle drug delivery system were evaluated in vitro.The main research contents and results are as follows:(1)Enrichment and identification of liver cancer stem cellsTumor sphere cells were enriched from human hepatocarcinoma cells HCCLM3using serum-free suspension culture.The results of flow cytometry showed CD90 and CD133 expression of tumor sphere cells were significantly up-regulated.Q-PCR result showed up-regulation of Nanog,Sox2,Oct4,CD133,CD44 and Ep CAM expressions in tumor sphere cells.Transwell assay demonstrated a stronger migration ability of tumor sphere cells.Colony forming experiment showed that tumor sphere cells formed more colonies than HCCLM3 cells,indicating the successful enrichment of LCSCs.(2)Construction and characterization of nanoparticle drug delivery systemUCNPs,salinomycin and IR780 were encapsuled by DSPE-PEG to construct a nanoparticle drug delivery system.Scanning electron microscope(SEM)and transmission electron microscopy(TEM)studies showed that the average particle size of UCNPs was 31.6 nm.Fourier transform infrared spectroscopy(FTIR)of the nanoparticle was consistent with DSPE-PEG and the Zeta potential of nanoparticles changed from-5.15 m V to-9.49 m V,indicated the successful capsule of nanoparticle by DSPE-PEG.Ultraviolet(UV)absorption spectrum showed new absorption peaks of the nanoparticle drug delivery system at 290 nm and 780 nm,indicated the successful loading of salinomycin and IR780.The concentration of salinomycin released from the drug-loaded nanoparticle solution reached 8μM in 48 h.Under the irradiation of1W/cm~2 NIR light,the temperature of the solution of the nanoparticle was increased about 8℃in 5 minutes.(3)Evaluation of in vitro anti-liver cancer effect of nanoparticle drug delivery systemCCK-8 assay showed that the nanoparticle without drugs had no effect on cell viability,indicated the good biocompatibility of the nanoparticle.The drug-loaded nanoparticle with 8μM salinomycin significantly down-regulated viability of liver cancer cells to 40%of the control group,and the cell viability was further down-regulated to 10%under irradiation of NIR light,which indicated that the combination of chemotherapy and PTT could further improve the inhibitory effect of the salinomycin-loaded nanoparticles on liver cancer cells.The drug-loaded nanoparticle with 2μM salinomycin significantly down-regulated viability of LCSCs to 25%of the control group,but had no effects on cell viability of LCSCs after irradiation by different power of NIR light,indicated that the thermal sensitivity of LCSCs may be different from those of liver cancer cells.The drug-loaded nanoparticle with 8μM salinomycin significantly down-regulated viability of LCSCs to 10%of the control group,indicated that salinomycin-loaded nanoparticles had stronger inhibitory effect on LCSCs.