Synergistic Inhibition Of Tumor By Co-delivery Of Dasatinib And All-trans Retinoic Acid Via Self-assembled Nanoparticles

Melanoma is one of the most common and malignant tumors.It has a high degree of malignancy,rapid development,extensive metastasis and poor prognosis.In recent years,the incidence of melanoma increased and threatened to human health.The traditional chemotherapy drugs have low selectivity,high toxicity and poor tolerance.Therefore,it is urgent to develop a new targeted therapy drug with high selectivity,high efficiency and low toxicity.Studies have shown that the expression of tyrosine protein kinase s(TPKs)are abnormal in melanoma,suggesting that TPKs may be new therapeutic target s for melanoma.TPKs are important proteins that control cell proliferation,migration and apoptosis.And the cell signaling pathways are closely related to the occurrence and development of tumor.More than 50% of the proto oncogene and oncogene products have TPKs activities,the abnormal TPKs can cause disorganized cell proliferation and tumor occurrence.Tyrosine kinase inhibitors(TKIs)could inhibit proliferation,migration and induce apoptosis of tumor cells through cellular signal pathways.They can overcome poor selectivities,adverse reactions and have broad prospects.However,the development of tumors are complex and multifactor network systems.Inhibition of the single signal pathway could not suppress tumor development.Cancer stem cells(CSCs),the “proliferative stem cells” in the tumor,are the source of tumor occurance,development and metastasis.CSCs have the characteristics of self-renewal,multi-directional differentiation,high tumorigenicity and drug resistance.At present,surgical resection,radiotherapy and chemotherapy are the main treatments for malignant tumors.These treatments could largely eliminate or kill tumor tissue and cells,and prolong the survival of patients with cancer.However,because of the lack of targeting of CSCs,it can not really solve the recurrence,metastasis and poor prognosis of the tumors,and can not get the best treatment effect.Based on the cellular signal pathways of tumor cells and the physiological characteristics of CSCs,we designed and prepared the self-assembled nanoparticles to inhibit TPKs and kill CSCs.It could improve anti-tumor effect and reduce the risk of recurrence.The first chapter was the background and objective for co-delivery of dasatinib(DAS)and all-trans retinoic acid(ATRA),providing theoretic support for this study.The second chapter of this study,we investigated the effects of dasatinib on the proliferation,migration and apoptosis of murine melanoma cell line B16F10 in vitro.MTT assay demonstrated that dasatinib could inhibit the proliferation of B16F10 cells in dose-dependent and time-dependent manners.Wound healing assay showed that dasatinib could markedly inhibit the migration of B16F10 cells in vitro.The cell morphology were changed and the nuclear were fragmented by treating dasatinib for 24 h.The apoptosis rates of different concentrations of dasatinib in B16F10 cells were(34.06 ± 0.83)%,(50.24 ± 1.66)% and(88.91 ± 0.96)%.In the third chapter,DAS/ATRA-NPs were prepared by probe ultrasound and the optimal preparation process were selected by a single factor experiment.The morphology of the DAS/ATRA-NPs were observed by transmission electron microscopy(TEM).The morphology of the DAS/ATRA-NPs were regular ellipsoids,the sizes were(130.350 ± 1.829)nm,particles dispersion index(PDI)were(0.247 ± 0.013)and zeta potentials were(26.617 ± 0.939)m V.The encapsulation efficiencies of dasatinib and all-trans retinoic acid more than 80% were measured by ultrafiltration and ultracentrifugation.In PBS buffer solution containing 10% ethanol,the cumulative release of dasatinib and all-trans retinoic acid in DAS/ATRA-NPs were detected less than the free drugs by dialysis.The sizes,PDI and zeta potential of the DAS/ATRA-NPs were not obviously changed at 4 ? for a month.In the fourth chapter,we studied the anti-tumor effect of DAS/ATRA-NPs in vitro.DAS/ATRA-NPs were labeled with coumarin-6.The fluorescence intensity of coumarin-6 were dectected by flow cytometry in dose-dependent and time-dependent manners.The DAS/ATRA-NPs localized in the cell cytoplasm.MTT assay showed that dasatinib and all-trans retinoic acid were able to inhibit the proliferation of B16F10 cells.DAS/ATRA-NPs had synergistic effects between dasatinib and all-trans retinoic acid.Wound healing assay and Transwell chamber assay demonstrated that the migration of the B16F10 cells were changed after the drug administration.The DAS/ATRA-NPs could effectively inhibit the migration of the cells,which were consistent with the anti-proliferation results.Cell apoptosis or death is an important factor in the treatment of tumor,the treated cells showed obvious chromatin condensation,nuclear membrane rupture and nuclear fragmentation.The results of flow cytometry demonstrated that DAS/ATRA-NPs could induce cell apoptosis and promote cell death.The cell apopto tic and dead rates were significantly higher than DAS group,ATRA group and DAS+ATRA group(P < 0.05).Then,this chapter made a preliminary investigation of the co-operative mechanism of DAS and ATRA in DAS/ATRA-NPs.Sphere formation experiment and side population cells(SP)experiment are used to detecting stem cells in vitro.Sphere formation experiment showed that B16F10 cells were able to form a sphere by the certain conditions,which had strong tumorigenicity and like cancer stem cells properties.DAS/ATRA-NPs could inhibit the sphere formation of B16F10 cells.DAS/ATRA-NPs also could significantly inhibit the like CSCs properties of B16F10 cells in side population cells experiment.Finally,dasatinib could inhibit the phosphorylation of TPKs.The expression of SRC P-Y418 and SRC in the B16F10 cells were detected by Western blot.And the expression of SRC P-Y418 in the cells were decreased after the drug administration.To sum up,DAS/ATRA-NPs could synergistically supress tumor via inhibiting like CSCs properties and the phosphorylation of TPKs.In the fifth chapter,the distribution of DAS/ATRA-NPs in vivo were studied.Subcutaneous tumor models were established in nude mice by inoculated with 5 × 106 cells.DAS/ATRA-NPs were labeled with DiD.IVIS Spectrum imaging system was used to dectect the distribution of DAS/ATRA-NPs in tumor bearing mice via tail vein injection.It showed that DAS/ATRA-NPs mainly distributed in liver,spleen and tumor.Tumor bearing mice were sacrificed at 2 h and 24 h,and tumor tissues were made into frozen sections.The tissue sections were observed by confocal laser scanning microscope.The results showed the Di D labeled DAS/ATRA-NPs were enriched in the tumor,and they had tumor targeting.There were few red fluorescence intensity in the tumor tissue after 24 h.In summary,this study explores a new way of tumor targeted therapy and enriches the theory of tumor cell therapy.The self-assembled nanoparticles have multifunctional anti-tumor effects and are expected to providing a new method for melanoma targrted therapy.