Experimental Therapy Of Melanoma By Transferrin Modified Platelets Loading With Doxorubicin

Melanoma is a common malignant tumor in skin cancer.In recent years,the incidence of melanoma has increased gradually due to the combined influence of environmental pollution,ultraviolet radiation,family history,dysplasia of mole,age and many other factors.Malignant melanoma is insensitive to chemotherapy drugs,and it has drug resistance and toxicity,it is necessary to explore new treatments for melanoma and targeted therapy is a hot topic in melanoma research at present.In recent years,the roles of platelet in tumor microenvironments have attracted more and more attention of researchers.While mainly confined to the blood,platelets can gain access to the tumor microenvironment via platelet-tumor and platelet-stromal interactions,Platelets can be recruited to shroud tumor cells shielding them from immune responses and promoting the proliferation and migration of tumor cells.OBJECTIVE To study the targeting and Experimental Therapy of Melanoma by Transferrin Modified Platelets Loading with Doxorubicin.METHODS This study constructed a platelet based drug delivery system by loading DOX in platelet cytoplasm and modifying the platelet surface with transferrin.Fluorescence microscope and flow cytometry were used to study the uptake and apoptosis analysis of platelet carriers by B16F10 cells.Tumor-bearing mice were randomly divided into four groups,5 mice in each group,including control group,DOX group,P-DOX group and Tf-P-DOX group,respectively.Near-infrared technology was used to monitor the distribution of near-infrared fluorescence in tumor-bearing mice,HE staining was used to observe the histological changes,and immunohistochemistry was used to study the effect of drug-loaded platelet carriers on caspase protein expression in mice tumor.RESULTS 1.Preparation and Characterization of Tf-P-DOX.The platelet-based drug delivery system was constructed by loading of DOX in platelet cytoplasm and modification with transferrin on platelet membrane surface.The morphology of platelets was observed under transmission electron microscopy(TEM),platelets without aspirin show many synapses that tend to aggregate to form thrombosis,and after adding aspirin,it form isolated pellets.The platelet distribution was uniform and the size did not change obviously after loading drug,indicating the stability of platelet carrier after drug loading.The loading of DOX in platelets and the binding of FITC-labelled transferrin on platelet membrane surface were validated using fluorescence microscope.The co-localization images indicate the transferrin was coupled successfully to the surface of platelets.At the DOX concentration of 0.05 mmol/l,the encapsulation efficiencies of P-DOX reached to 64.9%.The release of DOX from PDOX gradually increased in a time-dependent manner at pH 6.5 and pH 7.4 respectively,and more than 50% DOX was released from P-DOX at pH 6.5.2.Cell uptake and apoptosis analysis of B16F10 cells after being incubated with FITC/Tf-P-DOX.HUMSC cells and B16F10 cells with low/high expression of transferrin receptor were employed to test the targeting of Tf-P-DOX.Fluorescence microscope showed that there was no significant co-localization of FITC/Tf-P-DOX and HUMSC cells.In contrast,a significant increased uptake of FITC/Tf-P-DOX in B16F10 cells.B16F10 cells were treated with Tf-P-DOX containing different concentrations of DOX(0.125,0.25,0.5,0.75,1 and 2 ?M)to estimate the cytotoxicity of Tf-P-DOX on B16F10.The IC50 of DOX at 24 h was 0.73 ?M.Considering the encapsulation efficiency of Tf-P-DOX,1.13 ?M of Tf-P-DOX(equivalent to 0.73 ?M of DOX)was selected to measure their cytotoxicity to the B16F10 cells in the following test.Furthermore,Flow cytometry was used for examining the apoptosis effects of B16F10 cells.The result show that the cells total apoptosis rate of Tf-P-DOX treatment group(41.7%±3.24)increased significantly compared with that of control group(4.6%±0.42),P<0.01.3.The tumor targeting and anti-tumor effects of Tf-P-DOX in mice.Based on the preferential targeting accumulation and penetration of the platelets in tumors,we then further investigated the in vivo tumor targeting and anti-tumor effects of Tf-PDOX using the melanoma C57BL/6 mice model.After treatment,the weight of mice in DOX group(18.58±1.87)was significantly lower than that of control group(24.85±1.41)P<0.01,the weight of mice in P-DOX group(20.81±2.22)was reduced compared with that of control group(24.85±1.41)P<0.05,Tf-P-DOX group mice weight(23.14±2.22)had no significant difference compared with the control group(24.85±1.41).Tumor growth inhibition curves in mice showed that all treatment groups inhibited tumor growth after 12 days of treatment.The tumor volume(8.54±2.93)in DOX group decreased compared with the control group(27.17±3.90)P<0.001,P-DOX group(5.54±2.25)tumor volume significantly decreased compared to the control group(27.17±3.90)P<0.001,Tf-P-DOX group(2.97±1.89)tumor volume significantly reduced compared to the control group(27.17±3.90)P<0.001.In vivo imaging system showed that near-infrared fluorescence in tumor-tumor mice was mainly targeting to tumor site,and the volume of tumor in mice decreased gradually.Compared to P-DOX group,Tf-P-DOX group demonstrated an enhanced anti-tumor effect.Furthermore,biodistribution of the therapeutic platelets in major visceral organs was investigated.The fluorescence intensity of heart of Tf-P-DOX group(0.41±0.34)was weaker than that of P-DOX group(0.91±0.10),P<0.05.These morphological changes of heart tissue were observed,and the infiltration of inflammatory cells,the cavitations of myocardial fibers and the decrease of cardiomyocytes were found in DOX group,while the P-DOX group,the cardiomyocytes decreased,and the fibrous tissue hyperplasia.There was little difference in myocardial tissue between Tf-P-DOX group and control group.Moreover,it is should note that the morphological changes of tumor tissue in Tf-P-DOX group was serious compared with the control group,including the small nucleus,the insufficiency of cancerous nest,and the infiltration of inflammatory cells.Cytoplasmic and nuclear staining of caspase 9 protein on mice melanoma tissue was investigated.Cellular Localization of caspase 3 protein can be cytoplasmic and nuclear.The integral optical density(IOD)showed that compared with the control group,the levels of caspase 9 and caspase 3 protein expression were increased in DOX,P-DOX and Tf-PDOX groups.Caspase 9 protein expression of Tf-P-DOX group(84.23±24.97)was significantly higher than that of the control group(16.87±7.63)P<0.01.Caspase 3 protein expression of Tf-P-DOX group(74.66±22.08)was significantly higher than that of the control group(14.97±9.04)P<0.001.CONCLUSIONS The above results showed that the platelet carrier combined with transferrin receptors on tumor cell membranes to target tumors and induce apoptosis of melanoma cells.The establishment of this carrier makes Tf-P-DOX specific targeting tumor and then to be released in tumor microenvironment,reducing the accumulation of drugs in normal tissues and relieving the side effects of drug toxicity.