Study On The Synergies Of Artemisinin In Combination With Dacarbazine Used For The Treatment Of Melanoma B16 And The Preparation Of Their Co-Loading Liposomes

Dacarbazine(DTIC)is a classic drug for the treatment of advanced melanoma,but its therapeutic effect is limited.In recent years,DTIC has been conducting clinical research on the treatment of melanoma with other drugs such as interferon,tumor suppressor gene and protein.Therefore,in order to further expand the treatment plan of DTIC,this study will have a combination of artemisinin(Art)and DTIC,which is suggested have anti-melanoma activity and complementary mechanism of action,and the combination of artemisinin(Art)and DTIC.Co-loaded liposomes liposomes of the ART and DTIC are prepared to produce better therapeutic effects and its mechanisms are explored,The results are supposed to provide reference for the later research on the combination therapy based on nanocarriers.Method:(1)MTT assay was used to investigate the growth inhibition of B16 cells treated with Art and DTIC alone and combined with two drugs.The ratio of Art and DTIC was determined based on the best synergy by Gaddum model and Chou-Talalay analysis.The effect of Art,DTIC,Art combined with DTIC on B16 cells was observed under inverted fluorescence microscope by hoechst33258 staining method.Quantitative analysis of apoptosis of B16 cells was induced by Art,DTIC and Art combined with DTIC by Annexin V-FITC/PI double staining.(2)The preparation process of Art-DTIC co-loaded liposome(Art-DTIC-LP)was carried out by encapsulating Art and DTIC to form a double-loaded liposome using liposome as a carrier.The concentration of Art and DTIC drugs was determined by ultraviolet spectrophotometry.The preparation method of Art-DTIC-LP was optimized by orthogonal design.The quality of Art-DTIC-LP was evaluated by observation of its appearance,TEM,particle size and its distribution,potential,encapsulation rate,stability and release test.(3)A preliminary pharmacodynamics of Art-DTIC-LP in mice was studied.The tumor model of melanoma B16 mice was established.The changes of body weight,tumor weight and survival of the mice in each group were observed and compared.Experimental results and conclusions:(1)The IC50 values of Art B16 cells at 24 h,48h and 72 h were 558.63,192.23 and 105.61 μg/mL,respectively.The IC50 values of DTIC were 2053.29,204.89 and 104.11 μg/mL,respectively,and Art and DTIC were in effect.The IC50 value ratio was about 1:1 at 48 h.The parameter γ was calculated by Gaddum model formula to be 0.78 which displayed initially the synergy possibility of Art combination with DTIC.The ChouTalalay analysis showed that the ratio of Art to DTIC were 1:1,1:2,2:1.and The synergistic index CI values were 0.58,0.70,and 0.78,respectively,which suggested that the combination of the two drugs had some synergistic effects,and the synergistic effect is the best when the combination ratio of Art:DTIC was 1:1.Inverted fluorescence microscopy showed that the apoptosis characteristics of B16 cells treated with Art and DTIC were significantly stronger than those of ART and DTIC alone.The results of AnnexinV-FITC/PI double staining showed that the apoptosis of B16 cells was higher in the Art and DTIC combination group than in the other groups.The combination of Art and DTIC has synergistic proliferation inhibition effect on B16 cells,and its mechanism may be through synergistic induction of apoptosis.The synergistic effect is best when the ratio of Art to DTIC is 1:1.(2)The method for determining the content of Art and DTIC was established respectively by the two-wavelength ultraviolet method and ultraviolet spectrophotometry.Art-DTIC-LP was prepared by thin film ultrasonic-ammonium sulfate gradient method.The final Art-DTIC-LP was gotten by orthogonal experiment when the mass ratio of drug to phospholipid was 1:10,the ratio of phospholipid to cholesterol was 3:2,and the concentration of phospholipid was 30mg/mL.The prepared lipids had good body weight,and the encapsulation efficiency of Art and DTIC were(92.15±0.95)%,(75.93±2.58)%,the average particle size was(219.1±1.51)nm,and the zeta potential was(-54.3).±1.04)mV,multiple dispersion coefficient(PDI)was 0.342±0.02,and the stability was good after storage for two weeks at 4 °C.(3)The tumor model of melanoma B16 mice was successfully established.The results of the administration showed that Art-DTIC solution and Art-DTIC-LP suspension had a better therapeutic effect on mouse tumors compared with the blank group.ArtDTIC The tumor volume of the mice in the Art-DTIC-LP group was significantly lower than that in the other groups and the median survival time of the mice was 31 days.