The Efficacy And Mechanism Of A Novel Self-assembled Hybrid Nano-vaccine In The Prevention And Treatment Of Melanoma

ObjectivesA novel hybrid nano-vaccine MCL(Melittin-RADA₃₂-Cp G-Lysis)was successfully constructed by synchronously loading melittin（melittin）,tumor cell lysate（tumer lysate）and the immune activator Cp G,with self-synthesized short peptide RADA₃₂.The therapeutic and preventive effects of MCL、mechanism for melanoma were observed in vitro and in vivo.MethodsTo Synthesis a novel self-assembled hybrid nano-vaccine MCL and evaluate the safety of it.The self-synthesized novel polypeptide MR(Melittin-RADA₃₂)powder and、tumor cell lysate and the immune activator Cp G were dissolved in normal saline to form a novel hybrid nano-vaccine MCL(Melittin-RADA₃₂-Cp G-Lysis).The biocompatibility of nano-vaccine MCL was evaluated by hemolysis test.The safety of MCL was evaluated by dynamic monitoring the body weight and blood biochemical indexes after MCL injection.To evaluate the therapeutic and preventive effects of MCL in vivo1.Establishing a tumor therapy model to observe the therapeutic efficacy of MCL:C57BL/6 mice were subcutaneously inoculated with melanoma B16-Luc cells.When the tumor volume reached 20mm3,MCL(Melittin-RADA₃₂-Cp G-Lysis)、CL（Cp G-Lysis）,、MR(Melittin-RADA₃₂)and PBS was injected into the root of the tail and on the footpad or adjacent to the tumor,respectively.Calculating the tumor inhibition rate and the survivalrate by dynamically monitoring the tumor volume and recording the time of death in each grouprespectively.At the same time,in order to evaluate the anti-cancer efficacy of the drugs in vivo,We drewed the tumor growth curve and survival curve、performed imaging of small animals and and recorded the fluorescence intensity in mice2.Establishing a tumor prevention model to observe the preventive effect of MCL:MCL（Melittin-RADA32-Cp G-Lysis）、CL（Cp G-Lysis）,、MR（Melittin-RADA32）and PBS were given to the tail root and foot pad of C57BL/6mice three times.After that,melanoma B16-Luc cells were inoculated into the root of the thighs of mice.The tumor volume was monitored dynamically and the death time of each group was recorded to calculate the tumor inhibition rate and survival rate of each group.The tumor growth curve and survival curve were drawn to evaluate the efficacy of MCL in preventing tumor.To evaluate the immune Activation effect of MCL in vivo and vitro3.Primary mouse dendritic cells（dendritic cell,DC）were co-incubated with MCL,CL,MR,PBS in vitro,and the proportion of activated DC in each group was detected by flow cytometry.Subcutaneously transplanted melanoma model was established and treated with MCL,CL,MR,PBS respectively.When the tumor volume reached 500mm3,the lymph node size was observed by dissection of inguinal lymph nodes in mice.Opal multispectral tissue imaging technique was used to detect the number of activated DC in inguinal lymph nodes.Flow cytometry was used to detect the proportion of activated DC in inguinal lymph nodes and CD4⁺T,CD 8⁺T cells and IFN-γ⁺CD8⁺T cells in tumor tissues.ResultTo Synthesis a novel self-assembled Hybrid Nano-vaccine MCL and evaluate the safety of it.The colorless hydrogel could be formed by loading melittin with RADA32polypeptide.The hemolysis test of red blood cells showed that the hemolysis rate of MCL was 0 at 3.2μM,So it had good biocompatibility.There were no significant changes in body weight and liver、kidney function（AST,ALT,BUN,Cr）in each group before and after MCL administration.To evaluate anti-tumor effect of MCL in vivoThe tumor inhibition rates of MCL group,CL group and MR group were 90.74%,67.57%and 31.04%respectively on the 28th day after the treatment of foot pad and tail root of tumor-bearing mice.The survival rates on the 28th day after inoculation were 100%,83.33%and 57.14%,respectively,and the tumor growth curve showed that MCL could significantly inhibit tumor growth,and the tumor inhibition effect was better than that of CL and MR groups.Comparing with CL group,MR group and PBS group,MCL significantly prolonged the survival time of tumor-bearing mice.The tumor inhibition rates of MCL group,CL group and MR group were 99.91%,91.83%,50.42%,respectively after the treatment of paratumoral multipoint injection of tumor-bearing mice.and the tumor growth curve showed that MCL could significantly inhibit tumor growth,and the antitumor effect of MCL was better than that of CL and MR groups.The fluorescence intensities of MCL group,CL group,MR group and PBS group were0.94±1605*105a.u、17.62±1879*105 a.u、42.28±14116*105 a.u、264.46±82220*105a.u。on the 28th day,respectively.2.In the tumor prevention model,the tumor inhibition rates of MCL group,CL group and MR group were 99.36%,93.86%and 46.91%,respectively.The survival rates of MCL group,CL group and MR group were 87.5%,37.5%and 12.5%on the45th day after tumor inoculation,respectively,and the tumor growth curve showed that MCL could significantly inhibit tumor growth compared with CL group and MR group.The survival curve of mice showed that MCL significantly prolonged the survival time of tumor-bearing mice compared with the control group.To evaluate the immune Activation effect of MCL in vivo and vitroIn vitro,the percentage of activated DC in MCL,CL,MR,PBS group was91.76±0.36%、76.92±6.84%、60.04±4.00%、37.78±5.57%respectively;In vivo,the percentage of activated DC in inguinal lymph nodes in MCL,CL,MR,PBS group was6.98±1.32%、4.90±0.72%、3.4±0.41%、2.51±0.35%respectively;Opal multispectral tissue imaging showed that the number of activated DC in inguinal lymph nodes in MCL group,CL group,MR group and PBS group was 997.4±271.4、68.6±25.12、0±0and 2±1.41 respectively;In the tumor,the percentage of CD4+T in MCL group,CL group,MR group and PBS group were 37.58±0.68%、25.96±0.32%、20.41±0.98%、17.35±1.09%,respectively;The percentage of CD8+T cells in MCL group,CL group,MR group and PBS group were 26.52±0.42%、16.83±0.27%/15.87±0.57%、8.98±0.39%,respectively;The percentage of IFN-γ+CD8+T cells in MCL group,CL group,MR group and PBS group were 5.89±0.27%、3.10±0.09%、4.07±0.27%、1.92±0.07%,respectively.Conclusion:A novel hybrid nano-vaccine MCL(Melittin-RADA₃₂-Cp G-Lysis)containing melittin,tumor cell lysate and immune adjuvant was successfully synthesized.The vaccine can not only prevent and treat melanoma by direct killing and immunotherapy,but also has good biocompatibility and safety.It has important clinical transformation value.