The Preparation Of Different Nano-Vaccine Againstmultiple Myeloma And Study On Their Immune Response

MART-1_aa26-35*A27L cytotoxic-T lymphocytes?CTLs?can effectively lyse the multiple myeloma?MM?cells expressing HM1.24.However,this peptide vaccine is poor immunogenicity due to its short half-life,which cannot effectively induce CTLs immune response.Looking for an excellent carrier for vaccine peptides to protect them to reach antigen-presenting cells?APCs?quickly and further to be rapidly taken up and processed by APCs,it is promising directions and important strategies for improving the efficacy of antitumor vaccine.Liposomes?LP?and nonionic surfactant vesicles?NS?are the most commonly used nano delivery systems,which are biocompatible,biodegradable,targeted,and prolonged of blood circulation time,low toxicity and other advantages.More importantly,nano delivery systems simulated the size and morphology of tumor cells,can activate the anti-tumor immune response to eliminate tumor cells at the most extent.Therefore,this study investigated the effects and differences of these two nano-vaccines in inducing MART-1 _aa26-35*A27L specific CTLs immune response,which can lay the foundation for further research and promotion of tumor nano vaccines.Method:?1?An in vitro analytical method for the determination of MART-1_aa26-35*A27L was established by reversed-phase high performance liquid chromatography?RP-HLPC?,and MART-1_aa26-35*A27L liposome nano-vaccine(MART-1_aa26-35*A27L-LP)and MART-1_aa26-35*A27L nonionic surfactant vesicles nano vaccine(MART-1_aa26-35*A27L-NS)were prepared by film dispersion method.In addition,orthogonal experimental design was used to optimize the preparation and technology of nano vaccine to obtain high encapsulating efficiency and high drug loading of MART-1_aa26-35*A27L in LP and NS.Finally,the appearance,shape,particle size,Zeta potential,polydispersity indices?PDI?,encapsulation efficiency and drug loading of MART-1_aa26-35*A27L-LP and MART-1_aa26-35*A27L-NS were characterized.?2?Peripheral blood mononuclear cells?PBMNCs?were isolated from peripheral blood from healthy donors.A part of these was frozen for later use;the other part was cultured in vitro and induced to generate immature dendritic cells?imDCs?,and then MART-1_aa26-35*A27L-LP,MART-1_aa26-35*A27L-NS,LP-blank,NS-blank,MART-1_aa26-35*A27L-PBS,PBS and positive drug were added to induce the formation of mature DCs?mDCs?.After that,the mDCs of each group were co-cultured with T cells derived from PBMNCs to generate the specific T cells.Finally,the specific T cells of each group were harvested,and CD56^-CD8⁺T cells of each group were isolated.The number of specific CD8⁺CTL cells were detected and granzyme B were detected as well as the expression of maturation-related markers on the surface of DC were detected.Results and conclusions:?1?MART-1_aa26-35*A27L-LP were prepared by a film dispersion method,and orthogonal experimental design was used to optimize the preparation and technology of this nano vaccine.The results showed that the optimal preparation and technology for the liposomes were a final phospholipid concentration of 10?g/mL,a mass ratio of phospholipid to cholesterol of 6 to 1,a final peptides concentration of 1mg/mL and a hydration time of 30 min.?2?The in vitro detection-method of MART-1_aa26-35*A27L was established by RP-HLPC,and the characters of MART-1_aa26-35*A27L-LP and MART-1 _aa26-35*A27L-NS were detected by particle size analyzer and transmission electron microscope.The result showed that the average encapsulation efficiency was 49.4%and 52.89%;the average drug loading was 4.06%and 6.22%;and the average particle diameter was 192.4nm and207.50nm;the average Zeta potential was-9.41mv and-12.50mv;and the average PDI was 0.27 and 0.15,respectively.?3?The CTL immune response induced by MART-1_aa26-35*A27L-LP and MART-1_aa26-35*A27-NS as well as MART-1_aa26-35*A27L-PBS were detected by cells experiments in vitro.The result showed that both MART-1_aa26-35*A27L-LP and MART-1_aa26-35*A27-NS can significantly enhance the expression of CD80 on DCs surface,and can significantly enhance the induction of CD56^-CD8⁺CTLs number,and increase the release of granzyme B.