Basic Investigation Of Gold Nanoparticle-Based Vaccine With Different Morphologies On Lymph Delivery And Immune Response

Tumor immunotherapy has become the fourth therapy after surgery,chemotherapy and radiotherapy.Among them,cancer vaccine,as one of the effective immunotherapies against cancer,has attracted wide attention.Nanopaticle-based vaccine delivery systems have been developed to deliver antigens to lymphatic tissues to promote humoral or cellular immune response.The size,morphologies and surface properties of nanoparticles have significant impact on the immune efficacy in vivo.However,the effects of nanoparticles with different morphologies on antigen capture,lymphatic drainage,antigen presentation,immune response efficiency and other immune-related aspects are still unclear.Based on this,we selected gold nanoparticles as the model materials to synthesized gold nanocage(AuNC),gold nanorod(AuNR)and gold nanostar(AuNS)with the similar surface properties and size within a certain range,but different morphologies.They were combined with tumor cell lysate(TCL)to construct model nanovaccines AuNC-CL,AuNR-CL and AuNS-CL with different morphologies.The related effects of gold nanovaccines with different morphologies on tumor immunity were evaluated at the cellular and animal levels.The specific content and results of this research were as follows :1.Preparation and characterization of AuNC,AuNR and AuNS.AuNC was synthesized by replacement reaction using silver nanoparticles as templates;AuNR was prepared by seed growth method;AuNS was prepared by seed-mediated growth method without surfactant.The results of laser particle size analyzer and transmission electron microscope showed that the prepared AuNC,AuNR and AuNS had good dispersibility with size of 40-60 nm and potential of about-20 m V.The absorption peaks measured by UV-Vis spectrophotometer were all around 800 nm.2.Preparation and characterization of model nanovaccines AuNC-CL,AuNR-CL and AuNS-CL.The cell lysates(CL)of B16F10 melanoma cells were incubated with AuNC,AuNR,AuNS respectively.After centrifugation and washing,the model nanovaccines AuNC-CL,AuNR-CL and AuNS-CL were obtained.The hydrated particle size of gold nanoparticles increased,and the surface potential decreased to about-25 mV after incubation with CL.Transmission electron microscopy observed that the gold nanoparticles were uniformly dispersed in a protein halo.The above results showed that we have successfully prepared model nanovaccines AuNC-CL,AuNR-CL and AuNS-CL.BCA method and polyacrylamide gel electrophoresis experiments showed that the prepared gold nanoparticles can effectively capture tumor antigens,and the amount of antigens was AuNS-CL > AuNC-CL > AuNR-CL.Western Blot and liquid chromatography-tandem mass spectrometry indicated that AuNC and AuNS had better ability to capture tumor-associated antigens than AuNR,and AuNC had the highest types of antigen capture.The three antigens could capture tumor neoantigens and damage associated molecular patterns,but their relative abundance was different.3.In vitro anti-tumor activity of model nanovaccines.B16F10 melanoma cells,bone marrow-derived dendritic cells(BMDC)and RAW264.7 mouse macrophages were used as models to investigate the anti-tumor activity of the model nanovaccines in vitro.Cytotoxicity experiments showed that the prepared gold nanoparticles have good biological safety.BMDC and RAW264.7 uptake and DC maturation experiments showed that AuNC-CL,AuNR-CL,and AuNS-CL could be effectively taken up by antigen-presenting cell(APC)to promote the maturation of DC,thereby effectively presenting antigens.4.In vivo anti-tumor activity of model nanovaccines.The results of lymph nodes dissection,quantitative analysis of nanoparticles in lymph nodes,silver staining at different time points and flow analysis of nanoparticles in lymph nodes showed that the model nanovaccines could effectively drain from the inoculation site to the lymph nodes.Among them,the drainage ability of AuNS-CL was stronger than AuNC-CL and AuNR-CL,and AuNS-CL could be obviously retained in the lymphatic follicles;with the increase of time,all three could gradually enter the paracortical region of lymph nodes,and the distribution of AuNC-CL and AuNS-CL was slightly more than that of AuNR-CL.The determination of antibody concentration in serum showed that AuNS-CL and AuNC-CL played an immune protective role by promoting antibody production and triggering humoral immunity,and AuNS-CL had a stronger ability to promote antibody production The results of animal flow experiments and immune cytokine secretion further showed that AuNC-CL mainly activated CD8+T cells through MHC-I,while AuNR-CL and AuNS-CL mainly activated helped CD4+T cells through MHC-II.All of them could promote the production of TNF-α,IFN-γand IL-6 cytokines,thus promoting the anti-tumor immune response.The cytotoxic T lymphocyte killing experiment showed that all of them could activate T cells to specifically recognize and target the target cells.In vivo pharmacodynamic results showed that the model nanovaccines had anti-tumor preventive effect,which could inhibit tumor growth and prolong the survival time of mice.There was no significant difference in body weight,blood routine and blood biochemical analysis,which indicated that the nanovaccines had good biological safety.In summary,the subject provides theoretical research for the rational design of nanovaccines by evaluating the impact of gold nanoparticle-based vaccine with different morphologies on lymph delivery and immune response,which can guide the optimization process of nanoparticles to drive the antigen presentation response and generate effector cells/antibody mediated cell/humoral immunity for effective vaccination.