Investigation On The Construction Of An In Vivo Individualized Recombinant Nanovaccine For Enhancing Tumor Immunotherapy

Tumor therapeutic vaccine has become one of the promising immunotherapies because of its specific ability to activate the system immune response.Studies have shown that chemotherapy,radiotherapy and photothermal therapy(PTT)can damage tumor cells and induce the release of tumor-derived associated antigens(TDPAs),thereby exerting a vaccine-like immunotherapy effect.However,it is difficult to realize its full potential,since antigens are easily degraded and difficult to accumulate in the lymph nodesaccumulated less in lymph nodes.Based on the above problems,gold nanocages(AuNCs)loaded with simvastatin(SV)adjuvant(AuNCs/SV)were prepared,and then the AuNCs/SV was encapsulated by folic acid modifided temperature-sensitive liposomes(FA-TSL)to obtain FA-TSL/AuNCs/SV with tumor targeting effect.In the study,gold nanocages(AuNCs)loaded with simvastatin(SV)adjuvant(AuNCs/SV)was prepared,and folic acid modifided temperature-sensitive liposomes(FA-TSL)was used to encapsulate AuNCs/SV,then a nano drug delivery system(FA-TSL/AuNCs/SV)with tumor targeting effect was condtructed.When the drug delivery system reached the tumor site efficiently,a rise in the temperature produced by AuNCs mediated hyperthermia under 808 nm laser irradiation could peel off the external FA-TSL,and induceing tumor cells damage and the releaseing of TDPAs.Secondly,the internal AuNCs/SV captureds TDPAs through some forces,such as the Au-S bond,to form an in-situ recombinant nanovaccine(AuNCs/SV/TDPAs).The particle size of the recombinant vaccine was about 50 nm,which is beneficial to improve the targeting effect of the nano-vaccine on lymph nodes.It was worth noted that the dilation of blood vessels caused by hyperthermia could promote the accumulation of recombinant nano-vaccine in lymph nodes,which could further enhance the body’s immune response.The specific experiments and results are as follows:1.Preparation and characterization of FA-TSL/AuNCs/SVFirstly,the AuNCs was prepared by displacement reaction using silver nanoparticles as template.Secondly,SV was loaded into the cage of AuNCs by physical loading to form AuNCs/SV.Finally,AuNCs/SV was coated with FA-TSL by film dispersion method to prepare FA-TSL/AuNCs/SV nano drug delivery system.The entrapment efficiency of SV was about 32%.The results of ultraviolet-visible full-wavelength scanning,particle size,zeta potential,transmission electron microscopy(TEM)and photothermal properties showed the successful preparation of FA-TSL/AuNCs/SV.And the stability of FA-TSL/AuNCs/SV was good,which was conducive to the follow-up experiments.2.The protein adsorption performance and anti-tumor activity of FA-TSL/AuNCs/SV in vitroIn order to investigate whether the drug delivery system forms a kind of recombinant vaccine,murine B16F10 melanoma cells were used as a cell model to investigate the protein adsorption properties of AuNCs/SV and FA-TSL/AuNCs/SV after PTT in vitro.The co-incubation experiment of AuNCs/SV and protein showed that AuNCs/SV could adsorb protein in concentration-dependent manner.In addition,the experimental results of transmission electron microscopy,mass spectrometry and western blotting indicated the successful construction of in vitro recombinant vaccines.Cytotoxicity experiments showed that FA-TSL/AuNCs/SV was safely and could effectively inhibit tumor cells growth under laser irradiation.In addition,this study used bone marrow-derived dendritic cells(DCs)as another cell model.Cell uptake experiments and lysosomal escape experiments showed that in vitro recombinant vaccines could be successfully uptaken by DCs and escape from endosomes.DCs maturation experiments showed that the synergy of antigen and adjuvant was beneficial to the maturation of DCs.The above results indicated that the successfully reconstruction of effective vaccine that has a good immune activation ability in vitro.3.Study on the anti-tumor activity and immune response of FA-TSL/AuNCS/SV in vivoB16F10 melanoma cell tumor-bearing C57BL/6J mice were used as animal model.By observing the changes in tumor volume,changes in body weight,H&E staining and TUNEL of tissue sections,the pharmacodynamic and pathological characteristics of FA-TSL/AuNCs/SV drug delivery system were evaluated.The distribution in the body showed that the preparation could be gathered at the tumor site and targeted to the lymph nodes effectively after hyperthermia.The pharmacodynamic and pathological analysis results showed that the drug delivery system had good biological safety and could inhibit tumor growth.Flow cytometry results showed that the drug delivery system could promote the maturation of DCs in tumor sites and lymphatic sites for promoting the differentiation and formation of more CD4~+and CD8~+T cells to achieve tumor immunotherapy.Experimental results of bilateral tumor and lung metastasis models showed that the system could inhibit the growth of distant tumors and prevent lung metastasis.The research results showed that this project had successfully constructed an in vivo individualized recombinant nano-vaccine and could activate the systemic anti-tumor immune response effectively for enhancing immunotherapy.