Preparation Of Dendritic Cells Targeting Vaccine Based On Nanoparticles Loading Tumor Cells Antigens And Its Immunotherapy Effect On Melanoma

Aims:Whole tumor cell lysates(TCL)have been implemented as tumor antigens for cancer vaccine development;although clinical outcomes of TCL-based antitumor immunotherapy remain unsatisfactory.There is still more to improve in the field of immunotherapy tumor vaccine with tumor antigens based on tumor cell lysates.Therefore,new technology and method is needed for enhancing the efficacy of anti-tumor immunotherapy.In order to improve the efficacy of TCL-based vaccines,biomaterials have been employed to enhance antigen delivery and presentation.Here,we have developed chitosan nanoparticles(CTS NPs)with surface mannose(Man)moieties for specific dendritic cells(DCs)targeting(Man-CTS NPs).The Man-CTS NPs were then loaded with TCL generated from B16 melanoma cells(Man-CTS-TCL NPs)for in vitro and in vivo assessment.Methods:Firstly,thro?gh electrostatic adherence to prepare chitosan and tumor cell lysates mixture,then for the synthesis of mannose modified alginate.The Man-CTS-TCL NPs was formulated thro?gh electrostatic adherence of CTS-TCL NPs and Man-ALG.The structure was expressed by 1H-NMR and IR.Potency of the Man-CTS-TCL NPs as cancer vaccine was also assessed in vivo by immunization of mice with Man-CTS-TCL NPs followed by re-challenge with B16 melanoma cell inoculation.The effect of Man-CTS-TCL NPs on promoting bone marrow-derived dendritic cells(BMDCs)maturation and antigen presentation in vitro was tested by FACS for expression of surface markers of mature BMDCs,such as CD80,CD86,CCR7,MHCI,MHCII and CD40 and ELISA for releasing of cytokines that related to BMDCs maturation.The small animal imaging system was used for assaying the distribution of Man-CTS-TCL NPs after subcutaneous injection and its migration ability to the draining lymph nodes.Three different animal models(prophylactic tumor model,therapeutic tumor model and lung metastasis tumor model)were used for testing the anti-tumor effect of this kind of new nano-vaccine.Results:we successfully prepared the Man-CTS-TCL NPs and the structure was confirmed by 1H-NMR and IR.TEM result has shown that the Man-CTS-TCL NPs is in spherical shape.The antigens can be released in a more permanent manner at the pH value of 5.0.We have shown here that Man-CTS-TCL NPs can promote bone marrow-derived dendritic cells(BMDCs)maturation and antigen presentation in vitro.In vivo evaluation further demonstrated that the Man-CTS-TCL NPs were readily taken up by endogenous DCs within the draining lymph node(DLN)following subcutaneous administration accompanied by increasing in serum IFN-y and IL-4 levels.Tumor growth was also significantly delayed in mice primed with Man-CTS-TCL NPs vaccine,attributable at least in part to cytotoxic T lymphocytes response.Moreover,Man-CTS-TCL NPs vaccine also exhibited therapeutic effects in mice with melanoma.Results from the lung metastasis tumor model showed that Man-CTS-TCL NPs can inhibit the metastasis of tumor cells to the lung.The effect can be explained by the activation of cytotoxic CD8+T cells in peripheral blood of mice.Conclusion:Thus,we report here the Man-CTS-TCL NPs as effective anti-tumor vaccine for cancer immunotherapy.This vaccine with lower cytotoxicity,and is more prone to be up taken by antigen presentation cells.In vivo it can target to the APCs and migration in the draining lymph nodes.In vitro and in vivo experiment showed that Man-CTS-TCL NPs can promote the maturation of BMDCs and improve the cytokines release.The prophylactic and therapeutic model demonstrated that the vaccine can prevent the formation of melanoma and inhibit the growth and metastasis of tumor.This project successfully designs the new dendritic cells targeting chitosan nanoparticles vaccine and provide theoretical basis for the antitumor immunotherapy based on nanoparticles materials.