Preparation Of Self-adjuvant Hydrogel Vaccine And Its Application In Tumor Immunotherapy

Objective:As an immunotherapy strategy,tumor vaccines could achieve specific tumor cytotoxicity via stimulating immune system to produce immune response against tumor.However,low antigenicity and immunosuppressive tumor microenvironment(TME)limit the efficacy of tumor vaccines.In recent years,with the continuous breakthrough of biomaterial technology,researchers optimize design of tumor vaccines based on advantages of carrier or immunomodulation of biomaterials,and have developed many new types of tumor vaccines which bring hope to improve the effect of tumor immunotherapy.Hydrogel is a class of crosslinking system containing large amounts of water.Owing to its excellent biocompatibility and advantages of delivering hydrophilic drugs,proteins,nucleic acids,active molecules and cells,hydrogel is widely used for drug delivery,targeting therapy and tissue engineering.At present,many researchers are committed to developing tumor vaccines based on hydrogel materials and have made some progress.Nevertheless,few hydrogel materials with the functions of adjuvant,carrier and immunomodulatory have been reported.Focused on improving the immunogenicity of tumor vaccines and immunosuppression of the TME,we expected to develop a self-adjuvant hydrogel that possessed function of carrier and immunomodulation to overcome the limitations of traditional tumor vaccines and improve the effect of immunotherapy.Polyethyleneimine(PEI)was modified by reactive oxygen species(ROS)-sensitive crosslinker to synthesize ROS-sensitive PEI(PROS).Then based on the crosslinking of PROS and aldehyde hyaluronic acid(AHA)that was synthesized in our previous study,we prepared PROS-AHA(ROPA)hydrogel.The adjuvant effect of ROPA hydrogel was investigates.Tumor vaccines loaded with antigens or in situ vaccine were furtherly prepared for exploration of the anti-tumor effect and the synergetic effect of combined immunotherapy strategy based on ROPA hydrogel vaccine plus immune checkpoint blockade(ICB)on melanoma or triple-negative breast cancer(TNBC),respectively.Materials and Methods:1.Preparation and characterization of ROPA hydrogelHyaluronic acid was oxidized by sodium periodate to form AHA,and PROS was synthesized by adjusting the ratio of ROS-sensitive crosslinker and PEI.The chemical structure of AHA or PROS was analyzed by the proton magnetic resonance(1HNMR).PROS and AHA were mixed in a certain concentration and proportion to prepare ROPA hydrogel.Scanning electron microscope(SEM)and rotational rheometer were used to characterize morphology and gelation property of the hydrogel.The ROS responsiveness and ROS scavenging effect of ROPA hydrogel in vitro were evaluated by observing the morphological changes of ROPA hydrogel treated with H2O2 solution,and detecting the change of H2O2 concentration in the solution.In vitro cytotoxicity of ROPA hydrogel was evaluated by the thiazolyl blue tetrazolium bromide(MTT)assay,and in vivo biocompatibility of ROPA hydrogel was investigated by dorsal subcutaneous injection to BALB/c mice and the following pathological examination.2.Application of ROPA hydrogel vaccine loaded with antigens in immunotherapy of melanoma(1)Study of adjuvant function of ROPA hydrogelOvalbumin(OVA)loaded ROPA hydrogel vaccine(OVA/ROPA)was prepared using OVA as a model antigen,and its gelation property were characterized by rotational rheometer;The in vitro release performance and stability of antigens were evaluated by modified transwell method and Coomassie brilliant blue staining method.The extracts of OVA/ROPA were prepared,and their hydrodynamic sizes and zeta potentials were measured by Marlvern laser particle size analyzer.And microscopic appearance of OVA/ROPA extracts was observed by transmission electron microscopy(TEM).The effect of OVA/ROPA extracts on uptake of antigens by dendritic cells(DCs)was evaluated by flow cytometry and cell immunofluorescence-confocal laser scanning microscope(CLSM).Bone marrowderived dendritic cells(BMDCs)were isolated from the C57BL/6J mice,and maturation of BMDCs reduced by PROS in vitro was evaluated by flow cytometry.In order to verify the adjuvant function of ROPA hydrogel in vivo and evaluate the antigen-specific immune response induced by OVA/ROPA,mice were subcutaneously immunized with OVA/ROPA hydrogel vaccines.Then,levels of OVA-specific immune antibodies and interferon-γ(IFN-γ)in serum were detected by enzyme-linked immunosorbent assay(ELISA).(2)In vivo anti-tumor effect and mechanism of ROPA hydrogel vaccines on melanomaSubcutaneous xenograft models of B 16-OVA cells on C57BL/6J mice were established for evaluating the therapeutic effect and safety of OVA/ROPA hydrogel vaccine.The changes of TME after immunization and the synergistic effect of OVA/ROPA hydrogel vaccine and PD-1 antibody(aPD-1)were investigated;Mechanism of the combined immunotherapy was initially explored by flow cytometry and pathological methods.X-ray treated tumor cells(XTC)were chosen as antigen to form ROPA hydrogel vaccine containing XTC(XTC/ROPA);Subcutaneous xenograft models of B16-F10 cells on C57BL/6J mice were established for furtherly verifying adjuvant potency of ROPA hydrogel and evaluating the combined anti-tumor effect of XTC/ROPA hydrogel vaccine and aPD-1.3.Application of in situ vaccine composite system based on ROPA hydrogel in immunotherapy of TNBC(1)Preparation and characterization of ROPA hydrogel in situ vaccine composite systemDoxorubicin hydrochloride(DOX)and aPD-1 were co-loaded into ROPA hydrogel to prepare a hydrogel in situ vaccine composite system(DOX+aPD1/ROPA);The morphology and gelation property of the composite system were characterized by SEM and rotational rheometer.In vitro drug release was detected,and in vivo drug sustained release of ROPA hydrogel containing model drug was evaluated by IVIS Lumina system.(2)Detection of immunogenic cell death(ICD)induced by DOX in vitroExposure on cell surface of calreticulin(CRT)and release of high mobility group protein B 1(HMGB 1)of 4T1 cells were detected via flow cytometry,Western blot and immunofluorescence-CLSM;The DOX-treated 4T1 cells were co-cultured with BMDCs,and maturation of BMDCs was detected by flow cytometry.(3)Anti-tumor effect and mechanism of DOX+aPD-1/ROPA on postoperative model of TNBCPostoperative recurrence model of 4T1 cells on BALB/c mice was established for investigating anti-tumor effect of DOX+aPD-1/ROPA hydrogel in situ vaccine composite system.To investigate mechanism of anti-tumor effect,proliferation of tumor cells and exposure of CRT induced by DOX+aPD-1/ROPA in vivo were evaluated by immunohistochemistry and immunofluorescence assay,respectively.And changes of immune microenvironment of mice were detected via flow cytometry.(4)Study on the systemic antitumor immunity induced by DOX+aPD-1/ROPAAfter operation and local administration of DOX+aPD-1/ROPA to the primary tumor,the secondary tumor model was established on the opposite side of the primary tumor.In order to verify the systemic antitumor immunity induced by ROPA hydrogel in situ vaccine composite system,the growth of the secondary tumor was observed,and infiltration of CD8+T lymphocytes in the secondary tumor was analyzed by immunofluorescence assay.Results:1.Preparation and characterization of ROPA hydrogel(1)AHA and PROS were successfully synthesized by periodate oxidation method and condensation reaction,respectively,and their structures were verified by 1HNMR;ROPA hydrogel was prepared,and SEM and rheology test results confirmed morphology and gelation property of the hydrogel.(2)In vitro degradation of ROPA hydrogel was ROS-responsive.The concentration of H2O2 in the environment decreased during hydrogel degradation,which suggested that ROPA hydrogel may be used as a scavenger of ROS.(3)MTT results illustrated that compared with PEI and hydrogel that prepared by PEI and AHA,PROS and ROPA hydrogel had less cytotoxicity.The results of pathological examination showed that implantation of ROPA hydrogel had no obvious toxic effects on local tissues.ROPA hydrogel had good biocompatibility.2.Application of antigen loaded ROPA hydrogel vaccine in immunotherapy of melanoma(1)The results of rheology and in vitro antigen release assay illustrated that ROPA hydrogel could be a good carrier of protein antigen to form OVA/ROPA hydrogel vaccine.(2)The hydrodynamic size,zeta potentials and TEM results showed that OVA/ROPA hydrogel released nano vaccine during the degradation process,and its contribution for uptake of antigens by DCs was confirmed by flow cytometry and CLSM.(3)Flow cytometry results showed that PROS could up-regulate the expression of costimulatory molecules(CD80 and CD86)on surface of BMDCs,which suggested that PROS could induce the maturation of BMDCs.(4)The results of ELISA assay confirmed the increased levels of OVA specific antibodies and IFN-y in serum of normal mice immunized by OVA/ROPA hydrogel vaccine,which indicated that OVA/ROPA effectively induced antigenspecific immune response in vivo.(5)Animal experiments showed that OVA/ROPA hydrogel vaccine could inhibit growth of B 16-OVA tumor in vivo;The results of hematology and pathology reflected that OVA/ROPA hydrogel vaccine was safe in vivo.(6)OVA/ROPA promoted the infiltration of CD8+T lymphocytes in tumors,but also up-regulated expression of PD-1.The concurrent OVA/ROPA and aPD-1 could furtherly enhance the therapeutic effect,resulted in apparently proliferation inhibition and apoptosis promotion of tumor cells,and prolong survival time of tumor bearing mice;As shown in results of flow cytometry,the combined immunotherapy could promote the maturation of DCs in lymph nodes ipsilateral to the tumor,and the infiltration and activation of CD8+T lymphocytes in tumor.It also increased the content of CD4+T lymphocytes in spleen.All above suggested the combined strategy induced anti-tumor immune response in mice.(7)ROPA hydrogel could load tumor cells antigen to form XTC/ROPA hydrogel vaccine.Anti-tumor experiment results showed that XTC/ROPA had stronger immunogenicity compared with free XTC antigen,and combination of XTC/ROPA hydrogel vaccine and aPD-1 resulted in a strong in vivo anti-tumor effect on melanoma.3.Application of in situ vaccine composite system based on ROPA hydrogel in immunotherapy of TNBC(1)The DOX+aPD-1/ROPA hydrogel in situ vaccine composite system was successfully prepared,and ROPA hydrogel was proved to be good carrier for small molecule drugs and antibody drugs via rheological test,SEM and in vitro and in vivo drug release assay.(8)The results of flow cytometry,Western blot and CLSM showed the CRT exposure and HMGB1 release of 4T1 cells treated with DOX,which indicated that DOX could induce ICD of 4T1 cells that further stimulated maturation of BMDCs.(2)The postoperative recurrence model of 4T1 cell was successfully established.The in vivo results indicated that the DOX+aPD-1/ROPA could significantly inhibit the growth of recurrent tumor after surgery.Immunofluorescence images of CRT confirmed that DOX+aPD-1/ROPA could induce ICD of tumor cells in vivo.As shown in results of flow cytometry,the combined immunotherapy could promote the maturation of DCs in lymph nodes ipsilateral to the tumor,and facilitated the infiltration and activation of CD8+T lymphocytes in tumor partly due to the blocking of the PD-1/PD-L1 signal pathway.It also increased the content of CD4+T lymphocytes in spleen.On the contrary,combined immunotherapy reduced the content of immunosuppressive cells(MDSCs and Tregs)in tumor,thus alleviated the immunosuppression of TME to some extent.(3)The postoperative secondary tumor model of 4T1 tumor bearing mice was successfully established.The results showed that the operation and local administration of DOX+aPD-1/ROPA to the primary tumor could promote the infiltration of CD8+T lymphocytes in the secondary tumor,and significantly inhibit the growth of the secondary tumor.Conclusion:In this study,we focused on the dilemma of tumor vaccine development,designed and successfully prepared a self-adjuvant ROPA hydrogel.The study confirmed that ROPA hydrogel was a powerful adjuvant that could enhance the immunogenicity of tumor vaccine via effectively activating humoral and cellular immunity.ROPA hydrogel could possibly reduce the immunosuppression of TME to a certain extent by scavenging ROS.The protein vaccine and tumor cell vaccine based on ROPA hydrogel were prepared,and the anti-tumor effect of ROPA hydrogel tumor vaccines on melanoma was verified.The synergistic effect of ROPA hydrogel vaccines combined with aPD-1 and the initial mechanism were also investigated.In order to furtherly expand the application scope of ROPA hydrogel and explore the new strategy of TNBC postoperative immunotherapy,a DOX+aPD-1/ROPA hydrogel in situ vaccine composite system was constructed.In the story,the ICD effect induced by DOX formed a tumor vaccine in situ,which,combined with local administration of aPD-1,exerted a synergistic effect on inhibition of postoperative recurrence and metastasis of TNBC.In conclusion,ROPA hydrogel has simple preparation process,self-adjuvant effect,powerful carrier function and immune microenvironment regulation potentiality,as well as good biocompatibility.Tumor vaccines based on ROPA hydrogel have a certain application prospect in the field of tumor immunotherapy.This novel immunotherapy strategy based on biomaterials is an innovation and breakthrough,which provides a new idea for exploring the treatment of solid tumors.