Biomimetic Hybrid Nanovesicles And Liquid Crystal Formation System For Cancer Chemoimmunotherapy

Objective:Recently,cancer immunotherapy has achieved significant clinical outcomes along with advances in technologies,such as immune checkpoint blockades(ICB),chimeric antigen receptor-T cells(CAR-T)therapy,tumor vaccines and cytokines.However,only a subset of patients exhibits durable responses to immunotherapy,and immunotherapy alone is usually ineffective.The combination of chemotherapy and immunotherapy is considered to be a feasible strategy to improve and optimize the efficacy of immunotherapy,and appropriate and efficient drug delivery systems can further amplify the antitumor effect of chemoimmunotherapy.Therefore,to efficiently inhibit tumor growth or prevent postoperative tumor recurrence and metastasis,we proposed two different drug delivery strategies of chemoimmunotherapy for subcutaneous tumor transplantation models and postoperative recurrent tumor respectively,and further explored mechanisms by which the two delivery strategies enhanced the efficacy of chemoimmunotherapy.Methods:1.For the subcutaneous tumor transplantation models,we developed novel biomimetic hybrid nanovesicles(LINV)via fusing immunogenic tumor-derived nanovesicles(TNV)with artificial liposomes(LIP)and encapsulating immunogenic inducer doxorubicin(DOX)(DOX@LINV)for immunochemotherapy.(1)The LINV was fabricated via hydration extrusion method and characterized.The specific proteins of LINV were analyzed by western blot.(2)The drug-loading characteristics and stability of LINV were analyzed.The uptake of DOX@LINV by different tumor cells and the immunogenic cell death(ICD)effect induced by DOX@LINV were investigated.(3)Bone marrow-derived dendritic cells(BMDCs)were extracted to investigate the immunostimulatory activity of LINV.(4)Enzyme-linked immunodot detection(ELISPOT)and effector-target killing were analyzed to evaluate whether LINV could induce antigen-specific immune response.(5)The antitumor effect and mechanism of DOX@LINV were investigated by establishing subcutaneous tumor transplantation models of B16F10 melanoma and Lewis lung cancer(LLC).(6)The synergistic antitumor effects of DOX@LINV with immune checkpoint blockades(a PD-1)were investigated.(7)Triple negative breast cancer 4T1model was established to explore the antitumor effect of DOX@LINV on orthotopic tumor.2.For the treatment of postoperative tumor recurrence and metastasis,we employed an injectable liquid crystal formation system(LCFS),co-loaded with DOX and immunomodulator resiquimod(R848)(D/R@LCFS)for postoperative chemoimmunotherapy.(1)The precursors of liquid crystal formation system were prepared by mixing and stirring method.The rheological properties of the precursors were investigated by rheometer,and the internal structure of the precursors was explored by polarization optical microscope.(2)The in vivo storage effect and drug release characteristics of liquid crystal gel were investigated by optical in vivo image and frozen tissue sections,respectively.(3)BMDCs were extracted to investigate the immunostimulatory activity of R848 and immunogenic tumor cells.(4)A B16F10 melanoma surgical tumor model was established to investigate the antitumor effect of D/R@LCFS on postoperative tumor recurrence and its regulatory effect on tumor microenvironment(TME).(5)The synergistic antitumor effect and underlying mechanisms of D/R@LCFS with immune checkpoint blockade(a PD-1)to prevent postoperative recurrence and metastasis of B16F10 melanoma and 4T1 breast cancer were analyzed.Results:1.The constructed LINV,with uniform particle size and similar membrane components as the source cells,could efficiently encapsulate and deliver doxorubicin to cognate tumor tissue,provoking robust immunogenic cell death effect.Meanwhile,the LINV,preserving tumor antigens and immunogenic heat-shock proteins,could upregulate the expression of co-stimulatory molecules on dendritic cells(DCs)and elicit subsequent tumor antigen-specific immune response.DOX@LINV exhibited robust antitumor effects on both B16F10 murine melanoma and LLC,and significantly increased the infiltrition of effector CD8~+T cells and the secretion of Granzyme B,and reduced the frequency of immunosuppressive regulatory T cells in tumor tissue.Furthermore,the combination of DOX@LINV with a PD-1 further amplified antitumor response and prolonged survival time with 33.3%of the mice being tumor-free.In addition,the DOX@LINV displayed an excellent antitumor effect on orthotopic 4T1breast cancer with poor immunogenic.2.The LCFS precusor could undergo in situ phase transformation at the tumor resection site to form in situ gel.The gel could act as drug reservoir and release DOX and R848 in a spatiotemporal sustained manner.D/R@LCFS turned tumor into in situ vaccines via DOX-triggered immunogenic cell death effect accompanied with immune regulatory effect of R848,facilitating the maturation of DCs and the recruitment of effector CD8~+T cells,which favors antigen-specific T cell response.Meanwhile,D/R@LCFS provoked the polarization of immunosuppressive M2-type macrophages into immunostimulatory M1-type macrophages,and the transformation of myeloid-derived suppressor cells into tumoricidal antigen-presenting cells(M1 or DCs).The D/R@LCFS elicited strong immune response and long-term immune-memory effect in combination with a PD-1 to significantly prevent postoperative recurrence and metastasis of B16F10 melanoma and 4T1 breast cancer.Conclusion:In this thesis,we put forward two drug delivery strategies for chemoimmunotherapy based on the combination of immune-enhancing chemotherapeutic drug with immunogenic TNV,immunomodulators or ICB.These strategies effectively modulated tumor microenvironment and demonstrated excellent therapeutic efficacy or prevented postoperative tumor and matastasis,and may provide a new avenue for future clinical cancer treatment.Innovations:(1)The biomimetic LINV in the first combination chemoimmunotherapy strategy not only inherited the advantages of TNV,such as immunostimulatory effect and natural targeting ability,but also inherited the advantages of liposomes,such as efficient encapsulation of chemically different molecules,ease of functionalization and production.(2)The LINV can not only be used as an ideal targeted delivery carrier for chemotherapeutics,but also can intergrate its own immunostimulatory effect for synergistic chemoimmunotherapy against tumor.(3)The second combination strategy used the liquid crystal formation system with good biocompatibility and ease of fabrication,which can form liquid crystal gel and remain at the resection site for a long time.This combination strategy was the first to use LCFS for prevention of postoperative tumor recurrence and metastasis.(4)D/R@LCFS can transform postoperative residual or recurrent tumors into in situ vaccine and reverse the immunosuppressive microenvironment,thus effectively inhibiting the recurrence and metastasis of various postoperative tumors.This combination strategy based on LCFS is expected to provide a clinically available option for effective postoperative chemoimmunotherapy.