Supramolecular In Situ Nanovaccine Targeting STING Pathway For Colorectal Cancer Immunotherapy Research

Cancer statistics in 2023 show that colorectal cancer has the highest incidence and mortality rate,and there is an urgency to find powerful and effective treatment strategies.In recent years,immunotherapy,represented by immune checkpoint blockade,cell therapy,cytokine therapy and tumor vaccines,has given a strong boost to the clinical treatment of cancer.Among the above immunotherapies,tumor vaccines have received widespread attention due to their outstanding advantages of inducing tumor-specific immune responses with long-term immune memory and the promise of personalized tumor prevention.For traditional tumor vaccines,antigens need to be selected and obtained in advance,combined with adjuvants for entry into the organism,which in turn triggers the immune cycle.Commonly known antigens are usually difficult to elicit immune responses in all patients,and the process of acquiring new antigens is complicated and the frequency of expression is low.In contrast,in situ vaccines for patients with established tumors are another promising strategy for generating fully auto-antigens,but tumor in situ vaccines still have the disadvantages of difficult tumor targeting and failure to alleviate the immunosuppressive microenvironment.Based on the above research background,we show a tumor-targeted in situ nanovaccine strategy to achieve antitumor effects by initiating autoimmunity to relieve the immunosuppressive microenvironment.The main work of this thesis is to synthesize hyaluronic acid(HA)-graftedβ-CD and a conjugate(AD-SS-CPT)of amantadine(AD)with the herbal tumor-killing agent camptothecin(CPT),and to construct a supramolecular in situ nanovaccine(HCCSM)by self-assembly using the host-guest interaction of HACD with AD-SS-CPT and the STING pathway agonist MSA-2 to achieve specific delivery to colorectal cancer tissues,activating autoimmune responses through synergistic effects to achieve efficient immunotherapy against colorectal cancer.The specific research of this paper is as follows:Chapter 1:Preparation and characterization of HCCSMIn this section,HA-graftedβ-CD polymer(HA-CD)was synthesized by amidation reaction,the classical guest AD ofβ-CD was conjugated with the herbal tumor killing agent CPT(AD-SS-CPT)by disulfide bonding,and the immune adjuvant MSA-2 was combined,as well as the supramolecular in situ nanovaccine HCCSM was constructed by self-assembly using the host-guest interaction.The supramolecular in situ nanovaccine HCCSM was characterized by dynamic light scattering(DLS)and transmission electron microscopy(TEM),which showed that the supramolecular in situ nanovaccine HCCSM was homogeneous,spherical in shape,with particle size of 104.3 nm and high stability.The supramolecular in situ nanovaccine HCCSM contains disulfide bonds and is sensitive to glutathione(GSH),which can be responsively released by incubation at 10 m M GSH,and the drug release rate reaches 80%within 12 h.The above experimental results indicate that we have successfully prepared stable nanoparticles with dynamic responsive drug release,which provides strong support for subsequent cellular and in vivo experiments.Chapter 2:In vitro anti-tumor mechanism of HCCSMThe work in this section evaluates the ability of supramolecular in situ nanovaccines to increase drug uptake by tumor cells,to kill tumor cells in vitro,and to activate immunity.HA binds specifically to CD44 receptors highly expressed on the surface of CT26 tumor cells and can promote the uptake of HCCSM nanoparticles by CT26 cells.CPT is an inhibitor of cellular DNA topoisomeraseⅠwhich kills cells by It is shown that CPT not only kills tumor cells,but also induces immunogenic cell death(ICD)in tumor cells,which in turn triggers calreticulin(CRT)translocation to the cell membrane surface and nuclear High Mobility Group Protein 1(HMGB1)efflux,enhancing tumor immunogenicity.MSA-2 activates the STING pathway at tumor sites,which in turn increases IFN-βICD combined with STING activation promotes dendritic cell(DC)maturation and macrophage polarization to a tumor growth suppressive phenotype.It is predicted that supramolecular in situ nanovaccines can activate autoimmunity at the tumor site of the body and alleviate the tumor immunosuppressive microenvironment.Chapter 3:In vivo fluorescence imaging and in vivo antitumor effects of HCCSMThe scope of work in this section focuses on the use of in situ nanovaccine HCCSM for in vivo fluorescence imaging as well as in vivo antitumor analysis.Firstly,in vivo fluorescence imaging results showed that the supramolecular in situ nanovaccine HCCSM had the ability to actively target tumors and increased the fluorescence quantification nearly 3-fold compared to the control group.Secondly the fluorescence of organs also indicated that the in situ vaccine did have higher tumor delivery and less distribution in non-target organs.Finally high performance liquid phase assay showed a 2-fold difference in the accumulation of supramolecular in situ nanovaccine HCCSM in the tumor compared to the control and small molecule drugs.This demonstrates that the supramolecular in situ nanovaccine HCCSM actively targets tumor sites and enhances the accumulation of small molecule drugs at tumor sites.In the antitumor treatment of subcutaneous tumors as well as in distant tumors,the supramolecular in situ nanovaccine HCCSM therapeutic strategy showed significant therapeutic effects with significantly slower tumor growth,longer survival time,and high biosafety.Chapter 4:Immune mechanism study of anti-tumor of HCCSMThe work in this section focuses on exploring the mechanistic study of the anti-tumor of in situ nanovaccine HCCSM,and in vivo immune response analysis studies were performed.It was demonstrated that HCCSM-mediated combination therapy promoted DC maturation in draining lymph nodes,infiltration of CD8~+T cells in tumors,elevated cytotoxic T lymphocyte(CTL)ratio,and increased spleen memory CD8~+T cells in mice,as well as suppressed the expression of regulatory T cells(Treg),M2-type macrophages,and Myeloid-derived suppressor cells(MDSCs),alleviating the tumor immunosuppression microenvironment.To demonstrate the enhancement of antitumor immunity,we investigated the antitumor mechanism of the supramolecular in situ nanovaccine HCCSM in the CT26bilateral colorectal cancer mouse model and examined the immune infiltration of tumors on bilateral sides of the mice.Analysis of its induction of increased infiltration of tumor-associated CD8~+/CD4~+T cells,CTLs,inhibition of Treg cell and M2 cell development and,most importantly,treatment increased the proportion of effector memory T cells in the spleen of mice,confirming the potential of local immune memory induction and long-term immunity against tumor recurrence.To demonstrate that in situ vaccines induce vigorous tumor treatment effects by activating CD8~+T cells,the anti-tumor effects of administered mice were tested using anα-CD8 antibody to eliminate the inhibitory effect of CD8~+T cells on tumor growth.This further suggests that generalized tumor regression is caused by the immune response of the body.The supramolecular in situ nanovaccine HCCSM can effectively deliver drugs to tumor locations,achieve specific delivery to colorectal cancer tissues,and achieve efficient immunotherapy against colorectal cancer by synergistically activating anti-tumor immune responses as well as alleviating the immunosuppressive microenvironment of colorectal cancer.