Study On The Antitumor Effects Of Camptothecin-IDO Inhibitor Drug Conjugate And Its Nanotherapeutics

Chemotherapy is still the mainstay treatment of triple-negative breast cancer(TNBC),but the clinical efficacy is unsatisfactory.Chemotherapy not only cannot effectively kill cancer cells,but also increase the proportion of cancer stem cells(CSCs)and lead CSCs into dormancy and drug resistance.In addition,cancer can construct an immunosuppressive microenvironment to foster the survial,proliferation and metastasis of cancer cells and CSCs.How to efficiently ameliorate the tumor microenviroment and eliminate CSCs is of great clinical significance for the treatment of TNBC,but still faces many challenges.Killed by chemotherapeutic drugs like camptothecin,dead cancer cells have some extent of immunogenicity.And the immunogenic cell death(ICD)of cancer cells can promote the maturation and antigen presentation of dendritic cells,then activate T cell mediated immune response and elicit immune memory.However,it is inevitable for the dead cancer cells to elicite immunosuppression,which will greatly hinder the antitumor immune response and boost cancer immune escape.Thus,how to balance the immunogenicity and the immunosuppression is also a critical issue in cancer treatment.Indoleamine 2,3-dioxygenase(IDO)plays an important suppressive role in the tumor microenvironment.It can promote cancer stemness and induce the dormancy of CSCs.It can also enhance the suppressive function and induction of regulatory T cell,and increase the secretion of several suppressive cytokines,thus augment the immune suppression.In addition,IDO can also impair the function and differenciation of dendritic cells,and promote the anergy and exhaustion of T cells.Overall,IDO in the tumor microenvironment can inhibite the antitumor immunity and foster cancer immune escape by several ways.IDO inhibitor NLG919 can efficiently suppress IDO,and ameliorate the tumor microenviroment.Based on the background described above,a strategy that combins ICD induced by chemotherapeutic agents with IDO inhibition to modulate antitumor immunity in a multifaceted manner and eliminate CSCs for the treatment of TNBC was proposed.And by using camptothecin to induce ICD and NLG919 to inhibite IDO,a drug conjugate small molecule CN and its drug loaded nanoparticle CN@PHF were designed and synthesized.Lots of in vitro and in vivo asays were conducted to verify the effectiveness of the strategy,and to test the safty,efficacy and specific antitumor mechanisms of CN and CN@PHF.The main research contents and results are as follows:1.Preparation,characterization and in vitro activity evaluation of CN.To address the severe toxicity and poor solubility of chemotherapeutic drug camptothecin,a novel small molecule compound CN were synthesized by linking camptothecin to IDO inhibitor NLG919 by a 2,2’-dithiodiethanol.CN showed increased solubility and decreased toxicity,effective IDO inhibition and ICD induction,and promotion of the maturation of bone marrow-derived dendritic cells.2.In vivo antitumor activity of CN and its mechanism.4T1 subcutaneous tumor mice models were constructed to test the in vivo antitumor activity of CN and its mechanism by intratumoral administration.CN effectively inhibited the growth of mice TNBC 4T1 subcutaneous tumors,and its antitumor activity was significantly better than that of free CPT,free NLG919 and CPT combined with NLG919.CN showed effective promotion of the maturation of dendritic cells in paraneoplastic draining lymph nodes,reduction of tumor infiltrated Treg cells,relief of the cell cycle arrest of CSCs,elimination of CSCs,and reduction of the stemness of residual tumor cells.3.Preparation,characterization and activity evaluation of CN@PHF.In order to enhance the water solubility of CN,and to improve its tumor targeting ability with reduced toxicity and increased efficacy,a macromolecular nanocarrier polylactic acid-hydroxyethyl starchfolate was prepared in this study.By loading CN into the nanocarrier,the resultant CN@PHF was a spherical nanoparticle with a particle size of about 170 nm and uniform distribution,which was stable and could rapidly targete to tumor sites and be taken up by tumor cells.In a in vitro mudulated reductive cytoplastic condition,CN@PHF could rapidly disassemble and release the drugs to kill cancer cells efficiently.4.Antitumor activity of CN@PHF and its mechanism.The optimized dosage,antitumor efficacy and toxicity of CN@PHF were test in 4T1 subcutaneous tumor bearing mice by tail vein administration.CN@PHF showed dose-dependent antitumor efficacy,and the opimised dosage of CN in CN@PHF was choosen as 12 mg/kg.The toxicity,antitumor efficacy and mechanism of CN@PHF were test in 4T1 orthotopic tumor bearing mice by tail vein administration.CN@PHF could effectively inhibite the growth of orthotopic TNBC 4T1 tumors,and dramaticly prolong the median surrival of tumor bearing mice.The in vivo antitumor activity of CN@PHF was significantly better than positive control drugs and free CN.CN@PHF could effectively improve the tumor immune microenvironment by increasing the concentration of inflammatory cytokines IL-2 and IL-12,decreasing the concentration of immunosuppressive factors IL-6,IL-13,and TGF-β,and inhibiting indoleamine 2,3-dioxygenase to maintain a high tryptophan concentration in tumor tissues.CN@PHF treated mice exhibited effectively increased immunogenic cell death in the tumor,promoted DCs maturation,increased activation of effector T cells and memory T cells,reducd tumor-infiltrated regulatory T cells,and relieved CSCs dormancy,thus achieved effective elimination of CSCs and tumor cells.The strategy that combines immunogenic cell death induction with indoleamine 2,3-dioxygenase inhibition can effectively modulate Tumor immune microenvironment,promote antitumor immune response,alleviate immunosuppression,relieve CSCs dormancy and eliminate CSCs and cancer cells.CN has good ability to induce immunogenic cell death and inhibit indoleamine 2,3-dioxygenase activity,and can effectively regulate tumor immune microenvironment to improve the anti-tumor immune response,relieve CSCs dormancy,eliminate CSCs and inhibite tumor growth.The drug loaded nanoparticle CN@PHF is safe,effective and stable,with excellent tumor targeting ability and tumorspecific drug release.By improving the Tumor immune microenvironment and boosting the antitumor immune response,CN@PHF can effectively obliterate CSCs,inhibit tumor growth and prolong the survival of tumor-bearing mice.