Development Of New Camptothecin-and Ruthenium(Ⅱ)-based Anticancer Drug Candidates For The Treatment Of Malignancies

Background:The increased global burden of malignancies threatens the health and life of human.Chemotherapies based on the small molecules as the main treatment of malignancies are troubled with the severe side effects,low tumor selectivity and undesired drug resistance,which limits the clinical application.Thus,development of novel antitumor agents is promising to overcome the dilemma of chemotherapeutics and provides some new ideas for cancer therapy.Nano drug delivery system possesses the advances of improving water solubility,prolonging blood circulation,enhancing tumor accumulation and alleviating toxicity,thereby arousing more research interests in cancer treatment.Irinotecan(CPT-11)remains the standard-of-care drug for the treatment of malignancies,but only exhibits 0.1-1%of in vitro activities compared to its active metabolite 7-ethyl-10-hydroxycamptothecin(SN38)due to the low conversion rate.However,the clinical application of SN38 is limited by the poor solubility.Hence,it’s promising to construct novel SN38 nanomedicines by combination of nanotechnology and prodrug strategy.Additionally,in consideration of the side effects and drug resistance of the most widely applicated platinum-based drugs,it’s worthwhile to develop novel metallodrugs with highly efficiency and safety.Aim:The SN38 was covalently conjugated to oligo-ε-caprolactone(oligoCL)via an esterase-activatable linkage to obtain polyprodrugs,followed by encapsulation with amphiphilic polymer matrices to form a series of nanomedicines.The SN38 nanomedicines possessed desirable water solubility,long-term blood circulation,tumor-targeting capacity,responsive release profiles,which provided a new strategy for the treatment of colorectal cancer.Additionally,a series of ruthenium complexes were synthesized and the optimized molecule with excellent biological activities was screened,aiming to accelerate the development of metallodrugs.Methods:Section 1:The SN38 was ligated with several molecular-weight oligoCL via esterification to give a series of esterase-catalyzed polyprodrugs,and subsequently co-assembled with PEG10k-b-PCL10k through nanoprecipitation to form nanomedicines(termed oligoCLn-SN38 NPs,n=7,14,28).Dynamic light scattering and transmission electron microscopy were employed to characterize the size and morphology of nanoparticles.In vitro experiments were conducted to evaluate the stability and esterase-triggered release profiles of nanoparticles.The cytotoxicity of nanoparticles against several colorectal cancer cell lines was detected by CCK-8 assay,followed by the apoptosis investigation via AO/EB assay.Hemocompatibility of nanoparticles was testified by hemolysis assay.Xenograft mouse models and a chemically induced orthotopic colorectal cancer model were employed to determine the in vivo anticancer efficiency of nanoparticles.Pharmacokinetic assay was conducted to monitor the time-dependent plasma drug concentration.The systemic distribution and tumor-targeting capability of nanoparticles were testified through near-infrared(NIR)fluorescence imaging and tumor drug concentration analysis.Gastrointestinal tolerability of nanoparticles was evaluated by bloody diarrhea assay and mass spectrometry liquid chromatography coupled with tandem mass spectrometry.Finally,the multiplex cytokine assay was used to determine the immunotoxicity of nanoparticles.Section 2:A series of cyclometalated ruthenium(Ⅱ)complexes with multiple biological functions were synthesized and characterized by 1H NMR,13C NMR and electrospray ionization-mass.The cytotoxicity of complexes against several cancer cell lines was confirmed by MTT assay and the most active ruthenium complex was screened.AO/EB and EdU assays were conducted to investigate the ability of optimized complex in inducing apoptosis and inhibiting proliferation,respectively.Wound-healing assay and transwell assay were used to evaluate the antimetastasis capacity of optimized complex.The antiangiogenesis capability was demonstrated by the wound-healing assay and tube formation assay.Western blot assay was included to evidence the mechanism of optimized complex in inducing apoptosis,antiproliferation and antimetastasis.Finally,a metastatic ovary cancer xenograft mouse model was employed to demonstrate the therapeutic outcomes of optimized ruthenium complex.Results:Section 1:The oligoCL-tethered SN38 prodrugs were capable of constructing with PEG10k-b-PCL10k to form homogeneous and spherical nanoparticles.The particle sizes of oligoCL7-SN38 NPs,oligoCL14-SN38 NPs and oligoCL28-SN38 NPs were 63.8±19.3 nm,69.3±16.8 nm and 83.2±19.2 nm,respectively.The nanoparticles assembled by prodrugs with high-molecular-weight(n=14 and 28)were ultrastable in the phosphate-buffered saline(PBS)supplemented with 10%fetal bovine serum over one week,which indicated the superior stability.Meanwhile,the nanoparticles based on the prodrugs with high-molecular-weight exhibited slower release profiles.For example,only 21.8%of active drugs were released from oligoCL28-SN38 NPs in PBS conditions,while the oligoCL7-SN38 NPs released 47.2%of SN38.However,the release profiles were accelerated in the presence of 60 U/mL of esterase,and all the drugs were released from nanoparticles within 72 h.The esterase-activatable capability contributed to the rapid release in cancer cells.CCK-8 assay indicated that the cytotoxicity of nanoparticles was comparable to SN38 and significantly higher than CPT-11.AO/EB assay demonstrated that the efficacy of nanoparticles in inducing HCT-116 cell apoptosis was remarkably superior to CPT-11.Negligible hemolysis rate(<3%)of nanoparticles even at higher concentration suggested the desirable hemocompatibility.Nanoparticles produced superior therapeutic outcomes in two kinds of HCT-116 xenografts compared with CPT-11.Especially in a tumor-recurrent model,the tumor inhibition rates of oligoCL7-SN38 NPs,oligoCL14-SN38 NPs and oligoCL28-SN38 NPs were 65.5%,83.8%and 95.8%,respectively,which was markedly higher than that of CPT-11(34.0%).These results manifested that the nanoparticles based on the high-molecular-weight prodrugs displayed preferable antitumor efficacy,thereby screening the optimized oligoCL28-SN38 NPs.Meanwhile,the optimized nanoparticles significantly restrained the tumor number and sizes of orthotopic colorectal cancer which was failed to be cured by CPT-11,as well as alleviated the shortening of colons,showing superior anticancer efficiency.Pharmacokinetic analysis suggested that nano drug delivery systems markedly prolonged the blood circulation.The half-life times of oligoCL7-SN38 NPs,oligoCL14-SN38 NPs and oligoCL28-SN38 NPs were 12.83±1.69 h,22.70±1.42 h and 23.00±1.88 h,respectively,which were remarkably longer than that of CPT-11(2.11±0.17 h).NIR fluorescence imaging and tumor distribution assays elucidated that nanoparticles exhibited better systemic retention and tumor-targeting ability than small molecules.The optimized oligoCL28-SN38 NPs showed highest tumor accumulation.Finally,nanoparticles could alleviate gastrointestinal toxicity due to diminish the exposure of SN38 within colons,showing higher safety than CPT-11 and no obvious immunotoxicity was observed.Section 2:The cytotoxicity of ten synthesized ruthenium(Ⅱ)complexes(Ru1-10)bearing N-Heterocyclic carbenes was evaluated by MTT assay.The Ru8 was demonstrated to possess the highest bioactivities against several cancer cell lines,showing comparable toxicity to cisplatin.The EdU and AO/EB analysis confirmed that Ru8 significantly restrained the proliferation of A2780 cell lines and induced remarkable apoptosis.Western blot assay evidenced that Ru8 could upregulate the expression of c-PARP,c-Caspase 3 and c-Caspase 9 to induce apoptosis and disrupted the Cdc25c/Cdc2/cyclin B1 signaling pathway to arrest the cell cycle at G2/M phase for antiproliferation.The wound-healing,transwell and tube formation assays collectively suggested that Ru8 was capable of inhibiting the migration and invasion of cancer cells,as well as disrupting the formation of capillary networks of endothelial cells,showing the potential of antimetastasis and antiangiogenesis.In a metastatic ovary cancer model,after treatment with Ru8,the inhibition rate of orthotopic tumor reached to 91.4%,and average tumor weight was only 33.1%than that of cisplatin,suggesting the superior of Ru8 as chemotherapeutics.Conclusion:A series of esterase-activatable SN38 nanomedicines were constructed and evidenced that not only reversed the low conversion of CPT-11,but also possessed long-term systemic retention,tumor-targeting accumulation,responsive release kinetics,enhanced therapeutic effectiveness against colorectal cancer and improved safety.The optimized oligoCL28-SN3 8 NPs holds potentials as new nanotherapeutics for the treatment of malignances.In view of the wide application of metallodrugs,a series of ruthenium complexes were additionally synthesized and the optimized Ru8 was determined to possess cytotoxicity,antimetastasis and antiangiogenesis,holding potentials as candidates of novel metallotherapeutics.