The Construction Of Prodrug-based Liposomes And Polymeric Nanoparticles For Antitumor Study In Combination With Apatinib

Background:Malignancy is a life-threatening disease to the citizens in our country.Chemotherapy（i.e.,paclitaxel or camptothecin）is the one of the dominant treatment therapies for malignant tumors,whereas its clinical application has been substantially hindered by the constantly emergence of severe side effects and drug resistance in patients.Rational drug conjugation to traditional chemotherapeutics and subsequent delivery with nanoparticle platforms have now been the focus for studying anti-tumor medicines.Prodrug is formed by the specific chemical modification to parent component,which forms the temporal chemical conjugation and release active form via enzymatic or non-enzymatic mechanism.The fabrication of prodrug by chemical conjugation on chemotherapeutics increases the biocompatibility with liposomal or polymeric vehicles.Delivering prodrugs with nano-strategies could delay the drug release from scaffolds,enhancing the drug retention during systemic circulation and improving pharmacokinetics（PK）.Co-delivering of chemotherapeutics and molecular targeting agents of anti-angiogenesis is also a crucial regime in oncotherapy,which is supposed to efficiently exert synergistic tumoricidal effect,to overcome drug resistance,to reduce the dose of monotherapy and thus to alleviate drug toxicity.Aim:Section 1:Covalently conjugate taxane derivative cabazitaxel with unsaturated fatty acids docosahexaenoic acid（DHA）to synthesize prodrug DHA-Cabazitaxel and then co-assemble it with liposome compositions to fabricate stable cabazitaxel-incorporated liposomes（Lipoprodrug）.We are expected to evaluate the cytotoxicity of Lipoprodrug to tumor cells and explore the relative mechanism in vitro.The tumor targeting efficiency,drug distribution in tumor,PK,antitumor efficacy and biosafety will be assessed in multiple animal models.Section 2:Irinotecan active product 7-Ethyl-10-hydroxycamptothecin（SN38）is covalently conjugate with polymer polylactic acid（PLA）to synthesize prodrug p SN38.Then we explore the capability of p SN38 to formulate stable polymeric nanoparticles（NPs）in water when co-assembled with amphiphilic polymer m PEG-PLA.Anti-angiogenesis molecular targeting agents that could be stabilized in the cytotoxic polymeric nanoparticles via noncovalent interaction with p SN38 will be screened and chosen.The cytotoxicity of coloaded NPs to tumor cells and the effect of NPs on the function of HUVEC will be studied.Antitumor/antimetastasis efficacy and relative mechanism of the combination therapy in hepatocellular carcinoma（HCC）will be assessed in multiple mice models.Method:Section 1:Cabazitaxel was PUFAylated by DHA couple via ester bond to form prodrug DHA-Cabazitaxel.Then it was co-assembled with liposome compositions(lecithin,cholesterol and DSPE-PEG₂₀₀₀)to prepare Lipoprodrug via ethanol dilution.Liposomes were characterized by measuring size,zeta potential,morphology and drug release in vitro.We evaluated the cellular endocytosis of liposomes inside tumor cells.The cytotoxicity efficacy and mechanism of Lipoprodrug to tumor cells were assessed by MTT,flow cytometry of cell apoptosis/cell cycle,Western Blot and tubulin staining at cellular level.The tumor targeting efficiency and tumor distribution of Lipoprodrug were analyzed by in vivo imaging and biodistribution study in tumor xenograft bearing mice models.We evaluated PK of liposomes in SD mice.Antitumor potency and toxicity of liposomes were then evaluated in two CDX models（DU145 and A375 derived）and a melanoma PDX model.At last,the biosafety（i.e.,organ toxicity,myelosuppression and hepatorenal toxicity）of Lipoprodrug were assessed in healthy mice.Section 2:PLA was conjugated to SN38 via ester bond to construct prodrug p SN38and subsequent formulating cytotoxic NPs with amphiphilic polymer m PEG-PLA via nanoprecipitation.Then we screened the anti-angiogenesis tyrosin kinase inhibitor that could be stabilized in the cytotoxic NPs to form stable coloaded NPs,s TKI-p SN38.Molecular dynamic（MD）simulation was used to analyze the interaction between p SN38and small molecular inhibitor in the coloaded system.The characterization and drug release of NPs were performed in vitro.The cytotoxicity of s TKI-p SN38 to tumor cells were assessed by MTT.Tube formation assay,cell migration and cell invasion assay were performed to study the function inhibition on HUVEC by s TKI-p SN38.The chick chorioallantoic membrane（CAM）model was utilized to further explore the antiangiogenesis efficacy of s TKI-p SN38 in vivo.The antitumor efficacy was examined in Huh-7 derived xenograft mice model.The biodistribution of NPs and the inhibition on primary tumor and tumor metastasis was explored in H22 derived xenograft footpad mice model.Results:Section 1:DHA-Cabazitaxel is able to co-assemble with liposome compositions to fabricate stable liposomes with size of～130 nm and zeta potential of-29.33±7.24 m V.Transmission electron microscopy observation reveals monodisperse and spherical shapes of Lipoprodrug with characteristic liposomal particle sizes.The prodrug-based liposome achieved delayed drug release and could be effectively uptake by tumor cells.Lipoprodrug exerts the similar high cytotoxicity to tumor cells with free cabazitaxel and it induces cell apoptosis by introducing microtubule depolymerization inhibition,G2/M cell cycle arrest and activating classical Caspase signaling pathway.The in vivo study demonstrated that Lipoprodrug had excellent tumor targeting capacity and could efficiently accumulate in tumor.PK analysis demonstrated that,in comparison with free cabazitaxel,prodrug-based liposomal formulation significantly elongated the half-life period of cabazitaxel.The preferable antitumor efficacy and safety of Lipoprodrug was demonstrated in two CDX models and one melanoma PDX model.One out of five mice experienced complete tumor eradication and no significant body weight loss in the lipoprodrug-administered group in PDX model.Conversely,free cabazitaxel couldn’t bring to complete cure but elicited a rapid weight drop（～24%）at 8 days posttreatment.In vivo safety evaluation shows that Lipoprodrug increased the maximal tolerable dose of cabazitaxel by at least 10-fold and also substantially reduced organ toxicity,myelosuppression and hepatorenal toxicity of cabazitaxel.Section 2:After prodrug p SN38 was successfully synthesized,we screened and found that inhibitor Apatinib can be stably coloaded with p SN38 in m PEG-PLA assembled NPs.The resulting coloaded NPs（s TKI-p SN38）are characterized with size of～40 nm and size potential of-0.19±0.20 m V.s TKI-p SN38 achieved the sequential release of anti-angiogenesis agent Apatinib and subsequent cytotoxic SN38 in the presence of PBS.Interestingly,the release of cytotoxic SN38 was accelerated in the presence of cancer cell lysate.MD simulation shows that there areπ-πstacking and hydrogen-bond interactions existing between p SN38 and Apatinib.It is those non-convalent interactions that stabilize Apatinib in the cytotoxic polymeric NPs.Revealed by MTT assay,s TKI-p SN38 exert the similar high cytotoxicity to HCC cells with free SN38.In vitro analysis also demonstrated that s TKI-p SN38 simulates free Apatinib in inhibiting cellular function of HUVEC,including tube formation,cell migration and cell invasion,which is functioning through the inhibition of VEGFR2/ERK1/2 signaling pathway.Moreover,we found the vessel branches are substantially reduced after s TKI-p SN38 treatment in CAM model.Those results show that coloaded NPs is able to inhibit angiogenesis both in vivo and in vitro.The single injection of the s TKI-p SN38 exhibited potent tumor eradication efficacy in Huh-7 derived xenograft mice model,superior than that exerted by coadministration of two single-drug-loaded NPs.Near infrared imaging on footpad mice model suggest that s TKI-p SN38 have higher tumor targeting efficacy than the free drugs,which could not only target primary tumor lesions but also efficiently target adjacent lymph node.We found that the combination nanoparticulate strategy was able to simultaneously inhibit primary tumor growth and tumor metastasis to lymph node in footpad mice model,which is supposed to inhibit tumor development and metastasis and to some extent may could prevent the occurrence of drug resistance.Conclusions:This research developed a prodrug strategy to increase the compatibility of chemotherapeutics with nanocarriers.Chemotherapeutic agent cabazitaxel or SN38 was covalently conjugate to lipid DHA or polymer PLA to construct prodrug formulated liposomes（Lipoprodrug）or prodrug formulated polymeric NPs（s TKI-p SN38）.The stability of cabazitaxel was increased tremendously in the form of Lipoprodrug for drug delivery.Furthermore,prodrug-based liposomes greatly improve PK properties and drug accumulation of cabazitaxel in tumor,which renders elevated antitumor efficacy and alleviated drug toxicity.Polymeric NPs could not only realize the rational combination of anti-angiogenesis agent Apatinib and cytotoxic SN38,achieving the spatiotemporal drug release to exert synergistic HCC eradication effect,but also hinder the metastasis of HCC by targeting lymph node.The above two prodrug-based nanoparticle drug delivery systems and the drug combination strategy provide novel therapeutic thoughts and regimes for both HCC and other types of cancer.