Preparation Of Novel 7-ethyl-10-hydroxycamptothecin Nano-drug Delivery Systems And Their Preclinical Studies

Nanoparticulate drug delivery systems offer the possibility to improve the water solubility of traditional small molecular antitumor drugs,improve pharmacokinectics,increase the targeting ability and enhance the therapeutic activity.Nanoparticulate drugs could selectly accumulate in the tumor tissues during blood circulation via the enhanced permeability and retention(EPR).However,due to the complex biological process such as angiogenesis and vascular permeabilization,development of tumor heterogeneity and variable tumor microenvironment,The EPR effect is variable among different patients and tumor types.Therefore,EPR effect alone is not sufficient to favor the accumulation of drugs in the tumor.The surface decoration of nanoparticles with targeting ligands,such as antibody,peptide,aptamer,specifically binding to the receptors overexpressed on the surface of tumor vessels or tumor cells,increase the affinity of nanoparticles to tumor cells,and then enhance the tumor accumulation and the internalization of drugs.In addition,the pathological structures and metabolic features of tumor tissues provide the opportunity to design the stimuli-responsive drug delivery system,such as pH-responsive,oxidation-responsive,reduction(GSH)-responsive and enzyme-responsive,to enhance the controlled release of drugs in the tumor.Amphiphilic block copolymers or graft copolymers could be sef-assembled into polymeric micelles with a unique core-shell sturcture.Poly(ethylene glycol)(PEG)was selected as the hydrophilic part to improve the biocompability of the nanoparticles,avoid the reticuloendothelial system(RES)recognition and prolong the blood circulation.A hydrophobic drug as the hydrophobic part of the block copolymer would increase the drug loading content,minimize the use of other inert materialsand reduce the side effect.7-ethy-10-hydroxycamptothecin(SN38),the active metabolite of irinotecan(CPT-11),has 100-1000 fold more potent cytotoxicity in vitro compared with CPT-11.However,SN38 is extremely low soluble in any physiologically compatible and pharmaceutically acceptable solvents,which could be the hydrophobic part of polymeric micelle.Recently,our group have sucessfully synthesized an amphiphilic SN38 prodrug,named PEG-S-S-SN38,by conjugating PEG and SN38 with both disulfide bond and carbonic ester linkage as linkers.The amphiphilic PEG-S-S-SSN38 with a SN38 loading content of 15%wt.%could self-assemble into spherical nanoparticles with a diameter of about 73 nm in aqueous solution.We assumed that PEG-S-S-SN38 may be GSH/esterase/H2O2 triple-stumili responsive due to the introduction of disulfide bond and carbonic ester linkage.In the first part of this thesis,we will investigate the multiple stimuli-responsive characterization of PEG-S-S-SN38 and the in vitro and in vivo antitumor activities.The data demonstrated that PEG-S-S-SN38 nanoparticles were stable and have no initial burst release in PBS(pH 7.4),but released SN38 promptly and exhibited favorable stimuli-reponsive to GSH,esterase and H2O2 due to the presence of disulfide bond and carbonic ester linkage.The data from in vitro anticancer activity of PEG-S-S-SN38 demonstrated that PEG-S-S-SN38 nanoparticles could be quickly internalized into tumor cells and release active SN38.Moreover,PEG-S-S-SN3 8 exhibited more potent antitumor activity than CPT-11 and comparable anticancer activity as free SN38 against a panel of tumor cell lines.Most importantly,PEG-S-S-SN38 significantly inhibited the growth of tumors compared with CPT-11 in Bcap 37 xenograft tumors and no evident side effect was found.These encouraging data deserve further preclinical and clinical research on this novel SN38 delivery system.The second part of this thesis aims to design and develop a novel SN3 8 prodrug,which could actively target tumor and enhance the penetration of drug delivery systems.Firstly,we prepared an amphiphilic SN38 prodrug according to our previous systhesis route,named B-SN38,by using PEG as initiator and HEMASN38 as monomer through ATRP method.It have been known that CRGDK could specifically bind to neuropilin-1(Nrp-1)receptor overexpressed on the surface of tumor vessels and tumor cells,mediating cell internalization and tissue penetration.Subsequently,we coupled the amphiphilic block copolymer with CRGDK peptide to synthesize C-SN38.Through adjusting factors such as the type of water-miscible organic solvent or/and the block copolymer concentrations,both amphiphilic B-SN38 and C-SN38 prodrugs could self-assemble into spherical nanoparticles with sizes of 30 nm and 100 nm respectively.The detailed biological characterizations of B-SN3 8 and C-SN38 with size of 30 nm in vitro showed that both B-SN38 and C-SN38 nanoparticles were internalized via endocytosis.Importantly,significantly enhanced cellular uptake,as well as significantly increased cell-killing activity were observed in MDA-MB-231 and HT-29 cells overpressing Nrp-1 incubated with C-SN38 nanoparticles compared with B-SN38.Data from biological characterizations of B-SN38 and C-SN38 in vivo showed that C-SN38 nanoparticles possess significantly improved tumor accumulation and penetration capabilities than B-SN38 nanoparticles.Moreover,C-SN38 show dramatically increased in vivo anticancer activity compared with B-SN38 and CPT-11 in MDA-MB-231 and HT-29 xenograft tumors.In addition,two different nanoparticle sizes(30 nm and 100 nm)from the same prodrugs did not significantly affect the in vivo antitumor efficacy.In brief,We demonstrated for the first time that CRGDK functionalization could be a promising strategy for efficient delivery of SN3 8,and C-SN3 8 is a potent drug candidate for the treatment of neuropilin-1 overexpressing tumors deserving further research.