Construction And Characterization Of Camptothecin Nanodrug Delivery Systems For Cancer Therapy

Camptothecin(CPT)is a kind of alkaloid isolated from the bark and branches of camptotheca acuminate.It was used to treat cancer in the early stage by Chinese medicine personnel.However,the clinical application of CPT is hampered due to its obvious toxicity.For decades,a dozens of CPT derivatives were synthesized and brought to clinic market,including topotecan,irinotecan,bellotecan,hydroxyl camptothecin.9-amino camptothecin and other CPT derivatives have been listed in clinical trials.Camptothecin and its derivatives inhibit tumor growth by inhibiting DNA topoisomerase I(TOP I),which have a good effect on slow growing of solid tumors.Usually,the solubility of CPT and its derivatives is poor,and their lactone ring structure is unstable and easy to inactivate,resulting in large side effects.Nano drug delivery system provides an opportunity to overcome the defects of camptothecin derivatives and may expand their clinical applications.It is well known that tumor microenvironment displays weak acidity and high reducibility.By designing nanodrug delivery system of camptothecin being responsive to tumor environment,it is expected to inhibit lactone ring opening and deactivation,achieve localized drug release,improve bioavailability,reduce toxicity and enhance efficacy.Herein,the following studies is performed:(1)In the first part,redox responsive and core-crosslinked nanomicelles with high stability were developed for CPT delivery.Camptothecin-lipoic acid prodrug(CPT-LA)and two crosslinkable surfactants containing LA and were synthesized.The core-crosslinked nanomicelles containing CPT-LA(CPT-LA CNM)with a highly crosslinked core and a PEG hydrophilic shell were obtained by adding tetra-(3-mercaptopropionic acid)pentaerythritol ester(PETMP)using emulsification.Dynamic light scattering(DLS)characterization indicated that CPT-LA CNM possessed a narrow size distribution and negatively charged zeta potential(184.6±3.6 nm and-3.5±1.2 m V,respectively).The storage and physiological stabilities were further studied by measuring their size change.The results showed that CPT-LA CNM was extremely stable in different conditions.More importantly,a reduction responsive release of parent drug CPT was observed in the medium containing dithiothreitol(DTT).In addition,CPT-LA CNM had a much more rapid cellular uptake against cancer cells under confocal laser scanning microscopy(CLSM).Finally,the enhanced anti-tumor efficacy of CPT-LA CNM was further demonstrated by in vitro cytotoxicity and cell apoptosis assay.(2)In the second part,a redox-triggered double camptothecin(CPT)liposomal system was developed.Two amphiphilic CPT prodrugs(CPT-SS-3-GPC and CPT-SS-11-GPC,abbreviated as CPT-SS-GPCs)were synthesized by conjugating two CPTs to glycerylphosphorylcholine(GPC)via disulfide bond linker with different chain length.After that,CPT-SS-GPCs were assembled into liposomes using classic reverse evaporation method.The drug loading of CPT-SS-GPC_S liposomes was more than 40 wt.%.Morphology and size of the liposomes were characterized by transmission electron microscope(TEM),cryo-transmission electron microscope(cryo-TEM)and dynamic laser scattering(DLS)techniques.From the data,spherical CPT-SS-3-GPC and CPT-SS-11-GPC liposomes demonstrated uniform sizes of 194.2±3.8 nm and 160.1±2.5 nm.Moreover,the rapid release of parent CPT in reductive condition was verified through in vitro release and degradation experiments.By confocal laser scanning microscopy(CLSM),obviously high cellular uptake of CPT-SS-3-GPC and CPT-SS-11-GPC liposomes were observed using MCF-7 cell model.In vitro cytotoxicity and apoptosis assay against MCF-7 cells suggested that CPT-SS-11-GPC liposomes perform better anti-tumor activity than CPT and commercial irinotecan(Ir).(3)In the third part,a redox-triggered single 7-Ethyl-10-Hydroxycamptothecin(SN38)liposomal system was developed.SN38 was conjugated with lysophospholipid by using a cleavable disulfide bond linker.After that,the conjugate(SN38-SS-PC)was assembled into liposomes by thin film method.Dynamic light scattering(DLS)characterization indicated that SN38-SS-PC liposomes possessed a narrow size distribution(172.8±10.5 nm)and negative charged zeta potential(-8.9±0.3 m V).The results of storage and physiological stabilities showed that SN38-SS-PC liposomes was stable under different conditions.More importantly,a reduction responsive release of parent drug SN38 was observed in the medium containing glutathione(GSH).In addition,SN38-SS-PC liposomes had a much more rapid cellular uptake behavior against cancer cells.The enhanced anti-cancer efficacy of SN38-SS-PC liposomes was further demonstrated by in vitro cytotoxicity assay against MCF-7 and A549 cells.Under in vivo evaluation in 4T1 xenograft tumor model,SN38-SS-PC liposomes were observed to have lower systemic toxicity and higher tumor inhibition rate of 53.3%compared with the commercialized SN38prodrug Irinotecan(Ir).(4)In this chapter,dimeric artesunate-phosphatidylcholine conjugate(d ARTPC)-based liposomes encapsulated with irinotecan(Ir)were developed for anticancer combination therapy.First,d ARTPC featured with unique amphipathic properties formed liposomes by classical thin film methods.After that,Ir was encapsulated into d ARTPC-based liposomes(Ir/d ARTPC-LP)by the triethylammonium sucrose octasulfate gradient method.Physicochemical characterization indicated that Ir/d ARTPC-LP had a mean size around 140 nm and negative zeta potential of approximately-30 m V.Liposomes displayed an encapsulation efficiency of greater than 98%with a controllable drug loading of 4-22%.The in vitro release of dihydroartemisinin(DHA)and Ir from Ir/d ARTPC-LP was investigated by dialysis in different media.It was found that effective release of both DHA(65.42%)and Ir(77.28%)in weakly acidic medium(p H 5.0)after 48 hours was achieved in comparison to very slow release under a neutral environment(DHA9.90%and Ir 8.72%),indicating the controllable release of both drugs.Confocal laser scanning microscopy confirmed the improved cellular internalization of Ir/d ARTPC-LP.The cytotoxicity of Ir/d ARTPC-LP was evaluated in the MCF-7,A549 and Hep G2 cell lines.The results showed that Ir/d ARTPC-LP had significant synergistic efficacy in loss of cells growth.In vivo anticancer evaluation was performed by using a 4T1 xenograft tumor model.Ir/d ARTPC-LP had a high tumor inhibition rate of 62.7%without significant toxicity in comparison with an injection of Ir solution.In summary,three new camptothecin derivatives prodrug were synthesized,and assembled into unique redox-triggered nano drug delivery system.These nanosystems have high drug loadings,effective drug release,excellent stability and good antitumor effect.More importantly,they overcome the defect of drug is easy to leak and lactone ring is easy to open.Finally,a camptothecin derivative(Ir)was encapsulated into artesunate phosphocholine liposomes,which achieved synergistic inhibition of tumor growth.This paper provides new ideas for the development of camptothecin nanodrugs,and also lays a foundation for the preclinical research of camptothecin derivative nanodrugs in the future.