Research On Preparation And Targeting Anti-tumor Effect To Blood Cancer Of Platelets-based Drug Carrier System

PART 1 HUMAN PLATELETS REPURPOSED AS VEHICLES FOR IN VIVO IMAGING OF XENOTRANSPLANTSObjects:Human platelets were identified in tumors by many researchers,although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers.In this study,human platelets are repurposed as vehicles to target tumor cells within tumor microenvironment and image the tumor site in vivo by loading with imaging agent and surface-coupling proteins or antibodies.Methods:Firstly,cytoplasmic-loading of kabiramide(KabC),a potent inhibitor of actin polymerization and membrane protrusion,was determined by flow cytometry,confoal microscopy and electron microscopy.Secondly,KabC-platelets can accumulate high levels of other membrane-permeable cytoxins and probes,including epidoxorubicin,carboxyfluorescein di-ester and chlorin-e6.Flow cytometry and confoal microscopy were ultilized to optimize the reaction conditions of different cargos.Thirdly,mild reaction conditions were developed to couple tumor-targeting proteins(transferrin,Tf)and antibodies to KabC-platelets.The interactions between repurposed platelets and RPMI8226 cells or K562 cells were observed through confocal microscopy.Finally,the in vivo imaging system was used to determine the distribution of repurposed platelets within the mice bearing subcutaneous or intracranial xenotransplants.The excised xenotransplants from tumor bearing mice were further sectioned and observed under confocal microscopy.Results:KabC can inhibit platelet-aggregation in repurposing human platelets as vehicles for tumor-targeting.KabC-platelets can accumulate high levels of other membrane-permeable agents and be surface-labelled with proteins or fluorescent probes.Confocal microscopy studies showed KabC-platelets,surface-coupled with transferrin and Cy5,bound specifically to RPMI8226 and K562 cells,both of which over-express the transferrin receptor.Repurposed platelets with a feature of circulating for up to 144 hours increase their chance of interacting with target cells.KabC-platelets,surface-coupled with transferrin and Cy7,or chlorin-e6,and injected in immunocompromised mice were shown to accumulate specifically in sub-cutaneous and intracranial myeloma xenotransplants.The high-contrast,in vivo fluorescence images were recorded from repurposed platelets within early-stage intracranial RPMI8226 xenotransplants.Conclusions:Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help image special tumor cells in vivo.PART 2 HUMAN NANOPLATELETS AS LIVING VEHICLES FOR IN VIVO TUMOR-TARGETED IMAGING AND DELIVERY OF APOPTOSIS-INDUCING CYTOTOXINSObjects:In part 1,human platelets are repurposed to target tumor cells,but they are too stable to release cytoplasma-loaded drugs and can’t be endocytosed by targeted cells due to size limit.In order to utilize intact KabC-platelets to kill cells,KabC-platelets loaded with CE6,a kind of photodynamic therapy drug,could destroy the platelets membrane to release the drugs into tumor microenvironment.In this part,intact KabC-platelets are mildly sonicated to obtain KabC-nanoplatelets.Human nanoplatelets are engineered as living vehicles for tumor-targeted in vivo imaging and delivery of apoptosis-inducing cytotoxins.Methods:Nanoplatelets were generated from KabC-loaded human platelets via mild sonication and characterized by flow cytometry,dynamic light scattering and electron microscopy.Tumor-targeting and in vivo imaging functionalities are introduced to KabC-nanoplatelets by surface-coupling of transferrin or antibodies directed against a tumor marker,and near infra-red fluorescent(NIRF)probes(Cy5 and Cy7),respectively.High resolution fluorescence-imaging and flow cytometry are used to optimize the protocol of KabC-nanoplatelets preparation and detect the endocytosis of nanoplatelets by targeted cells.The apoptosis induction effect of functional nanoplatelets on RPMI8226 cells are analyzed by flow cytometry.In vivo NIRF-imaging and immuno-cytohistochemical analysis show the distribution of nanoplatelets in RPMI8226 cell-derived myeloma xenotransplants in immuno-compromised mice,as well as the apoptosis of RPMI8226 cells within xenotransplants.Results:Closed-membrane nanoplatelets,with an average size of 70～250 nm,are generated from KabC-loaded human platelets via mild sonication.KabC-nanoplatelets are shown to accumulate and retain membrane-permeable cytotoxins,including epidoxorubicin(EPI)and KabC.The high resolution fluorescence-imaging and flow cytometry analyses show that transferrin-coupled nanoplatelets loaded with EPI and Cy5 bind specifically to RPMI8226 cells,a human myeloma cell line over-expressing the transferrin receptor.The endocytosis of nanoplatelets by RPMI8226 cells is transferrin-dependent(2.5 times higher than nanoplatelets without transferrin),and induces their apoptosis.Nanoplatelets functionalized with EPI,Cy7 and transferrin are shown to accumulate in RPMI8226 cell-derived myeloma xenotransplants where they are used for high-contrast in vivo NIRF-imaging of early-stage tumors.Immuno-cytohistochemical analysis reveals extensive apoptosis of RPMI8226 cells in these tumors that is dependent on the injection of nanoplatelets functionalized with transferrin and EPI.Conclusion:Functionalized nanoplatelets represent a new class of living vehicle that has potential targeted imaging and drug delivery of therapeutic agents to tumors as well as other types of diseased tissue.