Identifying Of Prostate Cancer Cell-specific Internalizing Aptamers And Engineering Of Aptamer-targeted Nanoparticles For Prostate Cancer Therapy

Background:The advances of nanotechnology in the past several decades have brought tremendous revolution to numerous areas, including modern medicine. Due to their extremely small size and conveniently manipulation, nanomaterials possessed many unique properties for medical application, especially for controlled drug delivery. From the initial passive nanomaterials to the active targeting nanovectors, and then the multi-stage delivery system, a variety of nanocarrier based drugs and imaging agents have successfully entered widespread usage. Nanomaterials can serve as carriers for drugs, genes, and imaging agents that will bind to targets on injured or neoplastic tissue, which will play a critical role in the diagnosis and treatment of cancer and other diseases.Many investigations, including basic research and clinical trials, are focused on prostate cancers due to its relatively high prevalence and its amenability to the translation of nanomedicine from animal experiments to human application. Nanoparticles (NP) encapsulated with targeting ligands can enhance cellular uptake via receptor-mediated endocytosis and provide cell-targeting specificity. However, most ligands under development are isolated from well-characterized cancer antigens, and only limited number of antigens have been characterized for cancer cell recognition.Aptamers (Apts) recently emerged as a promising class of ligands for targeted nanoparticle delivery. Apts are single-stranded RNA or DNA oligonucleotides with low immunogenicity, relatively small size, and easy to be manipulated.Objective:(1) Isolating cell-specific internalizing aptamers against prostate cancer cells and evaluating their efficiency in vitro and in vivo.(2) Identifying the prostate cancer cell memberane proteins which can specificly bind to the aptamers.(3) Endineering aptamer-targeted nanoparticles for prostate cancer therapy.Methods:(1) Cell-uptake selection:Incubating prostate normal cells in the 2’-OMe-RNA pools (counter-selection), washing and collecting the unbound RNAs and presenting them to PCa cells (PC3 and LNCaP) for cell-specific binding and uptaking. Extracting the RNAs which bind to PCa cells (cell-uptake selection) and amplifying them via rtPCR. The rtPCR products were then transcribed into 2’-OMe-RNAs for the next round of selection and sequenced for Apt indentification in the last round selection.(2) Extracting PC3 cell memberane proteins and incubating them with biotin-Apts, the proteins binding to Apts were then collected use streptavidin beads. Isolating the protein products via SDS-Page electrophoresis and silver stain. The specific protein band was cut off and identified through use spectrography analysis.(3) Encapsulating docetaxel (Dtxl) in Apt-targeted NPs (PLGA-PEG-Apt-Dtxl) via single emulsion method. The drug loading efficacy and payload releasing profile of the NPs were analysised in vitro. PC3 cells were planted subcutaneously on SCID mice to establish the prostate cancer model. PLGA-PEG-Apt-Dtxl were applied to the SCID mouse to evaluate the therapy efficiency of the Apt-targeted NPs.Results:(1) Three Apts were isolated through cell-uptake selection:XE02 and XE09 can specificly bind to PC3 cells, and XE06 can specificly bind to LNCaP cells. The structure and sequence of the Apts were analysised. XE02 was further trunked and modified into XE02 mini, which with a smaller size (32 bp) and keep the targeting properties.(2) A group of memberane protein with molecular mass of about 75 kD were identified. Further analysis of their special structure and characteristics are still needed.(3) The size of PLGA-PEG-Apt NPs was around 180 nm and the zeta potential is around-20 mV. The Dtxl loading efficacy of PLGA-PEG nanoparticles was 14.76% and the drug loading efficiency can be up to 73.8%. In vitro and in vivo experiments demonstrated that XEO2 mini targeted NPs can specificly bind to PC3 cells and better therapy efficiency was observed in SCID mice PCa models in compare with non targeted NPs or Dtxl only.Conclusions(1) A group of Apts which can specifically target to PCa cells have been generated by a simple cell-uptake selection progress. Cell-uptake selection can be done without prior knowledge of target molecules and can be applied in a wide variety of onlogic diseases.(2) A PLGA-PEG-Apt-Dtxl targeted NP platform has been developed and demonstrated enhanced therapeutic efficacy in prostate cancer.(3) A group of PCa-specific memberane protein targets have been isolated, further characterization of them may lead to the discovery of clinically important PCa biomarkers.