Design of a Smart, Stealth Nano-system for Targeted Drug Delivery in Prostate Cancer Treatment

Abstract

There have been several polymeric drug delivery vehicles developed on the nanoscale for chemotherapeutic applications. However, the use of polymers without the implementation of design perspectives has led to limitations in efficacy, including rapid elimination, poor biocompatibility, and high off-site delivery of the therapeutic agent. In this study we developed polymeric nanoparticle systems with enhanced cell penetrating and targeting abilities with the aid of design and optimization. The ultimate goal was to fabricate an optimized system with improved properties and performance as a nanocarrier for the chemotherapeutic drug docetaxel to prostate cancer tissue. Poly (lactic-co-glycolic) acid provided the ideal polymeric matrix to entrap hydrophobic drugs with high efficacy. Chitosan provided a positively charged surface to the system, ensuring favorable interactions with negatively charged cell membranes. Poly ethylene glycol provided a shielding effect to increase in vivo residence time, while folate and anti-PSMA antibody acted as targeted ligands to bind to prostate specific membrane antigen, PSMA, a cell surface receptor that is overexpressed on prostate cancer cells. Fabrication approaches compared traditional and microfluidics-based method, and we were able to prepare uniform, reproducible nanoparticles using microfluidics. We used disulfiram and the plant- based bioactive quercetin as model bio-actives, and investigated chitosan and folic acid for their reported cell penetrating properties, using a design of experiments optimization. Finally, we used the drug docetaxel, with PLGA and PEG as matrix polymers and anti-PSMA antibody as a targeting ligand for its specific ability to bind to PSMA. All systems were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), Thermogravimetric Analysis (TGA), and X-ray powder diffraction (XRD). Drug loading and release were conducted by ultra-violet (UV) spectroscopy and high-performance liquid chromatography (HPLC). Using the microfluidics method and an intermediate PEG density, the disulfiram encapsulated nanoparticles were prepared with a size of 179±2.5 nm and maximum drug release of 70%. The targeted systems loaded with quercetin or docetaxel both displayed sustained pH dependent release profiles with maximum release of 78% quercetin and 72% of docetaxel at the tumour microenvironment- relevant acidic pH of 6. We evaluated in vitro toxicity of these systems on PSMA positive (LnCap) and negative (PC-3) cell lines as well as on a non-cancerous cell line (NIH-3T3), and thereafter conducted comparative fluorescence-based cellular uptake studies. Both targeted systems demonstrated higher cellular uptake in LnCap and an increase in toxicity of 66% for the quercetin-loaded and 60% for the docetaxel-loaded systems on LnCap compared to PC-3. The anti-PSMA targeted docetaxel-loaded system also selectively caused cell cycle arrest and 89,4±8,4% inhibition of cell migration in LnCap cells, but no inhibition on PC-3 cells, suggesting a PSMA-specific mechanism of action in these processes. Finally, we used an in vitro 3D LnCap multicellular tumour spheroid model to test for anti- cancer efficacy of the prepared docetaxel loaded nano-systems, and an in vivo nude mouse model to evaluate its biocompatibility. There was significantly more inhibition of spheroid growth by the non- targeted system (18,3±5,7%) and the targeted system (50,4±3,3%) compared to the untreated group after fifteen days of treatment, while all treatments showed no toxicity on the major organs of nude mice. Further studies are required to explore the potential of the PSMA targeted nano-system for its in vivo anti-cancer therapeutic potential.