The Investigation of Gold Nanoparticles as Radiosensitizers to Treat Prostate Cancer in 3D In Vitro models

Abstract

Prostate cancer (PCa) is the most prevalent cancer among Canadian men (1). Despite advancements in PCa treatment, castration resistant PCa (CRPC) and metastatic CRPC (mCRPC) remain difficult to treat with the currently limited treatment options, and radioresistance and recurrence persist (169,170). Radiation therapy and surgery are the primary treatments for localised PCas; however, if the cancer has metastasized, systemic treatments like chemotherapy may be used (26). To address the challenge of radioresistance, we investigated gold nanoparticles (GNPs) that leverage both the photoelectric properties of gold and the anti-cancer effects of phytochemicals, like curcumin (135,158,188). This research focused on synthesizing, characterizing and evaluating curcumin-coated gold nanoparticles (Curc-GNPs) for their efficacy as radiosensitizers for prostate cancer therapy using three-dimensional DU145 prostate cancer spheroids. In this study, two gold nanoparticle formulations, citrate-coated GNPs (Cit-GNPs) and Curc-GNPs were synthesized and characterized. Curc-GNPs were synthesized using an eco-friendly single-step green synthesis method, where curcumin acted as both a reducing and stabilizing agent (188,202). Cit-GNPs were synthesized using a citrate reduction method, known as the Turkevich method (203-205). Ultraviolet-visible spectroscopy was used to confirm the successful formation of spherical GNPs, where surface plasmon resonance peaks (SPR) were observed in the 520-530 nm range (188,202,204). Dynamic light scattering (DLS) analysis demonstrated that both formulations produced small nanoparticles with hydrodynamic diameters ranging from 18 – 25 nm. The average hydrodynamic diameter of Curc-GNPs and Cit-GNPs were 18.36 ± 0.27 and 22.55 ± 0.18 nm respectively. Both formulations had negative zeta potentials with Curc-GNPs measuring -44.5 ± 1.2 mV and Cit-GNPs measuring -40.0 ± 4.6 mV. Inductively coupled plasma optical emission spectroscopy (ICP-OES) showed that Curc-GNPs had a higher final gold concentration than Cit-GNPs, with measured concentrations of 361 ± 15 µg/mL and 20 ± 4 µg/mL, respectively. Overall, these results confirmed the successful synthesis and characterization of stable Curc-GNPs and Cit-GNPs suitable for downstream biological evaluation. In vitro studies supported the therapeutic efficacy of Curc-GNPs as radiosensitizers in three-dimensional DU145 prostate cancer spheroids, which better reflect the tumour-like architecture compared to conventional monolayer culture, and the therapeutic resistance seen in CRPC and mCRPC (148,188). Cellular uptake was quantified using ICP-OES after three-dimensional DU145 prostate cancer spheroids were exposed to different GNP formulations, concentrations and incubation times (148,188,202). The results demonstrated that nanoparticle uptake in spheroids was influenced by GNP coating type, dose, exposure time and the interaction of these variables. Curc-GNP uptake was generally greater than Cit-GNP uptake, with the strongest uptake observed for 100 µg/mL Curc-GNPs at 24 hours. At this concentration and time point Curc-GNP uptake was 4.76-fold higher than Cit-GNP uptake, suggesting that functionalizing curcumin onto GNPs improved nanoparticle internalization in DU145 spheroids (188,202). Clonogenic assay was used to evaluate the radiosensitizing potential of Curc-GNPs after exposure to clinically relevant 6 MV photon radiation doses of 0, 2, 4, and 6 Gy (33,137,188). Linear-quadratic model fitting demonstrated concentration-dependent radiosensitization, where with increasing Curc-GNP concentration, a progressive downward shift in survival was observed. This was supported by the increased α values, decreased β values, reduced D₅₀ values, increased sensitization enhancement ratios, and elevated α/β ratios (206-207). The D₅₀ decreased from 2.012 Gy in untreated controls to 1.295 Gy at 200 µg/mL Curc-GNPs, while the sensitization enhancement ratio (SER) increased from 1.000 to 1.554. These results suggest that Curc-GNPs were able to enhance radiation-induced cell killing through single-track DNA damage in DU145 spheroids, where the greatest radiosensitizing effect was seen at the highest concentration investigated 200 µg/mL (137,158,188). This thesis demonstrates the successful synthesis and characterization of Cit-GNPs and Curc-GNPs and supports the potential of Curc-GNPs as radiosensitizers for prostate cancer therapy. The findings show that curcumin functionalization improved GNP uptake in DU145 spheroids and enhanced radiation-induced cell death with increasing concentration. These results provide an important foundation for the development and optimization of Curc-GNPs as radiosensitizers, where future studies should investigate nanoparticle localization within tumour spheroids, confirm the biological mechanisms of radiosensitization, and optimize Curc-GNP formulations so they have improved uptake, stability, and localization in prostate cancer models (148,158,188). Overall, this work supports the continued development of Curc-GNPs as a promising radiosensitizers in prostate cancer.