Impact of PSMA Targeted Gold Nanoparticles on Prostate Cancer Radiation Bystander Effect

Abstract

Gold nanoparticles (GNPs) are effective radiosensitizers due to their ability to enhance ionizing radiation absorptance and deposit elevated dose in their vicinity. Radiation induced bystander effect (RIBE) expands radiation effects to non-targeted cells close to radiation-targeted cells, further improving radiation outcome. In this study, we investigated the impact of PSMA-targeted GNPs on RIBE. We hypothesize that the PSMA-targeted GNPs would intensify radiation effects by augmenting RIBE consequence as well as enhancing sensitivity of bystander cells to RIBE.Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PE Gylated GNPs through EDC/NHS chemistry. UV-Vis spectrophotometry and real time cell analysis were employed to evaluate the cellular uptake and biocompatibility, respectively, when LNCaP prostate cancer cells were treated with PSMA-targeted GNPs at varying concentrations. Then GNPs with biocompatible dosage were used for cancer cell treatments. The media transfer technique was performed to induce the RIBE effects on bystander cells. First, LNCaP cells pretreated with phosphate buffered saline (PBS) or GNPs for 24hrs were irradiated with 160kvP X-rays at varying doses (0∼8 Gy). The culture media from irradiated cells were collected and filtered (sterile 0.2µm polyethersulfone) to acquire PBS conditioned media (PBS-CM) and GNP conditioned media (GNP-CM). Then, the collected PBS-/GNP-CMs were transferred to non-irradiated LNCaP cells with/without GNPs for 24hrs to investigate RIBE effect. The final GNP-induced RIBE was evaluated via MTT cellular viability assay, clonogenic assay, γ-H2AX assay, as well as reactive oxygen species (ROS) assessment through 2',7'-dichlorofluorescein diacetate staining.PSMA-targeted GNPs showed minimal cytotoxicity in LNCaP cells up to 1mg/mL concentrations. Cellar uptake analysis demonstrated that GNPs were bound to cells within 30 mins, with maximum uptake at 250µg/mL. Compared to 2 Gy PBS-CM, 8 Gy PBS-CM demonstrated much higher RIBE response on the non-irradiated cells, showing the dose dependent RIBE in LNCaP cells. Compared to PBS-CM, GNP-CM exhibited much higher cell death in MTT assay, as well as demonstrated RIBE enhancement in γ-H2AX (133%) and clonogenic assays (120%) analysis. Moreover, GNP-CM showed elevated ROS expression (125%) than PBS-CM. In the presence of PBS-CM, the non-irradiated cells with GNPs showed increased cell death compared to that without GNPs, indicating that GNP would enhance the sensitivity of bystander cells to RIBE. This sensitivity enhancement ratio was also validated in γ-H2AX (120%) and clonogenic assays (120%).PSMA directed GNPs were associated with physical dose enhancement along with the enhanced RIBE, playing a critical role in increasing the overall radiosensitivity of prostate cancer cells. Our results also showed that PSMA-targeted GNPs enhanced the sensitivity of bystander cells to RIBE.