Ligand Shell Thickness of PEGylated Gold Nanoparticles Controls Cellular Uptake and Radiation Enhancement

The drive to improve the safety and efficacy of radiotherapies for cancers has prompted the development of nanomaterials that can locally amplify the radiation dose at a tumor without damaging the surrounding healthy tissue. Gold nanoparticles (Au NPs), in particular, exhibit promising radiosensitizing properties under kilovolt X-ray exposure, although the precise mechanism behind this enhancement is not fully understood. While most studies recognize the involvement of factors such as core composition, size, shape, and ligand chemistry in the effectiveness of Au NPs for radiation-induced cancer treatment, there is a scarcity of direct assessments that connect the photophysical properties of the nanomaterial with the observed cellular or biological outcomes. Despite previous evidence of low-energy electron (LEE) emission from Au NPs and their potential to initiate biological damage, to our knowledge, no studies directly correlate the secondary LEE emission with radiation-induced cell death. In this study we assessed Au NPs functionalized with polyethylene glycol (PEG) ligands of varying molecular weights and lengths (1, 5, and 20 kDa PEG) as potential radiosensitizers of A549 lung cancer cells using kilovolt X-ray source potentials (33-130 kVp). We assessed NP internalization using mass cytometry, radiation dose enhancement using clonogenic survival assays, and secondary LEE emission using a retarding field analyzer. Results reveal a statistically significant difference in cellular uptake and radiation dose enhancement for 5 kDa PEG-Au NPs compared to formulations using 1 and 20 kDa PEG, while analysis of secondary LEE emission spectra demonstrated that differences in the length of the PEG ligand did not cause statistically significant attenuation of secondary LEE flux. Consequently, we inferred increased cellular uptake of NPs to be the cause for the observed enhancement in radiosensitivity for 5 kDa PEGylated Au NPs. The approach used in this study establishes a more complete workflow for designing and characterizing the performance of nanomaterial radiosensitizers, allowing for quantification of secondary LEEs and cellular uptake, and ultimately correlation with localized dose enhancement that leads to cell death.