Platinum Nanoparticle Radioenhancement is Preserved Under FLASH Very High-Energy Electron Irradiation: An in vitro Tumor Spheroid Study

Introduction: Nanoparticle (NP) mediated radioenhancement has been widely studied under conventional dose rate (CDR) irradiation, but its interaction with ultra-high dose rate (UHDR) electron irradiation remains poorly explored. While conventional electron beams are limited to superficial tumors, very high-energy electrons (VHEE) enable the treatment of deep-seated tumors. However, the combination of VHEE with NPs has not yet been investigated. This study represents the first in vitro work combining UHDR irradiation, VHEE, and platinum nanoparticles (Pt NPs). Pt NPs were selected for their biocompatibility, enhanced radiation induced secondary electron emission, potential for faster renal clearance while maintaining sufficient enhanced permeability and retention, and functionalizable surface chemistry. The objectives are to evaluate and compare the radioenhancement effect of Pt NPs under CDR and UHDR irradiations using a tumor spheroid model. Methods: Three-dimensional HeLa spheroids were incubated for 12 h with 0.5 mM Pt NPs. Pt uptake was quantified by inductively coupled plasma mass spectrometry (ICP-MS). Radioenhancement was evaluated by clonogenic assays following irradiation with VHEE (195 MeV) under CDR (~0.4 Gy/s) and UHDR (5x10 8 Gy/s to 8× 10 8 Gy/s), at doses ranging from 0 to 10 Gy. Cell proliferation was assessed by Ki67 immunohistochemistry. Results: Clonogenic assays demonstrated the radioenhancement effect of Pt NPs under both irradiation modalities (SERs of 19% to 22% at 2 Gy and 42% to 56% at 8 Gy, and DEF values of 1.43 to 1.60 at SF=37%), characterized by an increase of directly lethal damage. The magnitude of radioenhancement was similar between UHDR and CDR, suggesting independence from dose rate at 195 MeV. Proliferation analyses revealed cytostatic effects of Pt NPs, consistent with radiation-induced mitotic catastrophe. Conclusion: This first experimental study combining UHDR, VHEE, and NPs shows that the Pt NP radioenhancement effect is preserved under ultra-high dose rate irradiation. These findings highlight the strong potential of this combination to improve tumor control in deep-seated tumors while expecting a possible normal-tissue sparing (FLASH effect) associated with UHDR. However, the normal-tissue sparing effect, which was not the objective of this study, remains to be validated in the experimental conditions of this work. In addition, given the limitations of the in vitro spheroid model, further studies are needed to validate these findings in vivo. Keywords: UHDR, Radioenhancers, VHEE, 3D cell model