From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

Brno University of Technology Department of Biomedical Engineering Technická 3082 12 61600 Brno Czech Republic

Czech Academy of Sciences Institute of Biophysics v v i Kralovopolska 135 612 65 Brno Czech Republic

Czech Academy of Sciences Institute of Biophysics v v i Kralovopolska 135 612 65 Brno Czech Republic Institute des Sciences Moléculaires d'Orsay Université Paris Saclay Université Paris Sud CNRS 91405 Orsay Cedex France

Department of Radiation Oncology Universitätsmedizin Mannheim Medical Faculty Mannheim Heidelberg University 68159 Mannheim Germany

German Cancer Research Center Im Neuenheimer Feld 280 69120 Heidelberg Germany

Institute des Sciences Moléculaires d'Orsay Université Paris Saclay Université Paris Sud CNRS 91405 Orsay Cedex France

Institute UTINAM UMR CNRS 6213 Université de Bourgogne Franche Comté 25020 Besançon Cedex France

Kirchhoff Institute for Physics University of Heidelberg Im Neuenheimer Feld 227 69120 Heidelberg Germany

Kirchhoff Institute for Physics University of Heidelberg Im Neuenheimer Feld 227 69120 Heidelberg Germany Department of Radiation Oncology Universitätsmedizin Mannheim Medical Faculty Mannheim Heidelberg University 68159 Mannheim Germany

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