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PURPOSE We investigated the dose enhancement and internalization of gold nanoparticles (AuNP) used as a radiosensitizer agent for rotational radiotherapy of breast cancer using a kilovoltage X-ray beam. METHODS Human breast cancer cells MDA-MB-231 were incubated with or without 100 μg/ml (4.87 nM) or 200 μg/ml (9.74 nM) 15 nm AuNPs and irradiated with 100 kV, 190 kV or 6 MV X-rays. To assess the toxicity of the AuNPs, we performed a Sulforhodamine B assay. Using atomic absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, and time-lapse optical microscopy (rate of 2 frames per minute), we carried out a quantitative assessment of the amount of gold internalized by MDA-MB-231 cells and a characterization of the static and dynamical aspects of this internalization process. RESULTS No effect of AuNPs alone was shown on cell viability. Time-lapse optical microscopy showed for the first time AuNPs cellular uptake and the dynamics of AuNPs internalization. Electron microscopy demonstrated AuNPs localization in endosomal vesicles, preferentially in perinuclear region. After irradiation at doses up to 2 Gy, cell survival fraction curves showed increased mortality with AuNPs, with respect to irradiation without AuNPs. The highest effect of radio-enhancement by AuNPs (at 9.74 nM AuNP concentration) was observed at 190 kV showing a dose enhancement factor of 1.33±0.06 (1.34±0.02 at 100 kV), while at 6 MV it was 1.14±0.06. CONCLUSION The observed radio-sensitization effect is promising for future radio-enhanced kilovoltage (kV) radiotherapy of breast cancer and quantitatively in the order of previous observations for 15 nm AuNPs. These results of a significant dose enhancement were obtained at 15 nm AuNPs concentrations as low as several nanomolar units, at dose levels typical of a single dose fraction in a radiotherapy session. Dynamical behavior of the 3D spatial distribution of 15 nm AuNP outside the nucleus of single breast cancer cells was observed, with possible implication for future models of AuNP sensitization. This article is protected by copyright. All rights reserved.