Electronic Supplementary Material (ESI) for Nanoscale This journal is The Royal Society of Chemistry Characterization of CeO2 NP suspensions

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1 % Vol. Electronic Supplementary Material (ESI) for Nanoscale Characterization of CeO2 NP suspensions CeO2 NPs show a strong tendency to aggregate as a function of ph, ionic strength, dilution and sonication processes. CeO2 NP aggregation states were characterized using Dynamic Light Scattering (DLS). Fig.1a shows the hydrodynamic diameter of NPs in pure water (ph=3.2), at 10 and 60 g/ml. These NP concentrations were selected in agreement with cytotoxicity studies. Hydrodynamic diameters at approximately 8 nm were measured at these concentrations, highlighting the good dispersion of NPs in pure water. Figure 1. Evolution of NP size distribution dispersed in pure water (ph ~ 3.2) as a function of NP concentration measured by DLS at a scattering angle of 73. (A). UV-visible absorption spectra of the CeO2 NPs in pure water at 10 µg/ml. Inset: Plot of the UV-visible absorption of increasing concentrations of CeO2 NPs (10 60 µg/ml) at 288 nm (B). The optical properties of CeO2 NP suspensions were investigated by UV/vis adsorption. The UV/vis spectrum of CeO2 NPs in pure water at 10 µg/ml is presented in Fig.1b. Absorbance linearity was controlled from 10 to 60 µg/ml (insert). The sample shows a strong absorption below 400 nm with an absorbance peak at around 288 nm. These data are similar to those obtained by Phoka et al 30, who observed an absorbance peak for CeO2 NPs (5-10 nm diameter) at 285 nm. Fluorescence emission of CeO2 NP suspensions could not be detected at any excitation wavelength. It is noteworthy that bacterial development might have a direct impact not only on cellular growth, but also on exogenous biomolecule synthesis. To avoid any possible bacteriological impact, the NP stock suspension (1 mg/ml) was sterilized by filtration through a 0.22-µm-pore filter (Supplementary Figure S1). No bacterial growth was observed at up to seven days using classic bacteriological tests (chocolate agar method, data not shown) befor filtration after filtration A b s Hydrodynamic diameter (nm) (A) Fig. 2. DLS measurement of the NPs suspension (1 mg/ml) before and after filtration (A). In both cases, the hydrodynamic diameters were 6 and 8 nm respectively. UV/vis absorbance measurement controls of both (B). (B)

2 XANES spectra XANES spectra are sensitive to the Ce redox state as well as the position of the surrounding atoms within the CeO2 structure. XANES results indicated firstly that no Ce3+ is formed due to protein interactions with NPs (Fig.3). XANES spectra of CeO2 NPs, with and without proteins, did not show any shift in energy, and in particular no contribution at ev corresponding to the Ce3+ absorption peak. Indeed, several authors have already demonstrated that cerium reduction can occur with organic compounds (nutritive media, bacteria, proteins )4. The comparison between CeO2 NPs + IgG, or CeO2 NPs + BSA, with bare CeO2 NP XANES spectra revealed that no major structural changes occurred for cerium at the NP surface. The three experimental XANES spectra were almost identical, even though slight modifications of the intensities of the main peaks at 5731 and ev (called the double white line) could be observed. This conclusion can also be drawn from the analysis of the EXAFS spectra, which indicate the evolution of the atomic structure around the Ce central atom (number, nature and distance of atoms surrounding Ce from 0 to approx. 5 Å) (Fig.3B). All experimental spectra can be superimposed, indicating that the atomic structure around the Ce atoms is not affected by the NPprotein interactions. Such local-scale stability suggests that the NP-protein surface interaction is not associated with surface complexation. Indeed, for such small particles, the fraction of Ce atoms at the surface represents ~ 30% of the total number of Ce atoms in the particle. If covalent bonds occur between protein functional groups and Ce surface atoms, the atomic environment of Ce at the surface is modified. As the proportion of Ce surface atoms is high (>30%), and the sensitivity of EXAFS to modification of cerium atomic structure is around 8-10%, we can estimate that the majority of the NP-protein interactions occurred through physical interaction (electrostatic interaction, steric stabilization ) rather than chemical surface complexation. Figure 3. A) Ce LIII edge XANES spectra and B) EXAFS spectra of nanoceo 2, nanoceo 2 in contact with BSA, Nano CeO 2 in contact with IgG and Ce 2 O 3 (Ce 3+ ) reference Spectrum. Figure 4. Zeta potential measurement of NPs and proteins in different media (ph 7.4). BSA and IgG were diluted in Tris-HCl buffet (20 mm).

3 Figure 5. UV/vis absorbance of the fractions at different wavelengths (with and without NPs). λ exc :488 nm; λ em :521 nm λ exc :647 nm; λ em :674 nm Fraction I0 I (I0-I)/I0 I0 I (I0-I)/I0 A A A A A A A Table 1: Fraction analysis by fluorescence measurement. I0: fluorescence intensities of the corresponding fraction in the control run; I: fluorescence intensities of the corresponding fraction in the test run; (I0-I)/I0: normalized fluorescence variations between both runs

4 Figure 6. Correlation function of DLS measurement in figures 1- C (main text): BSA (6 µm) in the presence of NPs at 10 and 60 µg/ml.

5 Figure 7. Correlation function of DLS measurement in figures 1-B (main text): IgG (2 µm) in the presence of NPs at 10 and 60 µg/ml.

6 Figure 8. Correlation function of DLS measurement in figures 5-A (main text): Stability of NPs (10 g/ml) suspensions in DMEM-F12-FCS culture medium after 1 min, 2, 6 and 24 h incubation time.

7 Figure 9. Correlation function of DLS measurement in figures 5-B (main text): Stability of NPs (60 g/ml) suspensions in DMEM-F12-FCS culture medium after 1 min, 2, 6 and 24 h incubation time.

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