V Baldim

F Bedioui

N Mignet

I Margaill

J.-F Berret

The enzyme-like catalytic activity of cerium oxide nanoparticles and its dependency on Ce 3+ surface area concentration

Cerium oxide nanoparticles are known to catalyze the decomposition of reactive oxygen species such as superoxide radical and hydrogen peroxide. Herein, we examine the superoxide dismutase (SOD) and catalase (CAT) mimetic catalytic activities of nanoceria and demonstrate the existence of generic behaviors. For particles of size 4.5, 7.8, 23 and 28 nm, the SOD and CAT catalytic activities exhibit the characteristic shape of a Langmuir isotherm as a function of cerium concentration. Results show that the catalytic effects are enhanced for smaller particles and for the particles with the largest Ce 3+ fraction. The SOD-like activity obtained from the different samples is found to superimpose on a single master curve using the Ce 3+ surface area concentration as a new variable, indicating the existence of particle independent redox mechanisms. For the CAT assays the adsorption of H 2 O 2 molecules at the particle surface modulates the efficacy of the decomposition process and must be taken into account. We design an amperometry-based experiment to evaluate the H 2 O 2 adsorption at nanoceria surfaces, leading to the renormalization of the particle specific area. Depending on the particle type the amount of adsorbed H 2 O 2 molecules varies from 2 to 20 nm -2 . The proposed scalings are predictive and allow determining SOD and CAT catalytic properties of cerium oxide solely from physicochemical features.

Introduction

Cerium oxide nanoparticles (nanoceria) exhibit a fluorite-like crystal structure with a facecentered-cubic unit cell and are made of divalent oxygen anions O 2-and tetravalent cerium cations Ce 4+ . Defects in the crystal lattice are associated with oxygen vacancies surrounded by two cerium ions in their reduced trivalent state, Ce 3+ . The coexistence of two oxidation states confers to these particles remarkable antioxidant and catalytic properties. [1][2][3] Nanoceria are hence non-stoichiometric particles and designated by CeO 2-x , where 2𝑥𝑥 is the Ce 3+ fraction. Nowadays, cerium oxide nanoparticles (CNPs) are present in several industrial processes and products. Applications include chemical-mechanical polishing, UV filtering and fuel cells, but also heterogeneous catalysis for the treatment of environmental contaminants and the reduction of diesel particulate emissions. In general, the catalytic processes consist in the adsorption of reactants at the nanoceria surface, followed by surface reaction of the adsorbed species and by the desorption of products. [START_REF] Trovarelli | Catalysis by Ceria and Related Materials[END_REF][START_REF] Davis | Fundamentals of chemical reaction engineering[END_REF] More recently, researchers have suggested that nanoceria catalytic activity may find applications in biology, primarily to scavenge exogenous reactive oxygen species (ROS), as recently reviewed by Xu and Qu. [START_REF] Xu | [END_REF] The targeted ROS molecules are superoxide radical anions (O ! !• ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH • ), nitric oxide (NO) and peroxynitrite (ONOO ! ). [7][8][9][10] In this context, nanoceria dispersions were assessed in vitro over a large variety of cell lines, e.g. lung epithelial cells, retinal neurons, colon cells, macrophages and cancer cell lines. [11][12][13][14][15][16][17][18] In most cases, nanoceria were found to act as an antioxidant by controlling the ROS level in cultures and by diminishing the cell mortality. In fewer cases, relevant prooxidant results were also reported. 2,[START_REF] Xu | [END_REF]19 Some authors studied CNPs in vivo in relation to pathologies associated with oxidative stress and ROS production. It was shown for instance that nanoceria can protect photoreceptor cells present in the retina and avoid peroxide-induced retinal tissue degeneration. 20 Nanoceria beneficial effects were also found in cardiomyopathy treatment, 21 in the regulation of the inflammatory responses in sepsis 22 and in the relief of multiple sclerosis progression. 23 More recently, Kim et al. have brought evidences that ceria nanoparticles can be also neuroprotective and diminish the infarction volume in cerebral ischemic stroke. 24 These findings strongly support the idea that CNP properties, both in vitro and in vivo are due to their ability to protect healthy cells and tissues against deleterious ROS effects. However, the mechanisms behind this catalytic activity are not yet fully understood and remain a matter of debate.

According to the early works of Baer and Stein, 25 Siegler and Masters 26 and Bielski and Saito 27 in the late 1950's, the reduction of Ce 4+ aqueous solutions by H 2 O 2 takes place in a two steps redox mechanism: Ce 4+ is first reduced by H 2 O 2 into Ce 3+ while hydroperoxyl radicals (HO !

• ) are produced. In a second reaction these radicals are also involved in the reduction of Ce 4+ into Ce 3+ . The overall reaction products are then Ce 3+ ions, protons, water and molecular oxygen. [25][26][27][28] In their pioneering work, Kitajima et al. reported that H 2 O 2 does react with supported metal oxide substrates 29 and produce the superoxide species O ! !• . Subsequently, Seal and coworkers proposed that nanoceria dismutate O ! !• and disproportionate H 2 O 2 in a similar way as the enzymes superoxide dismutase (SOD) and catalase (CAT). These authors suggested that the SOD-like activity of nanoceria was mainly dependent on the Ce 3+ fraction, while the CAT-like activity would depend on the Ce 4+ fraction. 7,8,30 By means of high-energy resolution X-ray spectroscopy, Cafun et al. proposed the "electron sponge" model in which delocalized negative charges over the entire particle volume is relevant to account for nanoceria chemical activity. 31 Several groups also examined the effect of the size, morphology and surface coating on the reactivity of CNPs towards H 2 O 2 and found that smaller particles have higher Ce 3+ fractions in general and are also more reactive. [32][33][34] Finally, recent studies supported the idea that oxidation of Ce 3+ into Ce 4+ induced by H 2 O 2 leads to the formation of stable intermediate species, such as peroxo or hydroperoxo species bound to the nanoceria surface. [35][36][37][38] The picture that emerges from the previous studies is that the mechanisms underlying nanoceria catalytic activities strongly depend on the particle properties (size, morphology, Ce 3+ fraction etc…), but a quantitative description of this dependence is still missing. In this work, we measured the SOD and CAT-like activities of four different particles of sizes 4.5, 7.8, 23 and 28 nm over extended cerium concentrations. These measurements reveal that for all particles the catalytic activities exhibit the characteristic shape of a Langmuir adsorption isotherm when plotted as a function of the Ce concentration. Moreover, the catalytic activity can be rescaled using reduced variables, leading to a generic behavior. For the SOD assays, the reduced variable involved in the scaling is identified as the Ce 3+ surface area concentration. For the CAT assays we found that the adsorption of H 2 O 2 molecules at the particle surface modulates the efficacy of the disproportionation process and should be taken into account in the scaling. To achieve this goal, we design an amperometry experiment and evaluate the adsorption of hydrogen peroxide at nanoceria surfaces. These assays provide the adsorption kinetics as well as the H 2 O 2 densities at the surface prior to its disproportionation.

II -Results and discussion

II.1 -Particle characterization

Transmission electron microscopy (TEM), X-ray photoelectron spectrometry (XPS) and electronic ultraviolet-visible spectroscopy (UV-vis) are used to characterize the nanoceria samples investigated in this work. Figs. 1a-d show representative TEM micrographs of the four nanoceria samples, together with the size distributions (insets). The size distributions are adjusted using log-normal functions, leading to median diameters of 4.5, 7.8, 23 and 28 nm respectively. In the following, the cerium oxide dispersions will be named CNP-5, CNP-8, CNP-23 and CNP-28 in reference to their TEM sizes. The images in Fig. 1 and those in Supplementary Information (Fig. S1) illustrate that CNP-5 particles are spherical nano-objects, whereas CNP-8 consist of small agglomerates of 2 nm crystallites. These latter findings are in agreement with our earlier determinations on this sample. [39][40][41] For the CNP-23 and CNP-28 samples, the particles are polyhedral and characterized by a broad size dispersity. The specific surface area 𝐴𝐴 ! of CNP-5, CNP-8, CNP-23 and CNP-28 nanoparticles is calculated from their median diameters and dispersities, according to:

𝐴𝐴 ! = 6 exp -2.5𝑠𝑠 ! 𝐷𝐷𝐷𝐷 ( 1
)
where 𝑠𝑠 is the dispersity, 𝐷𝐷 the diameter and 𝜌𝜌 the mass density. Details for the calculations are provided in Supplementary Information S2. 𝐴𝐴 ! is found to be 136, 101, 24 and 24 m 2 g -1 , respectively (Table I).

Studies have shown that besides the size, an important feature affecting nanoceria antioxidant properties is its Ce 3+ -fraction, noted 𝑓𝑓 !" !! in the following. 42 To determine 𝑓𝑓 !" !! , XPS experiments are performed on powder samples obtained after drying the particle dispersions. Fig. 1e shows the decomposed Ce3d XPS spectra for the four nanoparticles. Three pairs of peaks are associated to Ce3d electrons ejected from Ce 4+ , while two others pairs of peaks are due to Ce 3+ ions. The complete analysis of the XPS spectra including the peak assignment, the determination of the binding energies and of the Ce 3+ fraction is provided in the Supplementary Information (S3). For the CNP-5, CNP-8 CNP-23 and CNP-28 one gets 𝑓𝑓 !" !! = 43%, 14%, 9.0% and 9.5% respectively. This fraction is found to increase with decreasing nanoparticle size, a result that was noticed in earlier studies and that is related to the lattice strain induced by Ce 3+ and the presence of oxygen vacancies. 43 Wide-angle X-ray scattering (WAXS) performed on the 4 samples confirms the fluorite-like structure of the nanocrystals (Supplementary Information S4).

From the Rietveld MAUD analysis, the lattice constants were derived and found to increase linearly with the Ce 3+ fraction, a result that is attributed to the decrease of electrostatic force caused by valence reduction from Ce 4+ to Ce 3+ ions. 43 The overall characteristics of the cerium oxide samples are summarized on Table I.

Table I -Nanoceria characteristics.

The median diameter (𝐷𝐷), the dispersity (𝑠𝑠) and the morphology were obtained from transmission electron microscopy measurements. The lattice constant (𝑎𝑎) of the cerium oxide nanocrystals and the Ce 3+ fractions (𝑓𝑓 !" !! ) were determined from wide-angle X-ray scattering and X-ray photoelectron spectrometry respectively. The dispersity is defined as the ratio between the standard deviation and average size.

Another remarkable property displayed by nanoceria is its propensity to absorb UV light and to quickly change color when mixed with hydrogen peroxide (H 2 O 2 ). 42,[START_REF] Das | [END_REF] Fig. 2 shows UV-vis absorption spectra and photographs of CNP-5, CNP-8, CNP-23 and CNP-28 dispersions before and after H 2 O 2 addition. The spectral and visual color changes are much more intense for the smaller nanoparticles than for the larger ones, a result that was already noticed by many authors. 33 Following Lee et al., 42 we characterize the observed red shift by measuring the wavelength difference Δ𝜆𝜆 at the optical density 0.3 before and after peroxide addition and found Δ𝜆𝜆 = 90, 100, 17 and 10 nm for CNP-5, CNP-8 CNP-23 and CNP-28 respectively. This shift is regarded as a measure of the NP antioxidant capacity and allows simple comparison between samples. The colorimetric data show that increasing the particle size decreases the shift of the spectrum, a result that was attributed to the oxidation of Ce 3+ surface ions into Ce 4+ by H 2 O 2 42

or/and to coordinated peroxide species onto CNP surfaces. 35,37,46 Supplementary Information S5a displays absorbance results as a function of H 2 O 2 (from 0 to 882 mM) and confirms the saturation of the red shift at high concentrations. 34,37,42 Additional colorimetric assays on the absorbance kinetics after addition of hydrogen peroxide bring evidences of nanoceria catalytic cycling properties after a 6 day incubation (S5b). The results of the previous sections demonstrate that the nanoceria studied here display similar structural, chemical and optical properties than those of the literature. They were selected to cover a wide range of physicochemical features. In the next section we focus on their superoxide dismutase and catalase mimetic activities.

II. 2 -Superoxide dismutase mimicking activity of nanoceria

The SOD-like catalytic activity of nanoceria was investigated by a colorimetric assay using UV-Vis spectroscopy. 47,48 With this assay, superoxide radical anions are generated in situ by xanthine oxidase in a nanoceria dispersion. A water-soluble tetrazolium salt is then added to the solution, this latter being oxidized by the remaining superoxide radicals into a soluble formazan dye. The final product is then identified from its absorption property. 24,47,49 The SOD-like activity 𝐴𝐴 !"# is defined as the percentage of dismutated superoxide radicals at the end of the assay. Fig. 3a shows the effect of the particle type on 𝐴𝐴 !"# at cerium concentration 200 µM. 𝐴𝐴 !"# is found to decrease with increasing particle diameters, suggesting a potential size and Ce 3+ fraction effect. Fig. 3b illustrates the concentration dependence of 𝐴𝐴 !"# in the range 200 -2000 µM. Increasing the CNP concentration leads to increased dismutation rates for the 4 samples. To account for this dependence, the SOD results are displayed versus the surface area concentration (expressed in m 2 L -1 ):

𝑐𝑐 ! = 𝐴𝐴 ! 𝑐𝑐 (2)
where the specific surface area 𝐴𝐴 ! (Eq. 1) is in m 2 g -1 and the weight concentration in g L -1 (see also Supplementary Information S2). Fig. 3c shows that the SOD activity scales well for CNP-8, CNP-23 and CNP-28, the data being now well superimposed. This superposition is however not observed for CNP-5, i.e. for the particles with the highest 𝑓𝑓 !" !! . These data suggest that the surface density of Ce 3+ active sites plays a significant role on the SOD response. 7 Fig. 3d retraces the previous data as a function of the surface area concentration times the Ce 3+ fraction (𝑐𝑐 ! 𝑓𝑓 !" !! ). It is found that the SOD-like activity of the different nanoceria samples are well superimposed and follows a sigmoid-type behavior when plotted in semi-logarithmic scale (continuous curve in grey in Fig. 3d). These data are adjusted by a Langmuir-type adsorption isotherm of the form: 50

𝐴𝐴 !"# 𝑐𝑐 ! , 𝑓𝑓 !" !! = 1 1 + 𝑐𝑐 ! !"# /𝑐𝑐 ! 𝑓𝑓 !" !! (3)
where 𝑐𝑐 ! !"# (= 5.6 m 2 L -1 ) is the only adjustable parameter. Fig. S6 illustrates the Langmuir isotherm in linear scale together with the Eq. 1, showing in particular the existence of a linear regime at low 𝑐𝑐 ! 𝑓𝑓 !" !! followed by a saturation plateau. The findings of Fig. 3 substantiate the existence of a superposition principle once the surface area concentration and the Ce 3+ fraction are taken into account. 7,42,43 Here the multivariable dependencies of the SOD mimetic activity have been reduced into a single master curve and the Ce 3+ surface area concentration 𝑐𝑐 ! 𝑓𝑓 !" !! has been identified as the key parameter for the superposition principle. We suggest that this result is quite general and could be used to predict the catalytic activity of nanoceria, knowing its particle size and Ce 3+ fraction.

II. 3 -Catalase-like catalytic activity

The nanoceria catalase-like catalytic activity was investigated by spectrofluorimetry using the Amplex-Red reagent assay. 22,24,47 In this assay, cerium oxide dispersions at molar concentration 3×10 -1 to 3×10 4 µM are incubated with 5 µM hydrogen peroxide in 96-well plates. A mixture of horseradish peroxidase and Amplex-Red is then added, this latter being transformed into the fluorescent resorufin from the remaining H 2 O 2 . 24 The CAT-like activity 𝐴𝐴 !"# is defined as the percentage of decomposed H 2 O 2 at the end of the assay. Fig. 4a shows the effect of the particle size on 𝐴𝐴 !"# at cerium concentration 200 µM. 𝐴𝐴 !"# is found to be smaller for the large particles CNP-23 and CNP-28, suggesting again a potential size effect. When plotted in semi-logarithmic scale against the added cerium oxide, 𝐴𝐴 !"# exhibits a sigmoidal-shape dependence similar to that found for SOD (Fig. 4b). We also note some differences between the CNPs. For the smallest particles (CNP-5 and CNP-8), the H 2 O 2 percentages increase at concentration around 1 µM and display comparable responses. For CNP-23 and CNP-28, the sigmoids are shifted to higher concentrations, with a signal onset starting around 100 µM and a saturation above 10 4 µM.

Following the approach developed in the previous section, the H 2 O 2 disproportionation results have been rescaled using first the specific surface concentration 𝑐𝑐 ! (Fig. 4c) and second using the specific surface concentration times the Ce 3+ fraction, 𝑐𝑐 ! 𝑓𝑓 !" !! (Fig. 4d). After rescaling, the set of 𝐴𝐴 !"# -curves obtained have neared closer to each other, indicating that the size and the Ce 3+ fraction are here too key parameters in the activity of cerium oxide. The sigmoidal behavior was fitted by a Langmuir-type adsorption function of the form: 50

𝐴𝐴 !"# 𝑐𝑐 ! , 𝑓𝑓 !" !! = 1 1 + 𝑐𝑐 ! !"# /𝑐𝑐 ! 𝑓𝑓 !" !! ( 4
)
where 𝑐𝑐 ! !"# is the only adjustable parameter. In Supplementary Information S7 we show that the agreement between the results and Eq. 2 is excellent. The 𝑐𝑐 ! !"# values retrieved from the fitting are 0.57, 0.11, 0.61 and 3.57 m 2 L -1 for CNP-5, CNP8, CNP-23 and CNP-28 respectively. In contrast to the SOD-like activity, the data here do not superimpose, suggesting the existence of additional contributing factors.

II. 4 -Hydrogen peroxide adsorption at the nanoceria surface

To get some insight into the process occurring upon mixing H 2 O 2 and nanoceria, 35,37,38 we designed an amperometry experiments capable of monitoring the H 2 O 2 concentration based on the changes in electric currents. Briefly, a working Pt/Pt-black ultramicroelectrode (UME) and a reference Ag/AgCl electrode are placed inside a microplate well containing Tris-Cl buffer pH 7.5 and connected to a potentiostat. 51,52 A potential of 0.4 V is applied and the current is recorded while hydrogen peroxide and nanoceria dispersions are added consecutively to the solution. The representation in Fig. 5a illustrates the experimental set-up and provides the working conditions for the two electrodes. Fig. 5b shows the time evolution of the current during a typical amperometry monitoring. A base line is first recorded and is followed by a step increase of the current at 1 nA after H 2 O 2 addition. Five minutes later, CNPs are added to the previous solution, leading to a rapid decrease of the current and to its stabilization at long time. Fig. 5c displays further measurements aiming at calibrating the UME. Different volumes of a 15 mM H 2 O 2 solution are added stepwise to the initial solution, leading to steps in the current response. Similarly, additions of nanoceria (CNP-8, 60 mM) produce a step-like diminution of the electric signal in the proportion of the added volume. From the first part of the assay, a linear currentconcentration calibration characterized by a slope of 0.011 nA µM -1 is observed (Fig. 5d). The rapid decrease found upon CeO 2-x addition is caused by the decrease of the H 2 O 2 concentration consecutive to the adsorption of hydrogen peroxide at the nanoceria surface (Fig. 5e). It is followed by the formation of adsorbed oxygen species such as peroxide or hydroperoxide. [35][36][37][38] To our knowledge, this is the first report where chrono-amperometry was exploited to investigate and quantify such properties. Liu et al. also reported the adsorption of hydrogen peroxide to nanoceria surfaces and exploited it to develop a DNA fluorescence-based method for hydrogen peroxide titration. 53

Figure 5: Amperometric detection of hydrogen peroxide in the presence of nanoceria a) Experimental set-up used for the amperometric monitoring of hydrogen peroxide including the Pt/Ptblack working ultramicroelectrode and the Ag/AgCl reference electrode. b) Typical time evolution of the current upon H 2 O 2 and CeO 2-x addition. Note that the characteristic time scale for the [H 2 O 2 ] decrease is similar to that of the absorbance changes seen in UV-Vis spectrometry. c) Signal measurements following the addition of increasing volumes of a 15 mM H 2 O 2 solution and of a 60 mM CNP-8 dispersion, as indicated. d) Calibration curve between hydrogen peroxide concentration and current. The straight line is the result of least square adjustment, providing a slope of 0.011 nA µM -1 . e) Schematic representation of hydrogen peroxide adsorption at nanoceria surfaces.

To compare the CNPs with the amperometry technique, dispersions were prepared at fixed surface area concentration 𝑐𝑐 ! = 10 m 2 L -1 . Upon CNP addition, all experiments reveal a decrease of the H 2 O 2 concentration, as previously mentioned (Fig. 6a). This decrease depends however on the sample type, suggesting different adsorption site densities. The signal reduction is for instance stronger for CNP-8 compared to that of CNP-5. We estimate the surface density of hydrogen peroxide molecules from these experiments and find 12.0 ± 0.7 nm -2 , 16.7 ± 0.8 nm -2 , 5.3 ± 0.1 nm -2 and 2.3 ± 0.5 nm -2 for CNP-5, CNP-8, CNP-23 and CNP-28 respectively. These values are now used to define an effective specific surface for the CAT assay, noted 𝐴𝐴 !" . Here, we consider CNP-8 as a reference and recalculate the effective surface area density of active sites for the other particles using the 𝐴𝐴 !" values given in Table II. Fig. 6b displays the 𝐴𝐴 !"# percentages as a function of the effective surface concentrations 𝑐𝑐 !" times the Ce 3+ fraction in semi-logarithmic scale. The graph shows a good superimposition of the different curves, indicating that the nanoceria CAT-like activity depends mainly on three factors, the particle size, the Ce 3+ fraction and the H 2 O 2 adsorption properties. Replacing the concentration 𝑐𝑐 ! by the effective concentration 𝑐𝑐 !" , the data in Fig. 6 are adjusted using Eq. 2 and the parameters 𝑐𝑐 ! !"# are found to be 0.41, 0.11, 0.20 and 0.39 m 2 L -1 with increasing particle sizes. The persisting differences between samples can be attributed to the approximations made estimating the scaling factors and in particular the specific surface area. 𝐴𝐴 ! was indeed calculated assuming that the particles are spherical objects. Another factor could be the dependence of the catalytic activity on the exposed crystalline facets. 3,34,[54][55][56] The proposed superposition principle found here could be used to determine the CAT mimetic properties from the knowledge of the parameters mentioned above. Table II: Relevant parameters of the amperometric monitoring of hydrogen peroxide in the presence of nanoceria.

III -Conclusion

In this work we examined the enzyme mimicking activity of a series of cerium of different specific surface areas (from 20 to 140 m 2 g -1 ) and of different Ce 3+ fractions (from 9 to 43%). The physico-chemical features of the CNPs were obtained by extensive TEM, XPS, WAXS and UV-Vis measurements and analyze quantitatively. The superoxide dismutase and catalase-like activities were investigated versus cerium oxide concentration via the xanthine oxidase and Amplex-Red reagent assays respectively. Results show that the catalytic effects are enhanced for smaller particles and for the particles with the largest Ce 3+ fraction. In an effort to identify the key factors affecting the nanoceria catalytic activity and to provide a rationale about these dependencies, various types of scaling analysis were tested. We have found that once expressed in terms of the Ce 3+ surface area concentration, the percentages of dismutated superoxide radicals determined for the different CNPs were found to superimpose on a single master curve. Moreover, the data follow a Langmuir-type adsorption isotherm behavior. In particular, we could demonstrate the existence of a linear regime at low surface area concentration followed by a saturation plateau, the transition between the two regimes occurring around 5 m 2 L -1 . The observed Langmuir behavior indicates that the catalytic assays are probing surface phenomena related to the adsorption/desorption of test molecules. For the CAT-like activity, we found again Langmuir type isotherm variations for the 4 CNPs. However, the scaling using the Ce 3+ surface area concentration in abscissa failed and could not be observed at first. To get more insight in the interaction of H 2 O 2 with nanoceria surfaces, amperometry experiments were performed to monitor the H 2 O 2 concentration as a function of the time and in different working conditions. This technique provides the H 2 O 2 adsorption kinetics upon CNP addition, as well as the densities of the hydrogen peroxide molecules at the surface in the stationary regime. We found that the H 2 O 2 adsorption depends on the CNP used and that the specific surface area first derived from the TEM experiments ought to be renormalized. The surface density of hydrogen peroxide molecules was comprised between 2.3 nm -2 and 16.7 nm -2 . With these modifications, the CAT-like activity of cerium oxide was reexamined and the data coming from different CNPs could be superimposed into a single master curve. In conclusion, we show that the SOD and CAT mimicking catalytic activity obeys simple and generic behaviors for the different nanoparticles tested. We suggest that these scaling behaviors could be used to evaluate the catalytic performance of nanoceria solely from the nanoparticles physical and chemical properties.

IV -Materials and Methods

IV.1 -Materials CNP-5 and CNP-23 are aqueous dispersions at concentrations 200 g L -1 and 100 g L -1 (both at pH4) purchased from Sigma-Aldrich. The particles are positively charged, associated with zeta potentials ζ = +8 mV and +40 mV respectively, and according to the provider do not carry additives at their surface. CNP-8 originates from a 200 g L -1 low pH aqueous dispersion (pH 1.5) synthesized by Rhodia (Centre de Recherche d'Aubervilliers, Aubervilliers, France). 11,57,58 As shown in earlier reports, the CNP-8 are positively charged at acidic pH (ζ = + 40 mV) and aggregate above pH2. 40,41 The colloidal stability is ensured by electrostatic repulsion arising from positive surface charges, as discussed in Ref. 59 . In such a case, the surfaces are devoid of ligands or macromolecules. CNP-28 is a cerium oxide benchmark material (code name NM-212) synthesized for the OECD program "Testing a representative set of manufactured nanomaterials". [START_REF] Singh | Cerium Dioxide[END_REF] It was supplied as a fine powder by the Institute for Health and Consumer Protection (IHCP, Joint Research Centre of European Commission, Italy). In the report issued by the European Commission, the CNP-28 were extensively characterized with respect to their surface properties. The BET measurement gives a value of the specific surface area of 𝐴𝐴 ! = 27.2 m 2 g -1 , in good agreement with the TEM determination (𝐴𝐴 ! = 24 m 2 g -1 , see Table I). The positive zeta potential (ζ + = 33 mV) indicates the presence of positive charges at the surface. XPS results indicate that carbon and oxygen were detected, most likely associated with surface contamination or adsorbed carbon containing species. [START_REF] Singh | Cerium Dioxide[END_REF] Hydrogen peroxide (H 2 O 2 , 30 vol. %), hydrogen hexachloroplatinate(IV) solution, lead(II) acetate trihydrate and the superoxide dismutase kit assay (Kit #19160-1KTF) were bought from Sigma-Aldrich (Lyon, France). The Amplex® Red catalase assay kit (Cat # A22180) was purchased from Thermo Scientific (Illkirch, France). T

IV.2 -Ultraviolet-visible Spectroscopy (UV-vis)

A UV-visible spectrometer (SmartSpecPlus from BioRad) was used to measure the absorbance of nanoceria aqueous dispersions. The absorbance is related to the nanoparticle concentration by the Beer-Lambert expression 𝐴𝐴𝐴𝐴𝐴𝐴(𝜆𝜆) = 𝜖𝜖(𝜆𝜆)ℓ𝓁𝓁𝑐𝑐 where ℓ𝓁𝓁 is the optical length, 𝑐𝑐 the nanoparticle concentration, 𝜖𝜖(𝜆𝜆) the molar absorption coefficient. Absorbance data were used to determine the cerium oxide concentration for each batch. 39 IV.3 -Zeta potential Laser Doppler velocimetry was used to carry out the electrokinetic measurements of electrophoretic mobility and zeta potential (ζ) with the Zetasizer Nano ZS equipment (Malvern Instruments, Worcestershore, United Kingdom). Light scattering and zeta potential measurements were performed in triplicate at 25 °C after an equilibration time of 120 s. [START_REF] Oikonomou | [END_REF]

IV.4 -Transmission Electron Microscopy (TEM)

Micrographs were taken with a Tecnai 12 TEM operating at 80 kV equipped with a 1K×1K Keen View camera. Nanoceria dispersions were deposited on ultrathin carbon type-A 400 mesh copper grids (Ted Pella, Inc.). Micrographs were analyzed using ImageJ software for 200 particles. The particle size distributions are adjusted using a log-normal function of the form (Fig. 1):

𝑝𝑝 𝑑𝑑, 𝐷𝐷, 𝑠𝑠 = 1 2𝜋𝜋𝛽𝛽(𝑠𝑠)𝑑𝑑 𝑒𝑒𝑒𝑒𝑒𝑒 - 𝑙𝑙𝑙𝑙 2 (𝑑𝑑/𝐷𝐷) 2𝛽𝛽(𝑠𝑠) 2 (5)
In the previous equation, 𝛽𝛽(𝑠𝑠) is related to the size dispersity 𝑠𝑠 through the relationship 𝛽𝛽 𝑠𝑠 = ln (1 + 𝑠𝑠 ! ). 𝑠𝑠 is defined as the ratio between the standard deviation and the average diameter. [START_REF] Klein | Light Scattering, Principles and Developments[END_REF] For 𝛽𝛽 < 0.4, one has 𝛽𝛽 ≅ 𝑠𝑠. 39,62 IV.5 -X-ray Photoelectron Spectroscopy (XPS)

XPS data was collected using an Omicron Argus X-ray photoelectron spectrometer using a monochromated AlK ! (1486.6 eV) radiation source with a 300 W electron beam power. The emission of photoelectrons from the sample was analyzed at a takeoff angle of 45° under ultrahigh vacuum conditions (10 -8 Pa). The spectra were collected at pass energy of 100 eV for the survey scan and 20 eV for the high resolution scans. The XPS spectra were fitted using the software XPSPEAK41, applying a fixed Gaussian/ Lorentzian ratio for peaks of the same spectrum and constraining the full-width at half-maximum (FWHM) of each doublet to be equal. The fraction of Ce 3+ was determined through the ratio between the integrated intensities of the four peaks corresponding to Ce 3+ divided by the integrated intensities of all ten peaks, as detailed in Supplementary Information.

IV.6 -Wide-Angle X-ray Scattering (WAXS)

WAXS was carried out using an Empyrean (PANALYTICAL) diffractometer equipped with a multichannel PIXcel 3D detector and a Cu Kα X-ray source (1.54187 Å). Samples were deposited on a monocrystalline Si substrate, with a spinner movement (rotation time 1 s). A 1/16° divergence slit, a 1/8° anti-scatter slit and a 10 mm mask were installed before the samples. Typically, each pattern was recorded in the 𝜃𝜃 -𝜃𝜃 Bragg-Brentano geometry in the 10° -20° 2𝜃𝜃 range (0.0263° for 600 s), where 2𝜃𝜃 denotes the scattering angle.

IV.7 -Amperometric monitoring of H 2 O 2

A homemade Pt/Pt-black ultramicroelectrode (UME) was used as the hydrogen peroxide sensor. A Pt wire (diameter 25 µm) was put into a closed end glass capillary and then heated under vacuum until the glass was melted around the wire. Then, the capillary was filled with a conductive lacquer and a copper wire was finally inserted for connection with the potentiostat.

The UME surface was polished on emery paper with diamond solution and Pt-black was deposited on its surface by the reduction of a hydrogen hexachloroplatinate(IV) solution containing Lead(II) acetate trihydrate by applying a current density J of -8 mA cm -2 and a charge density Q of -240 mC cm -2 . The Pt/Pt-black UME was connected to a potentiostat (eDAQ Pty Ltd.) and immersed in 2 mL of Tris-Cl buffer pH 7.5 placed inside a well of a 24-well cell culture plate. The voltage was set at +0.4 V versus an Ag/AgCl reference counter electrode.

After the baseline stabilization, 50 µL of a H 2 O 2 solution were added to the well, followed by the addition of 50 µL of a nanoceria dispersion, while the current was recorded in real time.

IV.8 -SOD mimetic activity assay

The catalytic activity of nanoceria in the dismutation of superoxide radical anion was assessed by a colorimetric assay using UV-Vis spectroscopy (Kit #19160-1KTF). 47,48 Briefly, 20 µL of a nanoceria dispersion in Tris-Cl buffer pH 7.5 was added to a well of a 96-well plate and mixed with 200 µL of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt). The reaction was initiated with the addition of 20 µL of xanthine oxidase solution, prepared by mixing 5 µL of the enzyme in 2.5 mL of a dilution buffer provided. After incubating plate at 37°C for 20 min, the absorbance at 450 nm was measured using a microplate reader (EnSpire Multimode Plate Reader, Perkin Elmer). Final nanoceria dispersion concentration ranged from 200 to 2000 µM. The SOD-like activity, noted 𝐴𝐴 !"# is defined as the percentage of dismutated superoxide radicals after the 20 min.

IV.9 -CAT mimetic activity assay

The catalytic activity of nanoceria in the disproportionation of H 2 O 2 was assessed by spectrofluorimetry using the Amplex-Red reagent assay (Cat # A22180). 22,24,47 Briefly, 25 µL of a nanoceria dispersions in Tris-Cl buffer pH 7.5 was added to a well of a 96-well plate and mixed with 25 µL of a H 2 O 2 solution. Then, 50 µL Amplex Red reagent/HRP working solution was added and reactions are pre-incubated for 5 minutes. Amplex Red (10-acetyl-3,7dihydroxyphenoxazine) reaction with H 2 O 2 catalyzed by horseradish peroxide (HRP) produces the fluorescent molecule resorufin (excitation at 571 nm and emission at 585 nm). The fluorescence was measured after incubating for 30 min with protection from light. The H 2 O 2 concentration in each well was 5 µM, whereas those of nanoceria ranged from 3.5×10 -1 to 3.5×10 4 µM. The catalytic activity of nanoceria noted 𝐴𝐴 !"# is defined as the percentage of decomposed H 2 O 2 determined at the end of the assay.

TOC image

Figure 1 :

 1 Figure 1: Electron microscopy and X-ray photoelectron spectrometry resultsNanoceria characterization using transmission electron microscopy (a-d) and X-ray photoelectron spectrometry (e). For the TEM micrographs, the scale bars are 50 nm for (a) and (b) and 100 nm for (c) and (d). Insets: size distributions obtained from the analysis on 𝑛𝑛 = 200 particles. The distributions are adjusted using a log-normal function with median diameter 𝐷𝐷 and dispersity 𝑠𝑠 (see Supplementary Information S2 for details).The dispersity is defined as the ratio between the standard deviation and average size.[START_REF] Klein | Light Scattering, Principles and Developments[END_REF] e) XPS Ce3d spectra for CNP-5 (magenta), CNP-8 (black), CNP-23 (red) and CNP-28 (blue). The continuous thick lines display the sum of the different peak contributions adjusted according to the model described in S3.

Figure 2 :

 2 Figure 2: Absorbance properties of nanoceria with and without hydrogen peroxide UV-vis absorption spectra of nanoceria dispersions at the concentration of 1.6 mM before (black lines) and two minutes after addition of a concentrated hydrogen peroxide to the final concentration of 60 mM (red lines). a) CNP-5; b) CNP8; c) CNP-23 and; d) CNP-28. Insets: images of the nanoceria dispersionswith and without hydrogen peroxide. Only the smallest particles exhibit significant changes in the range 300 -500 nm.

Figure 3 :

 3 Figure 3: Superoxide dismutase-like activity of nanoceria a) Percentages of dismutated superoxide radicals 𝐴𝐴 !"# obtained for CNP-5 (magenta), CNP-8 (black), CNP-23 (red) and CNP-28 (blue) at cerium concentration 200 µM. In figures b, c and d, 𝐴𝐴 !"# is plotted respectively as a function of the cerium molar concentration [Ce], of the surface area concentration 𝑐𝑐 ! and of the surface area concentration times Ce 3+ fraction, 𝑐𝑐 ! 𝑓𝑓 !" !! . In d), the continuous line is obtained from Eq. 1 using 𝑐𝑐 ! !"# = 5.6 m 2 L -1 as a single adjustable parameter (see also S7).

Figure 4 :

 4 Figure 4: Catalase-like catalytic activity of nanoceria a) Percentages of disproportionated hydrogen peroxide 𝐴𝐴 !"# obtained for CNP-5 (magenta), CNP-8 (black), CNP-23 (red) and CNP-28 (blue) at cerium concentration 200 µM. In figures b, c and d, 𝐴𝐴 !"# is plotted respectively as a function of the cerium molar concentration [Ce], of the surface area concentration 𝑐𝑐 ! and of the surface area concentration times Ce 3+ fraction, 𝑐𝑐 ! 𝑓𝑓 !! !! . In these assays, the initial H 2 O 2 concentration was 5 µM.

Figure 6 :

 6 Figure 6: Catalase-like activity of nanoceria in rescaled units a) Chrono-amperogram showing the evolution of hydrogen peroxide concentration after and before addition of hydrogen peroxide (440 µM) followed by addition of CNP-5, CNP-8, CNP-23 or CNP-28

CeO 2-x nano- particles

		𝑫𝑫 nm	𝒔𝒔	𝒂𝒂 nm	Morphology	𝑨𝑨 𝑺𝑺 m 2 g -1	𝒇𝒇 𝑪𝑪𝑪𝑪 𝟑𝟑! %
	CNP-5	4.5	0.36	0.544572	Spherical	136	43
	CNP-8	7.8	0.17	0.541514	Agglomerate	101	14
	CNP-23	23	0.42	0.540858	Polyhedral	24	9.0
	CNP-28	28	0.31	0.540999	Polyhedral	24	9.5

Acknowledgment

We thank Jérôme Fresnais, Evdokia Oikonomou, Fanny Mousseau, Chloé Puisney for fruitful discussions. Dr. Sophie Novak from the "Plateforme Rayons X" at the University Paris-Diderot is acknowledged for carrying out the wide-angle X-ray scattering technique on nanoceria. Dr. Rémi LeBorgne from the "Plateforme de microscopie électronique" at the Institut Jacques Monod is acknowledged for his assistance and support with transmission electronic microscopy. Dr. Christophe Methivier and Dr. Christophe Calers from the Laboratoire de Réactivité de Surface at the University Pierre et Marie Curie, Paris 6 are thanked for carrying out the X-ray photoelectron spectrometry (XPS) experiments. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABX 086, ANR 11 IDEX 05 02. This research was supported in part by the Agence Nationale de la Recherche under the contract ANR-13-BS08-0015 (PANORAMA), ANR-12-CHEX-0011 (PULMONANO) and ANR-15-CE18-0024-01 (ICONS, Innovative polymer coated cerium oxide for stroke treatment).


Chloé Puisney

Evdokia K Oikonomou

Sophie Nowak

Alexandre Chevillot

Sandra Casale

Armelle Baeza-Squiban

Jean-François Berret
email: jean-francois.berret@univ-paris-diderot.fr

Armelle Baeza
email: baeza@univ-paris-diderot.fr

Brake wear (nano)particle characterization and toxicity on airway epithelial cells in vitro

Keywords: Air pollution, Brake wear particles, Dynamic light scattering, Inflammation, Metallic particle, Respiratory epithelium

Particulate air pollution results from different sources, among which those related to road traffic have a significant impact on human health. Combustion-derived particles emitted by thermal engines have been incriminated and are now better controlled. In contrast, non-exhaust emission sources related to car wear and degradation processes are not yet regulated. Here we report on brake wear particles (BWP) harvested in two test facilities operating in France, providing samples from different braking systems and driving/testing conditions. Using a combination of light scattering, X-ray fluorescence, optical and electron microscopy, the particle size and elemental composition are revealed. The BWP are shown to be in the nano-to micrometer range and to have a low carbonaceous content (6%), iron and copper being the main components (> 40%). To evaluate the toxicity potential of its nano-sized fraction, brake wear nanoparticles are isolated by sonication, filtration and ultra-centrifugation techniques, leading to stable colloidal dispersions. A significant outcome of this study is that the nano-sized fraction represents 26% by mass of the initial BWP. Human bronchial epithelial cells (Calu-3) are used as relevant target cells to investigate their cytotoxicity. We observe a clear short-term loss of viability associated to reactive oxygen species generation, but with limited pro-____________________________________To appear in Envriron. Sci. Nano

Introduction

Air pollution is now recognized as a major environment-related health threat. A number of epidemiological studies have shown the existence of an association between the exposure to air pollution and the increase of mortality and morbidity from cardiorespiratory diseases. 1,2 Toxicological studies have supported the biological plausibility of this association, especially for the particulate matter (PM) component. 3 PM air pollution results from different sources, among which road traffic has a significant part, impacting urban populations worldwide. Combustion-derived particles emitted by thermal engines are specifically held accountable, which has led state agencies to implement stricter regulations for the automotive industry, eventually resulting in the reduction of the PM 2.5 (particles with aerodynamic diameters ≤ 2.5 µm) in the last decades. 4 By contrast, nonexhaust emission sources related to wear mechanisms are not yet regulated. Wear and degradation processes concern the subcomponent moving parts in cars, including the clutch, tires, brakes etc. as well as the direct driving environments, such as roads or tarmac. Moreover, with the broadening of exhaust post-treatment (e.g. diesel particulate filters) and the emergence of electric vehicles, the proportion of wear particles in urban pollution is expected to soar in the coming years. 5,6 Recent studies have shown that the contribution of brake wear particles (BWP) can represent up to 55% by mass of the total non-exhaust traffic-related PM 10 emission and up to 21% by mass of the total traffic related PM 10 (particles with aerodynamic diameters ≤ 10 µm) emissions. 7,8 The two types of frictional braking systems available for light-duty and small vehicles are the drum and disc/pad brakes, whose emission have been quantified in specific contexts at a level of 30 mg km -1 and 9 mg km -1 of particles per vehicle, respectively. 9 There exists many different brands of braking systems on the market, and most of them comprise similar lining components, such as binders, fillers, reinforcing agents (fibers) and friction modifiers (abrasives and lubricants). 10 The filler and fiber components as well as some additives are mainly metallic, whereas the binders and additives are carbonaceous. 10,11 During braking, the friction between the pad and disc surfaces is used to convert the vehicle kinetic energy into heat, resulting in a significant alteration of the lining components and in a concomitant temperature increase. 8 The temperature there can grow to 400 °C and locally it may reach up to 700 to 1000 °C, depending on the testing or driving conditions. [12][13][14] In this process, metallic particles in the nano-to micrometer range are generated, together with carbon-based particles stemming from the condensation of evaporated organic compounds. 9 BWPs may have an impact on the environment and human health due to their composition and size. In particular, the nanoscale fraction of the BWPs could be preferentially retained in the vicinity of a roadway compared to larger more readily settleable materials. As such, the inhaled dose and exposure could be much higher than for larger particles. Traffic-related wear emissions are nowadays considered relevant for air quality policy development, however for now no specific regulations have been implemented.

More generally pertaining to human health, BWP emission leads to airborne (nano)particles for which the preferential entrance pathway is the respiratory tract. Besides their local impact on the lung epithelia, nanoparticles reaching the alveolar region may cross the air-blood barrier and accumulate in secondary organs. 15,16 So far, few studies have dealt explicitly with the toxicity of BWPs, and most of them were limited to in vitro assays, and not focused on the nano-sized fraction. In general it was found that their high content in transition metals and metallic compounds can generate various cytotoxic effects. 17,18 With the A549 alveolar cell line, BWPs were shown to induce oxidative stress 19 and to alter the tight junctions between the cells. 18 On peripheral blood lymphocytes, brake wear dust was found to cause chromosomal damages, likely produced by the metallic nanocrystals present in the dust. 20 Our present work focuses on pooled BWP samples collected on two test center facilities in France and corresponding to different braking conditions. The objectives are to characterize the BWPs in terms of size and composition, and to isolate its nano-sized fraction by a combination of sonication, filtration and ultra-centrifugation techniques. 21 The resulting nanoparticle dispersions in water are found to be stable over time and they are tested on a model of human bronchial epithelial cells (Calu-3). As a result, a short-term loss of viability associated to reactive oxygen species generation is observed. The nanosized fraction is found to exhibit lower toxicity and reactive oxygen species production than BWPs. In terms of effective dose however, the toxicity levels are indeed similar, suggesting that under the conditions of the study the cytotoxicity is particle size independent.

Materials and Methods

Brake wear particle collection

BWPs were obtained from two different sources, a motor vehicle testing center operating on light duty vehicles and a test bench working with an automotive brake dynamometer. In the latter case, the dynamometer is equipped with an environmental chamber, i.e. the brake system is operating in close mode. In both facilities, the brake wear dust was collected in a similar manner. The wheel was dismounted and the brake system opened. The dust was harvested in a 50 mL Falcon tube using a clean and sterile spatula. The disc and pad surfaces were not scrapped in the procedure. For the dust coming from transport vehicles, different types of driving conditions were realized, including tests with antilock braking and traction control system, park assist, wiper and accessories on different road surfaces. This multiple driving conditions allow retrieving a representative mixture of BWPs emitted by the current European car fleet and under real-life driving cycles. In both facilities, the dust was harvested on a regular basis and pooled with previous collected samples. As a result, it comprised both newly generated fresh particles and particles that aged in the braking systems and were hence subject to oxidation or decomposition. 19 The workers collecting the samples were trained to minimize contamination during the operation.

Brake wear particle characterization X-ray fluorescence analysis:

The BWP elemental analysis was carried out by X-ray fluorescence spectrometry. The as-received samples were deposited on a low absorption prolene membrane and analyzed on an Epsilon 3XL (Panalytical) spectrometer equipped with a silver X-ray tube. The analysis was performed using the Omnian software which detects and quantifies elements from sodium to uranium. Thermogravimetric analysis: Thermogravimetry was performed with LABSYS evo thermogravimetric analyzer (Setaram Instrumentations, Caluire-et-Cuire, France) under helium atmosphere. Approximately 50 mg of the as-received BWPs were placed in an alumina crucible and the temperature was raised from 30 °C to 1500 °C at a rate of 5 °C per minute.

Scanning electron microscopy: Scanning electron microscopy was performed on BWPs without metallization using a Zeiss SUPRA 40V SEM-FEG microscope (Oberkochen, Germany). The microscope was operated using an in-lens secondary electron detector working with a 2 kV acceleration tension and a 2 -3 mm working distance.

Optical microscopy: Phase-contrast images were acquired on an IX73 inverted microscope (Olympus Corporation, Tokyo, Japan) equipped with 20×, 40×, and 60× objectives. Seven microliters of a dispersion were deposited on a glass plate and sealed into a Gene Frame (ABgeneAdvanced Biotechnologies, Hamburg, Germany) dual adhesive system. 22 An EXi Blue camera (QImaging, Surrey, Canada) and Metaview software (Universal Imaging Inc., Bedford Hills, USA) were used as the acquisition system. ImageJ software was used for scale bar (http://rsbweb.nih.gov/ij/).

Brake wear dispersion and characterization

Nanoparticle dispersion: BWPs were suspended in water at 1 mg mL -1 , vortexed and sonicated for 1 to 10 minutes at maximum power (10.7 W) using a cup-horn equipped sonicator (Branson Ultrasonics, Shangai, China). For each assay, 7 vials each containing 2 mL of suspension were placed in the cup and sonicated over time. This procedure was optimized to establish a reliable protocol, in particular concerning the particle size, charge and long-term stability. In a second phase, the protocol was repeated to produce large volumes of dispersion required for the physico-chemical and toxicological studies. To separate the nano-sized fraction, the dispersions were hereafter filtered with a syringe equipped with 0.45 µm Nalgene™ polyether sulfone membranes (Thermo Fisher Scientific, Waltham, USA).

Light scattering: Scattering intensities 𝐼𝐼 ! and hydrodynamic diameters 𝐷𝐷 ! were measured using a Zetasizer NanoZS equipment (Malvern Instruments, Worcestershore, United Kingdom). A 4 mW He-Ne laser beam (𝜆𝜆 = 633 nm) was used to illuminate sample dispersion and the scattered intensity was collected at an angle of 173°. The second-order autocorrelation function was analyzed using the CONTIN algorithm to determine the average diffusion coefficient 𝐷𝐷 ! of the scatterers. The hydrodynamic diameter was calculated according to the Stokes-Einstein relation, 𝐷𝐷 ! = 𝑘𝑘 ! 𝑇𝑇/3𝜋𝜋𝜋𝜋𝐷𝐷 ! where 𝑘𝑘 ! is the Boltzmann constant, 𝑇𝑇 the temperature and 𝜂𝜂 the solvent viscosity. 22,23 Zeta potential: Laser Doppler velocimetry was used to carry out the electrokinetic measurements of electrophoretic mobility and zeta potential with the Zetasizer Nano ZS equipment (Malvern Instruments, Worcestershore, United Kingdom). Light scattering and zeta potential measurements were performed in triplicate at 25 °C after an equilibration time of 120 s. 24 Transmission electron microscopy coupled with X-ray dispersive energy: The BWP nanosized fraction was characterized by Transmission Electron Microscopy (TEM) using a JEOL 2010 microscope (JEOL Ltd, Tokyo, Japan) operating at 200 kV with a LaB6 filament and equipped with an Orius CCD camera (Gatan inc., Warrendale, PA, USA). Energy dispersive X-ray spectroscopy (EDX, PGT) investigations were performed to determine the elements from a single nanoparticle. To avoid parasitic contribution from copper on the EDX spectra, a molybdenum TEM grid and a beryllium sample holder were used. TEM image analysis was performed with ImageJ software, and the size distributions were obtained from 𝑛𝑛 = 200 particles. The distributions were adjusted using a log-normal function, leading median diameter and dispersity. 25

Toxicological studies

Cell culture: The human lung epithelial cell line Calu-3 has been shown to be relevant targets for the study of airborne particles on the respiratory tract. 26 Calu-3 cells (ATCC, HTB-55 TM , Manassas, VA, USA) were grown at 37°C under 5% CO 2 atmosphere in EMEM medium (Sigma Aldrich, Saint-Quentin Fallavier, France) supplemented with 10% foetal bovine serum (FBS, Eurobio, Les Ulis, France), 1% non-essential amino acids (Sigma Aldrich). Cells were trypsinized once a week (Trypsin-EDTA, Sigma Aldrich). Passages 25 to 29 were used.

WST-1 assays: Calu-3 cells were seeded at 40000 cells per well in 96-well plates. On the second day, cells were exposed for 24 h to BWPs and to its nano-sized fraction at increasing concentrations between 1 to 100 µg cm -2 , corresponding to weight concentrations 3 to 300 µg mL -1 . The cell viability was measured using the WST-1 assay performed using Cell Proliferation Reagent WST-1 (Roche, Basel, Switzerland) according to the manufacturer protocol. The experiments were performed in duplicates following the protocols developed by Vietti and coworkers. 27 After treatment removal, cells were rinsed twice with HBSS (Hank's Balanced Salt Solution, Invitrogen, Waltham, USA). A first plate was incubated with WST-1 dilution (1:10) in cell culture medium for 30 min, whereas the second one was treated with Triton X100 (0.1 wt. %) for 15 min before WST-1 addition. This second plate was used to evaluate potential interferences of adsorbed/internalized particles with WST-1. The absorbance signal reflecting the cell viability (mitochondrial activity) was measured at 450 and 630 nm with an ELx808 microplate reader (Biotek Instruments, Winooski, United States). The optic density at 630 nm was subtracted from the value at 450 nm to obtain the final signal.

Reactive Oxygen Species (ROS): Calu-3 cells were seeded at 40000 cells per well in 96well plate. Cells were first rinsed with HBSS then loaded with 25 µM solution of CM-DCFH 2 DA (Molecular Probes, Eugene, United States) in HBSS for 45 min at 37°C. Following a washing step with HBSS, cells were incubated with treatment during 0.5, 1, 2 and 4 h. Fluorescence was measured with FlexStation® 3 multi-mode microplate reader (Molecular Devices, Sunnyvale, United States) at 485/535nm. Fluorescence background at 535 nm was subtracted to the fluorescence signal at 485 nm. Positive control consisting in tert-butyl hydroquinone (TBHQ) and H 2 O 2 dilutions at 50 µm in cell culture medium was used according to manufacturer protocol.

Cytokine release: Calu-3 cells were seeded and treated in the same conditions as above. Supernatant were recovered and centrifuged (3000g, 4 °C) after treatment for quantification of IL-6, IL-8 and TNFα release in cell culture medium by ELISA (Enzyme-Linked Immunosorbent Assay). Absorbance signal reflecting cytokine release was measured at 450 and 630 nm with a ELx808 microplate reader (Biotek Instruments, Winooski, United States). Optic background density (OD) at 630 nm was subtracted to OD at 450 nm to obtain the final signal.

Results and Discussion

Brake wear particle characterization

The BWP samples collected from passenger vehicles and from an automotive brake dynamometer have the appearance of a fine black powder (Fig. 1a). The BWPs were characterized using different techniques including thermogravimetry, X-ray fluorescence, optical and scanning electron microscopy (Fig. 1). Fig. 1b shows thermogravimetry data obtained between room temperature and 700 °C. The mass variation reveals an initial 6% decrease that is attributed to the loss of bound water around 100 °C and to the degradation of the carbon-based binder, resin and additives. This amount is lower than expected, as binders in brake pads are known to account for 20 -40 % of the lining material. This result could be explained by the heat generated during braking that leads to high temperature and to the thermal degradation of the carbonaceous materials. 9,13,14 At the micrometer length scale, the particle size distribution was studied using phasecontrast optical microscopy. Fig. 1c highlights the presence of contrasted particles in the range 200 nm -10 µm, with a distribution peaked at 1.47 µm (median value) and a dispersity of 0.47 (Supplementary Information S1). Particles larger than 10 µm were also found but in a smaller proportion. The insets in Fig. 1c display close-up view of particles or aggregates of increasing size and contrast. X-ray fluorescence spectrometry was used to determine BWP elemental composition (Fig. 1d). The technique reveals 18 different elements at a level above 0.1%, including for the most prominent ones iron (Fe, 29%), copper (Cu, 17%), silicon (Si, 13%), aluminum (Al, 11%), zinc (Zn, 8%) and sulfur (S, 7%). The proportions and compositions obtained are consistent with those known for low and semi-metallic brake pads and drums typical for the European market. [7][8][9]11,19 They are also in line with the data received for the dynamometer test bench BWPs, indicating the weak impact of environmental driving conditions on the studied specimens (Supporting Information S2). The outcomes confirm the presence of metals such as iron, copper and zinc known to induce toxicity to living cells. We also note that antimony was not found from the elemental results and that barite was not used as filler in the brake pad formulations studied. Scanning electron microscopy (Fig. 1e) reveals that at the micro-and nanoscale the BWPs display a wide range of sizes and morphologies. 19,20,[28][29][30] The left-hand side images show fine particles with sharp edges and rough surfaces, as well as numerous pittings arising from material removal (arrows). Micron size objects appear as shavings and fragments generated by metallic surface friction. 7 At a sub-micron scale (right handside panels), the surfaces have either platelet packed solid structures or show a large number of smaller particles associated with the background debris. From these data, it is not clear whether the sub-micron particles adsorb during the braking and emission events or later during sample collection and treatment. For the separation and toxicity assays presented in the next sections, emphasis will be put on the BWPs from passenger vehicles. Data from the dynamometer test bench can be found in the Supplementary Information S2 and S3.

Figure 1: Brake wear particle characterization a) BWP sample obtained from a motor vehicle testing center operating on commercial transport vehicles. BWP composition was characterized using thermogravimetry analysis measured in helium atmosphere (b) and X-ray fluorescence for elemental analysis (d). To evaluate the size and morphology of the wear particles optical (c) and scanning electron (e) microscopy were performed. The insets in c) are 7×8 µm 2 fields.

Sonication and separation of brake wear particles in water SEM has shown the existence of sub-100 nm particles, and an important question pertaining to the toxicity assessment is that of the nano-sized fraction. 3 Sonication is a well-known technique used to break particle aggregates as well as to improve particle dispersability. BWP were dispersed in DI-water at 1 mg mL -1 , vortexed and sonicated as a function of the delivered sonication energy 𝐷𝐷𝐷𝐷𝐷𝐷 31,32 under conditions described in the Materials and Methods section. The suspensions were then filtered through a 0.45 µm membrane and characterized by static and dynamic light scattering. Fig. 2a show the scattered intensity 𝐼𝐼 ! (upper panel) and the hydrodynamic diameter 𝐷𝐷 ! (lower panel) against the 𝐷𝐷𝐷𝐷𝐷𝐷 for filtered and non-filtered BWP dispersions. 22 Prior to sonication, the dispersion exhibits a strong scattering intensity and diameters larger than 1 µm, in agreement with the optical and electron microscopy results. The filtered solution in contrast has a much weaker scattering, about 400 times smaller, indicating that as anticipated filtration did remove most suspended particles. With increasing 𝐷𝐷𝐷𝐷𝐷𝐷, the scattering intensity increases and displays a saturation plateau. In parallel the diameter decreases and levels off at 165 nm. The corresponding autocorrelation functions 𝑔𝑔 ! (𝑡𝑡) obtained from dynamic light scattering exhibit a quasi-exponential decay associated with a unique relaxation mode 23 and a distribution that refines with increasing 𝐷𝐷𝐷𝐷𝐷𝐷 (Fig. 2b). Note that at the highest input energy, the intensity for filtered and unfiltered dispersions are close to each other, suggesting that sonication was able to redisperse most particles. With a zeta potential of -26 mV, electrokinetic measurements confirmed that brake wear nanoparticles are negatively charged and that their stabilization is ensured by electrostatics (Fig. 2c). We also found that the surface charge does not depend on the sonication energy. The previous experiments were repeated in presence of bovine serum albumin (BSA) proteins at 1 g L -1 to evaluate whether adsorbed macromolecules could improve the dispersion stability. The figures in Supplementary Information S4 show that BSA has no significant effect on the particle dispersability, even at long term (> 1 month). To determine the proportion of nanoparticles in the BWP samples, the dispersions were evaporated in an oven and the residual dry matter weighed, revealing that 26 ± 1% of the initial BWP is indeed made up of nanoparticles. This percentage is consistent with the static light scattering results of Fig. 2a. 31,32 In the configuration used, a one-minute sonication translates into a total input energy of 30 J mL -1 . b) Second-order autocorrelation functions of the scattered intensity for the filtered solutions in a). With increasing 𝐷𝐷𝐷𝐷𝐷𝐷, the dispersity indexes (pdi) were 0.28, 0.29, 0.20 and 0.16, respectively. c) Intensity distribution as a function of the zeta potential 𝜁𝜁 for a sonicated and filtered dispersion showing that BWP nanoparticles are negatively charged.

Brake wear nano-sized fraction: determination of size, shape and composition

The BWP nano-sized fraction was further characterized by transmission electron microscopy coupled with X-ray dispersive energy spectroscopy. As shown in Figs. 3a and3b, nanoparticles have sizes comprised between 50 and 150 nm and a broad range of morphologies. The most abundant elements are copper and iron, in good agreement with the X-ray fluorescence data. Interestingly, EDX spectra from different brake wear nanoparticles show the presence of one dominant of metal per particle, with traces of others, indicating that the particles were generated from different parts of the pads and drums. Examples are provided in Supporting Information S5 for iron, silicon, zirconium, tin, bismuth and titanium. Fig. 3c displays a size distribution peaked at 66 nm (median value) and characterized by a dispersity of 0.50. Studied at different times after sonication, the BWP nanoparticles were found to be stable over time, with hydrodynamic diameters remaining constant over a month (Fig. 3d). In cell culture medium it was found that the nano-sized fraction slowly destabilizes as a function of time, giving rise to the formation of aggregates. 33 Aggregation is here primarily due to the adsorption of proteins and/or biological molecules at the particle surfaces that modify the interaction between particles. From repulsive in DI-water, the interaction becomes attractive in presence of salt or proteins, leading to the particle destabilization. [34][35][36] Fig. S6 displays the autocorrelation function and the size distribution after a 24 h incubation at 100 µg mL -1 . The data in Supporting Information exhibit three relaxation modes, one associated with the initial particle size (160 nm, 12%), the second and third modes being associated with medium (320 nm, 78%) and large (1200 nm, 10%) aggregates. The percentages in parenthesis indicate here the frequency of each contribution. These results will be exploited later in the manuscript to determine the effective dose administered to the cells using the in vitro sedimentation, diffusion and dosimetry model recently developed. 31,32 To conclude, we combined sonication and filtration to separate the BWP nano-sized fraction from the original dust. This protocol will be now applied for in vitro toxicity assessment studies.

Biological effects of brake wear particles and its nano-sized fraction on Calu-3 cells

The toxicity of BWP and its nano-sized fraction was investigated on human bronchial epithelial cells Calu-3. 26 The cytotoxicity was assessed using the WST-1 mitochondrial activity assay 37 for concentrations 1 to 100 µg cm -2 . After a 24 h exposure, the cell viability was found to decrease with the applied dose for both samples (Fig. 4a). However, the BWP exhibited a stronger effect, the viability being reduced by 81% at 100 µg cm -2 , this value becoming 35% for the nanoparticle dispersion. Zhao et al. observed that some brake wear samples reduce the viability of A549 alveolar cells, and that the decrease was comparable to ours. 19 Exposing the same cell line to an aerosol of freshly produced BWP however Gasser et al. did not notice any significant effect. This apparent discrepancy could be due to the different plating techniques used, cells being grown on inserts providing a denser epithelium compared to the conditions in plates, leading to lower particle dose per cell. 18 BWP being mainly composed of metal compounds (as shown in Fig. 1), this loss of viability may be related to the oxidative stress arising from the imbalance between intracellular oxidants and anti-oxidant molecules. 38 Metallic ions can generate ROS through the Fenton reaction for transition metals and by depleting thiol anti-oxidant molecules such as glutathione. 2 The intracellular redox state was assessed using the DCFH-DA probe that exhibits green fluorescence upon oxidation. A time-course study over 4 hours revealed a time dependent increase of the DCF fluorescence in cells treated with either BWP or with its nano-sized fraction. The fluorescence signal remains however lower than that of positive H 2 O 2 and T-BHQ controls (Supporting Information S7). Fig. 4b displays the ROS production as a function of the dose following a 4 h incubation together with the controls. Both BWP and nanoparticles exhibit a similar behavior, with a nearly two-fold increase at the highest dose. These results are in fair agreement with those of the literature for A549 cells treated in the same conditions. 18 The ability of BWP to induce a pro-inflammatory response was characterized by evaluating the release of the IL-6, IL-8 and TNF𝛼𝛼 cytokines after a 24 h exposure (Fig. 4c). While for the BWP IL-8 and TNFα releases were not significantly modified (Fig. S8), the IL-6 release was found to be dose dependent, with a deviation seen already at 10 µg cm -2 , i.e. at the concentration where cell viability is reduced by 50%. At the highest dose, the IL-6 release shows a 5-fold increase compared to the control. The situation is different for the nano-sized fraction for which IL-6 and IL-8 signals remain dose independent and similar to control. The toxicity, ROS production and pro-inflammatory response results in Fig. 4 reveal an overall lower toxicity for the brake wear nanoparticles compared to that of the initial fraction. We have checked whether this extra toxicity could be due to the soluble fraction that is present in the BWP dispersions (and not in the nanoparticle dispersions) and may contain harmful metallic ions. The cytotoxicity of the soluble fraction was evaluated by WST-1 assay and revealed a weak decrease of viable cells by 10% at the highest concentration, suggesting that the solubilized ions do not contribute significantly to the observed phenomenon (Fig. S9). From the above results, the nano-sized fraction appears hence to be less toxic than BWP. However the comparison was made on the basis of the nominal concentrations and not on the effective delivered concentrations. To examine this issue, we have applied the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) model recently developed by DeLoid and coworkers to improve dose metrics methodologies. 31,32 According to the ISDD model, the doses administered to the cells depend primarily on the particle sizes and on their densities. For brake wear nanoparticles we based our calculations on the light scattering data of Fig. S5, as suggested by the model. For the BWP, we used the size determined from optical microscopy (Fig. S1), the light scattering data being not reliable for micron size particles. The results of the ISDD simulations shown in Supporting Information S10 include the time dependencies of the deposited fraction and bottom concentration, and the concentration gradients in the well. It was found that the micron size BWP sediment rapidly and that the delivered dose reaches 79% after 24 h of exposure. For the nanoparticle in contrast, the dose delivered to cells was estimated to reach 7 % of the nominal dose in the same conditions. Considering the effective doses, the mitochondrial activity results of Fig. 4 can be re-interpreted. At 50 µg cm -2 for instance, the effective dose for nanoparticles at 24 h is around 4 µg cm -2 and the cell viability 70%. For the BWP, an effective dose of 4 µg cm -2 corresponds to a nominal dose of 5 µg cm -2 , for which the cell viability is 60%, so close to that of the previous value. In conclusion, the differences seen in the cell viability for regular BWP and for its nano-sized fraction are mainly due to dosimetric effects, demonstrating that BWPs are toxic, but the nano-sized fraction does not display extra toxicity. Similar findings have been identified for the pro-inflammatory response, whereas for the ROS production nanoparticles exhibit a more pronounced effect (Supplementary Information S11). Altogether these data suggest that under the conditions of the study, the brake wear macro-and nanoparticles exhibit similar cytotoxicity. We can speculate that the underlying mechanisms leading to cell death could be different, as indicated by the difference in ROS production.

Figure 4: Biological effects of brake wear particles and its nano-sized fraction on Calu-3 cells

Calu-3 cells were exposed to BWPs and to its nano-sized fraction at concentrations ranging from 1 to 100 µg cm -2 (corresponding to effective weight concentrations in the supernatant of 3 to 300 µg mL -1 ). a) Mitochondrial activity reflecting cell viability were assessed using WST-1 assay after a 24 h of exposure. b) Reactive oxygen species production was detected by DCFH probe oxidation after 4 h exposure. c) IL-6 cytokine release measured by ELISA was used to characterize the pro-inflammatory response after a 24 h exposure. Statistical analysis was performed using One-way ANOVA and Dunett's post-test to compare particles effects with no exposure. The symbols ( # ) and (°) refer to the BWP and to its nano-sized fraction respectively. Two-way ANOVA complemented by Holm-Sidak post test was performed to compare the BWP size effects (*).

Conclusion

We have shown that BWPs emitted from low-metallic brake pads used in passenger cars and coming from two different sources have typical sizes in the micron range and contain metallic compounds, such as iron, copper, silicon, aluminum and zinc under crystalline and amorphous forms. Scanning electron microscopy indicates that the initial BWPs appear as fragments or debris made from a large number of sub-micron particles. To isolate these sub-micron particles we set up a combination of sonication, centrifugation and filtration techniques. The first significant conclusion that emerges from this work is that the nanosized fraction represents around 26% by mass of the initial brake wear dust. The nanoparticles are also broadly distributed, with size comprised between 50 and 400 nm.

Their toxicity was assessed on human bronchial epithelial cells. The second important result is that BWPs display short-term toxicity and we do not find any enhanced effect from the nano-sized fraction. These results show finally the interest of isolating this fraction of brake wear dust and characterizing its in vitro behavior, in particular in the cell culture medium for accurate dosimetry and toxicity assessment. Corresponding authors: chloe.puisney@univ-paris-diderot.fr, baeza@univ-parisdiderot.fr, jean-francois.berret@univ-paris-diderot.fr

Supporting Information

Brake wear (nano)particle characterization and toxicity on airway epithelial cells in vitro

Figure 2 :

 2 Figure 2: Characterization of brake wear particles before and after sonication and filtration a) Scattering intensity (upper panel) and hydrodynamic diameter (lower panel) measured by light scattering on 1 mg mL -1 sonicated BWP dispersions as a function of the delivered sonication energy 𝐷𝐷𝐷𝐷𝐷𝐷.31,32 In the configuration used, a one-minute sonication translates into a total input energy of 30 J mL -1 . b) Second-order autocorrelation functions of the scattered intensity for the filtered solutions in a). With increasing 𝐷𝐷𝐷𝐷𝐷𝐷, the dispersity indexes (pdi) were 0.28, 0.29, 0.20 and 0.16, respectively. c) Intensity distribution as a

Figure 3 :

 3 Figure 3: Composition, size and stability of the brake wear nano-sized fraction a,b) left: TEM images of brake wear nanoparticles; right: EDX spectra of the particle region shown in the blue circles. c) Size distribution determined from TEM on the filtered particles. The median value is 66 nm and the dispersity 0.50. d) Hydrodynamic diameter as a function of the time for brake wear nanoparticle dispersions at increasing sonication energies. In the four instances, the particles are stable as the diameter remains unchanged for the period.

  ____________________________________To appear in Envriron. Sci.Nano March 2018

C. Puisney 1 , 2 *OutlineFigure S1 -

 12S1 Figure S1 -Size distribution from BWP found on commercial transport vehicles Figure S2 -Characterization of brake wear powder Figure S3 -Size distribution from BWP collected from an automotive brake dynamometer Figure S4 -Light scattering data for BWP dispersed and sonicated with proteins Figure S5 -Complementary TEM images and EDX spectra from brake wear nanoparticles Figure S6 -Dynamic light scattering results in cell culture medium Figure S7 -Reactive oxygen species production in Calu-3 cells Figure S8 -IL-6 and TNFα release by Calu-3 cells exposed to BWP or its nanosize fraction Figure S9 -Cell viability of Calu-3 cells exposed either to particles or the soluble fraction Figure S10 -Cell viability of Calu-3 cells exposed either to particles or the soluble fraction

Figure S10B :

 S10B Figure S10B: Table -List of input parameters injected in the in vitro sedimentation, diffusion and dosimetry model reported by DeLoid and coworkers (DeLoid G.M. et al. Nat. Protoc. 2017, 12, 355-371) for the brake wear nano-sized fraction. The size distribution refers to the light scattering data in Fig. S5c. a) The fraction of deposited particles as a function of the time shows a saturation around 7% of the total administered dose after 24 h. b) Bottom concentration as a function of the time. The initial concentration in the dispersion was set at 0.1 g L -1 (corresponding to 1 mg cm -3 ). c and d) Time evolution of the concentration as a function of the position in the sample. The abscissa 3 mm represents the bottom of the cell culture well. The different colored curves correspond to different snapshots of gradient concentration.

Acknowledgments

The authors thank DIM (Domaine d'Intérêt Majeur) Nano-K of Region Île-de-France for funding C. Puisney Ph. D. thesis at the University Paris-Diderot. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABX 086, ANR 11 IDEX 05 02. We acknowledge the ImagoSeine facility (Jacques Monod Institute, Paris, France), and the France BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, « Investments for the future »). This research was supported in part by the Agence Nationale de la Recherche under the contract ANR-13-BS08-0015 (PANORAMA) and ANR-12-CHEX-0011 (PULMONANO).

Conflicts of interest

There are no conflicts to declare.

Supporting Information Figure S1 -Size distribution from BWP found on commercial transport vehicles

Supporting Information Figure S2 -Characterization of brake wear powder

Figure S2: Characterization of brake wear powder originating from an automotive brake dynamometer a) Brake wear powder sample obtained from an automotive brake dynamometer for pad and drum testing. BWP composition was characterized using thermogravimetry analysis (b) and X-ray fluorescence for elemental analysis (d).

Note that for the sample coming from the motor vehicle testing center, the iron and copper proportions were 29% and 17% respectively, whereas the values here are 43 and 34 %. Moreover, this brake pad formulation contained Sb and Mo, most probably in form of sulfides (since content of S is 3.9%). Content of Ba (0.2%) suggests that Ba was not used as a filler. To evaluate the size and morphology of the wear particles optical (c) and scanning electron (e) microscopy were performed. The insets in c) are 24×40 µm 2 fields.

Supporting Information Figure S3 -Size distribution from BWP collected from an automotive brake dynamometer

Figure S3: Distribution of particle detected by optical microscopy obtained from an automotive brake dynamometer for pad and drum testing. The fitting curve is given by a log-normal function.

Supporting Information Figure S4 -Light scattering data for BWP dispersed and sonicated with proteins

Supporting Information Figure S6 -Complementary TEM images and EDX spectra from brake wear nanoparticles

Supporting Information Figure S7 -Reactive oxygen species production in Calu-3 cells

Figure S7: Reactive oxygen species production was detected by DCFH probe fluorescence in Calu-3 cells exposed for 4 h to BWP from 1 to 100 µg cm -². H 2 O 2 and TBHQ were used as positive controls

Supporting Information Figure S8 -IL-6 and TNFα α release by Calu-3 cells exposed to BWP or its nanosize fraction

Figure S8: IL-8 and TNFα cytokine release by Calu-3 cells exposed for 24 h to BWP and its nanosize fraction at different concentrations. Cytokine measured by ELISA was used to characterize the pro-inflammatory response after a 24 h exposure. Statistical analysis was performed using One-way ANOVA and Dunett's post-test to compare particles effects with no exposure. The symbols (#) and (°) refer to the BWP and to its nanosize fraction respectively. Two-way ANOVA complemented by Holm-Sidak post-test was performed to compare the BWP size effects (*).

Supporting Information Figure S9 -Cell viability of Calu-3 cells exposed either to particles or the soluble fraction

Figure S9: Cell viability of Calu-3 cells exposed for 24h to different concentrations of BWP, their nanosize fraction and the corresponding soluble fraction. Mitochondrial activity reflecting cell viability was assessed using WST-1 assay.

The soluble fraction was the supernatant recovered after BWP sonication, filtration and centrifugation.

Supporting Information Figure S10 -Application of the in vitro sedimentation, diffusion and dosimetry model to the brake wear nano-and microparticles