Alessandra Gianoncelli

Patrick Marmorato

Jessica Ponti

Lorella Pascolo

Burkhard Kaulich

Chiara Uboldi

François Rossi

D Makovec

M Kiskinova

Giacomo Ceccone
email: giacomo.ceccone@jrc.ec.europa.eu

Interaction of magnetic nanoparticles with

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The advent of ultrabright and tunable synchrotron light sources has lead to the development of high resolution X-ray imaging combined with micro X-ray fluorescence (XRF) elemental mapping, opening the unique opportunity for morphology and chemical imaging at submicron level, relevant for biological research. The characterization based on these methods, correlated to conventional approaches, becomes very attractive for investigating the biological effect of nanomaterials at single cell level. [START_REF] Ide-Ektesssabi | Applications of Synchrotron Radiation: Micro Beams in Cell Microbiology and Medicine[END_REF] Nanotechnology has a great potential to solve present and future challenges in several fields such as energy, material, environment, and medicine. After more than 2 decades of basic and applied research, nanotechnologies are reaching the commercial use and the number of producers offering basic nanomaterials is growing fast. [2,[START_REF]Cheaptubes.com, the source for Carbon Nanotubes[END_REF] In fact, nowadays more than 1300 products containing nanomaterials (e.g. tires, sporting goods and clothing, sunscreens, cosmetics, food and electronics) are on the market. [4] Notably, an increasing number of nanoproducts is considered in biomedicine as diagnostic, imaging, and drug delivery tools. [START_REF] Jain | [END_REF][6][START_REF]Roadmaps in Nanomedicine towards 2020[END_REF] For example, Ag NPs and ZnO NPs are added as antibacterial in several cosmetics, sunscreens, [START_REF] Noynek | [END_REF] and medical products, [9] whereas TiO 2 and SiO 2 NPs are used as food additives as well. [10] Furthermore, magnetic nanoparticles (MNPs), such as ferrites (Fe 3 O 4 ) and cobalt ferrites (CoFe 2 O 4 ), have found possible applications in biomedicine as drug carriers, as hyperthermia treatments in cancer therapy, and as magnetic resonance imaging contrast enhancement [11] and have also acquired importance in imaging relevant to cancer and diabetes diseases. [12] In the field of tissue engineering, MNPs have already been considered for many applications including cellular labeling, cell sorting and monitoring, targeted in vivo therapeutic delivery, stem cell replacement therapy, and welding tissue surfaces. [13] For instance, incorporating superparamagnetic NPs into mesenchymal stem cells for the regeneration of tissue damage in the central nervous system has enabled imaging and monitoring of cellular migration by external magnetic fields to the wound site and helped optimizing the number of cells needed for tissue regeneration and monitoring possible side effects. [14] Some superparamagnetic nanoparticles have been approved for imaging and therapeutic applications in humans, for example, Feridex IV W and Combidex W (Advanced Magnetics Inc., Lexington, MA, USA) [START_REF] Jain | [END_REF][6][START_REF]Roadmaps in Nanomedicine towards 2020[END_REF] and several other superparamagnetic nanoparticles are undergoing phases I and II clinical trials. [15] Although nanomaterials are key factors in nanotechnology innovations, their presence in commercial products raise concerns about possible adverse effects on environment, human health, and society (NanoEHS). In fact, even if the composition of nanomaterials is not considered as toxic, they may pose serious safety problems due to unpredicted interactions resulting from physicochemical properties controlled by their specific size, structure, and surface chemistry. However, despite the large amount of data reviewing the potential toxicity of nanoparticles, the evaluation and mechanisms of nanomaterials toxicity (nanotoxicity) are still poorly understood. In fact, there are conflicting results about toxicity of nanomaterials. For instance, SiO 2 NPs are found to induce cytotoxicity in some cell lines [16] but not in others, [17,18] whereas current studies of toxicity of carbon nanotubes and TiO 2 have reported controversial results. [19] Among the promising detection methods, synchrotron radiation XRF (SR-XRF) has already demonstrated its potential in biomedical research for exploring, among others, neurodegenerative disorders. [20][21][22][23] The application of SR-XRF with soft X-rays [24] has recently provided access to light elements, which are major constituents of the living matter and this set-up is developed and implemented for the first time in the TwinMic microscope operated at Elettra laboratory (Trieste, Italy). Using this set-up, which combines submicron soft X-ray microscopy with low energy XRF (LE-XRF) microspectroscopy, we recently investigated the effect CoFe 2 O 4 nanoparticles in mouse fibroblasts (Balb/3 T3 cells), showing that the accumulation of these nanoparticles can induce metabolic change. [25] In the present study, we investigated the effect of two iron-based nanoparticles in a new cell line monitoring morphological and chemical changes in the cells using the complementary X-ray imaging and LE-XRF capabilities of TwinMic beamline at Elettra Synchrotron in Trieste. [26] In particular, we have investigated the interaction of ferrites (Fe 2 O 3 , maghemite), and cobalt ferrites (CoFe 2 O 4 ) NPs with U87MG human glioblastomaastrocytoma cells.

Experimental

Ferrites (Fe 2 O 3 , maghemite) nanoparticles were synthesized in Josef Stefan Institute (Lubljana, Slovenia) using the wellknown co-precipitation of Fe 2+ and Fe 3+ salts using NH 4 OH in the presence of ricinoleic acid to obtain hydrophobic NPs surface. [27,28] Thermogravimetric analyses showed that the asprepared suspension consisted of 29wt% iron-oxide, 12wt% ricinoleic acid, and 59wt% methyl methacrylate (PMMA), whereas the X-ray diffraction data showed that maghemite NPs were obtained. The presence of the PMMA onto the nanoparticles was also confirmed by X-ray photoelectron spectroscopy analysis (data not shown).

Dynamic light scattering (DLS) measurements were carried out on 1 ml sample volume with the Zetasizer Nano Zs (Malvern Instruments, UK), following the measurement protocols indicated by ISO 13321:1996. Three measurements instead of the recommended six were performed after data quality assessment showing comparable results. The results of three measurements for each sample were averaged by the Zetasizer software.

The CoFe 2 O 4 NPs were supplied by Colorobbia S.p.A. (Italy) as suspension in 100% diethylene glycol (DEG). NPs were synthesized using the polyol method: cobalt acetate and iron acetate were solubilized in DEG at 110 C for 1 h, and then the solution was heated to 180 C. After 3 h, the product was air cooled to room temperature and then stored. [29] Cobalt concentration of 7mg/mL was measured by inductively coupled plasma mass spectrometry (PerkinElmer, ELAN DRC II SCIEXTM, Canada).

U87MG human glioblastoma-astrocytoma cells (ATCC number HTB-14) were prepared from deep-frozen stock vials, kept in a subconfluent state and maintained in complete culture medium (Modified Eagle Medium low glucose, 10%(v/v) fetal bovine serum (FBS) Australian origin, 1%(v/v) penicillin/streptomycin, Invitrogen, Milan, Italy) under standard cell culture conditions (37 C, 5% CO2, and 95% humidity, HERAEUS incubator, Germany).

The potential toxicity of Fe 2 O 3 and CoFe 2 O 4 NPs was evaluated by trypan blue exclusion dye test. The cytotoxicity of nanoparticles was tested after 24 h of exposure at concentrations ranging from 1 to 235 mg/ml. 7 Â 10 4 U87MG cells were cultured overnight into 24 well-plates and then incubated for 24 h with nanoparticles. At the end of the exposure time, cells were washed with phosphate buffer solution (PBS; Invitrogen, Italy), trypsinized and counted.

For the SR-XRF analysis, the cells were cultured on silicon nitride membrane (Silson Ltd, Northampton, UK) contained in six wells plate (Corning, Costar) at the density of 10 5 cells/well in 3 ml of complete culture medium. After 24 h, the medium was changed and cells were exposed to CoFe 2 O 4 or maghemite nanoparticles (concentrations 5-50 mg/ml range). After 24 h, samples were washed twice with PBS and fixed with 4% (v/v) of formaldehyde solution (Sigma, Italy) in PBS buffer.

The TwinMic instrument used for the present investigation works in the 0.4-2.2 keV energy range. [24,[START_REF] Kaulich | Proc. 8th Int. Conf. X-ray Microsc[END_REF] Soft X-ray microscopy can be performed both in full-field imaging and scanning transmission (STXM) modes. [START_REF] Gianoncelli | [END_REF] The data were acquired using the STXM mode, where the X-ray beam can be focused down to sub-mm spot size by zone plate diffractive optics and the specimen is raster-scanned across the X-ray probe for obtaining 2D images. The X-ray absorption and phase contrast images of transmitted X-rays were recorded by an Andor-Technology CCD camera providing simultaneously absorption and phase information. [START_REF] Gianoncelli | [END_REF] Simultaneous monitoring of the emitted fluorescence signal using eight silicon drift detectors distributed circularly in front of the sample provide the elemental maps covering K or L fluorescence emission of elements in the 150-2000 eV range (From B to P and metals as Mg, Fe, Mn, Cu, and Zn). [32] This simultaneous X-ray transmission and LE-XRF mapping allows direct correlation of the sample morphology to elemental composition.

The present experiments were carried out with photon energy 1.1 keV and spot size of 650 nm for XRF mapping, which was a good compromise for getting a sufficient fluorescence signal with acceptable spatial resolution. The elemental map was acquired in hdf5 format and then analyzed by the multiplatform program (PyMCA) developed by V. Sole and co-workers. [33] Interaction of magnetic nanoparticles with U87MG cells hydrophobic and prevent the agglomeration but not the protein corona formation. The Trypan dye exclusion test determined that U87MG cells maintain a viability from 100% to 80% when exposed from 10 to 235 mg/ml of CoFe 2 O 4 NPs. Differently, U87MG exposed to 5 mg/ml of Fe 2 O 3 NPs have shown a viability of about 30%, which is stable up to 75 mg/ml. A total of 5 and 50 mg/ml are the test concentrations chosen for XRF analyses and to compare morphological and chemical changes.

Results and discussion

The X-ray absorption and phase contrast images together with the C, Fe, O, and Na elemental maps of a control U87MG cell are shown in Fig 1 . At the photon energy used (1.1 keV), the differential phase contrast image is better than the absorption one in defining the morphology of the cell, enhancing the cell borders, and it adds a three-dimensional perception of the nuclear structure. On the other hand, the denser nucleoli inside the nucleus are appreciable in both the images. The maps of the endogenous macroelements C, O, and Na confirm the cell shape and density of the intracellular structures. The Fe map shows only a few small spots where the Fe is slightly higher than in the background. This indicates that the contribution to the Fe signal due to the Fe present in the cell is negligible. 4, the NPs are asymmetrically localized in the Golgi-like region or in the endoplasmic reticulum. However, at 50 mg/ml, the Fe 2 O 3 accumulation induces stress in the cells: the nucleus in the respective Fig. 3 (panels a andb) is almost contracted and the NPs are broadly distributed or aggregated inside the cell. This, in addition to the viability data, indicates an increasing cellular stress in the presence of NPs concentrations above 5 mg/ml. It should be also noticed that the cells exposed to CoFe 2 O 4 NPs show a different morphology compared with both the control and the cells exposed to the maghemites. In fact, both phase contrast and absorption images show the presence of a white corona around the nucleus (Figs. 4 and5) that is absent in the cells exposed to Fe 2 O 3 NPs (Figs. 2 and3) and in the control (Fig. 1). This may indicate a different type of interaction between cells and nanoparticles, different pathways of internalization and toxicity mechanisms. In fact, the difference in size of the nanoparticles when in contact with the culture medium might indicate a different uptake level of the ferrites compared with the CoFe 2 O 4 NPs. In the latter case, the presence of large agglomerates will probably reduce the number of nanoparticles internalized by the cells. However, at this stage, it is difficult to make a quantitative comparison of the two nanoparticles and further studies are needed to clarify this point. Moreover, it is also difficult to draw a conclusion only on the base of DLS measurements because it is well known that DLS data could strongly be influenced by the presence of a few large particles, aggregates or agglomerates that will mask the contribution of small nanoparticles. Moreover, the release of Co in the culture media from the CoFe 2 O 4 NPs that are not covered by polymers further complicate the system, making difficult to compare the behavior of the nanoparticles. The most interesting result from the comparison of the elemental maps is the clearly increase of C content in the perinuclear region in the presence of CoFe 2 O 4 NPs, partially co-localized with Fe (see the C and Fe colocalization maps). This interesting result is in agreement with our previous data indicating that the presence of CoFe 2 O 4 in Balb/3 T3 fibroblasts causes the accumulations of lipid droplets inside the cells, the effect being NPs concentration-dependent. [25] It is not surprising that the astrocytoma-derived cells show similar cell response, whereas the lack of a comparable metabolic change in the presence of Fe 2 O 3 NPs is a real novel result. The uptake of Fe-based NPs by a cell and its accumulation, particularly at high doses, may influence the cell electronic and/or ionic transport chains. For instance, a recent study using XANES technique has shown that the redox state of Fe in iron-oxide nanoparticles can induce oxidative stress in Escherichia Coli bacteria. [34] Thus, it is not surprising that 50 mg/ml causes pre-apoptotic conditions as resulting from the nuclear contraction in Fig. 3(a,b). On the other hand, it is highly plausible that a different availability of Fe to interact with cell structure in the presence or absence of Co in the NPs could be the reason for different toxicological pathways in astrocytoma-derived U87MG cells. In fact, the U87MG cells exposed to CoFe 2 O 4 NPs show different peculiar features. The absorption and phase contrast images [Figs. 4(a andb) and 5(a and b)] evidence the presence of a less absorbing circle delineating the cell nucleus. Both the white corona around the nucleus and some morphological changes of the cells (enlarged cell body and pale plasma membrane borders), suggest that the cell is under stress. At concentrations ≥ 60 mg/ml, the presence of bright vesicles in the cytoplasm becomes more evident. As explained in a previous publication [25] , the vesicles appear to be lipid droplet organelles and their number increases when the cells are exposed to the NPs. Moreover, in the same work, we have shown that in fibroblast cells exposed to CoFe 2 O 4 NPs at concentrations below 58mg/ml, Co and Fe are localized in cytosol, indicating that the NPs are preferentially localized in the perinuclear region. On the other hand, at higher concentrations (≥117 mg/ml), Fe and Co are also present in the nuclear region of the cell, with sensible accumulation of Co. A co-localization of P, Ca, and Fe at high concentration has also been observed indicating intracellular sequestration mechanisms as a response or in an attempt to reduce the nanoparticles toxic effects.

Nucleoli seem to be better defined in unexposed control U87MG cells (Fig. 1) than in the cells incubated in the presence of maghemite NPs (Figs. 2 and3), highlighting once more the stress induced by the interaction of U87MG cells with NPs.

Conclusions

Soft X-ray microscopy coupled with LE-XRF has proven to be a valuable and powerful tool for investigating biochemical processes at subcellular level. The combination of morphological and elemental information can provide useful insights on complex processes in biological matter occurring at the submicron length scales.

Even though the spatial resolution used in this investigation did not allow detecting single NPs, NPs clusters were easily visible through absorption, phase contrast, and XRF imaging. As discussed earlier, the present study has allowed extrapolating new insight in the interaction between cells and NPs. Further investigations at higher energies, allowing the detection of heavier elements such as P, S, and Ca, which are important constituents of the cells, are planned and they would help in evaluating the behavior of cells in the presence of NPs.

  Figures 2 and 3 together with Figs. 4 and 5 depict X-ray absorption and phase contrast images together with the C, Fe, O, and Na elemental maps of U87MG cells exposed for 24 h at two different concentrations (5 and 50 mg/ml) of Fe 2 O 3 NPs and CoFe 2 O 4 NPs (58 and 235 mg/ml), respectively. The limits of the cell structures and the presence of NPs, highly concentrated inside the cells, are clearly visible both in the absorption and differential phase contrast images. The localization is finally confirmed by Fe (and Co) maps. A clear increase of O content is also evidenced in correspondence of the NPs. At low NPs concentrations, the Fe maps indicate a distribution of both iron-based NPs maximal in the perinuclear region. As clearly shown in the Fe map in Fig. 2 and in the Fe and Co maps in Fig.

Figure 1 .

 1 Figure 1. Absorption (a) and differential phase contrast (b) images of a U87MG control (not exposed to nanoparticles) cell (40 mm  40 mm, 20 ms dwell time). Low energy XRF maps of C, Na, O, and Fe acquired on the area shown in panel a. The maps were collected on a 40 mm  40 mm area in an overall 60  60 pixel scan with 15 s/pixel acquisition time and spatial resolution of about 650 nm.

Figure 2 .

 2 Figure 2. Absorption image (a), differential phase contrast (b) image, and XRF maps of C, O, Co, Fe, and Na, with co-localization of Fe and C, all acquired at 1.1 keV on a U87MG cell exposed to 5 mg/ml Fe 2 O 3 NPs. The object field was 50 Â 36 mm 2 . The acquisition time of the 75 Â 54 pixels XRF maps was 12 s/pixel.

Figure 4 .

 4 Figure 4. Absorption (a) and phase contrast (b) images of a U87MG cell exposed to 58.75 mg/ml concentration of CoFe 2 O 4 NPs (40 Â 40 mm 2 size, 40 ms dwell time, 285 nm spot size). The other panels show the XRF maps of C, O, Fe, Co, and Na (40 Â 40 mm 2 size, 60 Â 60 pixels, 650 nm spot size) together with the co-localization of Fe and C, among the area indicated in red in panel a. All the images were acquired at 1.1 keV and 15 s/pixel was used for the XRF elemental maps.

Figure 3 .

 3 Figure 3. Absorption image (a), differential phase contrast (b) image, and XRF maps of C, O, Co, Fe, and Na, with co-localization of Fe and C, all acquired at 1.1 keV on a U87MG cell exposed to 50 mg/ml Fe 2 O 3 NPs. The object field was 66 Â 40 mm 2 . The acquisition time of the 100 Â 60 pixels XRF maps was 9 s/pixel.

Figure 5 .

 5 Figure 5. Absorption (a) and phase contrast (b) images of a U87 cell exposed to 235 mg/ml concentration of CoFe 2 O 4 NPs (40 Â 40 mm 2 size, 40 ms dwell time, 285 nm spot size). The other panels show the elemental distribution of C, O, Fe, Co, and Na (40 Â 40 mm 2 size, 40 Â 40 pixel, 650 nm spot size) and the co-localization of Fe and C among the area of panels a and b. All the images were acquired at 1.1 keV and 15 s/pixel was used for the XRF elemental maps.

Table 1

 1 reports the results of DLS analysis of the Fe 2 O 3 and CoFe 2 O 4 in different media. The DLS data indicate an average hydrodynamic diameter of 111.6 AE 5.9 in MilliQ water and 35.6 AE 1.1 nm in the stock solution for Fe 2 O 3 and CoFe 2 O 4 , respectively. The average diameter increases when the NPs are in contact with different biological media. In particular, the average diameter of Fe 2 O 3 increases to 150.5 AE 14.2 nm in cell culture media, whereas in the case of CoFe 2 O 4 , the average diameter increases to about 560 nm (Table1). These results indicate the tendency to agglomeration of the CoFe 2 O 4 NPs when in contact with the culture medium rich in proteins because the hydrophilic surface of CoFe 2 O 4 NPs nanoparticles is directly exposed to the proteins present in the culture medium. On the other hand, the Fe 2 O 3 NPs are covered with PMMA that makes them more

Table 1 .

 1 Hydrodynamic diameters of Fe 2 O 3 and CoFe 2 O 4 NPs obtained from dynamic light scattering experiments

	Sample	Media	Mean particle diameter
			(nm) AE SD by intensity
	Fe 2 O 3	MilliQ	112 AE 6
	Fe 2 O 3	MEM 10% FBS	150 AE 14
	CoFe 2 O 4	MilliQ 2%DEG	36 AE 1
	CoFe 2 O 4	MEM 10% FBS	567 AE 68

SD, standard deviation; DEG, diethylene glycol; FBS, fetal bovine serum.

Acknowledgements

This work has been carried out within the 'Nanobiosciences' Joint Research Center action. The European Commission and Friuli Venezia Giulia Region (Nanotox 0060 grant 2009) are gratefully acknowledged for the financial support. The authors would like to thank G. Baldi and co-workers for providing the cobalt ferrites nanoparticles. The authors are also grateful to Dr. G. Giudetti for the DLS measurements.


Chiara Uboldi

Guido Giudetti

Francesca Broggi

Douglas Gilliland

Jessica Ponti
email: jessica.ponti@jrc.ec.europa.eu

Franc ¸ ois Rossi

Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts

Keywords:

Although amorphous silica nanoparticles (aSiO 2 NPs) are believed to be non-toxic and are currently used in several industrial and biomedical applications including cosmetics, food additives and drug delivery systems, there is still no conclusive information on their cytotoxic, genotoxic and carcinogenic potential. For this reason, this work has investigated the effects of aSiO 2 NPs on Balb/3T3 mouse fibroblasts, focusing on cytotoxicity, cell transformation and genotoxicity. Results obtained using aSiO 2 NPs, with diameters between 15 nm and 300 nm and exposure times up to 72 h, have not shown any cytotoxic effect on Balb/3T3 cells as measured by the MTT test and the Colony Forming Efficiency (CFE) assay. Furthermore, aSiO 2 NPs have induced no morphological transformation in Balb/3T3 cells and have not resulted in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively. To understand whether the absence of any toxic effect could result from a lack of internalization of the aSiO 2 NPs by Balb/3T3 cells, we have investigated the uptake and the intracellular distribution following exposure to 85 nm fluorescently-labelled aSiO 2 NPs. Using fluorescence microscopy, it was observed that fluorescent aSiO 2 NPs are internalized and located exclusively in the cytoplasmic region. In conclusion, we have demonstrated that although aSiO 2 NPs are internalized in vitro by Balb/3T3 mouse fibroblasts, they do not trigger any cytotoxic or genotoxic effect and do not induce morphological transformation, suggesting that they might be a useful component in industrial applications.

Introduction

During the last 10 years, the use of nanomaterials (at least one dimension <100 nm) in industrial and biomedical applications has increased to the point where important questions on the health effects induced by exposure to nanoparticles (NPs) have arisen. In this context, the Joint Research Centre (JRC) of the European Commission has developed a strategy research plan on nanoparticle toxicology [1] and recently, in cooperation with the Organization of Economical Cooperation and Development (OECD) Working Party on Manufactured Nanomaterials [START_REF] Oecd | Working Party on Manufactured Nanomaterials[END_REF], has built up a Reference Nanomaterials program [START_REF]Reference Nanomaterials program[END_REF].

Silica NPs, which belong to the OECD list of interest and are among the most utilized nanomaterials in industrial nanotechnologies, can occur in a variety of forms: crystalline, simple amorphous or mesoporous. Mesoporous silica particles have a well-defined nanoporous structure and were shown to be good drug carriers [START_REF] Lu | Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs[END_REF] and nucleic acid delivery systems [START_REF] Xia | Polyethyleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of siRNA and DNA constructs[END_REF]. Amorphous silica nanoparticles (aSiO 2 NPs), on the other hand, are characterized by a non-crystalline structure and are currently employed in a wide range of industrial and biomedical applications. The relative ease by which aSiO 2 NPs can be produced in different sizes and with different surface modifications has led to their widespread use in cosmetics (sun creams), dentistry (toothpaste) and paints as well as food and animal-feed additives [START_REF] Dekkers | Presence and risks of nanosilica in food products[END_REF]. In biomedicine, silica nanoparticles are used as drug delivery systems [START_REF] Huang | Highly efficient cellular labeling of mesoporous nanoparticles in human mesenchymal stem cells: implication for stem cell tracking[END_REF][START_REF] Shi | Nanoparticle-based biocompatible and long-life marker for lysosome labeling and tracking[END_REF], in cancer therapy [START_REF] Hirsch | Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance[END_REF] and in cancer diagnostics [START_REF] Moghimi | Nanomedicine: current status and future prospects[END_REF].

Notwithstanding their extensive use, there is evidence of cytotoxicity induced by silica nanoparticles. Many in vitro studies have reported cytotoxic effects following exposure of different cell types to aSiO 2 NPs. Amorphous silica has been shown to reduce the viability in several cell types of human origin [START_REF] Choi | Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells[END_REF][START_REF] Napierska | Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells[END_REF][START_REF] Ye | Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line[END_REF][START_REF] Lin | In vitro toxicity of silica nanoparticles in human lung cancer cells[END_REF][START_REF] Yang | SiO2 nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells[END_REF] or increase the secretion of reactive oxidative species (ROS) [START_REF] Eom | Oxidative stress of silica nanoparticles in human bronchial epithelial cell, Beas-2B[END_REF][START_REF] Choi | Silica-based nanoparticle uptake and cellular response by primary microglia[END_REF][START_REF] Dutta | Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials[END_REF][START_REF] Park | Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro[END_REF][START_REF] Wang | Oxidative stress contributes to silica nanoparticle-induced cytotoxicity in human embryonic kidney cells[END_REF]. Furthermore, aSiO 2 NPs have also induced cytotoxicity in rodent-derived cells, where NPs have impaired the cell viability and induced the secretion of ROS and pro-inflammatory cytokines such as IL-1␤ [START_REF] Choi | Silica-based nanoparticle uptake and cellular response by primary microglia[END_REF][START_REF] Dutta | Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials[END_REF][START_REF] Fubini | Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells[END_REF][START_REF] Rabolli | Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types[END_REF][START_REF] Morishige | The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1beta production, ROS production and endosomal rupture[END_REF][START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF].

Currently, few studies have investigated the in vitro cell transformation and the genotoxic potential of silica NPs. Morphological transformation has been reported to take place in Syrian Hamster Embryo (SHE) cells exposed to amorphous silica in the form of diatomaceous earth NPs but not following exposure to pyrogenic aSiO 2 NPs [START_REF] Fubini | Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells[END_REF][START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF][START_REF] Elias | Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells[END_REF]. Genotoxic effects, measured as increased micronuclei formation [START_REF] Gonzalez | Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models[END_REF] or decreased DNA content [START_REF] Cha | Cytotoxic effects of nanoparticles assessed in vitro and in vivo[END_REF] or damaged DNA [START_REF] Gonzalez | Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models[END_REF], were observed in A549 and HEp-2 [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles[END_REF] cells in the presence of aSiO 2 NPs. Nevertheless, single cell gel electrophoresis (SCGE) performed on 3T3-L1 mouse fibroblasts has not shown evidence of genotoxicity following exposure to silica NPs [START_REF] Barnes | Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity[END_REF].

Due to the great interest in using aSiO 2 NPs, uptake and potential toxicity induced by fluorescent particles have also been investigated. Fluorescent aSiO 2 NPs were shown to affect HEp-2 cellular viability [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles[END_REF] and induce genotoxicity in A549 cells [START_REF] Jin | Toxicity of luminescent silica nanoparticles to living cells[END_REF]. In addition, fluorescent aSiO 2 NPs were observed in the nuclei [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles[END_REF] and in other intracellular compartments such as endosomes and lysosomes [START_REF] Jin | Toxicity of luminescent silica nanoparticles to living cells[END_REF][START_REF] Al-Rawi | Uptake and intracellular localization of submicron and nano-sized SiO2 particles in HeLa cells[END_REF][START_REF] Stayton | Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level[END_REF].

In this work, we have investigated the effects induced by aSiO 2 NPs in Balb/3T3 mouse fibroblasts cell line. Our study, in particular, has focused on the cytotoxicity, cell transformation and genotoxicity of aSiO 2 NPs, and to this end we have performed the Colony Forming Efficiency (CFE), the Cell Transformation Assay (CTA) and the Micronucleus test (MN), respectively. The goal of this study was to provide additional data on the toxic potential exerted in vitro by aSiO 2 NPs with the same crystalline structure (amorphous) but different sizes. In addition, this work had the aim of increasing knowledge about the ability of aSiO 2 NPs to induce morphological transformation and genotoxicity in vitro.

Materials and methods

All the in vitro experiments were performed on immortalized Balb/3T3 mouse fibroblasts clone A31-1-1 [START_REF] Kakunaga | A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB-3T3[END_REF][START_REF] Kakunaga | Cell variants showing differential susceptibility to ultraviolet light-induced transformation[END_REF]. Cells were distributed, free from bacteria, fungi and mycoplasma, at the passage number 12 by Hatano Research Institute (Japan) in the context of a pre-validation study [START_REF] Vanparys | ECVAM prevalidation of three cell transformation assays[END_REF]. Cells were cultivated up to 70-80% confluence in tissue culture-treated flasks (BD Falcon; Milan, Italy) and passaged weekly. All the assays were performed under normal cell culture conditions (37 • C; 5% CO2; 95% humidity).

Methods

Synthesis and characterization of silica nanoparticles

Five different types of unlabelled aSiO2NPs and one fluorescently-labelled were used in the present study (Table 1). NM-200 and NM-203 belong to the list of nanomaterials of interest to the OECD and they were delivered in powder form, while the synthesis of NRT-808, NRT-817, NRT-820 and NRT-944 has been performed in our laboratory using the following protocol. Briefly, to obtain monodispersed silicon dioxide particles in the range below 80 nm, a strategy based on the recent publication by Hartlen et al. [START_REF] Hartlen | Facile preparation of highly monodisperse small silica spheres (15 to >200 nm) suitable for colloidal templating and formation of ordered arrays[END_REF] has been adopted. In this method, aSiO2NPs were produced in a growth medium of water containing arginine or lysine catalyst. The water/amino acid mixture was placed in a glass vessel and a smaller volume of an immiscible organic liquid (cyclohexane) added and allowed to float above the aqueous phase. If necessary, this bi-phase solution is then heated and allowed to stabilize at the desired reaction temperature with slow stirring of the aqueous layer. Where additional heating was necessary, the reactions were done using CEM Discover S microwave heating system (CEM; Matthews; NC). The choice of microwave heating was found to give improved monodispersivity and better reproducibility when compared to conventional heating methods. At this point tetraethyl orthosilicate (TEOS) was slowly added to the organic phase where it preferentially dissolved. The system was then left under slow stirring for time period of a few hours to several days. During this time, there was a slow diffusion of TEOS from the upper organic layer into the aqueous phase where hydrolysis and condensation occurs with the nucleation and growth of aSiO2NPs with narrow size distribution. The microwave heating system used here permits a maximum batch volume of 100 mL and this was established as the standard volume for aSiO2NPs synthesis by the bi-phase method. An appropriate choice of temperature, catalyst concentration and reaction time was found to allow the production of near monodispersed aSiO2NPs with sizes controllable in the range 17-80 nm. Larger particles can be produced by this method but Scanning Electron Microscopy (SEM) analysis has shown that the solutions may be contaminated with a proportion of smaller particles.

For the production of fluorescent silica particles (aSiO2-Ru(bipy)3; NRT-944) the same basic method has been adopted except that a suitable quantity of the Ru(bipy)3 dye (Tris(2,2 -bipyridyl) dichlororuthenium(II) hexahydrate) has been added to the aqueous phase of the reaction mixture. The presence of the dye was found to significantly increase the final size of the particles when compared to those obtained under the equivalent experimental conditions without the dye. Following a detailed study of the effects of experimental conditions, it has been possible to extend the range of particle sizes which may be made by this method so as to be comparable with that previously achieved for pure unlabelled silica NPs. The studies have shown that particle size can again be effectively controlled by varying parameters such as reaction time, temperature (20-60 • C) and the amino acid catalyst concentration (150-3000 mg/L). By careful variation of these three parameters, it has been found to be possible to produce low polydispersivity fluorescent aSiO2NPs across the size range from 10 to 120 nm in a controlled and reproducible way.

Dynamic Light Scattering (DLS) and SEM were used to study the size distribution and morphology of the particles, respectively. Samples were obtained from 100 g/mL stock solutions. In addition, DLS has been used to determine the polydispersity index (PdI), which is an indicator of the polydispersivity/aggregation of the solution tested. NM-200 and NM-203 powder stocks were dispersed in ultrapure water to a stock concentration of 1 mg/mL as follows: after weighing 1 mg of NM-200 powder and 1.20 mg of NM-203 powder, the weighed powders were resuspended in ultrapure water to a final concentration of 1 mg/mL and then bath-sonicated. After 5 min of sonication, solutions were vortexed for 10 s and bath-sonicated again for 5 min. For DLS analysis, all stocks were diluted to a final concentration of 100 g/mL, which corresponds to the highest concentration used for the in vitro testing. To check for dispersant-dependent effects on stability/aggregation state, the aSiO2NPs were resuspended in ultrapure water and serum-free medium. After dispersion and before analysis, the samples in cell culture medium were incubated for 1 h at 37 • C to partially simulate the temperature conditions at which the biological testing was carried out. The DLS measurements were performed on a 1 mL sample volume on a Zetasizer Nano Zs (Malvern Instruments; Malvern, UK) following the protocols indicated by ISO 13321:1996 and ISO 22412:2008. After 2 min equilibration at 25 • C, each sample underwent 3 measurements. Read number and duration for each measurement were set on 'automatic' on the Zetasizer control software. Only intensity distribution data were considered for the analysis. Averaging the 3 measurements was done using Malvern's proprietary software.

SEM imaging and analysis were performed on samples dispersed in ultrapure water only, after spotting a volume of 5 L of the same samples prepared for DLS on aminated silicon wafers, letting it dry for 10 min and then eliminating the excess liquid. SEM was performed using a FEI Nova Nanolab 600I (FEI; Eindhoven, The Netherlands). Images were taken at low acceleration voltages of the electrons (5 keV) in immersion mode. Size distributions for the nanoparticles were then obtained by image analysis of micrographs using the free software Image-J. The images were treated using standard smoothing, background subtraction, binarization and edge detection. The analysis of the particles has been made by measuring each particle area and presenting the results by means of the radius using the spherical particle approximation.

Basal cytotoxicity: Colony Forming Efficiency (CFE) and MTT assay

As already published [START_REF] Ceriotti | Assessment of cytotoxicity by impedance spectroscopy[END_REF], on day zero 200 Balb/3T3 cells were seeded in 3 mL of fresh complete medium (Minimum Essential Medium (MEM) 1×; Invitrogen; Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum-Australian origin (Invitrogen; Carlsbad, CA, USA) and 1% (v/v) antibiotics (10,000 U/mL penicillin and 10,000 g/mL streptomycin; Invitrogen; Carlsbad, CA, USA) in each 60-mm Petri dish (3 replicates per concentration, 3 experiments performed). After 24 h of incubation under standard cell culture conditions (37 • C; 5% CO2; 90% humidity), the treatment suspensions of aSiO2NPs (1 g/mL-10 g/mL-100 g/mL) were added as aliquots to the cells. Untreated cells and Na2CrO4•6H2O (10 -3 M) were used as negative and positive controls, respectively. Cells were kept in contact with the treatment for 72 h after which the test compounds were removed and replaced with fresh complete medium. At day 9 after seeding, the colonies were fixed using a solution of 4% (v/v) formaldehyde (Sigma-Aldrich; Saint Louis, MO, USA) in Phosphate Buffered Solution (PBS) (Invitrogen; Carlsbad, CA, USA), then stained using 10% (v/v) of Giemsa (Sigma-Aldrich; Saint Louis, MO, USA) in ultrapure water. After air-drying, colonies (composed of at least 50 cells) were manually scored and counted using a stereomicroscope (Olympus; Milan, Italy). The data obtained were employed in order to define the dose-effect curve at 72 h and consequently to choose the concentrations to be tested in the cell transformation assay. The results were normalised to the untreated control and expressed as % CFE ((average of treatment colonies/average of solvent control colonies) × 100). The corresponding standard error of the mean (sem) has been calculated for 3 independent experiments and at least 3 replicates for each experimental point (sem = (standard deviation (SD)/ √ number of replicates)). The statistically significant differences for CFE values versus controls were calculated by the one-way ANOVA analysis (GraphPadPrism4 statistical software, GraphPad Inc.; La Jolla, CA, USA).

To perform the MTT assay, on day zero 7500 cells/well and 2500 cells/well, in a final volume of 100 L/well of media, were seeded into 96-well plate for 24 h and 72 h exposure to aSiO2NPs, respectively (6 wells per concentration, 3 experiments performed). After 24 h incubation, Balb/3T3 mouse fibroblasts were exposed to 1 g/mL, 10 g/mL and 100 g/mL of aSiO2NPs for 72 h. At the end of the incubation, cells were washed twice with fresh complete cell culture medium to remove unbound and non-internalized aSiO2NPs, and then cells were incubated with MTT (1:10 in complete cell culture medium; 100 L/well; MTT stock solution: 5 mg MTT/1 mL PBS; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich; Saint Louis, MO, USA). After a 2 h incubation (37 • C; 5% CO2; 90% humidity) 100 L/well of lysis solution, composed of 20 g sodium dodecyl sulphate (Sigma-Aldrich; Saint Louis, MO, USA) and 50 mL N,N-dimethylformamide (Sigma-Aldrich; Saint Louis, MO, USA) in ultrapure water, was pipetted and cells were incubated overnight. Absorbance was acquired the next day at 540 nm using the plate reader FLUOstar Omega (BMG Labtech GmbH; Offenburg, Germany). The results were normalized to the untreated control and expressed as % viability. The mean value was calculated for 3 independent experiments and at least 3 replicates for each experimental point (sem = (SD/ √ number of replicates). Statistical analysis was performed applying unpaired t-test (GraphPadPrism4 statistical software, GraphPad Inc.; La Jolla, CA, USA).

Cell transformation studies by Cell Transformation Assay (CTA)

On day zero, 2 × 10 4 Balb/3T3 cells were seeded in 6 mL of complete fresh medium Minimum Essential Medium (MEM) 1× supplemented with 10% (v/v) fetal bovine serum-Australian origin and antibiotics (10,000 U/mL penicillin and 10,000 g/mL streptomycin) in 100 mm-Petri dish (5 replicates per concentration). After 24 h, the treatment suspensions were administered in the desired concentrations by adding aliquots of NP preparations. Non-treated Balb/3T3 cells and cells exposed to 4 g/mL methylcholanthrene (Sigma-Aldrich; Saint Louis, MO, USA), which is a well-known carcinogenic compound, were used as negative and positive controls, respectively. At the end of exposure (72 h), medium containing aSiO2NPs was removed and replaced with Dulbecco's modified Eagle's medium/F12 (DMEM/F12) with high glucose, l-glutamine (365 mg/L) and sodium bicarbonate (1200 mg/L) (Invitrogen; Carlsbad, CA, USA) supplemented with 2% (v/v) fetal bovine serum-Australian origin, 2 g/mL insulin (Sigma-Aldrich; Saint Louis, MO, USA) and 1% antibiotics (Invitrogen; Carlsbad, CA, USA). The culture medium was changed twice a week, and on days 31-35 cells were fixed with 4% (v/v) of formaldehyde in PBS. Subsequently, cells were stained with a 10% (v/v) Giemsa solution in ultrapure water. The dishes were air-dried and observed using a stereomicroscope (Olympus; Milan, Italy) in order to detect and count only morphologically transformed colonies (type-III foci), as described from the International Agency for Cancer Research (IARC) Working-Group [START_REF] Iarc | Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: overview and recommended protocols[END_REF]. Transformation results were expressed as transformation frequency (T f ) (5 replicates per concentration, 3 experiments performed) using the following formula:

T f = [A/(B × C × D)],
where A = total number of type-III foci per treatment; B = CFE (%)/100; C = plating efficiency (%)/100; D = number of cells seeded × number of plates; plating efficiency (%) = number of colonies formed in the control × 100/200, and 200 is the total number of cells seeded in one CFE dish.

The probability of a neoplastic event occurring per surviving cell was determined by using the F-Fisher exact test and was considered statistically significant versus untreated control only when p < 0.05 [START_REF] Ponti | An optimised data analysis for the Balb/c 3T3 cell transformation assay and its application to metal compounds[END_REF].

Genotoxicity studies by Micronucleus (MN) test

On day zero, 3 × 10 5 Balb/3T3 mouse fibroblasts were seeded in 4 mL of complete medium Minimum Essential Medium (MEM) 1× supplemented with 10% (v/v) fetal bovine serum-Australian origin and antibiotics (10,000 U/mL penicillin and 10,000 g/mL streptomycin) in each well (1 replicate per concentration) of a 6 wells per plate. 24 h after seeding, the Balb/3T3 cells were treated with NP suspensions and incubated for 24 h. Non-treated cells and Balb/3T3 mouse fibroblasts exposed to 0.5 M mitomycin C (Kyowa Italiana Farmaceutici; Milano, Italy) were used as negative and positive control, respectively. At the end of the exposure period, the treatment was removed and each well washed twice with PBS. To block cell division, 4.5 g/mL cytochalasin B (Sigma-Aldrich; Saint Louis, MO, USA) was added to 4 mL of fresh complete culture medium per well. After a 24 h incubation (day 3), the cells were trypsinized and harvested in PBS. The cell suspensions were centrifuged (250 × g) and the pellets treated first with a prefixing (methanol:acetic acid, 3:5) and then with a fixing solution (methanol:acetic acid, 6:1). Fixed cells were dropped on glass slides and air-dried overnight. The slides (2 replicates for each sample) were stained with a 2% (v/v) Giemsa solution in ultrapure water and air-dried. The results (2 replicates per concentration, 3 experiments performed) were analyzed by calculating the frequency of binucleated micronucleated (BNMN) cells as the number of binucleated cells containing one or more micronuclei per 1000 binucleated cells. Statistical analysis was performed by F-Fisher exact test and differences versus solvent control were considered statistically significant at p < 0.05.

Uptake of fluorescent amorphous silica nanoparticles using microscopy studies

For microscopy studies, on day zero 1.5 × 10 5 Balb/3T3 cells were plated onto 4-chamber polystyrene vessel tissue culture-treated glass slides (BD Falcon; Milan, Italy) in 3 mL of complete Minimum Essential Medium (MEM) 1× and cultivated under standard culture conditions. After 1 day of cultivation, the cells were exposed for 6 h, 24 h and 72 h to 200 g/mL aSiO2NPs labelled with the fluorescent dye Ru(bipy)3. At the end of the exposure Balb/3T3 cells were fixed with 4% (v/v) of formaldehyde in PBS and nuclei stained using Hoechst-33342 (Invitrogen; Carlsbad, CA, USA) diluted 1:1200 in PBS. After staining, cells were washed in PBS/Tween-20 solution and mounted for microscopy using GelMount (DAKO; Glostrup, Denmark).

Images were acquired with an Axiovert 200M inverted microscope (Carl Zeiss; Jena, Germany) equipped with ApoTome slide module and AxioVision 4.8 software (Carl Zeiss; Jena, Germany), using 40×/1.0 objective lens. Images were acquired using a black&white AxioCam MRm (Carl Zeiss; Jena, Germany) and pseudocolors were applied after image acquisition.

Results

Synthesis and characterization of silica nanoparticles

DLS analysis of the solutions was first performed in ultrapure water at the final concentration of 100 g/mL. The DLS analysis of the dispersions in ultrapure water (Table 2) resulted in PdI numbers in agreement with a good monodispersity of the samples while the measured sizes were consistent with the values obtained from measurements of the stock suspensions. SEM imaging and subsequent statistical analysis performed on the samples have confirmed the DLS sizing for the NRT-817 sample (Fig. 1b), while the NRT-808 (Fig. 1a) and NRT-944 (Fig. 1f) samples have shown a difference in size in the range of 10 nm, but also a higher SD (Table 3). The NM-200 and NM-203 dispersions were exceptions to these considerations. The NM-200 dispersion has shown high PdI and an overall data quality too poor to allow for data fitting, which deemed the sample to be not suitable for DLS analysis. The high PdI value and the noisy baseline of the relative correlogram (Fig. 2) have indicated the presence of aggregates and/or agglomerates precipitating in the sample. This has also been confirmed by SEM images, clearly showing clumps of aggregated and/or agglomerated silica nanoparticles in the sample (Fig. 1d). Furthermore, the NM-203 dispersion has shown a PdI (Table 2) that is in agreement with a fairly monodispersed state of the NPs, but the calculated size does not correspond to the declared one (see Table 1), suggesting that the preparation procedure has generated a monodispersed sample but not to the original NPs size. In fact, SEM imaging has revealed the presence of clumps of nanoparticles also in this case (Fig. 1e).

DLS analysis of NRT-820 has revealed a very low PdI classifying it as monodispersed. In contrast to this, SEM imaging and statistical analysis (Table 3 and Fig. 1c) have shown clearly that NRT-820 is actually constituted of two distinct size populations of around 40 nm and 80 nm. This result in particular underlines the necessity of integrating DLS data with different characterization techniques, to comprehensively estimate the size of nanoparticles to be used in toxicological in vitro testing and then correctly correlating it with the eventually observed biological effects.

When the DLS analysis of the aSiO 2 NPs was performed in serumfree cell culture medium (Table 2), we have observed that particles maintained the same dispersity status as their counterparts dispersed in ultrapure water. The mean particle size of the samples was found to be only slightly increased, showing that they maintain reasonable stability even in a saline solution. Only the NRT-817 sample has shown a substantial change in dispersity, given by the appearance of a secondary population of differently sized NPs aggregates and/or agglomerates in the range of 200 nm. Size distribution analysis by volume has indicated that this population represents only 0.2% of the sample (data not shown).

An overall review of the characterization data has shown that there is little effect on stability of nanoparticles when dispersed in serum-free cell culture medium, and that the aSiO 2 NPs suspensions derived from dry powders (NM-200 and NM-203) were difficult to disperse back into a monodispersed form. Suspensions have maintained an aggregated/agglomerated state, thus greatly increasing the characteristic size at which they are biologically tested. In addition, an important issue arises when examining data from the NRT-820 sample, in which the DLS has been unable to separate the two size populations that are clearly visible in SEM imaging and the relative statistical analysis. This is due to a technical limitation of DLS, which is not well adapted to analysis of this type of multi-dispersed sample [START_REF] Calzolai | Separation and characterization of gold nanoparticle mixtures by flow-field-flow fractionation[END_REF]. The inability of the technique to detect the 40 nm particles in the presence of the 80 nm particles is primarily the result of the non-linear variation of light scattering as a function of size, which increases with the sixth power of their radius [START_REF] Berne | Dynamic Light Scattering with Applications to Chemistry[END_REF][START_REF] Filipe | Critical evaluation of Nanoparticle Tracking Analysis (NTA) by Nanosight for the measurement of nanoparticles and protein aggregates[END_REF] effectively masking the presence of the smaller particles. The implication of this consideration is that when using the DLS technique for nanoparticle size characterization, coupling it with one or more different characterization techniques, such as SEM, is a must.

Basal cytotoxicity studies by Colony Forming Efficiency (CFE) assay

The cytotoxic potential of water dispersed aSiO 2 NPs NRT-808, NRT-817 and NRT-820, and NM-200 and NM-203, which were delivered in powder form from OECD, has been investigated by CFE assay in Balb/3T3 mouse fibroblasts. 1 g/mL, 10 g/mL and 100 g/mL were selected as test concentrations and the exposure has been of 72 h.

As shown in Fig. 3, aSiO 2 NPs have not induced any significant impairment of CFE in Balb/3T3 cells in vitro, suggesting that neither the synthesis procedure nor particle size and particle agglomeration/aggregation has been responsible for cytotoxicity. Compared to the untreated control (100% CFE ± 1.3) 1 g/mL NM-200 resulted slightly toxic but the reduction in colony formation (92.99% CFE ± 4.86) has not been significant. Similarly, 100 g/mL NM-203 have induced a non-significant (93.5% CFE ± 6.61) cytotoxic effect in Balb/3T3 cells after 72 h exposure. The exposure of Balb/3T3 cells to the other aSiO 2 NPs investigated and at different concentrations has not induced any effect on CFE, with all the values obtained being in the range between 97.24% CFE ± 3.15 (10 g/mL NRT-808) and 106.28% CFE ± 5.18 (100 g/mL NRT-820), values which are not statistically significant compared to the negative control (untreated cells).

MTT assay (Fig. 4) has, in contrast, shown a slight but significant reduction of the viability of Balb/3T3 cells after 72 h in vitro exposure to NRT-820 (80 nm nominal size diameter). In fact, a concentration-dependent impairment of the viability of Balb/3T3 mouse fibroblast has been measured. Measurements of Balb/3T3 cells exposed to 1 g/mL, 10 g/mL and Fig. 3. Colony forming efficiency of Balb/3T3 cells exposed for 72 h to aSiO2NPs. Cells were exposed for 72 h to increasing concentrations of aSiO2NPs (1 g/mL-10 g/mL-100 g/mL). After fixation and counterstaining, colonies were manually counted. Data were analyzed in comparison to the untreated control and evaluated by one-way ANOVA. aSiO2NPs did not induce any cytotoxic effect in Balb/3T3 cells, suggesting that not size not their synthesis method are responsible for cytotoxicity. Mean of 3 independent experiments ± standard error of the mean (sem). Fig. 4. MTT assay on Balb/3T3 cells exposed for 72 h to aSiO2NPs. Cells were exposed for 72 h to increasing concentrations of aSiO2NPs (1 g/mL-10 g/mL-100 g/mL). At the end of the incubation period, cells were washed and then incubated with MTT solution (1:10 in complete cell culture medium). After 2 h incubation, lysis buffer was pipetted in each well and the next day absorbance was acquired at 540 nm with a plate reader. Data were analyzed in comparison to the untreated control and evaluated by unpaired t-test. Exposure to aSiO2NPs NRT-820 (80 nm declared size diameter) induced a slightly significant cytotoxic effect in Balb/3T3 cells. Mean of 3 independent experiments ± standard error of the mean (sem). *p < 0.05. The positive control (Na2CrO4 1000 M) induced complete cell death as expected and the histogram value is equal to 0. Fig. 5. Cell transformation assay on Balb/3T3 cells exposed to aSiO2NPs for 72 h. aSiO2NPs were tested at the fixed concentration of 100 g/mL. After exposure (72 h), cells were cultured up to day 35, then fixed and stained, and type-III foci were counted. Exposure to aSiO2NPs did not exert any morphological transformation in Balb/3T3 mouse fibroblasts. The statistical significance was evaluated by F-Fisher exact test (***p < 0.001) in respect to untreated control. Mean of 3 independent experiments ± standard error of the mean (sem). Methylcholanthrene, a well-known carcinogenic compound, was used as positive control (C+). The positive control (Na2CrO4 1000 M) induced complete cell death as expected and the histogram value is equal to 0. 100 g/mL NRT-820 showed viability of 81.44 ± 6.36, 75.96 ± 8.44 and 74.86 ± 8.10 respectively, when expressed as percentage of the untreated control (100% viability ± 2.13). In the presence of the other silica particles investigated in this work no reduced Balb/3T3 cells viability has been shown by MTT assay.

Cell transformation studies by Cell Transformation Assay (CTA)

Amorphous SiO 2 NPs were tested for their carcinogenic potential by CTA. aSiO 2 NPs, those selected from OECD list (NM-200 and NM-203) and those synthesized in water solution (NRT-808, NRT-817 and NRT-820) have not exerted any morphological transformation in Balb/3T3 cells after 72 h exposure (Fig. 5). When test samples were compared with untreated controls, F-Fisher exact test did not reveal any significant difference in the transformation frequency (T f ), which is an indicator of the carcinogenic potential exerted by NPs. Compared to the untreated cells (T f = 0.266 × 10 -4 ± 0.10) aSiO 2 NPs in water solution have not induced the formation of type-III foci in Balb/3T3 cells in vitro. In fact, the transformation frequency obtained for NRT-808, NRT-817 and NRT-820 corresponded to 0.1 × 10 -4 ± 0.1, 0.288 × 10 -4 ± 0.22 and 0.166 × 10 -4 ± 0.10, respectively. Similarly, compared to the negative control, NM-200 (T f = 0.246 × 10 -4 ± 0.12) and NM-203 (T f = 0.360 × 10 -4 ± 0.14) did not induce any significant morphological transformation in Balb/3T3 cells. In contrast, methylcholanthrene, which is a well-known carcinogenic compound, induced extremely significant formation of type-III foci (T f = 3.924 × 10 -4 ± 0.16).

These results indicate that, as already shown by CFE, size and synthesis method does not play any role in the carcinogenic potential of the aSiO 2 NPs we have selected for our study.

Genotoxicity studies by Micronucleus (MN) test

The genotoxic potential of aSiO 2 NPs has been investigated by the micronucleus assay. At the concentration of 100 g/mL, Fig. 6. Micronucleus assay on Balb/3T3 cells exposed to aSiO2NPs for 24 h. aSiO2NPs were tested at the fixed concentration of 100 g/mL. Compared to untreated control cells, aSiO2NPs did not induce genotoxicity in Balb/3T3 mouse fibroblasts. The statistical significance was evaluated by Fisher exact test (**0.001 > p > 0.05) in respect to untreated control. Mean of 2 independent experiments ± standard error of the mean (sem). Cytochalasin B (4.5 g/mL) was used as positive control (C+). BNMN = binucleated micronucleated cells; BN = binucleated cells. aSiO 2 NPs did not induce any significant formation of micronuclei in Balb/3T3 cells in vitro (Fig. 6). In fact, the frequency of binucleated micronucleated (BNMN) Balb/3T3 mouse fibroblasts per 1000 binucleated (BN) cells corresponded to 9 BNMN ± 3, 7 BNMN ± 1 and 9.5 BNMN ± 2.5 after 24 h exposure to NRT-808, NRT-817 and NRT-820, respectively. Therefore, compared to the untreated control (9.5 BNMN ± 0.5) aSiO 2 NPs did not trigger the formation of micronuclei, suggesting that neither the size diameter nor the particles' synthesis procedure induces genotoxicity.

Our results indicate that for aSiO 2 NPs, particle size does not play a role in genotoxicity as no significant differences were observed in Balb/3T3 cells exposed to aSiO 2 NPs of different sizes.

Internalization of fluorescent amorphous silica nanoparticles

To verify if the absence of any cytotoxic, cell transformation or genotoxic effect on Balb/3T3 cells by aSiO 2 NPs was simply because NPs were not internalized, the uptake and the in vitro internalization of 85 nm-sized aSiO 2 -Ru(bipy) 3 in Balb/3T3 cells has been investigated by fluorescent microscopy. We have chosen 85 nm-sized particles (NRT-944) because, as previously shown, for CFE, CTA and MN assays were performed using aSiO 2 NPs with equivalent diameter and surface charge (NRT-820).

Using optical microscopy we observed that after 6 h exposure (Fig. 7a-c) aSiO 2 -Ru(bipy) 3 particles accumulated in the cytoplasmic region, and the internalization of particles was confirmed by performing Z-stack series of the samples followed by a 3D reconstruction of the images.

Compared to 6 h exposure, after 24 h (Fig. 7d-f) a higher number of cells were observed under the microscope and after 72 h (Fig. 7g-i) cells were confluent. In addition, particles were taken up by Balb/3T3 cells and they were stored in the cytoplasm (Fig. 7b,e,h), with no evidence of entry in the nuclear compartment (Fig. 7a,d,g). To confirm that fluorescently-labelled aSiO 2 NPs did not induce Fig. 7. Uptake of fluorescent aSiO2NPs in Balb/3T3 cells. For studying particle internalization, Balb/3T3 cells were incubated in the presence of fluorescent aSiO2NPs (aSiO2-Ru(bipy)3; 200 g/mL) for 6 h (a-c), 24 h (d-f) and 72 h (g-i). After incubation, cells were fixed with 4% paraformaldehyde and nuclei stained with Hoechst-33342. aSiO2-Ru(bipy)3 were internalized and stored intracellularly in the perinuclear region and no particles were observed in the nuclei. Size bar: 20 m. (a + d + g) Hoechst-33342; (b + e + h) aSiO2-Ru(bipy)3; (c + f + i) merge. aSiO2-Ru(bipy)3 were tested by CFE for their potential cytotoxicity in Balb/3T3 cells (j). aSiO2-Ru(bipy)3 (200 g/mL) and the Ru(bipy)3 dye did not induce any toxic effect in Balb/3T3 cells after 72 h exposure. cytotoxicity in Balb/3T3 mouse fibroblasts, we performed CFE and, as shown in Fig. 7j, after 72 h exposure neither aSiO 2 -Ru(bipy) 3 nor the dye used to label particles impaired the formation of Balb/3T3 colonies.

Taken together, our data indicate that although fluorescent aSiO 2 NPs were internalized in Balb/3T3 mouse fibroblasts and stored in the cytosol, aSiO 2 -Ru(bipy) 3 do not induce any cytotoxic effect in Balb/3T3 cells. Studies to quantify the uptake of aSiO 2 -Ru(bipy) 3 as a function of the exposure time are currently ongoing.

Discussion

Due to the widespread use of aSiO 2 NPs in industrial and biomedical applications, studying the possible toxicological effects of these nanomaterials is of great importance. The aim of this study was to investigate the potential cytotoxicity, morphological transformation and genotoxicity induced by aSiO 2 NPs in Balb/3T3 mouse fibroblasts.

The greater part of the published results on the cytotoxic potential of aSiO 2 NPs have been obtained using colorimetric assays such as MTT, which is a test that measures the metabolic and mitochondrial activity of the cells and is generally accepted as a cytotoxicity test. MTT has been shown to generate reliable data for cytotoxicity studies with chemical compounds; nevertheless, it has been found to be less reliable in testing the potential cytotoxicity of NPs [START_REF] Worle-Knirsch | Oops they did it again! Carbon nanotubes hoax scientists in viability assays[END_REF][START_REF] Monteiro-Riviere | Challenges for assessing carbon nanomaterial toxicity to the skin[END_REF][START_REF] Casey | Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity[END_REF][START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF][START_REF] Horie | Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells[END_REF]. The impaired cellular viability we have detected by MTT assay in Balb/3T3 cells exposed to NRT-820 might not be due to a loss of the metabolic activity, but rather to the formazan dye interfering with NPs [START_REF] Worle-Knirsch | Oops they did it again! Carbon nanotubes hoax scientists in viability assays[END_REF][START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF]. To avoid this problem, in this work we decided to use the CFE. This assay has already been used and considered promising to assess the cytotoxicity induced by nanomaterials [START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF][START_REF] Ceriotti | Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system[END_REF][START_REF] Ponti | Colony forming efficiency and microscopy analysis of multiwall carbon nanotubes cell interaction[END_REF][START_REF] Ponti | Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts[END_REF], since it is a clonogenic assay in which the endpoint is the formation of colonies by those cells surviving after the exposure to the test compound. At the end of the exposure, solutions containing NPs are removed and cells maintained in culture with fresh medium. In this way interactions between the dye used to stain colonies and free test particles are eliminated, thus avoiding any misinterpretation of the experimental outcome [START_REF] Monteiro-Riviere | Challenges for assessing carbon nanomaterial toxicity to the skin[END_REF][START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF][START_REF] Monteiro-Riviere | Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line[END_REF].

Although aSiO 2 NPs are generally considered as non-toxic nanomaterials, a number of previous in vitro studies have reported time-, concentration-or particles' size-dependent cytotoxicity induced by aSiO 2 NPs. Time-dependent reduction of the cellular viability has been measured by MTT in human hepatic L-02 cells exposed to 21 nm SiO 2 NPs [START_REF] Ye | Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line[END_REF]. Moreover, the viability of A549 (human pulmonary epithelial cells) cells has been shown to decrease over the time of exposure (24 h-48 h-72 h) in the presence of 15 nm and 46 nm aSiO 2 NPs [START_REF] Lin | In vitro toxicity of silica nanoparticles in human lung cancer cells[END_REF]. Furthermore, particles' size has been reported as playing a role in the cytotoxicity of aSiO 2 NPs in human umbilical endothelial EAHY96 cells: aSiO 2 NPs with a size diameter of 14-60 nm have affected more rapidly and more severely the survival of EAHY96 cells as compared to aSiO 2 NPs with a diameter above 100 nm [START_REF] Napierska | Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells[END_REF]. In HaCaT cells (human keratinocytes) 15 nm and 30 nm aSiO 2 NPs induced dose-dependent cytotoxicity and apoptosis. In addition, a more significant cytotoxic effect has been detected, by CCK-8 assay, following the exposure of HaCaT cells to 15 nm silica NPs [START_REF] Yang | SiO2 nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells[END_REF]. The viability of human kidney HEK293 cells in the presence of 20 nm and 50 nm has revealed, by MTT assay, a dose-and time-dependent cytotoxicity and, once again, smaller aSiO 2 NPs were reported inducing higher toxicity compared to larger NPs [START_REF] Wang | Oxidative stress contributes to silica nanoparticle-induced cytotoxicity in human embryonic kidney cells[END_REF].

Our results have shown that size has not triggered cytotoxicity in Balb/3T3 cells. We have examined aSiO 2 NPs with a size diameter ranging from 16 nm to 300 nm and the cellular viability has been impaired only after exposure to 80 nm particles (NRT-820), which indeed has been shown by electron microscopy as being composed of NPs with size of 40 nm and 80 nm. This result has been observed not by CFE, but using the colorimetric MTT assay, whose ability in interfering with nanomaterials and, consequently, leading to a possible misinterpretation of the data has already been reported [START_REF] Monteiro-Riviere | Interactions of aluminum nanoparticles with human epidermal keratinocytes[END_REF][START_REF] Horie | Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells[END_REF][START_REF] Monteiro-Riviere | Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line[END_REF][START_REF] Davoren | In vitro toxicity evaluation of single walled carbon nanotubes on human A549 lung cells[END_REF][START_REF] Horie | Ultrafine NiO particles induce cytotoxicity in vitro by cellular uptake and subsequent Ni(II) release[END_REF]. Nevertheless, while MTT is a test able to give immediate information on the metabolic and mitochondrial activity of the cells exposed to a test compound (NPs), the CFE protocol might allow cells to recover and repair any damage induced by the exposure to NPs.

Following exposure to monodispersed aSiO 2 NPs with a larger diameter (150-200 nm) the cellular viability of mouse microglial cells has not been impaired [START_REF] Choi | Silica-based nanoparticle uptake and cellular response by primary microglia[END_REF]. Our data have shown that aSiO 2 NPs with size diameter of 200 nm (NM-200) and 300 nm (NM-203) could not induce cytotoxicity in Balb/3T3 cells. However, the absence of toxicity might be due to the fact that these NPs formed huge aggregates and cells were not able to internalize them.

It has been reported that cytotoxicity not only depends on the physicochemical properties of particles, but also varies with the cellular type investigated [START_REF] Rabolli | Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types[END_REF]. Therefore, even if our results are in contrast to other studies performed using different cell lines and different silica NPs, we are confident of the reliability and reproducibility of the CFE assay for testing NPs [START_REF] Ponti | Colony forming efficiency and microscopy analysis of multiwall carbon nanotubes cell interaction[END_REF][START_REF] Ponti | Comparison of impedance-based sensors for cell adhesion monitoring and in vitro methods for detecting cytotoxicity induced by chemicals[END_REF].

Little is known about the morphological transformation induced by aSiO 2 NPs. Amorphous SiO 2 NPs were reported as inducing morphological transformation in SHE cells [START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF]. Three different biogenic diatomaceous earths have induced a dose-dependent transformation of SHE cells and, interestingly, pyrogenic amorphous silica has not shown any carcinogenic potential [START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF].

Elias et al. [START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF] have postulated that the presence of metal impurities might be responsible for morphological transformation detected in SHE cells. In fact, iron and aluminium were found on the surface of the biogenic diatomaceous earth NPs, which have induced morphological transformation whereas pyrogenic aSiO 2 NPs, free of these elements, have not shown carcinogenic potential in SHE cells [START_REF] Elias | Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells[END_REF].

In addition, after being heated diatomaceous earth silica nanoparticles were shown to induce morphological transformation in SHE cells [START_REF] Elias | Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells[END_REF]. Compared to non-heated biogenic diatomaceous silica, which did not induce any effect, a dose-dependent increase has been observed in transformed SHE cells exposed to diatomaceous silica, which had been heated to up to 900 • C and 1200 • C [START_REF] Elias | Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells[END_REF]. As possible explanation, Elias et al. [START_REF] Elias | Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells[END_REF] have reported that heating of diatomaceous earth silica induces crystallization of the samples and the formation of free radicals and particle-derived ROS, which have already been demonstrated as the key factor in quartz-induced cell transformation [START_REF] Daniel | Direct interaction between crystalline silica and DNA -a proposed model for silica carcinogenesis[END_REF][START_REF] Daniel | DNA strand breakage, thymine glycol production, and hydroxyl radical generation induced by different samples of crystalline silica in vitro[END_REF][START_REF] Saffiotti | Mechanisms of carcinogenesis by crystalline silica in relation to oxygen radicals[END_REF] and biogenic earth silica-induced cell transformation [START_REF] Fubini | Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells[END_REF].

Our aSiO 2 NPs, in contrast, have not been able to induce morphological transformation in Balb/3T3 cells, irrespective of size and synthesis method. Neither small (16-80 nm) water dispersed aSiO 2 NPs nor larger (200 nm) silica particles nor pyrogenic aSiO 2 NPs (NM-203; 300 nm size diameter) have shown any carcinogenic potential in vitro in Balb/3T3 mouse fibroblasts, suggesting that other factors than the presence of metal residues on the particles' surface might be responsible for triggering morphological transformation in Balb/3T3 cells in vitro.

Only a few studies have investigated the potential genotoxicity of aSiO 2 NPs. Using the Comet assay Barnes et al. [START_REF] Barnes | Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity[END_REF] have demonstrated that alumina-coated silica NPs with size diameter ranging from 20 nm to 250 nm are not genotoxic in 3T3-L1 mouse fibroblasts. Furthermore, 50 nm luminescent silica particles have not exerted any genotoxicity in A549 cells [START_REF] Stayton | Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level[END_REF]. In fact, Comet assay, Western blot analysis of the DNA and DNA agarose gel have not shown any significant DNA damage or any increased DNA repair activity in A549 following exposure to SiO 2 NPs, leading the authors to suggest that SiO 2 NPs were not able to induce genotoxicity because NPs have not penetrated in the nuclei [START_REF] Jin | Toxicity of luminescent silica nanoparticles to living cells[END_REF].

Interestingly, a cell type-dependent, and not concentration-or size-dependent, genotoxicity (reduction of the DNA content) was observed by Cha and Myung [START_REF] Cha | Cytotoxic effects of nanoparticles assessed in vitro and in vivo[END_REF]: among the different cell lines examined, brain (A-172) and liver (Huh-7) cells have responded more quickly than stomach (MKN-1) and lung (A549) cells to aSiO 2 NPs.

In addition, sub-toxic doses of 16 nm, 60 nm and 104 nm amorphous silica NPs did not induce any significant formation of micronuclei in A549 [START_REF] Gonzalez | Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models[END_REF]. The smallest silica NPs (16 nm) were reported as inducing slightly higher micronuclei formation than the 60 nm and 104 nm aSiO 2 NPs, although the micronuclei formation was not significant [START_REF] Gonzalez | Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models[END_REF]. Accordingly, we have demonstrated that, independently of their size, following exposure to aSiO 2 NPs no micronuclei formation has been detected in Balb/3T3 cells, suggesting that under the experimental conditions we have applied aSiO 2 NPs are not genotoxic.

To verify whether or not aSiO 2 NPs induce cytotoxicity, morphological transformation and genotoxicity in Balb/3T3 mouse fibroblasts in vitro, the uptake of aSiO 2 NPs has been investigated.

After characterization, 85 nm fluorescently-labelled silica nanoparticles (aSiO 2 -Ru(bipy) 3 NPs) were selected for the study. From Jin et al. [START_REF] Jin | Toxicity of luminescent silica nanoparticles to living cells[END_REF] it is already known that 50 nm Ru(bipy) 3 -labelled silica nanoparticles do not induce cytotoxic effects in A549 human pneumocytes. Similarly, we have observed that 80 nm Ru(bipy) 3labelled aSiO 2 NPs have not exerted cytotoxicity in Balb/3T3 mouse fibroblasts in vitro.

In contrast, Chen and von Mikecz [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles[END_REF] have demonstrated that 40-70 nm fluorescent silica NPs exerted toxic effects inhibiting the replication and the cellular proliferation of HEp-2 human cells. Furthermore, 70 nm fluorescent SiO 2 NPs were shown to damage the integrity of the cell membrane and have induced cytotoxicity in HeLa cells incubated in the absence of serum proteins. In this case the authors have suggested that serum proteins covered the particles' surface, preventing the direct interaction, and therefore the damage, to the cell membrane [START_REF] Al-Rawi | Uptake and intracellular localization of submicron and nano-sized SiO2 particles in HeLa cells[END_REF].

The intracellular localization of silica NPs has also been investigated and the results are not conclusive. On one side, Jin et al. [START_REF] Jin | Toxicity of luminescent silica nanoparticles to living cells[END_REF] have reported that, after 6 h incubation, 50 nm Ru(bipy) 3 -labelled silica nanoparticles become localized in the cytoplasmic compartment of A549 human pneumocytes but have not crossed the nuclear membrane. In the same way we have observed that 85 nm aSiO 2 -Ru(bipy) 3 NPs have accumulated exclusively in the cytoplasm of Balb/3T3 cells and have not entered the nuclei, and that the internalization of aSiO 2 -Ru(bipy) 3 NPs in Balb/3T3 mouse fibroblasts has occurred already within 6 h exposure.

Similarly, 13 nm fluorescent silica-capped CdSe-CdS NPs were observed in the perinuclear region of A549 cells [START_REF] Stayton | Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level[END_REF], and 70 nm fluorescent SiO 2 particles were observed in the lysosomal compartment of HeLa cells, whilst sub-micron (200 nm and 500 nm size diameter) fluorescent SiO 2 particles were found in the cytoplasm and in the endosomes of HeLa cells [START_REF] Al-Rawi | Uptake and intracellular localization of submicron and nano-sized SiO2 particles in HeLa cells[END_REF].

In contrast, Chen and von Mikecz [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles[END_REF] have shown that 40-70 nm fluorescent silica NPs entered the nuclei of HEp-2 cells, while fine (0.2 m size) and coarse (5 m size) SiO 2 NPs accumulated exclusively in the cytoplasm. As possible explanation the authors have suggested that the size of nanoparticles contributes to the effects exerted by SiO 2 NPs: small SiO 2 NPs entering into the nuclei have inhibited gene expression in HEp-2 cells, whereas larger particles (>200 nm) have not crossed the nuclear membrane and have not exerted any effect.

Conclusions

In conclusion, we have shown that when dispersed in cell culture medium aSiO 2 NPs were colloidally stable, with the exception of NM-200 and NM-203. NM-200 and NM-203 solutions were prepared from powder stocks, for which the dispersion protocol was unable to break the NP aggregates formed. As a consequence, the redispersed samples were composed of particles with much larger mean diameter than that expected from the data declared by the suppliers. Aggregation/agglomeration of NM-200 and NM-203, however, does not seem to play a role as these particles were observed inducing a non-significant toxic effect in Balb/3T3 mouse fibroblasts as the monodispersed NRT-808, NRT-817 and NRT-820.

Using the CFE assay we have shown that aSiO 2 NPs do not induce cytotoxicity in Balb/3T3 mouse fibroblasts. Moreover, the CTA and MN test demonstrated that the investigated aSiO 2 NPs were not able to induce morphological transformation or genotoxicity in Balb/3T3 cells, respectively.

In addition, we have observed 85 nm Ru(bipy) 3 -labelled aSiO 2 NPs internalized and stored in the perinuclear region of Balb/3T3 cells, supporting the evidence that although aSiO 2 NPs were taken up by cells, they have not triggered any toxic effect.

While these results are encouraging regarding the low toxicity of aSiO 2 NPs, their very widespread use in industrial applications, such as cosmetics, and their potential use in biomedicine means there is still great relevance in performing additional studies to investigate any potential DNA strand breaks induced by aSiO 2 NPs, as well as the ability of aSiO 2 NPs to induce the secretion of proinflammatory cytokines. In addition, due to the potential use of aSiO 2 NPs in drug delivery and cancer therapy, further studies to better clarify the endocytotic pathway of aSiO 2 NPs and their intracellular fate (accumulation and/or recycling) are needed.

Fig. 1 .

 1 Fig. 1. SEM images of aSiO2NPs. aSiO2NPs (100 g/mL in ultrapure water) were analyzed by SEM to verify their morphology and their size. NRT-808 (a), NRT-817 (b) and NRT-944 (f) resulted monodispersed and the analysis of their sizes acquired by SEM confirmed DLS measurements. NRT-820 (c) clearly shows two distinct populations of different size (40 nm and 80 nm), while NM-200 (d) and NM-203 (e) formed aggregates/agglomerates and the image analysis could not be performed. Size bar: 100 nm.

Fig. 2 .

 2 Fig. 2. Agglomeration and/or aggregation of NM-200. The noisy baseline of the correlogram (indicated by the arrow) indicates the presence of agglomerate and aggregates of particles precipitating in the sample.

Table 1

 1 Amorphous silica nanoparticles: concentration, origin and declared size.

	Sample name	Nominal	Origin	Declared size
		concentration		(nm)
	NM-200	Powder form	OECD list of interest;	10-25
			JRC repository
	NM-203	Powder form	OECD list of interest;	5-30
			JRC repository
	NRT-808	1.69 mg/mL	JRC	35
	NRT-817	1.53 mg/mL	JRC	15
	NRT-820	1.54 mg/mL	JRC	90
	NRT-944	5 mg/mL	JRC	80

Table 2

 2 DLS analysis of aSiO2NPs (100 g/mL) in ultrapure water and serum-free cell culture medium.

		Polydispersity	Mean particle
		index ± SD	diameter (nm) ± SD
	Sample analysis in ultrapure water
	NM-200	0.506 ± 0.058	Data quality too poor
	NM-203	0.123 ± 0.021	185.1 ± 7.51
	NRT-808	0.016 ± 0.011	37.91 ± 0.27
	NRT-817	0.054 ± 0.011	17.42 ± 0.16
	NRT-820	0.043 ± 0.017	89.99 ± 0.87
	NRT-944	0.021 ± 0.023	81.63 ± 0.42
	Sample analysis in serum-free cell culture medium
	NM-200	0.623 ± 0.096	332.6 ± 11.42
	NM-203	0.125 ± 0.046	187.3 ± 7.76
	NRT-808	0.079 ± 0.008	39.39 ± 0.13
	NRT-817	0.526 ± 0.126	16.08 ± 0.81
	NRT-820	0.073 ± 0.008	99.69 ± 0.88
	NRT-944	0.052 ± 0.003	89.98 ± 0.89

Table 3

 3 SEM image analysis results of aSiO2NPs (100 g/mL) in ultrapure water.

	Particle	Mean particle	Mean particle diameter	Particle number
		diameter (nm) ± SD	(nm) ± SD (2)
	NRT-808	25 ± 7.3		1495
	NRT-817	16.4 ± 2.9		868
	NRT-820	38 ± 24	91 ± 25	2419
	NRT-944	70 ± 14		216

Acknowledgments

The authors would like to thank Dr. César Pascual García for SEM imaging and analysis. This work was conducted in part with support from activities in the European Commission funded collaborative project NanoReTox (FP7-NMP-2007-SMALL-1).

Conflict of interest

None.