Stefaan J Soenen

Bella B Manshian

Tangi Aubert

Uwe Himmelreich

Jo Demeester

Stefaan C De Smedt

Zeger Hens

Zeger Hens ‡ , ∫ Kevin Braeckmans

Cytotoxicity of Cadmium-Free Quantum Dots and Their Use in Cell Bioimaging

Keywords: nanotoxicology, cadmium-free particles, nanoparticles

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The ongoing progress in nanotechnology is rapidly opening the doors for a wide number of advanced biomedical applications. [START_REF] Barkalina | Nanotechnology in reproductive medicine: Emerging applications of nanomaterials[END_REF][START_REF] Ding | Nanotechnological strategies for therapeutic targeting of tumor vasculature[END_REF][START_REF] Dvir | Nanotechnological strategies for engineering complex tissues[END_REF] Novel nanosized materials (NMs), each with their own unique properties, are generated at an incredibly fast pace, stimulating the progress of a broad variety of biomedical research areas, including tissue engineering, neural conductance, targeted drug delivery, multimodal whole-body imaging and combinations of both diagnostics and therapy. [START_REF] Gu | Recent advances in design and fabrication of upconversion nanoparticles and their safe theranostic applications[END_REF][START_REF] Ge | Functional block copolymer assemblies responsive to tumor and intracellular microenvironments for site-specific drug delivery and enhanced imaging performance[END_REF] Among the different types of NMs, colloidal nanoparticles (NPs) take an important place either as (multimodal) imaging contrast agents or as therapeutic agents. [START_REF] Wu | Recent development of silica nanoparticles as delivery vectors for cancer imaging and therapy[END_REF] One type of NM that has attracted a lot of attention in particular are colloidal semiconductor quantum dots (QDots). QDots are nanocrystals in the range of 1-10 nm diameter that are most often composed of heavy metals from group 12 and group 16. The most common QDot types with the highest quantum yields are the cadmium-containing QDots such as CdTe or CdSe. For biomedical purposes, the QDot cores will typically be surrounded by a shell layer (e.g. ZnS) that passivates the core and enhances the optical properties of the QDots. [START_REF] Walling | Quantum dots for live cell and in vivo imaging[END_REF] QDots possess exceptional fluorescence properties, such as a high brightness and high photostability and broad excitation spectra combined with very narrow, size-dependent emission spectra enabling multiplexed imaging of cells, tissues and whole animals using a single excitation source. [START_REF] Medintz | Quantum dot bioconjugates for imaging, labelling and sensing[END_REF][START_REF] Michalet | Quantum dots for live cells, in vivo imaging, and diagnostics[END_REF] In contrast to classical organic fluorophores, these properties have made QDots interesting tools for fluorescence imaging of fixed cells and tissues where multiple markers can be analyzed simultaneously. Additionally, QDots can be used for the continuous follow-up of single particles over longer time periods, allowing to study the kinetics of mobile cellular markers such as cell membrane receptors. [START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF] The use of cadmium-containing QDots for biomedical purposes has, however, remained quite limited due to the high toxicity of free Cd 2+ ions and concerns on the potential toxic effects of these Cd 2+ -containing NPs. [START_REF] Su | The cytotoxicity of CdTe quantum dots and the relative contributions from released cadmium ions and nanoparticle properties[END_REF][START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Rzigalinski | Cadmium-containing nanoparticles: Perspectives on pharmacology and toxicology of quantum dots[END_REF] Studies on the toxic effects of QDots have generated a lot of disparate data, owing to, among others, differences in a) size of the QDot core, b) chemical composition of the QDots, c) surface coating, d) presence or absence of a shell layer or e) the experimental methodology used to evaluate QDot toxicity. [START_REF] Rivera-Gil | The challenge to relate the physicochemical properties of colloidal nanoparticles to their cytotoxicity[END_REF] The in vivo toxicity of Cd 2+containing QDots remains largely unknown. Although there is one initial study on primates where no toxic effects were observed under the conditions tested, this study did not take into account any possible long-term effects. [START_REF] Ye | A pilot study in non-human primates shows no adverse response to intravenous injection of quantum dots[END_REF][START_REF] Sealy | Primates show mixed response to quantum dots[END_REF] In contrast to this, most cell studies found that Cd 2+containing QDots were highly toxic, although large differences exist between different cell types, where under some conditions, particles have been described to be selectively toxic towards cancer cells. [START_REF] Soenen | Turning a frown upside down: Exploiting nanoparticle toxicity for anticancer therapy[END_REF] These differences are likely due to the intrinsic differences between classical in vitro and in vivo methods, highlighting the need for proper experimental toxicity tests. [START_REF] Tsoi | Are quantum dots toxic? Exploring the discrepancy between cell culture and animal studies[END_REF][START_REF] Joris | Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro-in vivo gap[END_REF] Based on the data obtained, some general conclusions concerning Cd 2+ -based QDot toxicity can however be drawn: a) the presence of a passivating shell layer typically reduces QDot toxicity, b) a polymeric coating that envelopes the QDots typically provides the best protection in contrast to short ligands and c) toxicity is for the most part caused by the induction of reactive oxygen species and as a result of (intracellular) QDot degradation and release of free Cd 2+ ions. [START_REF] Su | The cytotoxicity of CdTe quantum dots and the relative contributions from released cadmium ions and nanoparticle properties[END_REF] As the presence of Cd 2+ ions has been shown to be a major determinant in the toxicity of Cd 2+containing QDots, several strategies have been worked out to optimize the production and performance of Cd 2+ -free QDots. [START_REF] Mandal | Cadmium-free quantum dots as time-gated bioimaging probes in highly-autofluorescent human breast cancer cells[END_REF][START_REF] Moret | Cadmium-free quantum dots in aqueous solution: Potential for fingermark detection, synthesis and an application to the detection of fingermarks in blood on non-porous surfaces[END_REF] Originally, these QDots displayed inferior optophysical properties compared to classical Cd 2+ -containing QDots, but recent advances have enabled the generation of very bright QDots, mostly In 3+ -based, with high quantum yields that were stable in aqueous media. [START_REF] Xie | InAs/InP/ZnSe core/shell/shell quantum dots as near-infrared emitters: Bright, narrow-band, non-cadmium containing, and biocompatible[END_REF] These particles have demonstrated their potential use for biomedical applications in proof-of-concept studies as Near-InfraRed (NIR) emitters and for in vitro imaging of cancer cells or in vivo imaging of sentinel lymph nodes. [START_REF] Xie | InAs/InP/ZnSe core/shell/shell quantum dots as near-infrared emitters: Bright, narrow-band, non-cadmium containing, and biocompatible[END_REF][START_REF] Pons | Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity[END_REF][START_REF] Yong | Synthesis of ternary CuInS2/ZnS quantum dot bioconjugates and their applications for targeted cancer bioimaging[END_REF] However, for these materials to develop further as biomedical research tools, next to optimizing their optophysical properties and comparing them to Cd 2+ -containing QDots, their potential toxicity needs to be analyzed. Few studies thus far have looked into the toxicity of these materials, but some promising preliminary results have already been obtained, where toxic levels were found to be lower than for their Cd 2+ -containing counterparts. [START_REF] Pons | Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity[END_REF][START_REF] Brunetti | InP/ZnS as a safer alternative to CdSe/ZnS core/shell quantum dots: in vitro and in vivo toxicity assessment[END_REF] The present work evaluates the cytotoxicity of two different types of Cd 2+ -free QDots (ZnSe/ZnS and InP/ZnS respectively) according to a previously established multiparametric methodology. [START_REF] Soenen | Cellular toxicity of inorganic nanoparticles: Common aspects and guidelines for improved nanotoxicity evaluation[END_REF] This methodology enables an in-depth evaluation of cell-NP interactions employing reproducible protocols and will provide a concentration at which no toxic effects can be observed. [START_REF] Soenen | Unraveling the effects of siRNA carrier systems on cell physiology: a multiparametric approach demonstrated on dextran nanogels[END_REF] As accurate determination of NP concentrations is a timeconsuming and error-prone task, a direct comparison of non-toxic NP concentrations cannot be performed with high certainty. Therefore, at the concentration at which no toxic effects are observed, the functionality of the QDots will be evaluated with regards to their potential for visualization of live cells, as determined by visualizing the number of QDot-containing cells by fluorescence microscopy during extended culture times, as described previously. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] Then, their functionality at non-toxic levels can be compared directly to other NPs with similar functionality that have been previously studied under identical conditions using the same multiparametric methodology, such as various types of Cd 2+ -containing QDots or fluorescently tagged silica NPs. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | Fluorescent non-porous silica nanoparticles for long-term cell monitoring: Cytotoxicity and particle functionality[END_REF]

Experimental procedures

Nanoparticles

Two types of Cd 2+ -free QDots were purchased from Mesolight LLC (Little Rock, Arkansas USA). One type of QDots were ZnSe/ZnS core-shell QDots, the other QDots were InP/ZnS coreshell QDots. The particles were coated with 3-mercaptopropionic acid, which bestows them with a negative surface charge. The particles were provided as 9 µM QDot stock solutions in alkaline H2O, pH 11.

Nanoparticle characterization

Size determination by transmission electron microscopy

The size of the QDots was determined by transmission electron microscopy (TEM) using a Cs corrected JEOL 2200 FS microscope operating at 200 kV.

Elemental analysis

The chemical composition of the QDots was determined by energy dispersive X-Ray spectroscopy (EDX) using the same microscope as for the TEM observations.

Absorbance and emission spectra

Emission spectra of the QDots were recorded using a Quantifluor fluorometer (Promega, Belgium) with an excitation wavelength of 340 nm.

Dynamic light scattering and electrophoretic mobility measurements

The hydrodynamic diameter and -potential of both types of QDots were measured using a Nanosizer instrument (Malvern, Worcestershire, UK). For -potential measurements, the QDots were diluted (1/500) in phosphate buffered saline (PBS: 10 mM; pH 7.0) after which the measurements were performed (12 cycles/run) in triplicate. For determination of hydrodynamic size, the QDots were diluted (1/500) in full culture medium (see below, section "Cell culture") containing 10% fetal calf serum and 5% horse serum after which the measurements were performed (12 cycles/run) in triplicate. As a control, serum-containing media only were measured. Upon addition of the QDots, any peaks that were shifted in size or new peaks that emerged were considered for the calculation of the average size of the particles in order to exclude the pure serum proteins.

QDot sterility tests

Both types of QDots were provided as sterile stock suspensions. To rule out the effect of any biological contaminants such as endotoxins, the common endpoint chromogenic QCL-1000 ® LAL assay (Lonza, Verviers, Belgium) was performed according to the manufacturer's instructions.

QDot-cell interaction studies

Cell culture C17.2 neural progenitor cells and PC12 rat pheochromocytoma cells were cultured in high glucose containing Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 5% horse serum, 1 mM sodium pyruvate, 2 mM L-Glutamine and 1% penicillin/streptomycin (Gibco, Invitrogen, Belgium). C17.2 cells were passaged every 48 h and split 1/5. PC12 cells were passaged when reaching near 70% confluency and split 1/5 in tissue culture dishes (Greiner Bio-OneBA/BV, Wemmel, Belgium) which were coated with synthetic laminin peptide (Synthetic laminin peptide for Rat Neural Stem Cells, Millipore SA/NV, Brussels, Belgium). To establish non-proliferating cell cultures, cells were exposed with 60 mM Apigenin (Sigma-Aldrich, Bornem, Belgium). As particle uptake is linked with cell cycle progression, Apigenin treatment occurred immediately after cells had been incubated with QDots at the desired concentrations. After media removal, fresh media containing 60 mM Apigenin were used, where media were replaced for 50% every other day with fresh Apigenin-containing medium for the duration of the experiments.

Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial basal/growth culture medium (EBM-2/EGM-2, Clonetics, San Diego, CA) with medium changes every 48 h. Cells were passaged when reaching near 80% confluency by lifting the cells with 0.05% trypsin (Gibco) and were plated (1/5) onto tissue-culture flasks coated with collagen. To establish non-proliferating HUVEC cultures, cells were given endothelial cell serum-free defined medium (Cell Applications, Tebu-Bio, Le Perray en Yvelines, France) immediately after cells had been exposed to the QDots. Confluent HUVEC monolayers could then be maintained for at least one week.

Cell-QDot studies

A detailed experimental methodology can be found in the Supporting Information that accompanies this manuscript.

Statistical analysis

All data are expressed as mean + SEM unless indicated otherwise and analysed using one-way analysis of variance (ANOVA). Comparisons between particle-treated groups and untreated control groups were analysed using the Dunnett post-hoc analysis method. In all cases, the degree of significance is indicated when appropriate (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Results and Discussion

Nanoparticle characterization

Two types of core-shell QDots were used, either InP/ZnS QDots (QDotsInP) or ZnSe/ZnS QDots (QDotsZnSe). The chemical composition of the two types of QDots was evaluated by elemental analysis (Supporting Figure S1), revealing that the QDotsInP consist of approximately 13% InP core and 87% ZnS shell, whereas the QDotsZnSe consist of about 48% ZnSe core and 52% ZnS shell, which is in line with the descriptions given by the commercial supplier. These QDots both had a coating of 3-mercaptopropionic acid (MPA) which results in a negative surface charge of -32 + 2 and -27 + 2 mV for QDotsInP and QDotsZnSe, respectively. Particle diameters were assessed by transmission electron microscopy (TEM; Supporting Figure S1), revealing similar sizes for both types of QDots, being 5.9 + 0.7 nm and 5.6 + 0.6 nm for QDotsInP and QDotsZnSe, respectively. The apparent aggregation of the QDots seen in the TEM images is an artifact that stems from the high concentration of NPs in the stock suspension and the TEM procedure itself which uses a vacuum system. The QDotsZnSe furthermore are not as electron dense as the QDotsInP, making the former less easily visualized by TEM. Hydrodynamic diameters of the QDots exposed for 24 h in cell media (DMEM) were assessed by dynamic light scattering (DLS), being 115 + 12 nm for QDotsInP and 147 + 14 nm for QDotsZnSe (Supporting Figure S2). These data reveal that the MPA coating results in relatively stable particles at alkaline pH, where in cell culture media, some agglomeration is seen and small clusters of nanoparticles and surface-bound molecules are formed. The QDotsZnSe have a maximal emission at 425 nm (Supporting Figure S3B) and a photoluminescence quantum yield (PLQY) of 36%.

The QDotsInP have maximal emission at 598 nm (Supporting Figure S3A) and a PLQY of 27%.

Additionally, the stock suspensions were shown not to contain any endotoxins as evaluated by the chromogenic QCL-1000 ® LAL assay (data not shown).

Cellular QDot uptake

In the present study, cell experiments were performed on three different cell types, being: primary human umbilical vein endothelial cells (HUVEC), murine neural progenitor cells (C17.2) and rat pheochromocytoma cells (PC12). These cells have shown to be well suited for nanotoxicity studies [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | Fluorescent non-porous silica nanoparticles for long-term cell monitoring: Cytotoxicity and particle functionality[END_REF][START_REF] Soenen | Cytotoxic effects of gold nanoparticles: A multiparametric study[END_REF] as they have widely differing characteristics and therefore provide a nice and general overview of nanoparticle impact on cultured cells. Additionally, the HUVEC and C17.2 cells are frequently used in transplantation experiments for which cell labeling by nanomaterials for cell tracking purposes is a major application. [START_REF] Berman | Cell motility of neural stem cells is reduced after SPIO-labeling, which is mitigated after exocytosis[END_REF] Furthermore, the cells have been used in various studies, enabling a direct comparison of the results obtained in the current study to those obtained in previous studies for similar particles investigated under identical conditions. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] For biomedical applications, the particles will finally be internalized by the cultured cells, which normally results in their enclosure in the endosomal compartments of the cell. [START_REF] Ruan | Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding[END_REF] Figure 1A reveals a high level of colocalization between either type of QDot and green fluorescent protein (GFP)-expressing endosomal compartments within the cells, revealing that both types of QDots were indeed intraendosomally located. Based on fluorescence images, the number of distinct dots (= QDot clusters) was counted, as described previously, [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF] revealing slightly but non-significantly higher uptake levels for the QDotsZnSe in all three cell types (Figure 1B, Supporting Figure S4).

To carefully evaluate nanoparticle toxicity, it is essential to determine the number of QDots per cell in order to link the toxicity of the particles to their functional (= intracellular) concentration. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] As Zn 2+ ions are present within the cells, the use of inductively coupled plasma spectroscopy for determining cellular QDotZnSe levels is not that straightforward. Therefore, we used a previously validated methodology, [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] in which cellular QDot levels are determined. To this end, the cells were lysed in a low molarity buffer with alkaline pH (9.8), after which the concentration of the QDots was determined by comparing fluorescence intensity levels with the fluorescence of known QDot stock suspensions in the same solvent. Figure1C (Supporting Figure S4) shows the same trend as for the number of clusters per cell, where the number of QDotsZnSe is slightly higher than the number of QDotsInP, especially in the case of the PC12 cells.

The differences in cellular uptake are somewhat surprising, given the similar hydrodynamic size and -potential of the QDots that have an identical surface coating. This suggests that the actual core of the particles influences "cell vision", which is a parameter that represents how the cell handles foreign materials when they are subjected to the biological environment (such as serum components) that surrounds the cell. [START_REF] Laurent | Crucial ignored parameters on nanotoxicology: The importance of toxicity assay modifications and "cell vision[END_REF] As the difference in surface charge and (hydrodynamic) size are so minimal, and considering that both QDots consist of an outer shell of identical chemical composition (ZnS), the difference in the chemical composition of the core seems to play an important role. Possibly, the different core structures influence the crystal structure of the shell layer, which in turn influences the formation of the protein corona and how these particles will be processed by the cell. More in-depth research is needed in the future to evaluate this hypothesis. Overall, these data suggest that both types of QDots are taken up efficiently by all cell types and are finally located within the endosomal compartments of the cell. The QDotsZnSe are internalized slightly more efficiently than the QDotsInP which likely reflects minor differences in cell vision depending on the QDot core.

Effect of endosomal environment on QDot properties

One of the consequences of being located inside endosomes is that the QDots will be exposed to low pH levels (4.5 for lysosomes; 5.5 for late endosomes). To verify the effect of this low pH on QDot stability, both types of QDots were subjected to endosomal buffer systems of pH 7.4, 5.5 or 4.5, as described previously, [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF] after which the effect of the pH on their fluorescence intensity was determined. Figure 2 reveals high optophysical stability of the particles, where almost no decrease in fluorescence intensity could be observed over several days, even when the QDots were exposed to the lowest pH levels. Compared to previous studies on various types of Cd 2+ -containing QDots studied under identical conditions, the stability of the Cd 2+ -free QDots is substantially higher, where the fluorescence intensity of Cd 2+ -containing QDots dropped by, on average, 60% after 5 days exposure to pH 4.5. The higher optical stability of the Cd 2+ -free QDots likely also reflects a higher chemical stability of the particles compared to the Cd 2+ -containing QDots. For the latter particles, it has been shown that their exposure to low endosomal pH levels results in a degradation of the QDots themselves as a result of acid etching [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] and subsequent release of highly toxic Cd 2+ ions in their immediate environment, increasing nanoparticle cytotoxicity. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF][START_REF] Chen | The cytotoxicity of cadmium-based quantum dots[END_REF]

Acute and long-term toxicity

The cytotoxic effects of both types of QDots were determined using the Alamar Blue assay for QDot concentrations ranging from 0 to 100 nM. Figure 3A (Supporting Figure S5) shows significant cytotoxicity starting from 60 nM for QDotsZnSe and 80 nM for QDotsInP. The difference in toxicity between the two QDot types likely reflects the differences in cellular nanoparticle levels, which was also slightly higher for the QDotsZnSe. However, the differences in cellular uptake are only minimal, suggesting that other factors intrinsic to the different types of QDots played an additional role in their cytotoxicity, such as the type of heavy metal used in the chemical composition of the QDots. As QDot degradation results in the release of heavy metal ions which can be highly toxic, such as Cd 2+ , the intracellular presence of QDots can result in a slow but gradually increasing damage of the cells. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] To test this, cells were first labeled with various concentrations of either type of QDot, after which they were kept in culture under nonproliferative conditions as detailed elsewhere. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF] The halt in cell proliferation is essential to allow to accurately determine the effect of QDot degradation on particle cytotoxicity without the rapid and asymmetric dilution of cellular particle numbers which accompanies cell division. [START_REF] Summers | Statistical analysis of nanoparticle dosing in a dynamic cellular system[END_REF] Furthermore, under these conditions, the cells more accurately mimic the in vivo situation, where most cells are also not actively dividing. Figure 3B reveals no increase in cytotoxicity up to at least 7 days after initial cell labeling. Together with the high stability of the particle fluorescence in time, these data indicate a high chemical stability of the QDots, where environmentally-induced degradation appears to be minimal. This is in stark contrast to common Cd 2+ -containing QDots that have been found to degrade under endosomal pH conditions. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF] This may be explained by differences in the crystal structure of the QDots where some Cd 2+ -free QDots have been described to be very robust against environmental influences such as oxidation.

Oxidative stress and secondary effects

The use of nanomaterials, such as zinc-containing NPs, is known to be closely linked to their induction of oxidative stress, which, when it persists over longer time periods or reaches higher levels, can result in cell death. [START_REF] Setyawati | Nanotoxicology of common metal oxide based nanomaterials: their ROS-y and non-ROS-y consequences[END_REF][START_REF] George | Use of a high-throughput screening approach coupled with in vivo zebrafish embryo screening to develop hazard ranking for engineered nanomaterials[END_REF][START_REF] Ng | The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles[END_REF][START_REF] Setyawati | Effect of zinc oxide nanomaterialsinduced oxidative stress on the p53 pathway[END_REF] Figure 3C (Supporting Figure S5) shows a clear induction of reactive oxygen species (ROS), reaching significant levels at 30 nM for QDotsZnSe and 50 nM for QDotsInP. The higher level of oxidative stress for the QDotsZnSe suggests the important role of ROS in QDot toxicity given their higher cytotoxicity. The link between oxidative stress and cytotoxicity is quite unclear, as all cell types have different degrees of antioxidative capacity. [START_REF] Diaz | Assessing methods for blood cell cytotoxic responses to inorganic nanoparticles and nanoparticle aggregates[END_REF] Oxidative stress can affect cell viability through several possible mechanisms, such as the induction of mitochondrial damage, alterations in calcium fluxes, or DNA damage. Therefore, the effect of both types of QDots on secondary oxidative stress mechanisms was investigated.

Figure 4A (Supporting Figure S6) shows no significant increase in the number of double strand breaks for either type of QDot as revealed by phosphorylated -H2Ax staining, a common marker for DNA double strand breaks. [START_REF] Lamkowski | DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation[END_REF] Furthermore, cellular calcium levels were slightly but not significantly increased for both types of QDots at higher particle concentrations (Figure 4B, Supporting Figure S7). Changes in calcium waves are important signaling mediators in cell homeostasis, where alterations in cytoplasmic calcium waves can have drastic effects on many important cellular processes such as cell division or cell viability. [START_REF] Tang | Mechanisms of unmodified CdSe quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons[END_REF] Calcium waves however have only a short lifetime and die out rather fast so they can easily be overlooked in the case of minor alterations. [START_REF] Soenen | Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling[END_REF] Therefore, the effect of the QDots on alterations in mitochondrial membrane potential (m) was investigated as a more robust marker. Figure 4C (Supporting Figure S8) shows a clear and significant loss of m for both types of QDots, starting from 30 nM for QDotsZnSe and 60 nM for QDotsInP. These data are closely matched with the differences in oxidative stress levels induced by both types of QDots as well as their cytotoxicity and therefore suggest the importance of oxidative stress in the cytotoxic profile of both types of QDots.

The difference between QDotsZnSe and QDotsInP is somewhat strange as both types of QDots have the same shell layer in terms of composition (ZnS). Additionally, they are coated with the same organic coating and have a similar hydrodynamic size and -potential. Yet, similar as with cellular uptake, the differences in the core structure of the QDots appears to affect their final cellular effects. Thus far, the exact cause of this remains rather unclear. Given the high stability of the particles against degradation, it is rather unlikely that many ions from the particle core will be free in the environment to affect cell viability. Even if very low levels of degradation would occur in time, some Zn 2+ ions will be released from the shell layer in both types of QDots. For the QDotsZnSe, more Zn 2+ ions can be released from the core, elevating the total level of cellular zinc, an essential component in cell homeostasis, above toxic thresholds. For the QDotsInP, In 3+ will be released, which in itself may not reach the levels which are above the toxic threshold.

The total level of Zn 2+ ions will then likely also be lower for the latter QDots, where the contribution of In 3+ and Zn 2+ separately on inducing oxidative stress may be less outspoken than higher levels of Zn 2+ alone. To evaluate this in more detail, the effect of In 3+ and Zn 2+ ions on cell viability (Supporting Figure S9) and oxidative stress (Supporting Figure S10) have been investigated. These data clearly reveal that neither of the ions result in any significant cytotoxicity at concentrations up to 12 µM (which exceeds the level that can be released from the QDots), as observed for Cd 2+ -containing QDots. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] The level of cellular In 3+ or Zn 2+ ions were not assessed in the current study as for Zn 2+ ions, which are naturally present, determining the level of QDot-derived Zn 2+ in a cellular environment by ICP-MS is very challenging. The lack of any toxic effects for these ions under the conditions used are in line with other reports, where, for example, for freshwater shrimp, the LC50 of In 3+ ions was found to be 60 µM compared to 0.5 µM for Cd 2+ . [START_REF] Yang | Comparative acute toxicity of gallium(III), antimony(III), indium(III), cadmium(II), and copper(II) on freshwater swamp shrimp (Macrobrachium nipponense)[END_REF] Interestingly however, is the induction of oxidative stress for both ions (Supporting Figure S10). In 3+ ions, which are known ROS inducers [START_REF] Lin | Indium chlorideinduced micronuclei via reactive oxygen species in chinese hamster lung fibroblast V79 cells[END_REF] show a clear concentration-dependent induction of ROS that reaches saturation levels at higher In 3+ concentrations and then slowly drop to lower levels. Zn 2+ ions, on the other hand, initially do not affect cellular ROS levels up to a concentration of 10 µM, but then result in a sharp increase at higher level. The lack of any significant ROS inductions by low levels of Zn 2+ ions is in line with their status as potent antioxidants. [START_REF] Matsui | Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: Excessive increase in intracellular Zn 2+ concentration[END_REF] The higher toxicity and higher induction of ROS by QDotZnSe is in apparent contrast to these findings. However, the gradual release of Zn 2+ ions upon QDot degradation that can locally reach very high concentrations may result in different physiological effects than elevated Zn 2+ levels throughout the entire cell. Furthermore, although ZnCl2 has antioxidant capacity, it has also been shown that Zn 2+ ions can aggravate the cellular stress induced by other stimuli. [START_REF] Matsui | Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: Excessive increase in intracellular Zn 2+ concentration[END_REF] In the present study, the QDots themselves may induce oxidative stress, where low levels of free Zn 2+ may further increase cellular ROS levels, specifically in the case of QDotZnSe.

Any differences in toxicity may also be due to differences in the crystal structure of both types of QDots where QDotsInP might be more stable than QDotsZnSe. The crystal structure of the QDot core may furthermore also affect the stability of the shell layer, resulting in a lower level of ions being released from both the shell and core of QDotsInP.

Effects of QDots on cell functionality

Nanomaterials can affect cells in many different ways, requiring a large number of parameters to be analyzed to get a clear overview of potentially negative effects that these particles may exert. For functionally active cells, such as HUVEC or C17.2 cells that are actively being explored in cell transplantation studies, [START_REF] Berman | Cell motility of neural stem cells is reduced after SPIO-labeling, which is mitigated after exocytosis[END_REF] assessing cell functionality is an important aspect. It is important to note that dead cells will logically not have any more functionality and therefore only those conditions are selected at which no cytotoxicity was observed, being 60 nM for QDotsZnSe and 80 nM for QDotsInP. In the present study, the three cell types used offer optimal conditions for assessing important functional parameters. The widely spread HUVEC cells are ideally suited to investigate the effect of the QDots on cell morphology and cytoskeletal architecture. [START_REF] Soenen | Cytotoxic effects of gold nanoparticles: A multiparametric study[END_REF] For QDots that are ideally suited for bioimaging applications in live cells, it is essential that the intracellular presence of the particles does not affect cell structure. Some particles, including QDots, have been found to disturb cell morphology and cell cytoskeleton and some studies have shown direct effects on actin fibers. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Tay | Nanoparticles strengthen intracellular tension and retard cellular migration[END_REF] The HUVEC cells were therefore stained for F-actin and -tubulin, after which the spreading of the cells was analyzed by confocal microscopy and image analysis, as performed previously. [START_REF] Soenen | Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling[END_REF] Figure 5A shows representative images of the cells which display a clear spreading and nice cytoskeletal architecture for untreated control cells and cells labeled with low concentrations of QDots. At higher levels, the particles appear to affect cell spreading. Cell area distribution was therefore analyzed (Figure 5B), which shows a clear decrease in cell spreading at 40 nM for QDotsZnSe and 50 nM for QDotsInP. These effects may in part be due to the induction of ROS, but when cells were treated with 5 mM N-acetylcysteine (NAC), a free radical scavenger that reduced cellular ROS levels to near control values (data not shown), the decrease in cell spreading could only be partially overcome (Figure 5C). The results demonstrate that oxidative stress plays a major role in the cytotoxic effects of the QDots, but that for both types of particles, other mechanisms also played an important role, such as cytoskeletal rearrangements and reductions in cell spreading. For C17.2 cells, cell functionality can be evaluated by determining the efficiency of cell differentiation, where the neural progenitor cells can be differentiated into full neurons. Figure 5D shows a clear and significant loss in differentiation efficiency of the C17.2 cells when treated with 50 nM QDotsZnSe, whereas QDotsInP did not appear to have any significant effect. Treating the cells with 5 mM NAC could again only partially overcome the observed effects, suggesting that cell differentiation was affected through other non-ROS associated mechanisms. The differentiation of the C17.2 cells is a long process that is accompanied with high levels of cell death, which can harden any analysis of minor effects. [START_REF] Soenen | Cellular toxicity of inorganic nanoparticles: Common aspects and guidelines for improved nanotoxicity evaluation[END_REF] The PC12 cells present a more elegant approach for investigating nanoparticle effects on cell functionality as they can reversibly induce rapid neurite outgrowth that can easily be quantitated. [START_REF] Pisanic | Nanotoxicity of iron oxide nanoparticle internalization in growing neurons[END_REF] This process is also accompanied with only low levels of cell death and therefore allows picking up any effects with higher sensitivity than the C17.2 cells. Figure 5E shows significant reductions in neurite outgrowth efficiency at 10 nM QDotsZnSe and 20 nM QDotsInP.

Taken together, these data show that at subcytotoxic concentrations, both types of QDots still affect cell homeostasis, by impeding cell functionality and inducing oxidative stress. To evaluate the functionality of the particles themselves, further tests are performed at the concentrations of the particles at which no adverse effects were observed, being 10 nM for QDotsZnSe and 20 nM for QDotsInP. When compared to different types of Cd 2+ -containing core-shell QDots studied under identical conditions, these concentrations are approximately 10-fold higher (ranging from 0.5 to 2 nM). [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] This high level of particle concentrations that can be tolerated by the cells is likely caused by a combination of several factors, in part being the lower intrinsic cytotoxicity of both types of Cd 2+ -free QDots, which is in line with other studies reporting lower toxicity levels for Cd 2+ -free QDots. [START_REF] Brunetti | InP/ZnS as a safer alternative to CdSe/ZnS core/shell quantum dots: in vitro and in vivo toxicity assessment[END_REF] However, in order to accurately assess particle toxicity, the intrinsic toxicity of the particles must be considered. A direct comparison of the different particles is made impossible by differences in their size, surface coating and the associated changes in cellular interaction such as alterations in cellular uptake levels. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] Furthermore, a comparison of particle toxicity based on concentrations is prone to big errors as accurate determination of nanoparticle numbers in stock suspensions is extremely tedious. 14

Functionality of the QDots for cell imaging

Based on the determination of the safe concentrations of both types of QDots, their functionality can be assessed by evaluating how long they can be used for visualizing live, actively dividing cells, which would be one major application of the QDots. This then allows a much better comparison of different nanoparticles with the same functionality as it includes a large number of potential differences between particles such as differences in uptake levels, errors in determining stock concentrations or QDot PLQY. [START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] In practice, cells are seeded with either type of QDot at non-cytotoxic concentrations for 24 h (being 10 nM for QDotsZnSe and 20 nM for QDotsInP), after which cells are kept in culture. Upon every two cell divisions, cells are reseeded at their initial density and part of the cell population is kept for microscopy analysis, as described in detail in the Supporting Experimental Methodology. The reseeding of the cells is performed in order to avoid the need for low cell densities at initial time points (HUVEC and PC12 cell proliferation requires optimal cell densities) and to avoid cell cultures to become overpopulated which would impede cell division due to contact inhibition. Figure 6 shows that the QDotsZnSe can be used to visualize cells for approximately 4 cell cycles, whereas the QDotsInP allow cells to be tracked for approximately 6 cell cycles. For various types of core-shell Cd 2+ -containing QDots, cells could be tracked for approximately 2 to 4 cell cycles, indicating that the Cd 2+ -free QDots are approximately 1.5-fold better than some Cd 2+ -containing QDots previously tested. [START_REF] Soenen | The cytotoxic effects of polymer-coated quantum dots and restrictions for live cell applications[END_REF][START_REF] Soenen | The effect of nanoparticle degradation on poly(methacrylic acid)-coated quantum dot toxicity: the importance of particle functionality assessment in toxicology[END_REF] The difference in particle functionality is therefore far less than expected based on the concentrations of the particles only. This is likely caused by the relatively low brightness of the Cd 2+ -free QDots compared to some Cd 2+ -containing QDots. Additionally, this may be due to a lower concentration of QDots in the stock suspension than originally determined as well as the short organic ligands which may result in lower colloidal stability of the particles in cell medium and reduced cellular uptake. [START_REF] Kirchner | Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles[END_REF] Overall, the Cd 2+ -free QDots appear to be well suited alternatives for Cd 2+ -containing QDots for cell bioimaging purposes, where the QDotsZnSe appear to be slightly less suited than the QDotsInP, likely because of a higher intrinsic toxicity and lower brightness of the QDotsZnSe. This is further supported upon determining the cellular fluorescence intensity of the QDot-labeled cells, where QDotZnSe-labeled cells have a much lower fluorescence intensity than the QDotInP-labeled cells (Supporting Figure S11). At this stage, however, both types of QDots appear to be similar to common Cd 2+ -containing QDots. This is further supported by a recent study, where more advanced Cd 2+ -containing QDots (i.e. so-called gradient alloy QDots, with identical surface coating to the QDots evaluated here) that were found to have a PLQY of up to 90%, resulted in a similar duration of cell tracking at non-toxic conditions (up to 6 cell doublings). Therefore, the Cd 2+ -free QDots examined in the current study appear to be similar to more advanced Cd 2+ -containing QDots. In the future, further improvements in surface chemistry and PLQY of the Cd 2+ -free QDots may boost their functionality to higher levels, especially for the QDotsInP. At this stage, however, more research needs to be performed for these particles to be optimized and to reach their full potential in replacing current Cd 2+ -containing QDots.

Conclusion

The present work shows that the cadmium-free QDots, despite having a high chemical stability and low intrinsic toxicity, appear to only be slightly better than commonly used Cd 2+ -containing QDots in terms of cell visualization. This is primarily due to the relatively low PLQY and associated lower brightness of both types of cadmium-free QDots tested in this study. Although the QDotInP and QDotZnSe both possess a ZnS shell layer, are of similar size and have the same surface chemistry, they result in slight differences in cellular uptake levels, but significant differences in toxic effects, which are most pronounced for QDotZnSe. These data indicate the importance of the chemical composition of the QDot core in their final interactions with biological entities such as cells or whole organisms. The use of the multiparametric methodology enables to define non-toxic particle concentrations and sheds some insight into the mechanisms involved in the negative effects of the QDots. Additionally, using the same methods and cell types and investigating particle toxicity under well-defined and controllable conditions allows to compare the results obtained with results for other particles studied in the same way. This comparison reveals that cells can tolerate a higher level of cadmium-free QDots than cadmiumcontaining QDots, but when taking particle brightness and uptake levels into account, similar results are obtained, where cadmium-free QDots are only moderately more functional. This comparison shows the importance of particle functionality when assessing particle toxicity as comparing particle concentrations does not give an appropriate estimation of the true differences in particle toxicity, as many parameters such as errors in determining particle stock concentration, cellular uptake levels etc are not taken into account. The low intrinsic toxicity of the cadmium-free QDots offers a lot of potential for future applications, where more stable

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. The fluorescence intensity levels of QDotInP and QDotZnSe exposed to buffer systems

Figure 3 .

 3 Figure 3. (A) Viability of HUVEC as a function of QDot concentration (ranging from 20 to 100

Figure 4 .

 4 Figure 4. Secondary ROS effects of Cd 2+ -free QDots. (A) The level of phosphorylated -H2Ax,

Figure 5 .

 5 Figure 5. (A) Representative fluorescence images of HUVEC cells either untreated (top row) or

Figure 6 .

 6 Figure 6. (A) Representative fluorescence images of HUVEC cells exposed to non-cytotoxic

ASSOCIATED CONTENT Supporting Information. Elemental analysis and transmission electron micrographs; dynamic light scattering plots of the nanoparticles used; quantum dot emission spectra; cellular uptake of the quantum dots in C17.2 and PC12 cells; effects of the quantum dots on viability, ROS, DNA damage, calcium levels and mitochondrial health in C17.2 and PC12 cells and a full experimental methodology. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*(S. J. S.) Email: Stefaan.Soenen@med.kuleuven.be *(S. C. D.) Email: Stefaan.DeSmedt@ugent.be

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources

S. J. S. is a postdoctoral fellow from FWO-Vlaanderen. The authors acknowledge FWO-

Vlaanderen for financial support.

Notes

Any additional relevant notes should be placed here.


Tangi Aubert

∥ , ‡,£ Stefaan J Soenen

Daniel Wassmuth

Marco Cirillo

Rik Van Deun

Kevin Braeckmans

Zeger Hens

Bright and Stable CdSe/CdS@SiO 2 Nanoparticles Suitable for Long Term Cell Labeling

Keywords: quantum dots, fluorescent, microemulsion, bioimaging, theranostics

We report on the synthesis of luminescent CdSe/CdS@SiO2 nanoparticles and their application to cell labeling. The main novelty of these nanoparticles is the use of newly developed 'flash' CdSe/CdS quantum dots (QDs), which are obtained through a new fast and efficient synthesis method recently reported. These core-shell QDs are encapsulated in silica nanoparticles through a water-in-oil microemulsion process, resulting in CdSe/CdS@SiO2 nanoparticles with good morphology and controlled architecture. The main asset of these luminescent nanoparticles is their high photoluminescent quantum yield, which is equal to that of the original CdSe/CdS QDs and remains unchanged even after several months of storage in water. Thanks to the remarkable stability of their optical property in aqueous environment and to their low levels of toxicity, the high potential of these nanoparticles for long term cell labeling is demonstrated.

Introduction

The use of functional nanoparticles in the fields of biotechnology, medical diagnostic and therapy techniques has raised an increasing interest in the past years. [START_REF] Pelaz | The State of Nanoparticle-Based Nanoscience and Biotechnology: Progress, Promises, and Challenges[END_REF][2][START_REF] Zhang | Nanoparticles in Medicine: Therapeutic Applications and Developments[END_REF] Luminescent nanoparticles in particular are very useful for bioimaging and cell labeling. For these applications, semiconductor quantum dots (QDs) are an attractive alternative for classic organic fluorophores as light emitters since they exhibit a higher brightness thanks to large absorption cross-sections, a tunable narrow emission spectrum and a broadband absorption spectrum. [START_REF] Michalet | Quantum Dots for Live Cells[END_REF] Therefore, QDs are ideal potential nanoprobes for sensitive and long-term multiplexed bioimaging and biosensing. [START_REF] Medintz | Quantum Dot Bioconjugates for Imaging, Labelling and Sensing[END_REF][START_REF] Xing | Semiconductor Quantum Dots for Biosensing and In Vivo Imaging[END_REF][START_REF] Chan | Luminescent Quantum Dots for Multiplexed Biological Detection and Imaging[END_REF] For optimal properties, colloidal QDs are best synthesized at relatively high temperature (up to 350 °C) in apolar solvents. To be used in bioimaging, they should thus be transferred to biological media such as water or buffer solutions. For this purpose, the encapsulation of QDs in silica nanoparticles has been investigated in the past years. [START_REF] Mulvaney | Silica Encapsulation of Quantum Dots and Metal Clusters[END_REF][START_REF] Gerion | Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor Quantum Dots[END_REF][START_REF] Darbandi | Single Quantum Dots in Silica Spheres by Microemulsion Synthesis[END_REF][START_REF] Koole | On the Incorporation Mechanism of Hydrophobic Quantum Dots in Silica Spheres by a Reverse Microemulsion Method[END_REF][START_REF] Selvan | Non-Cytotoxic, Silica-Coated CdSe Quantum Dots with Efficient Photoluminescence[END_REF] Indeed, a silica matrix can provide nanocrystals with an enhanced colloidal stability in polar solvents and for a wide range of conditions (pH, ionic strength). [START_REF] Guerrero-Martínez | Recent Progress on Silica Coating of Nanoparticles and Related Nanomaterials[END_REF] In addition, silica is a relatively biocompatible material and its surface can be functionalized with a wide range of groups, including amines and thiols, [START_REF] Yan | Dye-Doped Nanoparticles for Bioanalysis[END_REF] that can thereafter be used for coupling the nanoparticles with biomolecules of interest and for specific targeting of cells or intracellular structures. 2,[START_REF] Knopp | Review: Bioanalytical Applications of Biomolecule-Functionalized Nanometer-Sized Doped Silica Particles[END_REF] Unfortunately, the properties of semiconductor QDs can quickly deteriorate in strongly oxidative environments such as water, which therefore limits their use in biological applications. In solution, reactive oxygen species (ROS) such as oxygen superoxide or singlet oxygen can cause photooxidation of the QDs resulting in serious deterioration of their optical properties, with in particular a decrease of their photoluminescence quantum yield (PLQY). [START_REF] Zhang | Time-Dependent Photoluminescence Blue Shift of the Quantum Dots in Living Cells: Effect of Oxidation by Singlet Oxygen[END_REF][START_REF] Manner | Role of Solvent-Oxygen Ion Pairs in Photooxidation of CdSe Nanocrystal Quantum Dots[END_REF][START_REF] Wang | Mechanism of Strong Luminescence Photoactivation of Citrate-Stabilized Water-Soluble Nanoparticles with CdSe Cores[END_REF] In the same way, the degradation of QDs can result in the release of toxic elements such as Cd 2+ ions, a known heavy metal toxicant. [START_REF] Chen | The Cytotoxicity of Cadmium-Based Quantum Dots[END_REF][START_REF] Kirchner | Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles[END_REF][START_REF] Hardman | A Toxicologic Review of Quantum Dots: Toxicity Depends on Physicochemical and Environmental Factors[END_REF] These issues were so far not fully solved by the encapsulation of the QDs in silica nanoparticles due to the high porosity of the silica matrix made by sol-gel approaches. Furthermore, in studies on the encapsulation of QDs in silica nanoparticles the effect of the process on the PLQY is not rigorously analyzed. The reported values are often obtained by measurements relative to a dye with a known PLQY, whereas this method cannot be accurate due to the scattering of light by such silica coated nanoparticles. In addition, the PLQY values are typically reported for freshly prepared samples and the long term stability of the PLQY of QD@SiO2 nanoparticles is usually not addressed. Thus, in addition to the long lasting problem of QD degradation, which has always limit their true application to bioimaging, the accurate monitoring of this degradation is also an open challenge.

In this study we overcome most of these limitations by the silica encapsulation of newly developed 'flash' CdSe/CdS QDs, which are obtained through a very fast and efficient method we recently reported. [START_REF] Cirillo | Flash" Synthesis of CdSe/CdS Core-Shell Quantum Dots[END_REF] These 'flash' QDs meet with state-of-the-art criteria of quality in terms of morphology and optical properties for such core-shell heterostructures, including good size monodispersity, narrow emission spectra, high PLQYs and a low blinking behavior, which are essential properties for biolabeling applications. In a second step, these QDs were encapsulated in silica nanoparticles through a water-inoil microemulsion process, a very powerful tool for the synthesis of functional silica nanoparticles with complex architectures and with sizes below 50 nm, [START_REF] Aubert | Functional Silica Nanoparticles Synthesized by Water-in-Oil Microemulsion Processes[END_REF] which has already been successfully employed for the silica encapsulation of hydrophobic nanocrystals, including semiconductor QDs, [START_REF] Darbandi | Single Quantum Dots in Silica Spheres by Microemulsion Synthesis[END_REF][START_REF] Koole | On the Incorporation Mechanism of Hydrophobic Quantum Dots in Silica Spheres by a Reverse Microemulsion Method[END_REF][START_REF] Selvan | Non-Cytotoxic, Silica-Coated CdSe Quantum Dots with Efficient Photoluminescence[END_REF] but also anisotropic nanorods. [START_REF] Pietra | Synthesis of Highly Luminescent Silica-Coated CdSe/CdS Nanorods[END_REF] The effect of the silica encapsulation and the subsequent transfer of the nanoparticles into water on the PLQY of the QDs was carefully investigated by using an integrating sphere and an improved calculation method as detailed in the Experimental Section and in the Supporting Information. With this method we demonstrate the high physicochemical stability of these 'flash' CdSe/CdS@SiO2 nanoparticles. Opposite from QDs synthesized using a classical 'SILAR' approach, [START_REF] Li | Large-Scale Synthesis of Nearly Monodisperse CdSe/CdS Core/Shell Nanocrystals Using Air-Stable Reagents via Successive Ion Layer Adsorption and Reaction[END_REF] they fully retain their PLQY even after long term storage in water and exhibit a high photostability under continuous UV irradiation. This enhanced stability and its careful monitoring represent considerable improvements to the field of QD@SiO2 nanoparticles and constitutes the major novelty of this work. In addition, the cytotoxic effects of the CdSe/CdS@SiO2 nanoparticles were investigated according to previously optimized methods for nanocytotoxicity evaluation, [START_REF] Soenen | Cellular Toxicity of Inorganic Nanoparticles: Common Aspects and Guidelines for Improved Nanotoxicity Evaluation[END_REF] proving effective protection against acute toxicity by the silica coating. Finally, taking the advantage of the high stability of their optical properties and their reduced toxicity, the usefulness of these nanoparticles for cell labeling in terms of particle uptake and long-term cell visualization is evidenced. We thus demonstrate that the combination of 'flash' CdSe/CdS QDs and a microemulsion based silica coating yields nanoparticles with a high physicochemical stability and photostability but also with a low toxicity. These results truly defeat the limitations restricting the bio-applications of QDs. This considerable breakthrough will pave the way for the development of efficient nanoprobes for long term cell tracking and labeling, but also for the further development of complex multifunctional nanoparticles.

Experimental Section

2.1. Chemicals. Cadmium oxide (CdO, 99.998% Puratronic) and tri-octylphosphine oxide (TOPO, 98%) were purchased from Alfa-Aesar. Octadecylphosphonic acid (ODPA, 98%) was purchased from PCI. Tri-octylphosiphine (TOP, 97%) and sulfur powder (S, 99.999%) were purchased from Strem. Selenium powder (Se, 99.999%), oleic acid (OA, 90%), tetraethyl orthosilicate (TEOS, 98%) and polyoxyethylene (4) lauryl ether (Brij30) were purchased from Sigma-Aldrich. n-Heptane (99.9%) was purchased from VWR. Ammonia solution (28-30% in water) was purchased from Merck.

Synthesis of the 'Flash'

CdSe/CdS QDs. The 'flash' CdSe/CdS QDs were synthesized according to a method we recently reported. [START_REF] Cirillo | Flash" Synthesis of CdSe/CdS Core-Shell Quantum Dots[END_REF] First, 4 nm sized wurtzite CdSe cores were prepared according to a procedure described in the literature. [START_REF] Carbone | Synthesis and Micrometer-Scale Assembly of Colloidal CdSe/CdS Nanorods Prepared by a Seeded Growth Approach[END_REF] Briefly, 0.06 g of CdO, 3 g of TOPO and 0.280 g of ODPA were mixed in a 25 mL three neck flask. The mixture was heated to 150°C under a vacuum for one hour. The solution was then put under a nitrogen atmosphere and heated to 345°C. Next, 1.8 mL of TOP were injected. After the temperature had recovered, a solution containing 0.058 g of Se dissolved in 0.360 g of TOP was injected. The reaction time was adapted to obtain CdSe QDs of 4 nm in size and the QDs were purified by several cycles of precipitation, centrifugation and resuspension. Next, the CdSe/CdS core-shell QDs were obtained through a seeded growth approach using the previously mentioned wurtzite CdSe QDs as cores. For this synthesis, 0.08 g of CdO and 1.6 g of oleic acid were mixed with 3 g of TOPO in a 25 mL three neck flask. The reaction mixture was heated to 120°C while flushing with nitrogen for one hour. The temperature was then increased to 330°C. As soon as the solution became colorless, 1.8 mL of TOP were injected. After the temperature had recovered, 1.8 mL of a solution containing 87 nmol of CdSe core QDs and 0.06 g of S in TOP were injected. After 3 minutes, the reaction was quenched by a sudden drop of the temperature using a water bath, followed by the injection of 10 mL of toluene. The nanocrystals were purified by the addition of isopropanol and methanol, centrifugation and redispersion in toluene. The purification was repeated three times.

Synthesis of the

CdSe/CdS@SiO2 Nanoparticles. The above mentioned CdSe/CdS QDs were encapsulated in silica nanoparticles through a water-in-oil microemulsion process. For this 25 nmol of CdSe/CdS QDs were mixed in 50 mL of heptane and 16 mL of Brij30. After 15 min of stirring, 2.5 mL of MilliQ water and 0.5 mL of ammonia solution were added dropwise. After 1 h of stirring to ensure stability and homogeneity of the microemulsion, 1.5 mL of TEOS were added. The reaction was left under stirring for 3 days, then the microemulsion was precipitated by addition of a large volume of ethanol (~ 30 mL) and the nanoparticles were collected by centrifugation (relative centrifugal force, RCF = 3000 g, 5 min). The nanoparticles were purified by repeated centrifugation cycles in MilliQ water (RCF = 10000 g, 30 min) and finally redispersed in MilliQ water. The pH of the QD@SiO2 nanoparticles solution was always comprised between 8 and 9.

Characterization Techniques.

Bright field transmission electron microscopy (TEM) images were taken using a Cs corrected JEOL 2200 FS microscope. Photoluminescence measurements were done on an Edinburgh Instruments FLSP920 UV-Vis-NIR spectrofluorometer, using a 450W xenon lamp as the excitation source. The signals were collected with a Hamamatsu R928P PMT detector, which has a response curve between 200 and 900 nm. All emission spectra were recorded for an excitation wavelength of 365 nm and were corrected for the detector sensitivity. The PLQY of the QD@SiO2 nanoparticles were measured for an excitation wavelength of 365 nm and by using an integrating sphere. The measurement method for measuring the absolute PLQY of these nanoparticles was adapted from the method described by Mello et al. [START_REF] De Mello | An Improved Experimental Determination of External Photoluminescence Quantum Efficiency[END_REF] which was improved in order to correct the measurements for the reflection on the cuvette inside the integrating sphere (see the Supporting Information for a complete description of the method and calculation details).

Toxicity and Cell Labeling

Studies. All toxicity and cell labeling studies were performed using primary human umbilical vein endothelial cells (HUVEC) as a representative cell model for both in vitro and in vivo evaluation of nanoparticle toxicity. [START_REF] Blechinger | Uptake Kinetics and Nanotoxicity of Silica Nanoparticles Are Cell Type Dependent[END_REF] For these studies the concentration in nanoparticles was estimated by weighing a dried extract of the initial solution and by assuming a density of 2 for silica. A full methodology on cell culture protocols and detailed experimental protocols on cell labeling studies can be found in the Supporting Information that accompanies this article.

Results and Discussion

Structure and Morphology

3.1.1. 'Flash' CdSe/CdS QDs and CdSe/CdS@SiO2 Nanoparticles. One of the main advantages of the 'flash' synthesis is the possibility to grow relatively thick CdS shells in no more than 3 minutes of reaction. The 'flash' CdSe/CdS QDs synthesized according to the procedure described in the Experimental Section are 9.5 ± 1.2 nm in size (Figure 1a), which corresponds to about 8 layers of CdS. As demonstrated elsewhere, [START_REF] Cirillo | Flash" Synthesis of CdSe/CdS Core-Shell Quantum Dots[END_REF] with this synthesis method the CdS shell grows epitaxially on the wurtzite CdSe cores and the CdSe/CdS QDs also have a wurtzite structure. Through the microemulsion process the QDs were successfully encapsulated in silica nanoparticles (Figure 1b). The CdSe/CdS@SiO2 nanoparticles show a good morphology with more than 99% of them having one single QD perfectly in its center and surrounded by a homogeneous silica layer. The CdSe/CdS@SiO2 nanoparticles have an average diameter of 33.8 ± 2.8 nm, corresponding to a silica shell thickness of about 12 nm. Figure 1. TEM images of the CdSe/CdS QDs (a), the CdSe/CdS@SiO2 nanoparticles obtained as described in the experimental section (b), and the CdSe/CdS@SiO2 nanoparticles obtained by using either 40 times less (c) or 25 times more (d) silica precursor.

Tuning the Silica Shell Thickness.

Being able to control the size of the nanoparticles is an important parameter when developing tools for biotechnology applications. With this microemulsion technique, the thickness of the silica shell could be easily tuned in a wide range of sizes just by varying the amount of silica precursor (TEOS) in the synthesis. Indeed, as demonstrated by other authors, [START_REF] Darbandi | Single Quantum Dots in Silica Spheres by Microemulsion Synthesis[END_REF][START_REF] Koole | On the Incorporation Mechanism of Hydrophobic Quantum Dots in Silica Spheres by a Reverse Microemulsion Method[END_REF] in a water-in-oil microemulsion, hydrophobic nanocrystals capped with amphiphilic ligands act as nucleation centers for the growth of the silica nanoparticles. During this process, both the nanocrystals and the silica precursor are initially located in the oil phase. The amphiphilic ligands are exchanged by hydrolyzed silica monomers, and the nanocrystals can then migrate to the aqueous inverted micelles where the growth of the silica shell will proceed. [START_REF] Darbandi | Single Quantum Dots in Silica Spheres by Microemulsion Synthesis[END_REF][START_REF] Koole | On the Incorporation Mechanism of Hydrophobic Quantum Dots in Silica Spheres by a Reverse Microemulsion Method[END_REF] As a result of this mechanism, each QD@SiO2 nanoparticle contains only one single QD core, and thanks to the full chemical yield of the silica formation there is a direct linear relationship between the amount of TEOS involved in the synthesis and the volume of the silica shell per nanoparticle. For illustration, Figure 1c and1d show CdSe/CdS@SiO2 nanoparticles with silica shell thicknesses of less than 2 nm and more than 30 nm respectively, which were obtained by using either 40 times less or 25 times more of TEOS compared to the synthesis described in the experimental section. Following this line of reasoning, the thickness of the silica shell could also be tuned by using the same amount of TEOS but with varying the amount of QDs. However, with this approach there is still the possibility of forming empty silica nanoparticles if the concentration of QDs in the microemulsion is too low (see Supporting Information, Figure S3a). In the same way, if the concentration of QDs is too high, it results in the formation of inhomogeneous and sometimes incomplete silica shells (see Supporting Information, Figure S3b). Thus, for the silica encapsulation of QDs, there is an optimal range of concentrations of these nanocrystals in the microemulsion, which will of course depend on the composition of the microemulsion (i.e., oil:water:surfactant ratios). Once this range of concentrations is found, the thickness of the silica shell is better tuned by adapting the amount of silica precursor.

Optical Properties

We use in this work 'flash' CdSe/CdS QDs with a welldefined and sharp emission peak centered at 630 nm (Figure 2a). The encapsulation of these CdSe/CdS QDs in silica nanoparticles did not induce any modification in neither the shape nor the position of their emission spectrum, even after more than 10 months of storage in water (Figure 2a). This observation is a first indication of the long term stability of these nanoparticles in water. Indeed, partial oxidation of the QDs would have resulted in a shift of the emission spectrum toward shorter wavelengths.

Stability of the Photoluminescence Quantum Yield.

The absolute PLQY of the CdSe/CdS@SiO2 nanoparticles was followed carefully in time by using an integrating sphere (see the Experimental Section for a complete description of the method and calculation details) and the values are reported in Figure 2b. As a benchmark, more 'conventional' CdSe/CdS/ZnS QDs prepared by the successive ionic layer adsorption and reaction (SILAR) method were also encapsulated in silica through the same microemulsion process (See the Supporting Information, for the synthesis and characterization details). The PLQY of these 'SILAR' CdSe/CdS/ZnS@SiO2 nanoparticles was also followed as a function of time of storage in water and the results are compared with those obtained for 'flash' CdSe/CdS@SiO2 nanoparticles in Figure 2b. The values at days 0 to 2 correspond to measurements of the PLQY during the microemulsion syntheses. On day 3, the nanoparticles were purified by centrifuga-tion and transferred into water. Thus, the values at day 3 are obtained after the nanoparticles have spent no more than a few hours in water. The Figure 2b shows that both QDs have an initial PLQY of about 40%. In the case of nanoparticles based on SILAR grown CdSe/CdS/ZnS QDs, the Figure 2b shows that their PLQY already decreases significantly during the microemulsion process. Upon transfer into water, the PLQY quickly dropped below 10% and keeps on decreasing with further storage in water. On the other hand, Figure 2b clearly evidences that the nanoparticles based on 'flash' CdSe/CdS QDs keep their PLQY unchanged with time. Even after 10 months of storage in water they remained perfectly bright, with a PLQY of about 40%. In addition, the preservation of the PLQY with 'flash' QDs appeared to be independent from the silica shell thickness. Indeed, QD@SiO2 nanoparticles with either very thin or very thick silica shells as those illustrated in Figure 1c and 1d also retained their PLQY in water. In the case of SILAR grown QDs, the decrease in PLQY can be explained by the action of the ROS that are present in water. It has been reported that defects in core-shell QDs, such as grain boundaries or incomplete shell layers, act as an inlet for oxygen to reach the core of the QDs. [START_REF] Van Sark | Bleaching, and Blinking of Single CdSe/ZnS Quantum Dots[END_REF] The oxidation of QDs typically results in a deterioration of their optical properties with notably a blue shift of the absorption and emission spectra. In a humid or aqueous environment, this effect is even more critical since the presence of water molecules broadens the lowest unoccupied molecular orbital (LUMO) states of oxygen and therefore facilitate the formation of ROS, [START_REF] Pechstedt | Photoluminescence of Colloidal CdSe/ZnS Quantum Dots: The Critical Effect of Water Molecules[END_REF] which will be even more aggressive toward the QDs and can critically decrease their PLQY. In this regard, 'flash' CdSe/CdS QDs which are synthesized at a relatively high temperature compared to SILAR QDs (330 °C and about 225 °C respectively), may have a much better crystallinity and thus a lower defect density resulting in a more efficient protection of the core from ROS. Chen et al. recently reported a method to prepare CdSe/CdS QDs with compact CdS shells through a slow continuous growth, which also retained a high PLQY upon transfer into water. [START_REF] Chen | Compact High-Quality CdSe-CdS Core-Shell Nanocrystals with Narrow Emission Linewidths and Suppressed Blinking[END_REF] The relatively thick CdS shells might also play an important role in preserving the PLQY of 'flash' CdSe/CdS QDs. So-called 'giant' CdSe/CdS QDs were al-ready reported to fully retain their PLQY in water, [START_REF] Chen | Giant" Multishell CdSe Nanocrystal Quantum Dots with Suppressed Blinking[END_REF] although the present 'flash' CdSe/CdS only have about 8 layers of CdS compared to the 15 or more CdS layers in 'giant' CdSe/CdS QDs. Thus, both the high crystallinity of the 'flash' CdSe/CdS QDs which are synthesized at high temperature and their relatively thick CdS shells could explain why these QDs are much more resistant than SILAR ones after silica encapsulation and transfer into water.

Silica

Passivation of the QDs. When 'flash' CdSe/CdS QDs were transferred into water using a S 2-ligand exchange procedure, i.e. without any silica encapsulation (see the Supporting Information for details on the procedure), their PLQY immediately dropped to 14%. This critical drop of the PLQY is most likely due to a poor passivation of the surface of the QDs by the sulfur ligands. Thus, the fully preserved PLQY of the QD@SiO2 nanoparticles indicates that the silica is intimately interacting with the QD surface, resulting in a good passivation and a high PLQY. It is worth noting that the thickness of the silica shell in itself does not play any role in preserving the optical properties. Indeed, CdSe/CdS@SiO2 nanoparticles with very thin silica shells, such as those illustrated in Figure 1c, also retained a PLQY of about 40% after transfer into water.

Improved Photostability.

To further assess the photostability of these 'flash' CdSe/CdS@SiO2 nanoparticles, a 3 ml solution of these nanoparticles in water was continuously irradiated with UV light simply by leaving the solution directly on top of a standard 4W laboratory UV lamp (model Benda NU-4 KL, λ = 366 nm). The solution was prepared so as to have an absorption of ca. 50% at 365 nm across an optical path length of 1 cm. Figure 2c shows the evolution of both the PLQY and the absorption of this solution as a function of the irradiation time. The reported absorption values are those determined directly during the PLQY measurements using an integrating sphere (see Supporting Information for calculation details). One should note that for every measurement the entire volume of the same solution was systematically analyzed and that its vicinity with the UV lamp typically increased the temperature of the solution to ca. 40 °C. After one week of continuous irradiation in these conditions, the PLQY of the QD@SiO2 nanoparticles solution decreased by about 40% and the absorption by about 15% of their initial values. Longer irradiation periods up to two weeks did not result in any further decrease of the PLQY or absorption values. The partial dissolution of the QDs, as indicated by the decrease in the absorption value, induced a shift of the emission spectrum of only 3 nm toward shorter wavelengths (see the Supporting Information, Figure S4). The dissolution of QDs upon UV irradiation is due to their photooxidation, which is a strongly solvent dependent process. [START_REF] Manner | Role of Solvent-Oxygen Ion Pairs in Photooxidation of CdSe Nanocrystal Quantum Dots[END_REF] For instance, the same experiment performed on these QDs dispersed in toluene without any silica coating resulted in the dissolution of 80% of the QDs in only a few days, with a concomitantly gradual decrease of their PLQY down to complete extinction (See the Supporting Information, Figure S5), whereas the QDs remained relatively unaffected when they were dispersed in heptane. In this regard, water constitutes a very aggressive solvent as ROS are easily formed due to interactions between water and oxygen molecules. As a matter of fact, when S 2- stabilized 'flash' CdSe/CdS QDs were transferred into water using a ligand exchange procedure, they fully dissolved under UV irradiation in a matter of a few tens of minutes (see Supporting Information, Figure S6). Thus, it appears that the silica encapsulation does not only provide the QDs with enhanced physicochemical stability, as demonstrated by the fully preserved PLQY after transfer into water, but also with enhanced photostability. Improved photostability was already reported for silica coated CdSe/CdS dot-in rods, which were prepared in a similar way as the 'flash' CdSe/CdS dot-in-dots involved in this study. [START_REF] Pietra | Synthesis of Highly Luminescent Silica-Coated CdSe/CdS Nanorods[END_REF] However, even if the silica coating appears to be essential for the photochemical stability of the QD@SiO2 nanoparticles, it is not a sufficient condition. Indeed, in the case of the CdSe/CdS/ZnS@SiO2 nanoparticles based on SILAR grown QDs and in spite of the silica shell, the QDs could still dissolve, leaving empty hollow silica nanoparticles behind (see the Supporting Information, Figure S2). Thus, even if the silica shell is of importance, it has to be combined with the good crystallinity of the 'flash' CdSe/CdS QDs, which are synthesized at relatively high temperature, to result in CdSe/CdS@SiO2 nanoparticles with high physicochemical stability and photostability.

Application to Cell Labeling

As these 'flash' CdSe/CdS@SiO2 nanoparticles constitute promising nanoprobes for bioimaging, several parameters relevant to cell labeling were evaluated in detail, including acute cytotoxicity, qualitative and quantitative uptake of the nanoparticles and their effects on cell morphology. Finally, the functionality of these nanoparticles at non-toxic concentrations for long-term cell visualization by fluorescence microscopy was investigated to have a first, straightforward indication of the suitability of these nanoparticles for cell tracking studies. These results are compared in details with our previous report on the toxicity and cell labeling studies of bare S 2-stabilized 'flash' CdSe/CdS QDs. [START_REF] Soenen | The Effect of Intracellular Degradation on Cytotoxicity and Cell Labeling Efficacy of Inorganic Ligand-Stabilized Colloidal CdSe/CdS Quantum Dots[END_REF]

Toxicity Study. Acute cytotoxic effects of the

CdSe/CdS@SiO2 nanoparticles on HUVEC cells were evaluated and compared to the toxicity of empty SiO2 nanoparticles of similar size without any inner QD core (See the Supporting Information, Figure S7 for the synthesis and characterization details of these empty SiO2 nanoparticles). To this end, we used previously optimized methods that assess nanoparticle toxicity under highly reproducible conditions and thus facilitate a comparison of data obtained for other materials (See the Supporting Information for details on the methodology). [START_REF] Soenen | Cellular Toxicity of Inorganic Nanoparticles: Common Aspects and Guidelines for Improved Nanotoxicity Evaluation[END_REF] Figure 3 shows a clear effect of the QD@SiO2 on the HUVEC viability only at the highest dose of 100 nM, whereas the emp-ty SiO2 nanoparticles did not elicit any toxic effects over the entire concentration range (2 to 100 nM). Thus, the toxic effect observed in the first case can be solely attributed to the QDs. Previous studies have reported cytotoxic effects of Cd 2+containing core-shell QDs to be noticeable from concentrations of 20 nM on. [START_REF] Kirchner | Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles[END_REF][START_REF] Soenen | The Cytotoxic Effects of Polymer-Coated Quantum Dots and Restrictions for Live Cell Applications[END_REF] We recently reported that S 2-stabilized 'flash' CdSe/CdS QDs were also found to induce cytotoxicity at levels as low as 2 nM. [START_REF] Soenen | The Effect of Intracellular Degradation on Cytotoxicity and Cell Labeling Efficacy of Inorganic Ligand-Stabilized Colloidal CdSe/CdS Quantum Dots[END_REF] The present results clearly indicate that the silica coating greatly reduces the toxicity of the QDs since the first significant effect only appears at a dose that is 50 times higher than for bare 'flash' QDs. This is consistent with other studies where embedding different types of hydrophobic nanomaterials in a silica shell has also been found to enhance their colloidal stability and to reduce their toxicity. [START_REF] Zhang | Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements[END_REF] Given the rather porous nature of the silica shell, this finding is however somewhat surprising as it would be expected that the bare QDs or silica-coated QDs would be exposed to their final microenvironment to a similar degree and therefore elicit toxicity to a similar extent. This finding is nevertheless in line with the results obtained above, where the CdSe/CdS@SiO2 nanoparticles in water remained very stable under continuous UV irradiation whereas the S 2-stabilized CdSe/CdS dissolved quickly in the same conditions. These preliminary results on the toxicity of 'flash' CdSe/CdS@SiO2 nanoparticles clearly indicate that the CdSe/CdS@SiO2 nanoparticles could be used for cell labeling at a concentration up to 50 nM. The stability of the CdSe/CdS@SiO2 nanoparticles was further demonstrated by performing two complementary control experiments, where the toxicity of free Cd 2+ ions on C17.2 cells was evaluated (see the Supporting information, Figure S8). The data reveal clear significant effects on cell viability at levels of 800 nM of free Cd 2+ . In order to evaluate whether these levels of Cd 2+ are representative for the Cd 2+ levels obtained from the CdSe/CdS@SiO2 nanoparticles, cells were also exposed to medium that was previously incubated with the nanoparticles for 24 h, after which the nanoparticles were removed by ultracentrifugation. Figure S9 (Supporting Information) reveals a clear concentration-dependent effect of Cd 2+ ions released from the nanoparticles on cell viability, but the extent of the effects compared to the full CdSe/CdS@SiO2 nanoparticles was much lower. The data reveal that for nanoparticle-derived Cd 2+ at 50 and 100 nM of nanoparticles, similar cell viability levels were obtained as for free Cd 2+ at 400 and 800 nM respectively, suggesting low levels of Cd 2+ release (approximately 8 ions per nanoparticle).

Cellular Uptake.

To evaluate the CdSe/CdS@SiO2 nanoparticles in terms of their potential for cell labeling, first the cellular uptake of the QDs was investigated. HUVEC cells were incubated with the CdSe/CdS@SiO2 nanoparticles and 5 µM of 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO). DiO is a lipophilic dye that can primarily stain endosomal structures when a sufficiently short incubation time is used. Figure 4a reveals a clear colocalization of the green-colored vesicles (endosomes) and red dots (CdSe/CdS@SiO2 nanoparticles) as assessed by confocal microscopy, indicating that the nanoparticles are internalized by the cell through means of endocytosis and that they reside in endosomal or lysosomal structures, which is common for most types of nanomaterials such as semiconductor QDs, [START_REF] Cho | Long-Term Exposure to CdTe Quantum Dots Causes Functional Impairments in Live Cells[END_REF] including the bare 'flash' CdSe/CdS QDs. [START_REF] Soenen | The Effect of Intracellular Degradation on Cytotoxicity and Cell Labeling Efficacy of Inorganic Ligand-Stabilized Colloidal CdSe/CdS Quantum Dots[END_REF] Next, the cellular uptake levels of the CdSe/CdS@SiO2 nanoparticles were quantified. As a first approximation, the number of nanoparticle-containing clusters (red dots) in an entire cell was counted for a total of 100 cells per condition (Figure 4b). However, when nanoparticles are endocytosed by the cell, they will eventually all localize in a limited number of endosomal structures, which each can contain from a few nanoparticles up to several hundreds or even thousands of them. Therefore, the number of nanoparticle-containing clusters per cell is not very informative from a toxicological point of view as it is impossible to link observed toxicity with actual QD numbers. To determine the actual number of nanoparticles that are internalized per cell, we labeled cells with the CdSe/CdS@SiO2 nanoparticles, after which 1×10 6 cells were collected and lysed in a low osmotic potassium buffer with alkaline pH (9.8). Upon cell lysis, the nanoparticles are released in the alkaline surroundings and their fluorescence intensity can then be measured spectrofluorometrically to determine their number. Figure 4c shows the average number of CdSe/CdS@SiO2 nanoparticles taken up by the HUVEC cells, revealing that even at the lowest dose, several tens of thousands of nanoparticles have been internalized. Thus, in addition to low toxicity effects, the CdSe/CdS@SiO2 also show high cellular uptake levels.

Effect on Cell Morphology.

As one of the prime applications of the CdSe/CdS@SiO2 nanoparticles would be the fluorescent visualization of labelled cells, one aspect that is very important is whether the internalization of the nanoparticles affects cell morphology and whether labelled cells still look identical to unlabelled cells. For this purpose, HUVEC cells were labelled with various concentrations of the CdSe/CdS@SiO2 nanoparticles after which the cells were stained for -tubulin and visualized by confocal microscopy. Based on these images, the cell spreading (i.e. the surface area covered by a single cell) can be determined as an indication of whether the internalized nanoparticles affect cell morphology. Additionally, cell spreading is an important indication of how "stressed" the cells are. Indeed, it has been shown that cellular geometry is an important indicator and mediator in determining cell life or death. [START_REF] Chen | Geometric Control of Cell Life and Death[END_REF] Figure 5 shows well-spread cells in either control conditions (Figure 5a) or when labelled with 10 nM (Figure 5b), 20 nM (Figure 5c) or 50 nM (Figure 5d) of CdSe/CdS@SiO2 nanoparticles. The internalized nanoparticles show a typical perinuclear localization in the most dense regions of tubulin. Based on these images, the nanoparticles do not appear to disturb the tubulin cytoskeleton network. This is in contrast with previous findings on various types of particles, including iron oxide particles, gold particles and QDs. [START_REF] Soenen | The Cytotoxic Effects of Polymer-Coated Quantum Dots and Restrictions for Live Cell Applications[END_REF][START_REF] Soenen | Cytotoxic Effects of Iron Oxide Nanoparticles and Implications for Safety in Cell Labelling[END_REF][START_REF] Soenen | Cytotoxic Effects of Gold Nanoparticles: A Multiparametric Study[END_REF] High intracellular, but non-toxic levels of these materials (ranging from 10 nM for QDs to 50 nM for gold particles), all showed to result in cytoskeletal rearrangements. Interestingly, in a previous study on dye doped silica nanoparticles, no cytoskeletal effects were previously observed either, [START_REF] Soenen | Fluorescent Non-Porous Silica Nanoparticles for Long-Term Cell Monitoring: Cytotoxicity and Particle Functionality[END_REF] suggesting that the silica coating of the CdSe/CdS@SiO2 nanoparticles is the dominant factor and overcomes cytotoxic effects that the core QDs would have on the cellular cytoskeleton. The reason behind the protective role of the silica coating is still unclear, Figure 6. Efficacy of the CdSe/CdS@SiO2 nanoparticles for longterm cell visualization. (a) Representative fluorescence images of HUVEC cells exposed to CdSe/CdS@SiO2 nanoparticles for 24 h at 50 nM. Images are taken after cells have undergone 0 (a1), 3 (a2), 7 (a3) or 9 (a4) cell divisions. The CdSe/CdSe@SiO2 nanoparticles appear as red and the cell nuclei are counterstained in blue by DAPI. (b) Percentage of HUVEC cells containing CdSe/CdS@SiO2 nanoparticles relative to the entire population of HUVEC cells. Cells were labeled for 24 h with CdSe/CdS@SiO2 nanoparticles at 10 nM (light grey), 20 nM (medium grey) or 50 nM (dark grey) after which they were kept in culture and representative samples were collected after cells had undergone 0, 1, 3, 5, 7 or 9 cell divisions. Data are expressed as mean ± SEM (n = 3). and more research is needed to elucidate the precise mechanism at play. When comparing the total cell areas of labelled cells with unlabelled control cells (Figure 5e-g), no distinct difference can be seen between the various conditions, suggesting that at non-cytotoxic conditions, the CdSe/CdS@SiO2 nanoparticles do not affect cell spreading. In comparison, bare 'flash' CdSe/CdS QDs were also found not to affect significantly cell spreading but in this case the study was limited to lower concentrations due to their higher toxicity. [START_REF] Soenen | The Effect of Intracellular Degradation on Cytotoxicity and Cell Labeling Efficacy of Inorganic Ligand-Stabilized Colloidal CdSe/CdS Quantum Dots[END_REF] The results reported here on high concentrations of CdSe/CdS@SiO2 nanoparticles clearly indicate that they can be efficiently used for fluorescent visualization of live cells.

Long Term Cell Visualization.

One key aspect in evaluating the potential of functional nanoparticles is how long they can be efficiently visualized in live (dividing) cells. To this end, HUVEC cells were labelled with various nontoxic concentrations of the CdSe/CdS@SiO2 nanoparticles, after which the cells were allowed to grow in culture and divide. At several points, small samples of the cells were taken and visualized by fluorescence microscopy to look for the presence of the nanoparticles (Figure 6a). Based on these images, the percentage of cells which contain at least one clearly distinguishable cluster of CdSe/CdS@SiO2 nanoparticles is then calculated. Figure 6b reveals that even for the lowest concentration of CdSe/CdS@SiO2 nanoparticles (10 nM), the cells can easily be visualized for up to 5 cell divisions with more than 50% of them being still positive for nanoparticles, before the majority of them loses their fluorescent labels due to dilution effects and possible degradation of the nanoparticles. [START_REF] Zhu | Stability of Quantum Dots in Live Cells[END_REF][START_REF] Summers | Statistical Analysis of Nanoparticle Dosing in a Dynamic Cellular System[END_REF] In addition, for the highest tested concentration (50 nM), more than 50% of the cells remain efficiently labelled with nanoparticles after 9 cell divisions, is a considerably long period of time for QD based nanoprobes. [START_REF] Soenen | The Cytotoxic Effects of Polymer-Coated Quantum Dots and Restrictions for Live Cell Applications[END_REF] This long duration signifies that: 1) the CdSe/CdS@SiO2 nanoparticles themselves are not very as high initial levels can be tolerated without any cytotoxic effects, 2) the emission properties of the nanoparticles appear to be relatively unaffected by the endosomal environment to which they are exposed. This is in clear contrast with bare 'flash' CdSe/CdS QDs which were found to quickly release Cd 2+ ions in the cells and could not be visualized for more than 2 cell divisions. [START_REF] Soenen | The Effect of Intracellular Degradation on Cytotoxicity and Cell Labeling Efficacy of Inorganic Ligand-Stabilized Colloidal CdSe/CdS Quantum Dots[END_REF]

Conclusion

In conclusion, we demonstrate that 'flash' CdSe/CdS QDs as synthesized using a seeded growth method at high temperature are very efficient light emitters for biolabeling applications. They can be encapsulated in silica nanoparticles through a water-in-oil microemulsion process, with a high control on the morphology of the nanoparticles and the thickness of the silica shell. This silica coating provides the nanocrystals with enhanced colloidal stability in polar solvents, while fully preserving their optical properties. Indeed, the PLQY of the nanoparticles remains high and unchanged even after several months of storage in water. They also showed greatly improved photostability in water under continuous UV irradiation compared to bare QDs. This enhanced stability of the optical properties, which were thoroughly investigated using an integrating sphere, is a key parameter in the development of QDs based luminescent nanoprobes as it determines their potential for bioimaging applications. In addition, the silica shell successfully reduced the cytotoxicity of the QDs, with first significant effects on HUVEC cell viability appearing only at concentrations 50 times higher than for bare QDs. The potential of these CdSe/CdS@SiO2 nanoparticles for cell labeling was evidenced by fluorescence confocal microscopy showing their rapid and high endosomal uptake by HUVEC cells. Furthermore, thanks to their reduced toxicity and to the high stability of their optical properties, these CdSe/CdS@SiO2 nanoparticles appeared to be particularly useful for long term cell visualization, with nanoparticles identifiable by confocal microscopy for up to 9 cell divisions. Taken together these data clearly demonstrate the extensive potential of these nanoparticles for biomedical applications. Possibly thanks to their high crystallinity, alloyed interfaces and relatively thick CdS shells, 'flash' QDs have a higher physicochemical stability and photostability than other QDs synthesized using more common strategies such as the SILAR method. Additionally, the presence of the silica shell provides an important level of protection against environmentally induced degradation of the QDs, making these 'flash' CdSe/CdS@SiO2 nanoparticles highly efficient luminescent markers with powerful optical properties combined with low toxicity levels. Their further development into even more complex architectures will lead to highly functional tools for advanced applications such as intracellular imaging and theranostics.

Figure 2 .

 2 Figure 2. (a) Emission spectra of the CdSe/CdS QDs in toluene and the CdSe/CdS@SiO2 nanoparticles after 10 months of storage in water (the emission spectra are normalized and slightly offset for clarity). (b) Evolution of the PLQY of the CdSe/CdS@SiO2 nanoparticles as a function of time and compared to CdSe/CdS/ZnS@SiO2 nanoparticles where the QDs were prepared by the SILAR method (the dashed line indicates the day the nanoparticles were transferred into water). (c) Evolution of the PLQY (solid symbols) and absorption at 365 nm (A%, open symbols) of a CdSe/CdS@SiO2 nanoparticles solution in water under continuous UV irradiation (4W UV lamp, λ = 366 nm). In (c) the values are normalized to their initial value (t = 0 hours).

Figure 3 .

 3 Figure 3. Relative viability of HUVECs cells exposed for 24 h to CdSe/CdS@SiO2 nanoparticles (a) or SiO2 nanoparticles (b) at various concentrations and for MTT and LDH assays (*: p < 0.05; **: p < 0.01).

Figure 4 .

 4 Figure 4. Cellular uptake of the CdSe/CdS@SiO2 nanoparticles. (a) HUVEC cells were exposed to CdSe/CdS@SiO2 nanoparticles for 30 min at 50 nM in the presence of the lipophilic dye DiO, which stains the endosomes. The fluorescence images show the DiO-stained cellular vesicles (green channel, a1), the emission of the CdSe/CdS@SiO2 nanoparticles (red channel, a2) and the merged image (a3). The degree of colocalization is given in the upper hand corner (a3). (b,c) Number of CdSe/CdS@SiO2-positive vesicles per HUVEC cell (b) and number of CdSe/CdS@SiO2 nanoparticles per HUVEC cell (c) upon exposure of HUVECs for 24 h to various non-toxic concentrations of the nanoparticles. Data are expressed as mean ± standard error to the mean (n = 3).

Figure 5 .

 5 Figure 5. Effect of the CdSe/CdS@SiO2 nanoparticles on the cell morphology. (a-d) Representative confocal images of HUVEC cells exposed to 0 nM (a), 10 nM (b), 20 nM (c) or 50 nM (d) of CdSe/CdS@SiO2 nanoparticles for 24 h, where the cells are stained for atubulin (left column; green) and show uptake of the CdSe/CdS@SiO2 nanoparticles (middle column, red). The images in the third column are merged images. (a-g) Histograms showing the cell area of control (dark bars) and CdSe/CdS@SiO2-treated (light bars) HUVEC cells exposed to the nanoparticles for 24 h at 10 nM (e), 20 nM (f) or 50 nM (g). The average cell area for control cells is indicated by § and for treated cells by *.

AUTHOR INFORMATION

Corresponding Author * E-mail: tangi.aubert@ugent.be (TA), zeger.hens@ugent.be (ZH).

Present Addresses

£ Department of Medicine, Biomedical NMR Unit, KU Leuven, Herestraat 49, 3000 Leuven, Belgium.

ACKNOWLEDGMENT

The authors acknowledge BelSPo (IAP 7.35, photonics@be), FWO-Vlaanderen (Project G.0760.12; KaN 1509012N), Ghent University (BOF12/GOA/015) and Hercules Foundation (project AUGE/09/024 "Advanced Luminescence Setup") for financial support.

ASSOCIATED CONTENT

Supporting Information. Details on measurement and calculation of the PLQY, synthesis and characterization of the CdSe/CdS/ZnS@SiO2 nanoparticles based on SILAR grown QDs, additional characterizations on 'flash' CdSe/CdS QDs and CdSe/CdS@SiO2 nanoparticles, synthesis and characterization of the empty SiO2 nanoparticles, methodology for toxicity and cell labeling studies, data of free Cd 2+ and nanoparticle-devoid media on cell viability. This material is available free of charge via the Internet at http://pubs.acs.org.