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Standard cellular testing conditions generate an exaggerated nanoparticle cytotoxicity profile. Bella B. Manshian, Uwe Himmelreich, and Stefaan J. Soenen Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00340 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Chemical Research in Toxicology

Standard cellular testing conditions generate an exaggerated nanoparticle cytotoxicity profile. Bella B. Manshian, Uwe Himmelreich, Stefaan J. Soenen* MoSAIC/Biomedical MRI Unit, Faculty of Medicine, KU Leuven, Herestraat 49, B3000 Leuven, Belgium. KEYWORDS quantum dots, nanotoxicology, bio-nano interactions, toxicity mechanisms

* Corresponding author: Address: O&N I Herestraat 49 - box 505, 3000 Leuven, Belgium; Email: [email protected]; Tel: +32 16 330034

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ABSTRACT Cellular internalization of nanoparticles (NPs) is key to many biomedical applications and serves as a model to investigate the potential toxicity of NPs on entire organisms. Large discrepancies between in vitro and in vivo nanotoxicity data however exist, suggesting that cellular systems may not be optimal for predictive in vivo toxicology. Here, we use validated multiparametric high-content imaging protocols to evaluate the impact of common cell culture conditions on NP cytotoxicity studies. The data show that high NP to cell ratios, typical for cellular studies, stress the cells by high endocytosis levels that overstimulate mitochondria, resulting in oxidative stressmediated mitochondrial damage, which induces autophagy. Using proliferation-restricted models, we show that lowering endocytosis levels overcomes most toxicity while resulting in higher final cellular NP numbers. The data suggest that many common NP cytotoxicity mechanisms may partially be an artifact caused by overstimulated endocytosis.


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INTRODUCTION Biomedical applications of NPs such as bio-imaging and drug delivery require a close interaction of the NPs with their biological environment. The level of success often relies on an efficient cellular internalization of NPs either by in vitro cultured cells or by targeted cells in vivo. For most applications, a high number of cellular NPs is desirable. Cellular NP uptake is however limited, with uptake efficiencies generally being only a few percent of the total number of NPs administered to the cells.1 Various strategies have been employed to better understand and modulate cellular uptake,2–6 but to achieve sufficient levels in target cells, excessive NP numbers are commonly used, despite a clear negative correlation in NP concentration and uptake efficiency.7 This high ratio of NP numbers per cell used for in vitro cell labeling studies is in contrast with the conditions found in live animals or humans, where upon administration NPs will immediately be diluted (in the blood), after which the NPs will distribute in different organs throughout the body (see Supp Section S1). This difference has likely contributed to the discrepancy observed between in vitro and in vivo NP toxicity,8–10 where the latter is commonly lower. For a profound understanding of bio-nano interactions, the use of in silico tools and in vitro data are essential. To date, mechanistic studies have often generated disparate data,10 questioning the validity of current in vitro studies for predictive in vivo analysis. Here, a validated multiparametric highcontent imaging approach11,12 was used to evaluate to what extent standard cell labeling conditions may influence common bio-nano interaction studies.

EXPERIMENTAL SECTION


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Cell culture Mouse embryonic fibroblasts (MEFs) and transgenic MEFs (MEF Atg5 KO) were grown in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM),supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-Glutamine and 1% penicillin/streptomycin (Gibco, Invitrogen, Belgium). The MEF cells were passaged upon reaching 80% confluence and reseeded at a ratio of 1:5. Mouse mesenchymal stem cells were provided by Prof. Catherine Verfaillie (KULeuven) and maintained in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 10% horse serum, 1 mM sodium pyruvate and 2 mM LGlutamine (Gibco, Invitrogen, Belgium). Cells were passaged when reaching nearly 80% confluence and reseeded at a density of 100,000 cells/flask in 75 cm² tissue culture flasks (Nunc, Belgium). Nanoparticles Carboxylated quantum dots (Quantum Dot Corporation, USA) with maximal emission of 655 nm and DsRed-containing 30 nm diameter NH2-functionalized SiO2 nanoparticles (Micromod Partikeltechnologie GmbH, Germany) were used in the present study. Cell-nanoparticle interaction studies For high-content imaging studies, all cell types were seeded at 7500 cells/well in 24 well plates (Nunc, Belgium). Cells were allowed to attach overnight in a humidified atmosphere at 37 °C and 5% CO2, after which the cells were incubated with the carboxylated QDots in full growth medium at the concentrations and times indicated in the respective sections. For evaluation of


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oxidative stress-mediated effects, cells were seeded similarly as described above, after which they were pre-incubated to 5 mM N-acetylcystein (NAC - a free radical scavenger, Sigma Aldrich, Bornem, Belgium) for 2 h, after which the media were removed and cells were exposed to the QDots in the presence of 5 mM NAC. For evaluation of autophagy-mediated effects, either Atg5 KO MEF cells or MSCs incubated with 1 mM 3-MA (Sigma Aldrich, Bornem, Belgium) were used. Each experiment was performed in three independent repeats anddata were analyzed using full data sets of the different repeats. A detailed methodology can be found in the Supplementary Information that accompanies this manuscript. Statistical analysis All data are expressed as the mean + standard error to the mean (SEM). For all experiments, any statistical significance between a single condition and untreated control cells were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test using GraphPad 6 software.

RESULTS AND DISCUSSION Quantum dot toxicity on cultured cells For this study, we used commercially available QDots (Invitrogen, Belgium) with maximal emission at 655 nm that have been frequently studied.12 The QDots were coated by a carboxylated amphiphilic polymer, generating negatively charged NPs (Supp Section S2). Next, murine mesenchymal stem cells (MSCs) and murine embryonic fibroblasts (MEFs) were exposed to a series of QDot concentrations, and the effect on various parameters was evaluated (Figure 1a). The data reveal significant concentration-dependent effects of 24 h QDot exposure


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on cell viability, cell membrane damage, mitochondrial ROS, mitochondrial health, cell morphology, cell skewness and autophagy, at concentrations of 14nM or more. No significant differences were obtained in the cellular responses between either proliferating or nonproliferating cells (Figure 1b, Supp Figs. S1-S4). These values are in line with previous reports from our and other groups using similar carboxylated QDots.13 Based on these data, the main mechanisms underlying cellular reactions to the QDots include oxidative stress linked with mitochondrial damage and induction of autophagy, which are common processes through which many NPs have been reported to affect cultured cells.14,15 Quantum dot uptake kinetics For the following experiments, two conditions were chosen, being 6 and 16 nM, (indicated as CL24, CH24, respectively) which represent non-toxic and significantly toxic levels, respectively. Firstly, the uptake kinetics of the NPs were evaluated (Figure 1c,d). The total fluorescence intensity level of both populations increases rapidly, but levels off after 6 hours exposure (Figure 1c). CH24 cells still slowly increase in fluorescence intensity over time. When looking at the individual cellular uptake levels, the results are quite different (Figure 1d). For both CL24 and CH24 cells, the number of cellular QDots decreases over time, likely because the rate of cellular NP uptake is surpassed by the rate of cell division. These results are in line with earlier kinetic studies that revealed that NP uptake levels change with time, where after a few hours the cellular NP level reaches a certain steady-state threshold, after which cellular NP levels decreased.16 To evaluate to what extent the QDots are internalized via active (= ATP-dependent) mechanisms, the cells were exposed to the QDots at 16°C, conditions under which metabolic processes in the cell have been found to be halted and ATP-dependent endocytosis mechanisms are inhibited.17 Supp Fig S5 shows a significant decrease in cellular uptake levels of the QDots, indicating that


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the QDots used display minimal passive uptake but are rather internalized via active endocytosis mechanisms, which is in line with literature.18 Quantum dot toxicity as function of uptake kinetics To investigate the effect of intracellular NP numbers versus NP internalization rates on cellular toxicity, proliferation-restricted cells were used. Both MSCs and MEFs were treated with mitomycin C (MMC) to reversibly inhibit cell division (Supp Fig. S6).19 Treatment of the cells with MMC did not affect any of the cellular parameters assessed in our studies (Supp Fig S7). The cellular internalization of the QDots by proliferation-restricted cells was slightly, but not significantly lower than the internalization rates for proliferation-potent cells, as demonstrated by comparing cellular fluorescence levels during the initial time points for CL24 and CL72 conditions (Figure 1d). Proliferation-restricted cells exposed to 6 nM QDots for 72 hrs (CL72) resulted in higher QDot levels than proliferation-potent CH24 cells (Figure 1c,d). The data show a clear gradual increase in cellular QDot levels in proliferation-restricted cells, which after approximately 48 hrs reaches similar levels of cellular fluorescence as proliferation-potent CH24 cells. The fluorescence intensity-based measurements were further supported by determination of total cellular QDot levels, as assessed by measuring total Cd2+ concentrations (Figure 1e). Confocal microscopy analysis (Figure 1f, Supp Fig. S8) reveals a clear endosomal localization of the NPs in all conditions tested, indicative of an active, ATP-consuming endocytosis process, which is the case for nearly all types of NPs.20 In terms of toxicity, CL72 cells displayed limited toxicity, significantly less than CH24 cells (Figure 2, Supp Fig S9-S11). For CH72 cells (72 h exposure at 16 nM), the toxicity caused by the QDots was too high and involved several secondary effects (Supp Fig S12) rendering it impossible to undertake any mechanistic study and this condition therefore was omitted from this study.


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Next, the kinetics of cellular toxicity were evaluated for CH24 cells, which displayed a clear sequential procedure (Figure 3, Supp Fig S13,S14). Firstly, mitochondrial health was affected (2 h), after which autophagy was induced (6 h) which correlated with cell death. The sequential nature suggests a cause-effect scenario, which was further supported using n-acetylcystein (NAC), an inhibitor of oxidative stress, which significantly restored mitochondrial health and reduced both the levels of autophagy and cell death (Supp Fig. S15c,d). Autophagy-inhibited cells (Atg5 KO or treated with 3 methyladenine (3-MA)), did not have any effect on mitochondrial health, yet significantly, but not completely, restored cell viability (Supp Fig. S15e,f). Effects of rapid quantum dot internalization of cellular metabolism To evaluate the impact of NP internalization on cellular metabolism, mitochondrial respiration was then studied immediately before, 3 and 6 hrs post cellular QDot exposure, in line with the kinetics of cellular toxicity. For both proliferation-potent and -restricted cells, no effects are observed for CL24 cells (Figure 4, Supp Fig. S16-S18). For CH24 cells, the sequential effects observed earlier are also observed here, where after 3 hrs, basal respiration, ATP production and proton leak are increased, while the spare respiratory capacity has gone down (Figure 4c). After 6 hrs, the basal respiration has returned to control levels, while the ATP production has dropped below control values and the spare respiratory capacity has further decreased. The latter effects are indicative of clear mitochondrial damage, possibly caused by the higher energy demands caused by the high rates of NP internalization. Interestingly, these observations were highly consistent between proliferation-potent and -restricted MSCs and MEFs, after normalizing the data for the overall reduced metabolic activity in the proliferation-restricted cells.


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Effects of rapid quantum dot internalization on cellular gene expression We next studied the effects of NP internalization on gene expression, where a total of 86 genes involved in cellular stress responses were evaluated in proliferation-restricted cells (Figure 5, Supp Tables S1, S2). The data reveal a clear difference in cellular responses to the NPs, where CH24 cells had the strongest response, where the genes affected were mainly associated with hypoxia, autophagy and oxidative stress. CL72 cells gave less changes, but also resulted in some oxidative stress, inflammation and apoptosis (Figure 5). Together, the data reveal a clear distinction in cellular responses based on cellular NP levels or cellular NP uptake kinetics. The big changes seen in CH24 cells likely start by the induction of low levels of oxidative stress by the QDots, which can affect mitochondrial metabolism.21 Mitochondria are vital in providing cellular energy and are also the main sources of reactive oxygen species (ROS).22 The increased energy demand due to NP internalization coupled with the induction of ROS by the QDots will result in an overall high level of oxidative stress. As all mitochondrial processes are highly interdependent,21 the oxidative stress will decrease mitochondrial respiration, resulting in damaged mitochondria and conditions mimicking hypoxia. The damaged mitochondria and hypoxia-linked genes can then in turn induce autophagy,23 which explains the sequential nature of our observations. Of the affected hypoxiarelated genes, Bnip3l has been described to induce direct autophagic cell death,24 suggesting that cell death is for a large part attributed to mitochondrial stress, and therefore only indirectly susceptible to the presence of the actual NPs. High levels of cellular NPs taken up slowly generate only minor levels of oxidative stress, and far less cell death was observed, mainly involving apoptosis. For Cd2+-containing NPs such as the QDots used in the present study, the long-term exposure of QDots has been reported to result in Cd2+ release due to acid etching,


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which in turn can lead to apoptosis.25 This was supported by our findings, where the cellular level of free Cd2+ ions was significantly higher for CL72 cells compared to CH24 cells (Supp Fig. S19). The mechanism underlying cell death therefore switches from apoptosis for high levels of internalized QDots to autophagic cell death for high levels of QDot endocytosis. Cellular NP toxicity has been linked with different paradigms, being mainly the induction of oxidative stress15 and, more recently, the induction of autophagy.16 Although both mechanisms have been associated with a broad variety of chemically diverse NPs in vitro, this has been less observed in vivo.9,10 The extent of both mechanisms can be influenced by altering NP properties such as chemical composition of the core, NP size or surface chemistry. These parameters, however, also influence cellular NP uptake levels. Considering the discrepancy between in vitro and in vivo data, and the occurrence of both mechanisms with such a broad library of different NP types, it is hard to link these effects to particular NP properties. As the only common factor in these studies is the high level of cellular NP uptake, our data suggests that these common mechanisms observed in cell cultures may, in part, represent an artifact elicited by the high ratio of NPs per cell and the overstimulation of cellular endocytosis. Our data are further supported by recent findings that indicated clear differences in NP toxicity profiles in acute versus chronic exposure studies,26,27 or when NPs were pneumatically driven into cells, bypassing any endocytosis.28 Where these studies explain the differences due to cellular adaptation or the importance of the precise intracellular microenvironment to which the NPs are exposed, we suggest that toxicity is in part caused by the high levels of NP uptake. Cells seem to have an intrinsic limitation to the stress they can handle caused by stimulated endocytosis, as suggested by the reduction in NP uptake kinetics after 6 hrs, regardless of the NP levels reached at that point. However, in combination with the actual toxicity exerted by the internalized NPs, the


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combination of the two mechanisms may exceed cellular defense capabilities, hereby resulting in cell death. Evaluation of the generic nature of the observed concept with silica nanoparticles To evaluate whether the observed effects for the QDots also apply to other types of NPs, amine-functionalized 30 nm diameter silica NPs were used. The particles were found to have a hydrodynamic diameter of 47 nm and a ζ-potential of 24 + 6 mV. SiO2 NPs themselves are selected as a more inert type of NP, where toxicity is not caused by acid etching of the NP surface and associated release of toxic metal ions, as is the case for Cd2+ ions and QDots, but toxicity is mainly attributed to oxidative stress.29,30 The high positive surface charge of the SiO2 NPs will result in a close interaction of the NPs with the cell surface and facilitate cellular NP uptake. Upon exposing the MSCs and MEFs to the SiO2 NPs for a broad concentration range, clear dose-dependent cytotoxicity was observed, which was similar in trend as for the QDots (Figure 6a). Proliferation-restricted cells displayed similar toxicity levels (Figure 6b) in 24 h exposure conditions. Similar as for the QDots, toxicity seemed to be caused mainly by oxidative stress and autophagy, which is in line with literature reports.29,30 Here, low subcytotoxic concentrations were defined at 40 µg/ml (CL24, CL72), while higher toxic concentrations were defined at 80 µg/ml (CH24). In terms of cellular NP uptake, prolonged exposure in proliferation-restricted cells (CL72) resulted in higher cellular NP levels than short exposure at higher NP levels (CH24) (Figure 6c, d), while uptake kinetics were lower for CL72 exposed cells. When comparing the toxicity levels of CH24 and CL72-exposed cells, similar results were obtained as for the QDots used, where despite having higher final cellular SiO2 NPs levels, no significant toxicity was observed for CL72


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exposed cells in contrast to CH24 exposed ones (Figure 6e, f). Together, these data suggest that while SiO2 NP induce ROS under the current exposure conditions, cytotoxicity is caused by both NP-elicited ROS and endocytosis-elicited ROS. Mechanistic understanding of NP toxicity The present work suggests that mechanistic NP cytotoxicity studies may suffer from interference by the high levels of NPs used for cell labeling, which in itself induces cellular stress. The concept found for QDot and SiO2 NP toxicity suggests a significant contribution of cellular endocytosis levels and associated generation of ROS and mitochondrial stress in the cellular toxicity mechanisms. At low levels of cellular NP uptake, ATP demand may slightly increase, which will not affect mitochondrial homeostasis. Any toxicity derived under these conditions therefore stem more from the NPs themselves, being the generation of ROS or release of metal ions upon acid etching of the NP surface (Figure 7).15,31 At higher NP uptake levels, ATP demand will increase, which in itself will inflict stress on the mitochondria. The higher levels of NPs will also generate higher levels of NP-associated ROS, which combined, will result in elevated levels of mitochondrial damage compared to when toxicity would only be caused by the NPs (Figure 7). The higher levels of mitochondrial stress may then elicit other toxicity mechanisms, such as autophagy, aimed at recycling the damaged mitochondria.24 Our data revealed that long-term cellular exposure to lower NP concentrations in proliferation-impeded cells enables one to study the true toxicity of the NPs and their mechanisms, without the contribution of cellular stress due to high endocytosis levels. It is important to bear in mind that any cytotoxicity caused by NPs is a fine interplay between intrinsic toxicity caused by the NPs and toxicity associated with cellular stress due to higher


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energy demands. The final outcome of any mechanistic cytotoxicity study will therefore be influenced not only by cellular NP levels, but also by the rate of cellular NP uptake. Rapid cellular NP internalization for longer time periods may induce cellular stress, while shorter time periods may be easily handled by the cells without any significant damage, as suggested by our kinetic data and previous literature reports.32 Next to uptake kinetics, the intrinsic toxicity of the NP to be tested will also play a big role. Rapidly degrading NPs such as ZnO will mainly elicit toxicity through the release of Zn2+ ions,33 and the contribution of cellular endocytosis will be minimal. The precise contribution of NP-induced toxicity and endocytosis-related toxicity is a fragile balance and hard to predict. More studies should be performed on different types of NPs to enable in silico analysis to investigate which NP-associated parameters (NP size, chemical nature, surface coating, NP shape etc) play a role and what the exact contribution of endocytosisrelated toxicity will be for these NPs in their observed toxicity profile. The kinetic profile of NP uptake and the associated toxicity caused by inducing cellular stress corresponds to the profile typically observed for cellular ROS induction by NPs, where typically, maximal ROS levels are observed between 4 and 24 h, after which cellular ROS levels decrease.34 These values therefore correspond nicely to the observed changes we notice in cellular stress while they correspond less to the level of cell-associated NPs, supporting our theory that cytotoxicity may in part be caused by cellular stress induced by NP endocytosis. Current in silico analysis studies have already significantly improved our current understanding of how NPs behave in a biological environment with regards to their toxicity and targeting efficacy.35,36 Specifically for QDots, the meta-analysis of QDot toxicity revealed the NP-associated parameters that most contribute to their toxicity profile.36 The data found in our study will not have any grave effect on the outcome of such studies, as NP-associated parameters


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governing toxicity are also linked with their cellular uptake rates. Our data may impact mainly mechanistic studies for particular types of NPs where any observed effect, as observed here for elevated autophagy levels may be influenced by the stress elicited by NP endocytosis. Additionally, the definition of safe NP concentrations used may be reconsidered, where it is important to link NP toxicity to both exposure concentrations as well as intracellular NP levels,12 as through the combination of both, one may be able to predict the influence of NP uptake kinetics. CONCLUSIONS The present work demonstrates the importance of in-depth studies of bio-nano interactions and the need for optimized realistic NP concentrations to mimic physiologically relevant exposure conditions. Although these studies focus on a single NP type, we present a generic model to perform mechanistic bio-nano interaction studies and suggest the need to use low NP concentrations for longer time periods to evaluate true NP-linked biological outcomes. The approaches outlined here may be of general value to bridge the gap between in vitro and in vivo bio-nano interaction studies, and possibly shed some light on the seemingly disparate nature of predictive toxicological studies. Funding Sources This work was supported by the FWO-Vlaanderen (S.J.S, KAN 1514716N to B.B.M.), the KU Leuven program financing IMIR (PF 2010/017), the Flemish agency for Innovation through Science and Technology (IWT-SBO MIRIAD, IWT-SBO NanoComit).


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Supporting Information. Theoretical calculations of NP/cell ratios in vitro and in vivo, NP characteristics, supporting data and full experimental methodology. This material is available free of charge via the Internet at http://pubs.acs.org. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Abbreviations CH24: high dose (16 nM), 24 h exposure; CL24: low dose (6 nM), 24 h exposure; CL72: low dose (6 nM), 72 h exposure; MEFs: murine embryonic fibroblasts; MMC: mitomycin C; MSCs: mesenchymal stem cells; NPs: nanoparticles; QDots: quantum dots; ROS: reactive oxygen species

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Figure 1. QDots display clear endosomal internalization, where low QDot concentrations can result in high cellular QDot levels upon prolonged exposure in proliferation-restricted cells. A,B) Heat maps of high-content imaging-based data for A) MSCs and MEF cells and B) proliferationrestricted MSCs and MEF cells exposed to varying concentrations of QDots for 24 h and analyzed for cell viability, cell membrane damage, mitochondrial ROS, mitochondrial health, area of the cell, skewness of the cell and the level of autophagy. Data are shown as relative values after z-normalization compared to untreated control cells where the fold-change (= 0 for control cells) is indicated by the respective color-code. Data have been acquired for a minimum of 5000 cells/condition which were gathered from three independent experiments. C) Histogram


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displaying the fold-increase in fluorescence levels of MSCs (compared to fluorescence levels at 1 hr exposure) exposed to the QDots at 6 nM (CL24), 16 nM (CH24) or at 6 nM in proliferationrestricted MSCs (CL72) for the time points indicated. D) Histogram displaying the fluorescence intensity normalized per 10,000 cells for MSCs exposed to the QDots at 6 nM (CL24), 16 nM (CH24) or at 6 nM in proliferation-restricted MSCs (CL72) for the time points indicated. E) Histogram representing the number of QDots per cell as derived from measuring total cellular Cd2+ levels by ICP-MS in MEF or MSC cells exposed at 6 nM for 24 h (CL24), 16 nM for 24 h (CH24) or 6 nM for 72 h in proliferation-restricted cells (CL72). Data are presented as mean + SD (n = 3). F) Representative confocal micrographs of MSC (top row) and MEF (bottom row) cells that were transduced with CellLight Lysosomes-GFP (green) and subsequently exposed to the QDots at 6 nM (L) or 16 nM (H) for 24 or 72 h. Yellow/orange dots represent colocalized QDots and lysosomes. Scale bars: 30 µm.


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Figure 2. Cellular responses reveal distinct cytotoxicity profiles that correlate more to cellular endocytosis levels, than cellular QDot levels. A,B) Histograms representing the relative cellular effect in A) MSC or B) MEF cells exposed to the QDots under the conditions indicated (CL24, CH24 (proliferation-potent), CL72 (proliferation-restricted). The following parameters were analyzed: cell viability, membrane damage, mitochondrial ROS, mitochondrial health, size of the cell, cell skewness, level of autophagy. The data are expressed relative to untreated control cells, where “100%” means no effect compared to untreated control cells (proliferation-potent (CL24, CH24) or proliferation-restricted (CL72)). Data are presented as mean + SD for a minimum of 5000 cells per condition (n = 3). Statistical significance is indicated when appropriate (*p < 0.05; **p < 0.01; ***p < 0.001). C) Representative InCell high-content images of control MEFs or MEFs


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exposed to the QDots at the indicated conditions (CL24, CH24, CL72) after which the cells were stained with dead cell green (top row, green), MitoTracker Red CMXROS (middle row, green), or actistain (bottom row, green). Scale bars = 100 µm, the area in the white rectangle is depicted in a magnified view at the right of the original images.


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Figure 3. Kinetic analysis reveals that high levels of QDot internalization causes mitochondrial stress, which in turn induces autophagy that finally results in cell death. A) Histograms representing the relative cellular effect in MSC (left) MEF (right) cells exposed to 16 nM QDots for the time points indicated. The following parameters were analyzed: cell viability, mitochondrial stress and level of autophagy. The data are expressed as fold-level changes from untreated control cells, where “1-fold” means no effect compared to untreated control cells. Data are presented as mean + SD for a minimum of 5000 cells per condition (n = 3). Statistical significance is indicated when appropriate (*p < 0.05; **p < 0.01; ***p < 0.001). B) Representative InCell high-content images of control MEFs or MEFs exposed to 16 nM QDots


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for 2, 4, or 6 h after which the cells were stained with dead cell green (top row, green), MitoHealth stain (top row, yellow), or LC3 (bottom row, green) and counterstained for their nucleus with Hoechst (blue). Scale bars = 100 µm.


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Figure 4. Kinetic analysis of mitochondrial metabolism reveals clear sequential steps in cellular responses to high levels of QDot internalization. A,B). Representative graphs of OCR outputs from the XF24 analyzer of MSCs exposed to A) 6 nM or B) 16 nM QDots obtained immediately prior and following 3 and 6 hrs of QDot exposure. C) Analysis of the OCR outputs gives the basal metabolism, the spare respiratory capacity, ATP production levels and the level of proton leak in MSCs exposed to 6 (grey) and 16 nM (black) QDots at the same time points. Data are expressed as mean + SD (n = 3). Statistical significance at 3 or 6 hrs compared to their respective control values (0 hr) is indicated when appropriate (*: p < 0.05).


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Figure 5. Clear differences in expression patterns of stress-related genes are observed between high acute and low prolonged exposure conditions. Gene expression levels of proliferationrestricted MSC or MEF cells treated with the QDots under the indicated conditions (CL24, CH24, CL72). Data are expressed as the net fold change in gene expression for QDot-exposed proliferation-restricted cells compared to untreated proliferation-restricted cells only exposed to PBS. From the entire panel of 84 genes tested, only those genes are shown where significant changes (>2-fold level difference from PBS-treated control cells) are observed.


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Figure 6. SiO2 NPs display similar behavior as the QDots studied. A,B) Heat maps of highcontent imaging-based data for A) MSCs and MEF cells and B) proliferation-restricted MSCs and MEF cells exposed to varying concentrations of SiO2 NPs for 24 h and analyzed for cell viability, cell membrane damage, mitochondrial ROS, mitochondrial health, area of the cell, skewness of the cell and the level of autophagy. Data are shown as relative values after znormalization compared to untreated control cells where the fold-change (= 0 for control cells) is indicated by the respective color-code. Data have been acquired for a minimum of 5000


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cells/condition which were gathered from three independent experiments. C) Histogram displaying the fold-increase in fluorescence levels of MSCs (compared to fluorescence levels at 1 hr exposure) exposed to the SiO2 NPs at 40 µg/ml (CL24), 80 µg/ml (CH24) or at 40 µg/ml in proliferation-restricted MSCs (CL72) for the time points indicated. D) Representative InCell highcontent images of MSCs or MEFs exposed to the QDots at the indicated conditions (control (= no NPs) CL24, CH24, CL72) after which the cells were stained with Caspase event dead cell green (top row, green), Scale bars = 50 µm. E, F) Histograms representing the relative cellular effect in E) MSC or F) MEF cells exposed to the SiO2 NPs under the conditions indicated (CL24, CH24 (proliferation-potent), CL72 (proliferation-restricted). The data are expressed relative to untreated control cells, where “100%” means no effect compared to untreated control cells (proliferationpotent (CL24, CH24) or proliferation-restricted (CL72)). Data are presented as mean + SD for a minimum of 5000 cells per condition (n = 3). Statistical significance is indicated when appropriate (*p < 0.05; **p < 0.01; ***p < 0.001).


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Figure 7. Schematic illustration of the observed cytotoxicity mechanisms. In conditions of low NP uptake kinetics, the ATP demand is low and cytotoxicity is mainly associated with direct NPassociated effects (ROS, leaching of toxic metal ions…). In conditions of high NP uptake kinetics, the ATP demand is increased, posing (oxidative) stress on the mitochondria. Coupled with the direct NP-associated effects, this results in increased levels of mitochondrial damage, which if it exceeds cellular defense capabilities, will result in mitochondrial damage which can then elicit autophagy or other toxicity mechanisms, which were not triggered by the NP themselves, even at higher intracellular NP concentrations.


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TOC figure.


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