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High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

Manshian BB, Munck S, Agostinis P, Himmelreich U, Soenen SJ - Sci Rep (2015)

Bottom Line: A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine.Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels.Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

View Article: PubMed Central - PubMed

Affiliation: MoSAIC/Biomedical MRI Unit, Faculty of Medicine, KU Leuven, Herestraat 49, B3000 Leuven, Belgium.

ABSTRACT
A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

No MeSH data available.


Related in: MedlinePlus

Effects of exposure to varying concentrations of QDots in MSCs and MEFs.(a) representative confocal micrographs of MSC (left) and MEF (right) cells that were transduced with CellLight Lysosomes-GFP (green) and subsequently exposed to 10 nM QDots (red) for 24 h. yellow/orange dots represent colocalized QDots and lyosomes. Scale bars = 25 μm. The area in the white rectangle is shown as a magnified view below the image. (b) Heat maps of high-content imaging-based data for MSCs and MEFs exposed to varying concentrations of QDots and analyzed for; cell viability, cell membrane damage, mitochondrial ROS, size of the mitochondrial network, area of the cell, skewness of the cell, and level of autophagy. Data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data have been acquired for minimum 5000 cells/condition which were gathered from three independent experiments. (c,d) Representative InCell high-content images of control MSCs or MSCs exposed to the QDots at 10 or 20 nM for 24 h, after which the cells were stained for (c) β-actin (green) or (d) mitochondrial ROS (green). Cells were counterstained with Hoechst nuclear stain (blue). Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
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f1: Effects of exposure to varying concentrations of QDots in MSCs and MEFs.(a) representative confocal micrographs of MSC (left) and MEF (right) cells that were transduced with CellLight Lysosomes-GFP (green) and subsequently exposed to 10 nM QDots (red) for 24 h. yellow/orange dots represent colocalized QDots and lyosomes. Scale bars = 25 μm. The area in the white rectangle is shown as a magnified view below the image. (b) Heat maps of high-content imaging-based data for MSCs and MEFs exposed to varying concentrations of QDots and analyzed for; cell viability, cell membrane damage, mitochondrial ROS, size of the mitochondrial network, area of the cell, skewness of the cell, and level of autophagy. Data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data have been acquired for minimum 5000 cells/condition which were gathered from three independent experiments. (c,d) Representative InCell high-content images of control MSCs or MSCs exposed to the QDots at 10 or 20 nM for 24 h, after which the cells were stained for (c) β-actin (green) or (d) mitochondrial ROS (green). Cells were counterstained with Hoechst nuclear stain (blue). Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.

Mentions: The QDots used are commercially available CdSe/ZnS core/shell nanoparticles (Invitrogen, Belgium) with maximal emission at 655 nm, which were coated using a carboxylated amphiphilic polymer, generating negatively charged NPs (Supp Section S1). When exposing mouse mesenchymal stem cells (MSCs) and mouse embryonic fibroblasts (MEFs) to these QDots, a clear endosomal localization can be observed, as the QDots are efficiently endocytosed and do not merely adhere to the plasma membrane (Fig. 1a, Supp Fig. S2), which is in line with other reports on carboxylated QDots28. Next, MSCs and MEFs were exposed to a series of QDot concentrations, and the effect on various parameters was evaluated (Fig. 1b–d). The data reveal significant concentration-dependent effects of the QDots on cell viability (Supp Fig. S3), cell membrane damage (Supp Fig. S4), autophagy induction (Supp Fig. S5), cell morphology (Supp Fig. S6), cell skewness (Supp Fig. S7), size of the mitochondrial network (Supp Fig. S8) and mitochondrial ROS (Supp Fig. S9), at concentrations of 15 nM or more. These values are in line with previous studies using carboxylated QDots28, suggesting that QDots appear to affect cell homeostasis through various mechanisms.


High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

Manshian BB, Munck S, Agostinis P, Himmelreich U, Soenen SJ - Sci Rep (2015)

Effects of exposure to varying concentrations of QDots in MSCs and MEFs.(a) representative confocal micrographs of MSC (left) and MEF (right) cells that were transduced with CellLight Lysosomes-GFP (green) and subsequently exposed to 10 nM QDots (red) for 24 h. yellow/orange dots represent colocalized QDots and lyosomes. Scale bars = 25 μm. The area in the white rectangle is shown as a magnified view below the image. (b) Heat maps of high-content imaging-based data for MSCs and MEFs exposed to varying concentrations of QDots and analyzed for; cell viability, cell membrane damage, mitochondrial ROS, size of the mitochondrial network, area of the cell, skewness of the cell, and level of autophagy. Data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data have been acquired for minimum 5000 cells/condition which were gathered from three independent experiments. (c,d) Representative InCell high-content images of control MSCs or MSCs exposed to the QDots at 10 or 20 nM for 24 h, after which the cells were stained for (c) β-actin (green) or (d) mitochondrial ROS (green). Cells were counterstained with Hoechst nuclear stain (blue). Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
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Related In: Results  -  Collection

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f1: Effects of exposure to varying concentrations of QDots in MSCs and MEFs.(a) representative confocal micrographs of MSC (left) and MEF (right) cells that were transduced with CellLight Lysosomes-GFP (green) and subsequently exposed to 10 nM QDots (red) for 24 h. yellow/orange dots represent colocalized QDots and lyosomes. Scale bars = 25 μm. The area in the white rectangle is shown as a magnified view below the image. (b) Heat maps of high-content imaging-based data for MSCs and MEFs exposed to varying concentrations of QDots and analyzed for; cell viability, cell membrane damage, mitochondrial ROS, size of the mitochondrial network, area of the cell, skewness of the cell, and level of autophagy. Data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data have been acquired for minimum 5000 cells/condition which were gathered from three independent experiments. (c,d) Representative InCell high-content images of control MSCs or MSCs exposed to the QDots at 10 or 20 nM for 24 h, after which the cells were stained for (c) β-actin (green) or (d) mitochondrial ROS (green). Cells were counterstained with Hoechst nuclear stain (blue). Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
Mentions: The QDots used are commercially available CdSe/ZnS core/shell nanoparticles (Invitrogen, Belgium) with maximal emission at 655 nm, which were coated using a carboxylated amphiphilic polymer, generating negatively charged NPs (Supp Section S1). When exposing mouse mesenchymal stem cells (MSCs) and mouse embryonic fibroblasts (MEFs) to these QDots, a clear endosomal localization can be observed, as the QDots are efficiently endocytosed and do not merely adhere to the plasma membrane (Fig. 1a, Supp Fig. S2), which is in line with other reports on carboxylated QDots28. Next, MSCs and MEFs were exposed to a series of QDot concentrations, and the effect on various parameters was evaluated (Fig. 1b–d). The data reveal significant concentration-dependent effects of the QDots on cell viability (Supp Fig. S3), cell membrane damage (Supp Fig. S4), autophagy induction (Supp Fig. S5), cell morphology (Supp Fig. S6), cell skewness (Supp Fig. S7), size of the mitochondrial network (Supp Fig. S8) and mitochondrial ROS (Supp Fig. S9), at concentrations of 15 nM or more. These values are in line with previous studies using carboxylated QDots28, suggesting that QDots appear to affect cell homeostasis through various mechanisms.

Bottom Line: A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine.Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels.Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

View Article: PubMed Central - PubMed

Affiliation: MoSAIC/Biomedical MRI Unit, Faculty of Medicine, KU Leuven, Herestraat 49, B3000 Leuven, Belgium.

ABSTRACT
A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

No MeSH data available.


Related in: MedlinePlus