Quantum Dots for Human Mesenchymal Stem Cells Labeling. A Size

Lately certain cytotoxicity of quantum dots (QDs) and some deleterious effects of labeling procedure .... detectable in both experiments at 96 h after...
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NANO LETTERS

Quantum Dots for Human Mesenchymal Stem Cells Labeling. A Size-Dependent Autophagy Activation

2006 Vol. 6, No. 12 2826-2832

Oleksandr Seleverstov,*,† Olga Zabirnyk,‡ Matthias Zscharnack,† Larysa Bulavina,§ Marcin Nowicki,‡ Jan-Michael Heinrich,† Maksym Yezhelyev,| Frank Emmrich,§ Ruth O’Regan,|,⊥ and Augustinus Bader† Center for Biotechnology and Biomedicine (BBZ), Institute of Anatomy, and Institute of Immunology and Transfusion Medicine, UniVersity of Leipzig, D-04103 Leipzig, Germany, and Winship Cancer Institute, Emory UniVersity, Atlanta, Georgia 30322 Received August 22, 2006; Revised Manuscript Received October 23, 2006

ABSTRACT Lately certain cytotoxicity of quantum dots (QDs) and some deleterious effects of labeling procedure on stem cells differentiation abilities were shown. In the present study we compared cytotoxicity and intracellular processing of two different-sized protein-conjugated QDs after labeling of the human mesenchymal stem cells (hMSC). An asymmetrical intracellular uptake of red (605 nm) and green (525 nm) quantum dots was observed. We describe for the first time a size-dependent activation of autophagy, caused by nanoparticles.

Introduction. The search for a reliable method for stem cell tracking is of a key importance. Conventional labeling techniques have significant drawbacks and lead to controversial results.1,2 Recently developed quantum dots (QDs) hold unique optical properties with potential advantages as fluorescent probes for stem cell labeling.3,4 The bright and narrow emission spectrum of QDs allows simultaneous multicolor visualization of labeled cells what is beneficial in real-time multiplex tracking of several stem cell subpopulations. Recently two different protein-conjugated QDs (565 and 655 nm) were used to study progenitor cell fusion in long-term cocultures.5 QDs have different emission wavelength and vary in shape, in diameter, and in the chemical composition. Although QDs gained popularity as a fluorescent labeling agent both for in vitro and for in vivo studies, QD-cell interactions are still poorly understood and require additional investigation. It was shown that small uncoated nanoparticles were more toxic and had different distribution rules than large particles.6 Different strategies of QD surface modification, the so-called “coating procedure”, were introduced attempting their biological functionalization.7 Additionally, the coating procedure is considered to equalize * Corresponding author. Phone: +49 341-9731350. Fax: +49 341-97 31359. E-mail: [email protected]. † Center for Biotechnology and Biomedicine (BBZ), University of Leipzig. ‡ Institute of Anatomy, University of Leipzig. § Institute of Immunology and Transfusion Medicine, University of Leipzig. | Winship Cancer Institute, Emory University. ⊥ Contributed equally as senior author. 10.1021/nl0619711 CCC: $33.50 Published on Web 11/08/2006

© 2006 American Chemical Society

stability of small and big particles.8,9 Cytotoxicity of QDs is still under thorough investigation. Cytotoxic mechanisms and the role of QDs in the intracellular metabolism are not clear yet. A release of shell components and reactive oxygen species are believed to be a key mechanism of QD cytotoxicity,6,10 whereby apoptosis and necrosis should play a central role in cell death mechanisms. Recently, autophagy was shown to be the mechanism responsible for intracellular bulk uptake and important selfdefending mechanism under stress.11 Autophagy potentially can arise in all eukaryotic cells containing a lysosomal fraction.12,13 It is also considered to be an important cell death mechanism. Recently, mouse embryonic stem cells were used as a model for autophagic machinery study. Autophagy can be quickly activated in these cells, as a response to starvation, and is “a major route for lysosomal protein degradation”.14 The aim of this study was to explore the suitability of QDs for stem cell labeling using two different-sized nanoparticles. Having identical chemical components QD525 and QD605 differ in size by almost a factor of 2.15 We examined the difference in the distribution of these two types of QDs in hMSC. Additionally, we explored cytotoxicity of QDs for hMSC and their suitability for differentiated stem cells labeling. Here for the first time to our knowledge we demonstrate activation of autophagy in response to intracellular introduction of QDs. We consider the size-dependent uptake of nanoparticles in living cells is autophagy mediated. Additionally, we speculate that autophagy is a mechanism of small-sized QD clearance, which causes rapid escape of QD525 from the cell.

Materials and Methods. A detailed description of the experimental procedures and materials used can be found in the Supporting Information. We should note that the purification of hMSCs from the bone marrow aspirate and differentiation experiments were performed as described earlier.16 To create a long-term model for tissue engraftment, cocultures of QD-labeled hMSC with unlabeled synovial cells, chondrocytes, and dermal fibroblasts were performed. The cells for cocultures were chosen, based on their fast growth ability in comparison to hMSC and their trend to polylayer development. We considered it to be an appropriate model for normal tissue regeneration, because of primary higher percentage of stem cells and gradually their decrease in maturating tissue. Cytotoxic/membrane integrity was analyzed via lactate dehydrogenase (LDH) release and proliferatiVe actiVity via bromodeoxyuridine (BrdU) incorporation. As metabolic tests, alamar blue17 and MTT assay were used. For cell labeling we applied Qtracker cell labeling kits 605 and 525 (Quantum dots Corp.). The coating procedure was done in accordance to manufacture’s instructions. The labeling concentrations between 0 and 10 nM for each QD were applied. The complete procedure was carried out in a cell suspension (300 000 cells) at 37 °C in 200 µL of whole culture media on a shaker for 45 min. The colabeling procedure was done in two settings: (1) a separate coating of each QD type with further mixing in complete media; (2) the first uncoated QDs were mixed together followed by incubation with a coating solution, then the coated QD mixture was added to the complete medium for labeling. We speculated that simultaneous coating and/or intrusion of two different-sized QDs into the same cell might impact intracellular uptake. For all other experiments, the cells were transfected with one type of QD only. Transmission electron microscopy (TEM) was done as described.18 For this set of experiments, 0 and 5 nM of each QD type were used. Many recent studies, involved cytotoxic research on stem cells with nanoparticles or other labeling agents, were concentrated on detection of necrosis and apoptosis2,6,10 as signs for cell damage. The interest to autophagy is still low in toxicological research and it is probably due to TEM recognition as a gold standard for its verification.11 Newly established methods, based on LC3 and its subforms LC3-I and LC3-II detection, present an additional screening method.19,20 Results. The cells labeled with QD605 retained their fluorescent signal for the whole period of observation time (52 days) either in continuous culture or after two to four passages, depending on used labeling concentration. However, in all long-term experiments with QD525 rapid loss of fluorescence signal (days 2-7 depending on labeling concentration) was observed in culture. Notably, after 48-96 h postlabeling, 2-5% of QD525 labeled cells appeared as bright lighting globes, which were considered to be dead cells. All other cells had normal morphological features, observed by light microscopy. We noted some interesting features for both QD distributions. First, immediately after the labeling procedure, QDs Nano Lett., Vol. 6, No. 12, 2006

were distributed diffusely throughout cell cytoplasm that resulted in the equal diffuse fluorescence from the whole cellular cytoplasmic area, but in few hours higher fluorescence was observed mainly in the perinuclear region. After 24-48 h QDs were visualized as perinuclear granulations. No fluorescent signal from the nuclei was observed. After colabeling with both green and red nanoparticles, QD605 maintained a stable fluorescence, whereas the QD525 signal rapidly diminished, suggesting the disappearance of green QD in accordance with previous data demonstrating rapid disappearance of QD525 after monochromatic transfection (Figure 1). Interestingly, that fluorescent signal from the protein-coated QD mixture (red and green) was detected as a yellow signal in the merge filter (Figure 1a,b). In contrast, consecutive labeling of hMSC with separately conjugated QD525 and QD605 had clearly distinguishable red and green granules, and only few overlapped yellow areas (Figure 1d,e). Additionally, only a red fluorescent signal was detectable in both experiments at 96 h after the labeling procedure (Figure 1c,f). No significant differences were found using described cytotoxic (LDH release), metabolic (Alamar blue and MTT), and proliferative (BrdU incorporation) assays in both QD labeled cells (see Supplemental Information, Figures S1S4). To assess the stability of QD transfection during stem cells differentiation, we transfected hMSC with two types of QDs followed by the induction of either osteogenic or adipogenic differentiation. In both osteogenic and adipogenic experiments, the morphology of the labeled cells has changed synchronic to the controls. Although, we did not achieve well-detectable fluorescence signal in each cell after 3 weeks of culturing with QD605 in adipogenic (Figure 2a) and osteogenic medium (Figure 2e), they were still present in cells, but green fluorescence of QD525 disappeared from hMSC equally quickly to the QD525 labeled cells (control) in normal growth medium (Figure 2b; Figure 2f). To confirm successful differentiations van Kossa (Figure 2g,h) and oil red O (Figure 2c,d) stainings were performed at the end point of experiments. We further studied post-trasfectional cellular ultrastructure using transmission electron microscopy. The concentration of QD and time point for TEM observation were based on our data from fluorescent microscopy in cell culture: bright fluorescence and no notable differences in both QD types of distribution at 24 h; weak or no fluorescence from QD525labeled cells at 72 h. We found no significant differences in the distribution of both QD types after 24 h of postlabeling (Figure 3a,e). In each cell a significant amount of both types QD was noted, which confirms the equal efficacy of transfection for both small and big nanoparticles. Although, some QD granules were found adjacent to the nuclear membrane, no intranuclear presence of QD was detected at this time point. A major portion of the QDs was localized in the perinuclear area in the form of granular inclusions. QDs localized inside of the intracellular structures with a regular round shape and similar diameter, which are the specific characteristics of endosomes. Moreover, a lot of 2827

Figure 1. Colabeling experiments. (a-c) Simultant conjugation of both QDs with coating protein (2.5 nM of each QD type). From the first observation well-detectable mixed signal, resulting in yellow spectra by overlay in a merge filter: (a) 24 h, (b) 48 h, (c) 96 h. (d-f) Simultant labeling with QD525 and QD605, separately conjugated with the same peptide. Well-distinguishable red and green fluorescence at 24 h (d) and 48 h (e) with only few yellow spectra by overlay and (f) 96 h. (c, f) 96 h, complete loss of green fluorescence with present red one in both conjugation settings. Magnification 400× for all.

these structures were colocalized with microtubular bundles that also confirm their primary endosomal localization and transport (Figure 3a). Additionally, QD aggregates were often colocalized with the lysosomes and with mitochondria. Single autophagic vacuoles were recognized, based on specific morphological features such as the presence of their double membrane and cytoplasmic content. In 72 h cultures labeled with QD525, the majority of cells presented cytoplasm free from the nanoparticles, but a significant number of autophagosomes was found. Only a few cells had specific opaque structures in free cytosol, which had a regular round form and similar size. Some QD aggregates were also seen in nuclei. A lot of mitochondria appeared swollen, and unstructured as well as Golgi cisterns were enlarged (Figure 3c). Numerous autophagosomes, containing organelles and giant QD aggregates, were present 2828

(Figure 3b,c). Approximately 2% of the cells exhibited features of autophagic cell death (Figure 3d). The QD605 in the 24 and 72 h cultures build up the large accumulations, consisting of numerous smaller aggregates (Figure 3e,f). They were not surrounded with membrane structures or included in vacuoles. Additionally, QD605 were localized in free cytosol without preferable colocalization, except for a few lysosomes. In contrast to QD525, a minimal number of autophagic vacuoles and damaged mitochondria were observed. After the 24 h postlabeling period we found dotlike structures positive for LC3 in hMSC containing QD525, which could represent LC3-II positive autophagic vacuoles (Figure 4a). In contrast, the cells labeled with QD605 (Figure 4b) or with the coating solution only (Figure 4c) contained a weak specific diffuse fluorescence signal without granularNano Lett., Vol. 6, No. 12, 2006

Figure 2. Differentiation experiments. (a-d) Adipogenic differentiation. (a) QD605, 3 weeks in adipogenic medium. Overlay (white contrast and red fluorescence). Fluorescence is present as well as lipid droplets. Not each cell has a well-detectable signal. (b) QD525, the same experiment. Overlay (white contrast and green fluorescence). No fluorescence is observed. (c, d) Oil red O staining. (e-h) Osteogenic differentiation. (e) QD605, 3 weeks in osteogenic medium. Overlay (phase-contrast and red fluorescence microscopy). Typical morphology, well-detectable fluorescence, but not in each cell. (f) QD525, the same experiment. Overlay (phase-contrast and green fluorescence microscopy). No fluorescence is observed. (g, h) van Kossa staining. Magnification 400× for a and b, 200× for e and f, 100× for c, d, g, and h.

ity suggesting the presence of a cytoplasmic fraction of LC3 (LC3-I) only. Additionally, nuclei stained with DAPI showed no apoptotic signs. There was a well-preserved QD fluorescence observed after the staining procedure. Cocultures were observed over 3 weeks, and welldetectable fluorescence from single cells labeled with QD605 Nano Lett., Vol. 6, No. 12, 2006

Figure 3. TEM. (a) QD525, 24 h after labeling, many nanoparticles are included in endosome-like structures, they are transported along microtubules (black arrow), many of them are co-localized with lysosomes (white arrow). Magnification 30000×. (b) QD525, 72 h after labeling, almost all nanoparticles are included in big vacuoles with double-spaced membranes (black arrow), enlarged endoplasmatic reticulum (white arrow), and destructed mitochondria (arrowhead). Magnification 7000×. (c) QD525, 72 h after labeling, excessive activation of autophagy, numerous vacuoles with doublespaced membranes and cytoplasmatic content (black arrow), the cytoplasm nearly free from QD, many lysosomes fuse with autosomes (white arrow), swollen and destructed mitochondria (arrowhead), and enlarged endoplasmatic reticulum suggest excessive damage. Magnification 12000×. (d) QD525, 72 h after labeling, autophagic cell death. Almost all nanoparticles are included in big vacuoles (black arrow), totally destructed cytoplasm with big vacuoles with and without content, absence of nucleus fragmentation and lyses. Magnification 3500×. (e) QD605, 24 h after labeling, mostly diffuse cytoplasmic QD scattering with no notable colocalisation with any organelles, except of few lysosomes. Building of small and big QD aggregates. No notable organelle damage. Magnification 7000×. (f) QD605, 72 h after labeling, no detectable morphological changes. Nanoparticles are developing big aggregates, consisting of many smaller aggregates. Few lysosomes are colocalized with nanoparticles. None of them are included in autophagosomes. Magnification 12000×.

was detected (Figure 5). The same experiments with QD525 showed rapid disappearance of fluorescence signal (data not shown). Discussion. In the present study we showed that the labeling of hMSC with protein-conjugated QD is a well2829

Figure 4. LC3 immunostaining. (a) 24 h after labeling with QD525 (green). Well-detectable dotlike structures (red) with preferable perinuclear localization. (b) 24 h after labeling with QD605 (red). No visible green fluorescence (anti-LC3). (c) the same experiment, exposition with coating peptide only. No visible red (anti-LC3) fluorescence. Magnification 400× for all.

tolerable procedure and brings a stable long-term fluorescence signal with big-sized particles (QD605). We propose that application of small-sized QD (QD525) for long-term stem cell tracking may be limited due to the rapid nanoparticles escape. This should be taken into consideration while attempting the multicolor tracking of living cells using different-sized QDs. Different cell division number and subsequent unequal dilution of labeling agent can explain the presence of both fluorescent and nonfluorescent cells in culture after differentiation induction. Also asymmetric division of stem cells21 and nonequivalent distribution of endosomes between daughter cells7 could be a reason. This fact should be also considered when proceeding with data quantification. The observation that coated-in-mixture QDs give overlapped yellow signal in contrast to the mixture of separately conjugated QDs, which show separated green and red granules, indicates that they build aggregates already during the conjugation procedure. Rapid disappearance of green particles from hMSC in both experiments regardless of coating protocol suggests possible involvement of QDs in 2830

some kind of intracellular processing mechanism, separating nanoparticles. Afterward, the shape and the diameter of the QDs play a crucial role in their fate. Both QD525 and QD605 form a mixture within endosomes after simultaneous transfection. Different diameters of aggregates, built by small (green) and big (red) particles, and formation of complexes with cellular proteins can be responsible for a higher toxicity of green QDs.6 We propose that other processes might also develop in the cell and play a critical role in selective clearance of cytoplasm from small particles. Frequently observed colocalization of introduced QD with lysosomes was also noted by others.7 This event occurred relatively early, before remarkable autophagy activation and organelle damage. Various coating strategies result in QD internalization by endocytosis;7 however, the delivery mechanisms are not well understood.22 Primary endo-lysosomal localization and transport23-26 of QDs also speak to their mainly perinuclear accumulation. This phenomenon was observed quickly after transfection and coincides with previously reported data.8,24,27 Obviously, lysosomes play a crucial role in postlabeling processing of a protein-coated QD surface. Since coating is known to stabilize and equalize properties of different-sized particles,8,9 we suppose that decoating/ surface modification might take a place inside the cell. Uncoated small particles were shown to have a higher permeability through cellular membranes6 and, apparently, decoated small-sized QDs can escape lysosomes. The process of green nanoparticle selection from the coated mixture within the endo-lysosomal compartment (in our colabeling experiments) and further selective clearance from the cell is considered to be indirect evidence of coating loss. There are no available analytic methods to evaluate surface coating stability within living cells up to now. Moreover the released nanoparticles might undergo second protein adsorption onto their surface in culture media.28 We propose that the application of different-sized nanoparticle combinations can be helpful for coating stability estimation and membrane permeability experiments; however, more studies are required. Since the “dimmed fluorescence“ was shown,26 we applied transmission electron microscopy to verify whether the loss of green fluorescence (QD525) was due to the declination of ability to give a signal or was a consequence of the particles’ disappearance from cell. Electron microscopy data demonstrated the strong evidence for nanoparticles related activation of autophagy in QD525-labeled cells, which obviously played an important role in their postlabeling processing and further hMSC self-cleaning from labeling agent. As demonstrated in our colabeling experiments, QD525 disappeared rapidly even in the presence of QD605 in the same cell. Autophagy is believed to be a nonselective process;13 however, our study suggests that this process might be size-dependent. In addition, it is still unknown why QD525 escape cytoplasm selectively, since endocytosis is believed to be a nonspecific process with the same mechanism for both QD types. However, some size-dependent rules in this process activity and efficiency were recently shown for various nanoparticles.25,28 Since some QD525 were found Nano Lett., Vol. 6, No. 12, 2006

Figure 5. Cocultures. (a, b) 3 days and 3 week cultures of 605QD labeled hMSC with dermal fibroblasts (c, d) the same with chondrocytes, and (e, f) with synovial cells. In each experiment well-detectable fluorescence was found throughout the whole observation period. No diffuse scattering of signal was noted. Magnification 100× for a, c, and e, 400× for b, d, and f.

in the cell nuclei, we suppose that the nuclear entrance with subsequent nuclear component interaction might lead to autophagy activation.29 Mitochondria are known to be triggering as well as targeted organelles in autophagy activation.11-13 We found increased number of morphologically changed mitochondria in the hMSC with remarkable autophagic signs after labeling with QD525. However, in the present study we are not able to address the question of whether mitochondria damage is a primary event, responsible for the autophagy activation or its consequences. Nano Lett., Vol. 6, No. 12, 2006

Having not found any significant metabolic and proliferative function impairments in labeled cells, we conclude that hMSC, carrying QD525, are able to replace damaged organelles and compensate for their functions well in parallel with severe autophagy activation. It is known that autophagy plays a bivalent role, representing a self-defending mechanism or mechanism of nonapoptotic programmed cell death.11,18 Our TEM findings of typical autophagic signs in dead hMSC, labeled with QD525, confirm that autophagy not only is a defensive mechanism but also, at the some 2831

advanced step, represents a mechanism of programmed cell death. The fact that at this step all remaining QD525 were already included inside autophagic vacuoles is strong evidence for this process to be initiated by QD themselves. It is important to notice results from another group showing a caspase-independent cell death caused by QD.30 A recently published study demonstrated activation of several genes as a response to QD labeling.31 Notably, the majority of the upregulated genes were responsible for intracellular vesicles formation and cell membrane associated and intracellular vesicular proteins involved in cellular response to stress. Nowadays little is known about a whole gene ensemble, responsible for autophagic cell death, and autophagy activation11,13 as well as origin of the autophagic membrane is still debated. The possible relation of these genes and their products needs to be further investigated in the terms of autophagy. It would be interesting to know if gene changes have a clear dose-dependent profile;31 however, other authors, similar to our results, did not find such dose dependency in proliferative activity or in necrotic-apoptotic damage. This fact strongly suggests the activation of the specific adaptive mechanism in labeled cells, which could have dose-dependent regulation. These data, together with other studies,32,33 where authors showed decrease in expression of specific differentiation markers but nonsignificant impact on other functions of hMSC, suggest that nanoparticle introduction leads to cellular stress. Autophagy activation might be a subsequent response. The practical aspect of these data is the fact that autophagy and, in particular, autophagic cell death, can be induced by small-sized nanoparticles in low differentiated cells. The present possibility to introduce nanoparticles into cells of interest in vivo34 opens an opportunity for targeted autophagy activation as a treatment method, for example in tumor cells, resistant for a standard therapy35,36 as well as in another situations, like neurodegenerative diseases, where autophagy activation could be an advantage.37 In conclusion, we first show that the size-dependent uptake of QD is autophagy mediated. Further investigations are necessary to determine the role of autophagy as a response to the different nanosized objects. Disclosure. The authors indicate no potential conflicts of interest. Acknowledgment. We highly appreciate technical skills of J. Craatz. We are thankful T. Rumpf and K. Lorenz for kindly providing cells. Supporting Information Available: Cell-culturing conditions, cell isolation and immunocytology methods, cytotoxicity, metabolic and cell proliferation assay descriptions. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Pearson, H. Nature 2006, 439, 519. (2) Burns, T. C.; Ortiz-Gonzalez, X. R.; Gutierrez-Perez, M.; Keene, C. D.; Sharda, R.; Demorest, Z. L.; Jiang, J.; Nelson-Holte, M.; Soriano, M.; Nakagawa, Y.; Luquin, M. R.; Garcia-Verdugo, J. M.; Prosper, F.; Low, W. C.; Verfaillie, C. M. Stem Cells 2006, 24, 1121-1127. 2832

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NL0619711 Nano Lett., Vol. 6, No. 12, 2006