Silicon-based quantum dots induce inflammation in human lung cells and disrupt extracellular matrix homeostasis.

Silicon-based quantum dots induce inflammation in human lung cells and disrupt extracellular matrix homeostasis Miruna-Silvia Stan1, Cornelia Sima2, Ludmila Otilia Cinteza3 and Anca Dinischiotu1 1 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, Romania 2 National Institute for Laser, Plasma and Radiation Physics, Bucharest-Magurele, Romania 3 Faculty of Chemistry, University of Bucharest, Romania

Keywords autophagy; cytokines; inflammation; matrix metalloproteinases; quantum dots Correspondence A. Dinischiotu, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independentei, Bucharest 050095, Romania Fax: +40 21 318 1575 Tel: +40 21 318 1575 E-mail: [email protected] (Received 24 March 2015, revised 11 May 2015, accepted 27 May 2015) doi:10.1111/febs.13330

Quantum dots (QDs) are nanocrystalline semiconductor materials that have been tested for biological applications such as cancer therapy, cellular imaging and drug delivery, despite the serious lack of information of their effects on mammalian cells. The present study aimed to evaluate the potential of Si/SiO2 QDs to induce an inflammatory response in MRC-5 human lung fibroblasts. Cells were exposed to different concentrations of Si/SiO2 QDs (25–200 lgmL1) for 24, 48, 72 and 96 h. The results obtained showed that uptake of QDs was dependent on biocorona formation and the stability of nanoparticles in various biological media (minimum essential medium without or with 10% fetal bovine serum). The cell membrane damage indicated by the increase in lactate dehydrogenase release after exposure to QDs was dose- and time-dependent. The level of lysosomes increased proportionally with the concentration of QDs, whereas an accumulation of autophagosomes was also observed. Cellular morphology was affected, as shown by the disruption of actin filaments. The enhanced release of nitric oxide and the increase in interleukin-6 and interleukin-8 protein expression suggested that nanoparticles triggered an inflammatory response in MRC-5 cells. QDs decreased the protein expression and enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and also MMP-1 caseinase activity, whereas the protein levels of MMP-1 and tissue inhibitor of metalloproteinase-1 increased. The present study reveals for the first time that silicon-based QDs are able to generate inflammation in lung cells and cause an imbalance in extracellular matrix turnover through a differential regulation of MMPs and tissue inhibitor of metalloproteinase-1 protein expression.

Introduction The use of quantum dots (QDs) in bioimaging and nanomedicine applications has been investigated intensively in recent years. The unique properties of these semiconductor nanomaterials, such as a high intensity self-fluorescence, broad excitation and narrow emission

spectra, compared to traditional organic dyes, provide advantages and have stimulated much interest in nanotechnology development [1]. Although many synthesis procedures and conjugation methods have been used to increase the stability of QDs in biological environ-

Abbreviations ECM, extracellular matrix; FBS, fetal bovine serum; IL, interleukin; LDH, lactate dehydrogenase; MEM, minimum essential medium; MMP, matrix metalloproteinase; QD, quantum dot; ROS, reactive oxygen species; TIMP, tissue inhibitor of metalloproteinase.

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ments, their compatibility with live cells has many unresolved issues and research in this field of nanotoxicology is still ongoing. As a result of their specific characteristics (e.g. small size, surface charge) and composition, nanoparticles can cross biologic barriers, including cell membranes, as well as endosomal–lysosomal and nuclear membranes, and interact with various biomolecules. The uptake and the subsequent outcomes are modulated, in part, by the nanoparticle biocorona formed in a biological compartment [2,3]. Typically, the nanoparticles are incorporated in cells via endocytosis [4,5], secondary pathways of internalization also being available depending on cell type and nanoparticle-intrinsec properties (passive diffusion, macropinocytosis or phagocytosis) [6–8]. Most of the endocytic pathways arrive at lysosomes that are able to sequestrate and degrade even the most biopersistent nanoparticles because of their acidic pH and hydrolytic enzymes [9]. In connection with the endo-lysosomal route of internalization, autophagy can be initiated by different types of nanoparticles as a common cellular response [10–12]. However, the mechanisms involved in the nanoparticle-mediated autophagy are still unclear, oxidative stress being a possible connection between cell death induced by nanoparticles and autophagy. As a result of the generation of reactive oxygen species (ROS) after exposure to nanoparticles within the lung, they can cause significant changes in membrane permeability followed by oxidative stress and cell death [13,14]. Besides ROS, reactive nitrogen species production, which includes nitric oxide (NO) and highly reactive peroxynitrite, may lead to a nitrosative stress associated with pulmonary inflammation [15,16]. Inflammation is a defense mechanism activated by organisms in response to the adverse effects induced by exogenous particles [17]. This physiological response involves the activation of some specific processes: the release of inflammatory mediators, such as chemokines and cytokines [interleukin (IL)-1b, IL-6, IL-8 and tumor necrosis factor-a], the attraction of immune cells and the development of tissue damage [18]. Nanoparticle exposure could induce other processes, such as tissue degradation and remodeling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs), which are regulated by specific inhibitors, such as tissue inhibitor of metalloproteinases (TIMP) [19]. Concerning the toxicology of silicon-based QDs after pulmonary exposure, little is known about the common physiological reactions triggered by QD-induced lung FEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS

Silicon quantum dots induce pulmonary inflammation

toxicity. In the present study, we investigated the potential of Si/SiO2 QDs to induce an inflammatory response in human lung fibroblasts (MRC-5 cell line), aiming to expand the limited information available. The uptake of QDs was monitored in response to protein adsorption on their surface and correlated with the level of cell membrane damage and the accumulation of lysosomes. As far as we are aware, the effects on ECM turnover and cytoskeleton organization, together with their implication in Si/SiO2 QDs-induced inflammation, are assessed and reported for the first time.

Results Si/SiO2 QDs properties As a result of their intrinsic properties (Table 1) induced by the method of synthesis [20], Si/SiO2 QDs dispersed in MilliQ water (Millipore, Billerica, MA, USA) exhibited aggregates with an average value of 196.7 nm (Table 2). This is in agreement with the morphology of QD aggregates in water, which was revealed by transmission electron microscopy images in a previous study [21]. Compared to water, the incubation in minimum essential medium (MEM) increased the hydrodynamic size of Si/SiO2 QDs, which was even higher in the presence of 10% fetal bovine serum (FBS). The Si/SiO2 QDs displayed moderately polydisperse size distributions in aqueous solutions and biological media, as shown by the polydispersity index (Table 2). The zeta potential value confirmed the electrostatic stabilization of QDs in water (Table 2). By contrast, the absolute value of zeta potential of QDs dispersed in MEM containing FBS was significantly lower compared to the other two suspensions. Table 1. Physical and chemical characteristics of Si/SiO2 QDs [21]. XRD, X-ray diffraction; SAED, selected area electron diffraction; EDX, energy-dispersive X-ray spectroscopy; HRTEM, highresolution transmission electron microscopy; TEM, transmission electron microscopy. Characteristics

Description

Method

Form of synthesis Composition

Powder

Pulsed laser ablation

Crystalline silicon Pure SiO2 > 99% Core-shell structure with a crystalline silicon core and an amorphous SiO2 shell (1–2 nm) Spherical 6–8 nm

XRD and SAED EDX

Purity Structure

Morphology Particle size

EDX HRTEM, TEM

TEM TEM

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Table 2. Hydrodynamic size, polydispersity index and zeta potential of Si/SiO2 QDs in various media. Data are expressed as the mean SD (n = 3).

Medium MilliQ water MEM MEM + 10% FBS

Hydrodynamic size (nm)

Polydispersity index

Zeta potential (mV)

196.7 46.40 348.2 47.32 622.8 42.84

0.309 0.02 0.322 0.01 0.344 0.01

29.72 2.08 26.30 0.79 13.46 0.70

Effects of the protein corona on Si/SiO2 QDs cellular uptake To examine how proteins attached to the surface of QDs influenced their behaviour and internalization into lung fibroblasts, the nanoparticles were suspended in culture media containing 10% FBS. First, the incubation of QDs in the absence of cells revealed differences in particle dispersion/agglomeration (Fig. 1A). The Si/SiO2 QDs remained fully dispersed in the absence of serum after 96 h, even at high concentrations of QDs (200 lgmL1), whereas the presence of FBS induced an agglomeration of particles confirmed by the pellet that settled at the bottom of the tube for which the size increased dependent on the amount of suspended QDs. Second, the protein affinity of Si/SiO2 QDs was confirmed by SDS/PAGE (Fig. 1B), which revealed an increase in adsorbed proteins in a concentration-dependent manner. The main adsorbed protein was BSA, which was the most abundant protein in FBS. It was identified based on its molecular mass of 66.5 kDa. The protein attachment at the surface of nanoparticles might explain the increase in hydrodynamic size and the decrease of dispersion and zeta potential absolute value in the presence of serum proteins (Table 2). Furthermore, fluorescence microscopy was used to highlight the uptake of QDs and their subcellular localization in lung fibroblasts (Fig. 1C). The Si/SiO2 QDs were internalized into MRC-5 cells and accumulated in the cytoplasm, especially in the lysosomes, without passing through the nuclear membrane into the nucleus after 72 h of incubation. Moreover, the effect of serum on the degree of uptake of QDs was investigated based on their most important property: self-fluorescence. The percentage of QDs inside cells during the 96 h of incubation was lower in the absence of FBS (Fig. 1D) compared to the culture media containing 10% FBS (Fig. 1E) for all concentrations of QDs tested. In addition, percentage uptake was inversely proportional to the initial amount of QDs added to the culture media. Practically, the amount of nanoparticles inside cells, represented by 2916

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multiplication of the percentage uptake of QDs with the initial concentration of nanoparticles added to the media, increased in a concentration-dependent manner until the saturation restricted continuous uptake (Fig. 1F,G). In the case of 200 lgmL1 QDs in the presence of FBS, the plateau phase was reached at 48 h of incubation, indicating the saturation point. Between these two types of media, and especially for 25 lgmL1 QDs, an exponential curve profile can be noted during the exposure time in the presence of serum in contrast to the flat curve without FBS. The spectra shown in Fig. 1H confirmed the stability over time of QDs suspensions. In addition, the presence of FBS in the culture medium did not induce any modification in the QDs fluorescence spectrum. Effects of Si/SiO2 QDs on cell membrane integrity Being released in the extracellular space upon membrane disruption, the amount of lactate dehydrogenase (LDH) was used as an indicator of cell membrane integrity. The cytotoxicity assay revealed a concentration-dependent increase in LDH release from cytosol in the cell culture medium compared to control cells (Fig. 2). After 24 and 48 h of exposure, the level of LDH release was almost similar for all concentrations tested. Furthermore, the cytotoxicity of high concentrations QDs (100, 150 and 200 lgmL1) was suggested by an increase of 65%, 77% and 109%, respectively, after 72 h compared to control. Effects of Si/SiO2 QDs on accumulation of lysosomes and autophagy induction The fluorescence microscopy images (Fig. 3A) revealed an accumulation of lysosomes especially at high concentrations (50 and 200 lgmL1) of QDs. By quantifying the fluorescence intensity of LysoTracker Green (Invitrogen, Carlsbad, CA, USA), a time- and concentration-dependent increase in lysosome level was observed (Fig. 3B). It was noted that the fluorescence intensity was elevated by 6.4- and eight-fold at 48 and 72 h, respectively, for the 200 lgmL1 concentration, which correlated with the colocalization of QDs with lysosomes shown in Fig. 1C and also with cellular uptake (Fig. 1E). Furthermore, the autophagic pathway is in tight connection with the lysosomal degradation pathway, as observed from the microscopy images in which autophagosomes were labeled with monodansylcadaverine. Figure 4A,B shows an induction of autophagy after exposure to QDs that was first observed at 24 h of incubation with 200 lgmL1 QDs (an increase FEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS

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Fig. 1. Cellular uptake of Si/SiO2 QDs in response to biocorona formation. (A) QDs were dispersed in serum-free MEM or MEM containing 10% FBS in the absence of cells. They were maintained at 37 °C in 5% CO2 atmosphere for 96 h. (B) SDS/PAGE gel of proteins adsorbed to QDs in the presence of 10% FBS after 96 h. Molecular weights are shown for corresponding bands in the molecular weight marker (MWM) lane. (C) Representative fluorescence images showing uptake of QDs in MRC-5 cells and colocalization with lysosomes after exposure to 200 lgmL1 Si/SiO2 QDs for 72 h. Green, lysosomes (LysoTracker Green DND-26); red, QDs (self-fluorescence); yellow, overlap of the green and red channels; blue: cell nuclei (Hoechst 33342). Scale bar = 10 lm. (D, E) Time-course of uptake of Si/SiO2 QDs in MRC-5 cells grown in MEM without FBS (D) or with 10% FBS (E). The cells were exposed to 25, 50 and 200 lgmL1 Si/SiO2 QDs up to 96 h. The quantitative analysis of fluorescence intensity measured was expressed as a percentage of QDs inside cells at each time point reading from the initial concentration of nanoparticles added. Values are the mean SD (n = 3). (F, G) The amount of Si/SiO2 QDs internalized into MRC-5 cells grown in MEM without FBS (F) or with 10% FBS (G). The cells were exposed to 25, 50 and 200 lgmL1 Si/ SiO2 QDs up to 96 h. The fluorescence intensity of QDs was measured and expressed after the multiplication of the percentage uptake of QDs with the initial concentration of nanoparticles added in the media. Values are the mean SD (n = 3). (H) Emission spectra of cell-free QDs suspensions in MEM and MEM containing 10% FBS freshly prepared (left) and after 96 h from the moment of preparation (right). The samples are at the same concentration and the intensity of emission is expressed in arbitrary units (AU).


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cells (revealed by the staining with propidium iodide in Fig. 4A) was concentration-dependent after 72 h. Effects of Si/SiO2 QDs on actin cytoskeleton morphology and organization

Fig. 2. LDH release from MRC-5 cells after exposure to 25, 50, 100, 150 and 200 lgmL1 Si/SiO2 QDs for 24, 48 and 72 h. Results are expressed as a percentage of the control that was not exposed to QDs. Values are the mean SD (n = 3). Statistical analysis was performed using Student’s t-test (*P < 0.05 and **P < 0.01 compared to control).

of 1.8-fold compared to control), being more extended for the 50 and 200 lgmL1 concentrations after 48 and 72 h (a four-fold higher level for 200 lgmL1 QDs at 72 h compared to control). The level of 200 lgmL1 QD-induced autophagy after 48 h was comparable to that resulting from exposure to 10 lM tamoxifen (Fig. 4C), which is an anti-tumoral agent known for its ability to activate the autophagic pathway. In addition, an increase in the number of dead

Figure 5 shows that, in control cells, the actin filaments were organized in thick bundles extending throughout the cytoplasm, starting from the cell surface. After exposure to concentrations of 50 and 200 lgmL1 QDs, an alteration of actin cytoskeleton was induced in a time-dependent manner. The filaments were poorly formed, without being organized into radial stress fibers and were found especially near the cell membrane and in the structure of lamelipodia and filopodia. Membrane ruffling (empty triangles) and small microspikes (filled triangles) were noted in fibroblasts incubated with 200 lgmL1 QDs for 24 h and punctate staining indicating nonfilamentous actin organized in bead-like structures was visible within the cells at 48 h for the same concentration of QDs. Furthermore, the parallel arrangement of linear F-actin fibers that was observed in control cells was mostly replaced by a diffuse matrix, as shown by the diffuse pattern of actin staining, and the remaining filaments were thin and irregularly disposed within the cells after 72 h of exposure to QDs.

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Fig. 3. Si/SiO2 QDs induced time- and dose-dependent lysosomes accumulation in MRC-5 cells. (A) Representative fluorescence images showing green lysosomes stained with LysoTracker Green and blue nuclei stained with Hoechst 33342 in cells exposed to 25, 50 and 200 lgmL1 Si/SiO2 QDs for 24, 48 and 72 h. Scale bar = 50 lm. (B) The green fluorescence intensity of lysosomes was quantified using IMAGEJ, version 1.48, and expressed relative to control cells. Values are the mean SD (n = 3). Statistical analysis was performed using Student’s t-test (*P < 0.05 and **P < 0.01 compared to control).


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Fig. 4. Si/SiO2 QDs triggered formation of autophagic vacuoles in lung fibroblasts. (A) MRC-5 cells exposed to 50 and 200 lgmL1 QDs for 24, 48 and 72 h were stained with monodansylcadaverine to detect autophagy and were counterstained with propidium iodide for cell death. Scale bar = 100 lm. (B) The green fluorescence intensity of autophagic vacuoles was quantified using IMAGEJ, version 1.48, and expressed relative to control cells. Values are the mean SD (n = 3). Statistical analysis was performed using Student’s t-test (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to control). (C) Representative fluorescence images showing MRC-5 control cells and MRC-5 cells exposed to 5 and 10 lM tamoxifen for 24 h, which were stained as described in (A). Tamoxifen was used as a positive control for autophagy induction. Scale bar = 100 lm.

Effects of Si/SiO2 QDs on lung inflammation The exposure to 25 lgmL1 QDs induced a timedependent increase in IL-6 and IL-8 protein levels by up to 6.5- and 11.2-fold, respectively, compared to control after 72 h (Fig. 6A,B). The release of both proteins was increased compared to control during the incubation with 50 lgmL1 QDs, although the maximum level was measured after 24 h for IL-6 and 48 h for IL8 (8.3 and 7.6-fold of control, respectively) (Fig. 6A,B). Regarding NO release from the cytosol into the cell culture medium, a time-dependent increase was noted after exposure to 50 lgmL1 QDs and no significant changes for 25 lgmL1 QDs were obtained (Fig. 6C).

Effects of Si/SiO2 QDs on ECM degradation The enzymatic activity of matrix metalloproteinases (MMPs) was determined from the culture supernatants after exposure to Si/SiO2 QDs using zymography (Fig. 7A). Although the MMP-9 activity decreased only by 5% and 20% compared to control after 24 h of incubation with 25 and 50 lgmL1 QDs, respectively, the proteolytic activity of this enzyme diminished by 65%


of control for both concentrations of QDs at 48 h, and by 67% and 73%, respectively, after 72 h (Fig. 7B). In the case of MMP-2, a decreased enzymatic activity compared to control was noted (Fig. 7C), which did not change significantly during exposure to QDs, activity being 65% and 55% of the control after 72 h of incubation with 25 and 50 lgmL1, respectively. The casein zymography indicated an inhibition of MMP-1 enzymatic activity for all concentrations used (Fig. 7D). The results of the western blotting analysis (Fig. 7E) indicated a diminished level of MMP-2 and MMP-9 protein expression compared to control after exposure to QDs (Fig. 7F,G). Incubation with 50 lgmL1 QDs decreased MMP-9 expression by 70% of control after 24 h and completely inhibited expression at 72 h of exposure. MMP-2 protein levels were diminished almost in the same manner as the enzymatic activity after 24 and 48 h, by 42% and 45%, respectively, for both concentrations. Incubation for 72 h with 25 and 50 lgmL1 QDs triggered a decrease in MMP-2 protein levels at 32% and 20% of control, respectively. By contrast to the inhibition of caseinase activity (Fig. 7D), protein expression of MMP-1 was up-regulated compared to control for all time intervals

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Fig. 5. Disorganization and decreased expression of actin filaments in lung fibroblasts after exposure to Si/SiO2 QDs. Representative fluorescence images showing green actin stained with FITCphalloidin and blue nuclei stained with 40 ,6-diamino-2-phenylindole in MRC-5 cells exposed to 50 and 200 lgmL1 Si/SiO2 QDs for 24, 48 and 72 h (empty triangles indicate areas of ruffling and filled triangles indicate microspikes). Scale bar = 25 lm.

(Fig. 7H). Furthermore, the activation of TIMP-1 protein expression in the cells exposed to QDs (Fig. 7I) could be one of the mechanisms responsible for the absence of MMP-1 enzymatic activity in treated fibroblasts, even in the context of an increased protein level as measured in the culture supernatants.

Discussion In the present study, the toxic effects triggered by Si/ SiO2 QDs on lung fibroblasts were examined to clarify the mechanisms of autophagy and inflammation induced by these nanoparticles. Generally, it is considered that doses lower than 25 lgmL1 silicon QDs are potentially ‘safe’ compared to other nanoparticles and thus high-dose toxicity effects require comprehensive investigation [22]. The concentrations of Si/SiO2 QDs (25–200 lgmL1) are comparable to those tested in previous studies regarding the toxic effects on lung cells induced by different types of nanoparticles [23– 25]. Although it is difficult to compare the in vitro measurements with the in vivo toxicity profile because of all the discrepancies between these two types of studies, the results obtained in the present study within this range of doses could provide valuable cell typespecific mechanistic information for predicting the potential animal health effects of QDs. Initially, the QDs were subjected to an analysis of their characteristics in physiological media aiming to better understand their behaviour prior to toxicity 2920

studies and to provide further useful information for the cytotoxicity and inflammatory potential of Si/SiO2 QDs. Thus, the selection of the type and concentration of culture media and serum for nanoparticle dispersion and measurement was carried out to coincide with the in vitro tests. The size measurements (Table 2) illustrated the tendency of QDs to form large agglomerates of approximately 200 nm, indicating that the nano-size identity of particles can suffer certain modifications in solutions compared to the primary size (6–8 nm) (Table 1), as also highlighted in previous studies [26,27]. In addition, the composition of the dispersion media significantly influenced the size and surface potential of the Si/SiO2 QDs (Table 2). The increasing trend of the hydrodynamic diameter suggested the interaction of QDs with amino acids, vitamins, inorganic salts and glucose from MEM, and proteins from FBS. The elevated size in the presence of serum indicated the tendency of QDs to adsorb proteins from FBS that form the biocorona on the surface of these nanoparticles, as revealed by SDS/PAGE (Fig. 1B). The zeta potential of approximately -30 mV suggested the stability and dispersity of QDs in water as indicated previously [28]. The two-fold reduction in zeta potential in the presence of FBS might be a consequence of a loss of stability of QDs in the presence of proteins that were also responsible for the diminution of repulsive forces between aggregates causing agglomeration (Fig. 1A). This effect was based on the finding that nanoparticles attracted oppositely charged bioFEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS

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Fig. 6. Inflammatory response in MRC-5 cells after exposure to Si/ SiO2 QDs. (A, B) The protein levels of IL-6 (A) and IL-8 (B) release in culture medium after 24, 48 and 72 h of incubation with 25 and 50 lgmL1 QDs were examined by western blotting and subjected to densitometric analysis. (C) NO release was measured as described in the Materials and methods. All results are expressed relative to control. Values are the mean SD (n = 3). Statistical analysis was performed using Student’s t-test (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to control).

molecules from serum and, as a result, the absolute value of zeta potential decreased compared to water or a basic media without FBS [27,29]. Taken together, it FEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS


could be suggested that size had an important influence on the reduction of the Si/SiO2 QDs zeta potential induced by protein corona, which is in accordance with the findings of previous studies [29,30]. The interactions between nanoparticles and macromolecules from biological fluids lead to the formation of biocorona, considered as the fingerprint of each nanomaterial [31]. Longer exposure periods were chosen in the present study not only with the aim of investigating the hard corona that represents the long-lived equilibrium state in a certain environment [32], but also because of the need for a more precise screening of the effects of QDs at different time intervals. The extent of protein adsorption on the surface of Si/SiO2 QDs was concentration-dependent, explaining the agglomeration trend observed in the tubes (Fig. 1A). The profile of adsorbed proteins revealed the albumin to be the most prominent protein attached on QDs (Fig. 1B), as also shown for other types of nanoparticles [33]. Because the biological identity of nanoparticles can dictate the bioreactivity of their surface and influence cellular uptake, accumulation and inflammation [34], an analysis of the internalization of Si/SiO2 QDs and uptake kinetics was performed. The majority of QDs were localized inside lysosomes, forming a perinuclear pattern (Fig. 1C), with their size being too high to pass through the nuclear membrane. This aspect might suggest a possible mechanism of internalization by endocytosis, with the cells recognizing them as exogenous particles that were further directed to lysosomes for sequestration and degradation. The pattern of distribution of QDs within MRC-5 cells was similar to that revealed by HarushFrenkel et al. [35] who reported the transition of anionic nanoparticles through the degradative lysosomal pathway. Besides the influence of surface charge on subcellular localization, the high amount of adsorbed BSA could play a key role in Si/SiO2 QDs guidance within cells because a previous study showed a localization of FITC-albumin in the lysosomes of alveolar type II epithelial cells [36]. In addition, the uptake of QDs could also be enhanced by BSA absorption because the fluorescence measurements indicated an elevated percentage of QDs inside cells in the presence of serum (Fig. 1E). Moreover, the difference between the uptake curves profile of the two types of media could be explained by the tendency of QDs to agglomerate in the presence of serum and to fall to the bottom of the culture dish under gravity (Fig. 1A) after a longer period of incubation (96 h), which might increase the chance of interaction with cell membranes. On the one hand, the amount of QDs inside cells, which increased in a concentration-dependent manner (Fig. 1E), explained the more evident cytotoxic effects

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Fig. 7. Exposure to Si/SiO2 QDs disrupted ECM homeostasis. (A) SDS/PAGE zymography of active MMPs secreted in the culture media of MRC-5 cells after 24, 48 and 72 h of incubation with 25 and 50 lgmL1 QDs. (B–D) The results of the densitometric analysis of the band intensity of MMP-9 (B), MMP-2 (C) and MMP-1 (D) were expressed relative to control. (E) Western blotting analysis of MMP-9, MMP-2, MMP-1 and TIMP-1 secreted in the culture media of MRC-5 cells after 24, 48 and 72 h of incubation with 25 and 50 lgmL1 QDs. (F–I) The results of the densitometric analysis of the band intensity of MMP-9 (F), MMP-2 (G), MMP-1 (H) and TIMP-1 (I) were expressed relative to control. Values are the mean SD (n = 3). Statistical analysis was performed using Student’s t-test. (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to control).

observed at higher concentrations (Fig. 2). On the other hand, the lower percentage uptake of QDs (calculated from the initial concentration suspended in the medium) noted for higher doses could indicate that agglomerations were developed, which, although they were not internalized because of an increased hydrodynamic size, could stick on cells, leading to cell membrane damage as a result of surface radicals generated throughout the method of synthesis of QDs. We suggest that the process of aggregation and the presence of proteins in the fluid composition were the critical factors for cell penetration rather than the difference between primary and hydrodynamic size. Hence, large aggregates of QDs could be ‘seen’ by cells as a material with an increased aspect ratio [37], stimulating cytoskeletal arrangements and membrane ruffling to facilitate internalization by macropinocytosis (Fig. 5). These results show that the protein corona promoted interaction with the cells and influenced the cell penetration rate, facilitating the internalization of a higher percentage of QDs. The toxic effects of QDs on cells occurred mainly as a result of their internalization or because of interaction with cell membranes whose integrity is impaired. The 2922

cell membrane damage shown by the concentrationdependent LDH release (Fig. 2) was consistent with previous studies [29]. The MRC-5 cells appeared to be more susceptible at high concentrations of QDs and longer time periods of exposure. An absent or decreased toxicity for low concentrations of QDs compared to other types of nanoparticles [38] supports the use of Si/SiO2 QDs for further in vivo studies. Taking into account that the amount of Si/SiO2 QDs inside MRC-5 cells increased during exposure in the media with FBS and accumulated in a manner dependent on the initial concentration of suspended nanoparticles (Fig. 1G), we could assume that the endocytosis of QDs increased with concentration, being reflected by an increment in the number and/or volume of lysosomes (Fig. 3). The larger population of lysosomes after exposure to QDs might also suggest an accumulation of altered molecules, oxidized proteins and damaged cell components in these organelles. Incorporation of QDs in lysosomal structures together with the accumulation of autophagic vacuoles (Fig. 4) reflected the mechanism involved in Si/SiO2 QD-mediated autophagy. As previously reported, the cytotoxicity of QDs was associated with the generation of ROS FEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS

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and oxidative stress [21], which could trigger the induction of autophagy as a cellular survival mechanism. The interaction of autophagosomes with endosomes/lysosomes can be an important step in this process because these cellular structures are intracellular sites of QD localization [10]. The QDs covered with protein corona that entered the cytosol of lung fibroblasts might be misidentified as damaged proteins, which are normally degraded by autophagy after incorporation in autophagic vacuoles that merge with lysosomes to ensure cell homeostasis and the turnover of macromolecules [39]. The biopersistence of nanomaterials might cause lysosomal dysfunction exhibited by lysosomal membrane permeabilization and an altered intracellular signaling and gene expression, and further might correlate with a blockage in the fusion of autophagosomes with lysosomes [9]. Also, an uncontrolled and prolonged autophagy might lead to autophagic cell death [40]. Therefore, the induction of autophagy by QDs may not only be seen as an adaptive response to cellular stress that allows self-clearance, but also as one of the mechanisms of toxicity responsible for the decrease of cell viability in the case of high concentrations of QDs. Furthermore, the uptake of nanoparticles by mammalian cells could affect the cytoskeleton structure because endocytosis involves the reorganization of cytoskeletal filaments [41]. Hence, the time-dependent disruption of actin cytoskeleton induced by Si/SiO2 QDs (Fig. 5) might be a consequence of their uptake kinetics in MRC-5 cells. Moreover, the internalization of QDs and the large number of vacuoles formed in the cells could induce steric impediments and changes in protein conformation after binding to nanoparticles, affecting the capacity of actin to anchor cells to the ECM by interacting with integrins [42,43]. Actin filaments are components of a highly dynamic and complex system organized as a network known as the actin cytoskeleton, which contributes to multiple essential cellular functions, such as cell adhesion, motility, division, cytokinesis and cell signaling [44]. By also mediating cell shape and stiffness, the modifications of actin cytoskeleton organization might be a cause of the altered morphology of lung fibroblasts as depicted previously by phase contrast microscopy [21]. The cytoskeletal arrangements and membrane ruffling after exposure to Si/SiO2 QDs can be explained throughout the S-glutathionylation of actin induced by the same concentrations of QDs [21] because this post-translational process regulates cytoskeletal assembly [45]. The changes in actin cytoskeleton to enable the formation of membrane ruffles involved in engulfing the submicron particles were characteristic of the high concentration of QDs tested in FEBS Journal 282 (2015) 2914–2929 ª 2015 FEBS


the present study, suggesting the macropinocytosis as a particular type of QDs endocytosis [46]. In response to the agglomeration of QDs at the cell surface, the actin cytoskeleton can reorganize, generating a diffuse pattern that destabilizes the tight junctions and leads to the cell permeability revealed by the leakage of LDH or other molecules in the extracellular medium during membrane disruption. Furthermore, the alteration of cytoskeleton architecture can represent a critical step in the autophagy defense mechanism triggered by QDs because a recent study indicated that actin remodeling is required for autophagosome–lysosome fusion in mammalian quality-control autophagy [47]. Besides oxidative stress, a common mechanism of nanoparticle toxicity is inflammation characterized by an increased production of cytokines involved in various signaling pathways [48]. The present study clearly showed an acute inflammatory response induced by Si/ SiO2 QDs, mediated by the release of IL-6, IL-8 and NO, which was differently modulated by the concentration of nanoparticles (Fig. 6). The progressive release of ILs during 72 h of exposure to 25 lgmL1 QDs compared to the rapid and transient release of higher amounts in the case of 50 lgmL1 QDs suggested that an elevated concentration of nanoparticles was more toxic and triggered the prompt up-regulation of these cytokines to prevent cells from injury. Also, the profile of uptake curve observed for these concentrations could match this pro-inflammatory cascade, although the appropriate mechanism responsible for this differently modulation in cytokine release remains to be determined. IL-6 and IL-8 play important roles in inflammation response and may have the potential to modulate the expression and activity of inducible NO synthase, which catalyzes the reaction of NO generation [49,50]. The time-dependent increased level of NO release after exposure to 50 lgmL1 QDs correlates with the presence of cytokine in the environment before NO generation. Although NO can express antioxidant and anti-inflammatory properties, and can also modulate vascular tone and play a pivotal role in intracellular signaling, it is a cytotoxic molecule involved in the induction of apoptosis in various cell types [51], as well as in the pathogenesis of tissue damage associated with acute or chronic pulmonary inflammation modulating cytokine gene expression and cyclooxygenase activity [15,52]. Other modulators of the inflammatory process are MMPs, which are involved in the degradation of ECM and other molecules associated with it [53]. Because of their role in establishing the architecture of ECM, MMPs are not only involved in essential cellular processes such as proliferation, differentiation and cell

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migration, but also in pathological situations, including inflammation, fibrosis and cancer. Zymography analysis revealed a decrease activity of gelatinases MMP-2 and MMP-9, which correlates with a lower protein expression compared to the control (Fig. 7). The up-regulation of MMP-1 protein expression followed by inhibition of its activity after exposure to Si/SiO2 QDs could be explained by the elevated level of TIMP-1 expression, an endogenous inhibitor of this enzyme [53]. Additionally, the release of IL-6 might be responsible for the upregulation of MMP-1 expression as reported previously [54]. These changes in MMPs expression and activity were similar to the molecular events involved in the pathogenesis of chronic rhino-sinusitis without nasal polyps, which involves high levels of IL-6 and IL-8 that trigger fibrosis by up-regulating TIMP-1 in fibroblasts [55]. In addition, the stimulation of TIMP-1 protein expression by IL-6, which was suggested in the present study, has also been reported in rat hepatocytes [56] and was indicated as a mechanism of cell growth inhibition [57], although a protective role of IL-6 associated with the induction of TIMP-1 was noted in hyperoxic acute lung injury [58]. The activation of MMPs takes place when the prodomain from the structure of zymogen form is cleaved by other proteases or when the interaction between the thiol group of a cysteine residue in the pro-domain and the zinc ion of the catalytic centre essential for proteolytic activity is disrupted [53]. The exposure to Si/SiO2 QDs could affect the process of activation by inducing chelating effects on zinc ions. Furthermore, ROS react with thiol groups, modulating the activity of MMPs. Generally, ROS activate MMPs by modifying the cysteine residue that stabilizes the inactive structure [59], although some studies supporting our results revealed a reduction of the activity of MMPs by tryptophan oxidation in the catalytic domain by ROS to restrain proteolytic activity during inflammation [60,61] or as a result of excessive protein oxidation, which led to protein degradation and altered the biological functions of MMPs. Because latent MMP-9 is proteolytically activated by MMP-1, the suppression of MMP-1 activity can be responsible for the decrease of MMP-9 activity together with the increased expression of TIMP-1 and high levels of NO [62]. Because the ECM plays a pivotal role in the cytoskeletal filament assembly, the changes induced by Si/SiO2 QDs on MMPs expression and activity correlate with the alteration of the actin cytoskeleton [63]. Therefore, this differential regulation of MMPs expression and decreased enzymatic activities may be a consequence of the oxidizing environment created as a result of ROS accumulation after exposure to QDs and indicate 2924

an imbalance in ECM homeostasis that might have an effect upon cell shape, proliferation and inflammation. The hydroxylation of the SiO2 surface could be expected based on the synthesis method (i.e. laser ablation) of QDs, providing the formation of silanol groups (Si-OH), which can be followed by the condensation of these hydroxyls into siloxane rings (Si-O-Si) according to the silica surface chemistry [64]. Recent studies have concluded that the specific groups on the silica particle surface could directly interact with the cell and lysosomal membranes inducing membrane damage and pro-inflammatory response [65,66]. Based on the similarities between these studies and our results on Si/SiO2 QDs, we suggest that cytotoxicity of QDs was very probably dependent on the synthesis pathway and more related to strained surface Si-O-Si rings than to the Si or SiO2 materials per se. The surface radicals could be responsible for modulating nanoparticle aggregation and interaction with macromolecules from the biological medium and subcellular components with further toxic consequences. In the future, specific surface modification of Si/SiO2 QDs may be able to create a bioactive coating improving their stability in physiological media and alleviating their cytotoxicity. In conclusion, the biopersistent Si/SiO2 QDs triggered an inflammatory response in human lung fibroblasts that was characterized by the release of pro-inflammatory cytokines responsible not only for stimulating the production of NO, but also for modulating MMPs, with consequences for the degradation and remodeling of actin cytoskeleton and ECM. Our findings provide insights for further research aiming to investigate the dynamics of nano-biointeractions involving cellular membranes and the cell death signaling pathways triggered by long-term exposure. The data accumulated in the present study significantly contribute to the expansion of knowledge relevant for the nanotoxicological characterization of QDs designed for applications in nanomedicine.

Materials and methods QDs The Si/SiO2 QDs tested in the present study were supplied by the Laser Department of the National Institute for Laser, Plasma and Radiation Physics (Bucharest-Magurele, Romania). The nanoparticles were made of a crystalline silicon core covered by an amorphous SiO2 shell, and were synthesized by pulsed laser ablation that yielded particles with a size distribution of 6–8 nm (Table 1). QDs showed red photoluminescence with a maximum intensity at approximately


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644 nm when were excited in UV light (325 nm) [67]. The synthesis method has been described previously [20].

Characterization of QDs in cell culture media The characterization of hydrodynamic size, polydispersity index and zeta potential of Si/SiO2 QDs was performed using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, UK). Dry powder of Si/SiO2 QDs was suspended at a concentration of 200 lgmL1 in ultrapure water and cell culture medium (MEM; Invitrogen) containing 10% FBS (Gibco, Invitrogen), or not, for 1 h at room temperature. The QDs suspensions were sonicated using a Sonics Vibracell VCX750 Ultrasonic Processor (Sonics & Materials, Newtown, CT, USA) at 200 W for 2 min at room temperature. The stock suspensions were diluted to 50 lgmL1 QDs in the respective medium and the measurements were carried out in triplicate with an equilibration time of 1 min at 25 °C using the refractive index of 1.52 corresponding to SiO2. To evaluate the capacity of QDs to adsorb proteins, different concentrations of nanoparticles (25, 50 and 200 lgmL1) were dispersed in culture medium containing 10% FBS and incubated for 96 h at 37 °C in the absence of cells. The suspensions were afterwards centrifuged at 14 000 g for 10 min and washed with PBS three times. The pellets obtained were loaded on a 10% SDS/PAGE and migrated under reducing conditions. The gels were stained using Coomassie Brilliant Blue G-250 (Merck Millipore, Darmstadt, Germany). The bands were visualized with a G: Box Chemi XR5 system (Syngene, Cambridge, UK) using GENESYS software (Syngene) and quantified with GELQUANT.NET software (provided by biochemlabsolutions.com).

Cell culture conditions and treatments MRC-5 human embryonic lung fibroblasts (ATCC CCL171) were cultured in MEM containing 10% FBS, 100 IUmL1 penicillin and 100 lgmL1 streptomycin, at 37 °C in a 5% CO2 humidified incubator. The medium was changed every 2 days and the cells were detached using 0.25% trypsin/0.03% EDTA solution (Sigma-Aldrich, St Louis, MO, USA). A stock suspension of 3 mgmL1 Si/SiO2 QDs was prepared by dissolving the powder in 0.9% NaCl and was sterilized in the autoclave for 20 min at 120 °C. The sterile QDs suspension was sonicated right before it was added to the serum-containing or serum-free medium of cultured cells, at concentrations ranging between 25 and 200 lgmL1 that corresponded to 3–24 lgcm2. The cells were maintained at 37 °C in a 5% CO2 humidified atmosphere for different periods of time up to 96 h. The culture supernatants were collected or removed at the indicated time points and the cells were washed with PBS and processed as described below.



Quantification of uptake of QDs The internalization of nanoparticles by MRC-5 cells was followed after 24, 48, 72 and 96 h, by measuring the fluorescence intensity of QDs that were initially added at concentrations of 25, 50 and 200 lgmL1 to the serum-free MEM or MEM containing 10% FBS. The measurements were performed on a FP-6300 spectrofluorometer (Jasco, Tokyo, Japan) using excitation and emission of 325 and 644 nm, respectively. The culture supernatants were carefully aspirated to exclude the nonspecific binding of QDs on the plastic bottom of culture dish and were used to measure the fluorescence of the nanoparticles that were not internalized. The quantity of QDs that were internalized was calculated as the difference between the fluorescence of cell-free culture media aliquots with the appropriate concentration of QDs and the fluorescence of culture supernatant, and expressed as a percentage of the initial QDs concentration. The fluorescence of the corresponding medium without QDs was subtracted from each measurement.

LDH assay The level of LDH release in culture media was measured using a commercially available LDH assay kit, (SigmaAldrich) in accordance with the manufacturer’s instructions. MRC-5 cells were seeded in 24-well plates at a density of 5 9 104 cellswell1 and incubated overnight to allow the attachment. Then, they were exposed for 24, 48 and 72 h to concentrations of 25, 50, 100, 150 and 200 lgmL1 QDs. Next, a volume of 50 lL of culture supernatant from each well was incubated with 100 lL of LDH reaction solution for 30 min at room temperature in a 96-well plate. The absorbance at 450 nm was measured using a microplate reader (Tecan GENios, Gr€ odig, Austria) and the results were expressed relative to control cells that were not exposed to QDs.

Fluorescence microscopy For fluorescence microscopy studies, the cells were seeded on coverslips mounted in Nunclon dishes with a culture area of 8.8 cm2 at a density of 105 cellsdish1 and allowed to attach overnight. QDs were added to the media at concentrations of 25, 50 and 200 lgmL1 QDs for 24, 48 and 72 h. The samples were prepared as described below and analyzed on an Olympus IX71 inverted epifluorescence microscope (Olympus, Tokyo, Japan) using the filters optimized for the different fluorochromes and a constant optimal exposure time. This microscope was used also to confirm the uptake of QDs by fibroblasts. No background QDs fluorescence that might interfere with the fluorochrome signal was observed under these imaging conditions. Lysosomes were stained with 100 nM LysoTracker Green DND-26 (excitation and emission wavelengths of 504 and 511 nm, respectively; Molecular Probes, Invitrogen) for

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30 min at 37 °C and 5% CO2, followed by the counterstaining of nuclei for 10 min at room temperature with 2 lgmL1 Hoechst 33342 (excitation and emission wavelengths of 350 and 461 nm, respectively; Molecular Probes, Invitrogen). The induction of autophagy in treated cells was assessed using the Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. The cells were analyzed by fluorescence microscopy for the detection of autophagic vacuoles (stained with monodansylcadaverine for 10 min at 37 °C) and dead cells (labeled by propidium iodide for 2 min at room temperature), respectively. In parallel, MRC-5 cells were incubated with 5 and 10 lM tamoxifen as a positive control for autophagy. The image analysis was performed with IMAGEJ, version 1.48 (NIH, Bethesda, MD, USA), which was used to quantify the fluorescence intensity of labeled lysosomes and autophagic vacuoles in control and treated cells using 50 fields per sample. The results were expressed as fold changes in fluorescence intensity over control. Actin filaments were stained with phalloidin conjugated with FITC (Sigma-Aldrich) after an initial step of cell fixation and permeabilization. Fibroblasts were fixed for 20 min at room temperature with 4% paraformaldehyde in PBS and subsequently washed three times with PBS. Fixed cells were permeabilized with 0.1% Triton X-100, 1.2% BSA in PBS for 1 h at room temperature and, after three washes with PBS to remove the detergent, the cells were incubated for 40 min with a 50 lgmL1 phalloidin-FITC solution for F-actin labeling. The nuclei were stained with a 2 lgmL1 40 ,6-diamino-2-phenylindole solution (Molecular Probes, Invitrogen) for 15 min at room temperature.

NO assay The concentration of NO in culture supernatants was determined using the method first described by Griess [68]. MRC5 fibroblasts, seeded onto 75-cm2 culture flasks at a density of 104 cellscm2 and incubated overnight for attachment, were exposed to 25 and 50 lgmL1 Si/SiO2 QDs for 24, 48 and 72 h. Samples, represented by serum-free culture supernatants collected at each time point, were transferred to a 96well plate. A stoichiometric solution (v/v) of 1% sulfanilamide (Sigma-Aldrich) and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride (Sigma-Aldrich) was prepared just before it was added to the sample wells in a 1 : 1 ratio (v/v). The absorbance of the colored product was measured at 485 nm using an Appliskan reader (Thermo Scientific, Waltham, MA, USA) and the concentration of NO was determined using a sodium nitrite (Sigma-Aldrich) standard curve.

Western blotting The protein levels of MMP-1, MMP-2, MMP-9, TIMP-1, IL-6 and IL-8 were determined up to 72 h of exposure to 25 and 50 lgmL1 QDs, using the serum-free culture

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supernatants collected as described above. Samples corresponding to 50 lg of protein were separated on a 10% SDS/PAGE under reducing conditions and transferred onto 0.4 lm poly(vinylidene difluoride) membrane (Millipore) in a wet transfer system (Bio-Rad, Hercules, CA, USA). The membranes were blocked with blocking solution included in the WesternBreeze Chromogenic kit (Invitrogen) for 30 min at room temperature. MMP-2 and MMP-9 protein detection was accomplished with primary rabbit polyclonal antibodies anti-MMP-2 and anti-MMP-9 (dilution 1 : 500; Chemicon, Temecula, CA, USA), whereas MMP-1, TIMP1, IL-6 and IL-8 preoteins were identified with primary mouse polyclonal antibodies anti-MMP-1, anti-TIMP-1, anti-IL-6 and anti-IL-8 (dilution 1 : 250; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the incubation with primary antibodies, the membranes were processed in accordance with the manufacturer’s instructions, using antimouse or anti-rabbit secondary antibodies coupled with alkaline phosphatase and 5-bromo-4-chloro-30 -indolyphosphate/nitroblue tetrazolium as the chromogenic substrate. The resulting bands were imaged with a G:Box Chemi XR5 system (Syngene) using GENESYS software and quantified with GELQUANT.NET software.

Gelatin and casein zymography The enzymatic activities of gelatinases (MMP-2 and MMP9) and caseinase (MMP-1) were detected on a 7.5% SDS/ PAGE containing 0.1% gelatin or 0.03% casein, using the same culture supernatants used for western blotting. Samples, corresponding to 10 lg of protein for gelatin zymography and 40 lg for casein zymography, were prepared in nonreducing conditions and were not thermally denatured prior to loading onto the gel. The total protein concentration in the samples was determined using the Bradford method [69]. After electrophoretic separation at 4 °C, the gels were washed with distilled water and incubated two times for 15 min with renaturing buffer (50 mM Tris HCl, pH 7.6, 2.5% Triton X-100). Next, the gels were washed with distilled water and incubated overnight at 37 °C under mild shaking with an incubation buffer (50 mM Tris HCl, pH 7.6, 10 mM CaCl2, 50 mM NaCl, 0.05% Brij 35) that allowed the enzymes to exert their catalytic activity. The gels were stained with Coomassie Brilliant Blue G-250 and the enzymatic activity of MMPs was detected as white bands on the blue gel after incubation with destaining solution. Next, the gels were scanned using a Vilber Lourmat imaging system (Vilber Lourmat, Marne la Vallee, France) and the bands were quantified with GELQUANT.NET software. Standards of MMP-9 (92 kDa) and MMP-2 (72 kDa) (Chemicon) were used to identify gelatinases. The enzymatic activity detected by casein zymography corresponding to MMP-1 was established based on its molecular mass of 54 kDa, being intensely expressed in pulmonary fibroblasts and MRC-5 cells [70,71].


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Statistical analysis Data are reported as the mean SD from three independent experiments and expressed relative to control. The results were analyzed for statistical significance using Student’s ttest. P < 0.05 was considered to be statistically significant.

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Acknowledgements M. S. Stan gratefully acknowledges the support of the European Social Fund through the contract POSDRU/159/1.5/S/133391. This research study was presented as a poster at FEBS-EMBO 2014 Conference, Paris, France.

Author contributions MSS performed the experiments, analyzed the data and drafted the manuscript. CS handled the quantum dot synthesis. LOC performed the nanoparticles characterization assays and analyzed the data. AD designed the experiments and wrote the manuscript.

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Quantum dots modulate intracellular Ca2+ level in lung epithelial cells.

ZnS quantum dots induce pro-inflammatory cytokine expression in mouse lung epithelial cells and macrophages.

Realgar quantum dots induce apoptosis and necrosis in HepG2 cells through endoplasmic reticulum stress.

ZnS quantum dots induce photodynamic effects and cytotoxicity in pancreatic cancer cells.

Chemically synthesized CdSe quantum dots inhibit growth of human lung carcinoma cells via ROS generation.

Red blood cells induce necroptosis of lung endothelial cells and increase susceptibility to lung inflammation.

CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility.

Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.

Amorphous silica nanoparticles impair vascular homeostasis and induce systemic inflammation.

Localisation microscopy with quantum dots using non-negative matrix factorisation.

MiR-26a modulates extracellular matrix homeostasis in cartilage.

Lung extracellular matrix and fibroblast function.

Imaging Live Cells Using Quantum Dots.

Semiconductor polymer dots induce proliferation in human gastric mucosal and adenocarcinoma cells.

Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation.

Progenitor Cells.

Bioactive extracellular matrix fragments in lung health and disease.

Sandwiched confinement of quantum dots in graphene matrix for efficient electron transfer and photocurrent production.

Structure and effects of annealing in colloidal matrix-free Ge quantum dots.

Synthesis of near-infrared quantum dots in cultured cancer cells.

Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment.

Ultrasensitive imaging in live cells using fluorescent quantum dots.

The uptake mechanism and biocompatibility of graphene quantum dots with human neural stem cells.

Can graphene quantum dots cause DNA damage in cells?