Research Article Received: 25 November 2013,
Revised: 6 January 2014,
Accepted: 16 January 2014
Published online in Wiley Online Library: 14 February 2014
(wileyonlinelibrary.com) DOI 10.1002/jat.2996
Carbon-based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape Martin Holzera, Peter Biharia, Marc Praetnera, Bernd Uhla, Christoph Reichelb, Janos Fentc, Minnamari Vippolad,e, Susan Lakatosc and Fritz Krombacha* ABSTRACT: Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg–1 body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg–1 body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte– endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs’ shape, but does not induce leukocyte–endothelial cell interactions or leukocyte transmigration. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: nanoparticles; carbon nanotubes; carbon black; thrombosis; platelets; inflammation; leukocytes; microcirculation; in vivo microscopy
Introduction
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*Correspondence to: F. Krombach, Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, 81377 Munich, Germany. E-mail: [email protected] a Walter Brendel Centre of Experimental Medicine, Ludwig-MaximiliansUniversität München, 81377 Munich, Germany b Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum der Universität München, 81377 Munich, Germany c Research Institute, Medical Centre, Hungarian Defence Forces, Budapest, Hungary d Department of Materials Science, Tampere University of Technology, 33101 Tampere, Finland e
Finnish Institute of Occupational Health, 00250, Helsinki, Finland
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Nanoparticles (NPs) are defined as those materials with at least one dimension in the range of 1–100 nm (Oberdorster et al., 2005b). Carbon-based nanomaterials (CBNs) include materials such as fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical carbon black (CB) (Terrones and Terrones, 2003). The unique properties of CBNs make them interesting for new industrial or medical applications, but on the other hand raise questions about potential health effects (Maynard et al., 2006). The administration routes of CBNs engineered for medical purposes are either local injection into the target tissue or intravenous injection. Moreover, it has recently been shown that inhaled carbon nanotubes, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time after exposure (Mercer et al., 2013). Once within the bloodstream, nanoparticles will interact with blood cells, serum proteins, and endothelial cells (Shi et al., 2011). Finally, they will be distributed throughout the body and, on their way, pass the microcirculation of tissues or organs (Oberdorster et al., 2005a; Nel et al., 2006). Differently shaped carbon-based nanoparticles such as spherical ultrafine carbon black (CB) or fiber-shaped SWCNT activate platelets in vitro. In addition, SWCNT are able to accelerate thrombus formation in the macro- and microcirculation in vivo (Bihari et al., 2010; Radomski et al., 2005). Although administration of CB has been found to induce fibrinogen and platelet deposition in postcapillary venules
(Khandoga et al., 2004, 2010), the impact of CB on microcirculatory thrombus formation has not yet been studied. In contrast, thrombotic process can influence inflammation (Wagner and Burger, 2003; Zarbock et al., 2007), and experimental data highlight the inflammatory potential of fiber-shaped CNTs in extracirculatory tissues after inhalation or intraperitoneal injection (Krug and Wick, 2011; Maynard et al., 2011; Poland et al., 2008). Although blood-borne nanoparticles will reach and pass the microcirculation, it is insufficiently investigated whether they have the potential to modulate leukocyte–endothelial cell
M. Holzer et al. interactions in microvessels. An inflammatory response is characterized by recruitment of leukocytes from the microcirculation to the site of trauma or infection (Ley et al., 2007). It is a complex cascade of different subsequent steps consisting of leukocyte rolling on the endothelial cell layer, firm adhesion mediated by integrins and adhesion molecules, and finally transmigration of the leukocytes through the vessel wall into the subendothelial tissue (Ley et al., 2007). Previous studies of our group did not detect increased leukocyte–endothelial interactions in hepatic microvessels upon intra-arterial injection or inhalation of CB (Khandoga et al., 2004, 2010). Whether systemically administered SWCNT have acute effects on leukocyte–endothelial interactions or leukocyte transmigration has not yet been investigated. Furthermore, there is no experimental data about the role of the shape of CBNs for proinflammatory and prothrombotic effects in microvessels, although shape is well known as an important factor for the behavior of nanomaterials in biological environments (Oberdorster et al., 2005a; Nel et al., 2006) . Thus, the aims of this study were to analyze and compare the early effects of two differently shaped CBNs (SWCNT and CB) (i) on thrombus formation as well as on leukocyte–endothelial cell interactions and leukocyte transmigration in the murine microcirculation in vivo and, (ii) on the activation of human platelets and leukocytes in vitro.
870 μl of dispersion before the addition of 100 μl of a 10× concentrated phosphate-buffered saline (PBS; Sigma–Aldrich, Schnelldorf, Germany) solution, yielding a pH of 7.4, and final concentrations of phosphate buffer and sodium chloride of 10 and 154 mM, respectively. To prepare dispersions at lower concentrations, dilutions of the stock solution were used. The final concentrations of nanoparticles in the dispersion were 0.002, 0.02 and 0.2 mg ml–1. The vehicle was prepared in the same way, but instead of nanomaterial the same volume of distilled water was added to the dispersion (Bihari et al., 2008b). To determine the quality of the dispersions, measurements of nanoparticle diameter, polydispersity index and zeta potential were conducted with a Zetasizer-Nano ZS instrument (Malvern, Malvern Hills, UK). Nanoparticle dispersions prepared by the above method were tested for the presence of LPS with the LAL (Limulus amoebocyte lysate) kinetic chromogenic assay (Lonza, Verviers, Belgium).
Methods
Analysis of Human Platelet Activation
Nanomaterials S-purified single-walled carbon nanotubes (SWCNT), outer diameter: < 2 nm, length: 1–5 μm were purchased from SES Research (Houston, TX, USA). Ultrafine carbon black particles (Printex 90; diameter: 14 nm) were obtained from Evonik-Degussa (Essen, Germany). Characterization of Nanoparticles The characterization of the nanomaterials included size and morphology analysis with scanning (SEM, Zeiss ULTRAplus FEG-SEM; Carl Zeiss NTS GmbH, Oberkochen, Germany) and transmission electron microscopy (TEM, Jeol JEM 2010; Jeol, Tokyo, Japan). The composition of the nanoparticles was determined by energy dispersive spectroscopy (EDS, ThermoNoran Vantage, Thermo Scientific, Breda, The Netherlands) attached to the TEM. The elemental composition is given as an average of five separate EDS measurements. The specific surface area of the CBNs used in this study was previously measured using the BET method with a Coulter Omnisorb 100 CX gas adsorption analyzer (Miami, FL, USA). The adsorbed gas was nitrogen and the temperature was 77.36 K. The specific surface area of SWCNT and CB was 436 and 265 m2 g–1, respectively (Vippola et al., 2009). Preparation of Particle Dispersions
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SWCNT or CB stock solutions were prepared at a concentration of 0.23 mg ml–1 in distilled water using sonication with 4.2 × 105 kJ m–3 specific energy. For in vitro experiments with human blood, 30 μl of 50 mg ml–1 human serum albumin (final concentration 1.5 mg ml–1) and for in vivo mouse experiments 30 μl of mouse serum (preparation see below) was added to
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Human Blood Sampling Human blood was collected from the cubital vein of 44 healthy volunteers into citrate anticoagulant-containing Vacuette test tubes (Greiner, Kremsmünster, Austria). All samples were obtained with the approval of the local Ethical Committee after the donor had given informed consent.
The effect of nanomaterials on platelet activation in human whole blood was determined by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA). 100 μl of citrated blood was either incubated with 100 μl of vehicle or 100 μl of SWCNT or CB dispersion at a concentration of 0.2 mg ml–1 for 10 min at room temperature. After incubation, samples were diluted five-fold with 0.35% bovine serum albumin containing Tyrodes’ buffer: BSA-Tyr (10 mM Hepes, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, 5.5 mM glucose, pH 7.4). As a positive control, vehicle-containing samples were incubated with 1 μM ADP (final concentration) for 10 min. Samples were stained according to the manufacturer’s instructions. To measure P-selectin expression on platelets, whole blood was incubated with PE-labeled anti-CD62P antibodies (Dako, Glostrup, Denmark) and FITC-labeled antiCD41 antibodies (Immunotech, Marseilles, France). To check non-specific binding of the antibodies, appropriate isotype control antibodies were used. After staining, samples were diluted 50-fold with BSA-Tyr buffer (500-fold final dilution of blood). To minimize the spontaneous activation of platelets, no washing steps were used. The CD41 platelet marker was used as a trigger signal for data collection. The platelets were gated on the FS-SS dot plot and the mean CD62P fluorescence intensity (MFI) was measured for CD41 positive events. To measure platelet-granulocyte aggregates, FITC-labeled anti-CD41 and PC5-labeled anti-CD15 (Beckman-Coulter, Krefeld, Germany) antibodies were used. Platelet-granulocyte aggregates were detected as double-positive events in the granulocyte gate. Here the CD15-PC5 granulocyte marker was used as a trigger signal. As a 500-fold dilution of blood was used in these measurements, the coincidence of platelets and granulocytes did not result in false-double positivity (Bihari et al., 2008a). The amount of platelet-granulocyte aggregates was determined as the percentage of CD41 positivity in the CD15-positive gate.
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Carbon-based nanomaterials accelerate arteriolar thrombus formation Surface Expression of Adhesion Molecules on Human Monocytes and on Granulocytes The effect of nanomaterials on the surface expression of CD11b, CD11c and CD18 on monocytes and on granulocytes in human whole blood was determined by flow cytometry (FACScan; Becton Dickinson). Next, 100 μl of citrated blood was incubated with 100 μl of a 0.2 mg ml–1 nanomaterial dispersion or vehicle for 60 min at room temperature. After incubation, samples were stained according to the manufacturer’s instructions. Briefly, 50 μl of samples was stained with the appropriate amount of monoclonal antibodies (PE-labeled anti-CD11b and FITC-labeled anti-CD11c: Dako, Denmark; FITC-labeled antiCD18: Immunotech, Beckman-Coulter; and PerCp-labeled antiCD14: Becton Dickinson) for 30 min at room temperature in the dark, then lysed with FACSLyse (Becton Dickinson) solution and washed twice with 0.5% bovine serum albumin containing phosphate buffer saline (pH 7.4). To check non-specific binding of the antibodies, appropriate isotype control antibodies were used. Data collection was triggered by the forward scatter (FS) signal. Monocytes were separated from granulocytes on the FS-SS dot plot based on their CD14 staining. Animals C57BL/6NCrl mice were purchased from Charles River (Sulzfeld, Germany). The experiments were performed with male mice with a body weight of 20–27 g. Animals were housed in Makrolon cages under constant temperature (22 ± 2 °C), humidity (55 ± 5 %) and light conditions (12-h cycle) with free access to pelleted food and water. All experiments were approved by the local authorities and performed according to the German legislation for the protection of animals.
Light/dye-induced thrombosis was performed as described earlier (Rumbaut et al., 2005) with slight modifications. The light intensity at 488 nm was daily measured by a photodiode at the exit of the light source and maintained at between 2.65 and 2.75 mA. After surgical preparation of the cremaster muscle, SWCNT or CB dispersions at concentrations of 0.01, 0.1 and 1 mg kg–1 body weight (in a volume of 5 μl g–1 body weight) or vehicle (control) were injected through the catheter. The nanomaterials were administered 10 min prior to induction of thrombosis in order to yield a uniform distribution of the particles in the microcirculation. Thereafter, 4 ml kg–1 body weight of a 2.5% solution of FITC-dextran (fluorescein isothiocyanate dextran 150 kD obtained from Sigma-Aldrich, Schnelldorf, Germany) was given. To verify comparable intravascular FITC concentrations among experimental groups, digital images were taken from each utilized vessel with the CCD camera mentioned above. Ten minutes after the application of nanomaterials, photoactivation was induced by exposing a vessel segment of 300 μm length to continuous epi-illumination with a wavelength of 488 nm. An Olympus water immersion lens (60×/NA 0.9) was used to focus the light onto the cremaster and to obtain fluorescent images. Thrombus formation was quantified in one arteriole (25–35 μm) and one venule (30–50 μm) by analyzing the time when platelets became adherent to the vessel wall (defined as time of onset of thrombus formation) and the time required for complete occlusion of the vessel (defined as time to cessation of blood flow). Cessation time in vessels without complete occlusion until the end of the recording was considered 20 min for venules and 40 min for arterioles.
Analysis of Leukocyte–Endothelial Cell Interactions and Leukocyte Transmigration
Preparation of Mouse Serum Mice were anesthetized with a ketamine/xylazine mixture (100 mg kg–1 ketamine and 10 mg kg–1 xylazine) administered by intraperitoneal injection. Blood was taken by heart puncture and allowed to clot. The blood was centrifuged with 1100 g for 20 min and the supernatant was taken. Serum samples were pooled and aliquots were stored at 20 °C until use. Surgical Procedure
Leukocyte recruitment was analyzed using the reflected-light oblique illumination technique (Mempel et al., 2003). A mirrored surface (reflector) was positioned directly below the specimen, its angle tilted relative to the horizontal plane. The reflector consisted of a round cover glass (thickness 0.19–0.22 mm, diameter 11.8 mm), which was coated with aluminum vapor (Freichel, Kaufbeuren, Germany) and brought into direct contact with the overlying specimen. For offline analysis of parameters describing the sequential steps of leukocyte extravasation CapImage image analysis software (Dr Zeintl, Heidelberg, Germany) was used. Rolling leukocytes were defined as those moving slower than the associated blood flow and were quantified for 30 s. Firmly adherent cells were determined as those resting in the associated blood flow for more than 30 s and related to the luminal surface per 100-μm vessel length. Transmigrated cells were counted in regions of interest covering 75 μm on both sides of a vessel over 100-μm vessel length. Leukocyte accumulation in each segment was quantified and shown as a proportion of the number of transmigrated leukocytes in the total area. Rolling velocities for three randomly-picked leukocytes per vessel and in total nine per experiment were obtained by timing the transit of the rolling leukocytes over a calibrated axial distance. At the beginning of each experiment, three post-capillary vessel segments in a central area of the spread-out cremaster muscle were chosen randomly by the observer among those that were at least 150 μm away from
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Light/dye-induced thrombosis in the cremaster microcirculation as well as observations of leukocyte recruitment were performed after surgical preparation of the cremaster muscle as originally described by Baez (1973) with minor modifications. Mice were anesthetized using a ketamine/xylazine mixture as mentioned above. The left femoral artery was cannulated in a retrograde manner for the administration of FITC-dextran and nanoparticles or vehicle. The right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally in a relatively avascular zone, using careful electrocautery to stop any bleeding, and spread over the pedestal of a custom-made microscopic stage. Epididymis and testicle were detached from the cremaster muscle and placed into the abdominal cavity. Throughout the surgical procedure and the intravital microscopy, the muscle was superfused with warm saline solution. At the end of each experiment, blood was collected from the heart for measurement of blood cell counts.
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Measurement of Thrombus Formation
M. Holzer et al. neighboring post-capillary venules and did not branch over a distance of at least 150 μm. After having obtained baseline recordings of leukocyte rolling, firm adhesion and transmigration in all three vessel segments, SWCNT or CB dispersions at a concentration of 1 mg kg–1 body weight (in a volume of 5 μl g–1 body weight), or vehicle (control) were injected through the catheter. Measurements, which took 5 min, respectively, were repeated at 5, 15, 30, 60 and 120 min after particle administration in the identical post-capillary vessel segments. Subacute effects were investigated in a separate set of experiments 4 h after particle application. As a positive control, TNF-α (recombinant murine tumor necrosis factor-alpha; R&D Systems, Wiesbaden, Germany) was locally administered by superfusion in acute experiments and intraperitoneally or systemically in subacute experiments (Zanardo et al., 2004). At the end of each experiment, centerline blood flow velocity was determined by measuring the distance between several images of one fluorescent bead (Invitrogen Corporation, Karlsruhe, Germany) under stroboscopic illumination. From measured vessel diameters and centerline blood flow velocity, apparent wall shear stress was calculated, assuming a parabolic flow velocity profile over the vessel cross-section (Tangelder et al., 1986). Finally blood samples were collected by cardiac puncture for the analysis of systemic leukocyte counts using a Coulter AcT counter (Coulter Corp., Miami, FL, USA). Statistical Analysis Data analysis of all experiments was performed with a statistical software package (SigmaStat for Windows, Jandel Scientific, Erkrath, Germany). The thrombus formation and the 4-h
leukocyte recruitment experiments in vivo were analyzed with Kruskal-Wallis one-way ANOVA on ranks followed by all pairwise multiple comparison procedures using the Student– Newman–Keuls method. The 120-min time-course experiments for leukocyte recruitment were analyzed with two-way repeated measures ANOVA followed again by all pairwise multiple comparison procedures using the Student–Newman–Keuls method. Data analysis of the in vitro experiments was performed with the t-test for paired samples. If the normality test failed, the Wilcoxon’s test on ranks was used instead. A P-value < 0.05 was considered as significant. Data are given as mean values ± standard error of mean (SEM).
Results Particle Characterization Scanning electron microscopy (SEM) and TEM revealed a typical fiber-like shape of SWCNTs and a spherical shape of CB (Fig. 1). The elemental composition of raw nanoparticle powder was measured with EDS and yielded 99 wt% of carbon with less than 0.9 wt% of residual metal catalyst (Fe, Co) for SWCNT whereas CB powder consisted of carbon ~100 wt%. The diameter and the polydispersity index of SWCNT and CB nanoparticles dispersed in physiological solutions as described in the Material and methods were determined by dynamic light scattering (Table 1). The average hydrodynamic equivalent diameters of dispersed nanoparticles were comparable at each concentration (~250 nm for SWCNT and ~130 nm for CB). The polydispersity index of SWCNT was higher than that of CB owing to the higher aspect ratio of these nanoparticles (Table 1). The zeta potentials
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Figure 1. Particle characterization using electron microscopy. Scanning electron microscopic images of (A) SWCNT and (B) CB, and transmission electron microscopic images of (C) SWCNT and (D) CB nanoparticles. Scale bar: 100 nm.
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Carbon-based nanomaterials accelerate arteriolar thrombus formation Table 1. Physical characterization of nanomaterials. Nanomaterials were prepared in distilled water at different concentrations (0.002–0.2 mg ml–1) by sonication and addition of serum albumin (final concentration 1.5 mg ml–1) and phosphate-buffered saline (PBS) (final concentrations of phosphate buffer and sodium chloride: 10 and 154 mM, respectively). Nanoparticle diameter, polydispersity index and zeta potential were measured with a Zetasizer-Nano ZS instrument. Results are given as means (± SEM). All measurements were done in triplicate. Particle SWCNT
CB
Concentration (mg ml–1)
Average diameter (nm)
0.2 0.02 0.002 0.2 0.02 0.002
231.90 ± 1.59 218.30 ± 0.50 250.87 ± 6.41 130.33 ± 0.78 129.00 ± 0.69 124.47 ± 2.18
were around –10 mV for both CBNs (Bihari et al., 2008b). The endotoxin content in SWCNT and CB dispersions was found to be below 0.5 EU ml–1. Light/dye-Induced Thrombosis in the Murine Cremasteric Microcirculation Microvascular thrombotic effects of nanoparticles were analyzed in the cremasteric microcirculation (Fig. 2). To assure comparable intravascular fluorescein isothiocyanate-dextran concentrations, intravascular fluorescence intensity was measured in each experiment. There were no significant differences in the fluorescent intensity among experimental groups (data not shown). Injection of SWCNT significantly decreased the cessation time in arterioles at doses of both 0.1 and 1 mg kg–1 body weight. Likewise, CB nanoparticles induced a decrease of the cessation time in arterioles, reaching statistical significance at the high dose. In contrast, injection of the two CBNs tested did not significantly alter arteriolar onset times. There were also no significant changes in the onset or cessation times measured in venules, suggesting that the prothrombotic effect of the CBNs was obviously too weak to further accelerate the already fast thrombus formation in venules. In Vitro Analysis of Human Platelet Activation For the assessment of human platelet activation by CBNs, platelet P-selectin expression (CD62P) and the amount of plateletgranulocyte complexes were measured (Table 2). SWCNT increased the mean fluorescence intensity of platelet P-selectin as compared with the control. Similarly, CB particles significantly enhanced platelet P-selectin expression. Moreover, SWCNT and CB significantly augmented the amount of platelet-granulocyte complexes as compared with the controls. ADP, a well-known platelet agonist, raised P-selectin expression as well as the amount of platelet–granulocyte complexes versus the control.
PdI 0.27 ± 0.01 0.38 ± 0.00 0.62 ± 0.07 0.14 ± 0.00 0.17 ± 0.00 0.34 ± 0.03
Zeta potential (mV) -9.09 ± 0. 15 -10.34 ± 0.49 -10.83 ± 0.92 -10.34 ± 0.26 -9.21 ± 0.67 -7.85 ± 0.58
rolling leukocytes initially increased. After 15 min, leukocyte rolling began to decrease, resulting in an even lower number of rolling leukocytes compared with baseline conditions (Fig. 3A). Superfusion of the cremaster muscle with TNF-α, a widely used stimulus for local induction of leukocyte recruitment (Zanardo et al., 2004), led to a slightly decreased number of rolling and significantly higher numbers of adherent and transmigrated leukocytes as compared with the control. Upon injection of SWCNT or CB at a dose of 1 mg kg–1 BW, however, no significant changes were detected in the numbers of rolling, adherent and transmigrated leukocytes during the whole course of the experiment. In a second set of experiments, the delayed effects of systemically injected SWCNT and CB on the different steps of leukocyte recruitment were analyzed 4 h after nanoparticle application (Fig. 4). Intraperitoneal injection of TNF-α, a cytokine commonly used to induce a systemic inflammation causing notably altered leukocyte traffic (Zanardo et al., 2004), resulted in an inflammatory response characterized by reduced numbers of rolling leukocytes, decreased rolling velocity, and significantly raised numbers of adherent and transmigrated cells. Similarly to TNF-α, injection of CBNs decreased the number of rolling leukocytes. However, in contrast to the action of TNF-α, where the decrease in the number of rolling leukocytes has been reported to be the result of reduced rolling velocities as well as of increased adhesion (Zanardo et al., 2004), CBN injection did not reduce rolling velocity nor increase adhesion or transmigration. Leukocyte Counts and Microhemodynamic Parameters After each experiment, blood was collected and systemic leukocyte counts and microhemodynamic parameters were measured. There were no statistically significant differences observed in systemic leukocyte counts, vessel diameter, mean blood flow velocity, or shear rate at 2 and 4 h after the administration of vehicle, SWCNT, CB, or TNF-α (Tables 3 and 4). In Vitro Analysis of Human Leukocyte Activation
In a first set of experiments, we assessed the immediate effects of systemically injected SWCNT and CB particles on leukocyte– endothelial cell interactions and leukocyte transmigration (Fig. 3). The surgical preparation of the cremaster muscle is well known to induce leukocyte recruitment in the cremasteric microcirculation (Kunkel et al., 1996). According to this, the numbers of
To determine leukocyte activation upon nanomaterial exposure, the expression of adhesion molecules was measured after incubation of human whole blood with CBNs in vitro by flow cytometry (Table 5). Lipopolysaccharide (LPS) as a positive control strongly increased the expression of CD11b, CD11c, and CD18 integrin proteins on granulocytes and monocytes. In
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Leukocyte–Endothelial Cell Interactions and Leukocyte Transmigration
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Figure 2. Light/dye-induced thrombosis in the cremasteric microcirculation. SWCNT and CB nanoparticles at doses of 0.01, 0.1, and 1 mg/kg body weight or vehicle were administered intra-arterially 10 min prior to the induction of thrombosis. Onset (black bars) and cessation times (white bars) are shown as means (± SEM) in cremasteric venules (A) and arterioles (C). Representative in vivo fluorescence microscopy images of a precapillary cremasteric arteriole undergoing light/dye-induced thrombosis show the onset thrombus formation (B, indicated by arrows) and cessation (D) of blood flow. Scale bar: 15 μm; n = 6; *p < 0.05 vs. vehicle.
Table 2. In vitro analysis of platelet activation (CD62P) and formation of platelet-granulocyte complexes (PGC). Human whole blood was incubated with PBS (control), ADP (1 μM), single-walled carbon nanotubes (SWCNT) or CB nanoparticles (0.1 mg ml–1) for 10 min at room temperature. Mean fluorescence intensity (MFI) of CD62P (P-selectin) on platelets (n = 29) and the percentage of platelet–granulocyte complexes (PGC) among granulocytes (n = 24) were measured by flow cytometry. Data are displayed as means ± SEM (*P < 0.05 vs. control). Control
SWCNT
CB
ADP
CD62P (MFI) 11.4 ± 0.9 21.4 ± 2.9* 22.2 ± 2.6* 62.3 ± 5.2* PGC (%) 30.8 ± 2.7 41.2 ± 4.5* 40.2 ± 4.6* 70.8 ± 3.3* contrast, incubation of whole blood with SWCNT or CB resulted in just a slight increase of expression of these activation markers on granulocytes and monocytes. Discussion
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The aim of this study was to analyze and compare the acute effects of two differently shaped CBNs (SWCNT and CB) in two different species (i) in the microcirculation of mice in vivo and (ii) in human whole blood in vitro. For analyzing prothrombotic effects in the murine microcirculation, the light/dye-induced thrombosis model was used (Rumbaut et al., 2005). Illumination of a vessel segment in the presence of a fluorescent dye leads to local generation of reactive oxygen species inducing prothrombotic changes in endothelial cells with subsequent
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growth of a luminal thrombus (Rumbaut et al., 2005). Arteriolar thrombus formation and vessel occlusion were accelerated in the presence of both CBNs, indicating that thrombus formation was enhanced independently of the CBN shape. Similar observations of prothrombotic effects driven by CBNs have been made by Nemmar et al. (2002, 2007) in larger vessels with multi-walled carbon nanotubes and also by Radomski et al. (2005) with SWCNT and CB. The doses used in our study (0.1 and 1 mg kg–1 body weight corresponding to about 2.5 and 25 μg per mouse) fall within the lower range of doses in previously published studies (Bai et al., 2010; Campagnolo et al., 2013; Radomski et al., 2005; Singh et al., 2006; Wang et al., 2013). The findings from our in vitro experiments with human cells corroborated the in vivo observations obtained in mice. In these in vitro measurements, platelet P-selectin expression and the number of platelet–leukocyte aggregates were increased after incubation of human whole blood with SWCNT or CB. The platelet-activating effect of carbon nanotubes has been reported to be caused by increased calcium influx (Lacerda et al., 2011; Semberova et al., 2009). As an explanation for an enhanced platelet aggregation, the formation of molecular bridges between platelets by SWCNTs has been suggested (Radomski et al., 2005). Moreover, when injected into the systemic circulation, NPs are immediately covered by a layer of blood proteins called protein corona (Aggarwal et al., 2009; Cedervall et al., 2007; Lynch and Dawson, 2008; Lundqvist et al., 2008; Tenzer et al., 2013). Adsorbed proteins influence the biological activity and the interaction of nanoparticles with the surrounding cells (Clift et al., 2010; Dutta et al., 2007). It has been shown that fibrinogen is one of the most frequently occurring proteins in the protein corona of SWCNTs (Meng et al.,
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Carbon-based nanomaterials accelerate arteriolar thrombus formation
Figure 3. Immediate effects of CBNs on the different steps of leukocyte recruitment in the cremasteric microcirculation. Leukocyte rolling (A), firm adhesion (B), and transmigration (C) were quantified in the cremaster muscle of mice by intravital microscopy. Measurements were carried out in three postcapillary venules at baseline and at 5, 15, 30, 60, and 120 min after intra-arterial administration of SWCNT or CB nanoparticles at a dose of 1 mg/kg body weight or vehicle. TNF-α (0.5 μg per mouse) added to the superfusion medium served as a positive control. Results are given as means (± SEM). A representative in vivo microscopy image shows a postcapillary venule of the cremasteric microcirculation (D; arrows indicate adherent, arrowheads transmigrated leukocytes counted in a defined region of interest covering 75 x 100 μm on both sides of the vessel; scale bar: 15 μm; n = 5; *p < 0.05 vs. vehicle, SWCNT, CB).
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Figure 4. Delayed effects of CBNs on the different steps leukocyte recruitment in the cremasteric microcirculation. SWCNT or CB nanoparticles at a dose of 1 mg/kg body weight or vehicle were administered intra-arterially. TNF-α (0.5 μg/mouse) was injected intraperitoneally 4h prior to observation of leukocyte behavior. Panels show results presented as means (± SEM) for leukocyte rolling (A), rolling velocity (B), firm adherence (C), and transmigration (D) (n = 7; #p < 0.05 vs. vehicle; *p < 0.05 vs. vehicle, SWCNT, CB)
M. Holzer et al. Table 3. Leukocyte counts and microhemodynamic parameters after administration of nanomaterials. Systemic leukocyte counts were measured in blood samples with a cell counter 2 and 4 h after application of vehicle, tumor necrosis factor-alpha (TNF-α) (20 mg kg–1 body weight), single-walled carbon nanotubes (SWCNT), or CB nanoparticles (1 mg kg–1 body weight). Venular diameter and centerline blood flow velocity (V mean) were obtained offline. Wall shear rate was calculated from vessel diameter and centerline blood flow velocity. Results are given as means ± SEM (n = 5–7).
Systemic leukocyte count (106 μl–1) Diameter (μm) V mean (mm s–1) Wall shear rate (s–1)
2h 4h 2h 4h 2h 4h 2h 4h
Vehicle
SWCNT
CB
TNF-α
4.4 ± 0.3 4.0 ± 0.5 25.8 ± 0.4 31.2 ± 0.3 1.3 ± 0.1 1.4 ± 0.2 1911.0 ± 201.0 2090.1 ± 249.7
3.8 ± 0.4 2.9 ± 0.2 25.3 ± 0.4 25.9 ± 0.8 1.3 ± 0.2 1.5 ± 0.2 2000.6 ± 324.8 2222.7 ± 313.3
4.4 ± 0.5 3.9 ± 0.8 25.2 ± 0.4 27.3 ± 1.8 1.4 ± 0.2 1.3 ± 0.2 2242.8 ± 277.4 1986.0 ± 167.6
4.1 ± 0.6 3.2 ± 0.4 26.3 ± 0.3 24.7 ± 0.5 1.5 ± 0.1 1.2 ± 0.2 2285.7 ± 164.5 1849.1 ± 233.6
Table 4. Surface expression of adhesion molecules on human monocytes and granulocytes upon incubation with carbon-based nanomaterials (CBNs). Human whole blood was incubated with phosphate-buffered saline (PBS) (control), lipopolysaccharide (LPS) (2 μg ml–1), single-walled carbon nanotubes (SWCNT), or CB nanoparticles (0.1 mg ml–1) for 60 min at room temperature. Expression of the leukocyte adhesion markers CD11b, CD11c, and CD18 were measured by flow cytometry. Averages (± SEM) of the mean fluorescence intensities (MFI) are given as a percentage of the corresponding PBS control values (n = 4–12; *P < 0.05 vs. control). Monocytes
CD11b (%) CD11c (%) CD18 (%)
SWCNT
CB
129 ± 4* 95 ± 4 99 ± 5
135 ± 5* 117 ± 8 122 ± 6*
Granulocytes LPS 188 ± 20* 219 ± 22* 155 ± 26*
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2005; Song et al., 2006) and CB (Kendall et al., 2011). Interestingly, inhalation as well as systemic administration of CB in mice resulted in fibrinogen and platelet deposition in postcapillary venules in the liver and heart (Khandoga et al., 2004, 2010). These findings suggest that fibrinogen-covered CBNs may accelerate thrombus formation through activation and binding of platelets. Recently, Guidetti et al. (2012) reported that both CB and multiwalled carbon nanotubes stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in integrin αIIbβ3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. As the authors also found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, they conclude from these findings that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. In a separate set of experiments, we investigated whether differently shaped CBNs given at the same concentration that accelerated arteriolar thrombus formation would also alter leukocyte recruitment, a complex cascade of subsequent steps consisting of leukocyte rolling on the endothelial cell layer, firm adhesion to endothelial cells, and transmigration of the leukocytes through the vessel wall into the subendothelial tissue (Ley et al., 2007). Interestingly, we did not found any effect of systemically administered SWCNT or CB on the different steps of the leukocyte recruitment cascade within the first 2 h after nanomaterial injection, although our in vitro experiments revealed slight leukocyte activation upon incubation of whole
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SWCNT
CB
124 ± 7* 99 ± 6 108 ± 3*
103 ± 3 115 ± 5* 141 ± 10*
LPS 271 ± 42* 164 ± 18* 220 ± 30*
blood with CBNs. Most probably, this effect is not strong enough or overwhelmed by other factors and, therefore, does not lead to increased leukocyte–endothelial interactions in vivo. In an additional set of experiments, we administered the CBNs 4 h prior to intravital microscopy, thereby allowing the upregulation of inflammatory genes of endothelial cells and leukocytes (Erdely et al., 2009). Again, injection of CBNs did not cause an inflammatory response in terms of an increase in leukocyte adhesion or transmigration. Interestingly, the model inherent, spontaneous leukocyte rolling was significantly attenuated 4 h after injection of SWCNT or CB, indicating that the presence of CBNs somehow interferes with this early step of the leukocyte recruitment cascade by a so far unknown mechanism. In contrast to the inflammatory stimulus TNF-α, however, this attenuation of leukocyte rolling was not accompanied by an increase in the subsequent steps of the leukocyte recruitment cascade, leukocyte adhesion and transendothelial migration. These findings are in good agreement with other studies on the biodistribution and biocompatibility of intravascularly injected CBNs that do not provide evidence for inflammatory processes either in short-term or in long-term observations (Al Faraj et al., 2011; Khandoga et al., 2004; Tang et al., 2012; Yang et al., 2008). In summary, our study shows that the systemic administration of two differently shaped CBNs of comparable size and surface area, SWCNT and CB, augmented arteriolar thrombus formation in the murine microcirculation in vivo and induced activation of human platelets in vitro. In contrast, the CBNs tested did not induce early leukocyte–endothelial cell interactions or leukocyte transendothelial migration in the murine microcirculation in vivo, and they only slightly activated human leukocytes
Copyright © 2014 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 2014; 34: 1167–1176
Carbon-based nanomaterials accelerate arteriolar thrombus formation in vitro. These findings suggest that CBNs may exert prothrombotic effects in the microcirculation and that these effects are independent of the CBNs’ shape. As a consequence, potential prothrombotic properties of CBNs should be carefully investigated when designed as systemically administered drug delivery systems. Acknowledgements This study was supported by European Commission grant NMPTCT-2006-032777 (NANOSH). The views and opinions expressed in this publication do not necessarily reflect those of the European Commission. Data presented in this manuscript are part of the doctoral thesis of M.H.
Conflict of Interest The authors declare that they are no conflicts of interests.
References
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J. Appl. Toxicol. 2014; 34: 1167–1176
Revised: 6 January 2014,
Accepted: 16 January 2014
Published online in Wiley Online Library: 14 February 2014
(wileyonlinelibrary.com) DOI 10.1002/jat.2996
Carbon-based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape Martin Holzera, Peter Biharia, Marc Praetnera, Bernd Uhla, Christoph Reichelb, Janos Fentc, Minnamari Vippolad,e, Susan Lakatosc and Fritz Krombacha* ABSTRACT: Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg–1 body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg–1 body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte– endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs’ shape, but does not induce leukocyte–endothelial cell interactions or leukocyte transmigration. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: nanoparticles; carbon nanotubes; carbon black; thrombosis; platelets; inflammation; leukocytes; microcirculation; in vivo microscopy
Introduction
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*Correspondence to: F. Krombach, Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, 81377 Munich, Germany. E-mail: [email protected] a Walter Brendel Centre of Experimental Medicine, Ludwig-MaximiliansUniversität München, 81377 Munich, Germany b Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum der Universität München, 81377 Munich, Germany c Research Institute, Medical Centre, Hungarian Defence Forces, Budapest, Hungary d Department of Materials Science, Tampere University of Technology, 33101 Tampere, Finland e
Finnish Institute of Occupational Health, 00250, Helsinki, Finland
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Nanoparticles (NPs) are defined as those materials with at least one dimension in the range of 1–100 nm (Oberdorster et al., 2005b). Carbon-based nanomaterials (CBNs) include materials such as fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical carbon black (CB) (Terrones and Terrones, 2003). The unique properties of CBNs make them interesting for new industrial or medical applications, but on the other hand raise questions about potential health effects (Maynard et al., 2006). The administration routes of CBNs engineered for medical purposes are either local injection into the target tissue or intravenous injection. Moreover, it has recently been shown that inhaled carbon nanotubes, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time after exposure (Mercer et al., 2013). Once within the bloodstream, nanoparticles will interact with blood cells, serum proteins, and endothelial cells (Shi et al., 2011). Finally, they will be distributed throughout the body and, on their way, pass the microcirculation of tissues or organs (Oberdorster et al., 2005a; Nel et al., 2006). Differently shaped carbon-based nanoparticles such as spherical ultrafine carbon black (CB) or fiber-shaped SWCNT activate platelets in vitro. In addition, SWCNT are able to accelerate thrombus formation in the macro- and microcirculation in vivo (Bihari et al., 2010; Radomski et al., 2005). Although administration of CB has been found to induce fibrinogen and platelet deposition in postcapillary venules
(Khandoga et al., 2004, 2010), the impact of CB on microcirculatory thrombus formation has not yet been studied. In contrast, thrombotic process can influence inflammation (Wagner and Burger, 2003; Zarbock et al., 2007), and experimental data highlight the inflammatory potential of fiber-shaped CNTs in extracirculatory tissues after inhalation or intraperitoneal injection (Krug and Wick, 2011; Maynard et al., 2011; Poland et al., 2008). Although blood-borne nanoparticles will reach and pass the microcirculation, it is insufficiently investigated whether they have the potential to modulate leukocyte–endothelial cell
M. Holzer et al. interactions in microvessels. An inflammatory response is characterized by recruitment of leukocytes from the microcirculation to the site of trauma or infection (Ley et al., 2007). It is a complex cascade of different subsequent steps consisting of leukocyte rolling on the endothelial cell layer, firm adhesion mediated by integrins and adhesion molecules, and finally transmigration of the leukocytes through the vessel wall into the subendothelial tissue (Ley et al., 2007). Previous studies of our group did not detect increased leukocyte–endothelial interactions in hepatic microvessels upon intra-arterial injection or inhalation of CB (Khandoga et al., 2004, 2010). Whether systemically administered SWCNT have acute effects on leukocyte–endothelial interactions or leukocyte transmigration has not yet been investigated. Furthermore, there is no experimental data about the role of the shape of CBNs for proinflammatory and prothrombotic effects in microvessels, although shape is well known as an important factor for the behavior of nanomaterials in biological environments (Oberdorster et al., 2005a; Nel et al., 2006) . Thus, the aims of this study were to analyze and compare the early effects of two differently shaped CBNs (SWCNT and CB) (i) on thrombus formation as well as on leukocyte–endothelial cell interactions and leukocyte transmigration in the murine microcirculation in vivo and, (ii) on the activation of human platelets and leukocytes in vitro.
870 μl of dispersion before the addition of 100 μl of a 10× concentrated phosphate-buffered saline (PBS; Sigma–Aldrich, Schnelldorf, Germany) solution, yielding a pH of 7.4, and final concentrations of phosphate buffer and sodium chloride of 10 and 154 mM, respectively. To prepare dispersions at lower concentrations, dilutions of the stock solution were used. The final concentrations of nanoparticles in the dispersion were 0.002, 0.02 and 0.2 mg ml–1. The vehicle was prepared in the same way, but instead of nanomaterial the same volume of distilled water was added to the dispersion (Bihari et al., 2008b). To determine the quality of the dispersions, measurements of nanoparticle diameter, polydispersity index and zeta potential were conducted with a Zetasizer-Nano ZS instrument (Malvern, Malvern Hills, UK). Nanoparticle dispersions prepared by the above method were tested for the presence of LPS with the LAL (Limulus amoebocyte lysate) kinetic chromogenic assay (Lonza, Verviers, Belgium).
Methods
Analysis of Human Platelet Activation
Nanomaterials S-purified single-walled carbon nanotubes (SWCNT), outer diameter: < 2 nm, length: 1–5 μm were purchased from SES Research (Houston, TX, USA). Ultrafine carbon black particles (Printex 90; diameter: 14 nm) were obtained from Evonik-Degussa (Essen, Germany). Characterization of Nanoparticles The characterization of the nanomaterials included size and morphology analysis with scanning (SEM, Zeiss ULTRAplus FEG-SEM; Carl Zeiss NTS GmbH, Oberkochen, Germany) and transmission electron microscopy (TEM, Jeol JEM 2010; Jeol, Tokyo, Japan). The composition of the nanoparticles was determined by energy dispersive spectroscopy (EDS, ThermoNoran Vantage, Thermo Scientific, Breda, The Netherlands) attached to the TEM. The elemental composition is given as an average of five separate EDS measurements. The specific surface area of the CBNs used in this study was previously measured using the BET method with a Coulter Omnisorb 100 CX gas adsorption analyzer (Miami, FL, USA). The adsorbed gas was nitrogen and the temperature was 77.36 K. The specific surface area of SWCNT and CB was 436 and 265 m2 g–1, respectively (Vippola et al., 2009). Preparation of Particle Dispersions
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SWCNT or CB stock solutions were prepared at a concentration of 0.23 mg ml–1 in distilled water using sonication with 4.2 × 105 kJ m–3 specific energy. For in vitro experiments with human blood, 30 μl of 50 mg ml–1 human serum albumin (final concentration 1.5 mg ml–1) and for in vivo mouse experiments 30 μl of mouse serum (preparation see below) was added to
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Human Blood Sampling Human blood was collected from the cubital vein of 44 healthy volunteers into citrate anticoagulant-containing Vacuette test tubes (Greiner, Kremsmünster, Austria). All samples were obtained with the approval of the local Ethical Committee after the donor had given informed consent.
The effect of nanomaterials on platelet activation in human whole blood was determined by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ, USA). 100 μl of citrated blood was either incubated with 100 μl of vehicle or 100 μl of SWCNT or CB dispersion at a concentration of 0.2 mg ml–1 for 10 min at room temperature. After incubation, samples were diluted five-fold with 0.35% bovine serum albumin containing Tyrodes’ buffer: BSA-Tyr (10 mM Hepes, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, 5.5 mM glucose, pH 7.4). As a positive control, vehicle-containing samples were incubated with 1 μM ADP (final concentration) for 10 min. Samples were stained according to the manufacturer’s instructions. To measure P-selectin expression on platelets, whole blood was incubated with PE-labeled anti-CD62P antibodies (Dako, Glostrup, Denmark) and FITC-labeled antiCD41 antibodies (Immunotech, Marseilles, France). To check non-specific binding of the antibodies, appropriate isotype control antibodies were used. After staining, samples were diluted 50-fold with BSA-Tyr buffer (500-fold final dilution of blood). To minimize the spontaneous activation of platelets, no washing steps were used. The CD41 platelet marker was used as a trigger signal for data collection. The platelets were gated on the FS-SS dot plot and the mean CD62P fluorescence intensity (MFI) was measured for CD41 positive events. To measure platelet-granulocyte aggregates, FITC-labeled anti-CD41 and PC5-labeled anti-CD15 (Beckman-Coulter, Krefeld, Germany) antibodies were used. Platelet-granulocyte aggregates were detected as double-positive events in the granulocyte gate. Here the CD15-PC5 granulocyte marker was used as a trigger signal. As a 500-fold dilution of blood was used in these measurements, the coincidence of platelets and granulocytes did not result in false-double positivity (Bihari et al., 2008a). The amount of platelet-granulocyte aggregates was determined as the percentage of CD41 positivity in the CD15-positive gate.
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J. Appl. Toxicol. 2014; 34: 1167–1176
Carbon-based nanomaterials accelerate arteriolar thrombus formation Surface Expression of Adhesion Molecules on Human Monocytes and on Granulocytes The effect of nanomaterials on the surface expression of CD11b, CD11c and CD18 on monocytes and on granulocytes in human whole blood was determined by flow cytometry (FACScan; Becton Dickinson). Next, 100 μl of citrated blood was incubated with 100 μl of a 0.2 mg ml–1 nanomaterial dispersion or vehicle for 60 min at room temperature. After incubation, samples were stained according to the manufacturer’s instructions. Briefly, 50 μl of samples was stained with the appropriate amount of monoclonal antibodies (PE-labeled anti-CD11b and FITC-labeled anti-CD11c: Dako, Denmark; FITC-labeled antiCD18: Immunotech, Beckman-Coulter; and PerCp-labeled antiCD14: Becton Dickinson) for 30 min at room temperature in the dark, then lysed with FACSLyse (Becton Dickinson) solution and washed twice with 0.5% bovine serum albumin containing phosphate buffer saline (pH 7.4). To check non-specific binding of the antibodies, appropriate isotype control antibodies were used. Data collection was triggered by the forward scatter (FS) signal. Monocytes were separated from granulocytes on the FS-SS dot plot based on their CD14 staining. Animals C57BL/6NCrl mice were purchased from Charles River (Sulzfeld, Germany). The experiments were performed with male mice with a body weight of 20–27 g. Animals were housed in Makrolon cages under constant temperature (22 ± 2 °C), humidity (55 ± 5 %) and light conditions (12-h cycle) with free access to pelleted food and water. All experiments were approved by the local authorities and performed according to the German legislation for the protection of animals.
Light/dye-induced thrombosis was performed as described earlier (Rumbaut et al., 2005) with slight modifications. The light intensity at 488 nm was daily measured by a photodiode at the exit of the light source and maintained at between 2.65 and 2.75 mA. After surgical preparation of the cremaster muscle, SWCNT or CB dispersions at concentrations of 0.01, 0.1 and 1 mg kg–1 body weight (in a volume of 5 μl g–1 body weight) or vehicle (control) were injected through the catheter. The nanomaterials were administered 10 min prior to induction of thrombosis in order to yield a uniform distribution of the particles in the microcirculation. Thereafter, 4 ml kg–1 body weight of a 2.5% solution of FITC-dextran (fluorescein isothiocyanate dextran 150 kD obtained from Sigma-Aldrich, Schnelldorf, Germany) was given. To verify comparable intravascular FITC concentrations among experimental groups, digital images were taken from each utilized vessel with the CCD camera mentioned above. Ten minutes after the application of nanomaterials, photoactivation was induced by exposing a vessel segment of 300 μm length to continuous epi-illumination with a wavelength of 488 nm. An Olympus water immersion lens (60×/NA 0.9) was used to focus the light onto the cremaster and to obtain fluorescent images. Thrombus formation was quantified in one arteriole (25–35 μm) and one venule (30–50 μm) by analyzing the time when platelets became adherent to the vessel wall (defined as time of onset of thrombus formation) and the time required for complete occlusion of the vessel (defined as time to cessation of blood flow). Cessation time in vessels without complete occlusion until the end of the recording was considered 20 min for venules and 40 min for arterioles.
Analysis of Leukocyte–Endothelial Cell Interactions and Leukocyte Transmigration
Preparation of Mouse Serum Mice were anesthetized with a ketamine/xylazine mixture (100 mg kg–1 ketamine and 10 mg kg–1 xylazine) administered by intraperitoneal injection. Blood was taken by heart puncture and allowed to clot. The blood was centrifuged with 1100 g for 20 min and the supernatant was taken. Serum samples were pooled and aliquots were stored at 20 °C until use. Surgical Procedure
Leukocyte recruitment was analyzed using the reflected-light oblique illumination technique (Mempel et al., 2003). A mirrored surface (reflector) was positioned directly below the specimen, its angle tilted relative to the horizontal plane. The reflector consisted of a round cover glass (thickness 0.19–0.22 mm, diameter 11.8 mm), which was coated with aluminum vapor (Freichel, Kaufbeuren, Germany) and brought into direct contact with the overlying specimen. For offline analysis of parameters describing the sequential steps of leukocyte extravasation CapImage image analysis software (Dr Zeintl, Heidelberg, Germany) was used. Rolling leukocytes were defined as those moving slower than the associated blood flow and were quantified for 30 s. Firmly adherent cells were determined as those resting in the associated blood flow for more than 30 s and related to the luminal surface per 100-μm vessel length. Transmigrated cells were counted in regions of interest covering 75 μm on both sides of a vessel over 100-μm vessel length. Leukocyte accumulation in each segment was quantified and shown as a proportion of the number of transmigrated leukocytes in the total area. Rolling velocities for three randomly-picked leukocytes per vessel and in total nine per experiment were obtained by timing the transit of the rolling leukocytes over a calibrated axial distance. At the beginning of each experiment, three post-capillary vessel segments in a central area of the spread-out cremaster muscle were chosen randomly by the observer among those that were at least 150 μm away from
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Light/dye-induced thrombosis in the cremaster microcirculation as well as observations of leukocyte recruitment were performed after surgical preparation of the cremaster muscle as originally described by Baez (1973) with minor modifications. Mice were anesthetized using a ketamine/xylazine mixture as mentioned above. The left femoral artery was cannulated in a retrograde manner for the administration of FITC-dextran and nanoparticles or vehicle. The right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally in a relatively avascular zone, using careful electrocautery to stop any bleeding, and spread over the pedestal of a custom-made microscopic stage. Epididymis and testicle were detached from the cremaster muscle and placed into the abdominal cavity. Throughout the surgical procedure and the intravital microscopy, the muscle was superfused with warm saline solution. At the end of each experiment, blood was collected from the heart for measurement of blood cell counts.
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Measurement of Thrombus Formation
M. Holzer et al. neighboring post-capillary venules and did not branch over a distance of at least 150 μm. After having obtained baseline recordings of leukocyte rolling, firm adhesion and transmigration in all three vessel segments, SWCNT or CB dispersions at a concentration of 1 mg kg–1 body weight (in a volume of 5 μl g–1 body weight), or vehicle (control) were injected through the catheter. Measurements, which took 5 min, respectively, were repeated at 5, 15, 30, 60 and 120 min after particle administration in the identical post-capillary vessel segments. Subacute effects were investigated in a separate set of experiments 4 h after particle application. As a positive control, TNF-α (recombinant murine tumor necrosis factor-alpha; R&D Systems, Wiesbaden, Germany) was locally administered by superfusion in acute experiments and intraperitoneally or systemically in subacute experiments (Zanardo et al., 2004). At the end of each experiment, centerline blood flow velocity was determined by measuring the distance between several images of one fluorescent bead (Invitrogen Corporation, Karlsruhe, Germany) under stroboscopic illumination. From measured vessel diameters and centerline blood flow velocity, apparent wall shear stress was calculated, assuming a parabolic flow velocity profile over the vessel cross-section (Tangelder et al., 1986). Finally blood samples were collected by cardiac puncture for the analysis of systemic leukocyte counts using a Coulter AcT counter (Coulter Corp., Miami, FL, USA). Statistical Analysis Data analysis of all experiments was performed with a statistical software package (SigmaStat for Windows, Jandel Scientific, Erkrath, Germany). The thrombus formation and the 4-h
leukocyte recruitment experiments in vivo were analyzed with Kruskal-Wallis one-way ANOVA on ranks followed by all pairwise multiple comparison procedures using the Student– Newman–Keuls method. The 120-min time-course experiments for leukocyte recruitment were analyzed with two-way repeated measures ANOVA followed again by all pairwise multiple comparison procedures using the Student–Newman–Keuls method. Data analysis of the in vitro experiments was performed with the t-test for paired samples. If the normality test failed, the Wilcoxon’s test on ranks was used instead. A P-value < 0.05 was considered as significant. Data are given as mean values ± standard error of mean (SEM).
Results Particle Characterization Scanning electron microscopy (SEM) and TEM revealed a typical fiber-like shape of SWCNTs and a spherical shape of CB (Fig. 1). The elemental composition of raw nanoparticle powder was measured with EDS and yielded 99 wt% of carbon with less than 0.9 wt% of residual metal catalyst (Fe, Co) for SWCNT whereas CB powder consisted of carbon ~100 wt%. The diameter and the polydispersity index of SWCNT and CB nanoparticles dispersed in physiological solutions as described in the Material and methods were determined by dynamic light scattering (Table 1). The average hydrodynamic equivalent diameters of dispersed nanoparticles were comparable at each concentration (~250 nm for SWCNT and ~130 nm for CB). The polydispersity index of SWCNT was higher than that of CB owing to the higher aspect ratio of these nanoparticles (Table 1). The zeta potentials
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Figure 1. Particle characterization using electron microscopy. Scanning electron microscopic images of (A) SWCNT and (B) CB, and transmission electron microscopic images of (C) SWCNT and (D) CB nanoparticles. Scale bar: 100 nm.
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Carbon-based nanomaterials accelerate arteriolar thrombus formation Table 1. Physical characterization of nanomaterials. Nanomaterials were prepared in distilled water at different concentrations (0.002–0.2 mg ml–1) by sonication and addition of serum albumin (final concentration 1.5 mg ml–1) and phosphate-buffered saline (PBS) (final concentrations of phosphate buffer and sodium chloride: 10 and 154 mM, respectively). Nanoparticle diameter, polydispersity index and zeta potential were measured with a Zetasizer-Nano ZS instrument. Results are given as means (± SEM). All measurements were done in triplicate. Particle SWCNT
CB
Concentration (mg ml–1)
Average diameter (nm)
0.2 0.02 0.002 0.2 0.02 0.002
231.90 ± 1.59 218.30 ± 0.50 250.87 ± 6.41 130.33 ± 0.78 129.00 ± 0.69 124.47 ± 2.18
were around –10 mV for both CBNs (Bihari et al., 2008b). The endotoxin content in SWCNT and CB dispersions was found to be below 0.5 EU ml–1. Light/dye-Induced Thrombosis in the Murine Cremasteric Microcirculation Microvascular thrombotic effects of nanoparticles were analyzed in the cremasteric microcirculation (Fig. 2). To assure comparable intravascular fluorescein isothiocyanate-dextran concentrations, intravascular fluorescence intensity was measured in each experiment. There were no significant differences in the fluorescent intensity among experimental groups (data not shown). Injection of SWCNT significantly decreased the cessation time in arterioles at doses of both 0.1 and 1 mg kg–1 body weight. Likewise, CB nanoparticles induced a decrease of the cessation time in arterioles, reaching statistical significance at the high dose. In contrast, injection of the two CBNs tested did not significantly alter arteriolar onset times. There were also no significant changes in the onset or cessation times measured in venules, suggesting that the prothrombotic effect of the CBNs was obviously too weak to further accelerate the already fast thrombus formation in venules. In Vitro Analysis of Human Platelet Activation For the assessment of human platelet activation by CBNs, platelet P-selectin expression (CD62P) and the amount of plateletgranulocyte complexes were measured (Table 2). SWCNT increased the mean fluorescence intensity of platelet P-selectin as compared with the control. Similarly, CB particles significantly enhanced platelet P-selectin expression. Moreover, SWCNT and CB significantly augmented the amount of platelet-granulocyte complexes as compared with the controls. ADP, a well-known platelet agonist, raised P-selectin expression as well as the amount of platelet–granulocyte complexes versus the control.
PdI 0.27 ± 0.01 0.38 ± 0.00 0.62 ± 0.07 0.14 ± 0.00 0.17 ± 0.00 0.34 ± 0.03
Zeta potential (mV) -9.09 ± 0. 15 -10.34 ± 0.49 -10.83 ± 0.92 -10.34 ± 0.26 -9.21 ± 0.67 -7.85 ± 0.58
rolling leukocytes initially increased. After 15 min, leukocyte rolling began to decrease, resulting in an even lower number of rolling leukocytes compared with baseline conditions (Fig. 3A). Superfusion of the cremaster muscle with TNF-α, a widely used stimulus for local induction of leukocyte recruitment (Zanardo et al., 2004), led to a slightly decreased number of rolling and significantly higher numbers of adherent and transmigrated leukocytes as compared with the control. Upon injection of SWCNT or CB at a dose of 1 mg kg–1 BW, however, no significant changes were detected in the numbers of rolling, adherent and transmigrated leukocytes during the whole course of the experiment. In a second set of experiments, the delayed effects of systemically injected SWCNT and CB on the different steps of leukocyte recruitment were analyzed 4 h after nanoparticle application (Fig. 4). Intraperitoneal injection of TNF-α, a cytokine commonly used to induce a systemic inflammation causing notably altered leukocyte traffic (Zanardo et al., 2004), resulted in an inflammatory response characterized by reduced numbers of rolling leukocytes, decreased rolling velocity, and significantly raised numbers of adherent and transmigrated cells. Similarly to TNF-α, injection of CBNs decreased the number of rolling leukocytes. However, in contrast to the action of TNF-α, where the decrease in the number of rolling leukocytes has been reported to be the result of reduced rolling velocities as well as of increased adhesion (Zanardo et al., 2004), CBN injection did not reduce rolling velocity nor increase adhesion or transmigration. Leukocyte Counts and Microhemodynamic Parameters After each experiment, blood was collected and systemic leukocyte counts and microhemodynamic parameters were measured. There were no statistically significant differences observed in systemic leukocyte counts, vessel diameter, mean blood flow velocity, or shear rate at 2 and 4 h after the administration of vehicle, SWCNT, CB, or TNF-α (Tables 3 and 4). In Vitro Analysis of Human Leukocyte Activation
In a first set of experiments, we assessed the immediate effects of systemically injected SWCNT and CB particles on leukocyte– endothelial cell interactions and leukocyte transmigration (Fig. 3). The surgical preparation of the cremaster muscle is well known to induce leukocyte recruitment in the cremasteric microcirculation (Kunkel et al., 1996). According to this, the numbers of
To determine leukocyte activation upon nanomaterial exposure, the expression of adhesion molecules was measured after incubation of human whole blood with CBNs in vitro by flow cytometry (Table 5). Lipopolysaccharide (LPS) as a positive control strongly increased the expression of CD11b, CD11c, and CD18 integrin proteins on granulocytes and monocytes. In
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Leukocyte–Endothelial Cell Interactions and Leukocyte Transmigration
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Figure 2. Light/dye-induced thrombosis in the cremasteric microcirculation. SWCNT and CB nanoparticles at doses of 0.01, 0.1, and 1 mg/kg body weight or vehicle were administered intra-arterially 10 min prior to the induction of thrombosis. Onset (black bars) and cessation times (white bars) are shown as means (± SEM) in cremasteric venules (A) and arterioles (C). Representative in vivo fluorescence microscopy images of a precapillary cremasteric arteriole undergoing light/dye-induced thrombosis show the onset thrombus formation (B, indicated by arrows) and cessation (D) of blood flow. Scale bar: 15 μm; n = 6; *p < 0.05 vs. vehicle.
Table 2. In vitro analysis of platelet activation (CD62P) and formation of platelet-granulocyte complexes (PGC). Human whole blood was incubated with PBS (control), ADP (1 μM), single-walled carbon nanotubes (SWCNT) or CB nanoparticles (0.1 mg ml–1) for 10 min at room temperature. Mean fluorescence intensity (MFI) of CD62P (P-selectin) on platelets (n = 29) and the percentage of platelet–granulocyte complexes (PGC) among granulocytes (n = 24) were measured by flow cytometry. Data are displayed as means ± SEM (*P < 0.05 vs. control). Control
SWCNT
CB
ADP
CD62P (MFI) 11.4 ± 0.9 21.4 ± 2.9* 22.2 ± 2.6* 62.3 ± 5.2* PGC (%) 30.8 ± 2.7 41.2 ± 4.5* 40.2 ± 4.6* 70.8 ± 3.3* contrast, incubation of whole blood with SWCNT or CB resulted in just a slight increase of expression of these activation markers on granulocytes and monocytes. Discussion
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The aim of this study was to analyze and compare the acute effects of two differently shaped CBNs (SWCNT and CB) in two different species (i) in the microcirculation of mice in vivo and (ii) in human whole blood in vitro. For analyzing prothrombotic effects in the murine microcirculation, the light/dye-induced thrombosis model was used (Rumbaut et al., 2005). Illumination of a vessel segment in the presence of a fluorescent dye leads to local generation of reactive oxygen species inducing prothrombotic changes in endothelial cells with subsequent
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growth of a luminal thrombus (Rumbaut et al., 2005). Arteriolar thrombus formation and vessel occlusion were accelerated in the presence of both CBNs, indicating that thrombus formation was enhanced independently of the CBN shape. Similar observations of prothrombotic effects driven by CBNs have been made by Nemmar et al. (2002, 2007) in larger vessels with multi-walled carbon nanotubes and also by Radomski et al. (2005) with SWCNT and CB. The doses used in our study (0.1 and 1 mg kg–1 body weight corresponding to about 2.5 and 25 μg per mouse) fall within the lower range of doses in previously published studies (Bai et al., 2010; Campagnolo et al., 2013; Radomski et al., 2005; Singh et al., 2006; Wang et al., 2013). The findings from our in vitro experiments with human cells corroborated the in vivo observations obtained in mice. In these in vitro measurements, platelet P-selectin expression and the number of platelet–leukocyte aggregates were increased after incubation of human whole blood with SWCNT or CB. The platelet-activating effect of carbon nanotubes has been reported to be caused by increased calcium influx (Lacerda et al., 2011; Semberova et al., 2009). As an explanation for an enhanced platelet aggregation, the formation of molecular bridges between platelets by SWCNTs has been suggested (Radomski et al., 2005). Moreover, when injected into the systemic circulation, NPs are immediately covered by a layer of blood proteins called protein corona (Aggarwal et al., 2009; Cedervall et al., 2007; Lynch and Dawson, 2008; Lundqvist et al., 2008; Tenzer et al., 2013). Adsorbed proteins influence the biological activity and the interaction of nanoparticles with the surrounding cells (Clift et al., 2010; Dutta et al., 2007). It has been shown that fibrinogen is one of the most frequently occurring proteins in the protein corona of SWCNTs (Meng et al.,
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Carbon-based nanomaterials accelerate arteriolar thrombus formation
Figure 3. Immediate effects of CBNs on the different steps of leukocyte recruitment in the cremasteric microcirculation. Leukocyte rolling (A), firm adhesion (B), and transmigration (C) were quantified in the cremaster muscle of mice by intravital microscopy. Measurements were carried out in three postcapillary venules at baseline and at 5, 15, 30, 60, and 120 min after intra-arterial administration of SWCNT or CB nanoparticles at a dose of 1 mg/kg body weight or vehicle. TNF-α (0.5 μg per mouse) added to the superfusion medium served as a positive control. Results are given as means (± SEM). A representative in vivo microscopy image shows a postcapillary venule of the cremasteric microcirculation (D; arrows indicate adherent, arrowheads transmigrated leukocytes counted in a defined region of interest covering 75 x 100 μm on both sides of the vessel; scale bar: 15 μm; n = 5; *p < 0.05 vs. vehicle, SWCNT, CB).
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Figure 4. Delayed effects of CBNs on the different steps leukocyte recruitment in the cremasteric microcirculation. SWCNT or CB nanoparticles at a dose of 1 mg/kg body weight or vehicle were administered intra-arterially. TNF-α (0.5 μg/mouse) was injected intraperitoneally 4h prior to observation of leukocyte behavior. Panels show results presented as means (± SEM) for leukocyte rolling (A), rolling velocity (B), firm adherence (C), and transmigration (D) (n = 7; #p < 0.05 vs. vehicle; *p < 0.05 vs. vehicle, SWCNT, CB)
M. Holzer et al. Table 3. Leukocyte counts and microhemodynamic parameters after administration of nanomaterials. Systemic leukocyte counts were measured in blood samples with a cell counter 2 and 4 h after application of vehicle, tumor necrosis factor-alpha (TNF-α) (20 mg kg–1 body weight), single-walled carbon nanotubes (SWCNT), or CB nanoparticles (1 mg kg–1 body weight). Venular diameter and centerline blood flow velocity (V mean) were obtained offline. Wall shear rate was calculated from vessel diameter and centerline blood flow velocity. Results are given as means ± SEM (n = 5–7).
Systemic leukocyte count (106 μl–1) Diameter (μm) V mean (mm s–1) Wall shear rate (s–1)
2h 4h 2h 4h 2h 4h 2h 4h
Vehicle
SWCNT
CB
TNF-α
4.4 ± 0.3 4.0 ± 0.5 25.8 ± 0.4 31.2 ± 0.3 1.3 ± 0.1 1.4 ± 0.2 1911.0 ± 201.0 2090.1 ± 249.7
3.8 ± 0.4 2.9 ± 0.2 25.3 ± 0.4 25.9 ± 0.8 1.3 ± 0.2 1.5 ± 0.2 2000.6 ± 324.8 2222.7 ± 313.3
4.4 ± 0.5 3.9 ± 0.8 25.2 ± 0.4 27.3 ± 1.8 1.4 ± 0.2 1.3 ± 0.2 2242.8 ± 277.4 1986.0 ± 167.6
4.1 ± 0.6 3.2 ± 0.4 26.3 ± 0.3 24.7 ± 0.5 1.5 ± 0.1 1.2 ± 0.2 2285.7 ± 164.5 1849.1 ± 233.6
Table 4. Surface expression of adhesion molecules on human monocytes and granulocytes upon incubation with carbon-based nanomaterials (CBNs). Human whole blood was incubated with phosphate-buffered saline (PBS) (control), lipopolysaccharide (LPS) (2 μg ml–1), single-walled carbon nanotubes (SWCNT), or CB nanoparticles (0.1 mg ml–1) for 60 min at room temperature. Expression of the leukocyte adhesion markers CD11b, CD11c, and CD18 were measured by flow cytometry. Averages (± SEM) of the mean fluorescence intensities (MFI) are given as a percentage of the corresponding PBS control values (n = 4–12; *P < 0.05 vs. control). Monocytes
CD11b (%) CD11c (%) CD18 (%)
SWCNT
CB
129 ± 4* 95 ± 4 99 ± 5
135 ± 5* 117 ± 8 122 ± 6*
Granulocytes LPS 188 ± 20* 219 ± 22* 155 ± 26*
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2005; Song et al., 2006) and CB (Kendall et al., 2011). Interestingly, inhalation as well as systemic administration of CB in mice resulted in fibrinogen and platelet deposition in postcapillary venules in the liver and heart (Khandoga et al., 2004, 2010). These findings suggest that fibrinogen-covered CBNs may accelerate thrombus formation through activation and binding of platelets. Recently, Guidetti et al. (2012) reported that both CB and multiwalled carbon nanotubes stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in integrin αIIbβ3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. As the authors also found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, they conclude from these findings that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. In a separate set of experiments, we investigated whether differently shaped CBNs given at the same concentration that accelerated arteriolar thrombus formation would also alter leukocyte recruitment, a complex cascade of subsequent steps consisting of leukocyte rolling on the endothelial cell layer, firm adhesion to endothelial cells, and transmigration of the leukocytes through the vessel wall into the subendothelial tissue (Ley et al., 2007). Interestingly, we did not found any effect of systemically administered SWCNT or CB on the different steps of the leukocyte recruitment cascade within the first 2 h after nanomaterial injection, although our in vitro experiments revealed slight leukocyte activation upon incubation of whole
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SWCNT
CB
124 ± 7* 99 ± 6 108 ± 3*
103 ± 3 115 ± 5* 141 ± 10*
LPS 271 ± 42* 164 ± 18* 220 ± 30*
blood with CBNs. Most probably, this effect is not strong enough or overwhelmed by other factors and, therefore, does not lead to increased leukocyte–endothelial interactions in vivo. In an additional set of experiments, we administered the CBNs 4 h prior to intravital microscopy, thereby allowing the upregulation of inflammatory genes of endothelial cells and leukocytes (Erdely et al., 2009). Again, injection of CBNs did not cause an inflammatory response in terms of an increase in leukocyte adhesion or transmigration. Interestingly, the model inherent, spontaneous leukocyte rolling was significantly attenuated 4 h after injection of SWCNT or CB, indicating that the presence of CBNs somehow interferes with this early step of the leukocyte recruitment cascade by a so far unknown mechanism. In contrast to the inflammatory stimulus TNF-α, however, this attenuation of leukocyte rolling was not accompanied by an increase in the subsequent steps of the leukocyte recruitment cascade, leukocyte adhesion and transendothelial migration. These findings are in good agreement with other studies on the biodistribution and biocompatibility of intravascularly injected CBNs that do not provide evidence for inflammatory processes either in short-term or in long-term observations (Al Faraj et al., 2011; Khandoga et al., 2004; Tang et al., 2012; Yang et al., 2008). In summary, our study shows that the systemic administration of two differently shaped CBNs of comparable size and surface area, SWCNT and CB, augmented arteriolar thrombus formation in the murine microcirculation in vivo and induced activation of human platelets in vitro. In contrast, the CBNs tested did not induce early leukocyte–endothelial cell interactions or leukocyte transendothelial migration in the murine microcirculation in vivo, and they only slightly activated human leukocytes
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Carbon-based nanomaterials accelerate arteriolar thrombus formation in vitro. These findings suggest that CBNs may exert prothrombotic effects in the microcirculation and that these effects are independent of the CBNs’ shape. As a consequence, potential prothrombotic properties of CBNs should be carefully investigated when designed as systemically administered drug delivery systems. Acknowledgements This study was supported by European Commission grant NMPTCT-2006-032777 (NANOSH). The views and opinions expressed in this publication do not necessarily reflect those of the European Commission. Data presented in this manuscript are part of the doctoral thesis of M.H.
Conflict of Interest The authors declare that they are no conflicts of interests.
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