Toxicology in Vitro 29 (2015) 1132–1139
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In vitro platelet activation, aggregation and platelet–granulocyte complex formation induced by surface modified single-walled carbon nanotubes János Fent a, Péter Bihari a, Minnamari Vippola b,c, Essi Sarlin b, Susan Lakatos a,⇑ a b c
Department of Pathophysiology, Scientific Research Institute, Military Hospital, Hungarian Defence Forces, Budapest, Hungary Department of Materials Science, Tampere University of Technology, Tampere, Finland Finnish Institute of Occupational Health, Helsinki, Finland
a r t i c l e
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Article history: Received 15 May 2014 Accepted 22 April 2015 Available online 5 May 2015 Keywords: Engineered nanomaterials Pegylated single-walled carbon nanotubes CD62P SWCNTs Surface modified nanotubes
a b s t r a c t Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet–granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1 mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet–granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The utilization of carbon nanotubes (CNTs) has been suggested for various fields of nanomedicine, such as specific delivery of bioactive molecules, drugs or contrast materials (Sakamoto et al., 2010; Zhang et al., 2012b). CNTs used for human therapy or diagnostics need to form a stable dispersion in physiological solutions and must not be toxic. Increased dispersibility can be achieved by attaching various functional groups to the surface of CNTs, like CONH2, COOH, OH, or PEG-molecules (Imasaka et al., 2009; Heister et al., 2010; Wang et al., 2012). However, any kind of Abbreviations: CNT, carbon nanotube; SWCNT, single-walled carbon nanotube; p-SWCNT, pristine single-walled carbon nanotube; COOH–SWCNT, carboxylated single-walled carbon nanotube; OH–SWCNT, hydroxylated single-walled carbon nanotube; CONH2–SWCNT, amidated single-walled carbon nanotube; PEG–SWCNT, pegylated single-walled carbon nanotube; MFI, mean fluorescence intensity; SE, standard error of mean; SEM, scanning electron microscope; TEM, transmission electron microscope; EDS, energy dispersive X-ray spectroscopy. ⇑ Corresponding author at: H-1134 Budapest, Róbert Károly krt. 44, Hungary. Tel.: +36 1 465 1800; fax: +36 1 465 1851. E-mail address: [email protected] (S. Lakatos). http://dx.doi.org/10.1016/j.tiv.2015.04.017 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
surface modification of CNTs may affect their pharmaco-kinetic properties (Lam et al., 2006; Zhang et al., 2012a) and thus their toxicity. Pegylation is a very effective tool in increasing the dispersibility of CNTs in aqueous media (Hadidi et al., 2011). Its effect has been studied the most among the many surface modification procedures as a mean to shield CNTs against phagocytosis and to lessen their toxicity (Bottini et al., 2011; Yang et al., 2012). The effect of CNTs either pristine or surface modified has been studied using various cell lines (Albini et al., 2010; Gutierrez-Praena et al., 2011; Jos et al., 2009; Montes-Fonseca et al., 2012; Pichardo et al., 2012). Importantly, most biomedical applications (Bottini et al., 2011; Chen et al., 2008; Cheng et al., 2011; Kolosnjaj-Tabi et al., 2010; Lam et al., 2006; Perán et al., 2012; Peretz and Regev, 2012) result in a close contact between blood cells and CNTs. One of the consequences of the interaction between blood cells and nanotubes is that certain CNTs affect the hemostasis (Sokolov et al., 2011) especially the platelet function (Guidetti et al., 2012; Radomski et al., 2005). Indeed, the in vivo thrombogenic potential of pristine CNTs in animal models has been reported (Bihari et al., 2010; Radomski et al., 2005). The CNTs also
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induce formation of heterotypic platelet-leukocyte aggregates, mainly via platelet activation. This kind of interaction plays an important role in platelet dependent granulocyte activation and migration (Kornerup et al., 2010), thus initiating crosstalk with the innate immune system (Li et al., 2000; Speth et al., 2013). Although functionalized CNTs have been shown to circulate longer in blood than their pristine counterpart (Hadidi et al., 2011; Heister et al., 2010), their effect on platelet function has been investigated less. Only a few data are available to date on functionalized CNTs in terms of their platelet activating potential (Burke et al., 2011; Meng et al., 2012; Sokolov et al., 2011). In addition, we could not reference any study dealing with the effect of the various functionalized carbon nanotubes exerted on the platelet– granulocyte complex formation. Thus the aim of our recent study was to judge the in vitro toxicity of several surface modified single walled CNTs through their potential to induce platelet activation, platelet aggregation and platelet–granulocyte complex formation. 2. Materials and methods 2.1. Single-walled carbon nanotubes studied Single-walled carbon nanotubes (SWCNTs) and its various surface modified forms were purchased from the following companies with characteristics as indicated on the technical sheets attached: pristine SWCNTs (p-SWCNTs, cat.# 900-1351, Outer diameter < 2 nm, length: 1–5 lm, purity: >90% CNTs and >50% SWCNTs) – SES Research (Houston, USA); pegylated SWCNTs (PEG–SWCNTs, cat.# 652474, diameter 4–5 nm, Length 0.5–0.6 lm, Purity: >80% carbon basis, 4–5% trace metals) and amidated SWCNTs (CONH2–SWCNTs, cat.# 685380, diameter 4–6 nm, length 0.7–1.0 lm, purity: >90% carbon basis, 5–8% trace metals) – Sigma–Aldrich; carboxylated SWCNTs (COOH–SWCNTs, cat.# 1288YJF, Average diameter 1–2 nm, length 5–30 lm, purity: >95% CNTs, >90% SWCNTs, –COOH: 2.59–2.87 wt%) – NanoAmor (Los Alamos, USA) and hydroxylated SWCNTs (OH–SWCNTs, cat.# SO-SL1, Outer diameter 1–2 nm, length: 3–8 lm, purity: >98 wt% SWCNTs, OH: 2–4 wt%) – NanoShel (Delaware, USA). 2.2. Characterization of nanotubes The morphology of studied nanotubes was characterized both with Zeiss ULTRAplus scanning electron microscope (SEM) and with Jeol JEM 2010 transmission electron microscope (TEM). In the case of SEM, very low accelerating voltage (3 kV) was used in order to analyze samples without any coating procedure. In the case of TEM, the accelerating voltage was 120 kV. Energy-dispersive X-ray spectroscopic (EDS) analyses were carried out with Thermonoran Vantage EDS-analyzer attached on the TEM. Theoretical detection limit of EDS analyzer is Z > 5 (boron) and element content > 0.1 wt%. Nanotube samples were prepared from each SWCNTs powder by crushing them gently between glass slides and mixing the powder either to ethanol or to distilled water and applying a drop onto copper grid or copper–holey carbon layer grid sample holder for scanning and transmission electron microscopy, respectively. 2.3. Preparation and characterization of nanotube dispersions Stock solutions of all the SWCNTs samples were prepared according to Bihari et al. (2008b) in distilled water using sonication with 4.2 105 kJ/m3 specific energy in three consecutive steps for 2 min each. After the first sonication human serum albumin at final concentration of 1.5 mg/ml was added followed by a second
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sonication with the same parameters as in the first time. Before the third sonication step, the physiological ionic strength of the sample was attained by adding a proper volume of 10 concentrated phosphate buffered saline solution (Sigma, cat.# P5493) to ensure a final phosphate buffer concentration of 0.01 M and a NaCl concentration of 0.154 M, pH 7.4. The final concentrations of stock nanotube dispersions were 0.2 mg/ml and 0.5 mg/ml for flow cytometry and for aggregometry measurements, respectively. Control sample was prepared in the same way as nanotube dispersions replacing nanotubes by distilled water. The 0.2 mg/ml stock nanotube dispersions were characterized by their polydispersity indices (PdI) and zeta-potentials measured by dynamic light scattering and by laser Doppler velocimetry, respectively, using a Zetasiter Nano-ZS instrument (Malvern, UK). The main component and the Z-average of the equivalent hydrodynamic diameters of various nanotubes were obtained from the dynamic light scattering data as well. 2.4. Human blood samples Human blood was collected from the cubital vein of healthy volunteers into Vacuette test tubes containing trisodium-citrate anticoagulant (Greiner, Austria, Kremsmünster) in accordance with the approval policy of the local Ethical Committee after the donor had given informed consent. Blood counts of samples involved in this study were in the normal range (160–320 109/L). 2.5. Flow cytometry 100 ll of citrated blood was incubated with 100 ll of 0.2 mg/ml nanotube dispersion at room temperature for 10 min followed by another 5-fold dilution with Tyrode-HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, pH 7.4) containing 5.5 mM glucose and 0.35% bovine serum albumin. For control measurements nanotube dispersion was replaced by control solution. 1 lM ADP (Chrono-Log, USA) served as positive control for platelet activation. Platelet activation was characterized by changes in the surface expression of CD62P (P-selectin), the platelet activation marker. Platelets in the whole blood were stained with PE-labeled anti-CD62P (Dako, Glostrup, Denmark) and FITC-labeled anti-CD41 (Immunotech, Marseilles, France) antibodies according to the manufacturer’s instructions. Aspecific staining of samples was checked by using appropriate isotype controls. After staining samples were diluted with Tyrode-HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, pH 7.4) containing 5.5 mM glucose and 0.35% bovine serum albumin in such an extent that a 250-fold final dilution of blood was attained. 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, and platelets were gated on the forward-side scatter dot plot and the mean CD62P fluorescence intensity (MFI) was measured. When the platelet–granulocyte complex formation was analyzed blood samples were treated in the same way as in the case of platelet activation experiments. Platelet–granulocyte complexes were measured as described previously (Bihari et al., 2008a; Fent et al., 2008). Briefly: whole blood was stained with FITC-labeled anti-CD41 (Immunotech, Marseilles, France) and Cy5-labeled anti-CD15 (BioLegend, San Diego, USA) antibodies. 500-fold dilution of blood was used in these measurements, thus coincidence of platelets and granulocytes did not result in false double positivity. The CD15 granulocyte marker was used as trigger signal for data collection. The amount of platelet–granulocyte complexes was determined as percentage of CD41 positive events in the
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granulocyte gate. All samples were run on a FACScan (Becton Dickinson, USA) flow cytometer. 2.6. Aggregometry In vitro aggregation of human platelets in whole blood was detected with a two-channel Chronolog Whole Blood Lumi-Aggregometer type 560 C (Chrono-Log, Havertown, USA) by measuring impedance change in plastic cuvettes at 37 °C with continuous stirring (1000 rpm). The reaction mixture contained 800 ll of citrated blood and 200 ll of control or 0.5 mg/ml stock nanotube dispersion. Since aggregation measurements are relatively long (30– 40 min) and the blood ages, so each time an age-matched control was measured with addition of collagen (3 lg/ml final concentration) to check the aggregation ability of blood. Data were collected both with a two-channel recorder and a computer. The nanotube induced aggregation was characterized by the impedance change with baseline correction detected at 30 min after the addition of the nanotube solution. 2.7. Statistical analysis All experimental values are presented as mean ± SE. The polydispersity indices and Z-average data of various SWCNTs were
compared by one-way ANOVA. Tukey test was used at all pairwise multiple comparison procedures. In platelet activation and platelet–granulocyte complex formation tests statistical significances were evaluated by the paired Student’s t-test against the matched controls. If normality test failed, Wilcoxon test (SigmaStat, ver. 3.0, SPSS Inc., USA) was used instead. Aggregation experiments were evaluated by the one sample Student’s t-test after subtracting controls. Normality was checked by the Kolmogorov–Smirnov test. A p < 0.05 value was considered statistically significant. 3. Results 3.1. Characteristics of nanotubes and nanotube dispersions The morphologies of the nanotubes studied by SEM and TEM are presented in Figs. 1 and 2. According to the images SWCNTs with amide and PEG modifications consist mainly of amorphous matrix material (with light elements) where the carbon nanotubes are embedded. Similar type of amorphous matrix material is also present in SWCNTs with OH modification. It can be concluded that the amount of surface modification material is high in these surface modifications and they contribute significantly to the physical appearance of carbon nanotube material. In the case of pristine SWCNTs and COOH–SWCNTs the carbon nanotube morphology appears conventional where material appears as entangled
Fig. 1. SWCNTs morphology by SEM.
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Fig. 2. SWCNTs morphology by TEM and elemental composition by TEM–EDS.
Table 1 Dynamic light scattering data (mean ± SE) of the SWCNT samples.
p-SWCNT (n = 38) CONH2–SWCNT (n = 18) COOH–SWCNT (n = 18) OH–SWCNT (n = 10) PEG–SWCNT (n = 32)
Main peak (d.nm)
% Of main peak area
PdI
Z-average (d.nm)
166.04 ± 3.01 68.49 ± 5.73 228.92 ± 11.29 340.42 ± 26.79 57.51 ± 4.37
95.79 ± 1.72 94.12 ± 1.07 97.01 ± 1.98 97.67 ± 2.33 98.38 ± 0.35
0.5 ± 0.02 0.55 ± 0.01 0.48 ± 0.05 0.72 ± 0.04* 0.48 ± 0.02
329.6 ± 13.5 378.23 ± 20.06 416.70 ± 29.98 813.10 ± 66.91§ 236.06 ± 16.6#
PdI: polydispersity index. * Significantly higher (p < 0.05) than all the other PdI values. § Significantly higher (p < 0.05) than all the other Z-average values. # Significantly lower (p < 0.05) than all the other Z-average values.
1 – potential (mV) (n = 3) 10.34 ± 0.49 12.17 ± 0.38 9.42 ± 1.18 11.47 ± 0.79 12.23 ± 0.55
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undetectable in the presence of 10-times diluted (final concentration: 0.01 mg/ml) SWCNTs (data not shown), with an exception of PEG–SWCNTs, which even at 0.01 mg/ml concentration induces well detectable platelet activation as well (Fig. 4). Fig. 5 shows the time dependence of platelet activation in the presence of 0.1 mg/ml PEG–SWCNTs for 8 donors. Although the extent of the platelet activation exerted by PEG–SWCNTs varies from donors to donors, its maximum is around 60 min for all of them. In case of CONH2–SWCNTs, COOH–SWCNTs and p-SWCNTs the time dependence of platelet activation has the same characteristics as in the case of PEG–SWCNTs (not shown). In the presence of OH–SWCNTs the expression of CD62P remains at the control level within the experimental error during a 2-h period. Fig. 3. Averages ± SE of CD62P mean fluorescence intensities for the CD41 positive events measured in whole blood in the presence of 1 lM ADP (striped columns) or in the presence of 0.1 mg/ml of various functionalized carbon nanotubes (filled columns), as indicated on the abscissa (n P 19). ⁄ denotes significant differences (p < 0.001) relative to the control (dotted columns) obtained from paired statistical tests.
nanotube clusters. EDS analysis indicated that all samples consist mainly of carbon (more than 90 wt%) but they also include various other residual elements in small quantities (Fig. 2). Due to detection limitations of EDS analyzer only qualitative elemental analysis of these residual elements was achieved. The main component and the Z-average of the equivalent hydrodynamic diameters, the polydispersity indices and the zeta-potentials of the nanotube suspensions studied are compiled in Table 1. PEG–SWCNTs have significantly (p < 0.05) the lowest Z-average diameter among the SWCNTs studied. The OH–SWCNTs have far the highest Z-average hydrodynamic diameter and polydispersity index. Both are significantly (p < 0.05) different from those characterizing all the other SWCNTs. The other polydispersity indices do not differ significantly from each other. The zeta potential values are practically the same for all the SWCNTs studied.
3.3. Formation of platelet–granulocyte complexes in the presence of various SWCNTs Fig. 6 shows the amount of platelet–granulocyte complexes as the percent of double (CD15 and CD41) positive events in the CD15 positive granulocyte gate. Every SWCNTs tested but OH substituted ones induced a significant increase in the amount of these complexes relative to the control. 3.4. Aggregation of platelets in the presence of various SWCNTs The aggregating effect of different SWCNTs involved in our study is summarized in Table 2. OH–SWCNTs and the CONH2–SWCNTs have practically no detectable aggregating effect at a concentration of 0.1 mg/ml, while those of the pristine
3.2. Activation of platelets in the presence of various SWCNTs In vitro activation of platelets in whole blood was characterized by changes in the surface expression of CD62P, an early and reliable platelet activation marker (Lu and Malinauskas, 2011). Fig. 3 shows the mean fluorescence intensities for CD62P (average ± SE) measured in the presence of various single-walled carbon nanotubes as indicated on the abscissa. ADP, the physiological activator of platelets was used as positive control. Each SWCNTs tested but OH substituted derivative induced a significant increase in the surface expression of CD62P on platelets at 0.1 mg/ml final concentrations (Fig. 3). The extent of platelet activation becomes
Fig. 4. Averages ± SE of CD62P mean fluorescence intensities for the CD41 positive platelets measured in whole blood in the presence of 1 lM ADP or in the presence of different concentrations of PEG–SWCNTs as indicated on the abscissa (n = 12). ⁄ denotes significant differences (p < 0.01) relative to the control obtained from paired statistical tests.
Fig. 5. Time dependence of CD62P expression on platelets (corrected with the respective controls) in the case of 8 donors in the presence of 0.1 mg/ml of PEG–SWCNTs. Experimental points were fitted with quadratic polynomials.
Fig. 6. Average per cents ± SE of granulocyte–platelet complexes among the granulocytes in the presence of 1 lM ADP (striped columns) or in the presence of 0.1 mg/ml of various carbon nanotubes (filled columns) as indicated on the abscissa (n P 13). ⁄ denotes significant differences (p < 0.001) relative to the control (dotted columns) obtained from paired statistical tests.
J. Fent et al. / Toxicology in Vitro 29 (2015) 1132–1139 Table 2 Whole blood aggregation measured as impedance change (mean ± SE) from the control baseline at 30 min after addition of collagen or various single-walled CNTs as stimuli.
*
Stimulus
N
Impedance change (ohm)
Collagen p-SWCNT CONH2–SWCNT COOH–SWCNT OH–SWCNT PEG–SWCNT
30 6 6 6 6 6
14.6 ± 0.7* 3.8 ± 0.8* 0.2 ± 0.5 3.9 ± 0.9* 0.2 ± 0.6 7.1 ± 1.4*
Denotes significant (p < 0.01) differences from zero.
Fig. 7. Typical impedance changes in the whole blood as a function of time upon addition of 3 lg/ml collagen (1) or 0.1 mg/ml PEG–SWCNTs (2). At the moment when aggregation was initialized a calibration signal (20 Ohm) was also recorded.
SWCNTs, COOH–SWCNTs and PEG–SWCNTs are well detectable, the latter having the most pronounced effect (Table 2). The aggregation curves have lag phases and are more elongated than that of induced by 3 lg/ml of collagen. The extent of aggregating effect exerted by pristine SWCNTs, COOH–SWCNTs and PEG–SWCNTs varies among the individual blood donors. Fig. 7 demonstrates a typical aggregation curve registered in whole blood in the presence of 0.1 mg/ml PEG–SWCNTs. 4. Discussion According to the literature and our previous data, pristine carbon nanotubes activate platelets (Guidetti et al., 2012; Lacerda et al., 2008; Semberova et al., 2009), promote platelet–granulocyte complex formation (Holzer et al., 2014) and have thrombogenic potential in animal models (Bihari et al., 2010; Radomski et al., 2005). Attaching various functional groups to the surface of CNTs aims to lessen their toxicity (Kunzmann et al., 2011) and at the same time to increase their dispersibility. In this work we tested 4 surface modified carbon nanotubes and a pristine one obtained from various manufacturers in terms of their in vitro effects exerted on platelets in whole blood. The dose of pristine and modified carbon nanotubes applied in our study is based on various literature data describing different exposure scenarios (Semberova et al., 2009; Radomski et al., 2005; Maynard et al., 2004; Brouwer, 2010; Nemmar et al., 2003; Czarny et al., 2014). According to the above literature data one can estimate that inhaled ambient carbon nanotubes can get into the blood circulation in concentrations around 0.05–0.2 mg/ml. Therefore, we used 0.1 mg/ml carbon nanotube concentrations in our in vitro study. Based on literature data on the half-life of SWCNTs in the circulation of various animals one can suppose that the concentration of SWCNTs is not diluted substantially in circulation in the first 10 min (Cherukuri et al., 2006; Shing et al., 2006). Furthermore,
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according to Holzer et al. a 10-min presence of p-SWCNTs in blood circulation of mice augmented the light/dye induced thrombosis (Holzer et al., 2014). Therefore, in accordance with our previous experience (Khandoga et al., 2010) we analyzed platelet activation in the whole blood upon SWCNTs treatments at ten minutes. The most interesting finding of our study is that PEG–SWCNTs have the most pronounced platelet activating and aggregating effects among the SWCNTs tested by us although according to literature data pegylation seems to be the most advantageous modification of carbon nanotubes in terms of pharmacokinetic and toxicological profiles (Bottini et al., 2011; Hadidi et al., 2011; Heister et al., 2010; Yang et al., 2012). PEG–SWCNTs strongly promoted the platelet–granulocyte complex formation in the human whole blood as well (Fig. 6). PEG–SWCNTs even at 0.01 mg/ml concentration induces CD62P platelet activation marker expression in whole blood (Fig. 4) which concentration is already a non-effective dose for all the other SWCNTs studied. It is noteworthy that all the carbon nanotube samples when added to whole blood contained HSA. Vakhrusheva and her coworkers (Vakhrusheva et al., 2013) claimed that HSA has protective effect against platelet activation of SWCNTs. The zeta potential of HSA in phosphate buffer between pH 6.4–8.4 is between 7 and 14 mV (Tousi et al., 2011). The zeta potentials of our samples are in the range of 10 mV (Table 1) suggesting that the surface all of our SWCNT samples are covered but with unknown amount of HSA. Vakhrusheva and her coworkers (Vakhrusheva et al., 2013), showed that PEG–SWCNTs adsorb much less (0.5 w/w) HSA than the carboxylated ones (>2.4 w/w). This feature of PEG–SWCNTs might be the reason of their extremely high platelet activation ability. Our aggregometric results support the view that platelet activation in the presence of SWCNTs if any is a time consuming multistep complex process (Guidetti et al., 2012; Semberova et al., 2009). ADP, one of the classical platelet agonists, induces platelet activation within several minutes (Milton et al., 1980) while in the presence of PEG–SWCNTs about 60 min are needed for the maximal activation (Fig. 5) which is reflected in the extended lag phases of platelet aggregations in the presence of PEG–SWCNTs (Fig. 7). The same results were obtained with p-SWCNTs and COOH–SWCNTs, but not with OH–SWCNTs or CONH2–SWCNTs which have no whole-blood aggregating effect (Table 2). This latter finding is hard to explain and needs further clarification since CONH2–SWCNTs itself induces CD62P expression and strongly promotes platelet–granulocyte complex formation (Figs. 3 and 6). Although no correlation could be demonstrated between the size parameters and parameters characterizing the platelet activating effect of our SWCNT samples there are still some remarkable data in Table 1. PEG–SWCNTs being most effective in terms of platelet activation (induction of CD62P expression) have far the lowest size parameters: the mean of the main component and Z-average of the equivalent hydrodynamic diameter. OH–SWCNTs which practically have no effects on platelets and on the platelet–granulocyte complex formation have the highest Z-average equivalent hydrodynamic diameter, the highest PdI as well as the highest mean equivalent hydrodynamic diameter of the main component. Neither the Z-average equivalent hydrodynamic diameters nor the main component diameters correlated with the length parameters of our samples provided by the manufacturer. According to SEM and TEM data significant amount of amorphous matrix is present not only in the OH–SWCNTs and CONH2–SWCNTs but in the PEG–SWCNTs as well; thus this amorphous matrix constituent might not be responsible for the differences experienced in the effects of various SWCNT derivatives exerted on whole blood. Furthermore, the very similar element composition of SWCNT derivatives detected by EDS (within
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detection limits of the analyzer, Fig. 2) does not give any hint to explain their different behavior either. Physico-chemical parameters (SEM, TEM, EDS, Z-average diameter, polydispersity index, zeta-potential) do not seem to be sufficient to predict the distinct effects of various single-walled carbon nanotubes in whole blood. According to our findings several methods (e.g. flow cytometry and aggregometry) need to be combined to judge their complex effects exerted on platelets. This is even more important since in spite of that, surface modifications of carbon nanotubes aim to increase biocompatibility the resulting products may still exhibit unexpected disadvantageous platelet activation in whole blood. Conflict of Interest The authors declare that are no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
In vitro platelet activation, aggregation and platelet–granulocyte complex formation induced by surface modified single-walled carbon nanotubes János Fent a, Péter Bihari a, Minnamari Vippola b,c, Essi Sarlin b, Susan Lakatos a,⇑ a b c
Department of Pathophysiology, Scientific Research Institute, Military Hospital, Hungarian Defence Forces, Budapest, Hungary Department of Materials Science, Tampere University of Technology, Tampere, Finland Finnish Institute of Occupational Health, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 22 April 2015 Available online 5 May 2015 Keywords: Engineered nanomaterials Pegylated single-walled carbon nanotubes CD62P SWCNTs Surface modified nanotubes
a b s t r a c t Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet–granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1 mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet–granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The utilization of carbon nanotubes (CNTs) has been suggested for various fields of nanomedicine, such as specific delivery of bioactive molecules, drugs or contrast materials (Sakamoto et al., 2010; Zhang et al., 2012b). CNTs used for human therapy or diagnostics need to form a stable dispersion in physiological solutions and must not be toxic. Increased dispersibility can be achieved by attaching various functional groups to the surface of CNTs, like CONH2, COOH, OH, or PEG-molecules (Imasaka et al., 2009; Heister et al., 2010; Wang et al., 2012). However, any kind of Abbreviations: CNT, carbon nanotube; SWCNT, single-walled carbon nanotube; p-SWCNT, pristine single-walled carbon nanotube; COOH–SWCNT, carboxylated single-walled carbon nanotube; OH–SWCNT, hydroxylated single-walled carbon nanotube; CONH2–SWCNT, amidated single-walled carbon nanotube; PEG–SWCNT, pegylated single-walled carbon nanotube; MFI, mean fluorescence intensity; SE, standard error of mean; SEM, scanning electron microscope; TEM, transmission electron microscope; EDS, energy dispersive X-ray spectroscopy. ⇑ Corresponding author at: H-1134 Budapest, Róbert Károly krt. 44, Hungary. Tel.: +36 1 465 1800; fax: +36 1 465 1851. E-mail address: [email protected] (S. Lakatos). http://dx.doi.org/10.1016/j.tiv.2015.04.017 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
surface modification of CNTs may affect their pharmaco-kinetic properties (Lam et al., 2006; Zhang et al., 2012a) and thus their toxicity. Pegylation is a very effective tool in increasing the dispersibility of CNTs in aqueous media (Hadidi et al., 2011). Its effect has been studied the most among the many surface modification procedures as a mean to shield CNTs against phagocytosis and to lessen their toxicity (Bottini et al., 2011; Yang et al., 2012). The effect of CNTs either pristine or surface modified has been studied using various cell lines (Albini et al., 2010; Gutierrez-Praena et al., 2011; Jos et al., 2009; Montes-Fonseca et al., 2012; Pichardo et al., 2012). Importantly, most biomedical applications (Bottini et al., 2011; Chen et al., 2008; Cheng et al., 2011; Kolosnjaj-Tabi et al., 2010; Lam et al., 2006; Perán et al., 2012; Peretz and Regev, 2012) result in a close contact between blood cells and CNTs. One of the consequences of the interaction between blood cells and nanotubes is that certain CNTs affect the hemostasis (Sokolov et al., 2011) especially the platelet function (Guidetti et al., 2012; Radomski et al., 2005). Indeed, the in vivo thrombogenic potential of pristine CNTs in animal models has been reported (Bihari et al., 2010; Radomski et al., 2005). The CNTs also
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induce formation of heterotypic platelet-leukocyte aggregates, mainly via platelet activation. This kind of interaction plays an important role in platelet dependent granulocyte activation and migration (Kornerup et al., 2010), thus initiating crosstalk with the innate immune system (Li et al., 2000; Speth et al., 2013). Although functionalized CNTs have been shown to circulate longer in blood than their pristine counterpart (Hadidi et al., 2011; Heister et al., 2010), their effect on platelet function has been investigated less. Only a few data are available to date on functionalized CNTs in terms of their platelet activating potential (Burke et al., 2011; Meng et al., 2012; Sokolov et al., 2011). In addition, we could not reference any study dealing with the effect of the various functionalized carbon nanotubes exerted on the platelet– granulocyte complex formation. Thus the aim of our recent study was to judge the in vitro toxicity of several surface modified single walled CNTs through their potential to induce platelet activation, platelet aggregation and platelet–granulocyte complex formation. 2. Materials and methods 2.1. Single-walled carbon nanotubes studied Single-walled carbon nanotubes (SWCNTs) and its various surface modified forms were purchased from the following companies with characteristics as indicated on the technical sheets attached: pristine SWCNTs (p-SWCNTs, cat.# 900-1351, Outer diameter < 2 nm, length: 1–5 lm, purity: >90% CNTs and >50% SWCNTs) – SES Research (Houston, USA); pegylated SWCNTs (PEG–SWCNTs, cat.# 652474, diameter 4–5 nm, Length 0.5–0.6 lm, Purity: >80% carbon basis, 4–5% trace metals) and amidated SWCNTs (CONH2–SWCNTs, cat.# 685380, diameter 4–6 nm, length 0.7–1.0 lm, purity: >90% carbon basis, 5–8% trace metals) – Sigma–Aldrich; carboxylated SWCNTs (COOH–SWCNTs, cat.# 1288YJF, Average diameter 1–2 nm, length 5–30 lm, purity: >95% CNTs, >90% SWCNTs, –COOH: 2.59–2.87 wt%) – NanoAmor (Los Alamos, USA) and hydroxylated SWCNTs (OH–SWCNTs, cat.# SO-SL1, Outer diameter 1–2 nm, length: 3–8 lm, purity: >98 wt% SWCNTs, OH: 2–4 wt%) – NanoShel (Delaware, USA). 2.2. Characterization of nanotubes The morphology of studied nanotubes was characterized both with Zeiss ULTRAplus scanning electron microscope (SEM) and with Jeol JEM 2010 transmission electron microscope (TEM). In the case of SEM, very low accelerating voltage (3 kV) was used in order to analyze samples without any coating procedure. In the case of TEM, the accelerating voltage was 120 kV. Energy-dispersive X-ray spectroscopic (EDS) analyses were carried out with Thermonoran Vantage EDS-analyzer attached on the TEM. Theoretical detection limit of EDS analyzer is Z > 5 (boron) and element content > 0.1 wt%. Nanotube samples were prepared from each SWCNTs powder by crushing them gently between glass slides and mixing the powder either to ethanol or to distilled water and applying a drop onto copper grid or copper–holey carbon layer grid sample holder for scanning and transmission electron microscopy, respectively. 2.3. Preparation and characterization of nanotube dispersions Stock solutions of all the SWCNTs samples were prepared according to Bihari et al. (2008b) in distilled water using sonication with 4.2 105 kJ/m3 specific energy in three consecutive steps for 2 min each. After the first sonication human serum albumin at final concentration of 1.5 mg/ml was added followed by a second
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sonication with the same parameters as in the first time. Before the third sonication step, the physiological ionic strength of the sample was attained by adding a proper volume of 10 concentrated phosphate buffered saline solution (Sigma, cat.# P5493) to ensure a final phosphate buffer concentration of 0.01 M and a NaCl concentration of 0.154 M, pH 7.4. The final concentrations of stock nanotube dispersions were 0.2 mg/ml and 0.5 mg/ml for flow cytometry and for aggregometry measurements, respectively. Control sample was prepared in the same way as nanotube dispersions replacing nanotubes by distilled water. The 0.2 mg/ml stock nanotube dispersions were characterized by their polydispersity indices (PdI) and zeta-potentials measured by dynamic light scattering and by laser Doppler velocimetry, respectively, using a Zetasiter Nano-ZS instrument (Malvern, UK). The main component and the Z-average of the equivalent hydrodynamic diameters of various nanotubes were obtained from the dynamic light scattering data as well. 2.4. Human blood samples Human blood was collected from the cubital vein of healthy volunteers into Vacuette test tubes containing trisodium-citrate anticoagulant (Greiner, Austria, Kremsmünster) in accordance with the approval policy of the local Ethical Committee after the donor had given informed consent. Blood counts of samples involved in this study were in the normal range (160–320 109/L). 2.5. Flow cytometry 100 ll of citrated blood was incubated with 100 ll of 0.2 mg/ml nanotube dispersion at room temperature for 10 min followed by another 5-fold dilution with Tyrode-HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, pH 7.4) containing 5.5 mM glucose and 0.35% bovine serum albumin. For control measurements nanotube dispersion was replaced by control solution. 1 lM ADP (Chrono-Log, USA) served as positive control for platelet activation. Platelet activation was characterized by changes in the surface expression of CD62P (P-selectin), the platelet activation marker. Platelets in the whole blood were stained with PE-labeled anti-CD62P (Dako, Glostrup, Denmark) and FITC-labeled anti-CD41 (Immunotech, Marseilles, France) antibodies according to the manufacturer’s instructions. Aspecific staining of samples was checked by using appropriate isotype controls. After staining samples were diluted with Tyrode-HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, pH 7.4) containing 5.5 mM glucose and 0.35% bovine serum albumin in such an extent that a 250-fold final dilution of blood was attained. 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, and platelets were gated on the forward-side scatter dot plot and the mean CD62P fluorescence intensity (MFI) was measured. When the platelet–granulocyte complex formation was analyzed blood samples were treated in the same way as in the case of platelet activation experiments. Platelet–granulocyte complexes were measured as described previously (Bihari et al., 2008a; Fent et al., 2008). Briefly: whole blood was stained with FITC-labeled anti-CD41 (Immunotech, Marseilles, France) and Cy5-labeled anti-CD15 (BioLegend, San Diego, USA) antibodies. 500-fold dilution of blood was used in these measurements, thus coincidence of platelets and granulocytes did not result in false double positivity. The CD15 granulocyte marker was used as trigger signal for data collection. The amount of platelet–granulocyte complexes was determined as percentage of CD41 positive events in the
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granulocyte gate. All samples were run on a FACScan (Becton Dickinson, USA) flow cytometer. 2.6. Aggregometry In vitro aggregation of human platelets in whole blood was detected with a two-channel Chronolog Whole Blood Lumi-Aggregometer type 560 C (Chrono-Log, Havertown, USA) by measuring impedance change in plastic cuvettes at 37 °C with continuous stirring (1000 rpm). The reaction mixture contained 800 ll of citrated blood and 200 ll of control or 0.5 mg/ml stock nanotube dispersion. Since aggregation measurements are relatively long (30– 40 min) and the blood ages, so each time an age-matched control was measured with addition of collagen (3 lg/ml final concentration) to check the aggregation ability of blood. Data were collected both with a two-channel recorder and a computer. The nanotube induced aggregation was characterized by the impedance change with baseline correction detected at 30 min after the addition of the nanotube solution. 2.7. Statistical analysis All experimental values are presented as mean ± SE. The polydispersity indices and Z-average data of various SWCNTs were
compared by one-way ANOVA. Tukey test was used at all pairwise multiple comparison procedures. In platelet activation and platelet–granulocyte complex formation tests statistical significances were evaluated by the paired Student’s t-test against the matched controls. If normality test failed, Wilcoxon test (SigmaStat, ver. 3.0, SPSS Inc., USA) was used instead. Aggregation experiments were evaluated by the one sample Student’s t-test after subtracting controls. Normality was checked by the Kolmogorov–Smirnov test. A p < 0.05 value was considered statistically significant. 3. Results 3.1. Characteristics of nanotubes and nanotube dispersions The morphologies of the nanotubes studied by SEM and TEM are presented in Figs. 1 and 2. According to the images SWCNTs with amide and PEG modifications consist mainly of amorphous matrix material (with light elements) where the carbon nanotubes are embedded. Similar type of amorphous matrix material is also present in SWCNTs with OH modification. It can be concluded that the amount of surface modification material is high in these surface modifications and they contribute significantly to the physical appearance of carbon nanotube material. In the case of pristine SWCNTs and COOH–SWCNTs the carbon nanotube morphology appears conventional where material appears as entangled
Fig. 1. SWCNTs morphology by SEM.
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Fig. 2. SWCNTs morphology by TEM and elemental composition by TEM–EDS.
Table 1 Dynamic light scattering data (mean ± SE) of the SWCNT samples.
p-SWCNT (n = 38) CONH2–SWCNT (n = 18) COOH–SWCNT (n = 18) OH–SWCNT (n = 10) PEG–SWCNT (n = 32)
Main peak (d.nm)
% Of main peak area
PdI
Z-average (d.nm)
166.04 ± 3.01 68.49 ± 5.73 228.92 ± 11.29 340.42 ± 26.79 57.51 ± 4.37
95.79 ± 1.72 94.12 ± 1.07 97.01 ± 1.98 97.67 ± 2.33 98.38 ± 0.35
0.5 ± 0.02 0.55 ± 0.01 0.48 ± 0.05 0.72 ± 0.04* 0.48 ± 0.02
329.6 ± 13.5 378.23 ± 20.06 416.70 ± 29.98 813.10 ± 66.91§ 236.06 ± 16.6#
PdI: polydispersity index. * Significantly higher (p < 0.05) than all the other PdI values. § Significantly higher (p < 0.05) than all the other Z-average values. # Significantly lower (p < 0.05) than all the other Z-average values.
1 – potential (mV) (n = 3) 10.34 ± 0.49 12.17 ± 0.38 9.42 ± 1.18 11.47 ± 0.79 12.23 ± 0.55
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undetectable in the presence of 10-times diluted (final concentration: 0.01 mg/ml) SWCNTs (data not shown), with an exception of PEG–SWCNTs, which even at 0.01 mg/ml concentration induces well detectable platelet activation as well (Fig. 4). Fig. 5 shows the time dependence of platelet activation in the presence of 0.1 mg/ml PEG–SWCNTs for 8 donors. Although the extent of the platelet activation exerted by PEG–SWCNTs varies from donors to donors, its maximum is around 60 min for all of them. In case of CONH2–SWCNTs, COOH–SWCNTs and p-SWCNTs the time dependence of platelet activation has the same characteristics as in the case of PEG–SWCNTs (not shown). In the presence of OH–SWCNTs the expression of CD62P remains at the control level within the experimental error during a 2-h period. Fig. 3. Averages ± SE of CD62P mean fluorescence intensities for the CD41 positive events measured in whole blood in the presence of 1 lM ADP (striped columns) or in the presence of 0.1 mg/ml of various functionalized carbon nanotubes (filled columns), as indicated on the abscissa (n P 19). ⁄ denotes significant differences (p < 0.001) relative to the control (dotted columns) obtained from paired statistical tests.
nanotube clusters. EDS analysis indicated that all samples consist mainly of carbon (more than 90 wt%) but they also include various other residual elements in small quantities (Fig. 2). Due to detection limitations of EDS analyzer only qualitative elemental analysis of these residual elements was achieved. The main component and the Z-average of the equivalent hydrodynamic diameters, the polydispersity indices and the zeta-potentials of the nanotube suspensions studied are compiled in Table 1. PEG–SWCNTs have significantly (p < 0.05) the lowest Z-average diameter among the SWCNTs studied. The OH–SWCNTs have far the highest Z-average hydrodynamic diameter and polydispersity index. Both are significantly (p < 0.05) different from those characterizing all the other SWCNTs. The other polydispersity indices do not differ significantly from each other. The zeta potential values are practically the same for all the SWCNTs studied.
3.3. Formation of platelet–granulocyte complexes in the presence of various SWCNTs Fig. 6 shows the amount of platelet–granulocyte complexes as the percent of double (CD15 and CD41) positive events in the CD15 positive granulocyte gate. Every SWCNTs tested but OH substituted ones induced a significant increase in the amount of these complexes relative to the control. 3.4. Aggregation of platelets in the presence of various SWCNTs The aggregating effect of different SWCNTs involved in our study is summarized in Table 2. OH–SWCNTs and the CONH2–SWCNTs have practically no detectable aggregating effect at a concentration of 0.1 mg/ml, while those of the pristine
3.2. Activation of platelets in the presence of various SWCNTs In vitro activation of platelets in whole blood was characterized by changes in the surface expression of CD62P, an early and reliable platelet activation marker (Lu and Malinauskas, 2011). Fig. 3 shows the mean fluorescence intensities for CD62P (average ± SE) measured in the presence of various single-walled carbon nanotubes as indicated on the abscissa. ADP, the physiological activator of platelets was used as positive control. Each SWCNTs tested but OH substituted derivative induced a significant increase in the surface expression of CD62P on platelets at 0.1 mg/ml final concentrations (Fig. 3). The extent of platelet activation becomes
Fig. 4. Averages ± SE of CD62P mean fluorescence intensities for the CD41 positive platelets measured in whole blood in the presence of 1 lM ADP or in the presence of different concentrations of PEG–SWCNTs as indicated on the abscissa (n = 12). ⁄ denotes significant differences (p < 0.01) relative to the control obtained from paired statistical tests.
Fig. 5. Time dependence of CD62P expression on platelets (corrected with the respective controls) in the case of 8 donors in the presence of 0.1 mg/ml of PEG–SWCNTs. Experimental points were fitted with quadratic polynomials.
Fig. 6. Average per cents ± SE of granulocyte–platelet complexes among the granulocytes in the presence of 1 lM ADP (striped columns) or in the presence of 0.1 mg/ml of various carbon nanotubes (filled columns) as indicated on the abscissa (n P 13). ⁄ denotes significant differences (p < 0.001) relative to the control (dotted columns) obtained from paired statistical tests.
J. Fent et al. / Toxicology in Vitro 29 (2015) 1132–1139 Table 2 Whole blood aggregation measured as impedance change (mean ± SE) from the control baseline at 30 min after addition of collagen or various single-walled CNTs as stimuli.
*
Stimulus
N
Impedance change (ohm)
Collagen p-SWCNT CONH2–SWCNT COOH–SWCNT OH–SWCNT PEG–SWCNT
30 6 6 6 6 6
14.6 ± 0.7* 3.8 ± 0.8* 0.2 ± 0.5 3.9 ± 0.9* 0.2 ± 0.6 7.1 ± 1.4*
Denotes significant (p < 0.01) differences from zero.
Fig. 7. Typical impedance changes in the whole blood as a function of time upon addition of 3 lg/ml collagen (1) or 0.1 mg/ml PEG–SWCNTs (2). At the moment when aggregation was initialized a calibration signal (20 Ohm) was also recorded.
SWCNTs, COOH–SWCNTs and PEG–SWCNTs are well detectable, the latter having the most pronounced effect (Table 2). The aggregation curves have lag phases and are more elongated than that of induced by 3 lg/ml of collagen. The extent of aggregating effect exerted by pristine SWCNTs, COOH–SWCNTs and PEG–SWCNTs varies among the individual blood donors. Fig. 7 demonstrates a typical aggregation curve registered in whole blood in the presence of 0.1 mg/ml PEG–SWCNTs. 4. Discussion According to the literature and our previous data, pristine carbon nanotubes activate platelets (Guidetti et al., 2012; Lacerda et al., 2008; Semberova et al., 2009), promote platelet–granulocyte complex formation (Holzer et al., 2014) and have thrombogenic potential in animal models (Bihari et al., 2010; Radomski et al., 2005). Attaching various functional groups to the surface of CNTs aims to lessen their toxicity (Kunzmann et al., 2011) and at the same time to increase their dispersibility. In this work we tested 4 surface modified carbon nanotubes and a pristine one obtained from various manufacturers in terms of their in vitro effects exerted on platelets in whole blood. The dose of pristine and modified carbon nanotubes applied in our study is based on various literature data describing different exposure scenarios (Semberova et al., 2009; Radomski et al., 2005; Maynard et al., 2004; Brouwer, 2010; Nemmar et al., 2003; Czarny et al., 2014). According to the above literature data one can estimate that inhaled ambient carbon nanotubes can get into the blood circulation in concentrations around 0.05–0.2 mg/ml. Therefore, we used 0.1 mg/ml carbon nanotube concentrations in our in vitro study. Based on literature data on the half-life of SWCNTs in the circulation of various animals one can suppose that the concentration of SWCNTs is not diluted substantially in circulation in the first 10 min (Cherukuri et al., 2006; Shing et al., 2006). Furthermore,
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according to Holzer et al. a 10-min presence of p-SWCNTs in blood circulation of mice augmented the light/dye induced thrombosis (Holzer et al., 2014). Therefore, in accordance with our previous experience (Khandoga et al., 2010) we analyzed platelet activation in the whole blood upon SWCNTs treatments at ten minutes. The most interesting finding of our study is that PEG–SWCNTs have the most pronounced platelet activating and aggregating effects among the SWCNTs tested by us although according to literature data pegylation seems to be the most advantageous modification of carbon nanotubes in terms of pharmacokinetic and toxicological profiles (Bottini et al., 2011; Hadidi et al., 2011; Heister et al., 2010; Yang et al., 2012). PEG–SWCNTs strongly promoted the platelet–granulocyte complex formation in the human whole blood as well (Fig. 6). PEG–SWCNTs even at 0.01 mg/ml concentration induces CD62P platelet activation marker expression in whole blood (Fig. 4) which concentration is already a non-effective dose for all the other SWCNTs studied. It is noteworthy that all the carbon nanotube samples when added to whole blood contained HSA. Vakhrusheva and her coworkers (Vakhrusheva et al., 2013) claimed that HSA has protective effect against platelet activation of SWCNTs. The zeta potential of HSA in phosphate buffer between pH 6.4–8.4 is between 7 and 14 mV (Tousi et al., 2011). The zeta potentials of our samples are in the range of 10 mV (Table 1) suggesting that the surface all of our SWCNT samples are covered but with unknown amount of HSA. Vakhrusheva and her coworkers (Vakhrusheva et al., 2013), showed that PEG–SWCNTs adsorb much less (0.5 w/w) HSA than the carboxylated ones (>2.4 w/w). This feature of PEG–SWCNTs might be the reason of their extremely high platelet activation ability. Our aggregometric results support the view that platelet activation in the presence of SWCNTs if any is a time consuming multistep complex process (Guidetti et al., 2012; Semberova et al., 2009). ADP, one of the classical platelet agonists, induces platelet activation within several minutes (Milton et al., 1980) while in the presence of PEG–SWCNTs about 60 min are needed for the maximal activation (Fig. 5) which is reflected in the extended lag phases of platelet aggregations in the presence of PEG–SWCNTs (Fig. 7). The same results were obtained with p-SWCNTs and COOH–SWCNTs, but not with OH–SWCNTs or CONH2–SWCNTs which have no whole-blood aggregating effect (Table 2). This latter finding is hard to explain and needs further clarification since CONH2–SWCNTs itself induces CD62P expression and strongly promotes platelet–granulocyte complex formation (Figs. 3 and 6). Although no correlation could be demonstrated between the size parameters and parameters characterizing the platelet activating effect of our SWCNT samples there are still some remarkable data in Table 1. PEG–SWCNTs being most effective in terms of platelet activation (induction of CD62P expression) have far the lowest size parameters: the mean of the main component and Z-average of the equivalent hydrodynamic diameter. OH–SWCNTs which practically have no effects on platelets and on the platelet–granulocyte complex formation have the highest Z-average equivalent hydrodynamic diameter, the highest PdI as well as the highest mean equivalent hydrodynamic diameter of the main component. Neither the Z-average equivalent hydrodynamic diameters nor the main component diameters correlated with the length parameters of our samples provided by the manufacturer. According to SEM and TEM data significant amount of amorphous matrix is present not only in the OH–SWCNTs and CONH2–SWCNTs but in the PEG–SWCNTs as well; thus this amorphous matrix constituent might not be responsible for the differences experienced in the effects of various SWCNT derivatives exerted on whole blood. Furthermore, the very similar element composition of SWCNT derivatives detected by EDS (within
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detection limits of the analyzer, Fig. 2) does not give any hint to explain their different behavior either. Physico-chemical parameters (SEM, TEM, EDS, Z-average diameter, polydispersity index, zeta-potential) do not seem to be sufficient to predict the distinct effects of various single-walled carbon nanotubes in whole blood. According to our findings several methods (e.g. flow cytometry and aggregometry) need to be combined to judge their complex effects exerted on platelets. This is even more important since in spite of that, surface modifications of carbon nanotubes aim to increase biocompatibility the resulting products may still exhibit unexpected disadvantageous platelet activation in whole blood. Conflict of Interest The authors declare that are no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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