Evidence That Polyhydroxylated C60 Fullerenes (Fullerenols) Amplify

Article pubs.acs.org/crt

Evidence That Polyhydroxylated C60 Fullerenes (Fullerenols) Amplify the Effect of Lipopolysaccharides to Induce Rapid Leukocyte Infiltration in Vivo D. M. Gonçalves and D. Girard* Laboratoire de Recherche en Inflammation et Physiologie des Granulocytes, INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Quebec, Canada H7V 1B7 ABSTRACT: Fullerenols C60(OH) have therapeutic potential, but there is debate regarding their toxicity. Here, we tested the hypothesis that C60(OH)n possesses a pro-inflammatory effect in vivo. Kinetic and dose-dependent experiments performed with the murine air pouch model of acute inflammation revealed that, unlike TiO2 used as a positive control in this model, C60(OH)n NPs were not pro-inflammatory in CD-1, C57BL/6, and BALB/c mice. However, after 3 h of treatment, C60(OH)n NPs were found to amplify the effect of lipopolysaccharides (LPS) causing a rapid leukocyte influx in which the major cells observed are neutrophils. The use of an antibody array assay to detect different analytes simultaneously indicates that the amplification effect is, at least partially, explained by an increased local production of several cytokines/chemokines in the exudates, including the pro-inflammatory cytokine IL-6. Using an ELISA to quantify the amount of IL-6 produced into air pouch exudates, we demonstrated that C60(OH)n increases the LPS-induced local production of this cytokine. Therefore, although C60(OH)n NPs alone do not exert proinflammatory activity under certain conditions, they can act in concert with other agents to cause inflammation, a situation that is likely to occur in vivo.

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INTRODUCTION Research in the field of nanotechnology has increased tremendously in the past few years, due to the expectation that nanomaterials may improve practically all types of products; more than 1000 nanoproducts already exist on the market.1 However, because of several properties, including their size, nanoparticles (NPs) are potentially biologically active and may interfere with normal biological systems and cell mechanisms. Water-soluble fullerenes were successfully synthesized about 20 years ago as polyhydroxylated C60, named fullerenols (“bucky balls”) or C60(OH)x (where x = 12−26).2 Together, they form a unique spherical cage structure to which side chains can be added to modify the physicochemical and/or pharmacological characteristics of the molecule.3,4 Unlike fullerenes, the fact that these hydroxylated “bucky balls“ are water-soluble makes it easier them to study in biological systems.5 Fullerenols have enormous therapeutic potential mostly because of their antioxidant properties.4,6 It has been shown that they can deactivate various reactive oxygen species (ROS), including singlet oxygen, superoxide anion, hydroxyl radicals, and nitric oxide.7−9 Because of their free-radical scavenging capabilities, C60(OH)x were reported to be able to act as neuroprotective agents against ROS-mediated neuronal death.8,10,11 Fullerene derivatives are capable of enzyme inhibition; they can inhibit enzymes such as the nitric oxide synthase or the human immune-deficiency virus protease, essential for the virus survival.12−14 Despite their enormous © XXXX American Chemical Society

potentials, there is little existing data about their biocompatibility and cytotoxicity, probably because most of the literature tends to demonstrate that fullerenols have no or very few cytotoxic properties. However, there are debates regarding the toxicity of fullerenols. For example, when tested at 1−100 μg/ mL, C60(OH)24 has a direct effect on human umbilical vein endothelial cells causing cell injury, cell death, and inhibition of growth.15 NPs or different engineered particles with various sizes can cause inflammation, one of the major toxic effect of NPs reported in the literature.16−19 Because exposure to NPs is increasing rapidly and no clear guidelines on the testing and evaluation of NPs are presently available, the use of in vitro experiments in nanotoxicology studies is becoming extremely relevant and important.1 However, it is impossible to recreate the complex inflammatory process in vitro, and therefore, in addition to in vitro assays, the use of in vivo models of inflammation is needed. The murine air pouch model of acute inflammation is probably the most versatile and simplest model to use for evaluating acute proinflammatory effects of NPs. This model was previously shown to be a convenient model for studying the behavior of lining cells involved in the formation of a structure similar to that of synovial lining cells.20−22 Effectively, in this model, administration of air under the skin produced a pouch wall that Received: July 18, 2013

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dx.doi.org/10.1021/tx4002622 | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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Characterization of C60(OH)n. Instead of determining the endotoxin level by the classical Limulus amebocyte lysate (LAL) assay for determining endotoxin level, a method in which NPs could interfere, we incubated the C60(OH)n in Lysogeny broth agar plates for up to 48 h and observed the presence or absence of colonies. As a positive control, we used a suspension of voluntary contaminated TiO2 NPs that we strictly reserved to perform this kind of analysis. The size distribution and surface charge (zeta potential) of C60(OH)n were determined by dynamic light scattering using a Malvern Zetasizer Nano-ZS (model ZEN3600) from Malvern Instruments Inc. (Westborough, MA). Measurements were performed with a solution of 100 μg/mL C60(OH)n in HBSS. Murine Air Pouch Model. CD-1 female mice (6−8 weeks of age) were obtained from Charles River Laboratories (St-Constant, Canada). A period of acclimation of 1 week was allowed to animals prior to initiation of experiments. On day 0 and 3, mice were anaesthetized with isofurane, and 3 mL of sterile air was injected subcutaneously, in the back, with a 26-gauge needle to form an air pouch as published previously.29 On day 6, 1 mL of HBSS (control) or an increasing concentration (50−500 μg/mL) of C60(OH)n was injected into the air pouch. Mice were killed by CO2 asphyxiation 3, 9, 12, or 24 h after the treatment, and the pouches were washed once with 1 mL and then twice with 2 mL of HBSS containing 10 mM EDTA. Exudates were centrifuged at 100g for 10 min at room temperature, and supernatants were collected and stored at −80 °C for further analysis. Cells were resuspended at 0.5 × 106 cells/mL, spread onto microscope slides, and stained with Hema-Stain (Fisher Scientific, Ottawa, Canada) for identification/quantification of leukocyte cell subpopulations. In some experiments, 1 μg/mL LPS (0111: B4 E. coli) (Sigma) or 100 μg/mL C60(OH)n was injected alone or mixed with C60(OH)n. For some experiments, female C57BL/6 or BALB/c mice (6−8 weeks of age) were used instead of CD-1. All experiments were performed as per protocols approved by Animal Use and Care Committees at INRS-Institut Armand-Frappier. Detection of Cytokines/Chemokines. Mouse antibody array kit was purchased from R&D Systems (Minneapolis, MN), and all of the steps for the simultaneous detection of 40 analytes were performed as per the manufacturer’s recommendation and as previously published.29,39 These analytes are listed in the corresponding figure. We selected the analytes based on the fact that the kit we used (Mouse Cytokine Array, Panel A array kit) is the one containing the most analytes related to inflammation. Exudates (n ≥ 4 mice) were harvested from HBSS-treated (control), C60(OH)n, LPS, or C60(OH)n + LPS-induced murine air pouches and pooled to probe the membranes, within two weeks following the in vivo experiments. This was performed because of the high cost that this will represent for doing antibody arrays for each animal in each group. Of note, we previously demonstrated the reproducibility of the technique by probing two membranes with the exudates from two distinct mice treated with LPS.29 The chemiluminescent signal from the bound cytokines/chemokines present in the exudates was detected on Kodak X OMAT-RA film. The signal intensity was normalized to the membrane’s positive control, and each analyte was done in duplicate. Results are expressed as ratios (fold increase/tested group/control). Ratios ≥ 1.2 were considered slightly positive.29,39 Protein array membranes were scanned, and densitometry analysis was performed using the Multi-Analyst program (Bio-Rad, Hercules, CA). Quantification of IL-6. The measurement of IL-6 production in the exudates was determined using a commercially available ELISA kit (Invitrogen Canada Inc., ONT) as per the manufacturer’s recommendation. Statistical Analysis. One way analysis of variance (ANOVA) followed by Bonferroni’s test (all pairs of columns) was used to evaluate for the significance between the control and each of the samples. Statistical analyses were performed using GraphPad Prism, version 5.00 for Windows (GraphPad Software, San Diego, California, USA). Statistical significance was set at p < 0.05.

is similar to a synovium since the inner lining is made up of macrophages and fibroblasts. This model is also used for evaluating the pro-inflammatory activity of a given product, including cytokines,23−26 directly administered into the air pouch at a large spectrum of concentrations (up to several hundreds of μg/mL/mouse). Injection of irritants into an air pouch induces an inflammatory response that can be quantified by the total protein concentration in the exudates, the infiltration of cells, the release of inflammatory mediators, etc. In this model, the three phases of inflammation occurred, namely, initiation, amplification, and resolution. For example, bacterial lipopolysaccharides (LPS), the major outer surface membrane components present in almost all Gram-negative bacteria, are extremely strong stimulators of innate immunity and are potent inducers of massive neutrophil recruitment into murine air pouches 6 h after a single administration.25,27 After 9−12 h, the number of attracted neutrophils start to decline, and after 18−24 h, the number of cells return to normal control values. This in vivo model can also be used for determining the anti-inflammatory activity of a given product on LPS (or other agent)-induced inflammation.27,28 Recently, we have used this in vivo model to determine whether TiO2 NPs are proinflammatory based on their ability to induce neutrophil influx into the air pouch as well as to increase the local production of several cytokines and chemokines.29 The vast majority of studies investigating the inflammatory properties of nanomaterials target pulmonary cells, airways, and lungs.30−32 However, NPs can also gain entry into human systems through exposition routes other than lungs, including ingestion, dermal routes, etc.18,33−35 If deregulated, acute inflammation can lead to chronic inflammatory disorders and diseases.36−38 A previous report has shown that pretreatment of mice with an intratracheal administration of fullerenol attenuated neutrophilic lung inflammation induced by quartz, a mineral well known for its pro-inflammatory properties.3 In an effort to increase our knowledge regarding the in vivo inflammatory properties of fullerenols, we evaluated their proinflammatory activity in vivo using the murine air pouch model where fullerenols were directly administered into the pouches at a concentration similar to those used in mice instilled intratracheally with fullerenols.3 We found that although fullerenol (C60(OH)x (x ∼ 18−22) did not induce acute inflammation in vivo (even at concentrations higher than those used by others), they can act in concert with bacterial lipopolysaccharides (LPS) in vivo to enhance and accelerate leukocyte infiltration.

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EXPERIMENTAL PROCEDURES

Fullerenols. Water-soluble polyhydroxylated C60 fullerenes (C60(OH)n; n ∼ 18−22 or fullerenols) were purchased from BuckyUSA (Houston, TX). TiO2 nanoparticles (anatase crystals) were obtained from Vivenano (Toronto, ONT) as an aqueous suspension of TiO2 NPs stabilized by polyacrylate sodium that are stable over a wide range of pH. The particles sizes are 1−10 nm (90%) as determined by transmission electron microscopy. Structure of the NPs is anatase crystal as confirmed by X-ray crystallography, according to the manufacturer. Both fullerenols and TiO2 NPs were dissolved in Hank’s balanced salt solution (HBSS) containing calcium and magnesium but without phenol red (pH 7.4) as detailed previously for in vivo experiments.3 Transmission Electron Microscopy. Fullerenol solutions (100 μg/mL in HBSS) were examined using a Hitachi H-7100 transmission electron microscope. B



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RESULTS Characterization of C60(OH)n. Since HBSS was the medium used for injection into air pouches throughout this study, the size and zeta potential were measured in HBSS alone at a concentration of 100 μg/mL C60(OH)n. As previously reported by others, aggregation of C60(OH)n was observed in culture media.40 In our hands, a bimodal distribution was observed in HBSS, the experimental condition used for in vivo administration: 97.8% with an average diameter of 572.5 nm and 2.2% with a large diameter of 2699 nm. The zeta potential was −18.8 mV ± 0.8 (mean ± SD). The results are representative of three different lectures. As indicated in Figure 1A, fullerenols aggregated into clusters, as shown in

As illustrated in Figure 2, administration of 50, 100, 200, or 500 μg/mL C60(OH)n into air pouches did not significantly increase

Figure 2. C60(OH)n do not enhance leukocyte infiltration in vivo. Murine air pouches were raised in CD-1 mice before the injection of 0−500 μg/mL C60(OH)n. Exudates were harvested after 3 h (A), 9 h (B), 12 h (C), or 24 h (D), and the total number of leukocytes was calculated as described in Experimental Procedures. Results are the means ± SEM (n ≥ 4). *P < 0.05 vs the control (0 μg/mL C60(OH)n).

Figure 1. Characterization of C60(OH)n. (A) Transmission electron microscopy was performed as described in Experimental Procedures. C60(OH)n were used at 100 μg/mL in HBSS cultured medium. Note that aggregates of fullerenols are observed. (B) An aliquot of a voluntary contaminated 100 μg/mL TiO2 NP suspension (positive control) or the solution of 100 μg/mL C60(OH)n to test were incubated in a Petri dish for up to 48 h. Colonies (arrows) could be observed in the positive control but not in sterile C60(OH)n.

the total number of attracted leukocytes. To eliminate the possibility that the lack of pro-inflammatory activity was specific to this CD-1 outbred strain of mice, we next performed a series of experiments with the inbred BALB/c and C57BL/6 strains of mice. As illustrated in Figure 3A and B, administration of C60(OH)n did not increase the number of leukocytes into the air pouches after 3 h and 9 h, whereas, as observed in CD-1 mice,29 100 μg/mL TiO2 also significantly increased the total number of leukocytes in both strains of mice. C60(OH)n Aggregates Act in Concert with LPS Causing Rapid Leukocyte Attraction. Since C60(OH)n aggregates are devoid of pro-inflammatory activity, we next verified whether they could augment the LPS effect of increasing the total number of leukocytes (mainly neutrophils) attracted into the air pouch, 3−9h following LPS administration.23,25,29 As illustrated in Figure 4A, after 3 h, LPS or C60(OH)n slightly, but not significantly (p = 0.98 and 0.69, respectively) increase the total number of leukocytes attracted in the air pouch. This was expected for LPS since the initiation and amplification phases of inflammation in air pouch occur somewhere after 3− 6 h when it is provoked by LPS.23,25,29 However, a strong proinflammatory effect was noted when LPS and C60(OH)n were mixed together. Moreover, the total number of leukocytes (means ± SEM, n = 8) recruited by C60(OH)n + LPS was also significantly increased (4.4 × 106 cells/pouch ±0.9) vs that of LPS alone (1.2 × 106 cells/pouch), suggesting that C60(OH)n NPs amplified the effect of LPS. Interestingly, identification of the leukocyte subpopulations indicated that more than 85% of cells recruited by C60(OH)n + LPS were neutrophils (Figure 4B). After 6 h, LPS increased the total number of cells (greater than ∼4 × 106 cells/pouch) vs the control, and this number was further increased, although not significantly (vs LPS), by

representative TEM images supporting the DLS analysis. Because C60(OH)n was administered in the presence or absence of LPS in some experiments, we have also determined the size distribution and zeta potential in these conditions. Again, a bimodal distribution was observed, and the results were similar in both conditions: 99% with a diameter of 748 ± 173 and 1% with 889 ± 1500 (n = 3) in the absence of LPS and 95.8% with a diameter of 682 ± 123.9 and 4.2% with 1712 ± 1488 (n = 3) in the presence of LPS. The zeta potential was −16.9 ± 0.4 mV and −17.9 ± 0.7 (n = 3). Therefore, no major changes were observed when C60(OH)n were mixed with LPS. Instead of performing the classical Limulus amebocyte lysate (LAL) assay for determining endotoxin level, a method in which NPs or engineered particles could interfere, we directly incubated the C60(OH)n solution in Lysogeny broth (LB) agar plates that, even though this approach does not indicate full sterility, is a very good indicator about potential contamination of a tested solution. As illustrated in Figure 1B, in contrast to the positive controls (arrows), no colonies were observed in the plate incubated with C60(OH)n indicating that the solution is sterile. C60(OH)n Do Not Induce Inflammation in Vivo. Because our laboratory is interested in classifying the degree of proinflammatory potential of a given molecule, including a NP or an engineered particle, according to its in vivo properties, we decided to determine here whether or not C60(OH)n caused leukocyte infiltration in vivo, using the murine air pouch model as previously performed for the pro-inflammatory TiO2 NPs.29 C



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ratio between 5.1 and 10; ratios for all of the other analytes were between 1.3 and 1.5 (Figure 6B). Among the ratios of 27/ 40 analytes increased by LPS, 16 were between 1.3 and 5; 5 between 5.1 and 10; and 6 between 10.1 and 15. Finally, the administration of C60(OH)n + LPS into air pouches after 6 h increased the ratios of 27/40 analytes: 17 between 1.3 and 5; 5 between 5.1 and 10; 4 between 10.1 and 15; and 1 between 15.1 and 20. From these results, it is clear that even if C60(OH)n did not increase significantly the number of leukocytes in the air pouches after 3 h of treatment, the production of several analytes was increased by fullerenols as compared to the control group, and most of them are chemokines known to attract several leukocytes. Of note, some other analytes are recognized as anti-inflammatory molecules, including the IL-1 receptor antagonist (IL-1Ra). Quantification of IL-6 in the Exudates in Response to C60(OH)n, LPS, and C60(OH)n + LPS. In order to support the concept that some analytes may be involved in the amplification effect observed with the mixture C60(OH)n + LPS, we decided to measure the concentration of the proinflammatory cytokine IL-6, based on the data obtained from the antibody array analysis where C60(OH)n or LPS only slightly increases its production, while the production of this cytokine was markedly increased when C60(OH)n and LPS were mixed together (Figure 6A, panels A−C). As illustrated in Figure 7, the results obtained by the ELISA confirm those obtained by the antibody array assay, in which C60(OH)n increased, but not significantly, IL-6 production from 25 ± 10.6 to 161 ± 58.1 pg/mL after 3 h of treatment. As expected, LPS also increased the IL-6 production (268 ± 85 pg/mL). Of note, the mixture of C60(OH)n + LPS significantly increased the production of IL-6 vs the control and also vs C60(OH)n or vs + LPS, supporting the enhanced effect of both agents when used together.

Figure 3. Lack of induction of leukocyte infiltration in vivo by C60(OH)n is not strain-specific. Murine air pouches were raised in BALB/c (A) or in C57BL/6 (B) mice before an injection of buffer (control), 100 (or 500) μg/mL C60(OH)n or 100 μg/mL TiO2 NPs for 3 h or 9 h. Exudates were harvested, and the total number of leukocytes was calculated as described in Experimental Procedures. Results are the means ± SEM (n ≥ 4). *P < 0.05 vs the control (0 μg/ mL C60(OH)n.

C60(OH)n + LPS. As expected, the effect of LPS, when used alone, declined dramatically after 9−12 h correlating with the resolution of inflammation with time.25 Detection of Analytes (Cytokines/Chemokines) in Exudates of Mice Exposed to C60(OH)n, LPS, and C60(OH)n + LPS. In order to better elucidate the mechanisms caused by C60(OH)n, we then used the collected murine air pouch exudates for the antibody array assay allowing for the detection of 40 distinct analytes (cytokines/chemokines) known to be important in inflammation. Figure 5 illustrates the different signals (raw data) obtained for each membrane used in the calculation of the different ratios as compared to the control. It is clear that C60(OH)n increased the production of a few analytes after 3 h as compared to the control, while the production of several analytes was increased by LPS. Interestingly, the mixture C60(OH)n + LPS increased only a few analytes (arrows for examples) that are not (or very weakly) detected by C60(OH)n or LPS alone. The complete analysis of the data is illustrated in Figure 6, in which the results are expressed as ratios (tested/the control). After 3 h, administration of C60(OH)n alone increased the ratio of 23/ 40 analytes (Figure 6A) in which 18 displayed a ratio (R) between 1.3 and 5 (1.3 ≤ R ≥ 5); 4, between 5.1 and 10 (5.1 ≤ R ≥ 10); and 1 between 10.1 and 15 (10.1 ≤ R ≥ 15). Administration of LPS alone increased the ratio of 38/40 analytes; 28 had a ratio between 1.3 and 5; 4 between 5.1 and 10; 5 between 10.1 and 15; and 3 between 15.1 and 20 (Figure 6A). C60(OH)n + LPS administration resulted in an increase of 34/40 analytes; 16 had a ratio between 1.3 and 5; 9 between 5.1 and 10; 8 between 10.1 and 15; and 1 between 15.1 and 20. After 6 h, the ratios of 11/40 analytes were increased by C60(OH)n where only one (the chemokine IP-10) possessed a

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DISCUSSION Using the murine air pouch model of acute inflammation, we established in three different strains of mice, namely, CD-1, BALB/c, and C57Bl/6 that C60(OH)n aggregates are not proinflammatory. Thus, our results attest to the existence of a selective mechanism occurring in this model in response to different agents, including NPs and engineered particles, since not all NPs act in a similar fashion. We have previously documented that TiO2 NPs are extremely pro-inflammatory as evidenced by a rapid recruitment of leukocytes into the air pouches, including mainly neutrophils.29 In addition, preliminary results suggested that other NPs possess a different degree of pro-inflammatory activity when tested with this in vivo model (unpublished observations). Although this is the first study to report that C60(OH)n are not pro-inflammatory in the murine air pouch model, others have demonstrated that fullerenols attenuate neutrophilic lung inflammation induced by quartz in BALB/cJ mice in which the number of neutrophils in BALs was significantly diminished, although this effect was not dose-dependent.3 Also, in agreement with our results, no signs of inflammation were reported by the authors, using a concentration of 0.02−20 μg/ mouse. Therefore, for all these reasons, the use of the murine air pouch model is suitable for evaluating the effect of an agent on the inflammatory process. The authors of this study also showed that intratracheal administration of 200 μg/mouse C60(OH)n had a pro-inflammatory effect, based on an increase of leukocytes in BALs as well as an increased production of D



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Figure 4. C60(OH)n enhance the pro-inflammatory effect of LPS in vivo. Murine air pouches were raised in CD-1 mice before the injection of buffer (control), 2 μg/mL LPS, 100 μg/mL C60(OH)n (C60), or a mixture of C60(OH)n + LPS (C60 + LPS). Exudates were harvested at the indicated periods of time. The total number of leukocytes was calculated (A), and the cell populations were identified by cytology (B) as described in Experimental Procedures. Results are the means ± SEM (n ≥ 4). *, P < 0.05 vs the control (0 μg/mL C60); #, P < 0.05 vs LPS or vs C60(OH)n . Black bars, proportion of polymorphonuclear cells (PMNs); open bars, proportion of monocyte-macrophage (monomac) cells.

macrophage inflammatory protein-2 (MIP-2). In our present study, C60(OH)n aggregates did not significantly increase the number of leukocytes in the air pouches, even at 500 μg/ mouse, a concentration 2.5 times greater than that used by Roursgaard and collaborators.3 In addition, MIP-2 was among the analytes tested in our antibody array assay; we found that C60(OH)n only minimally increased the production of MIP-2 by a ratio of 1.3 after 3 h and less than 1.2 after 6 h of treatment. In another study,41 pulmonary responses to C 60(OH)n were investigated 3 days after intratracheal administration in Sprague−Dawley rats. At 1 mg/rat, no adverse pulmonary toxicity was observed. These results support those of our present investigation. However, exposures to 5 or 10 mg/rat induced cell injury, oxidative and nitrosative stress, and inflammation, as assessed using BAL fluid biomarkers and pathological evaluation of lungs. Increased concentrations of IL-1β, TNF-α, and IL-6 in the BALs was measured by ELISA. We did not observe a significant leukocyte infiltration in the murine air pouch model in response to C60(OH)n alone after 3 h, and the antibody array assay revealed a slightly increased ratio for IL-1β, TNF-α, and IL-6. However, these three cytokines were associated with the amplified effect observed between LPS and C60(OH)n (see arrows in Figure 6A).

Figure 5. Detection of analytes in the murine air pouch exudates by antibody array assay. Exudates from different animals treated with medium alone (control), C60(OH)n, LPS, or C60(OH)n + LPS were pooled (n ≥ 4; for each conditions) and were used for the detection of the 40 different analytes listed in Figure 6. Some analytes are depicted (arrows) as examples to indicate that C60(OH)n altered the levels of expression of certain analytes induced by LPS alone.

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Figure 6. C60(OH)n increase the basal levels of expression of a few analytes present in the murine air pouch exudates but up-regulate the expression of others observed in C60(OH)n + LPS. Ratios for all analytes detected in the exudates after 3 h (A) or 6 h (B) of treatment were calculated as described in Experimental Procedures. The 40 different analytes are indicated in the y-axis, and the corresponding values of ratios are on the x-axis. Note that, unlike C60(OH)n alone, LPS strongly increased the levels of expression of a multitude of analytes with ratios (R) greater than 10 and that the profile of analytes detected in C60(OH)n + LPS is distinct compared to that of C60(OH)n or LPS alone. Gray arrows, IL-6.

Quantification of IL-6 by ELISA indicated that C60(OH)n alone could significantly increase IL-6 production, suggesting that a

weak increase observed with the antibody array assay can result in a greater concentration of a given analyte, at least for IL-6, F



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the past (IL-4, IL-15, IL-21, TNF-alpha, LPS, carrageenan, dieldrin, TiO2, etc.). One has to consider that, in the present study, we are studying acute inflammation and that the murine air pouch model allows one to investigate the first steps of a probable deregulation process of the inflammatory response. Importantly, NPs and engineered particles can gain entry into human systems also via ingestion or via dermal routes and can therefore reach the blood where neutrophils are the most abundant leukocytes in humans. Knowing that C60(OH)n does not cause inflammation in vivo by itself but can significantly enhance the response of other agents, including LPS (this article), highlights the need for future studies to include such an approach for a better comprehension of the toxicity and mode of action of a given NP or engineered particles of various sizes. This is further supported by the fact that during inflammation, leukocytes (including neutrophils) can be sequentially and/or simultaneously activated by a plethora of mediators, resulting in an exacerbation of inflammation when deregulation occurs. This is particularly important for particles with great therapeutic potential, including fullerenes and their derivatives, which represent promising candidates for use as inhaled drugs.41 Furthermore, water-soluble C60(OH)n can act as radical scavengers and as antioxidants in vitro and in vivo, thus representing interesting candidates for the development of novel anti-inflammatory drugs.42−45 Therefore, because fullerenes and their derivatives display interesting properties and gain increasing attention in pharmacology and biomedicine,6,46 evaluating the toxicological effects of C60(OH)n is of importance since potential contact of these particles with humans will certainly increase in the becoming years. Identification of a given particle as having no inflammatory effect by itself in inflammatory models has to be taken with caution since the particle could amplify the effect of other agents, a situation that is likely to occur in vivo.

Figure 7. Production of IL-6 is greater in C60(OH)n + LPS treated mice than in animals treated with C60(OH)n or LPS alone. Exudates were harvested for each animal (not pooled) and used to quantify the production of the pro-inflammatory cytokine IL-6 by ELISA after 3 h of treatment with buffer (control), C60(OH)n, LPS, or C60(OH)n + LPS. Results are the means ± SEM (n ≥ 4). *, P < 0.05 vs the control; #, P < 0.05 vs LPS and vs C60(OH)n.

than initially estimated. Nevertheless, the results obtained by ELISA, at least for IL-6, also fit very well with the amplified effect observed, confirming that IL-6 is associated with this phenomenon. Recently, priming of macrophages with LPS was found to activate carbon black nanoparticle-induced proinflammatory cell death inflammasome-dependent pyroptosis, based on caspase-1 activation and increased production of IL1β.42 In our present study, both LPS and C60(OH)n were injected simultaneously to observe an amplification effect, rather than a priming effect reported in their study. Whether or not the injection of LPS before C60(OH)n would induce inflammation in the murine air pouch model remains to be determined. Interestingly, in another study, LPS alone did not cause fibrosis in intratracheally instilled rats but was found to enhance multiwalled carbon nanotube-induced fibrosis after 21 days when LPS was administered 24 h before the NPs.43 However, unlike us, where LPS had no effect by itself as well as C60(OH)n when used alone, the multiwalled carbon nanotubes induced fibrosis by themselves. Also, when testing in parallel carbon black NPs that did not induce fibrosis when used alone in their model, the authors also did not observe a significantly increased fibrosis when LPS was administered prior to carbon black NPs. Therefore, to the best of our knowledge, the present study is the first to report that C60(OH)n and LPS, having no proinflammatory effects when used alone at a specific time point, become rapidly (3 h) proinflammatory when mixed together. In summary, the above observations attest to the necessity of using distinct models of in vivo inflammation for determining the toxicity of NPs, including their potential to induce acute inflammation. We are aware that the murine air pouch model is probably less representative of a real exposure scenario than inhalation models, for example, as the most susceptible way that a human can come in contact with NPs or engineered particle with sizes at the nanoscale is by inhalation. However, besides all of the advantages that the murine air pouch model presents (see Introduction), we and others have successfully used this model in the past to evaluate the global pro- or antiinflammatory potentials of many molecules and cytokines in

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AUTHOR INFORMATION

Corresponding Author

*Phone: 450-687-5010 (ext. 8847). Fax: 450-686-5309. E-mail: [email protected]. Funding

This study was partly supported by the Institut de recherche Robert-Sauvé en santé et en sécurité du travail (IRSST) and by Natural Sciences and Engineering Research Council of Canada (NSERC). D.M.G. holds a NSERC Ph.D. award. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mary Gregory for reading this manuscript. ABBREVIATIONS NPs, nanoparticles; LPS, lipopolysaccharides; TiO2, titanium dioxide; ROS, reactive oxygen species; HBSS, Hank’s balanced salt solution

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REFERENCES

(1) Arora, S., Rajwade, J. M., and Paknikar, K. M. (2012) Nanotoxicology and in vitro studies: the need of the hour. Toxicol. Appl. Pharmacol. 258, 151−165. (2) Chiang, L. Y., Upasani, R. B., and Swirczewski, J. W. (1992) Versatile nitronium chemistry for C 60 fullerene functionalization. J. Am. Chem. Soc. 114, 10154−10157.

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Evidence That Polyhydroxylated C60 Fullerenes (Fullerenols) Amplify

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