Biodistribution and toxicological study of PEGylated single-wall carbon nanotubes in the zebrafish (Danio rerio) nervous system.

YTAAP-13189; No. of pages: 9; 4C: Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

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Gisele E.B. Weber a,b, Lidiane Dal Bosco a,b, Carla O.F. Gonçalves c, Adelina P. Santos c, Cristiano Fantini d, Clascídia A. Furtado c, Gustavo M. Parfitt a,b, Carolina Peixoto a,b, Luis Alberto Romano e, Bernardo S. Vaz a,b, Daniela M. Barros a,b,⁎

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Article history: Received 6 March 2014 Revised 9 August 2014 Accepted 15 August 2014 Available online xxxx

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Keywords: SWNT-PEG Zebrafish Functionalization Biocompatibility Biodistribution Behavioral Oxidative stress Toxicity

Laboratório de Neurociências, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande, RS, 96210-900, Brazil Programa de Pós-graduação em Ciências Fisiológicas–Fisiologia Animal Comparada, FURG, Rio Grande, RS, 96210-900, Brazil c Laboratório de Química de Nanoestruturas, Centro de Desenvolvimento da Tecnologia Nuclear, Belo Horizonte, MG, 31270-901, Brazil d Instituto de Ciências Exatas, Departamento de Física, Belo Horizonte, MG, 31270-901, Brazil e Instituto de Oceanografía, Universidade Federal do Rio Grande, Rio Grande, RS, 96210-030, Brazil b

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Biodistribution and toxicological study of PEGylated single-wall carbon nanotubes in the zebrafish (Danio rerio) nervous system

Nanotechnology has been proven to be increasingly compatible with pharmacological and biomedical applications. Therefore, we evaluated the biological interactions of single-wall carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG). For this purpose, we analyzed biochemical, histological, behavioral and biodistribution parameters to understand how this material behaves in vitro and in vivo using the fish Danio rerio (zebrafish) as a biological model. The in vitro results for fish brain homogenates indicated that SWNT-PEG had no effect on lipid peroxidation, total antioxidant capacity, GSH (reduced glutathione) levels or GCL (glutathione-cysteine ligase) activity. However, after intraperitoneal exposure, SWNT-PEG proved to be less biocompatible and formed aggregates, suggesting that the PEG used for the nanoparticle functionalization was of an inappropriate size for maintaining product stability in a biological environment. This problem with functionalization may have contributed to the low or practically absent biodistribution of SWNT-PEG in zebrafish tissues, as verified by Raman spectroscopy. There was an accumulation of material in the abdominal cavity that led to inflammation and behavioral disturbances, as evaluated by a histological analysis and an open field test, respectively. These results provide evidence of a lack of biocompatibility of SWNTs modified with short chain PEGs, which leads to the accumulation of the material, tissue damage and behavioral alterations in the tested subjects. © 2014 Published by Elsevier Inc.

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Introduction

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Nanomaterials, including single-wall carbon nanotubes, possess chemical, physical and/or biological properties that are dependent on the nanostructure and that provide functional characteristics of interest for applications in the biomedical field (Oberdörster et al., 2005; Zhao et al., 2011). These nanomaterials can interact with biological systems at the molecular and supramolecular levels with high specificity (Smith and Mizumori, 2006; Gilmore et al., 2008; Cheung et al., 2010; Meng et al., 2012). In recent years, numerous studies have been conducted to evaluate the potential applications of carbon nanotube

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⁎ Corresponding author at: Laboratório de Neurociências, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Av Itália, Km 8, Rio Grande, RS, 96210-900, Brazil. E-mail address: [email protected] (D.M. Barros).

devices for diagnostics, the transport and controlled release of drugs and cell culture (Kam et al., 2005; Medina et al., 2007; Foldvari and Bagonluri, 2008; Liang and Chen, 2010; Vashist et al., 2011; Naderi et al., 2013). Strategies for carbon nanotube purification and for controlling their dispersion and stability in physiological media are required to optimize these nanoparticles for biomedical applications (Ajayan, 1999). Their high hydrophobicity and tendency to form aggregates in aqueous media are challenges that must be overcome. One of the main strategies to solving these issues is to modify the NT surfaces with appropriate chemical moieties, such as hydrophilic polymers (Liu et al., 2009). Thus, the incorporation of polyethylene glycol (PEG) has become an attractive option for biological applications, as it not only improves NT dispersibility and stability in physiological media but also provides the particles with a biocompatible and protective surface, increasing the blood circulation half-life and decreasing the liver uptake of these nanostructures (Liu, 2009; Yang et al., 2008, 2008b; Ilbasmiş-Tamer et al., 2010).

http://dx.doi.org/10.1016/j.taap.2014.08.018 0041-008X/© 2014 Published by Elsevier Inc.

Please cite this article as: Weber, G.E.B., et al., Biodistribution and toxicological study of PEGylated single-wall carbon nanotubes in the zebrafish (Danio rerio) nervous system, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.08.018

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et al., 2008; de Castro et al., 2009), including assessing the behavioral and biochemical effects of nanomaterials (Fako and Furgeson, 2009). Determining the biodistribution and accumulation of nanomaterials in the different organs and tissues of exposed organisms should also be considered necessary for a comprehensive toxicological study. NTs that reach the CNS are known to promote oxidative stress (Nel et al., 2006; Oberdörster et al., 2009). Additionally, NTs can accumulate in tissues and cause neuronal injury (Zhang et al., 2010), which implies functional damage to the exposed organisms. Depending on their surface modifications and physicochemical characteristics, NTs may exhibit distinct patterns of biodistribution, toxicity and accumulation in the organs of exposed organisms (Cheng et al., 2009; Kang et al., 2009; Johnston et al., 2010; Jain et al., 2011). Therefore, the aim of the present work was to investigate the possible in vitro and in vivo effects of SWNT-PEG in Danio rerio “zebrafish” (Teleostei, Cyprinidae). To evaluate if the SWNT-PEG exposition leads to oxidative damage or interacts with the antioxidant machinery, we used an in vitro system of the zebrafish nervous system. Hereafter, to evaluate the biodistribution and possible behavioral alterations, intact animals were exposed intraperitoneally to SWNT-PEG dispersions, and to characterize the consequences of the apparent bioaccumulation, histological analyses of the target tissues (organs of the abdominal cavity and the brain) were performed.

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Carbon nanotubes functionalized with polyethylene glycol (SWNTPEG) have various applications in the biomedical field; however, this 74 work is focused on their applications in nervous tissue. The neuropro75 Q14 tective effects of SWNT-PEG in vivo were first reported by Roman et al. 76 (2012), who demonstrated their actions on axonal repair and functional 77 recovery in rats after spinal cord injury. Therefore, neuroscience is a 78 promising field for nanotechnology applications, especially for devices 79 and materials based on NTs (Smith and Mizumori, 2006). Studies have 80 demonstrated the biocompatibility of NT-containing substrates based 81 on the ability of nerve cells to grow and differentiate (Mattson et al., 82 2000; Liopo et al., 2006; Galvan-Garcia et al., 2007). Moreover, changes 83 in the NT structure may modulate the development of neurons in 84 Q15 culture (Bekyarova et al., 2005; Sucapane et al., 2009). Although the 85 aforementioned studies may report exciting applications for NTs in 86 neuroscience, other reports have shown some drawbacks related to 87 their application. Because the internalization of nanoparticles, in some 88 cases, triggers reactions that lead to cell death (Nel et al., 2006; Porter 89 et al., 2007), an investigation of the potential toxic effects arising from 90 interactions between the NT and cellular constituents is of fundamental 91 importance for its medical application. 92 Because the neurochemical systems and neural structures of zebrafish 93 have many similarities to those of other vertebrates, it is an important bi94 ological model for studying neurotoxicology (Linney et al., 2004; Barros

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Fig. 1. Physical and chemical characterization of SWNT-PEG. The size distribution, surface coating and purities were characterized using the various techniques described in the Experimental Section.


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In vitro experiments

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Animals and housing. Five hundred adult “wild type” (short fin) zebrafish (D. rerio) were obtained through breeding and cultivation in aquariums in an aquatic bioterium room at the Institute of Biological Sciences ,ICB, FURG or from a commercial supplier (Red Fish, Porto Alegre, RS, Brazil). All fish were acclimated for at least 2 weeks in the experimental room, were housed in groups of 15 fish in 15 L and were kept in a recirculating water system equipped with a biological filter and disinfected using UV light with controlled temperature (28 ± 2 °C), pH (7) and a 14–10 h day/night photoperiod. Feeding was performed using a balanced artificial diet (Tetra Color Tropical Granules) offered ad libitum twice daily. All protocols were approved by the Institutional

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Preparation of brain homogenates. For organ dissection, the fish were sacrificed by immersion in 200–300 mg/L tricaine methanesulfonate, and then, the brains were extracted. The organs were homogenized (1:3) in Tris–HCl buffer (100 mM, pH 7.75) containing EDTA (2 mM) and Mg2+ (5 mM) and were centrifuged at 10,000g for 20 min at 4 °C, and the supernatant was exposed to SWNT-PEG (Ferreira et al., 2012). This supernatant was used for the measurement of lipid peroxidation, GSH (reduced glutathione) content, GCL (glutathione-cysteine ligase) activity and total antioxidant capacity. Before the biochemical analysis, the total protein content was determined through the Biuret method (Doles, Brazil) using a microplate reader (BioTek LX 800).

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Animal Care Committee (process number 029/2011, CEUA-FURG) and followed Brazilian legislation, the guidelines of the Brazilian Collegium of Animal Experimentation (COBEA) and the Canadian Council for Animal Care (CCAC) “Guide on the Care and Use of Fish in Research, Teaching and Testing.”

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Fig. 3. Total antioxidant capacity against peroxyl radicals in brain extracts after 4 h of exposure to 1, 10 or 100 mg/L SWNT-PEG. The data are expressed as the means ± standard error (n = 5). Higher bars indicate lower total antioxidant capacity against peroxyl radicals.

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In the present study, we used a commercial sample of single-wall carbon nanotubes (SWNTs) (Sigma-Aldrich, 652474, Lot MKBC 9435, St. Louis, MO, USA) synthesized via the electric arc discharge method and functionalized with polyethylene glycol (PEG, MW = 600 Da). The SWNT-PEG material provided was found to contain ~25 wt.% of grafted PEG, ~60 wt.% of SWNTs and ~20 wt.% of 20–40 nm diameter carboncoated catalyst particles (Ni–Y), as determined by thermogravimetric (TGA) (TA Instruments, SDT 2960, in dry air at a scanning rate of 5 °C min−1) and transmission electron microscopic (TEM) (FEI, Tecnai G2-20 SuperTwin, 200 kV) analyses. An SWNT-PEG dispersion was prepared in deionized water at a high concentration (greater than 2 mg/ml). To prepare a stable dispersion and remove excess unbound PEG, a multi-step dispersion process employing several cycles of sonication, high-shear mixing, centrifugation and ultracentrifugation was required. The procedure was adapted from Kalinina et al. (2011). The morphological characteristics of the dispersion were analyzed by TEM and high-resolution TEM (HRTEM) performed on a FEI Tecnai G2-Spirit 120-kV and a FEI Tecnai G2-20 SuperTwin 200-kV microscope, respectively. AFM (atomic force microscopy) observations were carried out with an NTegra Aura (NT-MDT Co.) microscope. The Zeta potential was determined using the electrophoretic light scattering technique on a Zetasizer Nano-ZS ZEN3600 system (Malvern Instruments). The final concentration of SWNTs in the dispersion was spectrophotometrically evaluated by performing optical absorption measurements using a Shimadzu UV–Vis–NIR spectrophotometer UV-3600 over the wavelength range of 190 to 1100 nm. A standard curve of the absorbance vs. SWNT concentration (based on the Lambert-Beer law) was generated by diluting the original dispersion in water and measuring the optical absorbance at 700 nm. The mass concentration was determined by drying and weighing a known volume of the original dispersion.

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Preparation of SWNT-PEG dispersion and sample characterization

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Experimental section

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Fig. 2. Concentration of thiobarbituric acid reactive substances (TBARS) in the brain extracts 1, 2 or 4 h after exposure to 1, 10 or 100 mg/L SWNT-PEG. The data are expressed as the means ± standard error. *Significant difference (p b 0.05) by an orthogonal contrast (n = 5).

Fig. 4. A) Reduced glutathione (GSH) content in brain extracts after 4 h of exposure to 1, 10 or 100 mg/L SWNT-PEG. The data are expressed as the means ± standard error. *Significant difference (p N 0.05). B) Glutathione-cysteine ligase (GCL)-specific activity in brain extracts after 4 h of exposure to 1, 10 or 100 mg/L SWNT-PEG. The data are expressed as the means ± SEM (n = 5).


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Measurement of biochemical parameters. The lipid oxidative damage was assessed through the thiobarbituric acid reactive substances 187 Q17 (TBARS) fluorimetric assay (Oakes and Van Der Kraak, 2003). This 188 method is based on the reaction of the most abundant final product of 189 lipid peroxidation, malondialdehyde, with thiobarbituric acid in an 190 acidic medium, which forms a fluorescent complex with excitation

and emission at 515 nm and 553 nm, respectively. The readings were carried out in a microplate reader fluorimeter (Perkin Elmer). The concentration of TBARS (nmols/g wet tissue) in the brain samples was calculated by employing a standard curve of tetramethoxypropane (TMP; Acros Organics). To determine the GCL activity and GSH content in the brain tissue, we followed the method described by White et al. (2003), in which the reaction of naphthalene dicarboxaldehyde (NDA, Invitrogen) with GSH or γ-glutamylcysteine (γ-GC) generates cyclic products that are highly fluorescent (excitation/emission at 485/530 nm). The GCL activity and GSH content of the homogenates were determined by employing a GSH standard curve and were read in a microplate reader fluorimeter (Perkin Elmer).

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Exposure to SWNT-PEG. The brain extracts were exposed in vitro for 1, 2 or 4 h to SWNT-PEG (1, 10 or 100 mg/L) in dark conditions at 28 °C. These concentrations were selected based on other in vitro studies performed with PC12 cells (Zhang et al., 2011). The untreated control group was also run in parallel. After each exposure time, the aliquots were stored at −80 °C for further biochemical analysis.

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Fig. 5. Raman spectra of Danio rerio organs after the injections of SWNT-PEG dispersions at different concentrations. The first line depicts a positive control of SWNT-PEG.

Fig. 6. The total distance traveled (A and C) and velocity (B and D) were recorded over 5 min for animals exposed to three concentrations of SWNT-PEG (0.01, 0.1 or 1.0 mg/ml).


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Fig. 7. Pictures A, B, C and D are photos of the peritoneal cavity of zebrafish 24 h after the intraperitoneal injection of different concentrations of SWNT-PEG. The other images are slides of zebrafish tissues stained with hematoxylin and eosin. E shows the exocrine pancreas, with a normal acini aspect in the peritoneal fat (20×); F depicts the midgut, with incipient inflammatory infiltrates in the submucosa and dense mononuclear infiltrates in the peritoneum (10×); G shows the intestines, with dense inflammatory infiltrates in the mucosa and submucosa and with superficial mucosal necrosis (40×); and H shows peritoneal tissue with dense inflammatory infiltrates, mainly by macrophages. Melanin deposits were observed in the macrophages (40×); I consists of brain cortex, with slight edema and normal clear cells (40×). J and K are from the cortex, with glial proliferation and focal edema (40×), and L shows a brain section with glial proliferation, edema, signs of neuronal distress with nuclear pallor and satellitosis (40×).

the brain and heart tissues were used, as the other organs were immersed in SWNT-PEG during the intraperitoneal injection. After removal, the tissues were homogenized in a lysis buffer (1% SDS, 1% Triton X-100, 40 mM Tris acetate, 10 mM EDTA, 10 mM DTT) through homogenization and sonication (1 min for each sample) (Liu et al., 2008). Shortly before the analysis in the equipment (Horiba T 64000 Raman spectrometer, laser excitation wavelength = 785 nm), the tissues were heated at 70 °C for 2 h to obtain a clear lysate for Raman spectroscopy readings.

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Open field test. The behavioral task was performed using an open field test. The apparatus consisted of a large rectangular opaque glass tank (12.3 cm height × 38.7 cm width × 47.3 cm length) filled with aquarium water to the level of 12 cm (Stewart et al., 2012). In the test session, 42 adult male zebrafish (Animals and Housing Section) were used. The animals were divided into groups as follows: saline (n = 9), 0.01 mg/ml SWNT-PEG (n = 11), 0.1 mg/ml SWNT-PEG (n = 11) and 1 mg/ml SWNT-PEG (n = 11). The animals received intraperitoneal injections

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Biodistribution of SWNT-PEG. For this experiment, adult male zebrafish were employed (described in Animals and Housing Section). The animals were injected intraperitoneally with a total of five doses of 10 μl of SWNT-PEG at different concentrations (0.01 mg/ml, 0.1 mg/ml or 1.0 mg/ml) on alternate days. The control group received saline. Forty-eight hours after the last injection, all the animals were sacrificed by immersion in 200–300 mg/L tricaine methanesulfonate for the subsequent tissue removal. For the biodistribution assays, only

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Finally, the total antioxidant competence against peroxyl radicals was analyzed through ROS determination by employing the fluorogenic compound H2DCF-DA (Invitrogen) in brain homogenates that were untreated or treated with a peroxyl radical generator (2,2′-azobis-2methylpropionamidine-dihydrochloride, ABAP, 4 mM, Sigma-Aldrich) (Amado et al., 2009). The readings were carried out in a microplate reader fluorimeter (Perkin Elmer) at excitation and emission wavelengths of 485 and 530 nm, respectively.

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Inflamatory infiltrates cells Necrosis Peritoneal inflamatory cells Peritonealmelanin Edema Glial proliferetion Cells with neural distress Cells with satelitosis

0/40× field None 0/40× field None None None 0/40× field 0/40× field

b2/40× field Rare and focal b2/40× field Small focal melanin Focal Small, focal area b2/40 × field b2/40× field

2–5/40× field Moderate 2–5/40 × field Moderate melanin Moderate Moderate 2–5/40× field 2–5/40× field

N5/40× field Abundant N5/40× field Abundant melanin Severe Severe N5/40× field N5/40× field

Intraperitoneal space Brain


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Histological examination. The animals were injected using the same protocol employed for the biodistribution assay. Forty-eight hours after the last injection, the animals were sacrificed by immersion in tricaine methanesulfonate (200–300 mg/L) for the subsequent tissue removal (organs of the abdominal cavity and brain). The organs were then collected and fixed in 4% neutral buffered formalin, processed routinely into paraffin, sectioned at 7 μm and stained with hematoxylin and eosin (H&E). In all the experimental groups, five to eight animals were used, and two histological sections were measured for each fish. The sections were evaluated by a single board-certified medical pathologist.

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Results and discussion

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Statistical analysis. In the in vitro assays, all variables were analyzed 256 by an analysis of variance (ANOVA) (Stewart et al., 2012). The assump257 tions of normality and homogeneity of variances were verified, and 258 when at least one of the assumptions was violated, mathematical trans259 formations were applied. Comparisons between the means were then 260 performed through the Newmann–Keuls post-hoc test. Orthogonal con261 trasts were applied in the TBARS assay (see Fig. 4). In all cases, the type I 262 error probability was fixed at 0.05 (α = 0.05). 263 In the in vivo open field test (OFT), the variables were analyzed by 264 repeated-measures ANOVA with a Bonferroni post-hoc test. Next, the 265 endpoint values were calculated by taking the sum of the activity that 266 occurred during 1–5 min for each individual fish; these data were 267 then analyzed by a one-way ANOVA, followed by a Newman–Keuls 268 post-hoc test. The data are presented as the mean ± standard error of 269 the mean (S.E.M.), with significance set at p b 0.05. 270 To evaluate the microscopic findings of the tissues, a semi271 Q18 quantitative grading scale adapted from Janovic, Whitley and Palic´ 272 (2014) was used. The significance of the differences between the histo273 pathology scores was evaluated using the Kruskal–Wallis test, followed 274 by Dunn's post-hoc test. The data are presented as the mean ± S.E.M., 275 with significance set at p b 0.05.

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To understand the activity of carbon nanotubes in biological systems both in vitro and in vivo, the physicochemical features (i.e., surface functionalization, metallic contaminants, size and aggregation state) of 280 the nanomaterials used in the experiments must be considered Q20281 Q19 (Warheit, 2012; Firme and Bandaru, 2010). Low-resolution TEM analy282 ses revealed that commercial PEG-functionalized SWNT material 283 formed large aggregates in water, even after prolonged bath sonication. 284 The sample contained a large amount of metallic catalyst as well as 285 some graphitic impurities (Fig. 1). The concentration of the final disper286 sion was estimated by measuring the intensity of light absorption at 287 700 nm. A concentration of 2.1 ± 0.2 mg/ml was obtained after the en288 tire process. 289 The SWNTs in the aqueous dispersion were found to exhibit a Zeta 290 potential of approximately − 60 mV. This result may be an indication 291 that the PEG-functionalized nanotubes still contained many unreacted 292 carboxylic acid groups (–COOH). Indeed, the Zeta potential of the 293 SWNT-PEG decreased to approximately −30 mV with increasing NaCl 278 279

concentration (up to 0.06 mol/L), indicating a screening of repulsive forces, as would be expected for charged particles. Due to their relatively high negative Zeta potential, we expected that the SWNT-PEG hybrids would be vulnerable to aggregation in biological systems because physiological fluids contain a mixture of salts, proteins and other macromolecules that can interact with negatively charged nanoparticle surfaces. Nance et al. (2012) demonstrated that nanoparticles with Zeta potentials less negative than −4 mV were able to diffuse throughout the nervous tissues of rats; however, particles with Zeta potentials lower than −6 mV were unable to diffuse through the same tissues. All of these complex interactions between nanoparticles and biological media constituents must be taken into account by performing in vitro analyses before performing studies in vivo. To assess their intrinsic behavior in biological environments in vitro, we introduced different concentrations of SWNT-PEG to the homogenized brain tissue of zebrafish and evaluated representative oxidative stress parameters to verify the interactions of these nanostructures with biomolecules. In toxicological evaluations, there are a number of different results that depend on the synthesis method and the physicochemical characteristics of the nanomaterial (Heister et al., 2010). Furthermore, certain properties, such as surface coating and solubility, may decrease or amplify the size effect, and the size, shape and aggregation are nanomaterial characteristics that can elicit reactive oxygen species (ROS) generation (Suh et al., 2009). Oxidative stress is possibly the main mechanism of nanomaterial toxicity, resulting in damage to lipids, proteins and DNA (Jones, 2006). We assessed lipid peroxidation as a sensible indicator of the injury level caused by ROS production (Jones, 2006). We observed a significant decrease (p b 0.05) in lipid peroxidation after exposure of the homogenized tissue to 1 mg/L SWNT-PEG for 4 h compared to the control group (Fig. 2). Wang et al. (2011) demonstrated that there was a significant increase in the levels of MDA in PC12 cells treated with different concentrations of SWNTs, which occurred in a time- and dose-dependent manner. This difference may be because the SWNTs used in this work were functionalized with PEG; similar nanomaterials were used in another study with PC12 cells in which the authors demonstrated that functionalization with PEG significantly reduces the side effects of SWNTs as well as the disturbance of oxidative stress-related gene expression (Zhang et al., 2011). Among the oxidative stress parameters studied in the tissue homogenates, we chose a time exposure of 4 h because the time condition was the only one for which a significant modulation in lipid peroxidation was observed. The next parameter analyzed was the total antioxidant capacity, which demonstrated no significant change (p N 0.05) (Fig. 3). These results indicate that the presence of SWNT-PEG did not disturb the antioxidant defense system in the brain homogenates. We also analyzed the GSH content, as GSH is a major intracellular low-molecular-weight thiol compound that plays a critical role in the cellular defense against oxidative stress through scavenging of ROS directly or indirectly in cells (Halliwell and Gutteridge, 2001), and GCL activity, as GCL is the enzyme responsible for catalyzing the synthesis of GSH (Maher, 2006). Our results indicated a significant decrease in the GSH content of the extract exposed to lower concentrations of SWNTPEG (Fig. 4), a finding that agreed with the results of other studies (Zhang et al., 2011; Valko et al., 2007). The depletion of GSH content induced by SWNT-PEG may be due to an increased production of reactive oxygen species (ROS). However, the other results of our study suggested that this increase in ROS was not sufficient to cause lipid damage in the brain extract and did not increase the activity of GCL (Fig. 4). In addition to the in vitro analysis, an in vivo study was conducted. We evaluated the biodistribution of the nanomaterial by Raman

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of 10 μl of SWNT-PEG 24 h before the test, and the tests were performed in the morning. The fish were placed into the tank and videotaped for 5 min (divided into five separate trials). The trials were recorded with a camera (Sony Handycam DCR-SR47, New York, NY) and were analyzed off-line using Ethovision XT7 (Noldus IT, Wageningen, Netherlands). Later, we analyzed the mean velocity (cm/s) and distance traveled (cm).

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Fig. 8. Histopathology scores. The semi-quantitative scores are representative of histopathologic features in the control and SWNT-PEG treatments. Comparisons of the mean histopathology scores for inflammatory infiltrate in the cells of the intestine (A), necrosis in the intestine (B), peritoneal inflammatory cells (C), peritoneal melanin (D), brain edema (E), glial proliferation (F), cells with neuronal distress (G) and cells with satellitosis (H) are shown. Values are expressed as the means ± SEM, n = 5–8. *p b 0.05 for a statistically significant effect (Kruskal–Wallis test, followed by Dunn's post-hoc test).


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This work was supported by Programa de Núcleos Emergentes (PRONEM/FAPERGS) (11/2037-9), DECIT/SCTIE-MS through Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Proc. 10/0036-5-PRONEX/Conv. 700545/2008), Nanotoxicology Network (MCTI/CNPq process number 552131/2011-3) and Instituto Nacional de Ciência e Tecnologia de Nanomateriais de Carbono (INCT-NanoCNPq). Daniela Martí Barros is a research fellow from CNPq, and Gisele Eva Bruch Weber received a graduate fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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spectroscopy; however, if the SWNT-PEG was distributed throughout the zebrafish organs, it reached tissues at concentrations below 10−5 mg/ml 357 (Fig. 5), as the Raman spectra of the SWNT-PEG G band was not observed 358 in the brain tissues. The SWNT-PEG G band was observed in the control 359 samples with SWNT-PEG concentrations as low as 10−5 mg/ml. Another 360 study, which was performed using mice and SWNTs noncovalently 361 functionalized with different PEG chains, also did not observe the 362 distribution of these nanomaterials to the brain. This study also 363 assessed particle distribution in the blood, liver and spleen, among 364 other sites, and in these tissues, the presence of PEGylated SWNTs 365 was verified (Liu et al., 2008). 366 To assess the indirect toxic whole-body effects caused by the nano367 tubes, we conducted an open field test on animals 24 h after exposure 368 to different concentrations of SWNT-PEG. Locomotor and exploratory 369 activities were evaluated in this test. Only the animals exposed to the 370 highest concentration of SWNT-PEG (1 mg/ml) demonstrated a signifi371 cant increase in the overall distance traveled (Fig. 6). Truong et al. 372 (2012) also observed abnormalities in the locomotor activity of animals 373 exposed to gold nanoparticles, supporting the validity of this test for 374 monitoring changes in the central nervous system. 375 Histological changes in the animals that received intraperitoneal in376 jections of SWNT-PEG were also analyzed (Fig. 7). In this experiment, 377 we observed increased tissue injury and inflammation in a dose378 dependent manner. In the abdominal cavity organs, we observed inju379 ries caused by direct tissue interaction with the nanomaterial. Although 380 nervous tissue damage occurred in an indirect way because no SWNT381 PEG was found in the brain, the animals presented an inflammatory re382 sponse, as demonstrated by the histological analysis. Furthermore, the 383 analysis of the semi-quantitative histopathology scoring of the tissues 384 (Table 1) shows differences between the control group and the treat385 ments, as shown in Fig. 8. These data show, in a dose-dependent man386 ner, an increase in inflammatory infiltrate cells in the intestine (A), 387 necrosis in the intestine (B), peritoneal inflammatory cells (C), perito388 neal melanin (D), brain edema (E), glial proliferation (F), cells with neu389 ronal distress (G) and cells with satellitosis (H). These results are 390 congruent with a study using fathead minnow, where intraperitoneal 391 injection of hydroxylated fullerenes was reported to induce hepatic 392 and renal tissue injury, with dose-dependent tissue injury and higher 393 Q21 cumulative histopathology scores as well (Janovic, Whitley and Palic´ 394 2014). 395 The results obtained in this study demonstrated that the SWNT-PEG 396 caused toxicity due to its lack of biocompatibility in vivo, which may 397 compromise its use for biomedical proposes. The in vitro experiment 398 showed that the SWNT-PEG produced changes in the oxidative stress 399 parameters analyzed, with a discrete antioxidant effect on lipid peroxi400 dation and an opposite pro-oxidant effect, as indicated by the depletion 401 of GSH. Additionally, it is important to consider the influence of contam402 inants present in the SWNT-PEG sample, such as Yttrium (Y) and Nickel 403 (Ni). Jakubek and co-workers have shown that these metals are capable 404 of inhibiting calcium channels, which could be the mechanism respon405 sible for a variety of toxic effects related to these metals (Jakubek et al, 406 2009). 407 It is possible to conclude that these nanomaterials have some limita408 tions. As demonstrated by the in vivo study, the SWNTs functionalized 409 with PEG 600 were not able to distribute to other organs, most likely 410 due to their instability in saline medium. In a biological medium, the ag411 gregation of the nanotubes was observed and resulted in damage to 412 blood vessels, leading to thrombus formation and eventual tissue necro413 sis. Thus, an extensive inflammatory process occurred once the 414 nanomaterials were trapped in the abdominal cavity after injection. Ad415 ditionally, in the central nervous system, several histological modifica416 tions were observed in addition to the behavioral changes observed in 417 the open field test. However, because these nanomaterials are promis418 ing in the biomedical area, further studies are necessary, especially 419 with SWNTs functionalized with larger PEG chains and denser PEG 420 coatings.

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Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio).

Chemical proteomic analysis of the potential toxicological mechanisms of microcystin-RR in zebrafish (Danio rerio) liver.

Manipulating galectin expression in zebrafish (Danio rerio).

Functional characterization of zebrafish (Danio rerio) Bcl10.

Short-term memory in zebrafish (Danio rerio).

Semicarbazide disturbs the reproductive system of male zebrafish (Danio rerio) through the GABAergic system.

The Control of Calcium Metabolism in Zebrafish (Danio rerio).

Genetic Analysis of the Touch Response in Zebrafish (Danio rerio).

Impairment of the immune system in GH-overexpressing transgenic zebrafish (Danio rerio).

dsRNA-loaded nanoliposomes.

Primary intestinal and vertebral chordomas in laboratory zebrafish (Danio rerio).

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

Effects of Atrazine on the Development of Neural System of Zebrafish, Danio rerio.

Transformation of tributyltin in zebrafish eleutheroembryos (Danio rerio).

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio).

dark preference in zebrafish (Danio rerio).

(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures.

Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

Mechanism of TiO2 nanoparticle-induced neurotoxicity in zebrafish (Danio rerio).

Cardiotoxicity evaluation of anthracyclines in zebrafish (Danio rerio).

A physiologically based toxicokinetic model for the zebrafish Danio rerio.

Vascular toxicity of silver nanoparticles to developing zebrafish (Danio rerio).

Establishment of Infection Models in Zebrafish Larvae (Danio rerio) to Study the Pathogenesis of Aeromonas hydrophila.

Brain Transcriptomic Response to Social Eavesdropping in Zebrafish (Danio rerio).