Letter pubs.acs.org/NanoLett
Graphite Nanoplatelets and Caenorhabditis elegans: Insights from an in Vivo Model Elena Zanni,†,⊥ Giovanni De Bellis,∥,‡,⊥ Maria P. Bracciale,§,⊥ Alessandra Broggi,§ Maria L. Santarelli,∥,§ Maria S. Sarto,∥,‡ Claudio Palleschi,*,†,∥ and Daniela Uccelletti†,∥ †
Department of Biology and Biotechnology “C. Darwin”, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy Department of Astronautic, Electrical and Energetic Engineering, Sapienza University of Rome, via Eudossiana 18, Rome 00184, Italy § Department of Chemical Engineering, Materials and Environment, Sapienza University of Rome, via Eudossiana 18, Rome 00184, Italy ∥ Research Center on Nanotechnology Applied to Engineering of Sapienza (CNIS), SSNLab, Sapienza University of Rome, P.le Aldo Moro, 5 Rome 00185, Italy ‡
S Supporting Information *
ABSTRACT: We evaluated the toxicity of graphite nanoplatelets (GNPs) in the model organism Caenorhabditis elegans. The GNPs resulted nontoxic by measuring longevity as well as reproductive capability end points. An imaging technique based on Fourier transform infrared spectroscopy (FT-IR) mapping was also developed to analyze the GNPs spatial distribution inside the nematodes. Conflicting reports on the in vitro antimicrobial properties of graphene-based nanomaterials prompted us to challenge the host−pathogen system C. elegans−Pseudomonas aeruginosa to assess these findings through an in vivo model. KEYWORDS: Graphene, graphite nanoplatelets, Caenorhabdtis elegans, antimicrobial agent, μFT-IR analysis, Pseudomonas aeruginosa
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reported,10,11 data on in vivo toxicity have not been extensively investigated yet. Here we report an in vivo toxicity study of graphite nanoplatelets on the well-known animal model Caenorhabditis elegans as well as the usefulness of the IR mapping technique for studying detailed spatial distribution of nanoscale materials inside such organisms. In addition, the antimicrobial properties of GNPs were investigated by in vitro and in vivo approaches. The experimental potential exploitable in C. elegans offers a system best suited for asking in vivo questions with relevance at the organism level. This free-living soil nematode can be easily cultivated in laboratory environment and reproduced in thousands of individuals essentially like microorganisms. The completion of the C. elegans genome has revealed that about 45% of genes have human homologues, including numerous disease-related genes.12,13 The use of C. elegans offers speed and relative easiness for discovering genetic modifiers of the studied phenotypes and for screening interacting chemical agents. Fluorescent nanoparticles have been used for in vivo imaging in C. elegans. Austin and co-workers 14 showed that the upconversion phosphors composed of Y2O3:Yb,Er can be
hough already investigated as early as 1947,1 graphene was not obtained as a single sheet until 2004, when 2010 Nobel prizes laureates Andre Geim and Konstantin Novoselov were able to isolate the first monolayer using a technique which will be thereafter known as the Scotch tape method.2 The whole scientific community has since then been exploring new applications of this outstanding material, taking advantage of its remarkable and unique properties. Graphene, a single sheet of sp2 hybridized honeycomb carbon, has proved to be among the most promising materials known to mankind due to its unique properties. It shows a Young’s modulus around 1 TPa,3 a thermal conductivity exceeding the values reported for both diamond and carbon nanotubes,4 and intriguing electronic properties, such as an extremely high carrier mobility and a linear dispersion relation.5,6 Besides these very exciting properties, recently graphene has been found to be a valuable material for applications in both biotechnology and nanomedicine. Different studies have in fact shown that graphene and graphene-related materials, such as graphite nanoplatelets (GNPs), can be efficiently used for a wide variety of useful applications, including drug delivery,7 photothermal anticancer activity,8 and biosensors and biofunctionalization.9 However, though several preliminary in vitro toxicity studies of both graphene and graphene oxide have been recently © 2012 American Chemical Society
Received: December 13, 2011 Revised: May 15, 2012 Published: May 21, 2012 2740
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imaged in the digestive system using a fluorescence microscope and a near-infrared laser as light source. The authors also checked the survival rate of the organism and discovered a little acute toxic effects of the material. In contrast, fluorescent nanodiamonds showed neither detectable in vivo toxicity nor any apparent side effects, like stress response.15 Serious reproductive senescence and early death of the organism were instead found for nanoparticles made of silver,16 silica,17 and fullerol.18 Our goal was to analyze the effects of GNPs intake by living organisms by utilizing the nematode C. elegans. The nanoplatelets were prepared as reported in a previous work19 and suspended in H2Odd. As confirmed by Fourier transform infrared spectroscopy (μATR-FTIR, see Supporting Information), as-synthesized GNPs are completely reduced and no residual oxygencontaining functional group was detectable. Moreover, the nanoplatelets showed a lateral size ranging from one to tens of micrometers as revealed by SEM analysis. Similarly, we observed thickness values from 1 to 20 nm, corresponding to a number of graphene layers varying between 3 and ∼60 (see Supporting Information for details). Typical as-produced GNPs are shown in Figure 1a,b. A toxicity study on adult C. elegans individuals treated with GNPs was then performed. After 3 h of GNPs exposure, worms were introduced on nematode growth medium (NGM) agar plates in the presence of E. coli OP50 strain as the feeding source. Afterward, the viability was examined with respect to untreated animals. For all tested concentrations, GNPs
treatment was not able to increase nematodes mortality (Figure 2a), demonstrating the absence of acute toxicity in vivo, in
Figure 2. (a) Acute toxicity assay of GNPs suspensions on C. elegans worms. Adult worms were incubated with different concentrations of GNPs for 3 h. Nematode survival was monitored for 3 days and is expressed as the percentage with respect to untreated worms. The results are the means of three independent experiments; the error bars indicate SD. The fullerol nanoparticles suspension (100 μg/mL) was used as a positive control of toxicity. (b) Average brood sizes per worm of nematodes exposed or not to suspensions of GNPs (250 μg/ mL) or fullerol (100 μg/mL) is reported. Worms were allowed to lay eggs, and all progeny was counted daily. The bars are the mean of three independent experiments. (c) Chronic toxicity assay of GNPs or fullerol suspensions on C. elegans worms. Newly hatched C. elegans were seeded onto NGM plates supplemented with E. coli and were treated or not with GNPs suspensions at the indicated concentrations. The lifespan analysis is reported. The error bars indicate SD.
contrast to fullerol nanoparticles used as a positive control.18 To deeper investigate GNPs bioassessment, we analyzed C. elegans reproductive potential as an indicator for chronic toxicity. Hence, a brood size assay for animals exposed for 3 h to GNPs was performed. As shown in panel b of Figure 2, the worms exposed to the highest GNPs concentration (250 μg/ mL) showed no deviation from the untreated controls for the progeny production. Almost identical results were also obtained
Figure 1. SEM micrographs of GNP flakes obtained after thermal expansion and sonication of GICs. 2741
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with GNPs suspensions concentrated at 100 and 50 μg/mL (not shown). To examine the effects of GNPs chronic exposure on the nematodes lifespan, newly hatched C. elegans were introduced onto NGM agar plates in the presence of OP50. Every 2 days animals were transferred to new plates, and 200 μL of GNPs suspensions, at the indicated concentrations, was distributed onto the plates, before nematodes seeding. Also in this case, no lifespan differences in GNPs-treated worms, in comparison with control animals, were observed (Figure 2c). By contrast, fullerol treated animals died 6 days before the untreated worm population. In order to demonstrate the effective GNPs intake and their distribution inside the animals, a μFT-IR analysis was carried out on adult worms fed for 24 h with the nanoparticles. In parallel, the analysis was also performed on the untreated nematode as the control. In order to observe the GNPs spatial distribution the peak centered at 865 cm−1, related to the C−C lattice mode, was monitored .20 Notably, untreated animals did not exhibit the GNPs-related peaks (Figure S3), and three independent experiments were performed to verify the spectra reproducibility. In Figure 3, the 2D (panel a) and 3D (panel b) mapping showed that the nanoplatelets are distributed along the nematode body. Moreover, the same GNPs-related peak was observed in the embryos laid by the adult, just after its mounting on the ZnSe window (Figure 3, panels c, d). This is a strong biological evidence demonstrating the transition of nanoparticles from the intestine to the gonads. In previous works FT-IR microspectroscopy was established as a powerful technique to obtain information on the secondary structure of proteins in complex biological systems including nematodes.21,22 Overall our data broadened the usefulness of IR mapping spectroscopy for the evaluation of the distribution of carbon nanomaterials in a whole living organism such as C. elegans. The ever-growing interest for graphene-based nanoparticles in potential applications such as nanosensors and nanomedicine prompted us to investigate the antimicrobial properties of GNPs. Conflicting reports have been published on the interactions between graphene-based nanoparticles and bacteria ranging from nonspecific enhancement of cell growth to efficient antibacterial activity.11,23−26 We therefore investigated the interaction between GNPs and Pseudomonas aeruginosa, an ubiquitous Gram-negative bacterium that causes systemic acute diseases in patients with weakened immune systems27 and establishes chronic infections in the lungs of cystic fibrosis patients.28 To this aim, a bacterial cell viability assay was carried out after a 5 h treatment with the nanoplatelets (see Supporting Information). We found that GNPs strikingly reduced the P. aeruginosa CFU count with respect to the untreated control, leading to a viability loss up to 69.5% (Figure 4a). In order to find out how GNPs kill bacteria, we explored the possibility that oxidative stress could be the major cause for the toxicity. It has been reported that oxidative stress mediated by graphenebased materials may come from reactive oxygen species (ROS) generation inside the cells.23 The ROS accumulation was thus monitored in bacteria after incubation with the nanoplatelets using the dihydrorhodamine staining method. However, no ROS production was observed in treated cells, suggesting that GNPs interaction did not induce oxidative stress in P. aeruginosa (data not shown). Since mechanical damages can be induced on bacterial cells after direct contact with graphenebased materials,23 a field-emission scanning electron micro-
Figure 3. Spectroscopic 2D and 3D images of the graphite nanoplatelets spatially distributed within the adult nematode (a, b) and the embryos (c, d). All spectra were collected in transmission mode with a 3 cm−1 spectral resolution, and the images were obtained by monitoring the 865 cm−1 peak; the absorption intensity was measured and expressed as arbitrary units.
scope (FE-SEM) was used to examine the interactions between the nanoplatelets and P. aeruginosa cells. Figure 4c,d shows that most of cells became crumpled on GNPs surface losing their cellular integrity. FE-SEM images give evidence that graphite nanoplatelets provoked severe mechanical damages at the bacterial surface. By contrast, in P. aeruginosa cells incubated 2742
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in vitro approaches, we then evaluated the in vivo antibacterial potential of GNPs by exploiting the unique opportunities of the P. aeruginosa−C. elegans infection model. In nematodes, in fact, the infection induces a number of defense mechanisms, some of which are similar to those seen in mammalian innate immunity. In addition, it has been demonstrated that several microbial virulence mechanisms required for full pathogenicity in mammals are also necessary for infection in nematodes.29 Therefore, C. elegans animals were infected for 16 h with a reference strain of P. aeruginosa (ATCC 15692) expressing the green fluorescent protein (GFP), resulting in bacteria appearing green under a fluorescence microscope.30 Infected animals were then exposed or not to the GNPs suspension, and GFP intensity was monitored as an indication of viable bacteria colonizing the worms. As shown in Figure 5, P. aeruginosa cells
Figure 5. Effect of GNPs treatment (250 μg/mL) on the colonization of P. aeruginosa cells within the nematode gut. Nematodes were infected with the GFP-expressing P. aeruginosa strain for 16 h and then exposed or not to GNPs. Fluorescence photomicrograph of a representative nematode after 30 min of treatment with GNP is reported. A representative untreated animal (Ut) was included for comparison.
were spread throughout the whole digestive tract of the untreated animals, with a higher accumulation in the anterior intestine (left panel). By contrast, an almost complete loss of the fluorescence was visualized, along the entire intestine, as a result of GNPs treatment (right panel). These data strongly support that effective killing of GFP-expressing Pseudomonas was taking place in the infected animals. The fluorescence reduction was detected already after the first 30 min of incubation with GNPs. The mechanical effect on the bacterial cell wall of GNPs would undoubtedly be affected by their dispersion state. GNPs, due to their hydrophobicity, can produce large agglomerates in aqueous solution like other graphene-related nanomaterials.31 Recently, highly dispersed preparations of graphene obtained in nonionic amphiphilic block copolymers resulted in modified biological outcomes, mainly improved biocompatibility.32 The possibility that GNPs, similarly dispersed, might mitigate their antibacterial effects or alter their distribution in the nematode should be acknowledged, and it will deserve further studies in future work. The use of a simple yet multicellular organism such as C. elegans allowed the in vivo investigation of the graphite nanoplatelets toxicity and their antimicrobial properties. Overall, our study pointed out the absence of any acute or chronic toxicity of GNPs on the animals. Moreover, the genotoxicity has been investigated, and no effect on C. elegans reproductive potential has been found. Our work highlighted the huge potential for the use of graphene and graphene-related
Figure 4. (a) In vitro antimicrobial activity of graphite nanoplatelets on Pseudomonas aeruginosa bacteria. Cell viability assay was carried out after incubation of P. aeruginosa cells (5 × 107 CFU) with the GNPs suspension (250 μg/mL) for 5 h. Cells survival was monitored by colony counting method and is expressed as the percentage with respect to untreated bacteria incubated with H2Odd. The error bars indicate SD (b) SEM images of cells after incubation with H2Odd for 5 h without graphite-based materials and (c, d) bacteria after exposure for 5 h with GNP suspension (250 μg/mL).
with H20dd for 5 h, the outer structures maintained their integrity. Given such interesting properties, highlighted by the 2743
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(14) Lim, S. F.; Riehn, R.; Ryu, W. S.; Khanarian, N.; Tung, C. K.; Tank, D.; Austin, R. H. Nano Lett. 2006, 6, 169−174. (15) Mohan, N.; Chen, C. S.; Hsieh, H. H.; Wu, Y. C.; Chang, H. C. Nano Lett. 2010, 10, 3692−3699. (16) Roh, J. Y.; Sim, S. J.; Yi, J.; Park, K.; Chung, K. H.; Ryu, D. Y.; Choi, J. Environ. Sci. Technol. 2009, 43, 3933−3940. (17) Pluskota, A.; Horzowski, E.; Bossinger, O.; von Mikecz, A. PLoS One 2009, 4, e6622. (18) Cha, Y. J.; Lee, J.; Choi, S. S. Chemosphere 2012, 87, 49−54. (19) De Bellis, G.; Tamburrano, A.; Dinescu, A.; Santarelli, M. L.; Sarto, M. S. Carbon 2011, 49, 4291−4300. (20) Nemanich, R. J.; Lucovsky, G.; Solin, S. A. Solid State Commun. 1977, 23, 117−120. (21) Ami, D.; Natalello, A.; Zullini, A.; Doglia, S. M. FEBS Lett. 2004, 576, 297−300. (22) Diomede, L.; Cassata, G.; Fiordaliso, F.; Salio, M.; Ami, D.; Natalello, A.; Doglia, S. M.; De Luigi, A.; Salmona, M. Neurobiol Dis. 2010, 40, 424−431. (23) Ruiz, O. N.; Fernando, K. A.; Wang, B.; Brown, N. A.; Luo, P. G.; McNamara, N. D.; Vangsness, M.; Sun, Y. P.; Bunker, C. E. ACS Nano 2011, 5, 8100−8107. (24) Park, S.; Mohanty, N.; Suk, J. W.; Nagaraja, A.; An, J.; Piner, R. D.; Cai, W.; Dreyer, D. R.; Berry, V.; Ruoff, R. S. Adv. Mater. 2010, 22, 1736−1740. (25) Das, M. R.; Sarma, R. K.; Saikia, R.; Kale, V. S.; Shelke, M. V.; Sengupta, P. Colloids Surf., B 2011, 83, 16−22. (26) Bao, Q.; Zhang, D.; Qi, P. J. Colloid Interface Sci. 2011, 360, 463−470. (27) Lyczak, J. B.; Cannon, C. L.; Pier, G. B. Microbes Infect. 2000, 2, 1051−1060. (28) Lyczak, J. B.; Cannon, C. L.; Pier, G. B. Clin. Microbiol. Rev. 2002, 15, 194−222. (29) Ewbank, J. J.; Zugasti, O. Dis. Models & Mech. 2011, 4, 300− 304. (30) Uccelletti, D.; Zanni, E.; Marcellini, L.; Palleschi, C.; Barra, D.; Mangoni, M. L. Antimicrob. Agents Chemother. 2010, 54, 3853−3860. (31) Schinwald, A.; Murphy, A. F.; Jones, A.; MacNee, W.; Donaldson, K. ACS Nano 2012, 6, 736−746. (32) Duch, M. C.; Budinger, G. R. S.; Liang, Y. T.; Soberanes, S.; Urich, D.; Chiarella, S. E.; Campochiaro, L. A.; gonzalez, A.; Chandel, N. S.; Hersam, M. C.; Mutlu, G. M. Nano Lett. 2011, 11, 5201−5207.
structures like GNPs as antibacterial materials, through both in vitro and in vivo experiments. We suggest that the data obtained by using the simple nonetheless differentiated animal model C. elegans could optimize the experimental validation procedures involved in safety issues. In fact, the combined effect of long-term biocompatibility, absence of genotoxicity, and antimicrobial activity demonstrated in C. elegans should push to study such properties directly in mammalian models, especially those highly predictive for human applications. We believe that the present study can represent a valuable support for the GNPs in the development of a wide range of bio-related applications, such as drug delivery, phototermal cancer therapy, biosensors, and nanocomposites for safer medical devices.
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ASSOCIATED CONTENT
* Supporting Information S
Detailed experimental protocols, bacterial and nematode strain used in this work, and GNPs SEM analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected]; Tel +39-0649912132; Fax +39-0649912351. Author Contributions ⊥
The authors contributed equally to the work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by MIUR-PRIN project entitled “Development and Electromagnetic Characterization of Nano Structured Carbon-Based Polymer CompositEs (DENSE)”, Prot. 2008NMRHJS, in part by Regione Lazio within the Project ULS003 “NANOLAB: A laboratory for the development of multifunctional innovative micro/nanostructured materials and devices” and in part by FIRB RBIN06E9Z8.
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REFERENCES
(1) Wallace, P. R. Phys. Rev. 1947, 71, 622−634. (2) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Science 2004, 306, 666−669. (3) Lee, C.; Wei, X.; Kysar, J. W.; Hone, J. Science 2008, 8, 321−358. (4) Balandin, A. A.; Ghosh, S.; Bao, W.; Calizo, I.; Teweldebrhan, D.; Miao, F. Nano Lett. 2008, 8, 902−907. (5) Bolotin, K. I.; Sikes, K. J.; Jiang, Z.; Klima, M.; Fudenberg, G.; Hone, J.; Kim, P.; Stormera, H. L. Solid State Commun. 2008, 146, 351−355. (6) Gierz, I.; Riedl, C.; Starke, U.; Ast, C. R.; Kern, K. Nano Lett. 2008, 8, 4603−4607. (7) Feng, L.; Liu, Z. Nanomedicine 2011, 6, 317. (8) Markovic, Z. M.; Harhaji-Trajkovic, L. M.; Todorovic-Markovic, B. M.; Kepić, D. P.; Arsikin, K. M.; Jovanović, S. P.; Pantovic, A. C.; Dramićanin, M. D.; Trajkovic, V. S. Biomaterials 2011, 32, 1121. (9) Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Trends Biotechnol. 2011, 29, 205. (10) Chang, Y.; Yang, S. T.; Liu, J. H.; Dong, E.; Wang, Y.; Cao, A.; Liu, Y.; Wang, H. Toxicol. Lett. 2011, 200, 201−210. (11) Akhavan, O.; Ghaderi, E. ACS Nano 2010, 4, 5731−5736. (12) Culetto, E.; Sattelle, D. B. Hum. Mol. Genet. 2000, 9, 869−877. (13) Brenner, S. Genetics 1974, 77, 71−94. 2744
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