Environ. Sci. Technol. 2006, 40, 6151-6156
Cytotoxicity of CeO2 Nanoparticles for Escherichia coli. Physico-Chemical Insight of the Cytotoxicity Mechanism A N T O I N E T H I L L , * , † O P H EÄ L I E Z E Y O N S , † OLIVIER SPALLA,† FRANCK CHAUVAT,‡ JERO ˆ M E R O S E , § M EÄ L A N I E A U F F A N , § A N D ANNE MARIE FLANK| CEA Saclay, Direction des Sciences de la Matiere, Departement de Recherche sur l’Etat Condense les Atomes et les Molecules, Laboratoire Interdisciplinaire sur l’Organisation Nanometrique et Supramoleculaire, 91191 Gif-sur-Yvette, France, CEA Saclay, Direction des Sciences du Vivant, Service de Biologie Moleculaire Systemique, 91191 Gif-sur-Yvette, France, CEREGE UMR 6635 CNRS-Universite Paul Cezanne Aix-Marseille 3, IFR Pole Mediterraneen des Sciences de l’Environnement, 112, Europole de l’arbois, 13545 Aix-En-Provence, France, and Swiss Light Source, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland
The production of nanoparticles (NPs) is increasing rapidly for applications in electronics, chemistry, and biology. This interest is due to the very small size of NPs which provides them with many interesting properties such as rapid diffusion, high specific surface areas, reactivity in liquid or gas phase, and a size close to biomacromolecules. In turn, these extreme abilities might be a problem when considering a potentially uncontrolled exposure to the environment. For instance, nanoparticles might be highly mobile and rapidly transported in the environment or inside the body through a water or air pathway. Accordingly, the very fast development of these new synthetic nanomaterials raises questions about their impact on the environment and human health. We have studied the impact of a model water dispersion of nanoparticles (7 nm CeO2 oxide) on a Gram-negative bacteria (Escherichia coli). The nanoparticles are positively charged at neutral pH and thus display a strong electrostatic attraction toward bacterial outer membranes. The counting of colony forming units (CFU) after direct contact with CeO2 NPs allows for the defining of the conditions for which the contact is lethal to Escherichia coli. Furthermore, a set of experiments including sorption isotherms, TEM microscopy, and X-ray absorption spectroscopy (XAS) at cerium L3 edge is linked to propose a scenario for the observed toxic contact.
1. Introduction Nanotechnologies are developing rapidly in numerous industries and in particular those related to the following: * Corresponding author e-mail:
[email protected]. † Laboratoire Interdisciplinaire sur l’Organisation Nanometrique et Supramoleculaire. ‡ Service de Biologie Moleculaire Systemique. § Universite Paul Cezanne Aix. | Paul Scherrer Institut. 10.1021/es060999b CCC: $33.50 Published on Web 08/31/2006
2006 American Chemical Society
computer sciences and information technologies; material sciences with optical, magnetic, or mechanical applications; medical sciencessdiagnostics tools (1) and drug delivery (2); and environmental applicationssclean energy (fuel cells), membranes, and catalysts. Indeed, the particular physical and chemical properties of nanoobjects (rapid diffusion, high specific surface areas, important reactivity in liquid or gas phase, size close to biomacromolecules such as DNA and other biological molecules) contribute to the positive expectations associated with their use. However, with these particular and promising properties, interrogations about their potential toxicity and environmental impact emerge (3-6). Indeed, it is suspected that their small size can bring new and unforeseen toxicity, and some aspects of recent history (e.g., asbestos fibers) just feed the mistrust of those materials. This suspicion of a direct toxicity is emphasized when considering the furtivity of fully dispersed nanoobjects. Past errors, and the application of a “principle of precaution” in many countries, underline the crucial need for intensive research on the risks associated with the increasing production of nanomaterials. Several papers related to this subject have been published very recently (7-10). The present work is dedicated to the impact of nanoparticles (thereafter called NPs) through a water path and describes the interaction between a water dispersion of nanoparticles and a model bacteria. We have selected cerium oxide as the model nanoparticles for our studies, and, on the other hand, E. coli was chosen as a widely used model organism. Our two main reasons for choosing these nanoparticles were as follows: they are relevant to the global initial question since cerium oxide is indeed used in its nanometric form as an exhaust gas catalyst, and, when they are diluted, they form a dispersion of individual NPs. Cerium toxicity has been previously studied in its CeIII form (11), and, more recently, a mechanism of ceria uptake by fibroblasts was proposed (12, 13). Moreover, not only the “negative” but also the “positive” effects of ceria NPs are now studied; indeed two recent studies showed that ceria NPs stabilized with polymers also display interesting neuro- and radiation protective properties on cells (14, 15).
2. Materials and Methods 2.1. CeO2 Nanoparticles and Bacterial Growth. Escherichia coli wild strain RR1 was used as a test organism. The bacteria culture was prepared at 37 °C overnight using LB (Luria Bertani) nutrient broth (16). Therefore, the bacteria are accompanied by a large amount of salt, organic molecules, and yeast extract. These raw cultures will be named “unwashed cultures” in the following. Taking into account that the NPs surfaces are known to adsorb very easily small organic materials (12, 17), their presence in the “unwashed cultures” can corrupt the interaction between bacteria and NPs. Thus, the culture was centrifuged at 5000 rpm for 3 min and resuspended in a water solution of KNO3, previously sterilized at 120 °C 1 bar for 20 min. This treatment was repeated three times. Three types of bacteria suspensions were prepared depending on the concentration of the KNO3 washing solutions A (10-3 M), B (10-2 M), and C (10-1 M). Then, depending on the experiment, the number of bacteria was adjusted either to 0.5 × 109 or 1.66 × 109 bacteria‚mL-1 as determined by CFU counting on LB Petri dishes. The final pH of the KNO3 cell suspension was around 6. CeO2 NPs were a gift from the chemical company Rhodia. They are obtained as a powder through precipitation of Ce4+(NO3-)4 salt at very low pH (19). When water is added to this powder, a stable solution forms spontaneously. This VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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solution contains perfectly dispersed ceria NPs, which are ellipsoidal monocrystallites with an average size of 7 nm (see Supporting Information) and a specific surface of 400 m2‚g-1 (18). The point of zero charge (PZC) of the NPs has been measured at a pH of 10.5, and the presence in acidic conditions of adsorbed nitrates helps their stabilization (18). These NPs can be easily dispersed in acidic medium (pH
