Nanomaterials: Risks and Benefits (NATO Science for Peace and Security Series C: Environmental Security)

Nanomaterials: Risks and Benefits NATO Science for Peace and Security Series This Series presents the results of scie...

Author: Igor Linkov | Jeffery A. Steevens

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Nanomaterials: Risks and Benefits

NATO Science for Peace and Security Series This Series presents the results of scientific meetings supported under the NATO Programme: Science for Peace and Security (SPS). The NATO SPS Programme supports meetings in the following Key Priority areas: (1) Defence Against Terrorism; (2) Countering other Threats to Security and (3) NATO, Partner and Mediterranean Dialogue Country Priorities. The types of meeting supported are generally "Advanced Study Institutes" and "Advanced Research Workshops". The NATO SPS Series collects together the results of these meetings. The meetings are coorganized by scientists from NATO countries and scientists from NATO's "Partner" or "Mediterranean Dialogue" countries. The observations and recommendations made at the meetings, as well as the contents of the volumes in the Series, reflect those of participants and contributors only; they should not necessarily be regarded as reflecting NATO views or policy. Advanced Study Institutes (ASI) are high-level tutorial courses intended to convey the latest developments in a subject to an advanced-level audience Advanced Research Workshops (ARW) are expert meetings where an intense but informal exchange of views at the frontiers of a subject aims at identifying directions for future action Following a transformation of the programme in 2006 the Series has been re-named and re-organised. Recent volumes on topics not related to security, which result from meetings supported under the programme earlier, may be found in the NATO Science Series. The Series is published by IOS Press, Amsterdam, and Springer, Dordrecht, in conjunction with the NATO Public Diplomacy Division. Sub-Series A. B. C. D. E.

Chemistry and Biology Physics and Biophysics Environmental Security Information and Communication Security Human and Societal Dynamics

http://www.nato.int/science http://www.springer.com http://www.iospress.nl

Series C: Environmental Security

Springer Springer Springer IOS Press IOS Press

Nanomaterials: Risks and Benefits Edited by

Igor Linkov

US Army Engineer Research and Development Center Concord, Massachusetts U.S.A. and

Jeffery Steevens US Army Engineer Research and Development Center Vicksburg, Mississippi U.S.A.

Published in cooperation with NATO Public Diplomacy Division

Based on the papers presented at the NATO Advanced Research Workshop on Nanomaterials: Environmental Risks and Benefits Faro, Portugal 27-30 April 2008

Library of Congress Control Number: 2008941252

ISBN 978-1-4020-9490-3 (PB) ISBN 978-1-4020-9489-7 (HB) ISBN 978-1-4020-9491 -0 (e-book)

Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. www.springer.com

Printed on acid-free paper

All Rights Reserved © Springer Science + Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

CONTENTS

Preface .................................................................................................................... ix Acknowledgements ................................................................................................ xi Part 1. Human Health Risks Human Health Risks of Engineered Nanomaterials: Critical Knowledge Gaps in Nanomaterials Risk Assessment ................................................................. 3 A. Elder, I. Lynch, K. Grieger, S. Chan-Remillard, A. Gatti, H. Gnewuch, E. Kenawy, R. Korenstein, T. Kuhlbusch, F. Linker, S. Matias, N. MonteiroRiviere, V.R.S. Pinto, R. Rudnitsky, K. Savolainen, A. Shvedova Disposition of Nanoparticles as a Function of Their Interactions with Biomolecules .......................................................................................................... 31 I. Lynch, A. Elder Assessment of Quantum Dot Penetration into Skin in Different Species under Different Mechanical Actions ...................................................................... 43 N.A. Monteiro-Riviere, L.W. Zhang Nanotechnology: The Occupational Health and Safety Concerns......................... 53 S. Chan-Remillard, L. Kapustka, S. Goudey Biomarkers of Nanoparticles Impact on Biological Systems ................................ 67 V. Mikhailenko, L. Ieleiko, A. Glavin, J. Sorochinska Nanocontamination of the Soldiers in a Battle Space ............................................ 83 A.M. Gatti, S. Montanari Part 2. Environmental Risk SMARTEN: Strategic Management and Assessment of Risks and Toxicity of Engineered Nanomaterials................................................................................. 95 C. Metcalfe, E. Bennett, M. Chappell, J. Steevens, M. Depledge, G. Goss, S. Goudey, S. Kaczmar, N. O’Brien, A. Picado, A.B. Ramadan Solid-Phase Characteristics of Engineered Nanoparticles: A Multi-dimensional Approach ........................................................................... 111 M.A. Chappell Nanomaterial Transport, Transformation, and Fate in the Environment: A Risk-Based Perspective on Research Needs .................................................... 125 G.V. Lowry, E.A. Casman

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Visualization and Transport of Quantum Dot Nanomaterials in Porous Media.................................................................................................... 139 C.J.G. Darnault, S.M.C. Bonina, B. Uyusur, P.T. Snee Developing an Ecological Risk Framework to Assess Environmental Safety of Nanoscale Products: Ecological Risk Framework........................................... 149 L. Kapustka, S. Chan-Remillard, S. Goudey Development of a Three-Level Risk Assessment Strategy for Nanomaterials ................................................................................................. 161 N. O’Brien, E. Cummins Classifying Nanomaterial Risks Using Multi-criteria Decision Analysis................................................................................................. 179 I. Linkov, J. Steevens, M. Chappell, T. Tervonen, J.R. Figueira, M. Merad Part 3. Technology and Benefits Nanomaterials, Nanotechnology: Applications, Consumer Products, and Benefits................................................................................................................. 195 G. Adlakha-Hutcheon, R. Khaydarov, R. Korenstein, R. Varma, A. Vaseashta, H. Stamm, M. Abdel-Mottaleb Risk Reduction via Greener Synthesis of Noble Metal Nanostructures and Nanocomposites............................................................................................. 209 M.N. Nadagouda, R.S. Varma Remediation of Contaminated Groundwater Using Nano-Carbon Colloids.......................................................................................... 219 R.R. Khaydarov, R.A. Khaydarov, O. Gapurova A Novel Size-Selective Airborne Particle Sampling Instrument (WRAS) for Health Risk Evaluation ................................................................................... 225 H. Gnewuch, R. Muir, B. Gorbunov, N.D. Priest, P.R. Jackson Nanotechnologies and Environmental Risks: Measurement Technologies and Strategies........................................................................................................ 233 T.A.J. Kuhlbusch, H. Fissan, C. Asbach Part 4. International Perspectives Processing of Polymer Nanofibers Through Electrospinning as Drug Delivery Systems .................................................................................... 247 E. Kenawy, F.I. Abdel-Hay, M. H. El-Newehy, G.E. Wnek Air Pollution Monitoring and Use of Nanotechnology Based Solid State Gas Sensors in Greater Cairo Area, Egypt........................................................... 265 A.B.A. Ramadan Advanced Material Nanotechnology in Israel...................................................... 275 O. Figovsky, D. Beilin, N. Blank

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Silver Nanoparticles: Environmental and Human Health Impacts ...................... 287 R.R. Khaydarov, R.A. Khaydarov, Y. Estrin, S. Evgrafova, T. Scheper, C. Endres, S.Y. Cho Developing Strategies in Brazil to Manage the Emerging Nanotechnology and Its Associated Risks ........................................................... 299 A.S.A. Arcuri, M.G.L. Grossi, V.R.S. Pinto, A. Rinaldi, A.C. Pinto, P.R. Martins, P.A. Maia The Current State-of-the Art in the Area of Nanotechnology Risk Assessment in Russia ................................................................................... 309 M. Melkonyan, S. Kozyrev Environmental Risk Assessment of Nanomaterials ............................................. 317 A.A. Bayramov Part 5. Policy and Regulatory Aspects Considerations for Implementation of Manufactured Nanomaterial Policy and Governance.................................................................................................... 329 F.K. Satterstrom, A.S.A. Arcuri, T.A. Davis, W. Gulledge, S. Foss Hansen, M.A. Shafy Haraza, L. Kapustka, D. Karkan, I. Linkov, M. Melkonyan, J. Monica, R. Owen, J.M. Palma-Oliveira, B. Srdjevic The Safety of Nanotechnologies at the OECD..................................................... 351 P. Kearns, M. Gonzalez, N. Oki, K. Lee, F. Rodriguez Nanomaterials in Consumer Products: Categorization and Exposure Assessment .................................................................................... 359 S. Foss Hansen, A. Baun, E.S. Michelson, A. Kamper, P. Borling, F. Stuer-Lauridsen Strategic Approaches for the Management of Environmental Risk Uncertainties Posed by Nanomaterials ........................................................ 369 R. Owen, M. Crane, K. Grieger, R. Handy, I. Linkov, M. Depledge Methods of Economic Valuation of the Health Risks Associated with Nanomaterials............................................................................................... 385 S. Shalhevet, N. Haruvy Nanomaterials: Applications, Risks, Ethics and Society ..................................... 397 A. Vaseashta Group Decision-Making in Selecting Nanotechnology Supplier: AHP Application in Presence of Complete and Incomplete Information..................... 409 B. Srdjevic, Z. Srdjevic, T. Zoranovic, K. Suvocarev Uncertainty in Life Cycle Assessment of Nanomaterials: Multi-criteria Decision Analysis Framework for Single Wall Carbon Nanotubes in Power Applications ........................................................................ 423 T.P. Seager, I. Linkov

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Knowing Much While Knowing Nothing: Perceptions and Misperceptions About Nanomaterials............................................................................................ 437 J.M. Palma-Oliveira, R.G. de Carvalho, S. Luis, M. Vieira Participants ......................................................................................................... 463 Author Index....................................................................................................... 471

PREFACE

Many potential questions regarding the risks associated with the development and use of wide-ranging technologies enabled through engineered nanomaterials. For example, with over 600 consumer products available globally, what information exists that describes their risk to human health and the environment? What engineering or use controls can be deployed to minimize the potential environmental health and safety impacts of nanomaterials throughout the manufacturing and product lifecycles? How can the potential environmental and health benefits of nanotechnology be realized and maximized? The idea for this book was conceived at the NATO Advanced Research Workshop (ARW) on “Nanomaterials: Environmental Risks and Benefits and Emerging Consumer Products.” This meeting – held in Algarve, Portugal, in April 2008 – started with building a foundation to harmonize risks and benefits associated with nanomaterials to develop risk management approaches and policies. More than 70 experts, from 19 countries, in the fields of risk assessment, decision-analysis, and security discussed the current state-of-knowledge with regard to nanomaterial risk and benefits. The discussion focused on the adequacy of available risk assessment tools to guide nanomaterial applications in industry and risk governance. The workshop had five primary purposes: Describe the potential benefits of nanotechnology enabled commercial products. Identify and describe what is known about environmental and human health risks of nanomaterials and approaches to assess their safety. Assess the suitability of multicriteria decision analysis for reconciling the benefits and risks of nanotechnology. Provide direction for future research in nanotechnology and environmental science to address issues associated with emerging nanomaterial-containing consumer products. Identify strategies for users in developing countries to best manage this rapidly developing technology and its associated risks, as well as to realize its benefits. The organization of the book reflects major topic sessions and discussions during the workshop. The papers in Part 1 review and summarize human health impact of nanomaterials. Part 2 includes papers on environmental risks. Part 3 presents benefits associated with nanomaterial enabled technologies over a wide range of applications. Part 4 encompasses a series of case studies that illustrate different applications and needs across nanomaterial development and use worldwide. The concluding Part 5 is devoted to policy implication and risk management. Each part of the book reviews achievements, identifies gaps in current knowledge, and suggests priorities for future research in topical areas. Each part starts with a group report summarizing discussions and consensus ix

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PREFACE

principles and initiatives that were suggested during the group discussions at the NATO workshop. The wide variety of content in the book reflects the workshop participants’ diverse views as well as their regional concerns. Simultaneous advances in different disciplines are necessary to advance nanotechnology risk assessment and risk management. Risk assessment is an interdisciplinary field, but progress in risk assessment has historically occurred due to advances in individual disciplines. For example, toxicology has been central to human health risk assessment, and advances in exposure assessment have been important for environmental risk assessment and risk management. Nanotechnology, however, ideally involves the planned and coordinated development of knowledge across fields such as biology, chemistry, materials science, and medicine. The workshop discussions and papers in the book clearly illustrate that while existing chemical risk assessment and risk management frameworks may provide a starting point, the unique properties of nanomaterials adds a significant level of complexity to this process. The goals of the workshop included the identification of strategies and tools that could currently be implemented to reduce technical uncertainty and prioritize research to address the immediate needs of the regulatory and risk assessment communities. Papers in the book illustrate application of advanced risk assessment, comprehensive environmental assessment, risk characterization methods, decision analysis techniques, and other approaches to help focus research and inform policymakers benefiting the world at large. U.S. Army Engineer Research and Development Center Concord, Massachusetts, USA

Igor Linkov

U.S. Army Engineer Research and Development Center Vicksburg, Mississippi, USA

Jeff Steevens

August, 2008

ACKNOWLEDGEMENTS

The editors would like to acknowledge Dr. Mohammed Haraza (NATO workshop co-director) and organizing committee members (Drs. Vicki Colvin, Delara Karkan, Abou Ramadan, Jeff Morris, Saber Hussain, Jose Figueira, Jose Palma-Oliveira and Carlos Fonseca) for their help in the organization of the event that resulted in this book. We also wish to thank the workshop participants and invited authors for their contributions to the book and peer-review of manuscripts. We are deeply grateful to Deb Oestreicher for her excellent management of the production of this book. Additional technical assistance in the workshop organization was provided by Elena Belinkaia and Eugene Linkov. The workshop agenda was prepared in collaboration with the Society of Risk Analysis Decision Analysis and Risk Specialty Group. Financial support for the workshop was provided mainly by NATO. Additional support was provided by the U.S. EPA, U.S. Army Engineer Research and Development Center, International Copper Association, American Chemistry Council and University of Algarve.

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HUMAN HEALTH RISKS OF ENGINEERED NANOMATERIALS Critical Knowledge Gaps in Nanomaterials Risk Assessment

A. ELDER Department of Environmental Medicine University of Rochester 575 Elmwood Avenue, Box 850 Rochester, NY 14642, USA [email protected] I. LYNCH Centre for BioNanoInteractions School of Chemistry and Chemical Biology University College Dublin Belfield, Dublin 4, Ireland K. GRIEGER Technical University of Denmark Department of Environmental Engineering Building 113 Kongens Lyngby 2800, Denmark S. CHAN-REMILLARD Golder Associates Ltd./HydroQual Laboratories Ltd. #4 6125-12th Street S.E. Calgary T2H 2K1, Canada A. GATTI University of Modena & Reggio Emilia Lab of Biomaterials Via Campi 213 A Modena 41100, Italy H. GNEWUCH Naneum Ltd. Canterbury Enterprise Hub Canterbury CT2 7NJ, UK E. KENAWY Polymer Research Group, Department of Chemistry Faculty of Science, University of Tanta Egypt

I. Linkov and J. Steevens (eds.), Nanomaterials: Risks and Benefits, © Springer Science + Business Media B.V. 2009

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R. KORENSTEIN Marian Gertner Institute for Medical Nanosystems Department of Physiology and Pharmacology, Faculty of Medicine Tel Aviv University 69978 Tel-Aviv, Israel T. KUHLBUSCH Institute for Energy and Environmental Technology Bliersheimer Street 60 Duisburg 47229, Germany F. LINKER Occupational Health Care Services, DSM ARBODienst DSM, Alert & Case Centre Kerenshofweg 200 NL-6167AE Geleen, The Netherlands S. MATIAS Instituto Superior Téchnico Universidade Téchnica de Lisboa Av. Rovisco Pais 1049-001 Lisboa, Portugal N. MONTEIRO-RIVIERE Center for Chemical Toxicology Research and Pharmacokinetics Department of Clinical Sciences, College of Veterinary Medicine North Carolina State University 4700 Hillsborough Street Raleigh, NC 27606, USA V.R.S. PINTO Rua Capote Valente 710 São Paulo 05409-002, Brazil R. RUDNITSKY Office of Space & Advanced Technology US Department of State OES/SAT, SA-23, 1990 K Street, NW, Suite #410 Washington, DC 20006, USA K. SAVOLAINEN Finnish Institute for Occupational Health, New Technologies and Risks Topeliuksenkatu 41 aA GI-00250 Helsinki, Finland

HUMAN HEALTH RISKS OF ENGINEERED NANOMATERIALS

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A. SHVEDOVA CDC/NIOSH 1096 Willowdale Road Morgantown, WV 26505, USA

Abstract. There are currently hundreds of available consumer products that contain nanoscale materials. Human exposure is, therefore, likely to occur in occupational and environmental settings. Mounting evidence suggests that some nanomaterials exert toxicity in cultured cells or following in vivo exposures, but this is dependent on the physicochemical characteristics of the materials and the dose. This Working Group report summarizes the discussions of an expert scientific panel regarding the gaps in knowledge that impede effective human health risk assessment for nanomaterials, particularly those that are suspended in a gas or liquid and, thus, deposit on skin or in the respiratory tract. In addition to extensive descriptions of material properties, the Group identified as critical research areas: external and internal dose characterization, mechanisms of response, identification of sensitive subpopulations, and the development of screening strategies and technology to support these investigations. Important concepts in defining health risk are reviewed, as are the specific kinds of studies that will quickly reduce the uncertainties in the risk assessment process.1 1.

Introduction

Nanomaterials are commonly described as having at least one dimension smaller than 100 nm. A broader definition, though, refers to those materials that are manipulated at the atomic, molecular, or macromolecular scales in order to achieve functionality that is different from that found in the bulk or molecular form [106]. Many consumer items are already available that contain nanomaterials, such as electronics components, cosmetics, cigarette filters, antimicrobial and stain-resistant fabrics and sprays, sunscreens, and cleaning products [115]. According to a recent survey of the Wilson Institute web site [29], there are at least 580 consumer products on the market, including four with FDA approval for therapeutic use. Although the potential for human exposures has not been fully evaluated and is likely to be low in many cases, the safety of nanomaterials at a wide range of doses and throughout the product life cycle should be characterized to ensure consumer, occupational, and environmental health. Critical components of a systematic safety assessment for engineered nanomaterials include: evaluation of exposure concentrations in occupational and

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Summary of the NATO ARW Working Group discussions.

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environmental settings; the physicochemical characteristics of the material at the portal of entry; the structure and function of epithelial barriers at the portals of entry; interactions of materials with biomolecules (proteins, nucleic acids, lipids); biodistribution and elimination kinetics and identification of possible target organs; characterization of dose-response relationships; elucidation of mechanisms of response; identification of target tissues for nanomaterials effects; and identification of human subpopulations with unique susceptibility to the effects of nanomaterials. These concepts are summarized in Figure 1. New products are rapidly emerging in the nanotechnology industry without a parallel development of critical information regarding their safety. Furthermore, risk assessments are currently proceeding in many cases without adequate methodologies to define risk. It should be noted that the assumptions used in assessing risks at the early stages of most emerging technologies are designed to be protective (precautionary principle) and to emphasize potential problems so that more attention is focused on managing or mitigating such risks. As the technology progresses through the product life cycle, more data becomes available and, thus, the assumptions used in risk assessment become more realistic [10, 94]. This article focuses on the critical knowledge gaps that impede the risk assessment process as well as strategies for rapid reductions in those uncertainties. 1

+ + ?

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(in gas or liquid)

4 2 2

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to blood, other organs? 5

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Figure 1. Key issues in assessing human health risk following nanomaterials exposures. (1) What is the nature of the nanomaterial at the portal of entry (e.g. agglomerated, charged, soluble, size?)?; (2) How do the physicochemical characteristics of nanomaterials change after deposition in the body (specific changes likely to depend on portal of entry)?; (3) Do nanomaterials penetrate epithelial barriers?; (4) Are nanomaterials transported away from the portal of entry to other organs (how much is transported? What are the target tissues?)?; (5) How do the nanomaterial properties changes as they are transported in the body (dissolution; protein/lipid binding)?; (6) How do responses at the cellular/tissue level affect transport of nanomaterials?


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Characterization of Nanomaterial Exposure

Although there is potential for occupational and environmental exposures to nanomaterials throughout their life cycle, very little is known about the concentrations of such exposures. Furthermore, the characteristics of nanoscale materials (e.g. size, shape, surface charge, agglomeration state, presence of secondary coatings from air or liquid carrier) as they might be encountered in the workplace or the environment are largely unknown. Workplace exposure data for nanoparticles is scarce. However, Maynard et al. [59] reported peak airborne levels of respirable particles of single-walled carbon nanotubes up to 53 μg/m3 in a small university laboratory. Han and colleagues [28] reported airborne levels of multi-walled carbon nanotubes during spraying, blending, and weighing operations in a research laboratory that ranged from undetectable levels to ~400 μg/m3. However, these data are from total particulate samples at the breathing zone and, thus, the total mass concentration was not comprised exclusively of nanotubes. Nevertheless, incorporation of control measures reduced the nanotube-containing dust concentrations to background levels. A recent leaflet from NIOSH regarding workplace exposures to nanomaterials states that current methods for controlling exposures are adequate, but that current measurement techniques “are limited and require careful interpretation” [69]. These somewhat contradictory statements reflect the need for personnel with extensive experience and specialized training in the handling and sampling of nanomaterials. Although NIOSH cites a lack of sufficient evidence as the basis for not recommending specific surveillance of nanoparticle-exposed workers, a framework for the safe exploitation of nanotechnology has recently been described that includes recommendations for methods and instrumentation to assess exposure levels, characterize particle size and surface area distributions, and to identify sources of nanoparticle release [58, 67, 68]. 2.1.

NANOMATERIALS CHARACTERIZATION

One critical research need is the development of methods and equipment for adequate nanomaterial characterization, as has been previously cited [4, 84, 95, 109, 110]. Nanomaterial properties may also be altered in both biotic and abiotic environments. Therefore, tools to detect and characterize chemical or physical modifications of nanomaterials in such environments are needed. There is also a pressing need to develop standardized assessments of particle characteristics including size, shape, size distribution, structure and surface area [70]. This would ensure that the same set of characteristics is described across studies, ultimately facilitating a comparison between materials and subsequent exposure. Another critical need is viewed to be the development of a set of reference nanomaterials that can serve as benchmarks for the investigation of other nanomaterials, thereby providing a basis for comparison. Reference materials are commonly used in traditional risk assessment frameworks for effects and exposure analyses. Significant efforts are being made in this regard, both by the National Institute of Standards

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and Technology (US) and the Institute of Reference Materials and Measurements (EU), although the initial focus is on reference materials for calibration of instrumentation with respect to size determination, rather than reference materials for benchmarking of potential toxicity. At present, the scientific community lacks a set of commonly accepted reference materials, including consensus on suitable positive and negative control nanoparticles for different testing systems. 2.2.

CHARACTERIZATION OF EXPOSURES

Assessing external human exposure to nanomaterials requires knowledge regarding the likelihood of exposure, changes in particle concentration over time, and identification and characterization of exposure directly prior to uptake. Workplace or ambient exposures to air- or liquid-suspended nanomaterials may occur. Although estimates have been reported for selected nanosized compounds [66], no data is available about actual levels of engineered nanomaterials in ambient environments, mainly due to the limitations of current measurement methods. There is clearly a need for a comparative exposure assessment which differentiates the routes and forms of exposure as well as the morphology of the nanomaterials. This section will mainly address inhalation exposures in the workplace, because this is currently seen as the most likely exposure scenario. However, skin and gastrointestinal tract exposures to gas- or liquid-suspended particles are also possible. Further details are provided in Kuhlbusch et al. [43] in this same edition. 2.2.1.

Measurement Methods

Measurement methods for detection of airborne (nano-) particles can be characterized as (1) online/offline detection methods that distinguish environmental from product materials, (2) methods for different matrices (gas/liquid/solid), (3) personal or fixed sampling methods, (4) methods for different exposure metrics (mass, surface area or number concentration (total and size-resolved), chemical composition, etc.), and (5) methods that predict lung regional deposited dose. No optimal method is currently available for measuring nanomaterials exposures, since, for example, the ideal metric is still a matter of debate. Certainly, the best method would be a personal sampler that determines all relevant physical and chemical properties in real time or near-real time within discrete particle size bins. This is, however, currently unavailable. Nevertheless, first steps towards simultaneously determining these properties are ongoing and are of extreme importance for realistic exposure assessment. Most exposure measurements have either used an online technique to determine particle size distribution [42, 46, 63, 114] or offline techniques like thermal or electrostatic precipitation or diffusion/impaction and subsequent particle characterization [23, 82]. The choice of using particle number-weighted, as opposed to mass-weighted, size distribution measurements is driven by the expense and availability of the equipment, the high sensitivity of number concentration measurements towards nanosized particles, the possible relevance of particle number concentration for health effects, and the requirement for speciation. Of


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similar importance with regard to linking particle properties to health may be the particle surface area, either as inhalable (Matter LQ 1-DC) or lung deposited fraction (TSI NSAM). An overview on measurement methods for nanoparticle detection can be found in Kuhlbusch et al. [44]. 2.2.2.

Measurement Strategies

One measurement challenge is the differentiation of environmental (background) from engineered nanoparticles. When deciding on measurement strategies and methods, the following points have to be taken into account. First, there is a need for a dynamic detection range, from a single particle to high number concentrations. Secondly, there is a need for particle physical and chemical characterization. Lastly, the time resolution (online/offline) must be considered. There are three particle concentration ranges in terms of number that can currently be evaluated [43]: single particle detection, a concentration of 1,000– 100,000 particles per cm3, and a concentration of more than 100,000 particles per cm3. Detection of single particles can be achieved using either single particle aerosol mass spectrometry (AMS) [72] or filter sampling with subsequent single particle analysis by TEM/EDX. Both techniques have their advantages and limitations, for example, the degree of chemical analysis that is possible. These methods would allow a differentiation of background from engineered nanoparticles. Detection of the source of particle concentrations >100,000 particles per cm3 should generally be easy since the source must be in the vicinity of the point of measurement. The source can either be visually identified or detected by determining spatial particle number concentration profiles. The difficulty in assessing nanoparticle exposure at levels between 1,000– 100,000 particles per cm3 is that background particle concentrations can be in the same concentration range. A first assessment of possible nanoparticle exposure can be conducted by concurrent measurements of ambient and workplace particle number concentrations and calculation of ambient particle penetration into the work area. This approach is possible for concentrations down to a few thousand particles per cubic centimeter [45]. Hence, clear differentiation of nanoparticles from environmental nanoscale particles can only be done by the methods described for single particle analysis. 2.2.3.

Levels of Exposure

The limited exposure measurements conducted thus far in the workplace generally show low levels or levels below the detection limits for exposure during normal production and handling of nanomaterials. However, the adequacy of existing detection instrumentation needs to be considered. The exposure-related measurements were conducted in all steps of production and handling from the reactor, to processing and handling/bagging of, for example, carbon black and titanium dioxide [38, 45]. Measurements conducted in the presence of a leak within the palletizing line showed high exposure values indicating that exposure can be

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possible, especially in cases where engineering controls fail or during cleaning and maintenance work in large scale nanomaterial production. Measurements of dustiness of powders containing nanomaterials were conducted by Dahman and Monz [14] in the framework of the NanoCare Project. This investigation showed that engineered particles below 100 nm were not normally released using a counter flow system. However, there were exceptions depending on the material investigated. This example shows that extrapolations from few measurements and generalizations to other materials should be done carefully. 2.2.4.

Future Tasks

Results are eagerly awaited from ongoing investigations focusing on possible human exposure during the life cycle of nanomaterials, from production, to their use in products, and during recycling. Several scenarios exist with different degrees of likelihood of possible release of nanomaterials into the environment and subsequent exposure. The following tasks are seen to bring advances in exposure assessments for nanoscale materials: the development of cost-effective screening methodologies for assessing exposure, the development of devices that measure personal exposure, evaluation of the adequacy of health surveillance protocols, strengthening current methods for assessing agglomerate stabilities in order to predict the potential for nanoparticle release during handling, the evaluation of nanoparticle aging during transport (e.g. airborne, in water), and improvements in the link between exposure assessments and dose metrics. 3.

Barrier Function of Skin, Gastrointestinal Tract, and Respiratory Tract

If it can be assumed that most exposures to nanomaterials will occur in air or via the food chain/drinking water, then the respiratory tract, skin, and gastrointestinal tract are the primary routes of exposure to nanomaterials. However, other routes such as intravenous, intradermal, and ocular are important to consider for specialized applications. A critical component in evaluating the health risks associated with nanomaterials exposure is knowledge regarding barrier function at the portal of entry. 3.1.

GASTROINTESTINAL TRACT

The gastrointestinal (GI) tract is not likely to be a primary route of exposure to nanomaterials. However, particles that deposit in the respiratory tract and taken up by alveolar macrophages are cleared via the mucociliary escalator and then expectorated or swallowed. Some of the particulate matter, then, that deposits in the lungs could be cleared to the GI tract (see following discussion about macrophage-mediated clearance of nanosized particles). However, the barrier function of the GI tract with respect to nanoparticles is somewhat equivocal. The transfer of nanoparticles into blood and subsequent tissue distribution is likely to be very dependent on particle surface characteristics because of the


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extreme shifts in acidity and the negatively charged mucous layer in the small intestine. Early work described the process of persorption, whereby micron-sized insoluble particles are transported from the intestinal lumen to the blood via paracellular pathways [113]. This process has been shown in in vivo studies to be size-dependent, with smaller particles (polystyrene microspheres, colloidal gold) being absorbed to a greater degree than larger ones [32, 35]. However, studies with highly insoluble radioactive metal nanoparticles have shown extremely low transfer into blood following GI tract exposures [41, 103], with some evidence for an inverse relationship between particle size and percent transfer as well as for negatively-charged particles having higher transfer rates [97]. Recent studies employing electron microscopy and elemental analysis have identified nanosized particulates, possibly from combustion sources or food, in human tissues such as liver, kidney, and colon [20–22]. Although it is not clear how the particles accumulated in these organs, both digestive and respiratory tact exposures are possible explanations. In vitro model systems are likely to have limited predictive power due to the absence of a mucous layer, which traps charged particles and potentiates their clearance via the feces. 3.2.

SKIN

Skin is the largest organ of the body. Its permeability to engineered nanomaterials with respect to depth of penetration and interactions with structural components as well as nanoparticle absorption into blood are not well understood. Recent in vitro studies have employed flow-through diffusion cells to assess nanoparticle penetration and absorption through skin. 3.2.1.

Potential for Nanomaterials Skin Penetration

Nanomaterials must penetrate the stratum corneum layer in order to exert toxicity in the lower cell layers. The quantitative prediction of the rate and extent of percutaneous penetration (into skin) and absorption (through skin) of topically applied nanomaterials is complicated due to many biological complexities, such as the diversity of the skin barrier function across species and body sites. The stratum corneum affords the greatest deterrent to absorption. Although the dead, keratinized cell layer itself is highly hydrophobic, the cells are also highly water-absorbing, a property that keeps the skin supple and soft as water is evaporated from the surface. Sebum appears to augment the water-holding capacity of the epidermis; however, its hydrophobic nature cannot be assumed to retard the penetration of xenobiotics, including nanomaterials. The rate of diffusion of topically-applied materials across the stratum corneum is directly proportional to the concentration gradient of the material across the membrane, the lipid/water partition coefficient of the material, and the diffusion coefficient of the material. It should be noted that organic vehicles may themselves penetrate into the intercellular lipids of the stratum corneum, thus affecting diffusion. Depending on the specific characteristics of the skin barrier, there is a functional molecular size/weight cut-off that prevents very large molecules from being passively absorbed across any membrane. The total

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flux of any material across the skin is also dependent upon the exposed area, with dose expressed as mass per square centimeter. In vitro studies of nanomaterial penetration of skin may only approximate the in vivo situation since a long period of time may be required to achieve steady state conditions and, thus, exceed the time constraints of in vitro evaluations. Transdermal flux (penetration) with systemic absorption of topically applied nanomaterials has obvious implications in toxicology and therapeutic drug delivery. However, knowledge of the depth and mechanism of particle penetration into the stratum corneum barrier is crucial. The skin provides an environment within the avascular epidermis where particles could potentially lodge and not be susceptible to removal by phagocytosis, yet be available for immune recognition through interaction with resident Langerhans cells. In fact, it is this relative biological isolation in the lipid domains of the epidermis that has allowed for the delivery of drugs to the skin using liposomal preparations. Several studies have evaluated the hypothesis that nanoparticles can get through or get lodged within the lipid matrix of skin. Zinc oxide (ZnO, 80 nm) and agglomerates of titanium dioxide (TiO2) smaller than 160 nm did not penetrate the stratum corneum of porcine skin in static diffusion cells [19]. Likewise, in vitro application of ZnO nanoparticles (26–30 nm) in a sunscreen formulation to human skin led to accumulation of nanoparticles in the upper stratum corneum with minimal penetration [13]. However, a pilot study conducted in humans about to undergo surgery showed penetration to the dermis of “microfine” TiO2 that was applied over a period of 2–6 weeks [105]. Block copolymer nanoparticles (40 nm) that were topically applied to hairless guinea pig skin in diffusion cells were able to penetrate the epidermis within 12 h [99]. Additional studies with spherical (QD565, the number refers to the fluorescence emission maximum) and elliptical (QD655) CdSe-ZnS semiconductor nanocrystals that were applied to porcine skin in flow-though diffusion cells showed that penetration is dependent on surface coating or charge. Polyethylene glycol (PEG)- and carboxylic acid-coated QD565 were localized primarily in the epidermis by 8 h, while the QD565 PEG-amine penetrated to the dermis. However, shape was also shown to be a determinant of nanocrystal localization by the fact that the carboxylic acid-coated elliptical crystals (QD655) did not penetrate into the epidermis until 24 h of exposure [88]. Studies have also reported that nanocrystal surface coatings and charge can influence their toxicity in human epidermal keratinocytes [89]. These results highlight the diversity in terms of size and composition of particles that could possibly penetrate the stratum corneum to reach the deeper, viable layers of skin. 3.2.2.

Factors that Affect Penetration Through Skin

Recent studies have demonstrated that mechanical action and perturbations of the skin barrier can affect the penetration of nanoparticles. For example, Tinkle et al. [108] reported that even large (0.5 µm) FITC-conjugated dextran beads could penetrate the stratum corneum of human skin and reach the epidermis following 30 min of flexing. However, the particles did not penetrate the skin at all if it was not mechanically flexed. Smaller amino acid-derivatized fullerene nanoparticles


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(3.5 nm) were able to penetrate to the dermis of porcine skin that was flexed for 60 min and placed in flow-through diffusion cells for 8 h; non-flexed control skin showed penetration that was limited to the stratum granulosum layer of the epidermis [65, 87]. QD655 and QD565 coated with carboxylic acid (hydrodynamic diameters of 18 and 14 nm, respectively) were studied for 8 and 24 h in flowthrough diffusion cells with flexed, tape stripped and abraded rat skin. No penetration occurred with the nonflexed, flexed, or tape-stripped skin. However, penetration to the viable dermal layer occurred in abraded skin. In some cases, retention of QD in hair follicles was observed in the abraded skin [117]. Another important consideration is the possible retention of nanoparticles in hair follicles, as has been alluded to above. Lademann and colleagues [48] showed that TiO2 microparticles and polystyrene nanoparticles could be localized near orifices in human hair follicles. Agglomerates of iron oxide and maghemite nanoparticles with organic coatings (primary particle sizes ~5 nm) have been shown to penetrate hair follicles and the epidermis of previously frozen human skin surgical samples, suggesting a potential capacity for nanoparticles to traverse the dermal barriers [6]. Other studies with TiO2 and methylene bis-benzotriazoyl tetramethylbutylphenol showed only 10% of the formulation remained in the furrows of the stratum corneum and infundibulum of the hair follicle of human skin [57]. QD621, nail-shaped PEG-coated CdSe-CdS nanocrystals that were topically applied to porcine skin in flow-through diffusion cells for 24 h penetrated the upper layers of the stratum corneum and were primarily retained in hair follicles and in the intercellular lipid layers, a situation also seen with carbon fullerenes [118]. Although it appears that only a small amount of the applied nanomaterial is retained in hair follicles, the kinetics of this retention and the possibility of subsequent systemic distribution must be evaluated. 3.2.3.

Potential for Nanomaterials Absorption into Blood from Skin

The evaluation of nanomaterial absorption into blood is a complex matter, so results from in vitro systems that do not have intact microcirculation should be carefully interpreted. Furthermore, human and porcine skin may react differently with respect to nanoparticle penetration as compared to smaller organic chemicals and drugs where, as described above, human and porcine skin are very similar. Nevertheless, most recent work has demonstrated that absorption into blood would not be predicted following topical application of nanomaterials to skin. For example, QD621 nanocrystals that were applied to porcine skin in flow-through diffusion cells were not found in the perfusate at any time point or concentration [118]. Likewise, studies with QD565 coated with PEG, PEG-amine, or carboxylic acid that were topically applied to human skin in diffusion cells for 8 or 24 h showed that all three QD preparations remained on the surface of the stratum corneum or were retained within hair follicle invaginations, but were not detected in the perfusate [64]. Similar observations were made by this same group with porcine skin exposed to the same particles [88]. A recent in vivo study, though, showed that nanosized TiO2 that was applied topically to pig skin in sunscreen

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formulation did not accumulate in lymph node or liver tissue following exposures for 5 days per week for 4 weeks [90]. These studies demonstrate the complexity of skin and the stratum corneum lipid barrier with respect to assessing nanoparticle penetration and absorption into blood. In most cases studied to date, topically applied nanoparticles have not been shown to be absorbed into the systemic circulation. However, penetration into the stratum corneum can occur in all animal species studied. This penetration could be significant relative to immunological and carcinogenic endpoints. Current findings suggest that surface coatings as well as nanoparticle geometry also seem to modulate penetration. All of these factors must be studied further if realistic risk assessments of manufactured nanomaterials are to be made. 3.3.

RESPIRATORY TRACT

3.3.1.

The Pulmonary Epithelial Barrier

Nanoparticles that are inhaled as singlets have high predicted deposition efficiencies via diffusional processes in all regions of the respiratory tract [34]. For singlet particles of ~20 nm, the highest fractional deposition occurs in the alveolar region, where bulk air flow is low or absent [93]. Nanosized particles are not efficiently taken up by resident phagocytic cells (alveolar macrophages) [1, 27] unless they are agglomerated, thus promoting their retention in the lung and increasing the likelihood of interactions with the epithelial barrier. The alveolar epithelial barrier has a large surface area (80–140 m2 in humans) [92] and is extensively vascularized. In a healthy lung, there are only a few cell types with which nanomaterials might interact in the alveolus: type I epithelial cells (which cover ~95% of the alveolar surface), type II epithelial cells, and macrophages. The basement membranes of the type I epithelial cells are continuous with those of endothelial cells in the pulmonary capillaries, so the total thickness through which nanoparticles have to travel to reach the blood is 0.3–2.5 μm, including the interstitial space [80]. The composition of lung lining fluid varies by region of the respiratory tract. In the alveolar region, the lining fluid consists of surfactants and an overlying aqueous phase. Pulmonary surfactant is ~90% lipids (mainly disaturated dipalmitoylphosphatidyl-choline and phosphatidylglycerol with smaller amounts of cholesterol) and 10% proteins, which are secreted by type II alveolar epithelial cells [26]. The alveolar lining fluid also contains plasma-derived proteins (e.g. albumin, transferrin, immunoglobulins) that are critical to host defense functions [39]. The degree to which nanomaterials might interact with these lipids and proteins in situ is largely unknown. 3.3.2.

Fate of Nanoparticles that Cross the Alveolar Epithelial Barrier

An important factor in assessing the toxicity of nanomaterials is their distribution throughout the body and persistence in tissues following exposure. Obviously, this


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is an issue that is difficult to fully address using in vitro model systems. Translocation to extrapulmonary tissues, including the liver and various brain regions (notably the olfactory bulb), has been demonstrated, albeit in small amounts, for inhaled nanosized poorly-soluble Mn oxide, 13C, Ag, and 192Ir [18, 41, 77, 78, 104]. In the case of the Mn oxide and 13C nanoparticles, the observed targeting of the olfactory bulb was reported to be due to transport along the olfactory nerve, which has projections terminating directly into the nasal cavity. In regards to targeting of neuronal structures, though, deposition in the nose or alveoli is not an absolute requirement. Studies by Hunter and Undem [33] showed that nodose and jugular ganglia of the vagus nerve could be targeted by the intratracheal instillation of dye tracer particles. Interestingly, Semmler and colleagues [96] showed that the retention and clearance kinetics of insoluble radioactive Ir nanoparticles (15–20 nm, count median diameter) was not different from reports in the literature for larger particles (polystyrene beads), although this was a mathematical exercise and not a direct comparison to larger particles with the same chemistry. However, later studies by this group showed that what was different was the degree of interstitialization of the nanosized 192Ir particles [98]. Oberdörster et al. [75] also reported that the interstitialization rates were ~10 times higher for nanosized TiO2 particles delivered to the lungs via intratracheal instillation as compared to larger particles of the same composition. More recently, Shvedova and colleagues [102] demonstrated that single-walled carbon nanotubes (SWCNT) delivered via inhalation exposure (deposited dose of 5 mg/mouse) resulted in the deposition of small SWCNT structures and the induction of cellular inflammation, LDH and protein release, and cytokine production that was two- to fourfold greater than responses that resulted from oropharyngeal aspiration exposure to larger agglomerated SWCNT structures. Morphometric evaluation of Sirius red-stained lung sections also showed that SWCNT inhalation caused a fourfold higher increase in fibrosis compared with that seen after pharyngeal aspiration, with collagen deposition in peribronchial and interstitial areas. Interestingly, Mercer et al. [60] demonstrated a fourfold greater fibrotic potency after pharyngeal aspiration of a well dispersed SWCNT compared to a less dispersed suspension. This potency difference was associated with a greater potential for smaller SWCNT structures to enter the alveolar walls and cause interstitial fibrosis. Overall, these results suggest that inhalation of dispersed SWCNTs leads to greater interstitialization and inflammation as compared to those delivered in an agglomerated bolus by aspiration. Thus, not only is the persistence or retention of the nanomaterials of importance, but so too is the distribution within an organ system. The liver, kidneys, and spleen have been shown to be the organs with the highest retention of nanoparticles that cross the alveolar epithelial barrier [96, 104]. It is not entirely clear, though, how primary particle size or in vivo dissolution may affect the accumulation of materials in extrapulmonary organs. Some studies have reported very rapid accumulation of nanoparticles, as determined via chemical means, in liver, kidney, and olfactory bulb following respiratory tract exposures [17, 85, 104]. In comparison to the respiratory tract, nanomaterials that

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are injected intravenously accumulate in almost all tissues that are harvested [12, 17], although this is somewhat size-and surface chemistry-dependent. Not surprisingly, surface coating has been shown to be an important determinant of nanoparticle tissue distribution. At least two studies have shown that the attachment of polyethylene glycol (PEG) to the surface of the semiconductor nanocrystals increases their circulatory half-life after intravenous injection [2, 5] due to lowered uptake efficiency by the liver and spleen (reticulo-endothelial system). Reduced efficiency of liver uptake has also been shown for PEGylated nanosized magnetite particles [52]. At least for CdSe-ZnS nanocrystals, the particle size has also been shown to be an important determinant of tissue retention following intravenous injection. Particles with hydrodynamic diameters smaller than ~5.5 nm are almost completely eliminated via urine within the first 4 h [12]. Partly due to the effective cut-off size of the kidney filter, somewhat larger particles are exclusively eliminated over time via the feces [98]. 4.

Nanomaterials Interactions with Biomolecules

Data from in vivo and in vitro studies suggesting lipid and/or protein oxidation as a result of nanomaterials exposure provides indirect evidence of interactions with biomolecules. For example, Oberdörster et al. [74] demonstrated lipid peroxidation, but not protein oxidation, in brain tissue obtained from largemouth bass that were exposed to aggregated nC60 fullerenes in tank water. Should such interactions be a surprise, though? It has long been known that implanted materials acquire a protein coating that ultimately determines the fate of the implant in terms of biocompatibility. While this is likely to be the case at the nanoscale, too, the challenge will be to identify those proteins, lipids, and other biomolecules that interact with nanoparticles in the target organs and then to characterize the kinetic nature of those interactions [54]. Progress along these lines has been made recently with detailed identification of the proteins bound to nanoparticles [8, 9, 16] and the first indications of inappropriate folding leading to protein aggregation in the presence of nanoparticles [50]. A further challenge will be to understand the predictive value of this information in the context of human risk assessment. 4.1.

INTERACTIONS WITH PROTEINS

Within the medical device community, it is now well accepted that material surfaces are modified by the adsorption of biomolecules such as proteins in a biological environment, and there is some consensus that cellular responses to materials in a biological medium reflect the adsorbed biomolecule layer, rather than the material itself [25, 55, 73]. Interestingly, recent studies suggest that nanomaterial surfaces, having much larger surface area than flat ones, are more amenable to studies to determine the identity and residence times of adsorbed proteins [9, 40]. The recently introduced concept of the nanoparticle-protein corona sees the adsorbed protein (biomolecule) layer as an evolving collection of


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proteins that associate with nanoparticles in biological fluids, and suggests that this is the biologically relevant entity that interacts with cells [53]. A recent systematic study of interactions of polystyrene nanoparticles with no modification (plain) or modified with positive (amine) or negative (carboxylic) charges indicates that the surface and the curvature (particle size) both influence the details of the adsorbed proteins, although in all cases, a significant fraction of the proteins bound were common across all particles [51]. The significance of this for safety assessment is clear, as it implies that detailed characterization of the nanoparticles in the relevant biological milieu is vital. Evidence is emerging in the scientific literature that coating of nanoparticles with specific proteins can direct them to specific locations – apolipoprotein E, for example, has been associated with transport of nanoparticles to the brain [61]. The binding of serum albumin to the surface of carbon nanotubes has also been shown to induce particle uptake and anti-inflammatory responses in a macrophage cell line [15]. However, there are several complicating factors, such as the fact that the biomolecule corona is not fixed, but is rather dynamic. The corona equilibrates with the surroundings, with high abundance proteins binding initially, but being replaced gradually by lower abundance, higher affinity proteins. Additionally, changes in the biomolecule environment, such as during particle uptake and distribution, will be reflected as changes in the corona. This makes for considerable difficulty in determining the nanoparticle biomolecule corona in-situ, as attempts to recover the particles for measurement by isolating them from their surroundings will by their very nature alter the subtle balance of the biomolecule corona. However, the situation is not all bad. A considerable portion of the biologically relevant biomolecules – the so-called “hard-corona” [51] – will remain associated with the nanoparticles for a sufficiently long time so as not to be affected by the measurement processes. First indications of a potential role for nanoparticles in misfolding and aggregation events [7, 50] as well as inhibition of misfolding [83] are emerging. A range of different nanoparticles, including polymer particles, cerium oxide, carbon nanotubes and PEG-coated quantum dots, enhanced the rate of fibrillation of the amyloidogenic protein β-2-microglobulin under conditions where the protein was in the slightly molton-globular state at pH 2.5 [50]. A mechanism based on a locally high concentration of the protein in the vicinity of the nanoparticle surface, thus increasing the probability of formation of a critical oligomer, was proposed. A recent report from Bellezza and colleagues [7] demonstrated the interaction of myoglobin (Mb) with phosphate-grafted zirconia nanoparticles. Adsorption induced marked rearrangements of Mb structure, particularly loss of the secondary structure (α-helices). Two distinct structures were observed: (i) globular aggregates, similar to those for the native protein, and (ii) very extensive, branching structures of Mb, with morphological properties similar to Mb prefibrillar aggregates. In this case, the authors suggest that the prefibril-like aggregates were always observed next to the zirconia nanoparticles, suggesting that these structures develop from the bound protein. Studies in animals have shown that C60 hydrated fullerene may have antiamyloidogenic capacity, as a single intracerebroventricular injection of a C60

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hydrated fullerene significantly improved the performance of a cognitive task in control rats, resulting from inhibition of the fibrillation of amyloid-beta 25-35 peptide [83]. These results may offer a significant therapeutic advantage towards diseases of the brain, which are often intractable, as well as raising the potential for risk. A recent review has summarized much of the current state-of-the-art in protein-nanoparticle interactions [54]. A major hope of this field of research is that it will be possible in the future to predict biological impacts of nanoparticles based on a screening of the proteins for which they have the highest affinity, and an understanding of the role of these proteins in nanoparticle uptake, trafficking and subcellular localization. 4.2.

INTERACTIONS WITH LIPIDS

There are almost no reports of the interaction between nanoparticles and lipids to date, although considerable work has been done to develop solid lipid nanoparticles for targeted drug delivery [36, 81] or using lipids such as phosphorylcholine or oleic acid to stabilize nanoparticles, including enabling their transfer from organic solvents to aqueous solutions [11, 24]. Several reports on the use of lipid coatings to reduce protein binding have also been published recently. Ross and Wirth [86] reported that laterally diffusible phosphocholine bilayers inside the pores of colloidal silica nanoparticles suppressed 93% of the binding of avidin relative to the unmodified silica colloidal crystals. Another recent report shows that gold nanorods can be coated with lipid bilayers and used as sensors for protein binding, but that the process is complex and requires issues such as membrane curvature and adhesion properties [3]. Some studies with the original aim of quantifying the binding of lipids to nanoparticles have been used as controls within broader studies of protein binding to nanoparticles. For example, a recent study of human serum albumin (HSA) binding to polymeric nanoparticles found that the thermodynamics of binding was very different in the presence and absence of oleic acid, which is a major binding ligand of HSA. Using isothermal titration calorimetry, the authors found that HSA binding to the polymeric particles is exothermic, whereas in the presence of oleic acid the adsorption is endothermic. Binding of oleic acid to the particles was found to be endothermic [49]. On the basis of the discovery that lipoproteins have a large affinity for nanoparticles of many different surface compositions, an obvious question that arises is whether the particles are actually binding the lipoprotein complexes. Thus, apolipoproteins in blood associate with lipoprotein particles, e.g. chylomicrons (>100 nm) and high density lipoproteins (8–10 nm), with diameters that are similar to engineered nanoparticles [56]. These lipoprotein complexes are composed of triglycerides and cholesterol esters in the core surrounded by proteins and a monolayer of phospholipids. A study of the binding of cholesterol and triglycerides to polymeric nanoparticles has shown that the ratio of bound cholesterol to bound triglyceride corresponds to the ratio in high density lipoprotein, suggesting that the nanoparticles bind the whole lipoprotein complex [31].


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Binding of lipoprotein complexes to nanoparticles could potentially explain why many of the nanoparticles that bind these proteins and complexes are not recognized by the body as foreign and as such do not elicit a toxic or immune response. However, it is early days yet, and considerably more research into nanoparticle-biomolecule interactions is needed. 5.

Mechanisms of Response to Nanomaterials

There is a plethora of studies in the literature regarding the in vitro and in vivo effects of engineered nanomaterials. However, much of this data is difficult to interpret because of inadequate particle characterization, exposure doses that are not well-justified in terms of realistic exposure conditions, or the elution of substances (impurities) of known toxicity (e.g. transition metals). Nevertheless, several studies have pointed to oxidative stress as an important mechanistic process related to nanomaterials toxicity. For example, Sayes et al. [91] showed that as nC60 fullerenes became more water-soluble through derivatization of the particle surface, toxicity was dramatically reduced. The reduction in cytotoxicity was correlated with a lowered oxygen radical production by the fullerenes. Nanoparticle oxidative capacity, as determined using acellular methods, has also been shown to correlate well with oxidant-sensitive reporter activity in cultured cells and acute in vivo inflammatory responses [76]. As mentioned above, Oberdörster’s study in bass [74] reported evidence of brain tissue lipid oxidation and a trend towards reduced glutathione depletion. Glutathione is an abundant tripeptide with broad antioxidant capacity and is gradually depleted in favor of the oxidized form as the severity of oxidative stress increases [71]. Shvedova and colleagues [101] exposed mice to singlewalled carbon nanotubes (SWCNTs) via oropharyngeal aspiration and showed dose-related increases in granuloma formation (in association with SWCNT aggregates in tissues), interstitial fibrosis (in areas where SWCNTs were not visible), neutrophilic inflammation, glutathione depletion, increases in 4-hydroxynonenal, and increases in soluble inflammatory mediators. Furthermore, in vitro studies using cultured human keratinocytes and murine macrophages supported the role of oxidant production in response to nanotubes, as evidenced by the intracellular formation of lipid peroxidation products and antioxidant depletion. The same studies also highlighted the role of trace amounts of iron from the synthetic process in the observed responses [37, 100]. This latter study, in particular, highlights the need to identify transition metals, either as contaminants or structural components, in nanomaterial preparations as part of a safety evaluation. In addition to the oxidative stress hypothesis, there is also compelling data regarding the role of surface coating or charge as a determinant of particle toxicity. Early studies using near micron-sized polystyrene micellar particles (~750 nm) demonstrated the principle that a negative surface charge was responsible for membrane depolarization and inflammatory cytokine induction in bronchial epithelial cells [112]. Likewise, a negative surface charge of micronsized ambient particulate matter from diverse sources was correlated with

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increases in intracellular calcium and cytokine induction [111]. These responses were thought to be related to the activity of acid-sensitive receptors on the cell surface, suggesting cell type specificity of response (e.g. neuroepithelial). RymanRasmussen and colleagues [89], though, recently showed that negatively-charged CdSe-ZnS semiconductor nanocrystals were more cytotoxic in human epidermal keratinocytes than positively-charged particles of the same size and composition. The extent to which these mechanisms may be involved in the responses of diverse cell types to nanosized particles remains to be determined. Following in vivo exposures, a combination of factors will ultimately determine the toxicity of a given material: oxidative capacity is likely to be related to acute responses and in vivo solubility; interactions with proteins and lipids may modify these processes (either increase or decrease toxicity) and also determine the biodistribution of the particles; and the persistence of the material will affect the long-term clearance and effects. 6.

Sensitive Subpopulations

Knowledge regarding the biodistribution of nanomaterials as well as the mechanisms of response to them will lead to reasonable hypotheses regarding subpopulations that might experience adverse effects following exposure where other individuals will not. For example, individuals with underlying cardiopulmonary disease are more susceptible to the effects of ambient particulate air pollution [47, 79, 107]. Pre-existing bacterial or viral infections or disease states (e.g. diabetes) can contribute to oxidant-antioxidant imbalance or the activation status of inflammatory cells such that nanomaterials exposure could lead to persistent and overwhelming oxidative stress and tissue injury. In addition, inflammatory disease states can affect epithelial barrier function [30, 62, 116], thus altering the distribution of nanomaterials that are deposited in the respiratory tract or that are circulating in the blood. Depending on the route of exposure and the characteristics of the nanoparticles, many studies have demonstrated accumulation in major organ systems and passage through epithelial barriers. This raises the possibility that nanosized particles can also accumulate in germ line cells or the placenta and perhaps be transferred to the developing fetus, although this is an issue that has not received a great deal of attention. 7. 7.1.

Summarizing Concepts ACCEPTABLE SCREENING STRATEGIES

In general, there are no commonly accepted screening assays for nanomaterials health effects. The American Society for Testing and Materials recently adopted a set a screening tests for the safety evaluation of nanomaterials intended for therapeutic use, including blood cell hemolysis, cytotoxicity in porcine kidney and human hepatocarcinoma cells, and the formation of mouse granulocyte-macrophage


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colonies. For nanomaterials that may be encountered in the workplace or the environment, though, any screening strategy needs to be related to known mechanisms of response and/or aspects of the material physico-chemistry that predict in vivo responses. Some examples could include measurements of the oxidative capacity of the particle surface and assessment of protein binding. However, these kinds of assays have not yet been validated. 7.2.

SUMMARY OF URGENT RESEARCH NEEDS

The most pressing research needs for the purpose of reducing the uncertainties in nanomaterials risk assessment are apparent from the preceding text. They include characterizations of external and internal exposures and identifications of mechanisms of response and sensitive subpopulations, all of which must be supported by thorough physicochemical characterization of test materials. This knowledge is likely to lead to useful screening approaches, as illustrated in Figure 2. Exposure Assessment

Target Organ Dose Nanomaterial Characterization

Mechanisms

Sensitive Subpopulations

Screening Strategies

Figure 2. Overview of the immediate research needs in regards to human health risk assessment of nanomaterials.

A full understanding of external exposure includes measurements of particle concentrations and physicochemical properties over time in gas or liquid carriers. In particular, the impact of agglomeration/deagglomeration behavior and soluble forms of the material need careful attention. Critical information for determining internal dose of nanomaterials includes an evaluation of the ability of the material to breech physiological barriers, the dose to and retention in target organs and cellular/subcellular structures, changes in the physicochemical properties of the material as it is distributed in the body, and how the interactions of the material with endogenous biomolecules ultimately affect target organ dose. Some of these efforts will require the development of new technologies, particularly for nanoparticle-containing aerosol characterization. Although it has presented a challenge for particle toxicologists in the past, in vivo-to-in vitro dose comparisons would be helpful not only in understanding the relevancy of in vitro test results, but also in the development of screening assays. Determinations of mechanisms of action also need to be clearly linked to realistic external and internal doses. However, it should be recognized that mechanistic

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information will be critical in identifying sensitive subpopulations that may have lower thresholds for responding to nanomaterials because of, for example, alterations in repair of tissue damage or oxidant/antioxidant imbalance. Lastly, it is imperative that there is strong global commitment to funding these essential research areas. It is more cost-effective in the long term to proactively address these critical knowledge gaps than to be reactive in regards to nanomaterials health risk assessment. Especially in light of significant scientific uncertainty and a lack of clear regulation, such an approach will allow the nanotechnology industry to flourish while increasing openness and transparency in decision-making processes.

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DISPOSITION OF NANOPARTICLES AS A FUNCTION OF THEIR INTERACTIONS WITH BIOMOLECULES

I. LYNCH Centre for BioNano Interactions School of Chemistry and Chemical Biology University College Dublin Belfield, Dublin 4, Ireland [email protected] A. ELDER Department of Environmental Medicine, University of Rochester 575 Elmwood Avenue, Box 850 Rochester, NY 14610, USA [email protected]

Abstract. This review focuses on emerging concepts in the fundamental understanding of how particle surfaces interact with components in biological fluids, with an emphasis on how these interactions may inform research regarding the biodistribution of nanosized materials from the portal of entry to other organ systems. The respiratory tract is given particular focus here because of expected occupational and environmental exposure scenarios. Information regarding the biodistribution of nanoparticles and how they might be altered during the process by their local environment is a critical part of a complete human health risk assessment. 1.

Introduction

Nanomaterials can be described as having at least one dimension smaller than 100 nm. More broadly, though, they are materials that are manipulated at the atomic, molecular, or macromolecular scales in order to achieve unique functionality [39]. Many consumer items are available that contain nanomaterials, as is a small number of FDA-approved therapeutic agents [42]. The likelihood of human exposures has not been fully assessed for the full product life cycle and is likely to be low in many cases (e.g. when the material is embedded in a solid). Nevertheless, the safety of these materials must be assessed in a systematic way to ensure standards of protection for consumer, occupational, and environmental health. In assessing human health risk from nanomaterials exposure, it is important to consider the likely routes of entry into the body. Such routes include the respiratory tract, gastrointestinal tract, and skin [7] for consumer, occupational, and environmental exposure scenarios. Determining the retained dose and effects at the portal


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I. LYNCH AND A. ELDER

of entry are critical components of nanomaterials risk assessment, as are the disposition of the material following exposure and effects in tissues distant from the portal of entry. The disposition of nanomaterials and their effects are likely to be dependent on properties of the surface, as this will determine interactions with biomolecules. This paper describes some of the current concepts and literature regarding how nanoparticles (NPs) interact with biomolecules and tissues. Common themes are highlighted, but not meant to be comprehensive, thus leaving room for new insights as this field grows into maturity. 2.

Interactions of Nanoparticles with Biomolecules

In the medical device community, it has long been understood than materials surfaces are covered by a layer of biomolecules immediately upon contact with physiological systems (e.g. upon implantation) which mediates the interaction of the material with the surrounding tissue [41]. It is likely that this phenomenon will also be the key to understanding much of the bio-nano-science world [25] and it has recently been argued that the effective unit of interest in the cell–nanomaterial interaction is not the NP per se, but the particle and its ‘corona’ of more or less strongly associated proteins from serum or other body fluids [26]. Ultimately it is this corona of more or less disrupted proteins ‘expressed’ at the surface of the particle that is ‘read’ by living cells. The high surface to volume ratio of NPs means that the adsorption potential is hugely amplified by the amount of surface exposed to living tissue (for example, there are 800 m2 surface area per litre solution at 1% concentration of 70 nm particles). There are additional complications relating to the particulate nature of NPs, and to the fact that (when sufficiently small) they can access almost every organ (see Section 3) and then be taken up into cells as opposed to interacting only with cell surface receptors, as is the case with the more traditional biomaterials. Thus, it is the nature of the organization of the adsorbed proteins and other biomolecules on the surface of NPs, and any subsequent colloidal instability of either the NPs (e.g. particle aggregation, flocculation, precipitation etc.) or the adsorbed proteins (such as protein aggregation, clustering, fibrillation etc.) that determines the initial biological responses to the presence of NPs. 2.1.

INTERACTIONS OF NANOPARTICLES WITH PROTEINS

Proteins constitute a major fraction of the dry mass of cells, and in fact represent about 18% (with water accounting for 70%) of a typical mammalian cell. Lipids (~5%), polysaccharides (~2%) and DNA and RNA are other macromolecular components of cells [2]. It is estimated that there are more than 1 million different proteins in the human proteome, while in plasma there are over 3,700 different proteins [28]. Thus, it is clear that the diversity of NP–protein interactions is enormous, and the potential impacts of NPs on protein functioning are significant. The recently introduced concept of the NP-protein corona sees the adsorbed

DISPOSITION OF NANOPARTICLES

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protein (biomolecule) layer as an evolving collection of proteins that associate with NPs in biological fluids based on abundance (initially) and affinity (higher affinity proteins are selectively enriched over time), and suggests that this is the biologically relevant entity that interacts with cells [25]. If our understanding of the risks associated with NPs and nanomaterials is to evolve, we need to begin to make serious connections between the nature of the NP-protein complex following different routes of exposure and the biological consequences of NP uptake and translocation. This has particular relevance in terms of the portal of entry of NPs, which in many cases is the lung (based on collective experience from ultrafine particles), and subsequent translocation to the systemic circulation and extrapulmonary organs, as the particles will have initial contact with biomolecules from the lungs, including surfactant proteins and lipids. The protein and lipid milieu of the respiratory tract exhibits considerable regional variability. In the alveolar region, the lining fluid consists of surfactants and an overlying aqueous phase. Pulmonary surfactant is ~90% lipids and 10% proteins. The main physiological role of surfactant is to keep both the alveoli and bronchioles patent during respiration. The lipid component is composed largely of disaturated dipalmitoylphosphatidylcholine and phosphatidylglycerol with smaller amounts of cholesterol. Surfactant proteins are also associated with the lipid layer and are secreted by type II alveolar epithelial cells [18]. The alveolar lining fluid also contains plasma-derived proteins (e.g. albumin, transferrin, immunoglobulins) that are critical to host defense functions [22]. For ultrafine pollution particles (whose dimensions are similar to those of many NPs), it has been shown that particles deposited in the hypophase may interact with lung surfactant proteins A and D or glycoproteins [21]. This study identified 13 mass fragments, diagnostic of the amino acids alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, serine, and valine, providing evidence that amino acids related to opsonin proteins adsorb to nonbiological particle surfaces exposed to human lung lining fluid. There is already considerable evidence that adsorbed biomolecules can (temporarily) mediate the effect of particles of known toxicity, such as quartz silica and kaolin clays. For example, binding of serum proteins to cristobalite (a form of crystalline silica) shifted the dose at which an inflammatory response was observed to a higher concentration; the same effect was found with titanium dioxide and asbestos [4]. Several studies have shown that simulated pulmonary surfactant delays the onset of cytotoxicity, DNA-damaging activity, and apoptosis induction by respirable quartz and kaolin in cultured cells [16, 17]. Another study also demonstrated that Diesel exhaust particles, but not carbon black, could bind to a pro-inflammatory molecule (interleukin-8) and that this binding was weakly inhibited with high serum concentrations [33]. Thus, there already exist clear linkages between adsorbed biomolecules and mediation of NP toxicity effects, and a systematic evaluation of the nature of the proteins that adsorb to NPs upon deposition in different sites in the lungs and correlation of this with biological effects would offer enormous potential for screening of the estimated >30,000 NPs that are under development in laboratories and industries worldwide.

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In order to really understand the implications of NPs on living systems, identification of the adsorbed proteins and their residence times is not sufficient, as the situation is dynamic and evolves as the NPs are transported around the system. As highlighted already in this volume [14], information is also required about the binding affinities and stoichiometries, and on the nature of the groups of proximate amino acid residues that are expressed at the outer surface of the adsorbed protein layer (the biological identity). This will require significant efforts from biophysicists and others over several years to really tease out these detailed (so-called) epitope maps [25]. However, there are several complicating factors, such as the fact that the biomolecule corona is not fixed, but is rather dynamic. The corona equilibrates with the surroundings, with high abundance proteins binding initially, but being replaced gradually by lower abundance, higher affinity proteins. Additionally, changes in the biomolecule environment, such as during particle uptake and distribution, will be reflected as changes in the corona. This makes for considerable difficulty in determining the NP biomolecule corona in-situ, as attempts to recover the particles for measurement by isolating them from their surroundings will by their very nature alter the subtle balance of the biomolecule corona. However, the situation is not all bad. A considerable portion of the biologically relevant biomolecules – the so-called “hard-corona” [44] – will remain associated with the NPs for a sufficiently long time so as not to be affected by the measurement processes. A recent review has summarized much of the current state-of-the-art in protein–NP interactions [45]. A major hope of this field of research is that it will be possible in the future to predict biological impacts of NPs based on a screening of the proteins for which they have the highest affinity, and an understanding of the role of these proteins in NP uptake, trafficking and subcellular localization. 2.2.

INTERACTIONS OF NANOPARTICLES WITH LIPIDS

Pulmonary surfactant is a phospholipid-protein complex, components of which are secreted by type II alveolar epithelial cells and Clara cells. Changes have been found in phospholipid concentrations in bronchoalveolar lavage fluid (BALF) of patients with pulmonary fibrosis, indicating that phospholipid is involved in fibrotic processes, such as occurs following prolonged, high-dose particulate exposures. Kuroda and colleagues [24] also demonstrated in rats that phospholipid concentrations in lung lavage fluid were increased significantly throughout a 6 month period following crystalline silica exposure and that the increases correlated with the severity of the inflammatory response. The interactions of fine particles (urban PM2.5) and surfactant removed from human lungs by bronchoalveolar lavage were studied using a surface analysis technique to identify which of the chemical components of lung lining fluid deposit on PM2.5. The most strongly associated mass fragment on PM2.5 surfaces exposed to BALF was di-palmitoyl-phosphatidylcholine, a component of lung surfactant [21].


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Thus, while there is significant literature on the interactions of ultrafine particles with lipids, evidence of engineered NPs interactions with lipids is only beginning to emerge [3]. A very recent study of the interaction of 15 nm gold NPs with semisynthetic pulmonary surfactant (dipalmitoyl-phosphatidylcholine (DPPC)/ palmitoyl-oleoyl-phosphatidyl-glycerol/surfactant protein B (SP-B) in the ratio 70:30) showed that low levels of gold NPs (3.7 mol% Au/lipid, 0.98% wt/wt) impeded the surfactant’s ability to reduce surface tension (gamma) to low levels during film compression and to re-spread during film expansion [3]. The authors concluded that gold NPs can interact with and sequester pulmonary surfactant phospholipids and, if inhaled from the atmosphere, could impede pulmonary surfactant function in the lung. Carbon nanotubes have also been shown to adsorb surfactant lipids and proteins, thereby modulating the function of pulmonary surfactant [33]. It is not yet clear if all nanomaterials can alter the concentration – as demonstrated for crystalline silica – or function of phospholipids in the lung or if lipid–NP interactions will change the “biological identity” of the particle surface. 3.

Disposition of Nanoparticles Following In Vivo Exposures

The physiological barriers with which nanomaterials are likely to interact – namely skin and the gastrointestinal and respiratory tracts – are diverse both structurally and physiologically. It is, therefore, somewhat unlikely that a single NP physicochemical property will explain all interactions with target tissues. For example, NPs that are taken up via the gastrointestinal tract are exposed to the highly acidic environment of the stomach and then interact with a mucous gel layer once they reach the intestines. These factors are not present in skin or lung, nor are there such extreme environmental shifts. For each of the barriers, it is important to understand how peculiarities of structure and physiology might impact interpretations regarding the physicochemical characteristics that are hypothesized to determine NP fate and effects following exposure. More details about the three different barriers are provided in Elder et al. [14]. Likewise, the unique protein and lipid environments at these barriers are likely to affect how NPs are initially, at least, transported in the body. This section will focus on the respiratory tract, as other papers in this book address skin and the gastrointestinal tract. 3.1.

THE RESPIRATORY TRACT BARRIER

It should be stated at the outset that in evaluating NP disposition, it is important to consider what it is that is being detected chemically or microscopically: the particle itself or a component of the particle (solute, tracer molecule). Obviously, the former is more desirable. Materials that lend themselves well to disposition and biokinetics studies are fluorescent (e.g. semiconductor nanocrystals, quantum dots, QDs) and electron-dense metal NPs. Nevertheless, physicochemical characteristics such as primary particle size, shape, surface coating, surface charge, hydrophobicity, agglomeration state in relevant solutions, and solubility are

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important parameters. QDs and metal NPs have been used extensively to evaluate the barrier function of the skin and respiratory tract in regards to nanomaterials. Another attractive feature of these two NP types is that it is possible to vary surface chemistry without changing particle core chemistry, but studies taking advantage of this are somewhat limited. There are many ways to deliver nanosized particles to the respiratory tract in order to gain information about their disposition, including intranasal and intratracheal instillation, oropharyngeal aspiration, intratracheal microspray, and nose-only, intratracheal, or whole-body inhalation. Both the dose rate and dose distribution are artificial with the instillation and aspiration methods. However, such delivery is acceptable for screening studies or when the exposure material is precious and follow-up studies are planned [12]. For nanosized particles delivered via inhalation as singlets, mathematical predictions suggest that they will efficiently deposit via diffusional processes in all regions of the respiratory tract, although the highest fractional deposition for particles of ~10–100 nm occurs in the alveolar region [20]. Two important anatomical features of this region of the respiratory tract are (1) the large surface area of the alveolar epithelium and (2) its high degree of vascularization. Deposition also occurs, though, in the tracheobronchial and nasopharyngeal-laryngeal regions, which contain projections of sensory nerves. Dendrites of the olfactory nerve, for example, project directly into the nasal epithelium. Clearly, then, given the different biological milieu represented by the various deposition sites (detailed in Section 2.1 above), a systematic investigation of the effects of NP size coupled to surface physicochemical properties and consequent adsorbed biomolecule coronas on NP biodistribution is a key direction for immediate research to begin to address one of the most significant gaps in current knowledge related to NP safety assessment. 3.2.

TRANSLOCATION OF NANOSIZED PARTICLES

In the alveolar region of the lung, where 10–100 nm diameter particles are predicted to deposit efficiently, there is a limited number of cells with which to interact in a healthy lung, namely alveolar macrophages and type I and type II alveolar epithelial cells. Particles that agglomerate and remain in that state in alveolar lining fluid may be taken up by alveolar macrophages and removed via mucociliary clearance. However, this clearance mechanism does not work well for NPs [1, 19], thus promoting their retention in the lungs and possibly leading to interactions with epithelial cells. Via mechanisms such as endocytosis and passive transcellular or paracellular translocation, NPs can gain access to the interstitial space and the blood. In the upper respiratory tract and in the tracheobronchial region, NPs can also be taken up by sensory neurons; the existence of sensory neurons in the alveolar region of the lung is somewhat controversial. Several studies have now shown that the alveolar epithelium, at least, permits transfer of nanosized particles into the interstitial space. Oberdörster et al. [29] showed that the interstitialization of nanosized TiO2 particles proceeded at a rate that was ~10 times faster than larger particles of the same composition that were


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delivered to the lungs via intratracheal instillation. Geiser et al. [15] reported that a substantial fraction (~20%) of inhaled nanosized TiO2 particles could be found in alveolar epithelial cells, the interstitium, and blood cells within 1 h of exposure. Other recent studies have also shown a high degree of interstitialization for nanosized 192Ir particles [35]. Evidence was also presented that suggested that the interstitialized particles could then be transported back to the airway epithelium for elimination. These studies have been done with spherical NPs, but work with single-walled carbon nanotubes, which are fibrous in nature, suggests that those particles that are better dispersed upon delivery to animals (e.g. via inhalation) have a greater interstitial fibrotic potential [27, 36]. Although the amounts of deposited NPs that leave the alveolar surface and travel into the interstitium and blood are small, several studies have demonstrated that they accumulate in extrapulmonary organs [13, 23, 30, 31, 37, 38]. NPs of various compositions were used (Ag, Au, Mn oxide, 13C, and 192Ir) and were generated in the gas phase and delivered via whole-body or intratracheal inhalation. It is important to note that it is likely that the NP itself and not a solute or label was being tracked in these studies. With the exception of Mn oxide and Ag NPs, which would be predicted to have limited in vivo solubility, the other particle types are very poorly soluble. In addition to tissues such as liver and spleen, the central nervous system was also shown to be a site of NP accumulation [13, 30, 31, 38]. It is proposed that NPs that are deposited onto the nasal epithelial surface after inhalation exposures are translocated to the olfactory bulb via the olfactory nerve and, possibly, to more distal brain structures [5, 6, 10, 11]. The liver, kidneys, and spleen have been shown to be the organs with the highest retention of NPs that cross the alveolar epithelial barrier [34, 38]. Similar to what was observed for NPs accessing the interstitium, it also appears that NPs accumulate very rapidly in extrapulmonary organs [38]. In comparison to the respiratory tract, nanomaterials that are intravenously injected accumulate in almost all tissues [9], although this is somewhat dependent on particle size and surface chemistry. It is to be expected that NPs will encounter very different protein and lipid milieu as they are transported from the lungs to extrapulmonary tissues, both in terms of distinct species and their relative abundances. The degree to which biodistribution depends on interactions of the NP surface with endogenous proteins and lipids is largely unknown, but is the subject of current research. One last issue is that the integrity of the epithelial barrier must be considered. This is likely to be of importance for two reasons. For one, inflammation can alter that the permeability of epithelial barriers [40, 43], thus potentiating transfer of NPs from the site of deposition to more distal organs. Indeed, a recent study by Chen and colleagues [8] showed that the transfer of radiolabeled nanosized, but not 200 nm, polystyrene beads into the blood following intratracheal instillation exposures in rats was potentiated by pre-exposure to bacterial lipopolysaccharide, a known inflammatory stimulus. Secondly, soluble inflammatory mediators that are present at the site of injury could become associated with the NP surface and either affect distribution or induce effects in tissues where the particles accumulate. Thus, the possibility exists that individuals with compromised barrier function


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(e.g. ongoing inflammation due to underlying disease or concurrent exposure to inflammatory stimuli) may be more susceptible to the effects of NPs. 4.

Summary and Concluding Remarks

Several important concepts were reviewed here in regards to the biodistribution of NPs that are deposited in the respiratory tract. First, translocation is dependent on particle size. Secondly, the organs in which NPs accumulate is likely to reflect the site of deposition in the lung and the physicochemical characteristics of the NPs (agglomerate state, solubility, e.g.). These parameters (deposition site and physicochemical characteristics) also determine the proteins and other biomolecules that adsorb onto the NPs immediately upon contact with the cells of the lung, and thus confer a “biological identity” to the NPs, which interacts with the cellular machinery and determines uptake and translocation pathways. The integrity of epithelial barrier is also a critical factor (e.g. lung inflammation). Lastly, it should be clear from the concepts reviewed herein that a well-done nanomaterials risk assessment requires a multidisciplinary approach, a global commitment to research funding, and a need for the development of new technologies such as screening assays and measurement tools. Such an approach should result in a rapid reduction in the uncertainties of the current risk paradigm and ensure the future success of the nanotechnology industry. Acknowledgements The authors are supported by the following funding: NIEHS Center grant P30 ESO1247, EPA STAR grant RD 83172201, DoD MURI FA9550-04-1-0430, NIH R01 CA134218, EU FP6 project NanoInteract (NMP4-CT-2006-033231), ESF Network EpitopeMap, IRCSET, SFI SRC BioNanoInteract (07/SRC/B1155) and HEA PRTLI4. References 1. Ahsan, F., Rivas, I. P., Khan, M. A., and Torres Suárez, A. I., 2002, Targeting to macrophages: role of physicochemical properties of particulate carriers - liposomes and microspheres - on the phagocytosis by macrophages, J. Control. Release 79: 29–40. 2. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D., 1994, Molecular Biology of the Cell, 3rd Edition. Garland Publishing, London. 3. Bakshi, M. S., Zhao, L., Smith, R., Possmayer, F., and Petersen, N. O., 2008, Metal nanoparticle pollutants interfere with pulmonary surfactant function in vitro, Biophys. J. 94: 855–868. 4. Barrett, E. G., Johnston, C., Oberdorster, G., and Finkelstein, J. N., 1999, Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar type II cell line, Toxicol. Appl. Pharmacol. 161: 111–122. 5. Bodian, D., and Howe, H. A., 1941b, The rate of progression of poliomyelitis virus in nerves, Bull. Johns Hopkins Hosp. 69: 79–85.


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23. Kreyling, W. G., Semmler, M., Erbe, F., Mayer, P., Takenaka, S., and Schulz, H., 2002, Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low, J. Toxicol. Environ. Health Part A 65: 1513–1530. 24. Kuroda, K., Morimoto, Y., Ogami, A., Oyabu, T., Nagatomo, H., Hirohashi, M., Yamato, H., Nagafuchi, Y., and Tanaka. I., 2006, Phospholipid concentration in lung lavage fluid as biomarker for pulmonary fibrosis, Inhal. Toxicol. 18: 389–393. 25. Lynch, I., Cedervall, T., Lundqvist, M., Cabaleiro-Lago, C., Linse, S., and Dawson, K. A., 2007, The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century, J. Colloid Interface Sci. 134–135: 167–174. 26. Lynch, I., Dawson, K. A., and Linse, S., 2006, Detecting crytpic epitopes in proteins adsorbed onto nanoparticles. Sci. STKE 327: 14. 27. Mercer, R. R., Scabilloni, J., Wang, L., Kisin, E., Murray, A. R., Schwegler-Berry, D., Shvedova, A. A., and Castranova, V., 2008, Alteration of deposition pattern and pulmonary response as a result of improved dispersion of aspirated single-walled carbon nanotubes in a mouse model, Am. J. Physiol. Lung Cell. Mol. Physiol. 294(1): L87–L97. 28. Muthusamy, B., Hanumanthu, G., Suresh, S., et al., 2005, Plasma proteome database as a resource for proteomics research, Proteomics 5: 3531–3536. 29. Oberdörster, G., Ferin, J., Gelein, R., Soderholm, S., and Finkelstein, J., 1992, Role of the alveolar macrophage in lung injury: studies with ultrafine articles, Environ. Health Perspect. 97: 193–199. 30. Oberdörster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Lunts, A., Kreyling, W., and Cox, C., 2002, Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats, J. Toxicol. Environ. Health 65: 1531–1543. 31. Oberdörster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., and Cox, C., 2004, Translocation of inhaled ultrafine particles to the brain, Inhal. Toxicol. 16(6– 7): 437–445. 32. Salvador-Morales, C., Townsend, P., Flahaut, E., Vénien-Bryan, C., Vlandas, A., Green, M. L. H., and Sim, R. B., 2007, Binding of pulmonary surfactant proteins to carbon nanotubes; potential for damage to lung immune defense mechanisms, Carbon 45: 607–617. 33. Seagrave, J., Knall, C., McDonald, J. D., and Mauderly, J. L., 2004, Diesel particulate material binds and concentrates a proinflammatory cytokine that causes neutrophil migration, Inhal. Toxicol. 16(Suppl. 1): 93–98. 34. Semmler, M., Seitz, J., Erbe, F., Mayer, P., Heyder, J., Oberdörster, G., and Kreyling, W. G., 2004, Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs, Inhal. Toxicol. 16: 453–459. 35. Semmler-Behnke, M., Takenaka, S., Fertsch, S., Wenk, A., Seitz, J., Mayer, P., Oberdörster, G., and Kreyling, W. G., 2007, Efficient elimination of inhaled nanoparticles from the alveolar region: evidence for interstitial uptake and subsequent reentrainment onto airways epithelium, Environ. Health Perspect. 115(5): 728–733. 36. Shvedova, A. A., Kisin, E. R., Murray, A. R., Johnson, V. J., Gorelok, O., Arepalli, S., Hubbs, A., Mercer, R. R., Keohavonf, P., Sussman, N., Jin, J., Stone, S., Chen, B. T., Deye, G., Maynard, A., Castranova, V., Baron, P. A., and Kagan, V. E., 2008, Inhalation vs aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress and mutagenesis, Am. J. Physiol. Lung Cell Mol. Physiol. 295(4): L552–565.


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37. Takenaka, S., Karg, E., Kreyling, W. G., Lentner, B., Möller, W., Behnke-Semmler, M., Jennen, L., Walch, A., Michalke, B., Schramel, P., Heyder, J., and Schulz, H., 2006, Distribution pattern of inhaled ultrafine gold particles in the rat lung, Inhal. Toxicol. 18(10): 733–740. 38. Takenaka, S., Karg, E., Roth, C., Schulz, H., Ziensis, A., Heinzmann, U., Schramel, P., and Heder, J., 2001, Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats, Environ. Health Perspect. 109(Suppl. 4): 547–551. 39. The Royal Society and the Royal Academy of Engineering, Nanoscience and Nanotechnologies: Opportunities and Uncertainties, The Royal Society, 2004. 40. Wagner, J. G., Hotchkiss, J. A., and Harkema, J. R., 2001, Effects of ozone and endotoxin coexposure on rat airway epithelium: potentiation of toxicity-induced alterations, Environ. Health Perspect. 109(Suppl. 4): 591–598. 41. Wilson, C. J., Clegg, R. E., Leavesley, D. I., and Pearcy, M. J., 2005, Mediation of biomaterial-cell interactions by adsorbed proteins: a review, Tissue Eng. 11: 1–18. 42. Woodrow Wilson International Center for Scholars, Project on Emerging Nanotechnologies, Consumer Products Inventory. http://www.nanotechproject.org/inventories/ consumer/. 43. Xiao, H., Banks, W. A., Niehoff, M. L., and Morley, J. E., 2001, Effect of LPS on the permeability of the blood-brain barrier to insulin, Brain Res. 896: 36–42. 44. Lundqvist, M., Stigler, J., Elia, G., Lynch, I., Cedervall, T., Kenneth A., and Dawson, K.A., 2008, Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. PNAS, 105(38): 14265–14270. 45. Lynch, I., and Dawson, K. A., 2008, Protein-nanoparticle interactions, Nano Today 3: 40–47.

ASSESSMENT OF QUANTUM DOT PENETRATION INTO SKIN IN DIFFERENT SPECIES UNDER DIFFERENT MECHANICAL ACTIONS

N.A. MONTEIRO-RIVIERE, L.W. ZHANG Center for Chemical Toxicology Research and Pharmacokinetics North Carolina State University 4700 Hillsborough Street Raleigh, NC 27606, USA [email protected]

Abstract. Skin penetration is one of the major routes of exposure for nanoparticles to gain access to a biological system. QD nanoparticles have received a great deal of attention due to their fluorescent characteristics and potential use in medical applications. However, little is known about their permeability in skin. This study focuses on three types of quantum dots (QD) with different surface coatings and concentrations on their ability to penetrate skin. QD621 (polyethylene glycol coated, PEG) was studied for 24 h in porcine skin flow-through diffusion cells. QD565 and QD655 coated with carboxylic acid were studied for 8 and 24 h in flow-through diffusion cells with flexed, tape stripped and abraded rat skin to determine if these mechanical actions could perturb the barrier and affect penetration. Confocal microscopy depicted QD621 penetration through the uppermost layers of the stratum corneum (SC) and fluorescence was found in the SC and near hair follicles. QD621 were found in the intercellular lipid layers of the SC by transmission electron microscopy (TEM). QD565 and 655 with flexed and tape-stripped skin did not show penetration; only abraded skin showed penetration in the viable dermal layers. In all QD studies, inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD did not detect Cd or fluorescence signal in the perfusate at any time point, concentration or type of QD. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, while QD565 and 655 penetrated into the dermis of abraded skin. The anatomical complexity of skin and species differences should be taken into consideration when selecting an animal model to study nanoparticle absorption/penetration. These findings are of importance to risk assessment for nanoscale materials because it indicates that if skin barrier is altered such as in wounds, scrapes, or dermatitis conditions could affect nanoparticle penetration deeper into the dermal layers and skin is an important organ and can serve as a potential route of exposure and should not be overlooked.


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1.

Background

Quantum dot (QD) nanoparticles have potential use in diagnostics, drug delivery and imaging in biomedicine or therapeutic applications due to their optical characteristics that result in strong fluorescence without photobleaching [1]. QD conjugated with streptavidin have been bound to cytoskeletal elements and surface receptors when visualized with monoclonal antibodies [2, 3]. A series of carboxylic acid coated QD with different emission wavelengths are now commercially available making QD a useful tool to mark certain proteins in cells. Prostate tumors in mice were imaged with a QD-antibody conjugate that provided a novel method of cancer labeling in vivo [4]. QD biocompatibility should be evaluated in cells and in tissues before incorporating them into structures for biomedical devices or implants. Currently, there is little information regarding QD permeability in skin. Their potential for toxicity and interactions with biological systems is needed before nanomaterial risk assessments can be made. QD565/655 contain a cadmium/selenide (CdSe) core with a zinc sulfide (ZnS) shell. By TEM, QD565 are spherical with a diameter of 4.6 nm, while QD655 are ellipsoid with a diameter of 6 (minor axis) X 12 nm (major axis). The hydrodynamic diameters for QD565 are 35 nm for the polyethylene glycol, (PEG), uncharged, 14 nm for the carboxylic acid (COOH), negatively charged, and 15 nm for the (PEG-amine (NH2), positively charged. The hydrodynamic diameters QD655 were 45 (PEG), 18 (COOH), and 20 nm (NH2) [5]. In comparison, QD621-PEG coated have a CdSe core and CdS shell coated with PEG polymer coils, and are nail shaped by TEM with the mean width of 5.78 ± 0.97 nm and length of 8.40 ± 1.9 nm with a hydrodynamic size of 39 ± 1 nm evaluated by sizeexclusion chromatography [6]. The heavy metals Cd and Se may have toxic effect on cells or tissues. QD have been shown to degrade in oxidative environments [7, 8]. Therefore, QD degradation and the potential Cd release in vivo may pose a toxic risk. Skin has been shown to be permeable to some engineered nanomaterials in commercial products, medicines, cosmetics and can serve as a portal of entry for localized or systemic exposure to humans, especially in an occupational scenario. Therefore, the objective of these studies was to assess if QD nanoparticles of different sizes, shapes, and surface coatings could penetrate the skin of different species under different mechanical actions. 2.

Interaction of Different QD Topically Applied to Intact Skin

Our laboratory investigated the effects of penetration by QD565 and 655 with diverse physicochemical properties in porcine flow-through diffusion cells. QD565 with PEG, PEG-NH2, or COOH showed penetration into the stratum corneum (SC) and localization within the epidermal and dermal layers by 8 h. PEG and PEG-NH2 coated QD655 were localized within the epidermal layers by 8 h. The penetration of COOH coated QD655 into epidermal layers was evident only at 24 h [5]. Recently, we have studied another type of QD, QD621 that has a

ASSESSMENT OF QUANTUM DOT PENETRATION INTO SKIN

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different shape (nail shaped) and different shell coating (CdS shell). QD621 was topically applied to porcine skin in flow-through diffusion cells to assess penetration. At the low concentration of 1 μM, QD621 were located primarily in the normal intact SC layers of the skin (Figure 1, left column). No QD621 fluorescence was detected in the stratum granulosum, stratum spinosum, or stratum basale layers of the epidermis. At the high concentration of 10 μM (right column) QD621 were primarily present in the SC layers or in between the stratum granulosum-corneum interface, although a small amount of fluorescence was detected in the upper epidermal layers. Occasionally, QD621 were seen in the outer root sheath of the hair follicle [9].

Figure 1. QD621-PEG applied to porcine skin flow-through diffusion cells for 24 h. Left column: 1 μM dose. Right column: 10 μM dose. Top row across: confocal-DIC images depicting the skin section by DIC. Middle row across: fluorescence indicating QD621-PEG. Bottom row across: overlay of DIC and fluorescence depicting QD on the surface and in the outer root sheath of the hair follicle (right). Bars = 100 μm.

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The penetration differences seen with QD565, QD655 and QD621 in flowthrough diffusion porcine skin cells showed how differences in composition, size, configuration, surface charge and other physicochemical parameters could influence penetration. PEG-QD621 with a hydrodynamic diameter of 39 ± 1 nm were capable of penetrating only the uppermost layers of the porcine SC after 24 h of exposure, while confocal microscopy showed that all three surface coatings of the QD565 penetrated at 8 and 24 h but only the QD655 COOH took 24 h to penetrate the skin. No fluorescence was detected in the perfusate. The above study showed that QD synthesized with the same core/shell with similar surface coatings and hydrodynamic diameters but different shapes and penetration rates can penetrate intact skin [5]. QD621-PEG was dissolved in water as stock solution, while QD565-PEG and QD655-PEG were in a borate buffer (pH 8.0) to prevent agglomeration and had a similar viscosity and similar pH with water. QD565 and QD655 penetrated through porcine skin faster and deeper than QD621. The SC layers remained intact and no other morphological alterations were noted by either laser scanning confocal microscopy or TEM due to pH effects that possibly could alter the skin barrier formation or cell morphology that would allow for penetration. Therefore, QD565-PEG, QD655-PEG and QD621-PEG penetration of porcine SC is independent of the vehicle or pH. These three QD have the same chemical composition including a “rigid” core and a “soft” surface coating. Penetration may not only be determined by size and charge, but also by the shape of the rigid core and durability of the coating. It has been reported that elastic particles were able to distribute through the epidermis faster, while rigid particles were found to remain on the surface of the upper SC [10]. The most common route of penetration in skin is via the intercellular lipid spaces between the corneocytes. Our previous study showed the diameter of porcine corneocytes to be 32 μm and the vertical and lateral gaps between corneocytes are 19 nm [11]. Therefore, the QD could potentially pass through the corneocytes lateral intercellular spaces since the QD621 has a rigid core length of 8.4 nm and width of 5.8 nm but overall size of 39–40 nm. It is theoretically possible that the outer PEG coating is a “soft” coating thereby allowing the QD621 to “squeeze” through the intercellular space and remain lodged within the SC lipid bilayers (Figure 2A, B). QD621-PEG penetration may be limited through the epidermis due to their large size and irregular configuration and this fact could explain the different behavior between the nail shaped QD621 (5.78 by 8.4 nm) and spherical QD565 (4.6 nm core) or elliptical QD655 (6 by 12 nm). Therefore, the 1 μM QD621 did not penetrate deep into the SC or epidermis, while the QD565 and QD655 (smaller and more regular in shape) would have less difficulty penetrating the lipid layers of the stratum corneum. QD nanoparticle studies in our lab reported on the penetration of QD into the SC layers or outer root sheath of hair follicles, but not within the deeper layers of skin [9, 12, 13] except for QD565/655 in porcine skin flow-through diffusion cells [5]. Other types of nanoparticles have been topically applied to the skin to assess penetration. TiO2 and ZnO nanoparticles are key ingredients that are added to sunscreens to protect the skin from UV induced damage. Cross et al., 2007 [34]


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Figure 2. TEM of QD621-PEG in the SC of flow-through porcine diffusion cells. (A) QD621-PEG in the intercellular lipid layers of the SC. Bar = 250 nm. (B) Higher magnification of the enlarged area of 2A depicting individual nail-shaped QD621 (arrow) and some small agglomerates. Bar = 50 nm.

reported that most micronized transparent ZnO nanoparticles of 26–30 nm in oil/water formulations topically applied to human skin in in vitro static cells for 24 h remained on the surface of the SC. Microfine ZnO of 80 nm and agglomerates of titanium dioxide less than 160 nm did not penetrate the porcine SC layer in in vitro static diffusion cells [14]. Maghemite nanoparticles of 5.9 nm have been shown to penetrate hair follicles and the SC layer of the epidermis, suggesting a potential route for nanoparticles to traverse the dermal barriers [16]. Polystyrene nanoparticles of 20 nm tend to distribute in human hair follicles [15]. Minoxidil loaded polymeric nanoparticles of 40 nm were able to penetrate the skin of hairy guinea pigs, probably through hair follicles [17]. Hair follicles are often envisioned as special channels for absorption of topical compounds, which could by pass the SC barrier [18]. If hair follicles are a route of exposure for QD then nanoparticles penetrating into the skin may be independent of particles size and may be a safety issue. All of the above studies have demonstrated that the penetration and distribution in skin for topical administration of QD or other nanoparticles are minimal. However, it will be interesting to investigate if smaller nanoparticles can penetrate deeper into the skin after repetitive applications and for longer durations. However, these types of studies will need to be conducted in vivo because there are limitations to in vitro cell systems. 3.

QD Penetration in the Skin via Mechanical Forces

Species differences may be a function of intercellular lipid structure or hair follicle density that could modify penetration processes [19]. Rat skin is sometimes used in toxicity studies and widely used due to the ease of handling and low cost. Pig and human skin have a sparse hair coat compared to that of rodent skin. Porcine skin is widely used in penetration studies because it is anatomically, biochemically

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and physiologically similar to human skin [20–22]. Skin from the back of pigs and abdomen of humans have 11 ± 1 hair follicles/cm2, while rat has a 289 ± 21 [23]. Our laboratory investigated the penetration of QD655 and QD565 coated with COOH in rat skin for 8 and 24 h in flow-through diffusion cells. Skin was flexed, tape stripped and abraded to determine if these mechanical actions could perturb the barrier and affect penetration. The hair was clipped on the back of rats 24 h prior to experiment. Skin from the back was removed and placed dermal side down on flow-through diffusion cells. QD655 or QD565 of 1 µM dosed for 8 or 24 h in intact skin showed both QD on the surface of the SC and on the hair without penetration into the epidermal layers. The irregular and uneven staining of QD on the surface of rat skin is probably due to the high hair follicle density that prevents QD from reaching the SC surface, thereby showing fluorescence on the surface of the hairs. Furthermore, QD655 or QD565 were found on the surface of the SC in a homogeneous and continuous pattern, but there was no difference in QD penetration between flexed skin and intact skin. Rat skin was tape stripped ten times to remove the SC layers. In tape-stripped skin, QD were deposited evenly and homogeneously on the surface of the viable epidermal layers. Rat skin was also abraded with sandpaper 60 times, until the skin was slightly red but not bleeding. This mechanical action removes the SC layers and viable epidermal layers so that penetration of QD can be facilitated through skin. QD655 and QD565 showed slight penetration into the dermis at both 8 and 24 h (Figure 3). Since QD consist of a Cd core, we evaluated for Cd leaching from the QD to detect absorption in the perfusate samples collected at different time points. No fluorescence was emitted or Cd detected in any of the perfusate samples at any time points. ICP-OES supported the fluorescence measurements that there was no evidence of absorption in the flow-through diffusion cells. These results suggested that barrier perturbation by flexion and tape stripping did not cause penetration of QD, but only abraded skin allowed QD to penetrate deeper into the dermal layers of skin. Additional studies in our laboratory with QD in tape stripped and intact human skin in flow through diffusion cells found similar results. QD penetration through human skin was minimal [13]. All of these observations demonstrate that there are species differences and these anatomical complexities may interfere with the penetration of QD in skin. Skin exposed to different mechanical actions such as tape stripping or abrasion is often used in skin pharmacokinetic research to study drug absorption in skin. Tape stripping of the SC facilitates the percutaneous absorption of a compound across skin providing a noninvasive procedure to predict human skin absorption for the compound [24]. Tape stripping has been used to assess the absorption of cosmetic products, heavy metals and other chemicals to determine the amount of a compound that has been absorbed [25–27]. Rat skin was tape stripped and investigated by its permeability of QD in flow-through diffusion cells. The macroscopic and microscopic results depicted tape stripping ten times removed most of the hairs and completely removed all the SC layers, and the effects of tape stripping showed QD565 or QD655 deposited evenly and homogeneously on the surface of viable epidermal layers without penetration into the epidermis at 24 h [12].


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Figure 3. QD565-COOH applied to abraded rat skin in flow-through diffusion cells for 24 h. Left column: 1 μM dose of QD565-COOH. Bar = 100 μm. Right column: higher magnification of the left column depicting QD565 in the dermal layers of the skin. Bar = 25 μm.

Skin abrasion is often used in clinical settings for skin resurfacing, drug delivery [28, 29] or to increase vitamin C absorption [30]. We abraded the skin and laser scanning confocal microscopy depicted penetration of QD565 and QD655 into the dermis at 24 h. Rat epidermis typically contains one to two layers of keratinocytes, and after abrasion, the epidermis was removed. In this study, QD penetrated into the abraded skin but not tape stripped skin indicating that the basement membrane had been partially removed so that QD could easily penetrate into the dermis without the basement membrane acting as another selective barrier. Flexed skin and its permeability to nanoparticles are of interest especially in an occupational setting. Skin flexion is a method that simulates flexing movements such as repetitive wrist bending. Polymeric nanoparticles coated with a 40 nm thick PEG block copolymer layer topically applied to hairless guinea pig skin for 12 h were able to penetrate the epidermis [17]. FITC-conjugated dextran beads of 0.5 μm penetrated the SC of human skin and reached the epidermis after 30 min of flexing [31]. Studies in our lab have shown that a fullerene amino acid-derivatized peptide nanoparticles of 3.5 nm were capable of penetrating the dermal layers of


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porcine skin flexed for 60 min and placed in flow-through diffusion cells for 8 h, while non-flexed control skin showed limited penetration to the upper epidermal layers [32, 33]. TEM found the derivatized fullerene was localized within the intercellular space of the stratum granulosum layer. In our study, rat skin was flexed at 45° with a frequency of 20 flexes/min similar to the porcine study above. The apparatus provides tension and compression that mimics repetitive skin movement. After 60 min of flexing, QD655-COOH and QD565-COOH were found on the surface of the SC in a homogeneous and continuous pattern in rat skin. Perhaps flexion of the skin enhanced the rate of QD penetration along the side of the hair shaft. Also, repetitive motions may alter the structural organization of skin and lead to an increase in penetration by compromising the permeability barrier. 4.

Conclusion

In summary, we showed that QD621 penetration into skin was minimal and limited to the uppermost SC layers and the outer root sheath of hair follicles. We did not detect any Cd in the perfusate by ICP-OES or QD by fluorescence indicating lack of dermal absorption or below the level of detection. When different mechanical stressors were applied to rat skin, QD showed no penetration in nonflexed control, flexed and tape stripped skin, but minimal penetration in abraded skin. The above studies provided a better understanding on the penetration of different types of QD with different types of surface coatings in different species. QD penetration depends on its size, charge, shape and other physicochemical parameters. Also, different mechanical actions on skin could alter the barrier properties that would effect nanoparticles penetration into skin and flexion could cause nanoparticles to penetrate deeper. This research suggests that there is risk for potential health care workers that suffer defects in their skin barrier such as atopic dermatitis, psoriasis or eczema on their hands and other parts of their body with a compromised skin barrier that could be susceptible to nanoparticle penetration. In addition, this study also provided information on nanoparticle absorption that could occur in abraded skin that could relevant in certain occupations exposure scenarios and potentially as a method of drug delivery. References 1. Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., and Weiss, S., 2005, Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307: 538–544. 2. Wu, X., Liu, H., Liu, J., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N., Peale, F., and Bruchez, M. P., 2003, Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 21: 41–46.


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3. Wang, J., Yong, W. H., Sun, Y., Vernier, P. T., Koeffler, H. P., Gundersen, M. A., and Marcu, L., 2007, Receptor-targeted quantum dots: fluorescent probes for brain tumor diagnosis. J. Biomed. Opt. 12: 044021. 4. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W., and Nie, S., 2004, In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22: 969–976. 5. Ryman-Rasmussen, J., Riviere, J. E., and Monteiro-Riviere, N. A., 2006, Penetration of intact skin by quantum dots with diverse physicochemical properties. Toxicol. Sci. 91: 159–165. 6. Yu, W. W., Chang, E., Falkner, J. C., Zhang, J., Al-Somali, A. M., Sayes, C. M., Johns, J., Drezek, R., and Colvin, V. L., 2007, Forming biocompatible and nonaggregated nanocrystals in water using amphiphilic polymers. J. Am. Chem. Soc. 129: 2871–2879. 7. Derfus, A. M., Chan, W. C. W., and Bhatia, S. N., 2006, Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 4: 11–18. 8. Chang, E., Thekkek, N., Yu, W. W., Colvin, V. L., and Drezek, R., 2006, Evaluation of quantum dot cytotoxicity based on intracellular uptake. Small 12: 1412–1417. 9. Zhang, L. W., Yu, W. W., Colvin, V. L., and Monteiro-Riviere, N. A., 2008, Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes. Toxicol. Appl. Pharmacol. 228: 200–211. 10. Honeywell-Nguyen, P. L., Gooris, G. S., and Bouwstra, J. A., 2004, Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo. J. Invest. Dermatol. 123: 902– 910. 11. Van der Merwe, D., Brooks, J. D., Gehring, R., Baynes, R. E., Monteiro-Riviere, N. A., and Riviere, J. E., 2006., A physiologically based pharmacokinetic model of organophosphate dermal absorption. Toxicol. Sci. 89: 188–204. 12. Zhang, L. W., and Monteiro-Riviere, N. A., 2008, Assessment of quantum dot penetration into intact, tape stripped, abraded and flexed rat skin. Skin Pharmacol. Physiol. 21: 166–180. 13. Monteiro-Riviere, N. A., and Inman, A. O., 2008, Evaluation of quantum dot nanoparticle penetration in human skin. The Toxicologist CD-An official. J. Soc. Toxicol. 102: S-1, 1029, 211. 14. Gamer, A. O., Leibold, E., and van Ravenzwaay B., 2006, The in vitro absorption of microfine zinc oxide and titanium dioxide through porcine skin. Toxicol. In Vitro 20: 301–307. 15. Alvarez-Roman, R., Naik, A., Kalia, Y. N., Guy, R. H., and Fessi, H., 2004, Skin penetration and distribution of polymeric nanoparticles. J. Control. Release 99: 53–62. 16. Baroli, B., Ennas, M. G., Loffredo, F., Isola, M., Pinna, R., and López-Quintela, M. A., 2007, Penetration of metallic nanoparticles in human full-thickness skin. J. Invest. Dermatol. 127: 1701–1712. 17. Shim, J., Seok, K. H., Park, W. S., Han, S. H., Kim, J., and Chang, I. S., 2004, Transdermal delivery of minoxidil with block copolymer nanoparticles. J. Control. Release 97: 477–484. 18. Monteiro-Riviere, N. A., 1998, Integument. In: Dellmann, H. D., and Eurell, J. A. (Eds.), Textbook of Veterinary Histology. Williams & Wilkins, Baltimore, MD, pp. 303–332. 19. Monteiro-Riviere, N. A., 2008, Anatomical Factors that Affect Barrier Function. In: Zhai, H., Wilhelm, K. P., and Maibach, H. I. (Eds.), Dermatotoxicology. CRC Press, New York, pp. 39–50. 20. Monteiro-Riviere, N. A., 1991, Comparative Anatomy, Physiology, and Biochemistry of Mammalian Skin. In: Hobson, D.W. (Ed.), Dermal and Ocular Toxicology Fundamentals and Methods. CRC Press, Boca Raton, FL, pp. 3–71.

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21. Monteiro-Riviere, N. A., and Riviere, J. E., 1996, The Pig as a Model for Cutaneous Pharmacology and Toxicology Research. In: Tumbleson, M. E., and Schook, L. B. (Eds.), Advances in Swine in Biomedical Research. Plenum, New York, pp. 425–458. 22. Monteiro-Riviere, N. A., 2001, Integument. In: Pond, W. G., and Mersmann, H. J. (Eds.), The Biology of the Domestic Pig. Cornell University Press, Ithaca, NY, pp. 625– 652. 23. Bronaugh, R. L., Stewart, R. F., and Congdon, E. R., 1982, Methods for in vitro percutaneous absorption studies. 2. Animal models for human skin. Toxicol. Appl. Pharmacol. 62: 481–488. 24. Schaefer, H., and Redelmeier, T. E., 1996, Skin Barrier. In: Schaefer, H., and Redelmeier, T. E. (Eds.), Skin Barrier: Principles of Percutaneous Absorption. Karger Basel, Switzerland, pp. 146–148. 25. Treffel, P., and Gabrad, B., 1996, Skin penetration and sun protection factor of ultraviolet filters from two vehicles. Pharmaceut. Res. 13: 770–774. 26. Hostýnek, J. J., Dreher, F., Pelosi, A., Anigbogu, A., and Maibach, H. I., 2001, Human stratum corneum penetration by nickel: In vivo study of depth distribution after occlusive application of the metal as powder. Acta. Derm. Venereol. 212(Suppl.): 5–10. 27. Fent, K. W., Jayaraj, K., Gold, A., Ball, L. M., and Nylander-French, L. A., 2006, Tape-strip sampling for measuring dermal exposure to 1,6-hexamethylene diisocyanate. Scand. J. Work Environ. Health 32: 225–240. 28. Orentreich, N., and Orentreich, D. S., 1995, Dermabrasion, in Dermatol. Clin. 15: 313– 327. 29. Grimes, P. E., 2005, Microdermabrasion. Dermatol. Surg. 31: 1160–1165. 30. Lee, W. R., Shen, S. C., Wang, K. S., Hu, C. H., and Fang, J. Y., 2003, Lasers and microdermabrasion enhance and control topical delivery of vitamin C. J. Invest. Dermatol. 121: 118–1125. 31. Tinkle, S. S., Antonini, J. M., Rich, B. A., Roberts, J. R., Salmen, R., DePree, K., and Adkins, E. J., 2003, Skin as a route of exposure and sensitization in chronic beryllium disease. Environ. Health Perspect. 111: 1202–1208. 32. Rouse, J. G., Yang, J., Barron, A. R., and Monteiro-Riviere, N. A., 2006, Fullerenebased amino acid nanoparticle interactions with human epidermal keratinocytes. Toxicol. In Vitro 8: 1313–1320. 33. Monteiro-Riviere, N. A., Inman, A. O., and Ryman-Rasmussen, J. P., 2007, Dermal Effects of Nanomaterials. In: Monteiro-Riviere, N. A., and Tran, C. L. (Eds.), Nanotoxicology: Characterization, Dosing, and Health Effects. Informa Healthcare, New York, pp. 317–337. 34. Cross, S. E., Innes, B., Roberts, M. S., Tsuzuki, T., Robertson, T. A., and McCormick, P., 2007, Human skin penetration of sunscreen nanoparticles: In-vitro assessment of a novel micronized zinc oxide formulation. Skin Pharmacol. Physiol. 20: 148–154.

NANOTECHNOLOGY The Occupational Health and Safety Concerns

S. CHAN-REMILLARD Golder Associates Ltd., and HydroQual Laboratories Ltd. #4, 6125 – 12th Street S.E. Calgary, Alberta T2H 2K1, Canada [email protected] L. KAPUSTKA LK Consultancy 8 Coach Gate Place SW Calgary, AB T3H 1G2 Canada [email protected] S. GOUDEY HydroQual Laboratories Ltd. #4, 6125 – 12th Street S.E Calgary, Alberta T2H 2K1, Canada

Abstract. Nanotechnology is a rapidly emerging field. There are currently over 500 consumer products available in the marketplace and the field of nanotechnology itself that will be worth over $1 trillion by 2012. However, with an increasing number of products emerging, there is also a consequent rise in ecological and human exposure. The risk and degree of exposure to nanoscale particles (NP) will vary depending on the form of the particle, for example, powder, liquid or encapsulated, when contact occurs. Although, general public exposure to NP is increasing due to the shear number of products available, the majority of human exposure still occurs in an occupational setting. Preliminary exposure studies demonstrate that NP may enter the body via the gastrointestinal, respiratory and integumentary systems and then translocate to other vital organs and systems (for example via the olfactory bulb). Historical data on ultrafine particles have shown a higher incidence of lung cancer and respiratory disorders associated with exposure. Due to these data and evidence emerging directly on NP, precautionary measures may be warranted to ensure worker safety. Regulatory agencies and manufacturers are beginning to consider standard practices that adequately protect workers from nanoscale particle exposure. The occupational hazards associated with exposure and the current safety recommendations will be discussed.


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1.

Introduction

The concept of nanoscale particles (NP) and processes in the nanoscale is not novel. Humans have been exposed to NP from the natural environment long before recorded history. However, it has only been in the last couple of decades where exposures to NP that are anthropogenic in origin, specifically the engineered forms, have become a potential health and safety issue. Nanotechnology has been compared to the industrial and computer revolutions for its ability to change/create many technologies and how we approach science. Many benefits may be realized through the integration of nanotechnology in existing and previously unattainable technologies. Although there is much excitement for nanotechnology within the scientific and commercial/industrial communities, there is a large gap in our knowledge regarding how NP exposure may impact living organisms. This lack of toxicity data may seriously hamper the progression and commercialization of this science. Nanoscale particles can be classified according to their source origin. Under this classification scheme they can be categorized as natural, incidental or engineered NP. Natural NP, as the name implies, are found naturally in the environment (e.g. viruses, products of bacterial processes, many of the functions that occur within living organisms are within the nanoscale), incidental NP are created as a function of industrial processes (e.g. combustion of diesel engines, welding fumes) and engineered NP have been specifically created for a function or property. As compared to natural NP, incidental and engineered forms are both anthropogenically introduced into the environment. Engineered NP are created with a specific chemical signature, homogeneous morphology, and size. Often times with specific functionality whereas incidental NP are a heterogeneous mixture for each of these characteristics. Due to the unique physical-chemical characteristics of engineered NP, our understanding of how they may react and defenses against these particles in biological systems is not very well known. Furthermore, the intrinsic toxicity of individual NP must also be factored into health impact assessments. 2.

Exposure

Although there are an increasing number of products with nanotechnology incorporated into them available on the consumer marketplace exposure to engineered NP is still occurring primarily within the occupational realm. Workers are exposed to the NP either through manufacturing of the particles directly or products that have these NP incorporated into them. Due to the lack of toxicity information on how NP react within biological systems key questions regarding the health and safety of frontline workers still remain. Do existing engineering controls and personal protective equipment guidelines adequately protect workers from NP exposure? Are the current tools/instruments used to measure exposure levels sensitive enough for measuring such small particulate matter?

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The degree of exposure to NP is highly dependent on the initial form of the particle and the organism that comes in contact with it. There are higher risks of exposure to NP that are within the dust or aerosolized forms due to their increased mobility, whereas exposure to particles that are immobilized within a liquid or a more rigid matrix (e.g. steel) will have a much lower risk of exposure [1]. However, it must be noted that exposure to NP in an immobilized structure may increase if the product breaks down resulting in the release of smaller particles into the environment. 3.

Routes of Entry

Nanoscale particles may enter into the body via three primary routes: inhalation, skin exposure and ingestion where the toxicity targets are the respiratory, integumentary and gastrointestinal systems respectively. 3.1.

RESPIRATORY SYSTEM

Epidemiological studies into ultrafine/incidental particles are beginning to demonstrate that there are some serious health effects associated with chronic exposure. Occupational workers exposed to particles from combustion engines or welding fumes for prolonged periods have higher incidences of lung cancer, chronic obstructive pulmonary disease, fibroids and cardiovascular diseases [3, 11, 13] as compared to the general population. Studies on chronic inhalation of ultrafine or incidental particles may provide some insight into the health impact of chronic exposure to engineered NP. The respiratory tract has three distinct regions: the nasopharyngeal, tracheobronchial and the alveolar. The regional deposition rate of NP within each of these compartments is highly dependent on the size of the particle. The deposition of NP that are 1 nm in dimension is primarily within the nasopharyngeal region whereas slightly larger particles (20 nm) deposit further down the respiratory tract in the alveolar macrophage regions where it is much more difficult for the body’s clearance mechanisms to remove these particles [23]. The respiratory tract has several clearance mechanisms for particulate matter. The respiratory tract has a thick layer of mucus that traps particles as they are inhaled. Within the tracheobronchial region, particles trapped in the mucus layer are removed via the mucociliary escalator. The particles are either expelled through expectoration or may enter the gastrointestinal tract through swallowing. The primary clearance mechanism within the alveolar region is a phagocytic activity through the action of alveolar macrophages. Alveolar macrophages phagocytose the particles and move them upwards into the tracheobronchial region where they are then removed by the mucociliary escalator. Under normal circumstances these respiratory clearance mechanisms are highly effective at clearing particulate matter that enters into the respiratory tract. However, due to the unique physico-chemical properties and the size and aspect ratio characteristics of NP the effectiveness of these clearance mechanisms is

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uncertain. The impairment of phagocytic activity and cytotoxic action towards alveolar macrophages exposed to several carbon based NP have previously been observed [10]. The impairment of respiratory defense mechanisms may consequently result in the persistence of NP in the lung, movement of the particles deeper into the lung tissue or translocation to other target organs. The presence of nanoscale carbon (black, nanofibers and multiwalled nanotubes) were toxic to human lung cells after only 24 h of exposure; toxicity increased with prolonged exposure (5 days). Morphological changes, such as decreased cellular contact, detachment from cellular matrix, condensation of nuclei and cytoplasmic retraction were observed in exposed cells [18]. The persistence of particles in lung tissue may result in an elevated inflammatory response and ultimately various lung diseases as observed in epidemiological studies on ultrafine particles [3, 11, 13]. Persistence of NP within the lung may increase the potential for translocation to other target organs. Translocation of NP into the blood stream can occur via the air/blood barrier, through the lymphatic system, move further into the lung tissue or interstitium or via the sensory neurons (e.g. olfactory bulb or the vagus nerve [24]). Nanoscale particles have been found to penetrate beyond the basement membrane into the capillary lumen and then attach directly onto red blood cells [29], which may explain the cardiovascular consequences of exposure. Inhaled NP have been detected in the liver and bladder [20, 23], heart and spleen [28], lymph nodes [24] and in the olfactory bulb and different regions of the brain [7] after varying periods of exposure. 3.2.

INTEGUMENTARY SYSTEM

Another major route of entry for NP is the integumentary system or the skin. The skin is made up of three distinct layers: the epidermis, dermis and the subcutaneous fatty layer. The epidermis is the top few layers of skin that includes the horny outer layer composed of dead keratinized skin cells (stratum corneum), prickle cell layer (stratum spinosum) and the basal cell layer (stratum basal). Collectively these three layers of the epidermis form a tight protective barrier for the underlying dermis that contains a rich blood supply, immune cells (macrophages/dendritic cells), lymph vessels and sensory nerve endings. Traditionally, the thick stratum corneum (0.5–1.5 mm thick) layer was thought of as a relatively impermeable barrier to many compounds as experienced in the pharmaceutical industry where creating effective topical medications with a high absorbance rate is a challenge. Skin flexion studies demonstrate that smaller particles (0.5–1 um) are able to penetrate into deeper skin layers than larger particles (2–4 um) suggesting a size dependent gradient for penetration [30]. However, due to the smaller size and unique physicochemical properties of NP is the epidermal layer as effective of a barrier against penetration by NP based formulations? The movement of NP into the dermal layer increases the chances of further translocation via the blood supply, lymph system, immune cells and sensory neurons to secondary target organs with potential unintended consequences.

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The most commonly used NP in topical applications are nanoscale titanium dioxide and zinc oxide in sunscreens. These NP have been used for over 2 decades. Prior to the use of NP in sunscreen formulations the application of sunscreen left a non-aesthetically pleasing white film on the skin. The advantage of using NP for sunblocks is that in the nanoscale range, titanium dioxide and zinc oxide still retain their UV blocking capabilities but are also transparent. This has a major impact on improving public health. Several studies have demonstrated that the epidermal layer is highly effective in preventing the passage of NP. Nanoscale titanium dioxide and zinc oxide did not penetrate beyond the stratum corneum of the protective outer epidermal layer [8, 6, 19, 27]. In a presentation to the FDA public forum on nanomaterials, the Cosmetic, Toiletry and Fragrance Association (CTFA) indicated that these particles have been rigorously tested and are deemed safe for human usage [26]. Nohynek et al. [22] also conclude that titanium dioxide and zinc oxide that are currently used in cosmetic preparation do not pose a risk to human health. However, caution should still be taken with other types of NP through dermal contact [4]. Another potential route for dermal penetration of NP is through the follicle. The follicular pathway may represent a route for NP to bypass the protective epidermal layer. The hair follicle penetrates deep into the dermal layer where there is a rich supply of blood and immune cell activity directly connected to the follicle. The potential for these particles to enter systemic circulation via the follicle is a mechanism that many topical drug delivery systems exploit. Lademann et al. [16] demonstrated that dyes carried by NP are able to penetrate deeper and persist longer in hair follicles than non-particulate counterparts. They also found that mechanical massage or motion aids the penetration of particles deeper into the follicle. The follicle not only acts as a transit point but also as a reservoir for topically applied medications. Although from a pharmaceutical perspective, this is advantageous. The unintended consequences of nonprescriptive particles depositing in the follicles may result in systemic circulation of particles with unknown sequelae. Follicular penetration studies using fluorescence microscopy found that larger particles remained in the upper regions of the follicle, whereas, 40 nm particles were found in the follicle as deep as the viable dermal layers. There was also increase immune cell sampling for 40 nm particles than larger particles [31]. Deposits of iron NP in and around the follicle were observed as far as 30–170 um below the viable epidermal layer [4]. With titanium dioxide (20 nm) deposition was observed as deep as 400 um into the follicle, however, no particles were found in the vital tissue or near sebaceous glands [17]. A key factor that may pose a challenge in the case of drug delivery systems or protection in the case of penetration of non-prescriptive NP is the opened or closed state of the follicle orifices. Follicles are open during periods of active sebum production or hair growth but during the resting phase a protective layer of sebum and desquamated cells covers the follicle opening [15, 25]. The open or closed state of the follicles may be a factor in penetration and persistence of NP inside the follicular orifice.

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3.3.


GASTROINTESTINAL SYSTEM

The third major route of entry for NP is via the gastrointestinal tract. Entrance of NP into this system may occur via direct ingestion or as previously discussed, via the mucociliary escalator. The entire gastrointestinal tract is approximately 6.5 m in length. It is divided into the two distinct regions, the upper (mouth, pharynx, esophagus and stomach) and lower gastrointestinal tract (large/small intestine and anus). Not only do absorptive/digestive/defecation processes occur throughout the tract, the digestive tract plays a prominent role in immune functioning via the gut associated lymphoid tissue (GALT) and is intricately associated with key accessory organs like the liver, gallbladder and pancreas. Similar to the respiratory tract, the gastrointestinal tract is lined with a thick mucus membrane that is in direct contact with the contents of the gastrointestinal tract. When inhalation or whole body NP exposure occurs, there is a high likelihood that these particles will be entering into the gastrointestinal tract. Considering the large surface area involved for absorptive/digestive processes the potential for ingested NP to disrupt this system may be considerable. What is the fate of these NP once in the gastrointestinal tract? Are they excreted, do they persist only in the intestinal tissue or do they translocate through the gut wall into other secondary organs? The efficacy of many oral pharmaceuticals is highly dependent on the ability of the gastrointestinal system to absorb the active ingredients. Much of our current understanding of how NP act at the gut wall/systemic circulation interface has arisen from research looking at the use of NP to enhance the delivery of drugs across the gut wall into systemic circulation. The implications of NP enhancing drug absorption and is great, however, the absorption of particles that are not physiologically relevant may have unintended consequences. The mucosal lining is composed of millions of villus lined with cells (enterocytes/epithelial) that are constantly and rapidly turned over. A key route to systemic access by NP is the ability to penetrate through this lining. Gold NP (4 and 10 nm) were able to gain access through the gut wall via tiny little pores that formed from enterocyte turnover through a process called persorption. The smaller the NP the further they were able to penetrate into the gut wall. Gold NP were observed on the apical and basolateral sections of the villi. There were also some particles observed near lymph vessels, suggesting another potential mechanism that NP may cross into the blood stream [12]. Through fluorescence imagery, nanoscale chitosan particles have been observed in epithelial cells lining the jejunum, duodenum and ileum. Chitosan were also found deeper in the lamina propria, suggesting movement of particles through the epithelium. Similar to the study by Hillyer and Albrecht, NP were also found in the peyer’s patches, which are key cells within the gut involved in immune surveillance [5]. The movement of gold nanoparticles was not isolated just to the deeper layers of the gut wall but was found in secondary organs. Four nanometer gold particles were isolated in the blood, brain, lung heart, kidney, spleen, liver, small intestine and stomach 7 days post exposure [12]. When compared to larger particles (58 nm),

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the 4 nm particles had the highest degree of translocation. Similar to the respiratory tract, this suggests a size dependent gradient for translocation across the gut wall. Yet others have found that ingested NP primarily transit through the gastrointestinal system and are excreted in fecal matter and urine [14, 28]. 4.

Occupational Health and Safety

The previous section discusses the impact of NP on the three key target exposure sites in humans illustrates the variability that the type of NP tested and the target sites evaluated can have on conclusions regarding physiological effects/responses. Although these studies contribute to the current database of information there is still not a large enough body of evidence or consensus on the ultimate effects NP have on living organisms to truly inform or regulate nanotechnology. This is one of the key issues surrounding the nanotechnology industry and how it is to be effectively regulated and the hazards and risks effectively managed. Until there is enough information to effectively inform regulatory agencies and industry, health and safety guidelines to the best of our knowledge must be developed and implemented to prevent human exposure to the potential health hazards of NP. Exposure to engineered NP will vary depending on the context of exposure. Currently, the majority of human exposure to NP is isolated to frontline workers, in the occupational setting, who are directly involved in producing or incorporating NP into products. Due to the rapid emergence of products containing NP available for consumer consumption, there will be a parallel increase in exposure levels among the general public. However, general public exposure will differ from occupational exposure in the form of the nanoparticle that exposure will occur. The majority of NP exposure to the general public will occur in the form of products where NP are bound within some sort of matrix. For example, titanium dioxide NP will be bound within the liquid matrix of a lotion or carbon nanofibers will be bound within the steel structure of an automobile frame. The matrix that binds the particles will confer a degree of protection to the consumer against exposure to free NP. Within the occupational setting, there is a higher likelihood of exposure to NP that are in the free form. The adequacy of health and safety protocols within an occupational setting remains uncertain due to our limited understanding of the toxicity of NP. 4.1.

NIOSH

There are several worldwide organizations (ASTM, NIOSH, ICON, SCENHIR) involved in assessing the safety protocols for handling nanomaterials. A key organization involved in this initiative is the National Institute of Occupational Safety and Health (NIOSH), an organization within the Centers for Disease Control and Prevention (CDC). The key mandate of NIOSH is to ensure that beneficial applications of nanotechnology are developed in a responsible manner with a high priority focus on the societal, human and environmental implications

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of nanotechnology. In 2004, the Nanotechnology Research Center (NTRC) was developed under the auspice of NIOSH. The NTRC was developed to specifically focus on nanotechnology research. Since 2005 NIOSH has internally redirected US$11 million of funding towards this initiative. The role of the NTRC is to (1) Determine whether NP and nanomaterials pose a human health risk to workers, (2) Conduct research on the use of nanotechnology to prevent work related injuries and illnesses, (3) Promote healthy workplaces through interventions, recommendations and capacity building and (4) Enhance global workplace safety and health through national and international collaborations on nanotechnology research and guidance. The research under the NTRC encompasses ten critical topic areas: (1) toxicity and internal dose, (2) risk assessment, (3) epidemiology and surveillance, (4) engineering controls and personal protective equipment, (5) measurement methods, (6) exposure assessment, (7) fire and explosion safety, (8) recommendations and guidance, (9) communication and education and (10) applications [1]. Currently, there is a knowledge gap in our understanding of the toxicity of NP to living organisms. Are NP toxic and at what dose or exposure level do NP pose a risk? Several different but complementary key critical topic areas have been established to answer these key questions. Scientists involved in the first critical topic area are investigating the physicochemical properties of NP that influence toxicity, determining the fate of NP once they have entered into biological systems and the short and long term effects of exposure to organ systems and tissues. Scientists involved in the exposure assessment group are evaluating possible inhalation and dermal exposure to nanomaterials, determining how exposure may differ by work process and determining the key factors that influence the production, dispersion, accumulation and re-entry of nanomaterials into the workplace environment. Scientists within risk assessment work stream are evaluating whether current exposure-response data for fine and ultrafine particles are adequate in assessing/identifying the hazards related to NP and are developing a risk-based framework for evaluating the potential hazards and occupational risk of exposure to NP. Researchers funded under the third critical topic area are involved in identifying what knowledge gaps can be filled with epidemiological studies to further advance our knowledge of NP. Scientists involved in the engineering controls and personal protective equipment stream of research have a twofold mandate. They are actively evaluating whether current engineering controls and personal protective equipment used are effective at protecting workers from NP exposure. The second mandate of this group involves looking at ways to enhance worker safety through incorporation of nanotechnology into personal protective equipment. Research from scientist working under the applications umbrella are also working on identifying ways to apply nanotechnology to enhance occupational health and safety. Scientists involved in the fifth work stream are actively involved in evaluating, developing and testing methods and validating sampling instruments to accurately measure airborne nanomaterials in the workplace. Due to the different physicochemical properties of compounds in the nanoscale range, there is a possibility that these materials may become flammable and

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explosive. An example of a compound that does not have explosive tendencies in the macro form but is highly explosive and volatile upon contact with air and water is zinc. One of the key research streams funded by the NTRC is identifying the physicochemical properties of nanoscale that contribute to their combustibility and flammability. This group then makes recommendations for alternative work practices that decrease or eliminate exposure to such situations. As previously discussed, the pace that nanotechnology products are emerging on the consumer marketplace is not paralleled by toxicity research. This poses a significant challenge to regulators and industry alike. A critical topic area under the mandate of the NTRC is to provide interim recommendations and guidance for workplace safety and health practices in handling nanomaterials using the current state of knowledge. To aid in the collection and dissemination of the most up to date information, the communication and education stream is actively involved in fostering international partnerships to ensure the sharing of research needs, approaches and results. 4.2.

ENVIRONMENTAL HEALTH AND SAFETY PLANS

There are many companies worldwide that are engaged in some level of nanotechnology development or usage. Since nanotechnology is a relatively new field of research, whether the health and safety plans of these organizations can adequately protect workers from the potential hazards of nanotechnology must be evaluated. In collaboration with the International Council on Nanotechnology (ICON), an interdisciplinary team of scientists from the University of California at Santa Barbara (UCSB) interviewed 64 organizations from private sector companies, research labs, university labs and consultant companies within North America, Australia, the Europe and Asia that claimed to work with nanotechnology in some capacity on their environmental health and safety (EHS) plans regarding nanotechnology [9]. Many of the respondents (38/64) to the survey had some sort of EHS program in place ranging from having guideline documents, using risk assessment approaches, EHS programs modeled after those for fine or ultrafine particles and more sophisticated programs that monitor actual exposure to NP. The EHS training programs included information on the safe handling and standard operating procedures for nanomaterials, the proper use of personal protective equipment, the hazards and toxicities associated with handling nanomaterials and engineering controls for decreasing exposure to nanomaterials. Fewer programs included information on emergency procedures to handle accidental exposure, proper waste handling practices, the potential for/implications of environmental release of nanomaterials, consumer protection, exposure monitoring and regulations governing nanomaterials. There seemed to be an association between the size of the company and the level of nano-specific safety training, with larger companies having more sophisticated health and safety programs. When asked why their organizations administered a nano-specific safety protocol, several organizations indicated that this was a safety precaution against unknown hazards that include potential toxicity, the

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minimization of employee exposure, a proactive approach to address potential risks from nanomaterials exposure or the unique hazards related to nanomaterials and compliance with safety regulations for fine particles. However, 26 out of the 64 companies surveyed did not have a nano-specific safety protocol in place. These companies sited reasons for not having EHS plans ranged from planning to implement a training protocol, employees were not in direct contact with the materials, they treated nanomaterials as a hazardous waste to the companies that deem the nanomaterials were not dangerous or there was not enough time or resources to implement a plan [9]. This report also describes internal and external barriers to implementing an EHS program. The respondents cited the major external barrier to implementing an EHS program was the lack of useful information and consistent guidelines regarding the safe handling of nanomaterials, while less frequently the ineffective techniques for detecting and measuring the presence of NP in the work place was also cited as a barrier. The most frequently cited internal barrier to instituting a health and safety plan was the costs that were associated with implementation. An interesting internal barrier to implementation cited was the attitudes of the workers towards EHS and nanomaterials risk. Workers either believed that implementation of these plans required too much effort and did not acknowledge the importance of safety protocols in handling nanomaterials, also described as the naïve approach or they had a cavalier approach where they felt that the safety protocols were ineffective and that there was little risk associated with handling the nanomaterials [9]. The knowledge gaps identified by the NIOSH research initiatives and the lack of toxicity data illustrates the many impediments that face regulators and industry in assessing the level of safety protocols that need to be implemented to protect occupational workers from being exposed to the potential hazards of handling NP. 4.3.

NANOMATERIALS – OCCUPATIONAL HEALTH AND SAFETY EXPOSURE CONTROLS

Although there are many barriers to implementing health and safety protocols, interim guidelines based on the most current information available have been developed to provide regulators and industry guidance on minimizing occupational exposure [1, 2, 9, 21]. The current guidelines to minimize work place exposure to NP suggest substituting or eliminating the hazardous material(s) from the process or when that is not possible to implement engineering and administrative controls. In the event that is not possible to implement or the effectiveness of engineering/ administrative controls is uncertain, the use of personal protective equipment (PPE) is recommended. Industrial hygiene specialists recommend the first line of defence against exposure to hazardous materials is to completely eliminate or substitute a compound for one that is less hazardous from a process. For example, the substitution of a powdered form of a NP, which is easily aerosolized and has a high likelihood of being inhaled or ingested, for a form that is bound within a liquid matrix would decrease the risk of exposure. In some instances, the complete elimination of the

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compound from the process would remove the likelihood of exposure to a toxic substance [1, 9]. However, in situations where elimination or substitution of a compound in a product or process is not a realistic option, the implementation of safety programs that incorporate engineering controls, administrative controls or PPEs are necessary to ensure worker safety. The use of engineering controls developed to control gases is the most effective means of controlling the movement of NP out of designated workspaces. The types of engineering controls that are recommended are local ventilation systems for the immediate work area (e.g. total enclosures [e.g. glove box], partial enclosures [e.g. chemical hoods, low flow vented balances], weigh hoods for dry materials, and exterior hoods located adjacent to workspace [e.g. receiving or draft hoods that draw in particles]), general exhaust ventilation (e.g. scrubbing systems, negative pressure), specific designation of a workspace by encapsulating/isolating the area as a nanomaterials zone or the use of specialized filters (e.g. HEPA filters). Encapsulation/isolation of work processes that involve nanomaterials may also be achieved through distance, physical separation/barriers or the use of isolation or control rooms [1, 21]. An important supplement to engineering controls is the use of administrative controls, which are driven by good laboratory practices and standard operating procedures, will also decrease the risk of occupational exposure to NP. The implementation of administrative controls involves extensive safety training of personnel exposed to processes that involve the use of NP. Important elements of administrative controls are the cleaning procedures that are used within a facility. The use of wet wiping procedures and HEPA vacuums systems but not blowers or fans to prevent the accumulation of NP within workspaces is recommended [1, 9, 21]. ASTM further suggests the use of surfactants during cutting/drilling to minimize dust production and the requirement for workspaces/equipment/furniture to be constructed of smooth, non-porous materials to simplify cleaning to further decrease the risk of occupational exposure. Another important facet of administrative controls is worker training and education. Worker education and training into the potential hazards of NP may help to decrease the previously described ‘naïve’ or ‘cavalier’ attitudes towards health and safety experienced by workers. Educating workers to the hazards associated with or suspected of NP may have a larger impact on attitudes towards personal health and safety within an occupational setting than simply advising on the need for protection. Educational programs should not only involve training/ education on proper handling procedures and safety issues surrounding NP, but should also include information on the prevention of transfer (e.g. no eating around NP workspaces, have designated lab coats/gloves/goggles, enclosed vessels), proper hazard labeling procedure, the availability of material safety data sheets and emergency response and medical surveillance procedures [1, 21]. The final method to control worker exposure to NP is through the use of PPEs. Personal protective equipment is recommended as the primary defense against exposure only in instances where engineering and administrative controls have been deemed ineffective at minimizing occupational exposure to NP. Types of PPEs used are respirators, eye protection and protective clothing and gloves that


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are specifically designated for NP use. Respirators fitted with N100 filters are recommended by NIOSH as being completely effective at blocking NP inhalation. However, the type of respirator (e.g. half or full faced mask) and proper fitting of the mask will affect the degree of protection offered by the respirator. If possible, the use of powered air purifying respirators fitted with a HEPA filter is recommended. When using the half faced masks, it is highly recommended that the type of eye protection used include at a minimum side shields around the eye. 5.

Conclusion

Many wonderful advances in science and technology have been or yet to be realized through the use and manipulation of material within the nanoscale range. In addition to the search for new applications for nanotechnology, there is also the responsibility to understand the impact these NP have on living organisms and to protect living organisms from potentially toxic exposure. The toxicity of these particles still remains relatively unknown. The new emerging science of nanotoxicology that studies how NP impact living organisms is not progressing at a parallel pace to product development. The lack of toxicity data introduces a serious gap in knowledge that may hamper our ability to further develop/introduce products, ensure consumer safety and effectively regulate these products. Until there is adequate toxicity data available interim safety guidelines based on the most current information have been developed. Proper education/training programs and implementation of EHS programs based on these guidelines will minimize the level of NP exposure to frontline workers. Further research needs to be conducted to enhance worker safety and to ensure consumer and environmental safety of nanotechnology. References 1. ASTM International (2007) Standard Guide for Handling Unbound Engineered Nanoscale Particles in Occupational Settings, E 2535-07. 2. Aitken, R.J., Creely, K.S., and Tran, C.L. (2004) Nanoparticles: An Occupational Hygiene Review, Institute of Occupational Medicine for the Health and Safety Executive. Research Report 274. Retrieved October 6, 2006, from http://www. hse.gov.uk/research/rrpdf/rr274.pdf 3. Attfield, M.D., and Kuempel, E.D. (2008) Mortality among U.S. underground coal miners: A 23-year follow-up, Am. J. Ind. Med. 51(4), 231–245. 4. Baroli, B., Ennas, M.G., Loffredo, F., Isola, M., Pinna, N., and Lopez-Quintela, M.A. (2007) Penetration of metallic nanoparticles in human full thickness skin, J. Invest. Dermatol. 127, 1701–1702. 5. Behrens, I., Vila Pena, A.I., Alonso, M.J., and Kissel, T. (2002) Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: The effect of mucus on particle absorption and transport, Pharm. Res. 19(8), 1185–1193.

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6. Cross, S.E., Innes, B., Roberts, M.S., Tsuzuki, T., Robertson, T.A., and McCormick, P. (2007) Human skin penetration of sunscreen nanoparticles: In vitro assessment of a novel micronized zinc oxide formulation, Skin Pharmacol. Physiol. 20(3), 148–154. 7. Elder, A., Gelein, R., Silva, V. Feiker, T., Opnanshuk, L., Carter, J., Potter, R., Maynard, A., Ito, Y., Finkelstein J., and Oberdorster G. (2006) Translocation of inhaled ultrafine manganese oxide particles to the central nervous system, Environ. Health Perspect. 114(8), 1172–1178. 8. Gamer, A., Leibold, E., and van Ravenzwaay, B. (2006) The in vitro absorption of microfine ZnO and TiO2 through porcine skin, Toxicol. In Vitro 20(3), 301–307. 9. Gerritzen, G., Huang, L., Killpack, K., Mircheva, M., Conti, J., Magali, D., Harthorn, B.H., Appelbaum, R.P., and Holden, P. (2006) A Report to ICON: Review of Safety Practices in the Nanotechnology Industry, 30–36. 10. Guang, J., Haifang, W., Lei, Y., Xiang, W., Rongjuan, P., Tao, Y., Yuliang, Z., and Xinbiao, G. (2005) Cytotoxicity of carbon nanomaterials: Single walled nanotubes, multiwalled nanotubes, and fullerenes, Environ. Sci. Technol. 39, 1378–1383. 11. Harber, P., Muranko, H., Solis, S., Torossian, S., and Merz, B. (2003) Effect of carbon black exposure on respiratory function and symptoms, J. Occup. Environ. Med. 45(2), 144–155. 12. Hillyer, J.F., and Abrecht, R.M. (2001) Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles, J. Pharm. Sci. 90(12), 1927–1936. 13. Järvholm, B., and Silverman, D. (2003) Lung cancer in heavy equipment operators and truck drivers with diesel exhaust exposure in the construction industry, Occup. Environ. Med. 60(7), 516–520. 14. Kreyling, W.G., Semmler, M., Erbe, F., Mayer, P., Takenaka, S., Schulz, H., Oberdorster, G., and Ziesenis, A. (2002) Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low, J. Toxicol. Environ. Health 65, 1513–1530. 15. Lademann, J., Otberg, N., Richter, H., Weigmann, H.J., Lindemann, U., Schaefer, H., and Sterry, W. (2001) Investigation of follicular penetration of topically applied substances, Skin Pharmacol. Appl. Skin Physiol. 14(Suppl. 1), 17–22. 16. Lademann, J., Richter, H., Teichmann, A., Otberg, N., Blume-Peytavi, U., Luengo, J., Weib, B., Schaefer, U.F., Lehr, C-M., Wepf, R., and Sterry, W. (2007) Nanoparticles – An efficient carrier for drug delivery into the hair follicles, Eur. J. Pharm. Biopharm. 66, 159–164. 17. Lekki, J., Stachura, Z., Dabros, W., Stachura, J,, Menzel, F., Reinert, T., Butz, T., Pallon, J., Gontier, E., Ynsa, M.D., Morretto, P., Kertesz, Z., Szikszai, Z., and Kiss, A.Z. (2007) On the follicular pathway of percutaneous uptake of nanoparticles: Ion microscopy and autoradiography studies, Nucl. Instrum. Meth. Phys. Res. B. 260, 174– 177. 18. Magrez, A., Kasas, S., Salicio, V., Pasquier, N., Seo, J., Celio, M., Catsicas, S., Schwaller, B., and Forro, L. (2006) Cellular toxicity of carbon based nanomaterials, Nano Lett. 6(6), 1121–1125. 19. Mavon, A., Miquel, C., Lejeune, O., Payre, B., and Morretto, P. (2007) In vitro percutaneous absorption and in vivo stratum corneum distribution of an organic and a mineral sunscreen, Skin Pharmacol. Physiol. 20, 10–20. 20. Nemmar, A., Hoet, P.H.M., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoyleaerts, M.F., Vanbilloen, D., Mortelmans., L., and Nemery B. (2002) Passage of inhaled particles into the blood circulation in humans, Circulation 105(4), 411–441. 21. NIOSH (2007) Progress Toward Safe Nanotechnology in the Workplace. Department of Health and Human Services, Centers for Disease Control and Prevention, National

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22. 23.

24. 25. 26. 27. 28.

29. 30. 31.

S. CHAN-REMILLARD ET AL. Institute of Occupational Health and Safety. DHHS (NIOSH), Publication No. 2007-123, June 2007. Nohynek, G.J., Lademann, J., Ribaud, C., and Roberta, M.S. (2007) Grey Goo on the skin? Nanotechnology, cosmetic and sunscreen safety, Crit. Rev. Toxicol. 37, 251–277. Oberdorster, G., Sharp, Z., Viorel, A., Elder, A., Gelein, R., Lunts, A., Kreyling, W., and Cox, C. (2002) Extrapulmonary translocation of ultrafine carbon particles following whole body inhalation exposure of rats, J. Toxicol. Environ. Health A 65, 1531–1543. Oberdorster, G., Oberdorster, E., and Oberdorster J. (2005) Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles, Environ. Health Perspect. 113(7), 823–839. Otberg, N., Richter, H., Knuttel, A., Schaefer, H., Sterry, W., and Lademann, J. (2004) Laser spectroscopic methods for the characterization of open and closed follicles, J. Laser Phys. 1(1), 46–49. Santamaria, A. (2006) Safety of nanoscale materials in personal care products, Presentation to the FDA Public Meeting on Nanomaterials, October 10, 2006. Available at: http://www.fda.gov/nanotechnology/meetings/santamaria.html. Schulz, J., Hohenberg, H., Pflucker, F., Gartner, E., Will, T., Pfeiffer, S., Wepf, V., Wendel V., Gers-Barlag, H., and Wittern, K.D. (2007) Distribution of sunscreens on skin, Adv. Drug Deliver. Rev. 54(Suppl. 1), S157–S163. Semmler, M., Seitz, J., Erbe, F., Mayer, P., Hayder, J., Oberdorster, G., and Kreyling, W.G. (2004) Long term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organ247s16, Inhal. Toxicol. 16, 453–459. Shimada, A., Kawamura, N., Okajima, M., Kaewamatawon, T., Inoue, H., and Morita T. (2006) Translocation of intratracheally instilled UF particles from lung into the blood circulation in the mouse, Toxicol. Pathol. 34(7), 949–957. Tinkle, S.S., Antonini, J.M., Rich, B.A., Roberta, J.R., Salmen, R., DePree, K., and Adkins, E.J. (2003) Skin as a route of exposure and sensitization in chronic beryllium disease, Environ. Health Perspect. 111(9), 1202–1206. Vogt, A., Combadier, B., Hadam, S., Stieler, K.M., Lademann, J., Schaefer, H., Autran, B., Sterry, W., and Blume-Peytavi, U. (2006) 40 nm, but not 750 nm or 1500 nm nanoparticles enter epidermal CD1a+ cells after transcutaneous application on human skin, J. Invest. Dermatol. 126, 1316–1322.

BIOMARKERS OF NANOPARTICLES IMPACT ON BIOLOGICAL SYSTEMS

V. MIKHAILENKO, L. IELEIKO, A. GLAVIN, J. SOROCHINSKA R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences 45 Vasilkivska Street 03022 Kyiv, Ukraine [email protected]

Abstract. Studies of nanoscale mineral fibers have demonstrated that the toxic and carcinogenic effects are related to the surface area and surface activity of inhaled particles. Particle surface characteristics are considered to be key factors in the generation of free radicals and reactive oxygen species and are related to the development of apoptosis or cancer. Existing physico-chemical methods do not always allow estimation of the nanoparticles impact on organismal and cellular levels. The aim of this study was to develop marker system for evaluation the toxic and carcinogenic effects of nanoparticles on cells. The markers are designed with respect to important nanoparticles characteristics for specific and sensitive assessment of their impact on biological system. We have studied DNA damage, the activity of xanthine oxidoreductase influencing the level of free radicals, bioenergetic status, phospholipids profile and formation of 1H-NMR-visible mobile lipid domains in Ehrlich carcinoma cells. The efficiency of the proposed marker system was tested in vivo and in vitro with the use of C60 fullerene nanoparticles and multiwalled carbon nanotubes. Our data suggest that multiwalled carbon nanotubes and fullerene C60 may pose genotoxic effect, change energy metabolism and membrane structure, alter free radical level via xanthine oxidase activation and cause mobile lipid domains formation as determined in vivo and in vitro studies on Ehrlich carcinoma cells. 1.

Introduction

Among engineered nanoparticles (NP) currently being produced, the most common are fullerenes C60 and carbon nanotubes (CNT). These materials possess nanostructure-dependent properties that may potentially lead to unusual biological activity which typically increases as the particle size decreases. Highly increased surface area of NP may be toxicologically relevant. Studies of mineral particles have demonstrated that the toxic and carcinogenic effects are mostly related to the surface area of inhaled particles and their surface activity [1]. Data yielded from I. Linkov and J. Steevens (eds.), Nanomaterials: Risks and Benefits, © Springer Science + Business Media B.V. 2009

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animal and cell culture studies pointed to an increase in pulmonary inflammation, oxidative stress incidence, an increased risk of carcinogenesis, inflammatory cytokine production, apoptosis, and activation of certain gene expression and cell signaling pathways [2]. A common mechanism of NP impact on cell damage is oxidative and nitrosative stress development. Fullerenes and CNT’s have been shown to produce superoxide and induce free radical damage to cells [3–6]. However, currently the potential adverse effects of engineered NP on human health and the environment can not be fully estimated due to insufficient knowledge of mechanisms of action and lack of standardized testing protocols. In our study we have develop a marker system to evaluate the toxic and carcinogenic effects of nanoparticles on cells in order to address their involvement into free radical processes, energy metabolism, membrane structural changes and genotoxic mechanisms. We studied DNA damage, the activity of xanthine oxidoreductase (XOR) influencing the level of free radicals, bioenergetics status, phospholipids profile and formation of 1H-NMR-visible mobile lipid domains (MLD) in Ehrlich ascites carcinoma (EAC) cells. Xanthine oxidoreductase is a complex molybdoflavoprotein identified as a terminal enzyme of purine catabolism, catalyzing the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. The XOR protein is apparently expressed as xanthine dehydrogenase form (XDH; EC 1.1.1.204) but partially can be converted, either reversibly or irreversibly, to xanthine oxidase form (XO; 1.1.3.22) by posttranslational modification [7, 8]. The forms of XOR enzyme are differentiated by the preference of oxidizing substrate, and generation of reactive oxygen species (ROS) – hydrogen peroxide and superoxide radical [7, 9]. XOR plays an important role in maintaining the balance of free radicals, takes part in NO circulation, decomposition of S-nitrosothiols and can be source of reactive nitrogen species (RNS) – NO and peroxynitrite [8, 10]. In fact, peroxynitrite can be produced by XOR itself [11]. At normal conditions, the XDH form predominates in vivo, producing the potent antioxidant uric acid. Conversion of XDH to XO form results in overproduction of the superoxide radicals in tissues and may cause intensification of lipid peroxidation (LPO) and production of additional quantities of hydrogen peroxide in tissues [12, 13]. Activation of XOR and conversion of its XDH form to XO form leads to apoptosis and death of the damaged cells at pathological processes [14]. At the same time connection between enzyme activation, ROS and RNS production, and subsequent damage of a genetic material which can cause tissues malignization was observed in a number of studies [15, 16]. 1 H NMR-visible MLD increased formation are reported as a peculiar feature of malignant cells in vitro and in vivo [17]. Cell membrane rearrangements coincident with malignancy and proliferation of tumor cells may contribute to the increase in the ratio of methylene (CH2 at 1.3 ppm) to methyl (CH3 at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance (1H NMR). Cellular origin of these resonances is related to lipid turnover and cell membrane structure and arises from the isotropically tumbling molecules, with sufficient molecular mobility. NMR signals from CH2 and CH3 groups originate mainly from mobile fatty acyl chains of tissue triacylglycerides with lesser contributions

BIOMARKERS OF NANOPARTICLES IMPACT ON BIOLOGICAL SYSTEMS 69

from free fatty acids and cholesteryl esters. The presence of NMR-detectable lipids in cells can originate from triacylglycerides in globular plasma membrane microdomains (22–28 nm in diameter) or intracellular lipid bodies, either adjacent to the plasma membrane or within the cytoplasm [18, 19]. Bioenergetic status of cells was characterized by 31Р-NMR spectroscopy by phosphorylated metabolites and their ratios: inorganic phosphate/β-nucleoside triphosphates (Рi/βNTP), inorganic phosphate/phosphocreatine (Рi/PCr), inorganic phosphate/phosphomonoesters (Pi/PME) that characterize the level of energy metabolism and phosphomonoesters/β-nucleoside triphosphates (PME/βNTP), inorganic phosphate/phosphomonoesters (Pi/PME) that indicate the level of hypoxia. The metabolism of membrane components was characterized by the phosphomonoesters/phosphodiesters (PME/PDE) ratio. The increase in the PME/PDE ratio indicates activation of membrane components synthesis, ratio reduction point to an intensive breakdown of cells membranes. Lipids profile was characterized by the contents of phosphor-containing lipids. It is known that cardiolipin is involved in apoptosis and oxidative phosphorylation, provides osmotic stability of mitochondria [20, 21]. Phosphatidylserine could affect the regulation of protein kinase C activity and apoptosis [22]. Decrease of phosphatidylinositol content may be caused by the processes of intensive degradation or by the inhibition of its synthesis. Degradation of phosphatidylinositol leads to the formation of such second messengers, as diacylglycerol and inositol1,4,5-triphosphate. Diacylglycerol is bound to the inner layer of the plasma membrane and participates in activation of proteine kinase C. Inositol-1,4,5triphosphate diffuses through the plasma membrane into cytoplasm and binds to the specific receptors on the endoplasmic reticulum causing the release of calcium ions into the cytosol. Alterations of the phosphatidylcholine/sphingomyelin (PtdCho/SpM) ratio points out to changes of the level of membrane structuring [23]. Ratio increases reflect reduction of membrane structuring and increased membrane permeability, whereas ratio decreases indicates increased membrane viscosity. The single-cell gel electrophoresis (or comet) assay is a rapid, simple and sensitive technique for visualizing and measuring DNA damage in individual cells. It is used as a primary method of screening for genotoxic compounds. The method is based on detection of various mobility damaged DNA contained in cells embedded in agarose gel and subjected to a constant electric field. Thus DNA migrates to the anode, forming a trace reminding a “tail of a comet” which parameters depend on the level of DNA damage [24]. The aim of this study was to develop marker system for complex evaluation the toxic and carcinogenic effects of nanoparticles on cells. Existing physicochemical methods, due to insufficient knowledge of mechanisms of action, do not always allow estimation of the nanoparticles impact on organismal and cellular levels. The proposed marker system is based on studies of DNA damage, the activity of XOR, bioenergetics status, phospholipids profile, and formation of MLD in cells and is hypothesized to reveal mechanisms of NP damaging effects on cells. The current marker system was used to test in vivo and in vitro the effects


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of C60 fullerene nanoparticles and multiwalled carbon nanotubes (MWCNT) on EAC cells. 2.

Materials and Methods

Investigations were carried out on white inbred male mice weighting 19–22 g, 2– 2.5 months old, bred by vivarium of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences of Ukraine (Kyiv, Ukraine). All experiments with animals were approved by the Regional Animal Ethics Committee. 2.1.

ANIMAL STUDIES

Ehrlich ascites carcinoma (EAC) obtained from the Bank of Cell Line of the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine). EAC were maintained and propagated by serial intraperitoneal transplantation of EAC cells in an aseptic environment. Cells of EAC (106 cells/mice) were injected intraperitoneally (i.p.) at the volume of 0.5 ml of physiologic solution. All the experiments on tumor bearing mice were conducted 6 days after the EAC transplantation. MWCNT’s suspension in physiologic solution was i.p. administered (0.5 ml per mouse) in concentrations of 0.5 and 1.5 mg/mouse for 24 h. 2.2.

CELL CULTURE

EAC cells were obtained from male mice with Ehrlich ascite tumor. Cells from ascites, after washing, were suspended in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS, Gibco Laboratory, Carlsbad, CA, USA) and maintained by culturing in a humidified atmosphere of 5% CO2 at 37°C for at least 12 h. EAC cells (7 105 cells/ml of DMEM) were treated for 24 h with carbon nanoparticles (CNP) suspensions: MWCNTs (0.07 0.035 and 0.017 mg/ml) and fullerene C60 (0.066 mg/ml). The percentage of living and dead cells was determined by trypan blue exclusion test. 2.3.

NANOPARTICLES

Two different type of CNP were examined in this study. MWCNT, obtained from Dr. J.I. Semencov and T.A. Alekseeva (TMSpetsmash Ltd., Kyiv, Ukraine), were acid treated to reduce the catalyst impurity, washed and resuspended in physiologic solution. Fullerene C60 was obtained from ALSI (Ukraine). All CNP suspensions were freshly sonified under 4°C before administration (6 x 30 s) to break up agglomerates.


2.4.

NMR ASSAY

Dual extraction of cellular lipids and water-soluble metabolites from tissue samples was made by the methanol-chloroform-water extraction method proposed by Tyagi et al. [25]. This method facilitates the simultaneous extraction of both the water-soluble metabolites and the organic-soluble lipid components from the same tissue sample. Water-soluble dried extract samples were re-dissolved in D2O, organic-soluble lipid dried extract samples were re-dissolved in CDCl3. 31Р-NMR spectroscopy allows simultaneous level assessment of the main phosphorylated metabolites. 31 P and 1H spectra of tissue extracts were acquired using Varian Mercury BB NMR spectrometer (Varian, Palo Alto, CA, USA), operating at frequency 300 MHz. All NMR measurements were carried out at temperature 20°C and all samples were spun at 20 Hz. Chemical shifts in 31P NMR spectra were recorded with respect to methylenediphosphonic acid, trisodium salt (MDP, Sigma, St. Louis, MO, USA), used as an external standard. The resonance of phosphocreatine was set at 0 ppm. Chemical shifts in 1H NMR spectra were recorded with use of 0.1% solution of sodium 3-trimethylsilyl[2,2,3,3-D4] propionate in D2O as a reference at 0 ppm. The acquisition parameters of 1H spectra of water-soluble metabolites: relaxation delay time 4 s; spectral width 6 kHz; number of points 7,218; 30° flip angle. The intense water resonance was partially suppressed by the use of presaturation of the residual water protons in the solvent. The acquisition parameters of 31P spectra of water-soluble metabolites: relaxation delay time 2.4 s; spectral width 6.5 kHz; number of points 6,503; 90° flip angle. Proton scalar coupling interactions were removed by using continuous low power proton coupling. The acquisition parameters of 31P spectra of phospholipids: relaxation delay time 5 s; spectral width 3 kHz; number of points 6,000; 90° flip angle. Proton scalar coupling interactions were removed by using continuous low power proton coupling. 2.5.

THE ALKALINE COMET ASSAY

EAC cells were washed in PBS and suspended in agarose gel (0.5 · 106– 0.7 · 106 cells/ml). Cells were then lysed, subjected to alkaline denaturation, and electrophoresis [26]. Slides were stained with acridine orange solution (20 μg/ml). Comet images were observed at 100x magnification with a fluorescence microscope connected to a video camera (CCD, Webbers, USA). One hundred images were randomly selected from each sample and analyzed by an image-analysis program “CometScore” (TriTek Corp, Sumerduck, VA, USA). The extent of DNA damage was estimated by the following parameters: Comet Area (AC) – the area covered by the whole comet; Tail Length (lT) – the horizontal distance from the centre of the head (start of tail) to the end of the tail; %DNA in Tail (DNAT) – the DNA percentage in the tail – %DNAT = 100DNAT/( DNAT+ DNAH); Tail Moment (MT) – the product of tail length and fraction of DNA in the


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tail – MT = lT% DNAT; Olive tail moment – the product of the proportion of tail intensity and the displacement of tail centre of mass relative to the centre of the head. 2.6.

MLD ASSAY

Cells were harvested and washed once with PBS then washed twice with PBS made with D2O to reduce protons signal from H2O. Cells (8–10 · 107 cells/ml) were suspended in a final volume of 0.6 ml of PBS-D2O, transferred to a 5 mm NMR tube and placed on ice until analysis. The percentage of viable cells, determined by Trypan blue exclusion test, ranged between 85% and 95%, both before and after NMR analyses. 1H NMR spectra were acquired using a 300 MHz Varian Mercury 300BB NMR spectrometer (Varian, Palo Alto, CA, USA) at 20°C, 90° flip angle, repetition time 10 s, 200 excitations, 16000K data points and 5 kHz spectral width. A glass capillary with 0.1% solution of TSP in D2O was used as a reference at 0 ppm for each sample. NMR spectra were obtained using presaturation of the residual water protons in the solvent, and samples were spun at 20 Hz to prevent settling of cells during the experiment. The standardized areas of the methylene and methyl protons resonances (at 1.3 and 0.9 ppm, respectively) were integrated using VNMR software (Varian, Palo Alto, CA, USA) and expressed in relative units. 2.7.

XOR ASSAY

Total XOR activity and activity of XO were examined in EAC cells [27]. Activity of XOR enzyme was estimated by the production of uric acid from xanthine (absorbance at 295 nm). Reaction kinetics were measured for 30 min at 26°С in special 96-well plates on the microplate reader Synergy™ HT (Bio-Tek Instruments, Winooski, VT, USA). In each well 250 μl of incubation mixture (50 mM sodium phosphate buffer with 0.3 mM EDTA, 0.5 mM xanthine, 0.5 mM NAD+ and 0.24 mM oxonic acid) and 3.6 · 105 EAC cells in 50 μl of 50 mM sodium phosphate buffer with 0.3 mM EDTA were added. Oxonic acid was used as uricase inhibitor [28]. XOR activity was expressed in nM uric acid formed by 1 . 106 AEC cells during 1 h. Total protein concentration was determined according to Greenberg and Craddock [29]. 2.8.

LPO ASSAY

Intensity of lipid peroxidation (LPO) was evaluated by spontaneous accumulation of malonic dialdehyde (MDA) and expressed in nanomoles of MDA per gram of cells per hour. The absorbance of the colored thiobarbituric acid-reactive substances was measured at 532 nm on a Diode-matrix UV-Vis spectrophotometer Agilent 8453 (Agilent, Santa Clara, CA, USA) [30, 31].


2.9.

STATISTICAL ANALYSIS

Statistical analysis was performed in cases when experiments were carried out at least in triplicate using Student’s t-test. Values are reported as mean ± standard error. 3.

Results and Discussion

The EAC cells used in our study are a well characterized biochemically and morphologicaly tumor model and is commonly applied in toxicological studies. The ascite form of EAC can be used for in vivo experiments and can be easy transferred into culture for in vitro studies. The EAC cells were treated with CNPs for 24 h in in vivo and in vitro experiments, after which the bioenergetics status, phospholipids profile, DNA damage, XOR activity, LPO level, and MLD formation were simultaneously quantified. 3.1.

CHARACTERISATION OF BIOENERGETIC STATUS AND PHOSPHOLIPIDS PROFILE OF EAC CELLS TREATED BY CNP

3.1.1.

Energy Metabolism of EAC Cells

The bioenergetic status of cells was characterized by 1H and 31P NMR spectroscopy. The quantity of individual phosphor-containing metabolites were determined: Glucose 6-phosphate (G6-P), Phosphoethanolamine (PE), Phosphocholine (PC), Inorganic Phosphate (Pi), Glycerophosphocholine (GPC), Glycerophosphoethanolamine (GPE), Phosphomonoesters (PME represented by PE + PC), Phosphodiesters (PDE represented by GPC + GPE), Nucleoside triphosphate (NTP), Nucleoside diphosphate (NDP), Choline (Cho), Creatine (Cr), and Phosphocreatine (PCr), as shown in Figure 1. Cells exposure to fullerene C60 caused a 2.6-fold decrease in the Pi/PME ratio and 1.4-fold decrease in the PME/PDE ratio. Thus, fullerene C60 activated energetic metabolism, leading to a decline in membrane component synthesis and reduced hypoxia level (Figure 2B). Exposure to fullerene C60 caused a decrease in lactate (1.2-fold) and taurine (1.3-fold) contents, as well as an increase in Cho + PC + GPC (twofold) and Cr + PCr (4.53-fold) contents. Such changes of lactate content indicated a decline in anaerobic glycolysis (data not shown). Treatment with low concentration of MWCNT caused 1.2-fold increase of the Pi/β-NTP and PME/β-NTP ratios, that reflected inhibition of energetic metabolism and intensification of hypoxia. High doses of MWCNT caused a 1.5-fold rise in the PME/β-NTP ratio which indicates an intensification of hypoxia, however a 1.6-fold decline in the Pi/PME ratio indicates activation of energetic metabolism.


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5

10

9 3

1

13

8

4

14 11 12

16 15

−2

PPM

PPM

2

8.0

−8.0

7.0

PPM

6

20

18

16

−9.0

PPM

PPM

7

14

12

10

8

6

4

2

−2

0

−4

−6

−8

−18.0

−18.5

−10 −12 −14 −16 −18

31

Figure 1. Typical P NMR spectra of water-soluble metabolites obtained by dual extraction of EAC cells. 1 – MDP standard, 2 – G6-P, 3 – PE, 4 – PC, 5 – Pi, 6 – GPE, 7 – GPC, 8 – PCr, 9 – γNTP, 10 – βNDP, 11 – αNDP, 12 – αNTP, 13 – NADP(H), 14 – UDP, 15 – DPDE, 16 – βNTP. B

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Figure 2. Energy metabolism level in EAC cells treated in vivo with CNP. (A) Effect of MWCNT on Pi/β-NTP, PME/β-NTP, Pi/PME and PME/PDE ratios in EAC cells – ■ – control cells; – MWCNTtreated cells (0.5 mg/mouse); □ – MWCNT-treated cells (1.5 mg/mouse). (B) Effect of fullerene C60 (0.066 mg/ml) on Pi/PME and PME/PDE ratios – ■ – control cells; – fullerene C60 treated cells. 1 – Pi/β-NTP, 2 – PME/β-NTP, 3 – Pi/PME, 4 – PME/PDE.

The PME/PDE ratio increased 1.6 and 2.9 times under the influence of low and high doses of MWCNT, respectively. Increase in the PME/PDE ratio pointed to the activation of membrane components synthesis (Figure 2A). Low and high concentrations of MWCNT caused a modest decline of taurine content. Exposure to low doses of MWCNT caused a 1.3-fold decrease of Cho + PC + GPC content and a 1.2-fold increase of lactate content. However, high concentration of MWCNT caused a 1.5-fold decline of lactate content (data not shown).


3.1.2.

Phospholipids Profile of EAC Cells

Phospholipids of EAC cells were characterized by the contents of phosphatidylcholine (PtdCho), plasmalogen phosphatidylcholine (PlPtdCho), phosphatidylinositol (PtdIns), sphingomyelin (SpM), phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), plasmalogen phosphatidylethanolamine (PlPtdEtn) and cardiolipin (Card) by 31Р-NMR spectroscopy (Figure 3).

Figure 3. Typical 31P NMR spectra of phospholipids obtained by dual extraction of EAC cells. 1 – Card, 2 – PlPtdEtn, 3 – PtdEtn, 4 – PtdSer, 5 – SpM, 6 – PtdIns, 7 – PlPtdCho, 8 – PtdCho.

Exposure to fullerene C60 decreased PtdCho (1.2-fold), PlPtdCho (1.4-fold), PtdSer (1.6-fold), PtdEtn (1.3-fold), and PlPtdEtn (1.5-fold). Treatment with fullerene C60 was followed by a 1.4-fold decrease in the PtdCho/SpM ratio (Figure 4A). Low concentration of MWCNT caused decreases in PtdCho (1.2-fold), PlPtdCho (twofold), SpM (1.6-fold), PtdSer (1.4-fold), and PtdEtn (1.4-fold) contents. The PtdCho/SpM ratio increased 1.3 times under the influence of low doses of MWCNT. High concentration of MWCNT caused an increase in SpM (1.2-fold), PtdSer (1.4-fold), PlPtdEtn (1.3-fold), and Card (1.5-fold). Treatment with high doses of MWCNT caused small decrease of the PtdCho/SpM ratio (Figure 4B). Thus, fullerene C60 caused decrease of almost all phospholipids content and increase of plasma membrane structuring as determined by the PtdCho/SpM ratio. However, low doses of MWCNT caused decrease of phospholipids content and increase of membrane permeability. On the contrary, high concentrations of MWCNT caused increase of phospholipids content and a small rise of plasma membrane structuring.


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Figure 4. Phospholipids level in EAC cells treated with CNP: (A) Effect of fullerene C60 (0.066 mg/ml) – ■, control cells; , fullerene C60 treated cells. (B) Effect of MWCNT – ■ – control cells; – MWCNT treated cells (0.5 mg/mouse); □ – MWCNT treated cells (1.5 mg/mouse). 1 – PtdCho, 2 – PlPtdCho, 3 – PtdIns, 4 – SpM, 5 – PtdSer, 6 – PtdEtn, 7 – PlPtdEtn, 8 – Card.

3.2.

ASSESSMENT OF MLD BY PROTON NMR

The ratio of CH2/CH3 signal intensity was moderately increased (1.2 times) in fullerene C60-treated EAC cells, but the choline resonance signal (at 3.2 ppm) decreased twofold as compared with untreated EAC cells. This effect may be related to apoptosis-associated changes in fullerene C60 treated cells. Exposure to MWCNT was accompanied by small decreases in the CH2/CH3 ratio and choline resonance signal (Figure 5A). 25 15 5 -5 %

-15 -25 -35 -45 -55

Cho

CH2/CH3

Figure 5. Levels of MLD and Cho in EAC cells treated with CNP. (A) Typical 1H NMR spectra of EAC cells. Peak assignments: 1 – CH3 signal mainly from protein residues and lipids at 0.9 ppm; 2 – (-CH2)n signal from mobile lipids resonate at 1.3 ppm, 3 – creatine at 3.03 ppm and 4 – choline-based metabolite signal at 3.23 ppm. (B) The ratio of treated to untreated cells for Cho and CH2/CH3, % ■, fullerene C60 treated cells (0.066 mg/ml); , MWCNT treated cells (1.5 mg/mouse). 1

H NMR analysis revealed an increase in MLD formation in fullerene C60treated cultured cells in contrast with MWCNT effect after administration into peritoneal cavity. Neither CNP caused any significant cytotoxicity in the range of concentrations used, as evidenced by trypan blue exclusion test.


3.3.

XOR ACTIVITY AND LPO LEVEL

The activity of XOR was studied in EAC cells. Formation of uric acid in samples did not depend on the presence of NAD+ in incubation mixture. These data indicate that nearly all XOR in cells was present in the oxidase form, and dehydrogenase form was absent [32]. Presence of fullerene C60 in cultural medium resulted in a moderate increase of enzyme activity (21.5%) and more distinct decrease of LPO intensity (49.2%). Treatment with MWCNT also increased XO activity in EAC cells (Figure 6). The maximum effect was observed at the middle concentration of MWCNT in cultural medium (0.035 mg/ml), resulting in a 91.8% increase in XO activity. At MWCNT concentrations 0.017 and 0.07 mg/ml XO activity was raised on 39.2% and 45.3%, respectively. When MWCNT were administered into peritoneal cavity in concentration of 1.5 mg/animal, the XO activation was lower (23.5%), and LPO level in EAC was decreased to 86.5%. 200 150

%

100 50 0 -50 -100

1

2

3

4

5

Figure 6. XO activity and LPO intensity in EAC cells treated with CNP (the ratio of XO activity in CNP treated to untreated cells, %). Experimental groups: 1 – fullerene C60 treated cells (0.066 mg/ml); 2 – MWCNT treated cells (1.5 mg/mouse); 3 – MWCNT treated cells (0.017 mg/ml); 4 – MWCNT treated cells (0.035 mg/ml); 3 – MWCNT treated cells (0.07 mg/ml). ■ – XOR activity; – LPO intensity.

Thus, effects of fullerene C60 and MWCNT on XOR activity and LPO intensity of the EAC had unidirectional character. The activity of XOR was raised and level of the LPO was decreased. The alteration of XOR activity depended on MWCNT concentrations. Effects of MWCNT on XOR activity of EAC was more pronounced in cell culture than in the peritoneal cavity of mice. Lower effect of MWCNT in peritoneal cavity is probably related to adhesion of a considerable amount of MWCNT on organs of experimental animals. The decrease in LPO was unexpected and needs further investigation since the majority of publications observe the opposite effect. Taking into account that such an effect was observed in parallel


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with the activation of XO, which is capable of generating superoxide radicals, hydrogen peroxide, NO. and peroxinitrite [7, 8, 10], the decrease in LPO was probably caused by elimination of free radical compounds due to binding to CNP surface [33]. 3.4.

DNA DAMAGE

The ability of CNP to induce the formation of DNA strand breaks was assessed using the comet assay and the obtained results were compared to untreated EAC cells (Figure 7). Treatment of EAC cells during 24 h with fullerene C60 (0.066 mg/ml) induced comet area threefold, the tail length 2.3-fold, and the tail moment and Olive tail moment twofold.

A B

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Figure 7. DNA damage in EAC cells treated with CNP by comet assay. (A) Comet area and (B) tail length. 1 – control cultured cells; 2 – fullerene C60 treated cells (0.066 mg/ml); 3 – MWCNT treated cells (0.07 mg/ml); 4 – MWCNT treated cells (0.035 mg/ml); 5 – MWCNT treated cells (0.017 mg/ml); 6 – control ascitic cells; 7 – MWCNT treated cells (0.5 mg/mouse); 8 – MWCNT treated cells (1.5 mg/mouse).

The effect of MWCNT on DNA damage was inversely related to doses used for cells treatment. Treatment of EAC cells with MWCNT (0.07 mg/ml) induced the moderate increase of DNA damage (1.2–1.3 times) compared to untreated cells. At the time of analysis most of cells (98%) were not stained with Trypan blue. Treatment cells with MWCNT at 0.035 mg/ml was followed by a moderate rise in comet area, tail length, Olive tail moment increase of 1.5-fold, and tail moment increase of twofold. The number of cells with comets was increased 17%. Treatment cells with MWCNT in concentration of 0.017 mg/ml caused the largest effect on DNA damage. The comet area was increased threefold, the tail length and tail moment twofold, and the Olive tail moment 1.6-fold. The number of cells with comets rose 38%. The DNA damage was also assayed when EAC cells were treated with MWCNT in vivo. Low concentration of MWCNT (0.5 mg/mouse) induced the increase of the comet area in 1.5 times and the tail length and tail moment 1.3fold. The Olive tail moment did not change significantly. The higher MWCNT dose (1.5 mg/mouse) induced an increase in the comet area of 1.6-fold, the tail length 1.3-fold, the tail moment threefold, and the Olive tail moment twofold. The


number of cells with comets increased 4–10% in 0.5 ng MWCNT/mouse and 1.5 mg MWCNT/mouse, respectively. 4.

Conclusion

Insufficient knowledge of NP mechanisms of action and large variety of interactions with biological molecules require new approaches to estimate nanoparticle impacts on organismal and cellular levels. The complex estimation of toxic and carcinogenic effects of NP on cells was carried out to address their involvement in free radical processes, energy metabolism, membrane structural changes and genotoxic damage in EAC cells exposed to CNP. The use of EAC model provided an opportunity to study CNP impact in vitro on cultured cells as well as in vivo when NP were administered i.p. to tumor-bearing mice. Our data suggest that MWCNT and fullerenes may pose genotoxic effect, change energy metabolism and membrane structure, alter free radical level via XO activation, and cause MLD formation, as determined in the in vivo EAC model and in vitro cell culture. Exposures of cells to CNP (fullerene C 60 and MWCNT) induced DNA damages both on in vivo and in vitro systems. MWCNT-induced DNA damage was inversely related to doses used for cells treatment. Cells exposed to MWCNT in vitro exhibited a greater degree of DNA damage than cells exposed in vivo. XOR enzymatic activity did not depend on the presence of NAD+ in incubation mixture, this indicates that nearly all XOR in cells was present in the oxidase form and dehydrogenase form was absent. The effect of fullerene C60 and MWCNT on XOR activity and LPO intensity of the EAC had a unidirectional character. The activity of XOR was raised and level of the LPO was decreased. The effects of MWCNT on XOR activity in EAC was more pronounced in cell culture than in peritoneal cavity of mice. This may be due to transperitoneal absorption of a considerable part of substance or its adhesion to organs. Decrease of LPO levels were unexpected, possibly caused by the elimination of free radical compounds due to binding to CNP surface. Treatment with MWCNT caused intensification of hypoxia and activation of membrane component synthesis, yet fullerene C60 caused the opposite effect. Fullerene C60 and high doses of MWCNT revealed activation of energy metabolism and a reduction of membrane permeability, however low doses of MWCNT caused opposite changes. Phospholipid metabolism decreased after treatment with fullerene C60 or low concentration of MWCNT but conversely exposure to high doses of MWCNT caused elevation of phospholipids content. The obtained results demonstrate a possible link between cells exposed to CNP and corresponding changes of the proposed markers. The use of a wide variety of indicators allowed us to acquire information about major mechanisms of NP damaging effect on organism. Thus, the developed system of biomarkers can be suggested as a sensitive and efficient approach for assessment of toxic and carcinogenic CNP impact on organism.

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NANOCONTAMINATION OF THE SOLDIERS IN A BATTLE SPACE

A.M. GATTI Laboratory of Biomaterials University of Modena and ReggioEmilia Via Campi 213 A 41100 Modena, Italy [email protected] S. MONTANARI Nanodiagnostics srl Modena, Italy

Abstract. The paper deals with the unusual pathologies some soldiers contracted after exposure in battle theatres in Iraq and in the Balkans, and considers the hypotheses the Authors developed to explain the origin of those diseases, that proved to be lethal in a few cases. The scenario of particulate nanopollution generated by high-temperature combustions characteristic of some weapons is described. The electron-microscopy observations carried out in 37 soldiers’ pathological tissues verified the internal dissemination of toxic metallic micro and nano-particles. The article considers the way of entrance of those nanopollutants: the lung for inhalation and the digestive system for the ingestion of polluted food. Battle theatre pollution is also discussed. 1.

Introduction

The actual number has never been published and probably is not known, but what is unquestionable is that veterans from the first Gulf War, and most of them are American and British, have come home ill and some of them died. In a few instances, their symptoms, seemingly unhomogeneous and never experienced together before, were not recognized as belonging to a definite pathology. For that reason, they were either ignored or underestimated or, in the best of circumstances, classed as the expressions of something new christened “Gulf Syndrome” [1–13]. Something similar and sometimes even absolutely superimposable occurred after the war fought in the Balkans. When that war was declared concluded, Italian troops were sent to former Yugoslavia as peacekeepers and returned ill or, more often, grew ill after having been repatriated.


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A.M. GATTI AND S. MONTANARI

As far as we know, that pathology or, to be more accurate, that collection of pathologies, looked to be shared by French, Hungarian, Danish, Spanish, Portuguese, Belgian, Dutch and Greek soldiers [14] and, being a collection of symptoms and pathologies, was called a syndrome: the “Balkans Syndrome”. The symptoms observed can evolve into more serious diseases like, for example, various forms of cancer, Hodgkin’s and non-Hodgkin’s lymphoma or leukaemia. Also pathologies involving the blood have been diagnosed, pathologies that did not look particularly important but that, after a certain lapse of time, could result in a myeloma. All those post-war illnesses started to appear in 1991, to show up again after the second Gulf War among the American veterans, but also soldiers engaged in another war theatre, Afghanistan, reported similar conditions. As a rule, those soldiers leave in perfect health (such condition is testified by medical report written before the mission begins and, in any case, a soldier on active service must be in good health condition), but after a comparatively short time of stay they may start to show symptoms, sometimes trivial, but growing more and more serious and even fatal. For information completeness’ sake it is necessary to mention how some American and British soldiers who fell ill after the first Gulf War showed also neurological symptoms, something not observed or, in any case, not reported in Italian Balkans veterans. This piece of evidence is particularly important as a clue, because it means that, though the activities undertaken were unquestionably similar, there was something that made them somehow different. According to the Nuclear Regulatory Commission, Depleted Uranium (DU) may not contain more than 0.711% of U235 and the one used to make DU ammunitions contains less than 0.2%. Its radioactivity is so low that it is only reasonable to rule it out as directly accountable for the pathologies observed. As a matter of fact, people employed to work that metal where DU weapons are manufactured do not show any of the symptoms reported by the veterans nor any other particular pathology attributable to radioactivity. Nevertheless, it is impossible to exclude and, rather, it is very reasonable to say that if radioactive particles are ingested or inhaled, they find themselves in a particularly restricted biological environment where they can easily induce adverse reactions. If we look at the reports issued by the UNEP in 2003 about “DU in Bosnia ed Erzegovina: Post Conflict Environmental Assessment “ by the United Nations Environment Programme, Switzerland 2003 (www.unep.org), we read that places exist where a residual radioactivity still persists (see map in the report [15]), particularly when unexploded darts remain stuck or buried in the ground, but nobody has ever checked and documented if Italian soldiers were stationed there or had ever had a chance to come in contact with them. In order to identify the causal agent of the pathologies we are dealing with, it is imperative to locate place and possibility of exposure. Once determined those two factors, it is necessary to verify if the hypothesis may fit to the same pathologies in other cohorts of subjects like soldiers operating in firing grounds, people living in proximity to those posts and, in particular, civilians and NGO effectives present


85

in war theatres. It is only by evaluating the whole situation that we can single a causal agent out shared by all those classes of subjects. DU has been chosen because of a few favourable characteristics, among which its hardness, high specific weight, high melting point, excellent armour-piercing penetration and pyrophoricity. When the projectile is launched, its pointed penetrator can pierce relatively thick armour plates or virtually any other mark, and the explosion ensuing has part of the material involved vaporize, as the temperature induced is in the range 3,036–3,063°C [16]. After sublimation, everything is present in the volume involved gives origin to an aerosol and than to condensation dust that, because of the very high temperature, is often of nanometric size. The chemical composition of those particles is the result of the fortuitous combination of the elements present in the occasional crucible represented by the target and, on a smaller scale, by the bomb itself. The main factor conditioning the size of that particulate matter (sometimes within the order of magnitude of the tens of nanometres) is temperature and, as a general rule, the higher it is, the smaller the particles are. As a consequence, the particles generated close to the core of the explosion will be smaller than those formed in a more peripheral area. Similar results occur when a great quantity of conventional ammunitions is used, an event common when weapons must be disposed of and that is done by setting them off, or when an explosion in an arsenal occurs out of control. Such an event has been reported, for example, in a site close to Baghdad [17]. The ultramicroscopic analyses showed the presence of micro- and nanoparticles with unusual chemical compositions, in all cases metallic. Among other compositions, we found alloys of Lead and Tin, Zinc–Iron–Titanium, Lead–Bismuth and Bismuth alone, Tin–Silver, Iron–Copper–Zinc, Titanium–Iron, Silicon–Zirconium, Strontium–Sulphur, Cadmium–Silicon and also Uranium–Thorium. All these compounds are toxic due to at least one of their components and, because of their morphology and dimension, they show a physical aggressiveness towards the organism. The formation of a brand new pollution, never experimented before, with a chemical composition that at times is certainly toxic as is composed by non biodegradable, non biocompatible heavy metals represents a novel stimulus to which the human body is not prepared to react in a positive way nor is likely to be capable of adapting [18]. Our organism needs Oxygen to live, along with a variety of nutrients, and without Oxygen our cells can survive only for a very short time. Particularly in modern warfare, because of high-temperature weapons, a novel, particulate pollution is created that permeates man, animals and the whole environment, and that form of pollution can be inhaled with the air and ingested with the vegetables grown under the inevitable fallout that ensues. The school of Leuven (Belgium) [19] demonstrated that inhaling 100-nm particles is risky for our health, since dust that size negotiates the alveolar barrier within 60 s reaching the blood stream and, within an hour, the liver and all other tissues and organs. As has been observed by our group, those particles are trapped in any tissues acting like any mechanical

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filter or can penetrate cell nuclei where can induce adverse effects both as foreign bodies and as being composed of toxic elements. The evidence we found consistently in the pathological-tissue specimens of the more than 100 cases of ill soldiers studied is the most irrefutable demonstration of this theory [20]. Other Authors chose to keep looking for a Uranium contamination in the soldiers’ urine [21]. To be sure, the measurements they carry out can verify a possible contamination from Uranium radioactivity, but do not take into consideration the unavoidable lack of information about the quantity of radiations each patient absorbed before a presumed exposure in a war theatre (zero reference). For that reason, the value found is at least partially independent of the Uranium that may have entered that organism. In addition to that, that value depends on the capability the subject’s kidneys have to get rid of Uranium as an ion resulting from the solution of materials present in the body, nor can we know whether those materials are of natural or anthropic origin, and, in the latter case, if they come from the use of weapons. And that hypothesis does not offer any explanation about how subjects showing similar excretion values come to suffer from different pathologies and, in particular, offers no explanation about the neurological diseases reported by American and British soldiers (no systematic observations exist for other nationalities). Symptomatology caused by radiations is very well-known and is amply described in medical literature dealing with Japanese subjects exposed to A bomb radiations in August 1945. The symptoms reported there do not coincide with those found in the veterans from the Balkans and the Gulf and, therefore, the hypothesis that those syndromes may be caused by radioactivity looks hard to accept. It is a fact that, if at the beginning the symptoms observed were hardly attributable to a single disease, as the different pathologies develop, the soldiers died for cancerous diseases of different districts of the body; but cancer is very frequent among the population (the incidence now is that it affects 1 subject out of 3) who was never exposed to Uranium radiations. It is well known that chemicals, but radiations as well, can cause cancer, but it sounds strange that in a battle theatre that group if pathologies is triggered only by radiation. These considerations should lead to search for a cause compatible with the objective data and the events occurred, and equally shared by soldiers, civilians and animals. The analyses completed in our laboratory on the bioptical and autoptical samples taken from American, French and Italian soldiers, those on the same kind of samples from soldiers and civilians active in firing grounds along with the environmental analyses carried out in war theatres and in firing grounds induce us to sketch out a different scenario and another possible causal agent, i.e. the submicronic pollution created by weapon and target together. As briefly described above, a temperature like the one brought about by DU explosions generates extremely fine inorganic particles that can be inhaled and ingested by men and animals alike. One of the peculiarities of such anthropic


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pollution is its small size, and to that size they owe their capability to penetrate so easily virtually any organ and tissue, none excluded, from the lymph nodes to the brain to the gonads. Our analyses on about 1,000 cases involving soldiers, civilians and workers busy in polluted sites is evidence of the presence of such particulate matter and its dissemination inside the organism. In some circumstances, the assessment of their morphology and chemical composition identifies unambiguously those particles as coming from random and very particular combustions like a high-temperature explosion. It is a matter of fact that those particles are neither biodegradable nor biocompatible and can interact in a noxious way with the organism. For a long time medical literature has described pathologies due to small foreign bodies: silicosis, the lung disease caused by inhalation of silica microparticles; asbestosis and mesothelioma from the exposure to asbestos dust; foreignbody granulomatosis of various tissues. On the other hand, toxicology relevant to the exposure to nanoparticles is a fairly new subject and is still a matter of tentative approach in all technologically advanced countries. Proof of that are the several American and European projects in the field of nanotoxicology in progress at the moment. From 2002 to 2005, one of the Authors of this text (Dr. Gatti) was the coordinator of a European project called Nanopathology (www.nanopathology.it) at is now the coordination of a second project called DIPNA (Development of an integrated platform for the nanoparticle risk assessment). Yet, a number of studies exist about the easiness with which nanoparticulate enters the organism and is disseminated once inhaled or ingested. Their entry in the brain may even be possible through the olfactory nerve as described by Öberdörster [22, 23]. As soon as they are in the brain, they can represent an irritative factor because of their characteristic of acting as electrical conductors and/or, occasionally, because of their magnetic properties. The whole of all those anomalous activities and their non biodegradability can be the cause of local toxicity. One of the characteristics of this kind of particulate is its capability of moving from pregnant mother to foetus. We did not have the chance to examine tissues taken from miscarried, malformed foeti, the offspring of veterans, but checked those from stillborn, malformed lambs whose mothers grazed in meadows occupied by firing grounds. Pregnant sheep fed on grass polluted by the dust created by explosions, and that dust, delivered to the embryo, was then found in the dead lamb. It is evident that particulate matter can be compatible with the development of an embryo, but that development is abnormal and in most cases incompatible with life outside the mother’s womb. Similar cases did we find in human malformed foeti, but, in those circumstances, the cause was attributable to industrial or urban pollution. A few other hypotheses have been put forward to explain the so-called Balkans and Gulf syndromes. One of them is the use of multiple, certainly too close in time, vaccinations. That could be taken into account, but only if in those cases where a temporal consequence can be demonstrated, i.e. when, immediately after the vaccines have been administered, the subject shows an ailment that grows worse.

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It is a well-known fact that so-called adjuvants (http://www.emea.europa.eu/pdfs/ human/vwp/13471604en.pdf) are added to vaccines in order to improve the immune response so a lesser quantity of drug is needed and enhance the organism reaction because of their pyrogenicity. Adjuvants can be inorganic matter that can be made up by heavy metals. Mercury was widely used before it was banned because of its obvious toxicity. As shown by our studies, inorganic, non biodegradable and non biocompatible particulate like that used in vaccines cannot been disposed of by the organism and the consequences of its introduction into the body are the ones mentioned above. As briefly reported, it is not just soldiers who suffer from the Balkans and Gulf syndromes or from similar collections of symptoms and diseases, but it is also soldiers active in firing grounds or civilians living in war theatres. Especially civilians were never subject to multiple vaccinations nor took cocktails of drugs the way soldiers, in some circumstances, do, and, therefore, it is really hard to blame the vaccines when only part of the patients were exposed to them. Nevertheless, ruling out the possibility that using vaccines and drugs in a way that is so concentrated and outside medical experience may be an aggravation and make the onset of the disease easier, does not look correct and the hypothesis deserves further investigation. Hypotheses like the one linked to the use of sprays against bacteriological war shows the same weak points as the theory above. In conclusion, the analysis on pathological tissues aimed at detecting particulate matter looks the most meaningful test to assess the exposure the subject underwent. 2.

The Contamination

The soldiers’ pathological tissues we analyzed showed the presence of micro- and, more commonly, nanoparticles. The chemistry we came across was sometimes unusual: Mercury–Selenium, Antimony–Cobalt, Zirconia. It was somewhat surprising to find inside soldiers’ tissues particles we thought to be confined in nanotechnological laboratories. Zirconia, for instance, as we found in a soldier’s spleen (see Figure 1), has a melting point of about 2,400°C and the generation of nanoparticles of that material, outside a nanotechnological laboratory, implies temperatures peculiar to special combustions. During the blast of high-technology weapons or of an accumulation of ammunition, a very high temperature is created that can cause the formation of aerosolized material that are disseminated in all the solid angle around the explosion site. As a consequence of the blast power and the meteorological conditions (presence of wind, rain, etc.) this fresh pollution can be disseminated to a distance of many kilometres from its origin. A different stratification in the space of the micro and nanoparticles is possible and logic, but no scientific data are available in a battle theatre.


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Figure 1. Zirconia micro and nanoparticles embedded in a spleen tissue in a patient affected by nonHodgkin lymphoma. The Energy Dispersive spectroscopy identifies the particles composed of Carbon, Zirconium, Oxygen, Chlorine, Iron.

Immediately after the explosions a fresh contamination of the environment occurs that can involve humans and animals for the pollution of air and soil. Grass can act as a repository for the fall-out of this pollution and since it is a food for animals, it can pollute them. That way, animals ingest biodegradable grass containing not-biodegradable and non-biocompatible particles. The analyses we carried out on malformed lambs born inside a firing range confirm the hypothesis of a pollution in the mother and its translocation through the fetal circulation to the embryo. Also the observations on cigarettes and tobacco leaves from Sarajevo immediately after the bombing confirm the existence of a characteristics war pollution on the flora. (The tobacco industry was the only manufacturing plant that still operated during the siege and bombing of Sarajevo because, unlike other industries, it did not need electricity that was very scarce.) Figure 2 shows the so-called war contamination of a tobacco leaf where a particle containing also uranium and thorium is visible. As a conclusion, it is indispensable that the new wars take into account the micro and nanopollution generated by the explosions [24].

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Figure 2. Tobacco leaf surface with particles of environmental dust. The whiter debris is a compound of Phosphorus, Oxygen Carbon, Cerium, Lanthanium, Neodymium, Silicon, Aluminum, Magnesium, Chlorine, Potassium, Thorium, Uranium and Iron.

References 1. Iversen A, Chalder T, Wessely S. Gulf War illness: lessons from medically unexplained symptoms. Clin Psychol Rev. 2007 Oct; 27(7):842–854. Epub 2007 Jul 17. Review. 2. McDiarmid MA, Engelhardt SM, Oliver M, Gucer P, Wilson PD, Kane R, Cernich A, Kaup B, Anderson L, Hoover D, Brown L, Albertini R, Gudi R, Jacobson-Kram D, Squibb KS. Health surveillance of Gulf War I veterans exposed to depleted uranium: updating the cohort. Health Phys. 2007 July; 93(1):60–73. 3. Horner RD, Grambow SC, Coffman CJ, Lindquist JH, Oddone EZ, Allen KD, Kasarskis EJ. Amyotrophic Lateral Sclerosis among 1991 Gulf War Veterans: evidence for a time-limited outbreak. Neuroepidemiology. 2008 June 6; 31(1):28–32.


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4. Verret C, Jutand MA, De Vigan C, Bégassat M, Bensefa-Colas L, Brochard P, Salamon R. Reproductive health and pregnancy outcomes among French gulf war veterans. BMC Public Health. 2008 Apr 28; 8:141. 5. Nelson C. Veterans’ mysterious maladies: studies continue to examine the effects of depleted uranium on returning soldiers. State Legis. 2008 May; 34(5):28–29. 6. Hokama Y, Empey-Campora C, Hara C, Higa N, Siu N, Lau R, Kuribayashi T, Yabusaki K. Acute phase phospholipids related to the cardiolipin of mitochondria in the sera of patients with chronic fatigue syndrome (CFS), chronic Ciguatera fish poisoning (CCFP), and other diseases attributed to chemicals, Gulf War, and marine toxins. J Clin Lab Anal. 2008 22(2):99–105. 7. Golomb BA. Acetylcholinesterase inhibitors and Gulf War illnesses. Proc Natl Acad Sci U S A. 2008 Mar 18; 105(11):4295–300. Epub 2008 Mar 10. 8. Hooper TI, Debakey SF, Nagaraj BE, Bellis KS, Smith B, Smith TC, Gackstetter GD. The long-term hospitalization experience following military service in the 1991 Gulf War among veterans remaining on active duty, 1994-2004. BMC Public Health. 2008 Feb 13; 8:60. 9. Cazoulat A, Lecompte Y, Bohand S, Castagnet X, Laroche P. Urinary uranium analysis results on Gulf war or Balkans conflict veterans, Pathol Biol (Paris). 2008 Mar; 56(2):77–83. 10. Pols H, Oak S. War & military mental health: the US psychiatric response in the 20th century. Am J Public Health. 2007 Dec; 97(12):2132–2142. 11. Levine PH, Richardson PK, Zolfaghari L, Cleary SD, Geist CE, Potolicchio S, Young HA, Simmens SJ, Schessel D, Williams K, Mahan CM, Kang HK. A study of Gulf War veterans with a possible deployment-related syndrome. Arch Environ Occup Health. 2006 Nov–Dec; 61(6):271–278. 12. Barach P, Brautbar N, Richter ED, Friedman L. Latency: an important consideration in Gulf War syndrome. Neurotoxicology. 2007 Sep; 28(5):1043–4; author reply 1044– 1045. 13. Ismail K, Kent K, Sherwood R, Hull L, Seed P, David AS, Wessely S. Chronic fatigue syndrome and related disorders in UK veterans of the Gulf War 1990-1991: results from a two-phase cohort study. Psychol Med. 2008 July; 38(7):953–961. 14. Gatti, A, Montanari S. Approccio bioingegneristico alla sindrome dei Balcani, Fisica in Medicina, 2004, 2:107–114. 15. DU in Bosnia ed Erzegovina in Post Conflict Environmental Assessment, United Nations Environment Programme, Switzerland 2003 (www.unep.org). 16. Technical Report of the Air Force Armament Laboratory – Armament Development and Test Center, Eglin Air Force Base, FL, USA, From October 1977 to October 1978, Project no. 06CD0101 17. Report of Parliamentary Committee of Inquiry into cases of death and serious illness among Italian Military personnel engaged in International peace missions and into the storage conditions of Depleted uranium and its possible use in military exercise on national soil”, 2004, XIV LEGISLATURA, Doc. XXII-bis, no. 4. 18. Report of the “Parliamentary Committee of Inquiry on the cases of death and severe illnesses affecting Italian personnel assigned to military missions abroad, firing ranges and the sites where munitions are stocked, as well as civilian populations in war zones and in areas adjacent to military bases on the national territory, with special attention to the effects of depleted uranium shells and of the dispersion in the environment of nanoparticles of heavy minerals produced by the explosion of warfare material”, 11 October 2006, Doc. XXII-bis, no. 2. Available at: http://www.senato.it/documenti/ repository/commissioni/uranio15/final_report.pdf

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19. Nemmar A., Hoet P.H.M., Vanquickenborne B., Dinsdale D., Thomeer M., Hoylaerts M.F., Vanbilloen H., Mortelmans L., Nemery B. Passage of inhaled particles in to the blood circulation in humans, Circulation. 2002; 105(4):411–441. 20. Gatti A, Montanari S. Nanopathology: The health impact of nanoparticles – Ed. By Pan Stanford - Singapore 2008 (www.worldscibooks.com /nanosci/v001.html). 21. Durakovic A, Horan, D, The quantitative analysis of depleted Uranium isotopes in British, Canadian and US Gulf war Veteran, Mil Med. 2002; 167(8):620. 22. Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W, Cox C., Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol. 2004 June; 16(6–7):437–445. 23. Elder A, Gelein R, Silva V, Feikert T, Opanashuk L, Carter J, Potter R, Maynard A, Ito Y, Finkelstein J, Oberdörster G. Translocation of inhaled ultrafine manganese oxide particles to the central nervous system. Environ Health Perspect. 2006 Aug; 114(8):1172–1178. Erratum in: Environ Health Perspect. 2006 Aug; 114(8):1178. 24. Gatti A, Montanari S, Nanopollution: the invisible fog of future wars, The futurist. 2008 May–June 32–34.

SMARTEN Strategic Management and Assessment of Risks and Toxicity of Engineered Nanomaterials

C. METCALFE Environmental and Resource Studies Trent University Peterborough, Ontario, Canada [email protected] E. BENNETT Intertox, Inc. Salem, Massachusetts, USA M. CHAPPELL, J. STEEVENS Environmental Laboratory U.S. Army Corps of Engineers Vicksburg, Mississippi, USA M. DEPLEDGE Peninsula Medical School Plymouth, UK G. GOSS Department of Biology University of Alberta Edmonton, Alberta, Canada S. GOUDEY HydroQual Laboratories Golder Associates Ltd. Calgary, Alberta, Canada S. KACZMAR O’Brien and Gere Engineers Inc. Syracuse, New York, USA N. O’BRIEN School of Agriculture, Food Science and Veterinary Medicine College of Life Sciences University College Dublin Dublin, Ireland A. PICADO Instituto Nacional de Engenharia Tecnologia e Inovação Lisbon, Portugal


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A.B. RAMADAN National Egyptian Environmental and Radiation Monitoring Network Cairo, Egypt

Abstract. Traditional risk assessment procedures are inadequate for predicting the ecological risks associated with the release of nanomaterials (NM) into the environment. The root of the problem lies in an inadequate application of solid phase chemical principles (e.g. particle size, shape, functionality) for the risk assessment of NMs. Thus, the “solubility” paradigm used to evaluate the risks associated with other classes of contaminants must be replaced by a “dispersivity” paradigm for evaluating the risks associated with NM. The pace of development of NM will exceed the capacity to conduct adequate risk assessments using current methods and approaches. Each NM product will be available in a variety of size classes and with different surface functionalizations; probably requiring multiple risk assessments for each NM. The “SMARTEN” approach to risk assessment involves having risk assessors play a more proactive role in evaluating all aspects of the NM life cycle and in making decisions to develop lower risk NM products. Improved problem formulation could come from considering the chemical, physical and biological properties of NMs. New effects assessment techniques are needed to evaluate cellular binding and uptake potential, such as biological assays for binding to macromolecules or organelles, phagocytic activity, and active/passive uptake processes. Tests should be developed to evaluate biological effects with multiple species across a range of trophic levels. Despite our best efforts to assess the risks associated with NM, previous experience indicates that some NM products will enter the environment and cause biological effects. Therefore, risk assessors should support programs for reconnaissance and surveillance to detect the impacts of NM before irreversible damage occurs. New analytical tools are needed for surveillance, including sensors for detecting NMs, passive sampling systems, and improved methods for separation and characterization of NMs in environmental matrices, as well as biomarker techniques to evaluate exposure to NMs. Risk assessors should use this information to refine data quality, determine future risk assessment objectives and to communicate interim conclusions to a wide group of stakeholders.1 1.

Introduction

Engineered nanomaterials (NM) are generally regarded as man-made materials with at least one dimension below 100 nm [6]. Nanoparticles can occur naturally (e.g. ash, colloids, large biomolecules), or can be produced unintentionally (e.g. diesel exhaust), but concern over the potential adverse environmental impacts of 1

Summary of the NATO ARW Working Group discussions.

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nanoparticles has been directed at engineered NMs. Engineered NM can be divided into four different classes: carbon-based materials (e.g., fullerenes), metalbased materials (e.g., gold or titanium dioxide nanoparticles), dendrimers (e.g., nano-sized polymers), and composites (i.e., mixtures of nanoparticles). Nanoparticles typically have different physico-chemical properties compared to their respective bulk material, including different optical properties, thermal behaviour, material strength, solubility, conductivity and catalytic activity [8]. Probably the most significant change in the properties of nanoparticles is the increase in surface to volume ratio [4]. The proportion of atoms at the particle surface increases inversely with particle size, so that the surface properties of nanoparticles can dominate the properties of the bulk material [43]. These particles can also transfer energy to nearby oxygen molecules, which leads to the formation of reactive oxygen species (ROS). Exposure to oxyradicals can lead to cell damage and death [12]. Nanoparticles are similar in size to biological macromolecules such as proteins, DNA and phospholipids, so it is possible that NMs can cause disruptions at the molecular and cellular level. Many other physical and chemical factors can influence the toxicity of NMs, including surface reactivity, the dissolution ratio, and particle shape [42]. 2.

Ecotoxicology and Risk Assessment Techniques

Ecotoxicology is an integrative field that includes evaluations of the environmental fate and the biological effects of chemicals. Assessments of environmental impacts are based on a weight-of-evidence approach that combines environmental chemistry, acute and chronic toxicity testing with single species, evaluation of biomarkers of exposure and effect, and studies of ecosystem-level responses. Laboratory-based bioassays are typically performed using model species representing different feeding strategies and positions in food webs. Multiple species toxicity tests are of value to identify sensitive species and to study the variations in toxic effects across taxonomic groups. Elements of ecotoxicology are also included in methods for ecological risk assessment. In these procedures basic data gathering involves an “Exposure Assessment” and an “Effects Assessment”. In the Effects Assessment, efforts are made to determine the thresholds for toxicity in organisms, and in the Exposure Assessment, efforts are made to determine the concentrations to which organisms may be exposed in the environment. Risk Characterization involves comparing the Exposure Assessment and Effects Assessment data to give an indication of the “risk” of toxic effects occurring among organisms exposed to a chemical. In the context of exposures to NM, risk assessments must be conducted to try to evaluate the environmental hazards associated with new NM products that are to be introduced into the marketplace, or to assess the hazards associated with existing NM products that may already be present in the environment. For new NMs that have not yet been introduced into the marketplace, there are no data regarding the concentrations in the environment, and so the predicted environmental concentrations must be estimated. For NM that have the same elemental or chemical composition,

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but differ in size, shape or surface properties, is not clear whether separate risk assessments will be required for each individual product. Various jurisdictions have called for integrated risk assessment procedures for nanomaterials [6, 35]. In this review, we present a case for a fundamentally different approach to risk assessments for NM released into the environment. The “SMARTEN” approach requires that several elements of traditional risk assessments be abandoned or reformulated in order to address the unique characteristics of NM. 3.

The “Nano-effect” Paradigm

The “nano-effect” may be defined as unique or enhanced NM properties, reactions or biological interactions that occur below a specific particle size threshold. This term implies that such effects are not observed with larger particle sizes. Enhanced properties are associated with decreasing particle size, as a function of increased particle surface area. Incorporation of the principles of the nano-effect into traditional environmental risk assessment procedures requires a paradigm shift from the concepts that are applied to “conventional” environmental contaminants, such as pesticides and industrial chemicals. Table 1 summarizes the novel characteristics of nanoparticles that must be considered in an environmental risk assessment, relative to the parameters that are considered in risk assessments of other classes of contaminants. TABLE 1. Characteristics of NM that must be considered for environmental risk assessments, relative to the characteristics considered for “conventional” classes of contaminants. Characteristic Distribution in water Distribution in porous media Biological availability Cellular uptake Toxic mechanisms Target trophic systems

Nanoparticles Dispersivity Filtration

Other contaminants Solubility Adsorption/desorption

Sorption? Vesicular transport? Steric hindrance, photo-chemical effects, oxidative damage, inflammation Bottom of the food chain?

Lipophilicity Passive or facilitated diffusion Interactions with cellular macromolecules and receptors, narcosis Top of the food chain

3.1.

DISTRIBUTION IN THE ENVIRONMENT

3.1.1.

Interactions

NM may interact in the environment in the following ways: (a) Flocculation: Most NMs tend to readily flocculate. NM dispersions may be temporarily stabilized by severe agitation, such as sonication, but this has the potential to introduce artifacts onto the NM surface. NM dispersions are also stabilized by derivatizing the particle surface by introducing charged groups. However, experimental evidence shows that adding very dilute salt is sufficient for NMs to again readily flocculate. NM dispersions are readily stabilized in the presence of dissolved humic substances. This behavior

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appears linked to the surfactive character of dissolved humic substances, which minimizes NM particle size and poly-dispersivity [9]. Aggregation of carbon nanotubes was inhibited by the addition of humic and fulvic acids [24]. Dissolved organic material commonly occurs at concentrations in natural aqueous systems that are capable of stabilizing NM dispersions [34]. These type of changes may alter the behavior of NMs in water and soils. Nanoparticulate FeO coated with sodium dodecylsulphate was stable in a soil suspension for 14 days, without changes in the particle size distribution [18]. In solid-gas systems (water-limited), the small size of NM makes them readily aerosolized. For example, Murr and Garza [31] showed that so-called cleanburning technologies produce extremely small-sized combustion products (i.e., carbon nanotubes) that easily form aerosols, compared to products created with older technologies. (b) Dissolution: Most NMs are highly insoluble, yet some material may dissolve in the presence of organic chelators, resulting in the release of its metallic constituents into the environment. For example, there is some evidence that FeO nanoparticles are dissolved in the presence of acetate/lactate [33]. (c) Sorption: NMs may readily sorb other constituents in the aqueous phase. For example, Madden et al. [29] observed that smaller FeO particles (7 nm) undergo greater specific adsorption by Cu2+ ions than larger FeO particles (25 nm). NMs themselves may also be sorbed onto soil surfaces. In a sense, NM sorption in a soil is analogous to flocculation of individual NM particles in which NMs are simply “added” to the bulk environmental solids. (d) Transformation and degradations: Most inorganic NMs are used in oxidized forms that are stable under ambient conditions. On the other hand, organic NMs may be degraded when exposed to the environment. For example, fullerenes appear to be spontaneously but slowly oxidized in solution. Ozone has proved much more reactive, however than molecular oxygen to fullerenes [10], which may be a relevant transformation mechanism in advanced treatment systems for water and wastewater. CNTs are highly resistant to degradation (analogous to soil black carbon). However, manufacturers appear to introduce limited functionalization in even so-called non-derivatized CNTs in order to facilitate separation and purification during manufacturing. There is little information available concerning the microbial transformation of NMs. Redox reactions are often mediated by microorganisms; either directly through enzymatic activity, or indirectly through the formation of biogenic oxidants or reductants [28]. Biological modifications, as well as degradation of the surface functionalization may result in modified NM structures and freed constituents. 3.1.2.

Distribution

Perhaps the most important paradigm shift that must be understood for risk assessments of NM relates to the concept of “solubility” of chemicals and the “dispersivity” of nanoparticles in aqueous media. The capacity of nanoparticles to disperse in aqueous media will govern their environmental fate. NM dispersed

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within the aqueous phase are more mobile, and aggregation of NM reduces mobility [17, 24]. This concept is fundamentally different from the solubility paradigm that drives our predictions of the environmental fate and biological availability of other classes of contaminants. Properties such as water solubility and octanol/water partition coefficients (i.e. log Kow) are the basic parameters used to assess the risks associated with exposure to contaminants that are governed by the solubility paradigm. Similar key properties have not yet been identified for risk assessment of nanoparticles, but the characteristics that influence the “dispersivity” of nanoparticles in aqueous media include particle size, charge, speciation, crystallinity, surface area, and adsorbed phase composition. These properties are reviewed elsewhere in this book. The distribution of NM in porous media, such as soils and sediments is also governed by the size, shape and charge distribution of the particles. Filtration of nanoparticles through porous media is influenced by electrostatic interactions between the particles and soil/sediment. However, physical interactions that “sieve” the particles within the media are also an important factor [13]. Data from laboratory experiments indicate that NM may be relatively immobile in soils [41], or they may be relatively mobile [26], depending on the characteristics and size of the NM. 3.2.

BIOLOGICAL AVAILABILITY AND UPTAKE

For small organic molecules, lipophilic compounds are more biologically available than hydrophilic compounds, and uptake of lipophilic compounds occurs through passive diffusion across the cell membrane. For metal cations, uptake occurs as a result of facilitated diffusion of metal-protein complexes across cell membranes. The factors governing the biological availability and cellular transport of NM are less well understood. For fish, it has been suggested that the first step governing biological availability is trapping of NM in the mucous layers of the skin, gills and gut epithelium [20]. It is unlikely that NM are transported by passive or facilitated diffusion across cellular membranes. Indeed, Moore [30] suggests that vesicular transport (i.e. endocytosis, pinocytosis) may be the most important mechanism of NM transport into cells. If this is the case, then some invertebrates (e.g. bivalves) that have a high capacity for vesicular transport within gastrointestinal tissues may be especially susceptible to uptake of NM. Fish are capable of greater uptake by endocytosis in the gut than higher vertebrates. It is clear that some NM can be transported through tissues, including the blood-brain barrier [25]. It is possible that this type of transport occurs through para-cellular routes, such as transport across tight junctions. However, much remains to be learned about the mechanisms of uptake and transport of NM in organisms. 3.3.

MECHANISMS OF TOXICITY

Once NM enter the tissues of organisms or are transported across cell membranes, toxicity is likely to occur principally through one or a combination of four mechanisms (Figure 1). The first mechanism involves the release of the chemical

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constituents from the NM, which produces toxicity through more or less “conventional” processes, such as the release of toxic cadmium ions from CdTe nanoparticles [11, 44]. The other three mechanisms of NM toxicity are typically not observed for the classes of contaminants that are considered when using traditional risk assessment methods. Thus, a second mechanism of NM toxicity is related to the size and shape of the particle, which produces steric hindrances or interferences with macromolecules such as phospholipids, nucleic acids and proteins. For instance, the penetration and toxicity of CdTe quantum dots in vitro in nerve and glial cells was more pronounced with small (2.2 nm diameter) positively charged CdTe than large (5.2 nm diameter), equally charged CdTe. [27]. A third mechanism involves the surface properties of the NM, such as photochemical properties, local electric fields, charge densities, and electronic semi-conductance. These surface properties may result in the formation of oxygen radicals that can damage macromolecules [3, 36], but it is also possible that the surface reactivity of NM could directly disrupt cellular processes, such as energy production in mitochondria [28]. In some cases, it is not clear whether damage as a result of the presence of oxyradicals is due to the direct effects of the NM (i.e. mechanism 3), or due to the indirect effects of macrophage and granulocytes involved in an inflammatory response induced by the presence of the NM in tissues (i.e. mechanism 2). Duffin et al. [16] observed that the extent of lung inflammation depended not only on the particle surface area, but also on the surface reactivity in rats exposed to nanoparticles. The fourth mechanism of toxicity is related to the capacity for NM to act as vectors for the transport of other toxic chemicals to sensitive tissues. In a study with fish (i.e. carp), cadmium accumulation was increased 2.5-fold when TiO2 nanoparticles were added concurrently with cadmium salts [45]. 3.4.

VULNERABLE LOCI IN TROPHIC WEBS

Organisms occupying particular loci in trophic webs may be at increased risk of nanotoxicity. Toxicity tests have been performed with NMs using a variety of test organisms, ranging from bacteria to algae to benthic invertebrates and fish [7, 28]. In many cases, bacteria, plants and invertebrates were the most sensitive organisms to the biological effects of NM. Adams et al. [1] evaluated the toxicity of three photosensitive NMs (FeO, TiO 2, SiO2) to two bacterial species, Bacillus subtilis and Escherichia coli, and the cladoceran, Daphnia magna. The most sensitive species was the suspension feeding cladoceran. In a study of the toxicity of ultrafine TiO2, a green alga, Pseudokirchneriella subcapitata, was the most sensitive species in comparison to rainbow trout and D. magna [42]. In addition, deposit feeders or filter feeders are the most likely organisms to accumulate NM from water, soil and sediments. There is considerable evidence that a range of NM exhibit anti-bacterial activity [32].

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Release of NM constituents

Other contaminants

Physical effects of size and shape

Effects on membranes

Effects on gene expression

Effects on macromolecules

Effects on enzyme activity

Inflammatory Response

Surface reactivity

Vector for other contaminants Light

Figure 1. Mechanisms of toxicity of nanomaterials in organisms.

These results indicate that the biological effects of NMs may be observed first in organisms from lower trophic levels. In conventional risk assessments, more weight is placed on toxicity testing using fish species, and special emphasis is placed on chemicals that show potential for bioaccumulation and biomagnification through food chains. For risk assessments of NM, it is logical to assume that biological effects will be observed among mainly invertebrate species and microorganisms at the lower levels of the food web, or organisms that are important in geochemical and nutrient cycling. For instance, Tong et al. [40] observed that fullerenes impacted the composition of soil microbial communities. 4.

The Strategic Management and Assessment of Risks and Toxicity of Engineered Nanomaterials (SMARTEN)

Risk assessments for NM will require a shift in approach from the methods of exposure assessment and effects assessment that have been used previously for other classes of contaminants [21, 37]. As discussed above, conventional risk assessment procedures are hampered by adherence to paradigms that focus on the solubility and partitioning of chemicals, and fate and exposure pathways that may not be relevant for NM. Extensive use of lethality data for toxicity endpoints may also be inappropriate as our greatest concerns for NM center around sublethal effects, such as genotoxicity and inflammatory responses. The diverse properties of NM and the lack of clearly defined approaches are currently a major impediment to risk assessment of these materials. Among companies producing NM products in Germany and Switzerland, 65% indicated that they do not currently conduct risk assessments [23]. At the moment, there is a relatively short list of NM products in commercial production that require environmental risk assessment, but there is looming on the horizon a much greater

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challenge. As we approach the next few decades, the large investments made in nanoscience around the world will yield a myriad of new products. The penetration of many new NM products into the marketplace will outstrip the ability to perform full risk assessments. Will a change of a single moiety on a given nanomaterial that alters its physical characteristics (e.g. water solubility) but not its functionality require a new risk assessment? Will the same NM product marketed over different size ranges require an individual risk assessment for each size class? The sheer magnitude of new materials will quickly outstrip the capacity of the regulatory agencies and industry to respond in a timely manner, resulting in reduced investment in the technology. Methods are needed to prioritize new NM products and target them for environmental risk assessment, while minimizing the potential for adverse environmental effects. An overall goal should be to provide industry with information on potential mechanisms of toxicity or biological interactions early in the product development cycle. Thus, the specific properties of the product can be tailored to minimize any unwanted effects, while maintaining the commercially desirable properties of the material. This “green-nano” approach will allow industry to develop their products using the best available information; regulators to prioritize particles of concern and provide industry with a structure under which they can introduce new products to the marketplace. One approach to effective risk assessment of new NM products is to more thoroughly examine the existing information available from the manufacturer, such as anticipated volumes of production, the product life cycle and the basic physical and chemical information available for the material. This conceptual model should accommodate non-traditional measures that provide evidence regarding the source, fate, expected media, exposure pathways, and potential receptors. As illustrated in Figure 2, some of this information can be used to make predictions about the likely transport processes into the environment, exposure pathways and receptors for biological effects. In spite of a lack of fate, transport and effects data normally associated with traditional risk assessment, this approach may allow predictions to be made of the environmental hazards of NM. To develop a strategy that provides this required information, it is absolutely necessary that toxicologists and physical scientists work together to identify the physical and chemical properties that make NMs hazardous. In a recent review, Handy et al. [21] identified the need to understand the biological implications of differences in NM shape, size, surface charge, coatings, attached functional groups, core metals, intracellular dissolution, etc. A logical effort will require specific manipulation of the physical characteristics of a singe base class of NM (and repeated for different classes of NMs), followed by toxicity testing with a number of in vitro and in vivo models. Moreover, similar testing should be crossvalidated in at least two independent laboratories to ensure the validity of the results. While this effort seems extensive (and expensive), a logical hypothesis based scientific approach offers a practical mechanism for the provision of baseline data in the near and far term to understand the nature of biological interaction with different NM classes.

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1. Sources

3. Exposure Pathways and Receptors

2. Media and Transport Processes Air

Engineered Nanodevices Reaction Intermediates Production Waste

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Figure 2. Conceptual framework for utilizing information from the NM manufacturer to make predictions about the environmental fate and effects of NM.

To address these issues, the SMARTEN approach involves thinking more carefully about the likely fate, exposure and effects of NM, such as considering the characteristics summarized in Table 1. For example, many NM with particle sizes in excess of 4 nm that enter aquatic environments are likely to aggregate and be deposited in sediments or floc layers that overly sediments. This suggests that it might be wiser to investigate the effects of these NM on deposit feeding organisms rather than species that live in the water column. Current laboratory methods that have been developed to provide the data for conventional risk assessments, such as the OECD test methods [14] may not be appropriate for evaluating NM. It may be necessary to develop new test methods, such as assays to evaluate binding to synthetic skin or nano-sensors, or uptake across biofilms. Test systems to evaluate phagocytosis or inflammatory responses as a result of exposure to NM may be more relevant endpoints than acute toxicity tests with whole organism models. 5.

Environmental Surveillance and Reconnaissance

The current regulatory frameworks in North America and Europe that require environmental risk assessments to be conducted prior to the introduction of new chemicals into the marketplace have only been in place for the past 15–20 years. However, there are now several examples of the failure of these regulatory approaches to predict the impacts of chemicals on ecosystems. For instance,

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perfluorinated substances used in fluoropolymer products are accumulating in the environment [38] including in humans [2], despite earlier evaluation of the risks that these substances might pose. Since it appears that current risk assessment protocols are not adequate for the surveillance of conventional chemical classes, it is reasonable to assume that some NM products will be approved for commercial production that will have impacts in aquatic and/or terrestrial ecosystems. Environmental surveillance and reconnaissance programs are required to safeguard against these eventualities. The traditional analytical approaches that are used to detect others classes of contaminants in the environment will not be appropriate and new analytical techniques will be needed for surveillance purposes [22, 39]. Modified and/or novel sampling devices may be needed, such as passive sampling devices or nano-sensors that can be deployed in water, air or soil to detect the presence of NM. Over the years, biomarkers have been developed that are efficient at providing an early warning of deleterious effects on biological systems [15]. Goldberg and Bertine [19] argued that the analysis of the detoxifying enzymes, cytochrome P-450 activity, metallothioneins and estrogenic responses can provide useful information on the effects of contaminants in the aquatic environment. It may be possible to develop a unique set of biomarkers that can be used in surveillance programs to monitor for the biological effects of NM. Toxicogenomics methods may be valuable biomarkers for evaluating exposure to NM. 6.

Overview

The SMARTEN framework can also be integrated into wider concepts of “Environmental Security”, which involves actions that guard against environmental degradation in order to preserve or protect humans and natural resources at scales ranging from global to local in a sustainable manner. Environmental security can best be viewed as a response to one or more of three categories of events: (a) Manmade gradual changes that slowly erode economic and environmental sustainability, and, in some cases, may even be irreversible. (b) Natural catastrophic events that, to some extent, may be predictable, so it is possible to plan response and protection measures. (c) Manmade catastrophic events, which are typically sudden and unpredictable. The different perspectives on environmental security are time, spatial scales (i.e. local, regional, national, trans-national, global). Nanotechnology risk management challenges may be viewed according to the rankings illustrated in Figure 3. The field of environmental security is changing rapidly. Government and academic research in western countries appears to be undergoing a process of reshaping on an annual basis as a result of public health scares and sudden tragic world events. Environmental security will continue to change and evolve as new threats, both manmade and natural, reveal themselves at local, national or international spatial scales. Frameworks for organizing environmental security programs for nanotechnology, therefore, must be flexible and must adapt as either current or

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new challenges and response to those challenges manifest in additional or unforeseen consequences. Assessment and Ranking of Risks Comprehensive, but qualitative Develop a list of potential threats and vulnerabilities

Qualitative to semi-quantitative Rank risks in terms of potential costs (e.g., $, injuries, fatalities, lost opportunities) and time scales over which risks occur

Semi-quantitative to quantitative Detailed analysis of ranked risks in order of ranking

Integrative Integration of risk analyses to identify shared attributes, Common variables, and risk synergies. Re-order Ranking accordingly

Figure 3. Rankings of risks associated with exposure to NM.

In addition to decision frameworks, new or improved technologies and environmental monitoring programs are needed to enhance prevention, response, and mitigation strategies and to anticipate or forecast when threats might occur. In future, environmental surveillance programs must: (a) support real-time decision making, (b) provide accurate and impartial data to avoid human interference, (c) provide for stable, long-term safekeeping of data, (d) support other environmental applications, and (e) support long-term planning schemes such as early warning systems. This paper presents a suggested framework for evaluating the potential environmental and ecological hazards of NM. This framework, referred to as SMARTEN outlines some ideas about the fundamental informational needs. However, with recognition of the wide range of physical and chemical properties of NM, their uses and the rate at which new products and applications are likely to be developed, it is not intended to be a comprehensive “check list” of required testing strategies that must be performed before NM products enter into commercial production. SMARTEN is intended as a starting point of an iterative process by which a NM product can be evaluated. The process features and emphasizes decision points where the new information is combined with and compared against previous information, as well as available data on similar materials. In each step, a conceptual model of the potential hazards of the material is refined, questions and data quality objectives are raised, and a decision is made as to the need for and scope of additional testing. This approach is intended to provide a degree of flexibility that reflects the current degree of uncertainty and the need to provide a means for the expedited evaluation of products of nanotechnology. References 1. Adams, L.K., Lyon, D.Y., McIntosh, A., and Alvarez, P.J. (2006) Comparative toxicity of nano-scale TiO2, SiO2 and ZnO water suspensions, Water Sci. Technol. 54, 327– 334.

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2. Apelberg, B.J., Goldman, L.R., Calafat, A.M., Herbstman, J.B., Kuklenyik, Z., Heidler, J., Needham, L.L., Halden, R., and Witter, F.R. (2007) Determinants of fetal exposure to polyfluoroalkyl compounds in Baltimore, Maryland, Environ. Sci. Technol. 41, 3891–3897. 3. Aryal, B.P., and Neupane, K.P. (2006) Metallothioneins initiate semiconducting nanoparticle cellular toxicity, Small 2, 1159–1163. 4. Banfield J.F., and Zhang, H.Z. (2001) Nanoparticles in the environment, Nanoparticles and the Environment 44, 1–58. 5. Borm, P.J.A., Robbins, D., Haubold, S., Kuhlbusch, T., Fissan, H., Donaldson, K., Schins, R., Stone, V., Kreyling, W., Lademann, J., Krutmann, J., Warheit, D., and Oberdoerster, E. (2006) The potential risks of nanomaterials: a review carried out for ECETOC, Part. Fibre Toxicol. 3, 12–22. 6. Borm, P.J.A., and Hreyling, W. (2004) A need for integrated testing of products in nanotechnology. In: Nanotechnologies: A Preliminary Risk Analysis on the Basis of a Workshop Organized in Brussels on 1–2 March 2004, Health and Consumer Protection Directorate General of the European Commission. Available online at http://europa. eu.int/comm/health/ph_risk/events_risk_en.htm (last accessed 31 July 2008). 7. Boxall, A.B.A., Tiede, K., and Chaudry, Q. (2007) Engineered nanomaterials in soils and water: how do they behave and could they pose a risk to human health? Nanomedicine 2, 919–927. 8. Burleson, D.J., Driessen, M.D., and Penn, R.L. (2004) On the characterization of environmental nanoparticles, J. Environ. Sci. Health Part A 39, 2707–2753. 9. Chappell, M.A., George, A.J., Porter, B.E., Price, C.L., Dontsova, K.M., Kennedy, A.J., and Steevens, J.A. (2008) Surfactive properties of dissolved soil humic substances for stabilizing multi-walled carbon nanotubes dispersions. In: Nanoparticles in the Environment: Implications and Applications, Proceedings of a workshop at Centro Stefano Franscini, Monte Verita, Ascona, Switzerland. 10. Chibante, L.P., and Heymann, D. (1993) On the geochemistry of fullerenes: stability of C60 in ambient air and the role of ozone, Geochim. Cosmochim. Acta 57, 1879–1881. 11. Cho, S.J., Maysinger, D., Jain, M., Roder, B., Hackbarth, S., and Winnik, F.M. (2007) Long-term exposure to CdTe quantum dots causes functional impairments in live cells, Langmuir 23, 1974–1980. 12. Choi, A.O., Cho, S.J., Desbarats, J., Lovric, J., and Maysinger, D. (2007) Quantum dotinduced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells, J. Nanotechnol. 5, 1–2. 13. Christian, P., Von der Kammer, F., Baalousha, M., and Hofmann, T. (2008) Nanoparticles: structure, properties, preparation and behaviour in environmental media, Ecotoxicology 17, 326–343. 14. Crane, M., Handy, R.D., Garrod, J., and Owen, R. (2008) Ecotoxicity test methods and environmental hazard assessment for engineered nanoparticles, Ecotoxicology 17, 421– 437. 15. Depledge, M.H., Amaral-Mendes, J.J., Daniel, B., Halbrook, R.S., Kloepper-Sams, P., Moore, M.N., and Peakall, D.B. (1992) The conceptual basis of the biomarker approach. In: Peakall, D.B., and Shugart, L.R. (eds.), Biomarkers: Research and Application in the Assessment of Environmental Health, NATO ASI Series H: Cell Biology 68, Springer, Berlin, pp. 15–29. 16. Duffin, R., Tran, L., Brown, D., Stone, V., and Donaldson, K. (2007) Proinflammogenic effects of low-toxicity and metal nanoparticles in vivo and in vitro: highlighting the role of particle surface area and surface reactivity, Inhal. Toxicol. 19, 849–856.

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17. Dunphy-Guzman, K.A., Finnigan, M.P., and Barfield, J.P. (2006) Influence of surface potential on aggregation and transport of titanium nanoparticles, Environ. Sci. Technol. 40, 7688–7693. 18. Gimbert, L.J., Hamon, R.E., Casey, P.S., and Worsfold, P.J. (2007) Partitioning and stability of engineered ZnO nanoparticles in soil suspensions using flow field-flow fractionation, Environ. Chem. 4, 8–10. 19. Goldberg, E.D., and Bertine, K.K. (2000) Beyond the mussel watch – new directions for monitoring marine pollution, Sci. Total Environ. 247, 165–174. 20. Handy, R.D., Henry, T.B., Scown, T.M., Johnston, B.D., and Tyler, C.R. (2008a) Manufactured nanoparticles: their uptake and effects on fish – a mechanistic analysis, Ecotoxicology 17, 396–409. 21. Handy, R.D., Owen, R., and Vadsami-Jones, E. (2008b) The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs, Ecotoxicology 17, 315–325. 22. Hasselov, M., Readman, J.W., Ranville, J.F., and Tiede, K. (2008) Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles, Ecotoxicology 17, 344–361. 23. Helland, A., Scheringer, M., Seigrist, M., Kastenholz, H.G., Weik, A., and Scholz, R.W. (2008) Risk assessment of engineered nanomaterials: a survey of industrial approaches, Environ. Sci. Technol. 42, 640–646. 24. Hyung, H., Fortner, J.D., Hughes, J.B., and Kim, J.H. (2007) Natural organic matter stabilizes carbon nanotubes in the aqueous phase, Environ. Sci. Technol. 49, 179–184. 25. Kashiwada, S. (2006) Distribution of nanoparticles in the see-through medaka (Oryzias latipes), Environ. Health Perspect. 114, 1697–1702. 26. Lecoanet, H.F., Bottero, J-Y., and Wiesner, M.R. (2004) Laboratory assessment of the mobility of nanomaterials in porous media, Environ. Sci. Technol. 38, 5164–5169. 27. Lovric, J., Bazzi, H.S., Cuie, Y., Fortin, G.R.A., Winnik, F.M., and Maysinger, D. (2005) Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots, J. Mol. Med. 83, 377–385. 28. Lyon, D.Y., Thill, A., Rose, J., and Alavarez, P.J. (2007) Ecotoxicological impacts of nanomaterials. In: Weisner, M.R., and Bottero, J-Y., (eds.), Environmental Nanotechnology: Applications and Implications of Nanomaterials, McGraw-Hill, New York, pp. 445–479. 29. Madden, A.S., Hocella, M.F.J, and Luxton, T.P. (2006) Insights for size-dependent reactivity of hematite nanomineral surfaces through Cu2+ sorption, Geochim. Cosmochim. Acta 70, 4095–4104. 30. Moore, M.N. (2006) Do nanoparticels present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 32, 967–976. 31. Murr, L.E., and Garza, K.M. (2008) Natural and anthropogenic environmental nanoparticulates: their microstructural characterization and respiratory health implications. In: Nanoparticles in the Environment: Implications and Applications, Proceedings of a Workshop at Centro Stefano Franscini, Monte Verita, Ascona, Switzerland. 32. Neal, A.L. (2008) What can be inferred from bacterium-nanoparticel interactions about the potential consequences of environmental exposure to nanoparticles? Ecotoxicology 17, 362–371. 33. Neely, B., Morris, P.J., Shields, J.P., Sutter, A.G., Bearden, D.W., and Bertsch, P.M. (2007) Microbial growth affects of zinc oxide nanoparticle structure and toxicity. Proceedings of the Annual Meeting of Society for Environmental Chemistry and Toxicology, North America, Milwaukee, WI, USA, 19–23 November 2007. 34. Nowack, B., and Bucheli, T.D. (2007) Occurrence, behavior and effects of nanoparticles in the environment, Environ. Pollut. 150, 5–22.

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35. Organization for Economic Cooperation and Development (OECD) (2006) Report on the OECD workshop on the safety of manufactured nanomaterials: building cooperation, co-ordination and communication. Available online at http://appli1.oecd. org/olis/2006doc.nsf/linkto/env-jm-mono(2006)19 (last accessed 31 July 2008). 36. Oberdorster, E. (2004) Manufactured nanomaterials (Fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass, Environ. Health Perspect. 112, 1058– 1062. 37. Owen, R., and Depledge, M.H. (2005) Nanotechnology and the environment: risks and rewards, Mar. Pollut. Bull. 50, 609–612. 38. Prevedouros, K., Cousins, I.T., Bick, R.C., and Korzeniowski, S.H. (2006) Sources, fate and transport of perfluorocarboxylates, Environ. Sci. Technol. 40, 32–44. 39. Teide, K., Boxall, A.B.A., Tear, S., Lewis, J., David, H., and Hasselov, M. (2008) Detection and characterization of engineered nanoparticles in food and the environment: a review, Food Addit. Contam. (in press). 40. Tong, Z., Bischoff, M., Nies, L., Applegate, B., and Turco, R.F. (2007) Impact of fullerene (C60) on a soil microbial community, Environ. Sci. Technol. 41, 2985–2991. 41. Turgittiplakorn, W., Cohen, C., and Kim, J.-Y. (2004) Engineered polymeric nanoparticles for soil remediation, Environ. Sci. Technol. 38, 1605–1610. 42. Warheit, D.B., Borm, P.J., Hennes, C., and Lademann, J. (2007) Testing strategies to establish the safety of nanomaterials: conclusions of an ECETOC workshop, Inhal. Toxicol. 19, 631–643. 43. Zhang, Y., Chen, W., Zhang, J., Liu, J., Chen, G., and Pope, C. (2007) In vitro and in vivo toxicity of CdTe nanoparticles, J. Nanosci. Nanotechnol. 7, 497–503. 44. Zhang, X., Sun, H., Zhang, Z., Niu, Q., Chen, Y., and Crittenden, J.C. (2007) Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticles, Chemosphere 67, 160–166.

SOLID-PHASE CHARACTERISTICS OF ENGINEERED NANOPARTICLES A Multi-dimensional Approach

M.A. CHAPPELL U.S. Army ERDC 3909 Halls Ferry Road Vicksburg, MS 39056, USA [email protected]

Abstract. The challenge associated with determining the environmental fate and risk of engineered nanomaterials lies in understanding the fundamentally associated solid-phase chemistry. The solid phase represents the most complex, most thermodynamically “powerful”, yet the least understood phase among the three phases (solid, liquid, gas) commonly present in environmental systems. This difficulty is compounded by the fact that the nanoparticle size range represents a frontier field in itself in solid-phase chemistry, being the smallest size particles, close to the solid-phase – macromolecule boundary yet the most chemically reactive fraction in solid mixtures. This chapter contains a brief review of some important properties or characteristics of solid phase particles. These properties are presented theoretically as directed interactions among multiple linkages of any single property to another. Selected properties discussed in this chapter include particle charge, crystal structure, surface and bulk speciation, surface area, and adsorption phase composition. This discussion is presented in the context of solid-phase characteristics that influence nanoparticle dispersion stability and potential bioavailability by controlling particle size. 1.

Introduction

The intended purpose of this paper is to review solid phase chemical properties of nanoparticles important for describing their reactivity in environmental systems. In doing so, it is important to realize that the current level of knowledge for solid-phase chemistry is far inferior to that of the chemical knowledge of liquid and gas phase systems. This knowledge gap is attributed to the higher order of complexity of the solid phase and the difficulty of probing these systems. When studying solids, one not only deals with the unique chemistry of the solids’ constituents, but the bonding and coordination environment among the constituents confers a super-molecular complex that exhibits its own unique dynamics, I. Linkov and J. Steevens (eds.), Nanomaterials: Risks and Benefits, © Springer Science + Business Media B.V. 2009

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structural order, vibration, electronic, and magnetic properties, to name a few. It is this ability that makes solid phase chemistry so formidable. Yet, it’s the power of this behavior that makes the solid phase the dominant phase controlling geologic and biological chemistry on the Earth’s surface. A major obstacle to deciphering this chemistry lies in probing the solid phase systems. While liquid and gas phases are easily captured and mobilized through elaborate analytical systems, environmental solids are neither fluid nor (typically) transparent. Numerous techniques have been developed to facilitate analysis of solid phases. Chief among these are extraction techniques designed to transfer the solid phase constituents to the liquid phase for ease of analysis. But aside from the elemental analysis and stoichiometries, most of the fundamental information is lost, particularly in terms of the constituent speciation (yet, there do exist some liquid phase techniques for this), coordination environment, long-term order, and macroscopic properties. More often than not, little is preserved of the solids’ inherent chemistry, often relegating atomic constituent speciation to arbitrary delineations based on the properties of the extractant. Adding to the complexity of solid phase analysis is the unique behavior of solids at their surface interface. A solid surface, typically defined as the first few atomic layers at the “outer edge” of the solid, is where most of the solid’s excess energy is exhibited, and potentials are created to promote chemical and physical work. While liquid and gas phase solutes enjoy high degrees of freedom of movement to alleviate energy excesses, the confining environment of solid phases forces these excesses to be transferred to the surface atomic layers. Three common means by which surface excess energy is reduced are (1) phase transitions, (2) surface reconstructions, and (3) adsorption of liquid or gas phases and associated solutes [1]. The relative small size of the surface compared to the rest of the material, termed the “bulk”, makes it difficult to probe as well. Solids exhibit a variety of seemingly unrelated or even contradictory properties so that adequate descriptions based on one or two properties are (if ever) adequate. And in addition, these properties can seem contradictory to each other. For example, a solid can be charged but behave hydrophically. Because the chief defining property of engineered nanomaterials is particle size, this review will focus on the relationship of solid phase properties to the particle size of nanomaterials. Figure 1 shows a theoretical chart proposing relationships between solid phase nanoparticle properties and linkages to particle size, and potentially stabilizing nanoparticle dispersions [2]. This list is by no means complete nor represents all of the properties of nanoparticles, but is presented to the reader as a guide for the following discussion of different solid phase properties. Notice in the schematic that particle size is predominantly represented as a property resulting from other properties. Thus, this review focuses on the other properties viewed as controlling nanoparticle size.

SOLID-PHASE CHARACTERISTICS OF ENGINEERED NANOPARTICLES 113 2

5

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7 Figure 1. Schematic showing hypothetical relationships between solid-phase properties discussed in this review and particle size (controlling dispersion potential). 1 = particle size, 2 = crystal structure, 3 = interior strain, 4 = species, 5 = surface area, 6 = surface charge density, 7 = surface electrical potential, and 8 = adsorbed phase. For this paper, edge sizes are all assumed = 1.

2. 2.1.

Particle Charge CHARGE DEVELOPMENT AND THE DDL

Perhaps, the best understood mechanism for influencing particle size is particle charge. Charge can develop on the surface through different ways, such as at the solid’s crystal edges or functionalization/degradation of the nanomaterial (NM) surface. For example, NMs may be functionalized with external COOH groups, which will deprotonate as the system pH is increased away from the functional group’s pKa. A material that develops charge in this way is termed “variablecharged” because the total charge of the solid phase is affected by the system pH. Charge that arises from internal deficits in the particle bulk composition (such as geologic isomorphous substitution) result in “constant-charge” materials. This review will focus solely on descriptions of variably charged materials. A variably charged surface experiences charge with a change in pH. Classical colloidal theory describes a situation at the solid-solution interface where charge developed on the surface is expressed out into the surrounding solution a certain distance away from the particle. This charged volume around the particle is called the “diffuse double layer” or DDL. The DDL is an electrical field driven by a surface electrostatic potential, ψo [3], which has formed to electro-neutralize the

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surface. In classical theory [4], the DDL is filled with oppositely charged counterions swarming around the wetted surface, attempting to establish electroneutrality – the distribution of cations and anions matching the electrical potential of the interfacial field. The DDL is generally divided into two layers (although more layers have been added on in modern times): The Stern layer and the diffuse layer (Figure 2). The Stern layer is a very thin volume of the solid-solution interface directly adjacent to the surface. The tight complexation of coordinating water and counterions makes the Stern layer rigid and compact, forming what is known as the plane of slippage or the plane of shear [5]. Beyond the Stern layer is a layer composed of moreloosely complexed counterions and coordinating water called the diffuse layer. Here, the influence of ψo pulls oppositely charged counterions toward the surface while diffusion forces allow some same-charged ions to approach the surface as well. Much of the charge-related chemistry that occurs at particle surfaces is controlled by the surface electrical potential and the distance in which that potential “influences” the bulk solution. The extent in which the surrounding solution “feels” ψo decays with distance from the surface. In the Stern layer, ψo decays linearly with distance d to ψd (known as the Stern potential). In the diffuse layer, ψo decays exponentially, beginning with ψd and approaching zero as d → ∞. The decay of ψ0 with distance (x) from the surface (see Figure 2) is mathematically represented as: ψ = ψoexp (-kx)

(1)

where ψ = the surface potential expressed in the DDL with distance from the surface (in nm) and k = inverse DDL thickness, which is equal to: 12

⎛ 8C O ⎞ ⎟⎟ k = ev ⎜⎜ ⎝ DKT ⎠

(2)

where, Co = bulk solution concentration, K = Boltzmann constant, T = absolute temperature, v = valence of ions in solution, D = medium dielectric constant, and e = elementary charge of an electron. Equation 2 predicts that increasing the concentration of counterions in solution (i.e., increasing the ionic strength) will increase k or compress the DDL. With the DDL compressed, the repulsive interactions from overlapping DDLs is minimized, thereby allowing the particle surfaces to come within the distance of the Stern layer, and flocculating through van der Waals forces. 2.2.

DISPERSION/FLOCCULATION PROCESSES

Inter-particle repulsive forces (RF), are influenced by ionic concentration and ionic valence. This response is described in the classical DLVO theory ([6], and references therein) as

SOLID-PHASE CHARACTERISTICS OF ENGINEERED NANOPARTICLES 115

RF =

64 ⎛ vFψ o ⎞ tanh ⎜ ⎟ C o RT exp (− k d ) K ⎝ 4RT ⎠

(3)

where, F = Faraday’s constant, R = molar gas constant, and d = separation between planar surfaces. Equations 1–3 predict that increasing k will reduce RF by reducing the DDL size. Note, however, in Eq. 3, the term CoRT represents the expression for osmotic potential or pressure, showing that DDL size is also affected by the osmotic pressure exerted by the constituents in the DDL. The osmotic potential of the DDL is controlled by the concentration of counterions adsorbed at the solid-liquid interface – which again is controlled by ψo – a property of the surface. Thus, the DLVO theory models changes in repulsive forces via two different mechanisms: One dependent on the properties of the solid, the other dependent on the bulk solution. The net result is the effect of ionic strength on the dispersion potential of differently charged colloids (e.g., one high charge, the other low charge). When increasing the ionic strength of the bulk

ψ°

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Diffuse layer

Low ionic strength

High ionic strength

Distance from surface (nm) % Dispersion

ZPC

σ2

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σ1

− 0 + Zeta potential (mV) or pH − pHo Figure 2. (Top) Theoretical plots showing the change in ψ(x) of charged NM particle with distance from the surface and changing solution ionic strength. This plot is overlain with a plot showing the distribution of counterions in the Stern and Diffuse layers. (Bottom) Relationship in the change in zeta potential (ξ) away from the ZPC (the rate of change with respect to surface charge density σ where |σ1| > |σ2|) and increase in particle dispersion and bioavailability. In both plots, it is assumed the solution phase does not contain any specifically adsorbing solutes.

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solution, Eq. 2 predicts the DDL size will reduce equivalently on both particles. Yet, the higher osmotic pressure exhibited in the DDL of the higher charged solid will enhance the pace of dispersion in the particles with higher ψo [7], as represented in Figure 2. 2.3.

ZETA POTENTIAL (ξ) AND ZPC

Zeta potential (ζ in mV) measurements are measures of the magnitude of charge (ψ in Eq. 1) expressed in the DDL at the plane of slippage (x = d). When the charged particle moves through aqueous media, all material within the plane of slippage (also called the plane of shear) “slides” along with it. The plane of shear/slippage is considered analogous to the Stern layer, which is roughly equivalent to the distance of the first hydration shell surrounding a surface. Zeta potential changes can be summarized by a property called the zero point-of-charge or ZPC, which is the pH in which NM zeta potential = 0. If we set ζ ~ ψ0 (given the very short distance of the plane of slippage from the surface), then the relationship between ζ and the ZPC can be simply represented as, ζ ~ ψ0 = 59.2 (pH0 – pH)

(4)

where pH0 = pH of the ZPC and pH = the pH of the bulk solution. For surfaces with mono-functionality, ZPC represents a minimum in which no charge is expressed, analogous a functional group pKa. For surfaces with poly-functional groups, ZPC represents the average of the different surface pKa values. It turns out that as a matter of convenience, solids exhibiting a ZPC will buffer the solution pH at that ZPC at equilibrium. The ZPC provides a simple parameter for estimating NM bioavailability. When ξ = 0, the pH is at the NM’s ZPC. Here, no charge is expressed at the plane of shear, and the particle essentially behaves as a hydrophobic solid. On the other hand, when ξ ≠ 0, the particle is charged at the plane of shear, and exhibits hydrophilic behavior. This information is directly relevant to the bioavailability of NM to organisms. Fully dispersed NMs are expected to exist at their minimum particle sizes in solution. In this form, NMs are expected to be the most bioavailable. At the ZPC, the material is expected to exhibit maximum flocculation. Thus, ZPC provides an appropriate estimation of the dispersion behavior, and bioavailability of NMs. 3.

Surface Charge Density/Distribution

While ZPC gives a relative sense of what pH a variably charged surface will exhibit charge at the plane of shear, the actual magnitude of charge controlling the surface potential is given by the specific charge density. Surface charge density is a measure of the total distribution of charged functional groups normalized to the total surface area of the material. The relationship between the surface electrical potential and the surface charge density (σ) is modeled as:

SOLID-PHASE CHARACTERISTICS OF ENGINEERED NANOPARTICLES 117

σ = [(2/π) CoDKT)]1/2 sinh(vFψ0/2RT)

(5)

For simplicity’s sake, this equation can be reduced by assuming ψ0 is small (C=O) , hydroxyl (-OH), and carboxyl (-COOH) group are formed on the surface of carbon particles. Thus the resulting NCC nanofluid is able to maintain its stability as long as the hydrophilic functional groups exist. 4.

NCC Preparation Technique

The electrolysis is executed by two stages: activation of the anode and the carbon nanoparticles generation. At the first stage the electrolyte has low conductivity, value of electric current is small, about 0.1–0.2 mA/cm2 and the oxidization reaction is slow. Duration of

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this stage is about 50 h and depends on the quality (density) of graphite. At this stage a voltage between electrodes can be high, about 60–100 V. As the reaction proceeds, the conductivity of the electrolyte is abruptly increased, electric current can increase up to 10 mA/cm2 and higher and the oxidization reaction is activated. As a result, the carbon is finely split, and then is covered by the carboxyl group. At the second stage the electric current between electrodes must be about 3–4 mA/cm2. If current density values greater than 8–10 mA/cm2, the rate of oxygen evolution is greater than the rate of its diffusion through the electrode; hence there is a pressure build-up within the electrode causing the electrode to disintegrate. NCC is not stable, in 2–3 weeks the precipitation of NCC is to be observed. Similarly, at current density less than 3–4 mA/cm2 the rate of oxygen evolution is such that, although some pressure builds up in the electrode, the gas is able to diffuse out of the electrode before disintegration occurred, small pieces of carbon broke off in the process to form colloidal carbon and very small amount of slurry. The NCC is stable during 150 days at least. The rate of diffusion of hydrogen at the cathode is such that little or no pressure built up within the electrode and therefore no colloid is produced at this electrode. The colloidal carbon is produced only at the anode and remains within the vicinity of the electrode, indicating that the carbon is negatively charged. Reversal of the electrode polarity results in the surrounding carbon migrating slowly to the new anode. Carbon nanoparticles are removed from the anode during the electrolyte stirring stage. Figure 1 demonstrates the process of carbon nanoparticles splitting. pH of the NCC is 2.8–3.1 and depends on the concentration of carbon nanoparticles; concentration of carbon nanoparticles is 150–400 ppm and depends on duration of the process; ion exchange capacity is 7.4 mmol/g for a monovalent cation. The typical TEM image for NCC obtained is shown on Figure 2.

Figure 1. Process of carbon colloids splitting.

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Figure 2. Typical TEM image of carbon colloids obtained (the scale is 200 nm).

5.

Water Decontamination by Carbon Nanoparticles

Removing heavy metal ions (Zn, Ni, Cu, Sb, Co, Cd, Cr, etc.) from water was studied. pH of the NCC was 2.8, concentration of carbon nanoparticles was 250 ppm Dependence of the distribution coefficient Kd for different ions at pH = 7.1 of the solutions is presented in Table 2. TABLE 2. Distribution coefficient Kd (ml/g) and percent adsorption P (%) for different ions (concentration of ions C0 = 10 mg/l, V = 50 ml, W = 0.5 g, pH = 7.1, contact time 1 h). Elements Cr(III) Co(II) Ni(II) Cu(II) Zn(II) Sr(II) Cd(II) Sb(II) Cs(I)

P >99 >99 >99 >99 >99 >99 >99 >99 60

Kd, 105 140 170 1,300 1,400 4,000 3,800 100 400 0.6

The results given in Table 2 demonstrate high ion-exchange potential of the colloidal carbons. In real water which contains different ions, the colloidal particles are coagulated within some time depending on the concentration of salts. Before and during coagulation process the nanoparticles as the ion exchangers react with

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cations. After coagulation water can be easily filtered from particles with attached cations. This means that the colloidal particles are quite effective for removal of the metal cations and may provide a useful alternative for example to method of flocculation of metal hydroxides and oxides. 6.

Conclusion

The described method allows producing NCC with concentration of carbon nanoparticles of 150–400 ppm and pH of 2.8–3.1. The concentration of nanoparticles depends on duration of the process and pH of NCC depends on concentration of carbon nanoparticles. The ion exchange capacity of carbon nanoparticles due to functional carboxyl groups is very high, 7.4 mmol/g for a monovalent cation. NCC can be used for effective removing metal ions (Zn, Ni, Cu, Sb, Co, Cd, Cr, etc.) from contaminated water. References 1. Bönnemann, H., and Richards, R. (2001) Nanoscopic metal particles – synthetic methods and potential applications, Eur. J. Inorg. Chem. 10, 2455 and 2480. 2. Coetser, S.E., Heath, R.G., and Ndombe, N. (2007) Diffuse pollution associated with the mining sectors in South Africa: A first-order assessment. Water Sci. Technol. 55: 9–16. 3. Theron, J., Walker, J.A, and Cloete, T.E. (2008) Nanotechnology and water treatment: applications and emerging opportunities, Crit. Rev. Microbiol. 34, 43–69. 4. Zhang, W. (2003) Nanoscale iron particles for environmental remediation: an overview, J. Nanopart. Res. 5, 323–332. 5. Peckett et al. (2000) Electrochemically oxidised graphite. Characterisation and some ion exchange properties. Carbon 38, 345–353.

A NOVEL SIZE-SELECTIVE AIRBORNE PARTICLE SAMPLING INSTRUMENT (WRAS) FOR HEALTH RISK EVALUATION

H. GNEWUCH, R. MUIR, B. GORBUNOV Naneum Limited, CEH, University of Kent Canterbury, Kent CT2 7NJ, UK [email protected] N.D. PRIEST Urban Pollution Research Centre, Middlesex University Queensway, Enfield, Middlesex EN3 4SA, UK P.R. JACKSON CERAM Queens Road, Penkhull Stoke-on-Trent, Staffordshire ST4 7LQ, UK

Abstract. Health risks associated with inhalation of airborne particles are known to be influenced by particle sizes. A reliable, size resolving sampler, classifying particles in size ranges from 2 nm–30 µm and suitable for use in the field would be beneficial in investigating health risks associated with inhalation of airborne particles. A review of current aerosol samplers highlighted a number of limitations. These could be overcome by combining an inertial deposition impactor with a diffusion collector in a single device. The instrument was designed for analysing mass size distributions. Calibration was carried out using a number of recognised techniques. The instrument was tested in the field by collecting size resolved samples of lead containing aerosols present at workplaces in factories producing crystal glass. The mass deposited on each substrate proved sufficient to be detected and measured using atomic absorption spectroscopy. Mass size distributions of lead were produced and the proportion of lead present in the aerosol nanofraction calculated and varied from 10% to 70% by weight. 1.

Introduction

The high health risk associated with the inhalation of airborne particles has been recognised and documented, see e.g. Brown et al. [1]; Pope et al. [15]. Many epidemiological studies have shown associations between exposure to particulate matter in the air and increases in morbidity and mortality [4]. There is a growing concern that health risk associated with airborne particles is influenced by size. Some studies indicate that nanoparticles (less than 100 nm in diameter) having I. Linkov and J. Steevens (eds.), Nanomaterials: Risks and Benefits, © Springer Science + Business Media B.V. 2009

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increased toxicity relative to larger particles composed of the same materials [5–7, 14]. Size-resolved sampling of airborne particles often requires various techniques to be employed [11]. There would be a significant benefit in a sampler that could reliably collect size resolved samples across the entire size range which is considered to be relevant to health effects. There are many technical difficulties in sizeresolved sampling of particles smaller than 100 nm – especially under real conditions of variable and/or high humidity. Pressure drop in low-pressure cascade impactors corrupts size distributions and causes condensation of water as well as other atmospheric constituents on substrates, e.g., Hart and Pankow [9] have estimated that the gas-particle mass exchange for PAHs could cause errors in measurements of up to 40%. Large mass changes were directly observed by Moor et al. [13] in experiments where atmospheric aerosol particles collected onto substrates of a cascade impactor were exposed to conditions with lowered partial pressures of semi-volatile compounds. 2.

Methods

In this paper, we describe a novel size-selective aerosol sampling instrument, WRAS (Wide Range Aerosol Sampler), based upon diffusion deposition coupled with inertial deposition in a single apparatus (www.naneum.com). This enables the size-selective sampling of aerosol particles in a wide range of airborne particle sizes without employing low-pressure cascade impactor technology. The WRAS instrument comprises an inertial unit similar to a May [12] cascade impactor having the lowest stage cut off diameter 0.25 μm and a specially designed diffusion deposition unit (nano-selector) for smaller particles. Operating flow rate is 20 l/min. First, aerosol particles are drawn into an isokinetic inlet of the cascade impactor where those greater than 0.25 μm in aerodynamic diameter are collected according to their aerodynamic diameter. Collection efficiency of particles in an inertial cascade impactor increases with size, therefore, the largest particles are removed from the flow by the first stage and the smaller particles are deposited onto following stages, stages 6–12 (Table 1). TABLE 1. The characteristics of the stages of the WRAS sampler.* Stage 1 2 3 4 5 6 7 8 9 10 11 12

Min. size (µm) 0.001 0.0015 0.005 0.015 0.06 0.25 0.5 1.0 2.0 4.0 8.1 20

Max. size (µm) 0.0015 0.005 0.015 0.06 0.25 0.5 1.0 2.0 4.0 8.1 20 ~35

Unit Nano-selector Nano-selector Nano-selector Nano-selector Nano-selector Cascade impactor Cascade impactor Cascade impactor Cascade impactor Cascade impactor Cascade impactor Cascade impactor

*Latest model of the WRAS sampler

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The flow emerging out of the cascade impactor contains particles smaller than 0.25 μm. These particles are collected by sets of Nylon nets. Collection efficiency of particles in a diffusion unit decreases with the size, therefore, the smallest particles are collected at the first net and the larger particles are deposited onto following nets according to cut off diameters shown in Table 1 (Sections 1–5). Nano-selector units have been designed to collect particles sequentially in the size range from 1 nm to 0.25 μm. In practice, this is achieved by selecting nets with differing fibre diameters, fibre densities and by varying the number of nets per stage and the flow velocity. The nano-selector deployed in the WRAS sampler has a cylindrical cross-section (ID = 4.7 cm) and contains five stages. From Table 1 the cut off diameters of the WRAS sampler covers the size range from 1 nm to 35 μm. Thus it is the first universal size-selective sampling apparatus which enables airborne particles to be collected in the entire aerosol size range that is of concern with respect to health risks. Artificial lead and tungsten aerosols were employed to calibrate the WRAS sampler according to the approach described by Sinclair et al. [16] and Cheng et al. [2]. The cut off diameters in the nanoparticle size range were calculated according to Cheng and Yeh [3] and were compared with the cut-off diameters found from the size distributions measured (with SMPS) before and after a net. The calculated and measured cut-off diameters were in good agreement (Table 2). TABLE 2. Calculated/measured cut off diameters of WRAS diffusion collector stages.* Stage

Di (nm) calculated

Di (nm) measured

2

17

16.5 + 1

3

80

80+ 5

4

120

110 + 10

*Previous model of WRAS sampler

A case study is presented here to illustrate the potential of the novel sampling system. The developed WRAS sampling unit (previous model) was employed to sample airborne particles at various working places in the crystal glass industry. Aerosol particles were collected in a range of sizes from 2 nm to 20 μm (11 size fractions) at a flow rate 20 l/min and at a controlled relative humidity of 80%. The sampling time employed varied from 2 to 24 h (according to aerosol concentration levels). The mass of lead collected was determined by atomic absorption spectroscopy [8]. Aerosol mass size distributions of lead were obtained from the samples collected. Size distributions are crucial for the evaluation of health risk. It is known that particle deposition efficiency in the human respiratory system is influenced by the size of particles. An example compiled from experimental and theoretical data is shown in Figure 4 [10]. The efficiency is a V-shaped function with a high degree of deposition for nanoparticles and for particles in micro-range (close to 100%). In

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the sub-micron range, the efficiency falls to about 10–20 %. Therefore, when the mass of airborne particles is concentrated in the sub-micron range (as in Figure 3) the dose of deposited particles is proportionally less than from an aerosol with a maximum positioned towards the nano size range (Figure 2). This shows the importance of size-resolved sampling for correct evaluation of health risk. 3.

Results

The total mass concentration of lead aerosols determined at working places ranged from 0.6 to 50 μg/m3. The nanoparticle mass fraction of aerosols (sizes less than 100 nm) was found to vary from 10% to 70%. A typical aerosol size distribution of lead (sampled at the glass smelting area at plant A) is shown in Figure 1. The size distribution has a maximum at about 0.35 μm. but there is a noticeable amount of Pb associated with nanoparticles in the range below 0.1 μm. Even more nanoparticle mass fraction was observed at plant B where melting of lead compounds also takes place (Figure 2). In contrast, at plant C, involved in glass polishing, airborne particle size distributions contain much less mass fraction in the nanoparticle range (Figure 3). Thus, size distribution of lead at workplaces may vary considerably depending on production processes.

Figure 1. Airborne lead particle size distribution obtained at a plant A involved in hot lead processing.


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Figure 2. Airborne lead particle size distribution obtained at a plant B involved into hot lead processing.

Figure 3. Airborne lead particle size distribution obtained at a plant C involved into hot lead processing.

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The size distribution of the number of particles deposited in the respiratory system Nl(D) is a product of the ventilation rate Q, exposure time t, airborne particle size distribution fa(D) and the deposition efficiency of particles E(D)

N l ( D) = Q t f a ( D) E ( D)

(1)

Nl(D), therefore, represents the mass distribution of particles that were deposited in the respiratory system. The fraction of particles captured by the respiratory tract calculated for distributions shown in Figures 1–3, and for the efficiency presented in Figure 4 showed that the fraction of deposited total particle mass varied from 16% (Figure 3) to 37% (Figure 1) to 51% (Figure 2). 100% 90%

Efficiency

80% 70% 60% 50% 40% 30% 20% 10% 0% 0

1

2

3

4

LogD, D - nm

Figure 4. Efficiency E(D) of airborne particles deposition in the human respiratory tract.

4.

Conclusions

The principles of inertial and diffusion deposition have been employed in the design and construction of a new instrument (WRAS) that was developed to sizeselectively collect aerosol particles across a wide aerosol size range relevant to health effects. The instrument developed does not require low pressure to collect nanoparticles and, therefore, can be employed to sample size-selectively aerosol particles across the entire aerosol size range down to nanometer-sized particles with minimal sampling artefacts caused by evaporation/condensation of volatile and semi-volatile compounds. Data shows that the size distributions of lead containing particles in the aerosol at workplaces are influenced by manufacturing processes in the crystal glass industry. The nanoparticle mass fraction of aerosols (sizes less than 100 nm) was found to vary from 10% to 60%. It was found that the fraction of mass of lead deposited in the respiratory system depends on the mass distribution of lead and


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varies from 16% to 51%. This result is important for air quality assessment and health risk evaluation. It shows that standard, non-size-resolving OH sampling techniques, used by the crystal glass industry and others will overestimate the total health risk considerably. References 1. Brown J. S., Kirby L. Z. and William D. B. (2002) Ultrafine particle deposition and clearance in the healthy and obstructed lung. Am J Resp Crit Care Med, 166:1240– 1247. 2. Cheng Y. S., Keating J. A. and Kanapilly G. M. (1980) Theory and calibration of a screen-type diffusion battery, J Aerosol Sci., 11:549–556. 3. Cheng Y. S. and Yeh H. C. (1980) Theory of a screen-type diffusion battery. J. Aerosol Sci. 11:313–320. 4. Dockery D. W., Pope C. A., Xu X., Spengler J. D., Ware J. H., Fay M. E., Ferris B. G. and Speizer F. E. (1993) An association between air pollution and mortality in six US cities. N Engl J Med 329:1753–1759. 5. Donaldson K., Li X. Y. and MacNee W. (1998) Ultrafine (nanometer) particlemediated lung injury. J Aerosol Sci 29:553–60. 6. Ferin J. (1994) Pulmonary retention and clearance of particles. Toxicol Lett 72:121– 125. 7. Ferin J., Oberdorster G. and Penny D. P. (1992) Pulmonary retention of ultrafine and fine particle in rats. Am J Resp Cell Mol Biol 6:535–542. 8. Gorbunov B., Priest N., Jackson P. R. and Cartlidge D. (2000) Aerosol size distribution of lead at working places. J Aerosol Sci 31(Suppl. 1):S520–521. 9. Hart K. M. and Pankow J. F. (1994) High-volume air sampler for particle and gas sampling. 2. Use of backup filters to correct for the adsorption of gas-phase polycyclic aromatic hydrocarbons to the front filter. Environ Sci Technol 28:655–661. 10. Hinds W. C. (1999) Aerosol technology. Properties, Behaviour and Measurement of Airborne Particles. New York: Wiley, pp. 233–259. 11. John W. (2001) Size Distribution Characteristics of Aerosols. In: Aerosol Measurement. Principles, Techniques and Applications. Ed. PA Baron and K Willeke. New York: Wiley, pp. 99–116. 12. May K. R. (1982) A personal note on the history of the cascade impactor. J Aerosol Sci 13:37–47. 13. Moore M., Gorbunov B. and Williams I. (1998) A new method to study interaction of semi-volatile compounds with aerosol particles. J Aerosol Sci 29(Suppl. 1):S887–888. 14. Oberdorster G., Ferin J. and Lehnert B. E. (1994) Correlation between particle-size, invivo particle persistence, and lung injury. Environ Health Perspect 102(Suppl. 5):173– 179. 15. Pope C. A., Dockery D. W. and Schwartz J. (1995) Review of epidemiological evidence of health effects of particulate air pollution. Inhal Toxicol 7:1–18. 16. Sinclair D., Countess R. J., Liu B. Y. H. and Pui D. Y. H. (1976) Experimental verification of diffusion battery theory. J Air Poll Control Assoc 26:661–663.

NANOTECHNOLOGIES AND ENVIRONMENTAL RISKS Measurement Technologies and Strategies

T.A.J. KUHLBUSCH, H. FISSAN, C. ASBACH Air Quality & Sustainable Nanotechnology Unit Institute for Energy and Environmental Technology (IUTA) e. V. Bliersheimerstr. 60 47229 Duisburg, Germany [email protected]

Abstract. Assessments of nanoparticle exposure are needed to enable risk assessments which are needed to achieve a sustainable development of nanotechnology including public perception. Therefore an overview of measurement techniques, needed data quality, comparability, and measurement strategies is given. Additionally some results of exposure related studies are summarized. Overall it is demonstrated that an integrated approach towards nanoparticle exposure assessments in workplaces, but also in the environment is needed, despite the current published results indicating mainly release of nanoparticle agglomerates in the size range larger than 100 nm. 1.

Introduction

Nanoparticles and nanoobjects, intentionally produced particles of nanoscale in three or two dimensions, have specific properties possibly altering the (eco-) toxicological potential when compared to larger particles or the corresponding bulk material. The detailed assessment of this potential risk is a prerequisite to accomplish sustainable nanotechnology since it directly influences the public perception. The risk is generally a function of potential hazard and exposure. The importance of the latter is given by (a) that no risk exists if no exposure, (b) the dose, leading to possible health effects, is directly linked to the exposure, and (c) correct exposure determination is also of importance for e.g. epidemiological studies. To assess exposure it is also necessary to clarify the areas (e.g. workplace, environment), subjects (e.g. humans, animals, ecosystems) and exposure media (air, liquid, solid) of interest. This paper focuses on the human exposure mainly in workplace environments since highest exposure can be expected in these areas. Also, measurement technologies and strategies can be evaluated and tested in workplace environments since possible sources and hence particle material is known and can be differentiated from ambient nanoscale particles.


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The currently discussed main exposure route of nanoparticles is via the airborne state by inhalation. Other discussed routes of exposure such as via the skin or gastrointestinal tract possibly leading to an uptake are currently seen as of minor importance [4] but still have to be investigated. Two major pieces of information are necessary for the assessment of exposure and possible nanoparticle implications: The exposure leading to a dose The hazard, influenced by the particle properties Hence, assessments of exposure to nanoparticles have a twofold task. One task is the general determination of an exposure and to quantify the ‘relevant’ aerosol property. The second task is the characterization of the nanoparticle properties since these may have been influenced or changed during the transport period after release. Any changes in these particle properties may have a significant influence on the possible hazard of nanoparticles. Spatial and time resolution of the measurements can therefore play a crucial role in the exposure determination and its evaluation. This background puts certain demands onto the measurement techniques for airborne nanoparticles as well as the measurement strategies. 2.

Measurement Techniques

Basically, various physical and/or chemical properties of nanoparticles and aerosols can be determined with especially particle size and concentration being physical properties of importance in the case of nanoparticles (

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