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In Vitro Toxicity Analysis of Nanoscale Aluminum: Particle Size and Shape Effects

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PAGE 1

IN VITRO TOXICITY ANALYSIS OF NANOSCALE ALUMINUM: PARTICLE SIZE AND SHAPE EFFECTS By MARIA PALAZUELOS JORGANES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Maria Palazuelos Jorganes

PAGE 3

3 To my husband Scott, my sister Amalia and my parents Pepa and Luis.

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4 ACKNOWLEDGMENTS I thank my mentor Dr. Kevin W. Powers for his unconditional support and guidance throughout what it has been an intense journey in science and personal growth. It was thank to him that I expanded my research experiences and that I practiced the Philosophy component of my doctoral degree in our endless conversations about ve ry different topics. To my advisor Dr. Richard B. Dickinson goes my sincere gratitude for chairing my advisory committee and always being there when I needed him. He has taught me to be rigorous about my work and I am truly appreciative for that. My research was possible thanks to the generous sponsorship of Dr. Brij M. Moudgil and the NSF. I consider myself very fortunate for the diverse and comprehensive formation that I have received from the Particle Engineering Research Center. I also thank the rest of my advisory committee members for their support, Dr. Spyros Svoronos and Dr. Yiider Tseng. I want to thank the whole PERC family for their help, encouragement and friendship. Special thanks are due to Gill Brubaker, Gary Scheiffele, Kathryn Finton, Jacqueline Gesner and Vanessa Kuder. My gratitude also goes to the administrative personnel for their helpful assistance. I am grateful to Dr. David Moraga for being a true catalyst for my research; he introduced me to tissue culture and biotechnology techniques needed in my investigation. I could have not asked for a better tutor. I specially thank Kerry Siebein fo r her wiliness to help me with HRTEM anytime. Thanks also go to Dr. Greg Erdos and Dr. Sharon Matthews for their help with EM microscopy in biological samples. Dr. Steve Roberts and the rest of the Nanotox group have been an important contribution to the common goals of our group and I sincerely thank each of them. My time in Gainesville has provided me with great friends and lots of memories that I will cherish forever. Thanks go to my friends and colleagues, Vijay Krishna, Rhye Hamey, Stephen Tedeschi, Anna Fuller, Marco Verwijs, Dauntel Sp echt, Milorad Djomlija and all the other with whom I shared this time with, for making this journey so much better.

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5 I owe this life-changing experience to Professor Brian Scarlett who was my mentor during some of the most important transitions in my professional and personal life. He became a dear friend who was an excellent example while providing guidance and security. I deeply miss him. I give gracias to my parents, Luis and Pepa. Their love and sacrifices for our family have been my biggest gift in life. I also thank my sister Amalia for always showing me the bright side of life. Last but certainly not least I thank my husband Scott; I have come this far thanks to him. He is my haven, keeping me sane and loving me every step of the way.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS................................................................................................................ .....4 LIST OF TABLES................................................................................................................. ................8 LIST OF FIGURES................................................................................................................ ...............9 ABSTRACT....................................................................................................................... ..................12 CHAPTER 1 INTRODUCTION................................................................................................................... .....14 1.1 Motivation and Research Outline...........................................................................................14 1.2 Nanotechnology: Definitions and Historical Context............................................................15 1.3 Nanotoxicology: A Discipline on Its Own.............................................................................18 1.4 Toxicity of Ultrafine and Nanomaterials: Literature Review.................................................20 1.4.1 History of Particle Toxicology: Ultrafine Particles.....................................................20 1.4.2 Guidelines to Risk Assessment....................................................................................21 1.4.3 Nanoparticle Interaction with the Lung.......................................................................22 1.4.4 Toxicity Review for Quartz and Titania......................................................................24 2 NANOSIZED ALUMINUM........................................................................................................30 2.1 What is Aluminum?.......................................................................................................... ......30 2.2 Aluminum Nanoparticles..................................................................................................... ...31 2.2.1 Economical and Social Impact.....................................................................................31 2.2.2 Synthesis of Nanoaluminum........................................................................................32 2.2.3 Regarding the Oxide Layer on Aluminum Nanoparticles...........................................36 2.3 Toxicology Profile of Aluminum...........................................................................................41 2.3.1 Sources of Aluminum..................................................................................................41 2.3.2 Aluminum Assimilation into the Body........................................................................43 2.3.3 Aluminum Distribution in the Body............................................................................43 2.3.4 Aluminum Distribution in the Cells.............................................................................44 2.3.5 Systemic Effects Induced by Aluminum.....................................................................44 2.3.6 Open Questions and Knowledge Gaps.........................................................................48 3 CHARACTERIZATION OF NANOPARTICulate SYstems......................................................51 3.1 Particulate Systems of Interest for this Research...................................................................52 3.2 Before Dosage: As Received..............................................................................................52 3.2.1 Sampling................................................................................................................. .....52 3.2.2 Density, Surface Area, and Porosity............................................................................55 3.2.3 Size and Shape........................................................................................................... ..58 3.2.3.1 Imaging techniques............................................................................................59 3.2.3.2 Light scattering techniques................................................................................61 3.2.4 Surface and Bulk Chemical Composition....................................................................64

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7 3.2.4.1 FTIR..................................................................................................................65 3.2.4.2 X-ray photoelectron spectroscopy (XPS)..........................................................66 3.2.4.3 Energy dispersive spectrometry (EDS).............................................................66 3.2.5 Zeta potential: Surface Charge.................................................................................68 3.2.6 Crystalline Phase........................................................................................................ ..70 3.2.7 Solubility............................................................................................................... .......71 3.3 In Physiological Media: As Dosed.....................................................................................72 3.3.1 Sampling................................................................................................................. .....72 3.3.2 Particle Size Distribution.............................................................................................73 3.3.3 Surface Chemistry,.......................................................................................................73 3.3.3.1 Zeta potential in media......................................................................................74 3.3.3.2 Protein adsorption on aluminum nanoparticles.................................................74 3.3.4 Solubility ICP in media................................................................................................76 3.4 In Physiological Environment: After Dosage.....................................................................77 4 REACTIVITY MEASUREMENTS............................................................................................95 4.1 Isothermal Heat-Conduction Microcalorimetry Technique...................................................95 4.2 Aluminum Reaction in Aqueous Media: Size and Shape Effects..........................................96 4.3 Aluminum Reactivity in Physiological Media.......................................................................99 4.4 Aluminum Reactivity in Acidic Physiological Environments.............................................100 5 IN VITRO BIOASSAYS...........................................................................................................105 5.1 Cell Line: A549............................................................................................................ ........105 5.2 Issues When Dealing with Nanoparticles and Classical In vitro Bioassays.........................106 5.3 Bioassays: Cell Death and Possible Mechanisms................................................................106 5.3.1 Cytotoxicity Detection (LDH)...................................................................................108 5.3.2 Apo-ONE Homogeneous Caspase-3/7.......................................................................109 5.4 Transmission Electron Microscopy: Sample Preparation....................................................109 5.5 Experimental Approach...................................................................................................... ..110 5.5.1 Toxicity Framework for Aluminum Nanoparticles...................................................110 5.5.2 Size and Shape Effect................................................................................................113 5.5.3 Particle-Cell Contact Effect.......................................................................................115 5.5.4 Fully Oxidized Particles: Particle Loading Effect.....................................................116 5.5.5 Ph Enzymatic Activity.............................................................................................117 6 CONCLUDING REMARKS.....................................................................................................132 6.1 Summary.................................................................................................................... ..........132 6.2 Conclusions................................................................................................................ ..........133 6.3 Recommendations For Future Work....................................................................................133 LIST OF REFERENCES............................................................................................................. ......136 BIOGRAPHICAL SKETCH............................................................................................................ .153

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8 LIST OF TABLES Table page 3-1 Common errors associated with powder sampling....................................................................79 3-2 Absolute density measurements of the powders investigated....................................................79 3-3 Specific surface area and calcula ted mean particle diameter.....................................................79 3-4 Size range and shape of the particles from image analysis........................................................80 3-5 Elemental surface composition from XPS analysis...................................................................80 3-6 Isoelectric points and zeta potentials ( ) in different environments..........................................80 3-7 Crystalline phase identifie d experimenta lly by XRD................................................................80 3-8 Particle solubility of aluminum nanoparticles incubated in cell culture media (ppm) during two different time intervals................................................................................................... .....81 3-9 Mass spectrometry results: most abundant prot eins found adsorbed to the surface of Al 2 particles...................................................................................................................... ................81 4-1 Heat of reaction in water for the different aluminum powders investigated............................102 5-1 Differences between necrosis and apoptosis............................................................................119

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9 LIST OF FIGURES Figure page 1-1 Length scale for objects in the nano and micron worlds............................................................27 1-2 Possible mechanisms by which nanomaterials interact with biological tissue..........................28 1-3 Biokinetics of nano-sized particles........................................................................................ ....29 2-1 Top-down and bottom-up approaches to nano-synthesis in the context of man-made processes as well as in the physiological environment..............................................................49 2-2 Atomic model of the face centered cube Al lattice and the adsorption of an oxygen molecule on the surface........................................................................................................ .....49 2-3 HRTEM of an aluminum nanoparticle.......................................................................................50 3-1 Particle size distributions of Al 3 measured by light laser diffraction.......................................82 3-2 High Resolution TEM images of NanoTek TiO2.......................................................................82 3-3 High Resolution TEM images of P25 TiO2...............................................................................83 3-4 Scanning Electron Microscope images of Min-U-Sil 5 quartz..................................................83 3-5 High Resolution TEM images of Al 1.......................................................................................84 3-6 High Resolution TEM images of Al 2.......................................................................................84 3-7 Scanning Electron Microscope images of Al 3..........................................................................85 3-8 Scanning Electron Microscope images of Al 4..........................................................................85 3-9 Electron Microscope images of Al 5......................................................................................... .86 3-10 Particle size distributions of the TiO2 and quartz powders as received by laser diffraction..86 3-11 Particle size distributions of the different aluminum powders as received measured by laser diffraction.............................................................................................................. ............87 3-12 Particle size distributions of the aluminum and quartz powders as received measured by dynamic light scattering....................................................................................................... ......87 3-13 Infrared (IR) reflection-absorption spect ra of the different aluminum samples........................88 3-14 Typical EDS spectrum from the TiO2 particles..........................................................................88 3-15 Typical EDS spectrum from the quartz particles.......................................................................88 3-16 Typical EDS spectra obtained form the different aluminum powders.......................................89

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10 3-17 Distribution of hydrolysis products (x, y)................................................................................ ..90 3-18 Particle size distributions as % number for Al 1 and Al 2 in water and in media.....................91 3-19 2D-gel showing a typical gel electrophoresis result from the exposure of Al 80 nm particles to culture media in under different experimental conditions.....................................................92 3-20 A549 cell exposed to Al 2 (80 nm) for 24 hrs...........................................................................93 3-21 Nanoparticles Al 2 outside an A549 cell in a 24 hrs exposure..................................................93 3-22 Image from TEM of some Al 1 nanoparticles outside the cells after a 12 hrs exposure...........94 4-1 Thermal Hazard Technology RC calorimeter design............................................................103 4-2 Heat output from the reaction in water of the different aluminum powders tested.................103 4-3 Heat output from the reaction in culture media (RPMI 1640) of the different aluminum powders tested................................................................................................................. .........104 4-4 Heat flow from the reaction of 50 nm aluminum in different media.......................................104 5-1 A549 cell in normal growth conditions under the light microscope at 63X magnification. A partial section of some neighboring cells can be observed......................................................120 5-2 Experimental protocol used for the bioassay test.....................................................................120 5-3 Release of LDH from the time course experiments.................................................................121 5-4 Release of LDH from the concentration course experiments..................................................121 5.5 Caspases 3/7 activity at 4 hours exposure in concentration course.........................................122 5.6 Caspases 3/7 activity at 48 hours exposure in concentration course.......................................122 5-7 Electron Microscope images of nanoparticle uptake by A549 cells........................................123 5-8 Representation of the major forms of cellular endocytosis......................................................123 5-9 Histopathology of A549 cells after nanoparticle uptake..........................................................124 5-10 Typical results of LDH release from a time course for A549 cells exposed to 250g/ml of different size aluminum particles.............................................................................................124 5-11 Typical results of LDH release from a concentration course after a 48 hrs exposure to different size aluminum particles.............................................................................................125 5-12 Electron Microscope images showing aluminum nanoparticle phagocytosis by A549 cells..125 5-13 Electron Microscope images showing aluminum nanoparticle accumulation inside A549 cells.......................................................................................................................... ................126

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11 5-14 Electron Microscope images of A549 cells after exposure to aluminum particles..................126 5-15 Endocytosis of aluminum flakes by A549 cells.......................................................................127 5-16 Electron Microscope images showing the uptake of quartz particles by A549 cells...............127 5-17 Cross section of a cell culture well showing the filter insert used to isolate the nanoparticles from the cells surface......................................................................................................... ......128 5-18 Release of LDH from cell exposure to the aluminum reaction products trough a filter insert.128 5-19 Release of LDH at after 48 hrs exposure to the unreacted and reacted particles.....................129 5-20 Transport mechanism of endocytosed materials from the extracellular fluid to the lysosomes inside the cell................................................................................................................ ...........129 5-21 Release of LDH for particle exposure with different Bafilomycin A1 treatments after 24 hrs exposure....................................................................................................................... ............130 5-22 Release of LDH for particle exposure with different Bafilomycin A1 treatments after 48 hrs exposure....................................................................................................................... ............130 5-23 Transmission Electron Microscope images confirming endocytosis after baf A1 treatment..131 6-1 Sketch of the in vitro toxicity mechanism for aluminum nanoparticles established in this research....................................................................................................................... .............135

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IN VITRO TOXICITY ANALYSIS OF NANO SIZED ALUMINUM: PARTICLE SIZE AND SHAPE EFFECTS By Maria Palazuelos Jorganes May 2007 Chair: Richard B. Dickinson Major: Chemical Engineering Nanostructured materials promise to revolutionize many key areas of science and technology. As our ability to manipulate matter at the nanoscale increases, there is a need to assess the effects of these materials on human health and the environment. Materials at the nanoscale are interesting and useful because they possess properties that are diffe rent from the equivalent bulk or molecular scale. These same properties can make toxicological profiles very different from those of the same materials on a different scale. There is a rising c onsensus that toxicity analysis of nanomaterials should start from a thorough physicochemical characterization of the materials under investigation in order to be able to establish a proper correlation between the nanoparticles characteristics and their effects and behavior in physiological environments. This research is a clear example of the necessity of comprehensive studies when investigating the toxicity of nanomaterials. Aluminum nanoparticles are being extensively used for their very unique energetic properties. These materials offer a very promising market that is fostering many startup companies which are expected to consolidate on strong technological positions. Aluminum is generally recognized as a non-toxic material to humans and it is widely used for applications which imply direct human contact. The effect of aluminum nanoparticles in human health is still an unknown. My research consisted of an in vitro toxicity screening of aluminum materials from nano to micron size, including spherical irregularly shaped pa rticles. Several issues relating to size, shape,

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13 detection and characterization of nanoparticles in the different environments relevant to in vitro toxicity analysis were addressed and suitable pr otocols were developed. Lung human epithelial cells were exposed to different concentrations of these materials and the effects were analyzed by means of various toxicity tests. Some of the materials i nvestigated caused elevated in vitro toxicity. Cells endocytosed the particles and a clear correlation between the particle size, shape and the effects observed was established. The hypothesized toxicity mechanism was explored using different analytical techniques. The detected toxicity of aluminum nanoparticles was demonstrated to be a direct effect of their reactivity inside the cells.

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14 CHAPTER 1 INTRODUCTION 1.1 Motivation and Research Outline The very rapid growth of nanotechnology in th e last few years promises great technological advances and application of nanomaterials in very diverse fields. Almost every scientific field and industrial sector is currently looking to the potential of materials at the nanoscale (Roco, 2005; The Royal Society & The Royal Academy of Engineering, 2004). With the implementation of new nanosize products the uncertainties about their effect on human health and the environment greatly justify the research efforts in this field (Dreher, 2004; Hoet et al., 2004; The Royal Society & The Royal Academy of Engineering, 2004; Thomas and Sayre, 2005). The research here presented was motivated by the lack of information about the inte raction and potential toxicities of nanoparticles at the cellular level. A comprehensive study was desi gned to establish the correlation between particle characteristics and their potential toxicity on an in vitro model. The rest of Chapter 1 includes a review of the current state and trends in nanotechnology and the field of nanotoxicology. Some of the more im portant findings regarding the toxic effects of nanomaterials are covered by an extensive literature review with a specific focus on inhalation and lung interactions relevant to this investigation. Interest in aluminum was stimulated by the Air Force which is exploring the use of nanoscale aluminum powders for energetic applications. Chapter 2 describes in detail the special properties of these materials, the different processes used for their production as well as what is known about the toxicity of aluminum. Knowledge of the materials tested is a prerequisite to fully understand their behavior in physiological environments. Chapter 3 illustrates the diverse techniques and methods used for the characterization of the different aluminum powders studied during this research. Aluminum is a reactive element passivated by an oxide coating that is less protective as size decreases into the nano range. However, once introduced in physiological media the particles are further protected (proteins and phosphate adsorbed onto the particle surface) and stay unreacted. When testing for in vitro toxicity it was found that aluminum nanoparticles were penetrating the cells and ending in cellular compartments (endosomes and lysosomes) meant for transport and digestion inside the cell. A correlation between particle size, shape and toxicity was established and it was hypothesized that the unreacted aluminum nanoparticles were being up taken by the cells and exposed to the acidic and catalytic activity of the enzymes contained in

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15 the lysosomes triggering the aluminum reaction inside the cells. This would ultimately be the cause for the observed cell death. To test this hypothesis microcalorimetry was c onducted to assess the reaction of aluminum in water vs. media (simulating extracellular environment) vs. acidic media (simulating the lysosome). Chapter 4 explains how these experiments were performed and the results collected. To further test on the hypothesis for in vitro toxicity the cell metabolism was modified to avoid the acidification of the endosomes and ultimately the enzymatic activity. Chapter 5 describes how the different biological experiments were designed and performed and the results obtained. Cell toxicity was reduced by suppressing the acidification of the endosomes and the enzymatic reactions despite the fact that aluminum nanoparticles were still penetrating the cell. Proof of hypothesis Q.E.D. Chapter 6 summarizes the findings of this research as well as suggestions for further investigations in this topic. 1.2 Nanotechnology: Definitions and Historical Context Nanotechnology definitions are as abundant as references to this topic. The National Nanotechnology Initiative (NNI) defines nanotechnolog y as the understanding and control of matter at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel applications. Encompassing nanoscale science, engineering and technology, nanotechnology involves imaging, measuring, modeling, and manipulating matter at this length scale (National Nanotechnology Initiative, 2007). The Royal Society and The Royal Academy of Engineering in the UK distinguish between nanoscience and nanotechnologies in their report Nanoscience and Nanotechnologies: Opportunities and Uncertainties published in July of 2004. In this report, nanoscience is defined as the study of phenomena and manipulation of materials at atomic, molecular and macromolecular scales, where properties differ significantly from those at a larger scale. The definition for nanotechnologies states that they are the design, characterization, production and application of structures, devices and systems by controlling shape and size at the nanometer scale. The word nanometer has its origin on two Greek expressions, nanos (dwarf) and metron (a measure). It describes the length unit equal to one billionth of a meter (1nm = 10-9m). Nanoscale

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16 things are naturally present in our environment and as artificial products of nanotechnology (see Figure 1-1.) Matter in this size range exhibits physical, chem ical, and biological properties that differ from those shown by individual atoms, molecules or bulk material. Properties of materials can be different at the nanoscale for two main reasons: larger relative surface area than the same mass of the same material in a larger form, and the surfacing of quantum effects at the lower end of the nanoscale. These two phenomena can cause nanomaterials to be more chemically reactive and potentially affect their optical, magnetic and electrical behavior. The proportion of atoms present at the surface compared to those in the bulk is much larger in nanomaterials giving them unique properties. Nanotechnology research and development are directed toward understanding and creating improved materials, devices, and systems that exploit these new properties. The first historical mention of nanoparticles and their use can be found more than 2000 years ago when ancient Chinese and Egyptians made carbon black as a byproduct of combustion and used it for its colorant properties. The definition of nanomaterials includes biomolecules like DNA and some other biopolymers; therefore, life itself is related to nanotechnology. However, it was not until 1959 when the concept of nanotechnologies was first laid out by the physicist Richard Feynman, in his lecture There Is Plenty of Room at the Bottom (Feynman, 1959). Feynman foresaw the possibility of manipulating and controlling things on a small scale. The term nanotechnology was not used until 1974 by Norio Taniguchi who described it as the ability to process, separate, consolidate, and deform materials by one atom or one molecule (The Royal Society & The Royal Academy of Engineering, 2004). Later on, in 1986, Drexler outlined the long-term potential for nanotechnology and the possible implications for humanity. From a bottom-up approach, the author builds universal assemblers from a proper arrangement of atoms in his book Engines of Creation (Drexler, 1986).

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17 Since then, the evolution of nanotechnology has been predicted to follow certain trends and several phases of development can be distinguished (Hood, 2004; Roco, 2005; The Royal Society & The Royal Academy of Engineering, 2004). At this time the most widespread use of these materials is in the form of passive nanostructures or simple particles designed to perform one task. Nanomaterials are being applied today in cosmetics, paints, clays, cutting tools and polymer composites to give a few examples. The second one entails active nanostructure prototypes being use for drug delivery, sensors, transistors and other special devices. This phase is being implemented currently and should fully develop in the next five to ten years. In the longer term more advanced devices that will respond to their environment with thousands of interacting components are envisioned in diagnosis and therapeutic tools, implants, nano-engineered filters and military equipment. The potential benefits that nanotechnologies could have to humankind are being considered and revised by leading experts in industry, academia and government and though concerns about the safety of engineered nanomaterials have been raised, several fundamental innovations are expected from nanotechnology (Roco et al., 2005d): Interdisciplinary teams of scientists, engineers and social experts are being fostered under the umbrella of nanotechnology research facilitating interchange of knowledge and findings. Nanotechnology could enable increased social connectivity by providing improvements in computing, sensing, communications, data storage, and display capabilities. Energy independence for major industrial nations cau sed by a host of efficiencies facilitated by nanotechnology is a feasible possibility. The trend in nanotechnology indicates that affordable nanoscale medical diagnostic and treatment devices will be available as well as advanced biomedical solutions to chronic diseases, and visualization of biological processes within the human body. Protection equipment for hazardous environments will benefit from nanosensors incorporation enabling them to adjust to vital signs as well as to exposure levels of toxic agents. Nanotechnology will greatly contribute to general economic growth. It is already a multimillionaire industry that is only expected to grow. Some nanomaterials used on information and

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18 biotechnologies could be worth $1 million/ton. Annu al production of these products could reach 1010,000 tons a year (The Royal Society & The Royal Academy of Engineering, 2004). 1.3 Nanotoxicology: A Discipline on Its Own With the rapid advancement in the field of nanotechnology, there has been increased concern regarding the potential risks associated with the widespread use of engineered nanomaterials (Dreher, 2004; Hoet et al ., 2004; The Royal Society & The Royal Academy of Engineering, 2004; Thomas and Sayre, 2005). Because of the increasing num ber of nanomaterials and the wide range of applications, research associated with the potentia l risk of nanoparticles to biological organisms has become a discipline on its own referred as nanotoxicology (Barnard et al., 2006; Donaldson et al., 2004; Hoet et al., 2004; Holsapple et al., 2005; Nel et al., 2006; Oberdrster et al., 2005b). Conducting reproducible and reliable toxicological studies with nanostructures is complicated by the behavior of particulate matter in biological settings and the difficulty in making in situ measurements of properties such as size, shape and surface chemis try. Because of this complexity, risk assessment of nanomaterials requires the close collaboration of experts in different fields like, toxicology, materials science, chemistry, medicine and molecular biology. Due to the wide variety of nanomaterials available, any extrapolation of the attributes of a particulate system to similar materials for the same or different organisms has to be made with caution. There are few attempts at constructing general principles that help the researcher approach these issues (Moghimi et al 2005; Powers et al., 2006; Roberts et al., 2004; Zhi et al., 2006,). The unusual physicochemical properties of nanomaterials are linked to their small size, chemical composition, surface structure, solubility, shape and aggregation. It is reasonable to believe that these unique properties will cause unidentified effects in biological systems. Figure 1-2 depicts the possible mechanisms of interaction between nanoscale materials and biological organisms and how different materials properties, like surface chemistry or solubility, can affect those interactions (Nel et al ., 2006). In fact, investigation in this

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19 field is splitting in two directions, possible toxi c effects as well as therapeutic and diagnostic applications of nanomaterials (Hoet et al., 2004; Moghimi et al., 2005). Particles in the nanosize range can enter the human body via several routes: respiratory system, gastrointestinal tract (GIT), skin and the circulator y systems. Contact with nanomaterials can occur in occupational exposures, by parenteral administrati on in medical applications and from ambient water and the food chain depending on the final fate of these materials in the environment. Nanotoxicology is also concerned with the possible translocation of these materials once they enter the organism. Because of their small size these nanoparticles and nanostructures are very likely to interact with cells, body fluids and proteins that can help in their migration throughout the human body (see Figure 1-3) to finally accumulate in target organs or to be eliminated through the normal excretion pathways (Donaldson et al., 2006; Oberdrster et al., 2005a). Until now there has been little clinically relevant evidence of engineered nanomaterials causing toxicity and the abundant studies found in the literature are still too scattered to draw meaningful conclusions. It is still to be determined if the distinctive properties of nanomaterials will introdu ce new mechanisms of injury and whether these will result in new pathologies. Biological systems respond to multiple pathways of injury in a limited number of pathological outcomes, such as inflamma tion, apoptosis, necrosis, fibrosis, hypertrophy, metaplasia and carcinogenesis (Holsapple et al., 2005; Nel et al., 2006). Even in the case scenario that nanomaterials will not cause new pathologies, they could introduce new mechanisms of injury that will require special tools, a ssays and approaches to assess their toxicity. While new tools and methods are being developed as the field matures, the investigation of potential toxicities of nanomaterials should not be delayed. It could start by applying the conventional study methods traditionally used to assess chemical toxicity. The community involved in nanoscience and nanotechnology has come to a consensus about the need of understanding and addressing the interaction of nanomaterials with biological systems. So far, the range of approaches and methods used to reach conclusion regarding the effects of

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20 manufactured nanomaterials and ultrafine particles has led to different results. This inconsistency indicates the urgent need for standardized tests in order to get comparable results in screening nanomaterials for potential adverse effects (Oberdrster et al., 2005a). Nanotoxicology should establish the principles a nd procedures that will ensure the safety of this technology for workers, consumers and the environment. Considering the large number of different nanomaterials produced and tested a nd the trend that this industry is following, toxicological studies should be predictive and prag matic. The goal of nanotoxicity research should be to develop a series of tests and assays that could predict the possible outcomes of interaction of a new material with biological systems. Ideally, a sounded database of information about the new materials tested will be collected and will allow a much fast er and economic classification of nanomaterials as safe or as possible hazards. In terms of regula tion of nanomaterials it is recommended (Balshaw et al., 2005; Nel et al., 2006) that decisions will be taken based on scientific evidence of toxicity, which should consider specific products or product lines and the likelihood of an exposure risk. 1.4 Toxicity of Ultrafine and Nanomaterials: Literature Review Although the term nanotoxicology and its use is relatively new, particle toxicology is a mature science that has investigated the effects of particulate matter on environmental and human health for the last 25 years (Donaldson et al., 2000). It is almost an impossible task to summarize the abundant literature existent about particle toxicity. Nonetheless, in this section a historical overview of the topic is given with a center of attention on the inhalation route of exposure and the in vitro cell interactions that ultimately relate to the investigation presented in this dissertation. 1.4.1 History of Particle Toxicology: Ultrafine Particles The first meeting presenting research in this field was held in Cardiff in 1979. The research done at that time mainly involved in vitro cytotoxicity (toxic effect on cells) analysis focusing on asbestos and other mineral dusts as well as ep idemiological studies of the affected population. Exposure to high concentrations of particulate matter in those years was mostly in mines and metal

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21 industries employees where workers developed respirat ory diseases like metal fume fever, asbestosis and silicosis (Wagner et al., 1982). Since then, the interest in particle toxicology has focused more on the effect that ultrafine particles (aerodynamic diameter <0.1 m) have on human health. The definition of ultrafine particles roughly overlaps with that of nanoparticles." The first expression, ultrafine particles, refers specifically to the aerodyn amic behavior of the particulates and it is mostly used for nanoparticles generated in an uncontrolled fashion. Meanwhile, the second term, nanoparticles, entails a true physical dimension of the particles and it generally refers to engineered nanomaterials. Air pollution is generated from natural sources like volcanic activity and forest fires and manmade sources from activities like heating, cooking, industrial manufacturing and the use of internal combustion engines. (The Royal Society & The Royal Academy of Engineering, 2004). Ultrafine particles are a component of urban envi ronmental air pollution. Epidemiological studies of the effect of air pollution show a link between morbidity and mortality and the amount of particulate matter. Laboratory-based studies through in vivo animal exposures and in vitro cell studies report findings of increased pulmonary inflammation, cytokine and chemokines release, production of white blood cells, oxygen-free radical production in the lungs, endotoxin mediated cellular and tissue responses, stimulation of irritant receptors and m odification of key cellular enzymes in response to ultrafine particles exposure (Nel, 2005; Oberdrster et al., 2005a). 1.4.2 Guidelines to Risk Assessment One of the qualities of nanomaterials is that they can be synthesized in highly homogenous forms with desired sizes, shapes and surfaces properties. What is an advantage in terms of application is a challenge in terms of their risk assessment. Risk assessment of any material traditionally is an evaluation of the toxicity inherent to that materi al, the probability of exposure, and the dose-response data available for that material (Balshaw et al., 2005; Environmental Protection Agency, 1993). Dealing with the unique properties of nanomaterials means an unfeasible prediction of their possible

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22 effects in biological systems based on the knowledge available for classical bulk materials. Limited research has been done up to this day in terms of correlating biological effects of engineered nanomaterials with properties like size, shape or surface charge. Smaller particles result in a larger surface area and number of particles per unit mass, increasing their potential for biological interactions (Hoet et al., 2004; Oberdrster et al., 2005a). In terms of probability of exposure of nanoparticles, it is important to define measures for exposure that describes their hazards (Donaldson et al., 2001). Donaldson et al., provide possible candidates for measures of exposure to ambient particulate matter (PM10) considering inhalation as the route of exposure: Physical measures Total airborne mass concentration ( g/m3) Particle number concentration (number of particles/m3) Specific surface area (m2/m3) Chemical measures Polycyclic aromatic hydrocarbon (PAH) concentration ( g/m3) Sulphate concentration ( g/m3) Factors to consider for risk management How does PM10 exert its harmful effects on the lungs? How practical is to carry out the measurement routinely? What health endpoint is of interest? In the case of nanomaterials, other factors like state of dispersion, surface charge, ability to deliver transition metals, solubility, particle-cell interactions, and possibility of migration throughout the body, to mention a few examples, are being examined within the disciplines of nanotoxicology and nanomedicine. A better understanding of the biological interactions of nanoparticles will allow a more predictive risk assessment of these materials instead of a recompilation of epidemiological data from harmful exposures that could had been avoided otherwise. 1.4.3 Nanoparticle Interaction with the Lung One of the possible routes of exposure to nanoparticles for humans is inhalation, lungs are the largest surface-area organ the in the organism. The lungs consist of two different parts: airways (transporting the air in and out the lungs), and alveoli (where the gas exchange occurs). Human lungs

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23 contain about 2300 km of airway and 300 million alveoli. The surface area of the lungs is about 140 m2 in adult humans. The airways are an active epithelium protected with a viscous layer of mucus and behave like a robust barrier. The air in the alveoli, on the contrary, is only separated from the blood stream by a 500 nm layer of epithelial cells and extracellular matrix. Spherical solids can be inhaled when their aerodynamic diameter is less than 10 m (PM10). The smaller the particles, the deeper they can travel into the lungs. When particles are smaller than 2.5 m (PM2.5), they can reach the alveoli. Nanoparticles are deposited mainly in the alveolar region. Fibers are defined as solid materials with a length to diameter radio of at least 3:1. Their penetration into the lung depends on their aerodynamic properties. Fibers with a small diameter will penetr ate deeper into the lungs, while very long fibers (>>20 m) are predominantly stuck in the higher airways (Hoet et al., 2004). The likelihood of retention or clearance of inhaled particles is dependent on several factors: (1) the site(s) of particle deposition; (2) the quantity of particles deposited; (3) the physicochemical characteristics of the particles; (4) and the partic le-cell interactions. Particles deposited in the upper airway will be more rapidly cleared than those accumulated in the alveoli (Tran et al., 1999). Clearance mechanisms are different for the different s ections of the respiratory airway. The different mechanisms are due to two processes: (1) physical clearance (mucocilary movement, macrophage and epithelial phagocytosis, interstitial translocation, lymphatic drainage, blood circulation and sensory neurons); (2) and chemical processe s (dissolution, leaching and protein binding) (Oberdrster et al., 2005a). The phagocytosis of fibers and particles, happening in the deep alveolar region, results in the activation of macrophages and induces the release of cytokines, chemokines, reactive oxygen species (ROS), and other mediators that can result in sustained inflammation and eventually fibrotic changes. The physical and chemical properties of the solid materials reaching the alveoli can affect the phagocytosis efficiency, thus increasing the retention half time of those materials and allowing

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24 interaction with the pulmonary epithelial cells (Hoet et al., 2004). Research regarding the interaction of nanoparticles with lung tissue is being actively pursued. 1.4.4 Toxicity Review for Quartz and Titania In the context of the research concerning this dissertation, two materials other than nanosize aluminum were used as reference controls. Quar tz and titania are commonly used as positive and negative controls respectively for toxicity studies In this section, a literature review about the biological effects found for these two materials is presented. Quartz: Silica (SiO2) can occur in non crystalline (amorphous) or in crystalline forms. Crystalline silica may be found in more than one form being alpha form the most abundant. This form is so abundant that the term quartz is often used in place of the general term crystalline silica. Quartz is a component of nearly every mineral deposit. Exposure to silica has been reported from many different industries and activities like, agriculture, mining, milling, construction, glass, cement, abrasives, ceramics, foundries, machinery, rubber and plastics, paint, etc. The exposure to respirable crystalline silica is associated with silicosis (a type of nodular pulmonary fibrosis) and other silica related diseases such as pulmonary tuberculosis, lung cancer, and chronic obstructive pulmonary disease (National Institute for Occupational Safety and Health, 2002). The International Agency for Research on Cancer (IARC) and the National Toxicology Program (NTP) have both listed respirable crystalline silica as a carcinogen to humans. The NI OSH exposure limit for respirable crystalline silica is 0.05 mg/m3. Numerous epidemiological studies are available that relate cumulative crystalline silica exposure data to the incidence of silicosis and other silica-related diseases. For example, from these studies (National Institute for Occupational Safety and Health, 2002), 1 to 7 silicosis cases are predicted per 100 workers to occur at concentrations of 0.025 mg/m3 over a 45year working lifetime. Despite the evidences of a correlation between respirable crystalline silica and the higher rate of respiratory diseases associated with it the exact mechanisms of the quartz toxicity are still unknown. Castranova (2000) reviewed in vitro and in vivo studies that reported possible

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25 mechanisms involved in the development of lung diseases associated with long term exposure to coal mine dust and crystalline silica. The results found supported four basic mechanisms of toxicity: Direct cytotoxicity of coal dust or silica, resulting in cell damage, release of lipases and proteases, and eventual lung scarring. Activation of oxidant production by pulmonary phagocytes, such as alveolar macrophages. When oxidation production exceeds antioxidant defenses, lipid peroxidation and protein nitrosation occur, resulting in tissue injury and consequent scarring. Activation of mediator release from alveolar macrophages and alveolar epithelial cells. Chemokines recruit leukocytes and macrophages from the pulmonary capillaries into the air space. Once there, these leukocytes are activated by proinflammatory cytokines to produce reactive species increasing oxidant injury and lung scarring. Secretion of growth factors from alveolar macrophages and epithelial cells that stimulate fibroblast proliferation and induces fibrosis. The surface reactivity and characteristics of the quartz particle has been linked to the toxic effects observed (Borm et al., 2001). The use of freshly fractured quartz fine sized particles, like Min U Sil, as a positive control for in vitro cytotoxicity studies is a widely accepted practice (Tsuji et al., 2005). Titania: Titanium dioxide (TiO2), also named titania, is a noncombustible, white, solid, crystalline, odorless powder. TiO2 can occur in different crystalline forms, rutile and anatase are two of the most common ones. TiO2 does not absorb visible light but strongly absorbs ultraviolet (UV) radiation. Titania is used mainly in paints, varnishes, lacquer, paper, plastic, ceramics, rubber, and printing. It is widely used as white pigment b ecause of its high refractive index. The occupational exposure to TiO2 is regulated by OSHA (Occupational Safety and Health Administration) under the permissible exposure limit (PEL) of 15 mg/m3 as total dust (over and 8 hour period) and 5 mg/m3 as respirable dust (National Institute for Occupa tional Safety and Health, 2005). In summary, few TiO2-related health effects have been identified in case reports. Lung particle analysis indicated that workers exposed to respirable TiO2 can accumulate particles in their lungs that may persist for year after the exposure has ended. Titania deposited in the lungs was often contaminated with other

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26 agents, most commonly silica, at much lower concentration than TiO2 particles. The chronic tissue reaction to lung deposited titania is distinct from chronic silicosis. Most cases presented a local macrophage response with associated fibrosis that wa s generally mild, but of variable severity, at the site of deposition. Overall, the available epidemiological studies of TiO2-exposed workers present a range of environments, from industry to population based. In general, these studies provide no clear evidence of elevated risk of lung cancer mortality or morbidity among those workers exposed to titania dust (National Institute for Occupational Safety and Health, 2005). On the contrary some animal studies have shown very different results in term of lung overload, inflammation and cytotoxicity depending on the animal model used (Bermudez et al., 2004). The lung clearance rate and nano P25 TiO2 toxicity in rodent species seems to follow an inverse relationship. Faster pulmonary clearance was correlated to less lung toxicity. Multiple studies suggest tumor response in rats exposed to ultrafine TiO2. The mechanism associated seems to be a secondary genotoxicity involving chronic inflammation and cell proliferation rather than direct genotoxic effect of the TiO2 particles (National Institute for Occupational Safety and Health, 2005). Lung clearance of particles in humans is greater than in rats. The origin of the lung cancer in rats indicates particle lung overloads not relevant for humans though the possibility of migr ation of the nanoparticles to other organs is a greater concern for humans (Borm et al., 2004, Oberdrster et al., 2005). The species differences become critical when extrapolating results from animal models to humans. There are studies indicating the greater inflammatory response in animal studies to ultrafine than to fine titania particles (Baggs et al., 1997, Ferin et al., 1992, Oberdrster et al., 2005). Nevertheless, TiO2 particles are found to be rather benign in terms of cell death ad metabolic activity and are commonly used as a positive control for in vitro toxicity (Oberdrster et al., 2005).

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27 Figure 1-1. Length scale for objects in the nano and micron worlds. Nanometer components are found in nature as well as products of engineered fabrication. Adapted from original provided by the Office of Basic Energy Sciences (BES) http://www.er.doe.gov/bes/scale_of_things.html Last accessed March 4, 2007.

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28 Figure 1-2. Possible mechanisms by which nanomaterials interact with biological tissue. Examples illustrate the importance of material composition, electronic structure, bonded surface species (e.g., metal-containing), surface coatings (active or passive), and solubility, including the contribution of surface species and coatings and interactions with other environmental factors (e.g., UV activation). Reproduced with permission of AAAS from Nel, A., Xia, T., Madler, L., and Li, N. (2006). Toxic potential of materials at the nanolevel. Science 311 624.

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29 Figure 1-3. Biokinetics of nano-sized particles. While many uptake and translocation routes have been demonstrated, others still are hypothetical and need to be investigated. Largely unknown are translocation rates as well as accumulation and retention in critical target sites and their underlying mechanisms. These as well as potential adverse effects will be largely dependent on physicochemical charact eristics of the surface and core of nanosized particles. Both qualitative and quantitative changes in nano-sized particles' biokinetics in a diseased or compromised organism need also to be considered. Reproduced with permission of Creative Commons Attribution License from Oberdrster, G., Maynard, A., Donaldson, K ., Castranova, V., Fitzpatrick, J., Ausman, K., Carter, J., Karn, B., Kreyling, W., Lai, D., Olin, S., Monteiro-Riviere, N., Warheit, D., and Yang, H. (2005b). Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Particle and Fibre Toxicology 2: 8.

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30 CHAPTER 2 NANOSIZED ALUMINUM The main purpose of the research presented in th is dissertation is to investigate the correlation between size and shape of aluminum nanoparticles and their effects on toxicity in vitro in a human lung cell line. In this chapter, nanosize aluminum relevance is reviewed in depth, from the different methods of manufacturing and possible applications to the potential toxicities of this metal. 2.1 What is Aluminum? Aluminum is the most abundant metal and the third most abundant element, after oxygen and silicon, in the earths crust. It is element 13 on the periodic table (second row of Group III) and it has a molecular weight of 26.98 g/mol, which makes it a very light metal. It is widely distributed and constitutes approximately 8% of the earths surf ace layer. Aluminum however is a very reactive element and it is never found as free metal in nature. It is found combined with other elements, most commonly with oxygen, silicon, and fluorine. These chemical compounds are normally found in soil, minerals (e.g., sapphires, rubies, and turquoise), rocks (especially igneous rocks), and clays. These are the natural forms of aluminum rather than the silvery metal. The metal is obtained from aluminum containing minerals, primarily bauxite. Small amounts of aluminum are also found in water in dissolved or ionic form. The most commonly found ionic forms of aluminum are complexes with hydroxyl ions (ATSDR, 1999). Aluminum has low density, high electric and thermal conductivities, high reflectivity and high corrosion resistance. Many of the common applications of aluminum rely on the durability of the material after the aluminum atoms on the surface of the metal quickly combine with oxygen in the air to form a thin, strong and protective coating of aluminum oxide or alumina. Because aluminum is very soft it is usually mixed with small amounts of other metals to form aluminum alloys, which are stronger and harder. This allows the use of aluminum in beverage cans, pots and pans, airplanes, siding and roofing, and foil. Aluminum compounds are found in many consumer products such as

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31 antacids, astringents, buffered aspirin, food additives, and antiperspirants. Aluminum metal powders were often used in explosives and fireworks (ATSDR, 1999) and more currently they are a main component of rocket propellants, munitions and composites with unique properties (Lowe, 2002). 2.2 Aluminum Nanoparticles 2.2.1 Economical and Social Impact There are three big subcategories in the nanomaterials business; nanoclays or nanocomposites, nanoscale metals and oxides, and carbon nanomaterials as nanotubes (Wood et al., 2002). Most of the optimistic predictions for the nanotechnology re volution can already be seen in the current successes and trends of nanometals. Metals are the worlds oldest technological materials and due to their wide applications and enhanced properties when in nanosize form, they were among the earliest nanomaterials to be commercialized. The nanometal s industry includes nanoparticles, nanolayers and thin films, nanofibers, and bulk nanostructured metals and alloys. The development of metal nanoparticles had been decelerated by the fact that their high surface energy and reactivity makes them dangerous to produce and handle. Only recent advances in the technology allow for the production and safe handling of these materials in significant quantities (Lowe, 2002). The market is dominated by simple metal oxides, such as silica, alumina and titania, and nanoscale metals such as aluminum. These particles are about 83% of the world nanoparticle market with an estimated value of around $900 millions (Wood et al., 2002). Aluminum powders and granules (diameters 1mm) worldwide annual sales are estimated to be 200K tons per year. The major businesses involved in this market are the metallurgical, chemical, and paint and pigment industries. The value of aluminum powders is so dependant on size that the difference in price between the bulk material and the nanoparticles is over two orders of magnitude from roughly $1/lb to $700/lb respectively (Kearns, 2004). These prices are expected to drop as demand increases and production processes are scaled up. The promising market and applications of

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32 these materials is fostering many new startup companies, which are expected to consolidate under bigger firms with a strong technology position (Wood. et al 2002). One of the unique properties of nanoaluminum is the vast quantity of energy stored in this material. According to the Director of the Weapons and Materials Research at the US Army Research Laboratory, energetic materials and ingredients that are produced on the nanoscale have the promise of increased performance in a variety of ways including, sensitivity, stability, energy release and mechanical properties (Miziolek, 2002). Thus, the areas of greatest interest for the application of nanoaluminum are fuels for space and naval vehicles and propellants for the military. Aluminum powder is used today in solid rocket boosters and there is an ongoing drive to reduce launching costs and increase payload. The increase in burn rate is between 2 and 10 times, when using nano versus regular aluminum powder according to the claim from Argonide that their nanoaluminum Alex doubles the burning rate and increases maneuverability and thrust compared to standard 20 m sized spherical aluminum powder (Kearns, 2004; Rai et al., 2004). In the military context, there is interest in the potential of the turbulent reaction of finely divided aluminum in contact with water to propel super-cavitating naval vehicles and weapons and in the super-thermitic reactions for pyrotechnics, primers and detonators (Lowe, 2002; Kearns, 2004). 2.2.2 Synthesis of Nanoaluminum There are several industrial methods for manufacturing of nanomaterials that can be subdivided in two categories: top-down, and bottom-up. The first one refers to the size reduction of larger size material fragments and/or devices by means or methods such as grinding or etching. Nano thin aluminum is an important component of computer chips and new nanodevices like nanoelectro-mechanical system (NEMS). The second one involves the synthesis of larger structures from chemical precursors or assembly of molecules. The manufacturing of nanoparticles usually requires a bottom-up approach (The Royal Society and The Royal Academy of Engineering, 2004). The current technology used for manufacturing is mostly a combination of both approaches in which bulk

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33 materials are reduced to the atomic level to then grow the nanoparticles and/or nanostructures from those atomic units by condensation, agglomeration, cr ystallization, etc. This evolution is illustrated in Figure 2-1 which also depicts the presence of nanomaterials in biological environments and the manmade manufacturing being inspired by the smaller molecules in living organisms. The chemical synthesis is generally used to produce nanoparticles, the process can occur in two different phases: gas, or liquid. The focus materials of this research consist of aluminum, which is normally transformed into nanoparticles using gas-phase techniques. Due to its very high reactivity any liquid phase method results in an aluminum oxide/hydroxide layer around the particles. Particle synthesis by reaction in the gas phase starts with seed generation, usually by a burst of homogeneous nucleation when reactions produce condensable products or when volatile products are quenched. This may be physical nucleation of a single speci es, but often involves complex chemical reactions. Once the seed particles are present, they may grow by condensation or physical vapor deposition, chemical vapor condensation, or coagulation. Coagulation dominates when the number concentration of particles in the nucleation burst is large, which is normally the case in industrial powder synthesis reactors (Masuda et al., 2006). The production market for nanomaterials is highly competitive and has originated many companies and patents around specific manufacturing processes. In the case of nanoparticles there is also a very dynamic demand on these products that can rapidly change. Until now, the scale-up in production of nanoparticles is limited for several reasons. Any new process or technology must be able to exceed (in terms of economic value) what is already in place, and it must be of value (or perceived value) to the consumer. The technology used in current industrial processes is already generally very advanced, and so nanotechnologies will only be used where the benefits are high. There are also technical barriers that start with the difficulty of scaling a process up from the laboratory to an industrial setting. These barriers in clude inadequate characterization and measuring tools and capabilities to enable on-line and in-line monitoring and processing control based on

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34 nanoscale features. Along side the technical barriers there are those related to regulation such as classification and standardization of nanomaterials and processes, and the management of any health, safety and environmental risks that may emerge (The Royal Society and The Royal Academy of Engineering, 2004). Different companies have manufactured the different size aluminum nanoparticles used for this research. The specific details about the production steps used by each company are proprietary but general outlines of their processes are provided as follows: QSI-NanoTM Process by Quantum Sphere Inc.: This company uses an adaptation of the gas phase condensation method. Metal is vaporized using resistance heating at a temperature above the boiling point of the material, until a sufficient rate of vaporization is achieved. By computer control of the metal flux, chamber pressure, temperature and gas flow, nanopowders having the desired size and particle distribution can be easily made at production rate desired. According to the company the resistance heated vapor condensation method provi des the best quality powder having the lowest level of agglomeration and fewest impurities (Quantum Sphere, 2006). Pulsed Plasma Process by Nanotechnologies Inc.: Aluminum rods, such as alloy 1350 or greater purity, are the starting material. These electrodes are placed inside a sealed vessel end-to-end with a small gap between the ends. The sealed vessel is filled with carefully control atmosphere of inert gases such as helium or argon. A very high electrical current (> 5 kV and >50 kA) is pulsed through the electrodes for about a millisecond. The ends of the electrodes ablate into aluminum plasma with a +2 or more energy state, which expands rapidly into the gasses. As the plasma ball expands it cools and falls back to natural valence state, reforming aluminum nanoparticles. The aluminum nanoparticles are collected while they are still in the inert atmosphere, because the particles at this point are extremely pyrophoric. Small amounts of oxygen are then added very slowly to create a passivation layer of aluminum oxide on the surface of the particles. This layer allows the production workers to handle the particles in normal air, though still with caution. If the oxygen is

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35 added to quickly, the resulting exothermic oxidation causes the particle to burn. Adding the oxygen slowly allows the particle oxidation rate to be diffusion-limited (Nanoscale, 2006). Inert Gas Atomization by Valimet Inc. and Toyal America Inc.: Both companies use variations of the same method to produce micron size high purity aluminum particles. Atomization is the process used commercially to produce the largest tonnage of metal powders. The raw material is melted and then the liquid metal is broken into individual particles. To accomplish this, the melt stock, in the form of elemental, multi-element metallic alloys, and/or high quality scrap, is melted in and induction, arc, or other type of furnace. After the bath is molten homogenous, it is transferred to a tundish which is a reservoir used to supply a constant, controlled flow of metal into the atomizing chamber. As the metal stream exits the tundish, it is struck by a high velocity stream of the atomizing medium (water, air or inert gas as in the case of aluminum). The molten metal stream is disintegrated into fine droplets that solidify during their fall through the atomizing tank. For aluminum atomization a small amount ( 3%) of oxygen is added to the atomizing gas in order to produce a passivated surface on the powder being produced (Antony et al., 2003, Masuda et al., 2006). Vacuum Deposition Technology by Sigm a Technologies International Inc.: High aspect ratio aluminum flakes, nano size in height, are produced with this technology. The vacuum deposition involves simultaneous deposition of polymer and various metal and ceramic coatings, on a rotating drum. Thousands of layers are deposited at high speed forming a multilayer nanocomposite material. The bulk nanocomposite is removed from the drum and it is reduced into a fine polymer/metal or polymer/ceramic powder, which for some applications may be further reduced to a nanoflake pigment. The polymer is designed to dissolve in a solvent, leading to the production of metal pigment (usually aluminum), composed of individual flakes, that have a cross sectional diameter of 5-20 m and a thickness of about 20-30 nm (Sigma Technologies Inc., 2006).

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36 2.2.3 Regarding the Oxide Layer on Aluminum Nanoparticles When reducing particle size to the nanometer range the ratio of surface area to volume for the same mass basis is significantly increased. Thus, surface physical and chemical properties are decisive in understanding the behavior of nanomateria ls. Aluminum is a very reactive element, with a high heat of combustion ( -31 kJ/g), and some of its reactions occur with explosive violence. On the other hand, the rapid formation of a thin oxide layer prevents the further attack by oxygen and retards chemical reactions of the aluminum such as with acids (Ramaswamy et al., 2005). Many researches interested in the combustion and energetic applications of nanoaluminum agree that the characteristics and integrity of this oxide layer defines most of the thermo chemical behavior of this material (Aunmann et al., 1995; Gromov et al., 2006; Ramaswamy et al., 2004, 2005; Schultze et al., 200). The thickness, composition, and structure of the oxide layer on the particles are responsible for the main differences in powder performance due to heterogeneous oxidation. The thermo chemical activity of the powders is related to the method used for powder production, the storage time and conditions, the particle size distribution, the specific surface area, the oxide layer thickness on the particles and the total metal content in the powder (Gromov et al., 2006). Generally the oxide layer is considered to be nonporous, very different from iron metal, which forms a porous oxide layer or rust, easily penetrable by water and allowing corrosion beneath the superficial layer of rust (Ramaswamy et al., 2005). Aluminum metal oxidizes very easily due to the large free energy of formation for the oxide ( -378 kJ/mol of Al2O3) (Askeland, 1989). The type of oxide film determines the rate at which oxidation occurs and weather the oxide causes the metal to be passive. The relative volumes of the oxide and the metal define three different types of behavior. This ratio is described by the PillingBedworth equation for the following oxidation reaction: m nm n2 2O M O M (2-1)

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37 ) )( ( ) )( ( atom per volume metal atom metal per volume oxide ratio B Poxide metal metal oxideM n M (2-2) Where M is the atomic or molecular mass, is the density, and n is the number of metal atoms in the oxide as defined in equation 2-1. If the Pilli ng-Bedworth (P-B) ratio is less than one the oxide occupies a smaller volume than the metal from which is formed. Tensile stresses develop in the oxide film, causing the film to crack and become porous. Oxidation can then continue rapidly. If the ratio is equal to one, the volumes of the oxide and the metal are equal and the filmed formed is considered to be adherent, nonporous and protective. In most cases, the oxide film tends to be protective until the P-B ratio exceeds about two. If the P-B ratio is greater than two, the oxide volume is greater than that of the metal and as the thickness of the film increases, high compressive stresses develop in the oxide. The oxide may flake from the surface, exposing fresh metal, which continues to oxidize (Askeland, 1989). In the case of the aluminum, the P-B ratio depends on the density of the possible oxides forms around the metal core. Considering the following values for molecular masses and densities in equation (2-2): Moxide = 101.963 g/mol, Mmeta l= 26.982 g/mol, metal = 2.7 g/cm3, and the density of aluminum oxide varying from 3.0 g/cm3 for low density amorphous Al2O3, to 3.98 g/cm3 for the crystalline phase of Al2O3 (Gutierrez et al., 2002), the P-B ratio values range from 1.7 to 1.3. According to the P-B ratio equation (2-2) then, the oxide layer around aluminum will be of more protection in the crystalline form than in the amorphous phase. For application, passive films should be stable Nonetheless, a typical feature is their variability under various conditions. According to Schultze et al (2000), the most important processes on metal oxides are given by: Growth (transfer of oxygen from the electrolyte or surrounding gas into the oxide) Corrosion (transfer of metal ions from the oxide into the electrolyte) Reduction (possible at very negative potentials or in reductive environments)

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38 Chemical dissolution (when the growth by oxidation equals the degradation by corrosion) Modification (intended and controlled, or not controlled as a result of changing conditions) Electron transfer reactions (hydrogen or oxygen evolution) Capacitive charging In the context of toxic effects of nanoparticles in biological environments as humans, animals or ecosystems it is important to consider the possibility that the passivation from further reaction for metals like aluminum might not be adequate once the nanoparticles are exposed to the unique conditions of physiological fluids. Modern and more advanced analytical techniques have allowed a more detailed observation of the atomic layer around the aluminum nanoparticles and a deeper understanding of the mechanisms involved in its formation, growth and implications in the reactivity of these materials. Following, a summary of a model proposed by Ramaswamy et al. in 2005 is included to illustrate the phenomena occurring at the atomic level on the surface of aluminum nanoparticles as oxidation occurs. Aluminum has a face-centered cubic lattice with the unit cell parameter a of 4.05 According to this model oxygen adatoms (adsorbed atoms) are able to move through the aluminum interatomic space to fill the lattice until electric char ge equilibrium is obtained and no further atoms can penetrate. The face-centered cubic unit cell of crystalline aluminum consists of single Al atoms located at the eight corners of the cube plus another atom centered in each face of the unit cubic cell. This structure leaves available octahedral sites ( 1.20 in diameter) with enough space for an oxygen molecule (2.4 in total length, or 4 oxygen atomic radiuses (0.6 )) to enter the surface on a vertical orientation. When a clean aluminum metallic surface (free from oxygen and oxide) is exposed to air or oxygen, oxygen molecules will a ttach themselves by physical adsorption (van der Waals forces) almost instantaneously (Figure 2-2). The oxygen molecule becomes clamped and permits the second phase of the reaction, a combined chemisorption and dissociation of the oxygen

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39 molecule, to take place. The oxygen thus combines with the metallic basis by sharing electrons. The high temperatures typical in the nanoparticle production processes provide the energy required by the oxygen to undergo this step. These same temperatures also excite the aluminum atoms to a state that let them shift in the lateral direction enough to allow the oxygen atom to move into the lattice. The oxygen adatom can fit snuggly into the next octa hedral interstitial site and when a new oxygen molecules deposits on the surface it can push it down. Furthermore the presence of oxygen atoms seems to have an autocatalytic effect for further oxidation exciting the surrounding aluminum atoms by combination reactions. As the oxygen atoms fill a unit cell, further oxygen molecules deposited on the surface can push the oxygen atoms farther dow n until a 2.5 nm oxide is created. This is the equivalent to the saturation of 6 unit cells. No further oxidation occurs when the interatomic space is so filled up with oxygen atoms that there is not extra space for any additional ones to move into after electric charge equilibrium has been reached. This model justifies the difficulty of forming single crystalline oxide on the surface of nanoparticles, but rather an overall amorphous or part-crystalline structure is created. The single molecular sheets produced following this model agree with high resolution TEM micrographs that show molecular layers appearing to superimpose at the surface of the nanoparticles with some crystalline mismatch. Figure 2-3 shows an example of the TEM images taken for the materials used in this research in which the layered structure of the oxide layer can be observed. The crystalline areas of the surface oxide are believed to be -aluminum oxide. The main phase composition of the oxide, forming upon aluminum combustion in oxygen, depends on the conditions of its formation and under usual combustion conditions it is -alumina, the most stable phase of alumina. The phase of alumina has a rhombohedral lattice that can grow into crystals with a platelet morphology, which may explain the obs erved laminar layers on the nanoaluminum surface.

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40 Another important observation made by Ramaswamy et al (2004, 2005) is the presence of water molecules on the surface of the aluminum nanoparticles. This was confirmed by Prompt Gamma Neutron Activation Analysis (PGAA), which detected hydrogen before and after drying of the samples. A large amount of hydrogen was found in the nanoparticles after the adsorbed water was released under vacuum. This means that the nanoaluminum coating is porous and some of the water, which is deposited into the coating, reacts to form aluminum hydroxide. The inner surface layer is thus considered to consist of -aluminum oxy-hydroxide Al-O (OH) and the hypothesis is confirmed with the results from PGAA for hydrogen content and from the X-ray diffraction data. This model explains the formation and some of the properties of the oxide-hydroxide layer that is found in aluminum nanoparticles. It also elucidates how the passivation layer might in fact fail to protect the nanoparticle core from further reaction under extreme environments such as high temperatures or extreme pH environment in which the integrity of the coating can be compromised (Kolics et al., 2001). When a metal is oxidized at high temperature and then allowed to cool the difference in thermal expansion coefficients between the metal and the oxide is another factor that can affect its stability. Typically the oxide film has a lower expansion coefficient, when the particles cool down the metal contracts a greater amount than the oxide and the compressive stresses imposed on the oxide may cause it to fail, specially is the P-B ratio is already high (Askeland, 1989) Another possible reason for the failure of the oxide layer in aluminum nanoparticles to protect them from further reaction is that the decreasing radius of curvature, for smaller size particles, could affect the mechanical stability of the oxide layer making it more porous and fragile (Schultze et al., 2000, Ratko et al., 2004 a, 2004 b). In a comparison between aluminum nanoparticles and flakes Ramaswamy et al., (2004, 2005) found the coating of the nanoparticles to be much more porous than the one on the flakes surface. Thus water adsorbed onto the surface can penetrate deeper on the nanoparticles than on the flakes.

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41 2.3 Toxicology Profile of Aluminum In this section a thorough review of the literature available on aluminum toxicity is presented. Even though the investigation of possible toxic effects from aluminum in diverse forms is well established there is very little information about the consequences that exposure to aluminum nanoparticles have on biological organisms. Aluminum is ubiquitous element extensively used in modern life. It is a nonessential metal that was previously considered virtually innocuous to humans (Sorenson et al., 1974). In the last two decades several studies suggest that aluminum ha s some toxicity towards plants, some aquatic animals and humans (Ganrot, 1986; Nayak et al., 2002; Pineros et al., 2001; Yokel, 2001). 2.3.1 Sources of Aluminum Environmental Exposure: Natural aluminum occurs in the soil and makes about 8% of the earths surface (Sposito, 1996). Higher concentrations may be found in soil surrounding waste sites associated with certain industries such as coal combustion and aluminum mining and smelting (ATSDR, 1999). The largest source of particle-borne aluminum is the dust from ores and rock materials. Dust particles are released onto the environment by both natural processes (weathering of aluminosilicate crystal material) and human activities (mining and agriculture) (Nayak, 2002). In the atmosphere aluminum is usually found as aluminosil icates associated with particulate matter and the background levels of aluminum generally range from 0.005 to 0.18 mg/m3 (Sorenson et al., 1974). The presence of aluminum in natural water is normally small. Acid rain leaches the abundant aluminum from the soil and contributes significantly to the environmental inputs (Harris et al., 1996). Dietary Exposure: Many natural foods contain aluminum that has been transferred from the soil to the plant roots when the pH is lower than 4.5-5.0 (Matsumoto, 2000). Aluminum is also present in many manufactured foods and is added to drinking water for purification purposes. Aluminum containing additives are common in processed cheese, baking powders, cake mixes, frozen dough, pancake mixes, etc. (Nayak, 2002). Leaching of aluminum from beverage cans and

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42 cookware can also occur. It has been estimated that about 20% of daily aluminum intake comes from cooking utensils made of aluminum (Greger et al., 1985; Lin et al., 1997). The daily average of aluminum in the daily diet is approximately 3.5 mg in Japan (Matsuda et al., 2001) and 3.4 mg in the United Kingdom (Ysart et al., 2000) Iatrogenic Exposure: Some medications commonly used contain aluminum and in some cases they are a direct source of aluminum into the blood stream. Acute aluminum intoxication cases in clinical practice are uncommon but have been found to occur, with a fatal outcome in some instances (Nakamura et al., 2000). There are several nonprescription drugs containing aluminum that are commonly used and increase aluminum exposure in a large population. Examples of these drugs are some antacids, buffered aspirins, antidiarrheal products, douches and hemorrhoidal medications (Nayak, 2002). Some patients consume as much as 5 g of aluminum daily in antacids and buffered aspirins (Lione, 1983; Flaten, 2001). Another source of aluminum introduced directly onto the bloodstream is the use of aluminum adjuvants in vaccine products. The FDA limit of aluminum in each vaccine dose is 0.85 mg. Occupational Exposure: Certain occupational groups are among the highest aluminum exposed populations. They are workers of aluminum refining and metal industries, people employed in printing and publishing, in automotive dealership s and service stations, and individuals involved in fabricating metal products (U.S. Public Health Service, 1999). Several studies have reported cognitive changes, possible impairments, and other occupational hazards in relation to exposure to aluminum dusts and fumes (Bast-Patterson et al., 1994; McLachlan, 1995; Rifat et al., 1990; White et al., 1992). A significant finding is the accumulation and long-term retention of aluminum within the respiratory tract of individuals repeatedly exposed in occupational settings (Schlesinger et al., 2000)

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43 2.3.2 Aluminum Assimilation into the Body Aluminum absorption seems to be very low, but many factors can enhance its assimilation in animals and humans (Deng et al., 1998). Even though intestinal absorption of aluminum is very poor there are many organic dietary components that are potential chelators of aluminum and may enhance its absorption (Deng et al., 2000; Venturini-Soriano et al., 2001, Dayde et al., 2003). Examples of these compounds present in normal diets are: tartaric acid, glutamic acid, malic acid, and succinic acid. They are common acids in fruits, in industrial foods and beverages. Miners, smelters and other metal workers are mainly exposed to aluminum through dusts and aerosols. It has been suggested the accumulation of aluminum in the brain via absorption through the olfactory system (Exley et al., 1996; McLachlan et al., 1980; Roberts, 1986) or systemized through the lung epithelia (Gitelman et al., 1995), and through the gastrointestinal (GI) tract when particulates are swallowed (Rollin et al., 1993). Aluminum absorption appears to be more efficient in the respiratory system than in the GI tract (Yokel et al., 2001). Dermal applications of aluminum compounds in cosmetic, antiperspirant, and health care products generally do not induce harmful effects on skin or other organs (Flaten et al., 1996; Sorenson et al., 1974). Aluminum absorption from in vivo animal studies is very low (1%) and seems to be sensitive to the amount of aluminum intake (Greger, 1983). To enter the body aluminum has to cross a layer of epithelial barrier. Despite of the abundant information collected over the years about the health effects of exposure to aluminum the interactions of GI, olfactory, pulmonary, and dermal epithelia with aluminum are not well understood (Nayak, 2002). 2.3.3 Aluminum Distribution in the Body The total aluminum content in healthy humans is approximately 30-50 mg (ATSDR, 1999). The total body aluminum is a flux between different systemic compartments (Exley et al., 1996).

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44 Unequal aluminum distribution throughout the va rious tissues has been reported in normal, aluminum-exposed humans and in aluminum-treated experimental animals (Yokel et al., 2001). From the total body aluminum about one half is in the skeleton and about one fourth is in the lungs (Ganrot, 1986). The brain is an important accumulation organ regardless of the route of exposure (McLachlan et al., 1980; ATSDR, 1999). The route, dose, and duration of the exposure characterize the distribution of the metal among the different target organs (Ding et al., 1997). In terms of the temporal pattern of pulmonary clearance of aluminum compounds from the lungs or the potential translocation to other organs little is known (Schelesinger et al., 2000). With increasing age, the aluminum loadings in lungs, liver, kidneys and brain have been found to be increased (ATSDR, 1999), which is an indication of the possible accumulation of aluminum overtime. 2.3.4 Aluminum Distribution in the Cells The same manner that aluminum distributes unevenly in the body accumulating in different target organs, aluminum distributes within the cells. Aluminum ions accumulate in the lysosomes, cell nucleus, and chromatin (Karlik et al., 1980). A correlation between the intranuclear aluminum accumulation and aluminum neurotoxicity has been suggested (Uemura, 1984). It has also been reported that aluminum present in the lysosome may be associated with dementia (van Rensberg et al., 1997). Aluminum has been found in the cytosolic, mytochindrial, lysosomal, and nuclear components. Accumulation of aluminum in the different cellular compartments has been found to be specific to the cell lines investigated (Exley et al., 1996; Julka et al., 1996). 2.3.5 Systemic Effects Induced by Aluminum Toxic effects of aluminum in the brain, liver, skeletal, muscles, heart and bone marrow are well established but the mechanisms of action are poorly understood (Flaten et al., 1996). There is also a lack of information on its cellular sites of action (Levesque et al., 2000). Neural System: Despite off the little information available regarding the molecular cytotoxicity of aluminum there are evidences in literature of its neurotoxicity (Suwalsky et al., 1999).

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45 In vivo studies in rats have found higher levels of aluminum in the brain of young aluminum-exposed rats than in those of older aluminum-exposed rats (Gomez et al., 1997) and prenatal and postnatal development inhibition of the brain (Yumoto et al., 2001). In humans there are several neurological manifestations attributed to aluminum intoxication. They include the following: memory loss, tremor, jerking movements, impaired coordination, sluggish motor movement, loss of curiosity, ataxia, myoclonic jerks and generalized convulsions (Crapper et al., 1980; Zatta, 1994). Some of the neuro-pathological conditions associated with elevated aluminum levels in the brain are Alzheimers type senile and presenile dementia, Down syndrome with manifested Alzheimers disease, amyotrophic lateral sclerosis affecting spinal cord and brain, Parkinsons dementia with neurofibrillary degeneration, dialysis encephalopath y, striatonigral syndrome alcohol dementia with patchy demyelination senile plaques of Alzheimers disease and aged brain (Crapper et al., 1980; Zatta, 1994). However the relationship between thes e pathological disorders and aluminum is still controversial. In terms of acute exposure to aluminum in adults some of the symptoms reported are agitation, confusion, myoclonic jerk, grand mal seizures, obtundation, coma and death (Bakir et al., 1986). Musculoskeletal System: From patients with renal dysfunction under dialysis treatment there are studies of chronic aluminum poisoning, in these cases the skeletal is the main target (Kerr et al., 1992). In animal studies, tracer analysis shows that the liver, kidney, muscle, and heart also accumulate aluminum (Walker et al., 1994). Rodriguez et al (1990) showed a decreased osteoblast surface, increased osteoid accumulation, and cessation of bone formation after aluminum administration to rats. In their study, aluminum wa s toxic to osteoblasts and inhibited mineralization even when osteoblasts were not decreased in number. For humans osteomalcia, bone pain, pathological fractures, proximal myopathy and failure to respond to vitamin D3 therapy are the common outcomes of aluminum induced musculoskeletal toxicity (Alfrey, 1984).

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46 Respiratory System: In the case of inhalation exposure the effects of aluminum are mainly localized on the respiratory system. Workers in the aluminum industry develop symptoms like asthma, cough, lung fibrosis, or decreased pulmonary function but whether these effects are only due to aluminum exposure is questionable. The working conditions and external factors other than aluminum affecting these workers complicate the isolation of the cause for these conditions (ATSDR, 1999). In animal studies aluminum exposure causes macrophages proliferation in broncho-alveolar lavage fluid and granulomatous reactions. Granul omatous reactions were characterized by giant vacuolated macrophages, which were associated with pneumonia in some cases (ATSDR, 1999). Cardiovascular System: Accumulation of aluminum in the heart is another outcome from aluminum exposure observed in the literature. He modialysis patients develop cardiac hypertrophy that in some cases has been related to aluminum accumulation (Zatta, 1994). Myocardial cells can accumulate aluminum in lysosomes (Gallet, 1987) and aluminum buildup in the myocardium has been associated with cardiomyopathy. Hemodialysis patients show a higher prevalence of arrhythmia and sudden death than other populations with normal renal function (Zatta, 1994). Hepatobiliary System: Evidences of the adverse effect of aluminum on the liver have been documented for animal studies in the literature (Stein et al., 1987). Despite high accumulation of aluminum, liver function is seldom affected due to biliary excretion. Abnormalities in hepatic function associated with aluminum include increased serum bile acid concentration and glucuronyl transferase activity and reduced mixed-function oxidase level and bile flow. In addition, decreased taurine conjugation with bile acids which may be associated with cholestasis (blocked bile flow) have been reported (Klein et al., 1989). It has also been observed that acute administration of aluminum adversely affects hepatic drug metabolism and protein synthesis (Cherroret et al., 1995; Jeffrey et al., 1987).

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47 Endocrine System: Autopsy and histological tissue studies from patients of encephalopathy associated with dialysis have shown numerous intracytoplasmic black colored fine granular inclusions in endocrine tissues (pituitary, para thyroid, and adrenal) suggesting accumulation of aluminum in these organs (Reusche et al., 1994). Aluminum has also been reported to concentrate in lysosome-like structures of parathyroid gland withou t alteration of the cellular ultra structure (Galle, 1987). High levels of parathyroid hormone are suggested to be associated with the preferential deposition of aluminum in the brain, bone, and parathyroid gland (Burnatowska-Hledin et al., 1983). Parathyroid hormone levels are disrupted by aluminum in animals and humans (Fernandez et al., 2000; Jeffrey et al., 1996). It has been speculated that aluminum induced neurological disorders, bone disease, and anemia may indirectly cause in many dialysis patients, parathyroid hormone toxicity (Mayor et al., 1983). The role of aluminum in the malfunctioning of the parathyroid gland is not clear but hypertrophy is often associated with aluminum poisoning (Galle, 1987). Urinary System: Urological dysfunction can both cause and result from aluminum accumulation though impaired renal function is not a prerequisite for increased tissue aluminum burden (Mayor et al., 1986). Because of their function the kidneys can rapidly concentrate aluminum but they can also get rid of it by excreting through the urine (Flaten et al., 1996). Nevertheless there are animal studies that have found significant lyso somal damage in response to aluminum (Stein et al., 1987). Blood and Hemopoietic System: There are several indicators of the toxicity of aluminum to the hemopoietic system like: microcytic, hypochromic anemia, or decreased numbers of red blood cells (OHare et al., 1982). Even in the absence of signs of anemia, ingested aluminum may depress hematopoiesis by affecting red blood cell production and cell destruction (Garbossa et al., 1996). Despite of the literature available suggesting a c onnection between aluminum toxicity to the blood the exact mechanism of aluminum-induced anemia is still debatable.

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48 Reproductive System: In animal studies, the testes have been found a target organ for accumulation of aluminum for long-term exposed rats (Gomez et al., 1997). In the same way, numerous intracytoplasmic black-colored fine granular inclusions were found in testes tissue collected from dialysis patients (Reusche et al., 1994). 2.3.6 Open Questions and Knowledge Gaps Despite the abundant available knowledge about the possible effects and diseases caused by aluminum exposure there are still severa l questions unresolved (Nayak 2002): What are the organ-specific variations in aluminum toxicity? What is the role of differential aluminum kinetics in different organs? How does aluminum enter the body by different routes? What are the molecular mechanism(s) of aluminum toxicity that may characterize features of aluminum toxicity common to all target organs? The studies reported here were conducted on forms of aluminum other than nanoparticles of pure metal. During the time invested in this investigation no studies were found with regard to the biological effects of aluminum nanoparticles. The research presented in my dissertation a ddresses some of the possible toxicities and mechanisms of aluminum nanoparticles at the cellular level in a lung epithelial cell model that could ultimately be affected by aluminum nanoparticles after inhalation.

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49 Figure 2-1. Top-down and bottom-up approaches to nano-synthesis in the context of man-made processes as well as in the physiological environment. Currently both approaches overlap for most of the nanomaterials being produced. Figure 2-2. Atomic model of the face centered cube Al lattice and the adsorption of an oxygen molecule on the surface. Generated with CAChe software from Fujitsu.

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50 A B Figure 2-3. HRTEM of an aluminum nanoparticle. (A) A multilayer oxide/hydroxide layer is observed around the aluminum nanoparticle. (B) Higher magnification shows mismatch in the molecular layers of the surface oxide that could justify their tendency to easily exfoliate.

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51 CHAPTER 3 CHARACTERIZATION OF NANOPARTICULATE SYSTEMS In order to establish any correlation between nanoparticle characteristics like size and shape, and their biological effects, a thorough characterization of the particles is essential. In this chapter, the techniques used for the physicochemical characterization of these materials and the results obtained for the systems of interest, are described. The chapter follows a logical sequence used in the characterization of nanomaterials for biological applications: as received, in physiological environment, and after dosage. The importance of knowing, to the best of the available capabilities for analysis, the material to be tested for toxicity has been widely recognized in the research community working in this field and several recommendations can be found in the literature (Bucher et al., 2004, Oberdrster et al., 2005a, 2005b, Powers et al., 2006). Complete characterization of nanoparticles includes such measurements as density, size, size distributi on, shape and other morphological features as, crystallinity, porosity and surface roughness, chemistry of the material, solubility, surface area, state of dispersion surface chemistry, and other physicochemical properties (Powers et al., 2006). Some of those nanoparticle properties like shape, density, and porosity for example are not expected to change when in a biological environment. At the other hand, others like particle size distribution and surface chemistry are very likely to change in physiological media due to the adsorption of biomolecules onto the surface and th e different states of dispersion that the physiological conditions will promote. Thus, the importance of taking into account the very likely changes by characterizing the materials as close as possible to the conditions of before, during, and after their interaction with biological organisms. An exhaustive material characterization is time consuming, expensive and complex. The extent to which it should be carried on depends on the objectives of the study.

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52 The main objective of this investigation is to an alyze the effect that size and shape of certain commercially available aluminum nanopowders have on pulmonary in vitro toxicity. The limitations of the available techniques and the complexity of the physiological environments are here discussed, as well as the protocols that have been developed during this research. 3.1 Particulate Systems of Interest for this Research The material of main focus of this investigation is aluminum in different sizes and shapes, ranging from tens of nanometer to tens of micron, a nd from spherical particles to irregular flake like morphologies. As mentioned in chapter 1 (1.4.4) other materials other than aluminum were tested in order to establish a toxicity reference frame. Th e particulate materials used were crystalline quartz and titanium dioxide. For reference purposes the names used hereafter in this chapter for the different samples investigated are listed bellow: NanoTek: TiO2 from Nanophase Technologies Corp. P25: TiO2 from Degussa Quartz: quartz Min-U-Sil 5 from US Silica Al 1: QSI-Nano aluminum from Quantum Sphere Inc. Al 2: 80 nm aluminum from Nanotechnologies Inc. Al 3: H-2 aluminum from Valimet Inc. Al 4: x-81 aluminum from Toyal Inc. Al 5: aluminum flakes from Sigma Technologies International Inc. Al 6: AlCl3 x 6H2O from Fisher Chemical, Fisher Scientific. 3.2 Before Dosage: As Received All the particulate materials used in this research were acquired from the different manufacturers that make them as powder systems commercially available. They were received in dry state and the total amount available varied depending on the powder, from few micrograms to the order of kilograms. Upon arrival powders were processed in several steps for characterization. 3.2.1 Sampling Reliable powder sampling constitutes the first step of most powder characterization and processing procedures. Sampling particulate matter entails collecting a small amount of powder from

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53 the bulk, such that this smaller fraction best repres ents the physical and chemical characteristics of the entire bulk (Holdich, 2002; Jillavenkatesa et al 2001). Samples were prepared taking into account the issues listed by the National Institute of Standards and Technology (Jillavenkatesa et al 2001): Quantity of powder from which samples are being obtained Amount of sample required Powder characteristics, including but not limited to flow characteristics of the powder, shape and size of the particles, tendency to segregate, surface chemistry that may cause the powder to be hygroscopic, etc. Mechanical strength of the powder, i.e., are the particles friable and thus, likely to fracture during transport or during sampling Mode by which powders are transported Possibility of powder contamination, a nd acceptable limits of contamination Duration of time needed to conduct the sampling procedure As general guidelines for powder sampling one can refer to the golden rules of sampling (Allen, 1981): 1. A powder should be sampled in motion 2. The whole stream of powder should be taken for many short intervals of time in preference to part of the stream being taken for the whole of time. Powder batches can vary from several tons to a few grams, which is the case for many nanomaterials. There are several techniques and devices that have been developed to aid in representative sampling of powders. Their design in corporates the golden rules to the greatest possible extent (Jillavenkatesa et al 2001). Several of the powders used in this investigation were received in bulk quantities in the order of kilograms or hundreds of grams. This was the case for NanoTek, P25, Al 2, Al 3, and Al 4. For easier handling and consistent sampling in further analysis, the sample sizes were reduced to the order of tens of grams and stored in hermetically sealed containers throughout the length of this

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54 research. For sample sub-division of the bulk amounts scoop sampling was used. The nanopowders investigated exhibited, with exception of the P25 and the aluminum SIGMA flakes, very poor flow characteristics and strong adhesive forces that did not allow the use of more reliable sampling devices like the spinning riffler. The scoop sampling technique is a widely used, simple method for sample division. An operator, using a scoop, extracts the laboratory samples from some portion of the bulk sample. This technique is only appropriate for materials that are homogenous and exhibit poor flow characteristics, which is the case of th e nanopowders used for this research. One of the drawbacks of this technique is that all the bulk material does not go through the sampling process. The other drawback is the dependency on the operator to decide where to scoop the material and what quantity of the sample to extract. This sampling technique is largely influenced by segregation. In order to minimize this problem samples were not taken from the surface of the bulk and the material was shaken in the container before scooping the subsample (Jillavenkatesa et al 2001). Despite off all the precautions mentioned above, there are several sources of error in the process of sampling both systematic and random. While these errors cannot be eliminated, standard protocols were implemented to minimize their influence in the final results. Some of the common errors associated with sample preparation, as well as the protocols implemented during this research to minimize those errors are summarized in Table 3-1. One of the powders, Al3, was noticed to be more susceptible to sampling variability than the others due to its very polydisperse particle size distribution and the cohesiveness of its particles. As it can be observed in Figure 3-1 different subsamples of this powder can result in large differences, up to one order of magnitude, in the mean particle size. As expected, the particle size distribution based on particle numbers was more sensitive to this phenomenon than the particle size distribution based on volume. This is due to the fact that a number size distribution is more skewed towards the size range which the more abundant number of particles on it whereas the volume distribution will be skewed towards the size range with the largest par ticles (and or agglomerates). Thus whenever more

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55 nanoparticles were subsampled the mean particle size for the number size distribution was closer to that of the smaller particles found in the powder Al 3. From the SEM images taken of this powder sample the presence of particles in the nanosize range around the bigger 2-3 m particles was observed and found to be frequent (Figure 3-7). For the materials of interest to this research the subsamples were collected from different regions of the bulk and mixed together in smaller containers (stocks) used to hermitically store the powders. Some of the characterization techniques required dry samples. In those cases appropriate amounts of powder were collected from the stock specimens prepared for this research. For the characterization techniques requiring dispersion of the powders standard protocols were developed and will be detailed in following sections. 3.2.2 Density, Surface Area, and Porosity Density is simply the mass of a quantity of matter divided by the volume of that same quantity. There are three densities associated with powders. Th e absolute density, also referred as the true or skeletal density, which excludes both the pores that may be in the particles and their interparticle spaces; the envelope or apparent density, includes the pores but excludes interparticle spaces; and the bulk density which includes both pores and interparticle spaces (Webb et al., 1997). For powders the bulk density depends on vibration and applied forces. It is not an intrinsic property of the material and it is not relevant for in vitro toxicity studies. The absolute and apparent densities are identical for a nonporous object. Absolute density by definition excludes all the open pores, i.e., the pores that have access to the outside. The apparent density includes pore spaces up to the plane of surface. In this research the absolute density of all powders was measured using a Quantachrome Ultrapyc 1000 gas pycnometer. A known amount of dr y powder is placed in a sealed chamber of known volume and exposed to a series of elevated and then released gas pressures to flush away atmospheric gases and vapors. Then the gas is released into another chamber of known volume. The

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56 pressures of both chambers are measured before and after the gas expansion, which allows calculating the sample volume. The gas used by this instrument is helium that with an atomic radius of 31 pm penetrates and fills all open spaces including pores open to the surface. The result of dividing the known mass by this measured volume is the absolute density. Results of the absolute density measurements are detailed in Table 3-2. The density values measured for the titania and quartz powders matched the reference values found in the literature. In the case of the aluminum nanoparticles the values measured were closer to that of pure aluminum for the larger size particles of samples Al 3 and Al 4 than for the smaller particles. As has been explained in chapter 2 the aluminum particles exhibit an oxide layer due to the very high reactivity of aluminum. Considering the total density of a coated particle as: layer surface total layer surface core total core total V V V V (3-1) Where are the densities and V are the volumes of each component. According to equation 31, the density of the smaller nanoparticles will be more influenced by the density of the surface layer than the density of the larger particles for which the ratio of surface layer to total volume is much smaller. This explains higher density values for the smaller sizes of aluminum nanoparticles and the aluminum flakes which higher surface areas are expected to be covered by some form of aluminum oxide/hydroxide. Surface Area is a measurement of the exposed surface of a material. There is an inverse relationship between particle size and surface area. For the same mass of materials the smaller the particles the larger is the surface area. The large su rface area is one of the main features that make nanoparticles so desirable for many different appli cations. Actual particles of whatever size if examined at the molecular scale, display planar regions, but also are likely to include lattice distortions, dislocations, and cracks. This means that the actual exposed surface of particles is greater than would be calculated assuming any one geometric shape (Webb et al., 1997).

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57 Surface area is usually measured by gas adsorption. In this research the instrument used to measure specific surface area was the Quantachrome NOVA 1200. Dry sample is evacuated of all gas and cooled to the temperature of liquid nitrogen, 77 oK. At this temperature, inert gases such as nitrogen, argon and krypton will physically adsorb on the surface of the sample exposed to the gas. An adsorption isotherm (one temperature) is usually recorded as volume of gas adsorbed versus relative pressure (i.e., sample pressure / saturation vapor pressure). One or more data points of the adsorption isotherm must be measured and the BET (after Brunauer, Emmett and Teller) equation is used to give specific surface area from this data. The BET equation is used to calculate the volume of gas needed to form a monolayer on the surface of the sample. The actual surface area can be calculated from knowledge of the size and number of the adsorbed gas molecules. Nitrogen was used measure BET surface for all the samples except for the quartz. The surface area of this powder is very low, and krypton was used instead of nitrogen for a more sensitive measurement because of its lower saturation vapor pressures at liquid nitrogen temperature. BET surface area measurements can also be used to derive the mean diameter of the particles forming a powder (Holdich, 2002). SSA X 6 (3-2) Where X is the mean particle diameter in m, SSA is the specific surface area in m2/g and is the absolute density of the powder in g/cm3. This technique is a reliable way to estimate a mean particle size of the particles in a powder, especially for nanoparticles for which the surface area is so high. Results from the BET measurements are summarized in Table 3-3 for every powder. Because not enough sample of Al 1 was available to experimentally measure the density with the gas pycnometer the reference density value of pure aluminum was taken to calculate the mean particle diameter from BET. The mean diameter values calculated from the BET data match the values given

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58 by the manufacturing companies as mean particle sizes for the nanometer samples (NanoTek, P25, Al 1 and Al 2). This is expected as SSA measurements by BET is the technique mostly used by manufacturing companies to calculate and report the mean particle size of their products Porosity can be estimated by analysis of EM images (SEM and TEM) at a high enough magnification to reveal particle surface morphology (see Figures 3-2, 10). This technique is only valid to detect pores open to the particle surface, unless ultra sectioning of the particles is performed to obtain cross section of the particles that would show their core topography. In the case of toxicity research one should be more concern about open pores that could affect the surface properties of the nanomaterials in terms of accessible surface, surface roughness and adsorption of molecules onto the surface. As specified by the manufactures all the nanom aterials used in this research were found to be non porous. 3.2.3 Size and Shape Knowledge of particle size, shape, and their distributions, of a powder is a prerequisite for a more accurate interpretation of any biological in teraction observed for nanopowders. Particle size and particle size distribution (PSD) have a very significant effect in properties of powders and finished objects like: bulk density, mechanical stre ngth, optical, electrical, and thermal properties. As discussed in previous chapters, many researchers have investigated the possible correlation between size and shape of nanoparticles and their effects in biological systems. Nonetheless, in most cases conclusions were based in mean particle diamet ers reported by manufacturers that do not totally describe the complexity of real particulate systems. In order to conduct an accurate particle size analysis, one ideally should have a representative sample, a well dispersed system, and a physical measurement technique that is carefully selected to produce data relevant to the intended use. For example, if a test material is administered as an aerosol, it would be logical to pick an aerosol technique. One has to measure an adequate number of particles across the entire breadth of the size distribution for statistical validity. For a monodisperse system this is relatively easy. However, as

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59 polydispersity increases it may be necessary to measure a large number of particles to accurately portray the size distribution (Masuda and Inoya 1 971). This number becomes larger still if the interest is focused on the tails of the distribution rather than on the mean or the median. An ensemble method of measurement (such as laser diffraction, centrifugal sedimentation, impaction, etc.) is normally preferred because the parameter measured (scattering pattern, mass) is generated by large numbers of particles. Counting techniques (such as microscopy and image analysis) should include enough particles to reach the desired statistical reliability. For dry asreceived powders, BET surface area is often used to estimate average size (based on a nonporous spherical model) and has the added advantage of providing a direct measurement of specific surface area (SSA). (Allen 2004, b) It should be noted, however, that this common method of reporting particle size assumes a monodispersed spherical system and cannot be used to determine the breadth or shape of the size distribution (see Figure 3-1). 3.2.3.1 Imaging techniques Microscopy-based techniques for particle size characterization provide a powerful tool for characterization of particle size, size distribution and morphology. They involve direct observation of particles and the consequent determination of size based on a defined measure of diameter. If compared with other available techniques for particle size analysis, a significant advantage of microscope-based techniques is the ability to determine particle shape or morphology in addition to make a direct measurement of size. Typically, the calculated sizes are expressed as the diameter of a sphere that has the same projected area as the projected image of the particle. The calculated sizes or size distribution can then be converted to, or expr essed in, different measures (area, mass or volume distributions) with suitable precautions (Jillavenkatesa et al 2001). Different types of microscopes can be used for size/shape analysis of powder materials by image analysis. Optical and electronic microscope s (EM) like the scanning electron (SEM) and the transmission electron (TEM) microscopes are the traditional choices depending on the powder

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60 properties. Magnification and resolution limits of these instruments determine which one to use in every case. In the case of nanomaterials EM are requ ired as the lower resolution limit for an optical microscope is about 1 m. This implies higher cost in sample preparation and analysis time (Masuda et al., 2006). The common errors in imaging techniques are: (1) errors associated with sample/specimen preparation; (2) errors associated with instrumentation/equipment and image analysis; (3) errors due to human and other factors. Another important consideration when dealing with imaging techniques for particle size characterization is the number of particles to be analyzed in order to obtain a statically reliable result. To estimate the number of particles to be analyzed for a specific powder, the National Institute of Standards and Technology refers to the mathematical theory developed by Matsuda and Iinoya in 1971 (Jillavenkatesa et al 2001). According to the equations developed in that theory, the total number of particles to analyze depends on the standard deviation of the particle sizes, the shape of the particle size distribution, the type of distribu tion (i.e., number, area, and volume) and the desired range for error. In order to get a mass median diameter within 5% error with 95% probability for a powder with a typical standard deviation of 1.60 about 61000 particles are required (Matsuda et al., 1999). While this number would be dramatically reduced to about 15000 particles for a 10% error, the elevated number of particles to be analyzed for a reliable particle size distribution measurement demotes the use of EM imaging for that purpose. Nonetheless EM images are required for shape assessment as well as to detect the presence of large particles, which are indistinguishable from agglomerates when using ensemble techniques for particle sizing. All the powders of interest for this research were examined under a JSM 6335 F SEM and a 2010 F scanning TEM, both from JEOL. Samples were prepared by dispersing a little amount of the powders in pure ethanol. The dispersions were sonicated in a 275 CREST Ultrasonics bath for 30 seconds and a drop of the suspension was poured on a sample stub for SEM, or a carbon-cupper grid

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61 for TEM samples. Samples were dried and in the case of the SEM samples coated with a nano thin coat of gold to aid electron conductivity. During the collection of the pictures the samples were manually scanned and representative images for particle size and shape assessment were recorded. The different instruments used were properly calibrated. Some of the images collected for every sample are shown (see figures 3-2, 10) and a summary of the size range and shape assessment is listed in Table 3-4. High Resolution TEM (HRTEM) also allows imaging the atomic crystalline lattice when the sample conditions are proper. In some instances a clear difference in structure between the particles core (crystal lattice) and surface can be appreciated in the image for the aluminum powders. 3.2.3.2 Light scattering techniques As observed in the images collected for all the sa mples investigated in this research the reality of most commercial powders is some degree of size polydispersity and particle shapes that can be very different from an ideal sphere. Particle sizing methods that analyze a sample on its entirety and rely on statistics to deconvolute the measured information into some sort of particle size distribution are known as ensemble techniques. One of the most common phenomena used to measure particle size is the interaction between light and matter (Jillavenkatesa et al 2001). Light has properties that can be used for determining particle size and particle size distributions. In the case of diffraction instruments, the angle at which the light is diffracted depends upon the wavelength of the light and the particle size. The angle of diffraction is measured to determine size. Another feature of light that can be used for determining the particle size is the frequency. Frequency change or shift information is used in Dynamic Light Scattering for particles that are very small compared with the wavelength of the light. The instruments used in this research differ on the type of light interaction and principle measured. One of them uses laser diffraction and the other one dynamic light scattering. Samples were prepared in the same manner for both analysis and when suitable measurements were taken in

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62 parallel. A small quantity of the powder was dispersed in deionized water and sonicated for 30 minutes in a 275 CREST Ultrasonics bath with an ultrasonic frequency of 2 kHz and sonic power average of 135 W. In order to determine an optimum ultrasonication time particle size distributions were measured after each 5 minutes interval until a minimum size was achieved repeatedly. At that point it was assumed that the particles had b een properly dispersed. Because the size range, sensitivity, configuration, principle measured a nd deconvolution algorithms of both instruments are very different, results were taken as complimen tary instead of comparable. By combining both results a better idea of the whole size distributions is acquired for nanosize powders. Beckman Coulter LS13 320 multi-wavelength laser diffraction particle size analyzer: This laser diffraction instrument offers a rapid analysis with a relatively easy sample preparation, measurements are relatively inexpensive, and it allows dry and liquid dispersion powder analysis. This instrument is capable of measuring particle sizes from 40 nm to 2mm. A 750nm laser is used for analysis in the range from 400 nm to 2mm and polarization intensity differential scattering (PIDS) assembly sizes from 40 to 400 nm and improves resolution in the 400 to 800 nm range. Regarding its limitations, the instrument performance is highly dependent on instrument design (e.g., laser sources of different wavelengths, different number and positi on of the detectors), the state of dispersion, and the knowledge of the sample optical properties. Laser diffraction techniques are based on three basic assumptions (Jillavenkatesa et al 2001): (1) particles scattering light are spherical in nature; (2) there is little to no interaction of the light scattered by different particles; (3) the scattering pattern collected on each detector is the sum of the individual patterns generated by each particle in the sample diffracting the laser at that angle Laser diffraction methods cannot distinguish betw een dispersed and agglomerated particles. The previous image analysis provided a qualitative assessment of the larger particle size for each powder. This evaluation is helpful to determine if the larger sizes observed in a particle size distribution measured by a light scattering instrume nt could be single particles or the effect of

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63 agglomeration. Results from this instrument for every powder as received are plotted as relative % number and relative % volume distributions in Figur es 3-10, 11. Several observations can be pointed out from these measurements: The large difference in the mean sizes from the number and volume particle size distributions for each powder. This is to be expected due to the wide particle size range observed in the micrographs as well as for the agglomeration occurring that could not be totally avoided in the experimental conditions of close to neutral pH and no dispersion aids used. Only a powder made of perfectly dispersed spheres of the sa me size would have identical number and volume size distributions. Both TiO2 powders were confirmed to have very narrow particle size distributions and very small difference in size between the two different manufacturers. The quartz powder contained a considerable fraction of nanoparticles (100 nm or smaller) as noticed in the particle size distribution by number and the images analyzed (see Figure 3-4 (B)). The aluminum powders were found to cover a very wide particle size range. Al 3 with a mean particle size of about 2 m according to the manufacturer was found to contain a very large fraction by number of particles in the nanosize range and that observation was confirmed by image analysis of the micrographs taken. The smaller size powders, Al 1 and Al 2 showed agglomeration after the dispersion protocol by sonication. Some of the particles in these powders are expected to hardly agglomerate during the manufacturing process and therefore remain aggregated after sonication. Better dispersion was achieved for the Al 2 powder as indicated by the volume particle size distribution. The particle size distribution of Al 5 should be only taken as a qualitative measurement of size. This powder consist of flake like particles and as previously explained light scattering methods for particle size measurements are based on the assumption that the particles are perfect spheres, thus the validity of the data obtained has not been proven. In this case image analysis was considered the primary method for particle size assessment. Microtrac UPA 150 ultrafine particle analyzer: Dynamic light scattering (DLS) theory is a well established technique for measuring particle size over the size range from a few nanometers to a few microns. When a coherent source of light (such as a laser), having a known frequency is directed at moving particles, undergoing random thermal (Brownian) motion, the light is scattered at a different frequency. The shift is termed a Doppler shift or broadening, and the concept is the same for light when it interacts with small moving particles. For the purposes of particle measurement, the

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64 shift in light frequency is related to the size of the particles causing the shift. Due to their higher average velocity, smaller particles cause a greater shift in the light frequency than larger particles. It is this difference in the frequency of the scattered light among particles of different sizes that is used to determine the sizes of the particles present (Jillavenkatesa et al 2001). The light source of this instrument is a 780 nm laser, it measures particles within a size range of 3.2 nm to 6.4 m, and it requires a very small sample volume. These two properties are very attractive for nanoparticles size measurements. At the other hand, the fact that the calculations for size from the velocities measured are better suited for mono modal distributions can affect the reliability of the results for the real powders analyzed. As seen on the microscopic images and the PSD obtained from laser diffraction the powders here investigated present wide particle size distributions and bimodality in some cases. To avoid the effect of very large particles or agglomerates that fall outside the measurable range by this technique, measurements were taken over long periods of time (180 seconds). This allowed for the big particles and/or agglomerates to settle at the bottom of the instrument without being taken in the calculations. Due to the valid size range from this instrument, it was not used to analyze sample Al 4. The typical size distributions measured after dispersion by soni cation for the different aluminum powders and the quartz are depicted in Figure 3-12. This technique provides better size resolution in the smaller size range of the particle size distribution allowing to better describe the lower size tails of the particle size distributions obtained from laser diffraction. On the other hand the upper size limit for this technique does not include the larger size particles a nd/or agglomerates that were detected by laser diffraction and image analysis. 3.2.4 Surface and Bulk Chemical Composition In order to understand the biological effects observed from nanoparticles it is essential to know the chemical composition of the powder. Of special importance is the chemical composition of the

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65 surface as this is the first and most dominant area of interaction for nanomaterials. Most of the techniques used for this purpose are based in an energy analysis of the radiation coming from a sample after excitation with a specific source. The photons or x-rays emitted from a particular specimen are intimately related to the atomic and molecular composition of that sample. 3.2.4.1 FTIR Infrared (IR) spectroscopy is a useful techni que for characterizing materials and providing information on the molecular structure, dynamics, and environment of a compound. When irradiated with IR light (photons) a sample can transmit, scatter, or absorb the incident radiation. Absorbed IR radiation usually excites molecules into higher-energy vibrational states. This can occur when the frequency (energy) of the incident light matches the energy difference between two vibrational states (or the frequency of the corresponding molecular vibration). Obtaining an IR spectrum requires detection of intensity changes as a function of wavenumber or frequency. Fourier transform infrared spectroscopy (FTIR) uses an interferometer to modulate the intensity of each wavelength of light at a different audio frequency. The instrument used for this analysis is an FTIR-Raman/FTIR Microscope Magna 760 from Thermo Electron that has a resolution of up to 0.1 cm-1 and it covers mid-IR (4004000 cm-1) as well as most of the NIR (11,700-2,000 cm-1) (ASM Handbook, 1986). It is a useful technique to identify organic and inorganic materials and to identify molecular species adsorbed on surfaces. The technique however provides very little elemental information, requires for the molecules to be detected to exhibit a change in dipole moment in one of its vibrational modes upon exposure to IR, and needs the matrix to hold the sample to be relatively transparent in the spectral region of interest. The spectra collected from the different aluminum samples are shown in Figure 3-13. The AlO-Al stretching and bending vibrations ( 1000 cm-1) were unsplit and broad. Significant absorption bands can be observed in the ranges 700-1300 cm-1 (Al-OH bend), 1300-1800 cm-1 (H-OH bend) and 3000-3800 cm-1 (AlO-H stretch). These peaks confirm the presence of an oxide/hydroxide layer on

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66 the surface of all the aluminum materials used in this research as well as the absence of foreign contaminants on the surface. The shift of the peaks observed as well as their broadness for each sample can be explained by the different rate of crystallinity at the surface (Geiculescu and Strange, 2003) depending on the manufacturing process and the aging process of the different aluminum samples. 3.2.4.2 X-ray photoelectron spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) spectra ar e obtained by irradiating a material with a beam of x-rays while simultaneously measuring the kinetic energy and number of electrons that escape from the top 1-10 nm at the surface of the materials being analyzed. XPS is a chemical analysis technique that can be used to analyze the chemistry of the surface of a material. It is a valid to detect all elements except hydrogen and helium. The detection limit for this technique is in the parts per thousand (ppth) range (ASM Handbook, 1986). The results from the XPS analysis for all the powders tested during this research are summarized in Table 3-5. The data confirmed the elemental surface elemental composition expected fo r the powders. The presence of carbon in this analysis is common due to adsorption on the particles of organic species containing carbon from the environment as well as from possible contamination of the samples by residual carbon present in the instrument during the measurement. Because of the nature of the analysis the quantitative results obtained should be taken as approximate values more than as an accurate measurement of the amounts present at the surface. Nonetheless the anal ysis confirmed the absence of foreign species within the detection limits of this technique. 3.2.4.3 Energy dispersive spectrometry (EDS) This technique is available in electron microscopes by using a dedicated detector (spectrometer) for the collection of x-rays emitted from a sample after the interaction with the beam of electrons used for imaging. These x-rays are ch aracteristic of the atomic species with which the electrons have interacted. This technique allows for detection of elements as light as carbon (Z 6).

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67 Even though quantitative analysis of the results can be performed, it is only valid for elements with an atomic number 11 and it is limited by the efficiency of the detector and its ability to resolve xrays of similar energy (ASM Handbook, 1986). Energy dispersive spectrometry (EDS) spectra of all the samples characterized for this research were acquired at the same time that TEM images were collected. Some typical examples of the spectra collected are shown in Figures 3-14, 16. It is important to point out that the presence of carbon in these spectra could come from different sources other than the sample. The particles are supported by a copper grid with a carbon lacey that holds the particles in place for analysis. Both elements, C and Cu can therefore appear on the spectrum depending on how close to the grid and lacey the examined particles are and on the angle of the projected x-rays with respect to the detector. Another possible source for C in the spectra is th e deposition of organic contaminants present in the microscope sample chamber at the time of analysis. Thus the C and Cu found for most of the samples was not considered to be relevant for the analysis but a source of error. The spots for analysis were selected in the neighborhood of the particle edge, so the contributions to the spectra would be mainly due to the elements on the surface layer, as well as in areas in the middle of the particles, so the results would mainly indicate the composition of the particle core. The presence of oxygen was detected for all the particles analyzed. The only exception were the very large particles > 500 nm were the aluminum peak was much more intense and no significant amount of oxygen was detected. This is explained by the fact that the ratio of oxide layer to aluminum core for the larger particles is much smaller for the big particles than for the smaller ones. The oxide layer around the particles acts as a passivation coat more effective for the large particles so larger proportions of pure aluminum are expected to be found in their core (Ramaswamy et al., 2004, 2005).

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68 3.2.5 Zeta potential: Surface Charge When nanoparticles are introduced in a liquid medi um charge may arise at their surface due by any of several possible mechanisms. The most important mechanisms for charge generation on colloidal materials are: (1) surface dissociation; (2) ion adsorption from solution; and (3) crystal lattice defects. The surface charge of nanoparticles is important for two reasons: (1) it is a major factor in determining the particle dispersion characteristics and (2) it will influence the adsorption of ions and biomolecules onto the particle surfaces, which may change how the cells see and react to the particles (Powers et al., 2006). The development of a net charge at the particle surface affects the distribution of ions in the neighboring interfacial region, resulting in an incr eased concentration of counter ions (ions of opposite charge to that of the particle) close to the surface. Thus an electrical double layer is formed in the region of the particle-liquid interface. Th e double layer consists of an inner region, which includes ions bound relatively strongly to the surface (including specifically adsorbed ions), and an outer, or diffuse, region in which the ion distribu tion is determined by a valance of electrostatic forces and random thermal motion. The potential in this region, therefore, decays as the distance from the surface increases until, at sufficient distance, it reaches the bulk solution value, conventionally taken to be zero. The potential at the boundary (surface of shear) between the particle with its most closely associated ions and the surrounding media is known as the zeta potential The zeta potential is a function of the surface charge of the particle, any adsorbed layer at the interface, and the nature and composition of the surrounding medium in which the particle is suspended. It is usually, but not necessarily, of the same sign as the actual potential at the particle surface but unlike the surface potential, the zeta potential can be measured experimentally. It reflects the effective

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69 charge on the particles and is therefore related to the electrostatic repulsion between them, which makes it relevant to dispersion and stability control of colloids (Hunter, 2001). The surface charge and potential of an insoluble metal oxide, which is the case of all the materials used in this investigation (considering the Si as a metalloid), is determined in part by the pH in the solution in which is immersed. For such systems the H+ and the OHions are the potentialdetermining ions, as a result of reactions like the one represented by equation 3-3. O H O M OH M OH MOH K H K2 2 (3-3) The presence of hydroxyl groups on metal surf aces has been amply demonstrated by IR spectroscopy. They can often be removed by heating to high temperatures but gradually return when the surface is exposed to water, either liquid or vapor (Hunter, 2001). An important value to consider when measuring the zeta potential is the point of zero charge (p.z.c.). Because the p.z.c is not measurable experime ntally, the isoelectric point (i.e.p.) is used as a valid replacement. The i.e.p is defined as the pH at which the zeta potential at the shear plane is zero ( =0). The resultant zero charge means no electrostatic repulsion among the particles, which will then come close enough for the van der Waals attractive forces to take over and cause particle agglomeration. In order to evaluate the electrical potential of the materials investigated as received when in a liquid suspension the zeta potential of the powders was measured in water using two different instruments: the AcoustoSizer II from Colloidal Dynamics, and the Zeta Reader Mark 21. The first instrument applies a high frequency electric field to a colloidal dispersion and the electric field induces and oscillation of the suspended particles. The oscillation of the particles generates sound waves that correspond to a particle size distribution and zeta potential. The zeta potential is measured as a function of pH as the suspension is automatic ally titrated to acidic or basic values using 0.1N HCl or 0.1 N NaOH respectively. This instrument al lows to easily finding an experimental value for

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70 the isoelectric point of the particles suspended. The second instrument uses a microscope and digital image capture to look at the particles streaming past the light source and displays the motion on the computer monitor. A big advantage of this last in strument is that allows to qualitatively estimating a zeta potential distribution because the particles measured are being visualized. It also allows measurement in very dilute samples and small volumes which in the case of nanoparticles can be a limiting factor for applying other techniques. On th e other hand it only allows to measure single point zeta potentials and titrations have to be made manua lly making more laborious to fully determine the zeta potentials versus pH relationship for each material. After the particles were suspended in deionized water, the electrolyte background was set at 0.01 N KCl and the pH was brought to 7.4 (physiological pH of cell culture media) by adding the proper amount of either HCl or NaOH. The zeta potential was then measured and the values found are listed in Table 3-6. In order to measure the i.e.p the same sample preparation was followed but the pH was not adjusted. The suspensions were then titrated and the found i.e.p. values found are summarized in Table 3-6. 3.2.6 Crystalline Phase Several studies have recognized the higher toxic ity of crystalline forms compared to the same chemical composition of an amorphous form of materials like silica (Fenoglio et al., 2003, Fubini et al., 2004, Warheit et al., 2003, Wierzbicki et al., 2003). A crystalline material is a three dimensionally periodic arrangement of atoms in space. Describing a unit cell as the smallest fraction of sample having all the fundamental properties of the crystal as a whole best depicts this arrangement. The location of the atoms within the unit cell depends on the types of atoms, the nature of their bonds, and their tendency to minimize the free energy by a high degree of organization (ASM Handbook, 1986). X-ray Powder Diffraction (XRD) is an efficient analysis technique to identify and characterize unknown crystalline materials. Monochromatic x-rays are used to determine the interplanar spacings

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71 of the unknown materials. X-ray diffraction peaks are produced by constructive interference of monochromatic beam scattered from each set of lattice planes at specific angles. The x-ray diffraction pattern is a fingerprint of periodic atomic arrangements in a given material (ASM Handbook, 1986). Samples were prepared by attaching a small amount of the powder to a glass slide and analyzed with a Philips XRD 3720 instrument that emits monochromatic Cu x-rays. The results obtained are in agreement with the specifications given by the manufacturers and the crystalline structures of the different powders are summarized in Table 3-7. The phases listed describe the crystalline structure of the bulk rather than the surface of the particles due to the operation conditions of this instrument and its interaction with the samples. 3.2.7 Solubility An important property of nanomaterials is the very high surface area compared to larger size particles. When a material is partially or totally soluble the rate at which that solubilization occurs is usually increased due to the larger contact area between solvent molecules and the surface of the particles. When investigating toxicity of nanomat erials an important aspect is the possibility of solubilization of the particles under the different p hysiological conditions of pH that the particles might find throughout a biological organism. Aluminum hydroxide is practically non soluble at around neutral pH (6.5-7) but it is also amphoteric which means its solubility increases at acidic and basic pHs (see Figure 3-17). From the plotted dist ribution of hydrolysis products it can be observed the very low solubility of Al ions. This phenomena is even more emphasized at pHs between 6 and 8 with a minimum solubility of about 10-6.5 m Al+3 at pH 7 (Baes and Mesmer, 1976) corresponding to the close to neutral pH found in media and other physiological fluids.

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72 3.3 In Physiological Media: As Dosed By definition physiological media is any environment in which a specific organism lives and thrives. In the case of tissue culture it refers to buffered solution of salts, amino acids, vitamins and other molecules required for the normal metabolism of the cultured cells. Usually these buffered solutions are supplemented with appropriate add itives to optimize cell growth and preserve cell culture development over extended periods of time. Hereafter complete media will refer to the mixture of RPMI 1640 media + 5% Fetal Bovine Serum (FBS) and 1 % antibiotic-antimycotic. 3.3.1 Sampling In order to dose the cells with the nanomaterials the particles were first suspended in liquid. The sampling of nanomaterials from liquid suspensions is more reliable assuming that the particles are properly dispersed and that sedimentation of bi gger particles and/or agglomerates is avoided by keeping the liquid under stirring conditions. However b ecause all the materials investigated in this research were received in dry state all the issues referring to sampling and detailed in section 3.2.1 applied in this case. The particles were first suspended, from the dry state, in ethanol (200 proof) and left overnight under the UV light of the incubator with the purpose of sterilizing the particles before exposing the cells to them. This was done with the purpose of avoiding toxicity caused by potential pathogens carried by the particles before dosing the cells. Next particles were resuspended in deionized sterile water according to the following protocol: 1. Centrifuged particles out of the ethanol for 15 minutes at 21000 rcf 2. Extracted the alcohol (supernatant) and added sterile deionized water 3. Vortex tubes for 30 seconds and repeated steps 1 and 2 4. Vortex again and repeated 1 and 2 for a 3rd time 5. Particles were left in the 3rd wash 6. Applied sonication in an ultrasound bath (2kHz) for 30 minutes Final concentration of the stock water suspensions were measured by a gravimetric method. The liquid of 1 ml of suspension was evaporated in a known weight plate and the residual dry

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73 particles were weighted. The stock water suspensions were then diluted in complete media to a final concentration of 500g/ml. 3.3.2 Particle Size Distribution Particle size distributions of the particles in media as dosed were measured using DLS. Figure 3-18 shows and example of the typical partic le size distributions obtained for Al 1 and Al 2 plotted as differential % number distributions. For comparison purposes the same plot includes the particle size distribution of the same powders measured in water. Agglomeration was assessed visually and quantified by an increasing mean of the particle size distributions measured in water and media. A similar tendency for agglomeration was observed for all of the materials. In the case of the titania the zeta potential of the particles at a pH of 7.4 is only about 20 mV, which indicates a low electrostatic barrier to agglomeration. The IEP valu es of the titania samples are very close to the neutral pH found in the in vitro growth media which explains their tendency to agglomeration in neutral physiological fluids. The agglomeration in complete media could also be due to the compression of the double electric layer caused by the high ionic strength of the physiological media which would result in reduced electrical repulsion. 3.3.3 Surface Chemistry, Nanoparticles have been found to adsorb proteins on the surface when suspended in physiological fluids (Bousquet et al., 1999; Lundqvist et al., 2004; Surve et al., 2006). The mechanism by which this phenomenon occurs is depending on the nanoparticle surface properties. In some instances the interaction of nanoparticles with proteins has been also linked to their toxic potential and final fate (Borm et al., 2006b; Hoet et al., 2004). In an attempt to characterize the surface of the nanomaterials tested once they enco unter the biomolecules and salts present in the culture media two different parameters were quantified, zeta potential and protein adsorption.

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74 3.3.3.1 Zeta potential in media The nanomaterials were suspended in complete media and the zeta potential was measured by electrophoresis in the Zeta Reader Mark 21. The values obtained are detailed in Table 3-6. As expected due to the high ionic strength of the physiological media the values of zeta potential for the different powders were very low, in many cases close to zero. This explains the tendency to agglomeration observed when the nanoparticles were suspended in media. 3.3.3.2 Protein adsorption on aluminum nanoparticles As a basic approach to a analyze proteins adsorbed to aluminum nanoparticles after exposure to in vitro culture conditions the following experiment was performed. Cells were cultured as described in Chapter 5 (5.1) and plated in 12 well pates. To minimize erroneous results in the protein analysis from cellular debris around the particles after in vitro exposure, plates with a filter insert (400 nm pore size) in each well were used. This plate design separates the bottom of the well were the cells grow adherently and the culture media. Cells were plated and incubated at 37 C for 48 hours before nanoparticle exposure. This allows cells to fully adhere to the plates and begin growing exponentially and reaching approximately 60-70 % confluency. Al 2 nanoparticles were dosed to the cells at 500 g/mL in culture media and incubated at 37 C for 60 hr. The particles were dosed inside the filters so that they would not touch the cells surface. Thus, particles inside the filter chamber were isolated from the cellular debris generated after cell death but they were exposed to the metabolites produced by the cells as they grew on the bottom of the well. Proper controls were run in parallel. After an exposure time of 60 hrs the particles were recovered from the plates and the culture media was washed five times using phosphate buffer saline by vacuum filtration through the filter inserts. Sample s were stored at -80 C for further analysis by gel electrophoresis and mass spectrometry. Gel Electrophoresis: Electrophoresis is the transport of charged molecules through a solvent by an electrical field. Any charged ion or group will migrate when placed in an electric field. As

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75 most biological polymers carry a net charge at any pH other than their isoelectric point, they will migrate at a rate proportional to their charge density. The mobility of a molecule through the electric field will depend on the strength of the field, the net charge, the size and shape of the molecule, and on the ionic strength, viscosity, and temperature of the medium in which the molecules are moving. In gel electrophoresis proteins are run through a polymeric matrix (the gel) driven by an electromotive force and they separated based on their charge and velocity traveling through the gel and compared to a known marker for several proteins. Gel electrophoresis was performed using the Invitrogen NuPAGE Bis-Tris Electrophoresis Syst em. Samples were prepared with 1X sample buffer, which desorbs the proteins, and 1X reducing agent, which further denatures the protein. The samples were then micro centrifuged at 5,000 g for 5 min and the supernatant was used for analysis. Samples without nanoparticles were prepared accord ingly diluting at 1:10. All samples were heated at 70 C for 10 min before use. Electrophoresis was performed at 200 V for 55 min. The gel was stained with the Invitrogen Colloidal Blue Staining Kit as directed. In order to have an in-gel digestion for mass spectrometry, a gel was run for only 5 min as stated, but was stained with 100 ml of Coomassie Blue + 20% methyl alcohol for 4 hr and then de-stained using 100 ml of deionized H2O + 25% methyl alcohol overnight with a sponge pa d. A typical gel collected after this type of experiments is shown in Figure 3-19. The particles were found to adsorb a very wide range of proteins directly from the complete media, fourth column on the gel from the left, as well as form the exposure to cell activity, second and third columns on the gel from the left. This observation suggests that the adsorption of proteins onto the particles is a dynamic process in which the surface does not get permanently saturated of serum proteins but an interchange with the proteins present in the media at any time happens. Mass Spectrometry: Mass spectrometry is a technique that measures the mass of individual molecules that have been converted into ions (i.e., molecules that have been electrically charged). The mass unit of measurement is the Dalton (Da) and is defined as 1 Da = 1/12 of the mass of a single

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76 atom of the isotope of carbon-12 (12C). This follows the accepted convention of defining the 12C isotope as having exactly 12 mass units. A mass spectrometer does not actually measure the molecular mass directly, but rather the mass-to-charge ratio of the ions formed from the molecules. Thus the charge of an ion is denoted by the integer number z of the fundamental unit of charge, and the mass-to-charge ratio m/z therefore represents Daltons per fundamental unit of charge. In many cases, the ions encountered in mass spectrometry have just one charge (z = 1) so the m/z value is numerically equal to the molecular (ionic) mass in Da. For this analysis, one dimensional (1D) high performance liquid chromatography (HPLC) interfaced to electro spray ionization (ESI) quadra pole time-of-flight (TOF) tandem mass spectrometry (MS and MS/MS) was utilized. The proteins underwent tryptic digestion before analysis and the results were analyzed using the MASCOT database from Matrix Science. Results can be seen in Table 3-9. Conclusion from this experiment were that the 80 nm aluminum nanoparticles adsorb mainly the more abundant serum proteins (albumin, hemoglobin and other serum proteins) after exposure to complete media in what seems to be a non-specific adsorption. 3.3.4 Solubility ICP in media In order analyze the potential solubility of aluminum nanoparticles in physiological environments particles of every aluminum powder were suspended in complete growth media, RPMI 1640 + 5% (FBS), and incubated in the same 5% CO2, 37 C environment that the cells are cultured in. After 24 hrs and 48 hrs incubation times the par ticles were filtered out of the suspensions through 0.2 m syringe filters and the remaining supernatants were analyzed. The concentration of the relevant Al +3 ions found in solution was measured by using Induced Coupled Plasma Spectroscopy (ICP). The ICP operates In the principle of atomic emission by atoms ionized in the argon plasma. Light of specific wavelengths is emitted as electron return to the ground state of the ionized elements,

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77 quantitatively identifying the species present. The de tection limit for this technique is less than 1 ppm. The results are summarized in Table 3-8. The starting concentration of the particle suspensions was 500 g/ml and the found concentrations of aluminum ions were below 1 ppm so it is concluded that solubilization of the nanoparticles investigated in the physiological media is insignificant. The results obtained from Al 6 (soluble aluminum) also proof the very low solubility of Al in neutral pH. 3.4 In Physiological Environment: After Dosage Imaging Techniques: Once the nanoparticles have entered the in vitro system the array of tools available for their characterization is very mu ch reduced to imaging techniques. Tissue cultures are complex systems in which the living cells are fed with enriched salt solutions supplemented with vitamins, proteins and other essential molecules. The in situ characterization of nanoparticles in this environment requires the use of an involved sample preparation process and TEM to capture the images. Observations from the images collected are valid to locate the nanoparticles inside the cells and appreciate the state of dispersion in vitro Nonetheless, as can be appreciated in Figures 320, 21 a more detailed characterization of the nanoparticles themselves becomes very difficult in this type of images. In some instances particles appeared to be coated with a foreign substance that could be proteins adsorbed to their surface, as protei ns had been confirmed to adsorb onto the surface (3.3.3.2). The sample processing involves protein fixation that could theoretically preserve these structures around the particle. In some instances the particles around the cells were captured in a well dispersed stated allowing a qualitative characterization of their morphology in the physiological environment around the A549 cells. Figure 3-22 shows a group of Al 1 particles outside the cell after a 12 hrs exposure. The particles appear to have conserved an external coating around them that is hypothesized to keep the aluminum core non-reacted up to that point. However, the very limited amount of pictures that is feasible to acquire with this technique does not allow making a reliable

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78 assessment about the nanoparticles after dosage. Mo re TEM images of the tissue sections as well as further discussion about the endocytosis phenom ena observed will be discuss in Chapter 5.

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79 Table 3-1. Common errors associated with powder sampling Error in powder sampling Protocols for improvement Non-representative sampling / Improper dispersion during sample preparation Use of specially designed instruments li ke spinning riffling when possible If scooping, mix sample before and take several subsamples across the bulk Agglomeration of primary particles during sample preparation If possible sample from a liquid suspension Use of dispersant aids when measuring the primary particle size* Contamination or introduction of artifacts during sampling / storage Use of clean instrumentation and containers Store in properly sealed containers Specimen degradation during storage Store in inert gas and avoid extreme temperatures, pressures and light exposure Check stability of properties over time until sure that powder is stable Only used to determine the primary particle distribution. It should not be used for toxicity testing unless it is a biocompatible surfactant present along the exposure route. Table 3-2. Absolute density measurements of the powders investigated Samples Density (g/cc) Reference Value* (g/cc) NanoTek 4.06 P25 3.95 Anatase TiO2 3.84 Rutile TiO2 4.26 Quartz 2.66 Crystalline SiO2 2.64-2.66 Al 1 ----Al 2 3.04 Al 3 2.77 Al4 2.74 Al 5 3.91 Aluminum 2.70 -alumina Al2O3 3.97 -alumina Al2O3 3.5-3.9 Monohydrate Al2O3.H2O 3.01 *From CRC handbook of Chemis try and Physics, 1970-1971. Both TiO2 powders used are approximately 80% anatase and 20% rutile. The reference value calculated for this ratio is 3.924. Not enough powder available of this material to m easure density with a gas pycnometer. Density of several aluminum compounds likely to be on the surface layer of the aluminum nanoparticles is listed for reference purposes. Table 3-3. Specific surface area and calculated mean particle diameter. Samples SSA (m2/g) Calculated Mean Particle Diameter* ( m) NanoTek 36.69 0.041 P25 45.41 0.038 Quartz 5.75 0.401 Al 1 70.75 0.031 Al 2 27.26 0.072 Al 3 8.08 0.268 Al4 2.25 0.973 Al 5 47.07 0.033 *According to equation 3-2, using the density values reported in table 3-1. Using the reference density value for aluminum metal of 2.7g/cc

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80 Table 3-4. Size range and shape of the particles from image analysis. Samples Size range* Shape NanoTek 10-500 nm Spherical P25 10-50 nm Rounded/ irregular Quartz 100 nm-12 m Angular/ irregular Al 1 20-50 nm Spherical Al 2 30-125 nm Spherical Al 3 100 nm-5 m Spherical Al4 2.560 m Spherical Al 5 100 nm-5 m/ 10-20 nm Flakes (high aspect ratio) *Diameter of the smallest and largest particles observed in the analysis. Generic shape descriptor based on the dominant morphology of the particles. Flake like particles: cross sectional diameter/ thickness Table 3-5. Elemental surface composition from XPS analysis. Samples O 1s (%) C 1s (%) Ti 2p3 (%) Al 2p (%) Si 2p (%) NanoTek 47.31 29.64 23.05 P25 47.74 25.32 28.94 Quartz 43.69 19.66 36.65 Al 1 43.54 21.69 34.77 Al 2 43.84 22.81 33.35 Al 3 40.09 27.13 32.78 Al4 37.32 36.02 26.66 Al 5 37.11 33.74 29.15 Table 3-6. Isoelectric points and zeta potentials ( ) in different environments. Samples IEP* (mV) in water at pH = 7.4 (mV) in media at pH = 7.4 NanoTek 6.3 -20.2 -0.8/-1.3 P25 6.6 -25.1 -0.2/-1.1 Quartz 1.3 -28.5 -8.4/-10.7 Al 1 +15.7/+23.6 -12.1/-16.7 Al 2 9.3-9.5 +18.2/+20.3 -1.1/-4.3 Al 3 +10.2/+12.7 -7.9/-10.3 Al4 +21.3/+23.5 -2.1/-3.4 Al 5 +34.2/+38.5 -0.5/-3.7 *Isoelectric point: pH at which = 0 Table 3-7. Crystalline phase identified experimentally by XRD Samples Crystalline Phase NanoTek Anatase/rutile 80/20 (tetragonal) P25 Anatase/rutile 80/20 (tetragonal) Quartz -quartz (hexagonal) Al 1 FCC (face centered cubic) aluminum Al 2 FCC (face centered cubic) aluminum Al 3 FCC (face centered cubic) aluminum Al4 FCC (face centered cubic) aluminum Al 5 FCC (face centered cubic) aluminum

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81 Table 3-8. Particle solubility of aluminum nanoparticles incubated in cell culture media (ppm) during two different time intervals Samples Initial Concentration 24 hrs 48 hrs Al 1 500 0.98 0.26 Al 2 500 0.16 0.15 Al 3 500 0.38 0.13 Al4 500 0.36 0.31 Al 5 500 0.14 0.16 Al 6 250 0.29 0.90 Table 3-9. Mass spectrometry results: most abundant proteins found adsorbed to the surface of Al 2 particles Mascot Hit MW* Score Protein name 1 71244 889 Albumin [bos taurus] 2 68083 645 Albumin 3 59720 639 Keratin 10, type I, cytoskeletal human 4 66149 580 Keratin, type II cytoskeletal 1 (cytok eratin 1) (K1) (CK 1) (67 kDa cytokeratin) (hair alpha protein) 5 133442 495 Thrombospondin 1 precursor 6 133321 486 Thrombospondin 1 precursor 7 66110 426 Epidermal cytokeratin 2 [homo sapiens] 8 54986 416 Keratin, 54K type I cytoskeletal bovine 9 62320 398 Cytokeratin 9 [homo sapiens] 10 133555 300 Thrombospondin [mus musculus] 11 70611 274 Serum albumin precursor (allergen Fel d 2) 12 65059 245 similar to Keratin, type II cytoskeletal 1 (cytokeratin 1) [rattus norvegicus] 13 516677 248 Apolipoprotein B-100 precursor human 14 36015 173 Apolipoprotein E [Bos taurus] 15 39193 210 Alpha-2-HS-glycoprotein precursor (fetuin-A) 16 67857 201 Albumin [canis familiaris] 17 47249 201 Apolipoprotein B 100 [alces alces] 18 29888 196 Keratin B1 [xenopus laevis] 19 51010 189 Keratin type 16 20 71447 147 Keratin complex 2, basic, gene 17; keratin complex 2, gene 17 [mus musculus] *MW = molecular weight in Da

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82 Figure 3-1. Particle size distributions of Al 3 measured by light laser diffraction. The curves correspond to two different subsamples of the same powder and represent the effect that polydispersity can have in sampling for cohesive powders. A B Figure 3-2. High Resolution TEM images of NanoTek TiO2. A) 25kX area showing the size polydispersity characteristic of this powder. B) 800kX magnification showing the crystalline lattice of the particle, a very thin surface layer of about 1nm is distinguished around the particles.

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83 A B Figure 3-3. High Resolution TEM images of P25 TiO2. A) 50kX area showing a smaller size range than for the NanoTek powder, and a more irregular shape of the particles. B) 200kX magnification, a polyhedral shape with some angular corners can be observed. A B Figure 3-4. Scanning Electron Microscope images of Min-U-Sil 5 quartz. A) 3.5kX magnification, a very wide size range can be observed. B) 11kX magnification showing the very sharp and angular geometry of these particles.

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84 A B Figure 3-5. High Resolution TEM images of Al 1. A) 50kX area showing the spherical shape of the particles and the relatively narrow particle size range. B) 200kX are where the oxide coating of about ~2.5-3 nm in thickness around the particles is clearly observed. A B Figure 3-6. High Resolution TEM images of Al 2. A) 50kX representative example of the size and shape of the particles in this powder. B) 100kX magnification showing the very uniform oxide layer around the particles of about 3-5 nm in thickness.

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85 A B Figure 3-7. Scanning Electron Microscope images of Al 3. A) 5kX image showing the spherical shape of the particles and the size polydispersity of this powder. B) Detail at 13kX showing the presence of nanoparticles around and in between the larger micron size particles. A B Figure 3-8. Scanning Electron Microscope images of Al 4. A) 430X area where the very wide particle size distribution of this powder can be appreciated. B) 700X image that shows the rounded shape of the particles that despite not being perfect spheres project a close to circular area.

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86 A B Figure 3-9. Electron Microscope images of Al 5. A) 5,5kX area showing the wide range of size in cross section of the flakes and the thickness estimated to be ~75 nm. B) 200kX magnification showing the oxide layer around the flakes. Figure 3-10. Particle size distributions of the TiO2 and quartz powders as received by laser diffraction.

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87 Figure 3-11. Particle size distributions of the different aluminum powders as received measured by laser diffraction. Figure 3-12. Particle size distributions of the aluminum and quartz powders as received measured by dynamic light scattering.

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88 Figure 3-13. Infrared (IR) reflection-absorption sp ectra of the different aluminum samples. The regions of the spectra where the bond vibrations expected for these materials are indicated in the plot by vertical lines. Figure 3-14. Typical EDS spectrum from the TiO2 particles. The picture shows the particle and location of the beam when the spectrum was collected Figure 3-15. Typical EDS spectrum from the quartz particles. The picture shows the particle and location of the beam when the spectrum was collected.

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89 A B Figure 3-16. Typical EDS spectra obtained form th e different aluminum powders. A) spectrum taken from the smaller size particles, the presence of oxygen was detected when directing the beam to the surface as well as towards the center of the particle. B) for larger particles the presence of oxygen was not as evident indicating a smaller ratio of oxide with respect to aluminum for the same interaction volume.

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90 Figure 3-17. Distribution of hydrolysis products (x, y) at I = 1 m and 25o in (a) 0.1 m Al (III), (b) 10-5 m Al (III) and (c) saturated solutions of -Al(OH)3. The dashed curves in a and b denote supersaturated regions with respect to -Al(OH)3; the heavy line in c is the total concentration of Al (III). Reproduced from Baes and Mesmer 1976 with permission of John Wiley and Sons Inc.

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91 Figure 3-18. Particle size distributions as % number for Al 1 and Al 2 in water and in media showing the typical agglomeration observed when these nanomaterials were resuspended in complete media.

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92 Figure 3-19. 2D-gel showing a typical gel electrophoresis result from the exposure of Al 80 nm particles to culture media in under different experimental conditions. Particles exposed to the A549 cell culture over a 60 hr period seem to adsorb a very wide range of proteins with higher abundance of albumin and other lighter weight proteins like insulin.

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93 Figure 3-20. A549 cell exposed to Al 2 (80 nm) for 24 hrs. Particles can be seen inside the cells in what appears to be enlarged endosomes. Figure 3-21. Nanoparticles Al 2 outside an A549 cell in a 24 hrs exposure. The particles appear agglomerated and seem to be coated by what could be proteins adsorbed to the surface.

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94 Figure 3-22. Image from TEM of some Al 1 nanoparticles outside the cells after a 12 hrs exposure. A coating around the particles can be observed.

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95 CHAPTER 4 REACTIVITY MEASUREMENTS 4.1 Isothermal Heat-Conduction Microcalorimetry Technique Calorimetry is a well known and widely used technique for measuring heat resulting from reactions occurring in a given system. In the case of systems in which the heat rate is very small, e.g. case of in vitro metabolic reactions, heat-conduction calorimetry is recognized as the best option to analyze the system (Kemp, 2000; Lewis et al., 2003). Isothermal heat-conduction microcalorimetry (IHCMC) is a type of heat-conduction calorimetry in which the detection sensitivity is very high (of the order of 0.1 W and the test sample has a small mass (typically 1-3 g) or a small volume (20-30 ml). IHCMC consists on differential measurement of the temporal changes in the enthalpy (in J) between a test material-test medium system and a reference material-reference medium system. The system is kept at a constant temperature by different means of cooling-heating. The instrument used for this research was a RC calorimeter from Thermal Hazard Technology. The design of this instrument allows heat measurements in very small volume samples, 1.5 ml or less and it provides a very low baseline noise in the order of 0.5 W. The test sample and the reference material are contained in two separate identical ampoules and ar e kept at a constant temperature in separate, identically constructed wells of the calorimeter (Figur e 4-1). The heat flow of the sample material [Q (in W)] is measured over time and the integral of th is curve is directly proportional to the exothermic or endothermic heat resulting from the process/es occurring in the sample vial. Another advantage of IHCMC is the possibility of collecting heat information of biological processes from tissue cultured in vitro (Bttcher et al., 1997; Kemp, 2000, 2001; Lisowska et al., 2004). It is important to point out that despite the simple experimental implementation of this technique for in vitro metabolism measurements, the complexity of the natural reactions occurring in living cells and their dependence on environmental factors make the interpretation of isolated microcalorimetry experiments rather difficult.

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96 One of the hypothesized toxicity mechanisms fo r aluminum nanoparticles in this research is that the reaction of aluminum nanoparticles around or inside the cells would disturb their normal cycle enough to cause cell death in a process that could be consider necrotic. In order to prove this hypothesis the chemical reactivity of the different aluminum powders was evaluated in several environments relevant to the in vitro cellular environment. 4.2 Aluminum Reaction in Aqueous Media: Size and Shape Effects Despite the complicated formulation of complete media it is an aqueous solution the bulk of which is water. Thus when investigating the possible reaction/s of aluminum powders in the physiological environment the first logical step is to study their reactivity in water. Based on a great body of literature and their own experimentation, Ratko et al (2004a), proposed the overall process of the aluminum water reaction to be as follows: O xH O Al O xH O Al2 3 2 2 3 2 where x = 2, 3 )] )( ( [ 2 2 ) 7 (2 4 2 2 3 2O H OH Al OH O H x O xH O Al The hydration of the oxide film affects it homogeneity and this favors the access of water to the metal surface and allows hydrolytic chemical reactions which are characterized by an increase in pH. OH ] )O [Al(H O H ] O) (H [Al(OH) ] O) (H [Al(OH) O H O Al OH ] O) [Al(H O H Al(OH) H Al(OH) O H Al 3 3 6 2 2 3 3 2 3 3 2 3 2 9 3 2 3 3 6 2 2 6 3 2 3 3 2 2 6 2 When this reactions occur at low temperatures <100 C as is the case for the in vitro experiments (37 C) relevant to this investigation the final product will be an amorphous Al (OH)3. In order to test the influence of particle size on the rate of reaction and the importance of the oxide layer on delaying and/or totally avoiding the reaction of the particles in water the following experiment

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97 was performed. A known amount of powder, 250g/ml was suspended in deionized sterile water and gently dispersed by 30 seconds of bath ultrasonication. A reference cell was prepared by adding 1 ml of the same water to an identical glass vial, both cells were carefully placed in the microcalorimeter and the temperature was equilibrated at 37 C. After 45 minutes required for the instrument to reach equilibrium state the heat output was measured and collected over extended periods of time, of up to 72 hours, in order to quantify the heat involved in the reaction of the particles. Experiments were repeated 2 times and the measurements were found to have very good repeatability. Results from these experiments are plotted in Figure 5-2. From the data collected several qualitative observations can be made: The rate of reaction is directly linked to the mean size of the particles. The smaller particles react more rapidly. The larger particles react later and over longer periods of time and for the largest micron size particles no reaction is observed over the 72 hrs the samples were under observation. Shape has also and influence in the rate of reaction. The aluminum flakes react at about the same time that the 80 nm aluminum powder. Secondary heat pulses were observed in the case of the flakes after the first exothermic reaction had happened. The amount of heat released is also directly related to the size of the particles. No reaction was observed for the quartz, which (being crystalline SiO2) is not expected to react in water. All the above observations are consistent with the studies about the oxide layer that have been reported in the literature (Schultze et al., 2000, Ramaswamy et al 2004, 2005, Ratko et al., 2004 a, 2004 b). The efficacy of the oxide layer in protecting the aluminum core is reduced for the smaller flakes and the irregular shapes as discussed in Chapter 2 (2.2.3). The sensitivity of this technique allows for more quantitative analysis of the aluminum reactivity. ) ( 2 2 3 ) ( 3 ) ( 2 3 ) ( g H s Al(OH) l O H s Al (4-1) Equation 4-1 describes the chemical reaction of the particle core, made of aluminum metal, with water. The standard heat of this reaction (Sposito, 1996) can be calculated as:

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98 mol kJ f(Al) H 0 mol kJ f) (Al(OH) H 25 12883 mol kJ f. O) (H H 83 2852 mol kJ f) (H H 02 mol kJ reagents products HR76 430 ) 83 285 ( 3 ) 25 1288 ( For 250 g of pure aluminum the heat of reaction would be equal to: J mol kJ mol g g 99 3 ) 76 430 ( 98 26 10 2506 (4-2) As described in Chapter 3 all the aluminum particles used for this research were coated by and oxide/hydroxide layer of about 2.5 nm in thickness. The aluminum contained in that layer will not react to produce a hydroxide thus it should not be taken into account in the calculation of heat produced by the reaction of the particles. A 2 nm thick layer around a, for example, 50 nm particle in diameter means a reduction of total reactive aluminum mass of about 20 % according to the following calculation: 778 0 ) 25 ( ) 2 25 (3 3 1 2 V V (4-3) Therefore the total heat output of 250 g of aluminum powder of 50 nm primary mean size is expected to be a 20% less of that of 250 g of pure aluminum. From equation 4-2 the heat experimentally measured from the reaction of th is sample should be about 3.99 x 0.778 = 3.11 J. From Figure 4-2 the integral of the area under the peak for the reaction of aluminum 50 nm was calculated as being 3.54 J 0.05, which confirms the total reaction of the aluminum powder during the first 5 hours after dispersion in water. The difference from the theoretical value could be due to several reasons: (1) polydispersity of the original powder particle size distribution affects the calculation of oxide to particle mass ratio; (2) thickness of the oxide layer taken as an average of 2.5

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99 for every particle when in reality thickness can vary among particles of the same sample; (3) particle aging during storage can cause a thickening of the oxide layer around the particles. A summary of the heat of reaction for the different size aluminum powders is presented in Table 4-1 with a comparison of the theoretical heat to be expected out of a hypothetical monodisperse sample of the same primary mean size as the tested sample with a 2 nm non reactive layer around the particles. The ratio of aluminum metal in the core to total mass of the particle is calculated using equation 4-3. The results from this experiment verified the direct connection between particle size and shape with the rate of reaction of aluminum nanomaterials in water. 4.3 Aluminum Reactivity in Physiological Media When investigating cell interactions in vitro, the aluminum particles encounter a complex environment rich in salts, vitamins, proteins and other biomolecules that could potentially modify their behavior and reactivity. For this reason severa l experiments in different fluids were design to simulate the possible different environments that the particle is exposed to in a cell culture. The first experimental approach taken was to measure the rate of reaction of aluminum nanoparticles in the culture media RPMI 1640 used to culture the lung cells investigated in the context of this research. Particles were prepared the same way as the experiment in water. However cell culture media was used instead of water. The pH of the suspensions in this buffered media was pH 7.4 (physiological). Samples were kept in the microcalorimeter to constantly monitor the heat flow. The results are shown in Figure 4-3. No significant reaction was observed from the aluminum spherical particles when the plots where compared to the heat flow output of the reaction in water. However the aluminum flakes did exhibit a unique pattern of incremental exothermic steps. The non reactivity of aluminum nanoparticles in physiological media was postulated to be due to the formation of an extra passivation layer around the native aluminum oxide/hydroxide consisting of phosphate bound to the surface. The rapid and e ffective adsorption of phosphates species onto

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100 aluminum hydroxide and oxides surfaces has been widely reported in research fields like microelectronics (Scandurra et al., 2001) pharmaceutics (Tang et al., 1997) and environmental chemistry (Tanada et al., 2003). In fact a chemical dipping into an organic bath containing phosphating agents is been demonstrated to provide a very efficient passivation of aluminum surfaces by the formation of one or two monolayers of orthoand polyphosphates directly grafted onto the alumina surface (Scandurra et al., 2001). These findings would suggest the hypothesis of aluminum nanoparticles gaining and extra layer of passivating material around their native oxide /hydroxide coating that would inhibit the reaction of the aluminum in physiological media (with a concentration of phosphates of 0.8 g/l) In the case of the flakes the step like progression of the reaction in media could be explained by the exfoliation of the passivating layers in areas of uneven coating due to the irregular surface topography of these particles shown in Chapter 3 (Figure 3.9). 4.4 Aluminum Reactivity in Acidic Physiological Environments From the lack of reaction over extended periods of time in physiological media the potential for aluminum nanoparticles to maintain an unreacted aluminum metal core when dosed to the cells was established. The pH in culture media, cell surroundings and the cytoplasm is known to be a constant physiological pH of around 7.2 (Alberts et al., 2002). Despite the fact that pH is highly buffered and controlled in physiological environments, it is not a unique value across the whole organism. The normal metabolic cycle of a cell requires the presence of acidic compartments known as late endosomes and lysosomes in which potentially particles could be found after interaction with living cells (Albert et al., 2002). Due to the complexity in chemical composition and activity of these acidic intracellular compartments a simple experime ntal approach was taken in order to characterize the reactivity of aluminum nanoparticles within thos e compartments. The pH of the particle in media suspensions prepared for the previous experiment was adjusting to pH 4 by addition of 1N HCl. 1 ml of this acidic particle suspension was placed in the sample holder of the microcalorimeter and the

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101 same volume of the dispersion media without the par ticles was used as a reference. Heat flow was monitored over extended periods of time (up to 72 hrs). No reaction was observed for any of the aluminum nanomaterials tested in acidic RPMI 164 0. Figure 4-4 depicts the typical reaction pattern for the aluminum nanoparticles represented by the Al 50 nm powder. The exothermic reaction observed for the particles in water is inhibited when in physiological media. No reaction was observed for the particles in media at neutral or acidic pH. From the results it seems that the passivation of the particles aluminum metal core holds stable under the acidic conditions that the particles could encounter inside the cell. The results from this experiment should be interpreted with caution. Because of the chemical complexity known to characterize the lysosomes inside eukaryotic cells th e simulated media used for this experiment is rather simple and might not be a true representation of the reactivity exhibit by aluminum nanoparticles in a real lysosome.

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102 Table 4-1. Heat of reaction in water for the different aluminum powders investigated. Samples Experimental Heat (J)/250 g Calculated Heat (J)/250 g Al 1 3.54 0.12 3.11 Al 2 2.83 0.25 3.42 Al 3 3.14 0.31 3.97 Al4 Unreacted 3.99 Al 5 2.25 0.15 2.93

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103 Figure 4-1. Thermal Hazard Technology RC calorimeter design (from http://www.thermalhazardtechnology.com ). The electrical Peltier cooling/heating system allows for very rapid adjustments and lower noise levels than in calorimeters containing other type of heat sinks, i.e. water baths. Figure 4-2. Heat output from the reaction in water of the different aluminum powders tested

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104 Figure 4-3. Heat output from the reaction in culture media (RPMI 1640) of the different aluminum powders tested Figure 4-4. Heat flow from the reaction of 50 nm aluminum in different media.

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105 CHAPTER 5 IN VITRO BIOASSAYS 5.1 Cell Line: A549 As discussed in Chapter 1 (section 1.4.3) the largest surface in the human body likely to interact with nanoparticles is the lungs. The epith elial lining is believed to be involved in lung clearance mechanisms of proteins and other macromolecules (Hastings et al., 2004). The pulmonary epithelium is composed of two major cell types, Type I and Type II. Approximately 96% of the surface area of the pulmonary epithelium is covered by Type I cells which are unable to divide. Type II cells, while covering a smaller surface area, are present in larger numbers and have distinct functions. They are the cells responsible for lung surfactant production and storage in the lamellar bodies. They are also believed to be the progenitors of Type I cells (Foster et al ., 1998). Another important characteristic of this cell type is its involvement in inflammatory processes and the regulation of the immune response by the production of interleukin-6 (Crestani et al., 1994). In this research an in vitro model of human Type II cells was chosen to investigate cell-nanoparticles interactions (Foster et al ., 1998). The selected cell line is the A549 human lung adenocarcinoma epithelial Type II. This line was initiated in 1972 by D.J. Giard, et al through explanted culture of lung carcinomatous tissue from a 58-year-old Caucasian male and has characteristic features of Type II cells of the pulmonary epithelium (ATCC). A549 cells were obtained from the American Type Culture Collection, (Rockville, MD) and maintained in RPMI-1640 medium containing 2mM LGlutamine, supplemented with 10% fetal bovine serum (heat inactivated), and 1% of an antibioticantimycotic mixture (Cellgro, Mediatech, Inc.). This supplemented medium is hereafter referred as complete medium. The following culture protocol was followed. The cells were grown adherently in microtiter plates or cell culture flasks and we re subcultured using a 0.25% (w/v) Trypsin/ 0.53 mM EDTA solution for detachment (Cellgro, Mediatech, Inc.). The culture medium was replaced every 23 days and cells were split 1:10 before reaching total confluency (surface covered by the cells). The

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106 cells are squamous cells (flat cells which form the surface of an epithelium) that grow in a monolayer. In normal culture conditions they double every 20-24 hours and are about 30-50 m in diameter when attached to the surface. A healthy looking cell can be observed in Figure 5-1. In order to be able to screen the effect of all the different materials investigated in several concentrations on a time course cells were subcultured in 96-we lls microtiter plates (200 l/well of a 1.2 x 104 cells/ml stock) for 48 hours before nanoparticle exposure. This allowed cells to fully adhere to the plates, begin exponential growth rate, and reach approximately 60% confluency at the time of exposure to the particles. 5.2 Issues When Dealing with Nanoparticles and Classical In vitro Bioassays Several different cell health indicators or endpoint s were looked at in this investigation. It is important to mention the fact that most of the classical bioassays used in the fields of toxicology and cell biology have been developed to study the effect of liquid or soluble substances. In general they consist of some sort of reagent /s that, when adde d to the culture, reacts with a specific substrate/s to give a measurable change in color and/or fluorescence in the medium. When investigating the effect of non soluble nanoparticles the dosed materials remain in culture after the exposure. Consequently, the protocols given by the manufactures to process the tissue culture and reagents were modified to eliminate, or reduce the effect of solid particles on the measurements. This step is essential to avoid the artifacts that the nanoparticles may cause in the measurements by scattering and/or absorbing the light used to quantify the color and/or fluorescence change. In the case of this research high speed centrifugation steps were introduced in the protocols and will be described for each assay. 5.3 Bioassays: Cell Death and Possible Mechanisms In order to establish cellular response to poten tial toxins there are numerous parameters and/or endpoints that can be measured. Choosing the most suitable assays can be a challenging task (Riss et al ., 2004). In this investigation a basic approach to determine cellular response to nanoparticles was taken. Cytotoxicity of the nanomaterials was linked to cell death and cell viability and quantified by

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107 means of three different endpoints, LDH for cytotoxicity, Caspase 3/7 for apoptosis and microscopy for necrosis. Typically cells were subcultured in 96 wells micro plates, exposed to the particle suspensions, incubated and processed to quantify the endpoint of interest. The protocol scheme used is illustrated in Figure 5-2. Cytotoxicity is the property of killing cells of a chemical compound (such as food, cosmetic, or pharmaceutical) or a mediator cell (cytotoxic T cell). Cytotoxicity does not indicate a specific cellular death mechanism. Cell death can occur by either two distinct mechanisms (Roche, 2004): Necrosis (accidental cell death): is the pathological process which occurs when cells are exposed to a serious physical or chemical insult. Apoptosis (normal or programmed cell death): is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Table 5-1 summarizes the main differences between the two cell death mechanisms and highlights possible indicators to identify each cell death type (Roche, 2004). Differentiation between necrosis and apoptosis can be rather complicated. The cells experience a different sequence of events that distinguish both events but the final result is the same in both cases, cells are lysed and their contents are their contents are either phagocytosed by other cells or dissolved in the surrounding media. Experimental protocol: All the experiments utilizing bioassays for toxicity interrogation started with the nanoparticles sterilization and su spension method described in Chapter 3 (3.3.1). The 96-well plates used for the nanoparticle exposure and bioassays were seeded at the same time to achieve a more homogenous cell population in terms of cells number and passage or dell generation. Following the incubation period, between 24 hrs and 48 hrs, in which the cells reach a stable equilibrium and approximately 60% confluency (estimated by observation of the wells under optical microscopy) the growth media was replaced with the particle suspension in complete media and particle concentration ranging from 500 g/ml to 32.21 g/ml. The chosen concentrations

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108 covered a very wide range of particle concentrations that have been reported in the literature (Green, 2000; Hussain et al ., 2005). At the selected time points (6, 12, 24, 48, and 72 hrs) the plates were processed for analysis with the bioassay of interest in each case. Up to now classical bioassays for toxicity have been developed to analyze the effects of chemicals or biological agents. Every bioassay kit came with detailed instructions that had to be adapted to avoid artifacts due to the presence of nanoparticles in the culture media. 5.3.1 Cytotoxicity Detection (LDH) The LDH kit was acquired from Roche. It is a colorimetric assay for the quantification of cell lysis and cell death based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. Cytotoxicity is calculated as a ratio of the LDH activity measured for exposed cells over the normal LDH activity found in healthy control cells using equation 5-1. The result represents the cell death rate in % over the control cells. 100 ) LDH LDH (Max ) LDH (LDH (%) ty Cytotoxicicells untreated cells treated cells untreated lls treated ce (5-1) The experimental protocol developed for this bioassay was as follows: Cells were incubated in the presence of the particles for the desired length of time Plates were centrifuged at 250 rcf for 10 minutes 50 l/well of supernatant were carefully transferred to a fresh 96 micro-well plate 50 l/well of freshly mixed LDH reagent was added Plates were incubated at room temperature, protected from light, for 30 minutes Reaction was stopped by adding 25 l of 1N HCl 50 l/well of 2% Triton X-100 in media was added to the plate containing the cells Plates were sonicated for 1 minute and incubated at 37 C for 3 hours Plates were centrifuged at 2500 rcf for 10 minutes 25 l/well of supernatant were carefully transferred to a fresh 96 micro-well plate 25 l/well of fresh media was added 50 l/well of freshly mixed LDH reagent was added Plates were incubated at room temperature, protected from light, for 30 minutes Reaction was stopped by adding 25 l of 1N HCl Absorbance was measured at 490 nm in a Molecular Devices Emax microplate reader Results were recorded and processed according to equation 4-1

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109 5.3.2 Apo-ONE Homogeneous Caspase-3/7 Apoptosis assay reagents were obtained from Promega Corp. This assay utilizes a profluorescent caspases-3/7 substrate (DEVD) w ith an optimized bifunctional cell lysis/activity. Caspases-3/7 play relatively early key effector role s in apoptosis in mammalian cells. Detection of an increase in the cellular activity of th ese proteases with respect to the control cells can be used as an indicator of the induction of apoptosis. The experimental protocol developed for this bioassay was as follows: Cells were incubated in the presence of the particles for the desired length of time At that time a ratio 1:1 in volume of freshly mixed reagent was added to the wells Plates were mixed for 30 seconds and incubated at room temperature for 4 hours Plates were centrifuged at 2500 rcf for 10 minutes 75 l/well of supernatant were carefully transferred to an clean plate Fluorescence was measured at 485 nm excitation and 530 nm emission wavelengths Results were recorded and directly compared to the activity measured for the controls 5.4 Transmission Electron Microscopy: Sample Preparation In parallel with every different experiment designed to investigate different particle-cell interaction scenarios and their toxicological out come from the bioassays samples were fixed and processed for TEM. Images collected provided and insight into the ultimate location of the particles and the morphology of the cells after exposure to the nanomaterials for different time intervals. In this research the tissue fixation protocol us ed was as follows. Cells were grown in 6 well plates which allowed a larger surface area a more cells per sample to be collected. The cultures were exposed to particles in the same manner than those prepared for toxicity tests using classical bioassays. Once the exposure time of interest had pass, 12, 24 or 48 hrs, the cells were gently rinsed three times with Na cacodylate or Tyrodes physio logical buffers to keep the morphological integrity of the cells as much as possible. That way, the excess particles around the cells were removed. After that the primary fixation was achieved by immersing the cells in a 2% paraformaldehyde, 1% glutaraldehyde solution in the same buffer used for rinsing. The cells were kept in the buffer

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110 anywhere from 1 hour to overnight at 4 C. The secondary fixation consisted on 2 rinses in 0.1 Na cacodylate with a 2% OsO4 for 20 minutes each at room temperature. Following samples were dehydrated in a concentration series of ethanol (from 30% to 100%) and embedded in low viscosity aliphatic epoxy resins (TAAB or Spurr). Sample s were scraped off the cell culture plates and centrifuged at 2500 rcfs for 10 minutes to move the cells towards the bottom of the pellets. The resin was cured overnight and the pellets were then trimmed as blocks in the appropriate shape for ultrathin sectioning. Ultra-thin sections were carefully collected and post-stained with a 4% uranyl acetate solution for about 15 minutes. The sections were then carefully placed on top of a Cu square mesh grid. Samples were observed under a Zeiss EM10A TEM. Images collected are following presented. Some of the sections were also observed under a JEOL 2010 F for high resolution imaging. 5.5 Experimental Approach 5.5.1 Toxicity Framework for Aluminum Nanoparticles Due to the lack of available data about the effect of aluminum nanoparticles on in vivo or in vitro models the first experiment was design to elucid ate if aluminum nanoparticles were toxic or not to A549 human lung epithelial cells. The aluminum materials tested for this study were Al 2, and two other materials were added as positive (crystalline quartz), and negative (titanium dioxide) toxicity controls (Tsuji et al., 2005, Oberdrster et al ., 2005). Cells were cultured and exposed to different concentration of nanoparticles as explained previously (5.3). The results are summarized in Figures 5-3, 6. Values plotted are the average of 3 independent tests, and the error bars represent the standard error of the replicate data points from their mean. This applies to every plot in the results unless otherwise specified. Figures 5-3, 4 represent the time and concentration courses for all the materials at 250 g/ml and 48 hrs respectively. The LDH results (assessment of membrane integrity) are expressed as value of cytotoxicity over the control. The results confirmed observations by other authors (Hussain et al ., 2005, Soto et al ., 2005, Warheit et al ., 2005) indicating that TiO2 nanoparticles do not cause

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111 significant in vitro toxicity. The total cytotoxiciy induced by the TiO2 nanoparticles used in this study only reached values below 5% even for the longe st time of exposure and highest nanoparticle concentration. Although both samples of TiO2 (P25 and NanoTek) showed very similar results, the P25 consistently caused slightly higher cell death. The difference however, was not statistically significant. Figure 5-7 shows two TEM micrographs of the A549 cells internalizing the TiO2 by endocytosis. All the materials tested in this experiment were found to be endocytosized by the cells at every time point from the 12 hrs. In the case of TiO2 nanoparticles, clathrin-coated pits were observed at the point of endocytosis on the cell membrane (Figure 5-7, right). The routes that lead inward from the cell surface to lysosomes start with the process of endocytosis, by which cells take up macromolecules, particulate substances, and, in specialized cases, even other cells. In this process, the material to be ingested is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form an endocytic vesicle containing the ingested substance or particle. As depicted in Figure 5-8 there are several forms of endocytosis depending on the size and selectivity of the vesicles formed. Two main types of endocytosis are distinguished on the basis of the size of the endocytic vesicles formed. One type is called phagocytosis (cellular eating), which involves the ingestion of large particles, such as microorganisms, dead cells, or particulate matter via large vesicles called phagosomes (generally >250 nm in diameter). The other type is pinocytosis (cellular drinking), which involves the ingestion of fluid and solutes via small pinocytic vesicles (about 100 nm in diameter). Most eucaryotic cells are continually ingesting fluid and solutes by pinocytosis (Alberts et al., 2002). Small, well dispersed nanoparticles are potentially internalized in the pinocytosis process large particles are most efficiently ingested by specialized phagocytic cells like macrophages. However endocytosis is found in most human cells as a basic process of their life cycles. The endocytic part of the cycle often begins at clathrin-coated pits. These specialized regions typically occupy about 2% of the total plasma membrane area. The lifetime of a cl athrin-coated pit is short: within a minute or so

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112 of being formed, it invaginates into the cell and pinches off to form a clathrin-coated vesicle. It has been estimated that about 2500 clathrin-coated vesicles leave the plasma membrane of a cultured fibroblast every minute (Alberts et al., 2002). The coated vesicles are even more transient than the coated pits: within seconds of being formed, they shed their coat and are able to fuse with early endosomes. Since extracellular fluid is trapped in clathrin-coated pits as they invaginate to form coated vesicles, any substance dissolved in the ex tracellular fluid is internalizeda process called fluid-phase endocytosis (Alberts et al. 2002) In the case of quartz and aluminum, the cytotoxicity measured reached maxima between 15% and 25% for the longest exposures at the highest co ncentrations of exposure. A noticeably different trend in the concentration dependence curves for these two materials was found. The quartz shows a monotonic increase in cytotoxicity with concentration whereas the cytotoxicity caused by aluminum reaches a maximum at 125g/ml. This type of response might be explained by the saturation of some solubility product of the Al nanoparticles. In aqueous solution aluminum has a very low solubility at near neutral pH (Ksp~8.0 x 10-6). Therefore, very little dissolution of the oxide coating would be expected and reaction of the bulk metal would be expected to result in the precipitation of aluminum hydroxide. Although the physiological environment in a living organism is highly buffered, it is not constant. Particularly, the lysosomes are acidic compartments and could be causing more dissolution of the particles and the production of Al(OH)2 + + H2(g) from the reaction of Al0 and water at low pH. This would explain the dense mass of material observed in the lysosomes containing Al and it could be a factor in the mechanism of the cell death observed. The quartz caused the highest cell death of all the materials tested in this study and was also found being phagocytosed by the A549 cells. Possible induction of apoptosis from particle exposure was assessed by measuring the caspases-3/7 activity. Figures 5-5, 6 show representative results versus concentration and versus time for this assay. The values measured were normalized with respect to the values obtained for the control cells. Values higher than the control w ould mean apoptotic cell death. The results indicate

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113 that apoptosis regulated by caspases activation is not a major mechanism of cell death over the 72 hour course of exposure, especially at higher partic le concentrations. The exposed cells showed a net reduction of caspases activity compared to the control cells that was most significant in the case of quartz and aluminum nanoparticles. A reduction of apoptosis was manifested at longer exposures. The results from the apoptosis assay were in contradiction with signs of apoptotic nuclei found in some of the TEM micrographs (Figure 5-9, right). Even though caspases are linked to most apoptotic events there are evidences in the literature that refer to apoptosis without caspases activation (Borner et al ., 1999) and this could be a possible explanation for these contradictory results. However signs of apoptosis were rarely observed and did not appear to be a common pathology in most of the cells. Over time, Al nanopowder caused the largest reduction on caspases-3/7 activity relative to the control values. This was interpreted as an indication of necrotic cell death mechanism which could be expected from the damage caused by the exposure to nanomaterials at high concentrations. 5.5.2 Size and Shape Effect Once the aluminum nanoparticles tested were found to be toxic in terms of cell death of A549 lung epithelial cells the following question was raised is size and shape a critical factor on the effect of aluminum particular matter? In order to find an answer, the next experiment was designed to include a wide range of sizes and different shapes of aluminum nanomaterials extensively described in Chapter 3. The objective was to study the potential effect of size and shape of aluminum particles on toxicity towards A549 cells. The quartz was in cluded as positive control for toxicity and the toxicity assessment was made by measuring LDH ac tivity and histological observations of the tissues cultured and exposed to the different materials. The previous experiment indicated an independent effect of the aluminum nanoparticles with respect to particle concentration (Figure 5-4) for the three higher concentrations tested. This observation leaded the hypothesis that the toxicity could be a result of some saturation product from the aluminum nanoparticles inside or around the cells. Therefore a soluble form of aluminum

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114 was added to the experiment to assess the effect of soluble aluminum. The aluminum form chosen was the salt Al2Cl3 6H2O and the dosage was calculated based on the stoichiometric aluminum content of this compound. Because the previous experiment did not result in positive values of apoptosis induction by aluminum no more measurements of caspases 3/7 activity were done and the analysis was based on the results from LDH activity. The results from this experiment are summarized in Figures 5-10, 11. Several trends were observed from these tests: Aluminum flakes were the most toxic form of all the different aluminum powders tested causing as much cell death as the quartz (Figures 5-10, 11) No significant toxicity was observed from the larger aluminum particles in the micron size range or from the soluble aluminum tested (Figures 5-10, 11). The maximum toxicity was observed from the smallest particle size (30 nm) powder and toxicity peaked at an earlier time point than for the next larger size (80 nm) (Figure 5-10). The cell death caused by the 80 nm aluminum powder was consistently higher than that triggered by the 30 nm particles (Figure 5-11). The tissue sections analyzed from these test confirmed that all the materials tested had been phagocytosed by the cells up to certain extent. The amount of internalized particles found by the cells increased with time exposure for all the materials tested (Figure 5-12) confirming and accumulation process inside the cells. The aluminum particles seem to carry and/or produce some sort of debris around them inside the endosomes (Figure 5-13). Only smaller particles from the larger mean size powders were found inside the cells. Particles as big as 3 m were observed phagocytosed (Figure 514). Despite their irregular shape aluminum flakes were consistently found inside the cells. The endocytic membranes around them appeared swelled and at higher magnifications the flakes showed different electronic contrast regions. This could be due to their irregular shape and position with respect to the TEM beam after microtome sectioning (Figure 5-15). The micrographs observations however supported the hypothesis of a potential reaction of the aluminum particles inside the cells.

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115 High Resolution TEM (HRTEM) was attempted, however the delicate nature of biological sections (beam damage) did not allow a successful EDS analysis of the elemental composition inside the cells. The high voltage (200keV) required for HRTEM was destructive for this type of samples. Figure 4-16 shows how quartz particles were also phagocytosed by A549 cells. Accumulation of particles over time was also confirmed for the quartz uptake. 5.5.3 Particle-Cell Contact Effect Once the toxicity observed had been correlated to the size and shape of the particles tested the next series of experiments was designed to bette r understand the toxicity mechanism. The hypothesis at this point was that the cell death observed after exposure to aluminum nanoparticles was caused by the reaction of the aluminum core of the particles inside the lysosomes (acidic compartments) of the cell. The results from the previous set of experiments suggested a direct correlation between amount of unreacted aluminum in the particle core, their shape, and the reaction kinetics of the different materials. According to this hypothesis, the 80 nm aluminum which contained more unreacted aluminum that the same mass of 30 nm aluminum would cause higher cell death. Toxicity reached a maximum at different exposure times which correlated to particle size, the larger particles reacting more slowly. This was confirmed ex vivo in simulated cell culture media and lysosomal fluid through microcalorimetry. (See Chapter 4) In order to test this hypothesis, the first step was to verify that the potential reaction and/or dissolution of the particles in the cell surroundings were not causing the toxicity. The following test was performed. Particles were suspended as previously described in Chapter 3 (3.3.1). Instead of directly dosed on top of the cell surface, the particles were isolated from the cell culture. A permeable 200 nm filter insert was used to keep the particle suspension away from the cells surface as depicted in Figure 5-17. This set-up allowed for the interchange of soluble products between the cells and the nanoparticles as well as of any potential heat of reaction in case the

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116 nanoparticles were to react. No phagocytosis was allowed as the cells never came in direct contact with the nanoparticles. No toxicity was observed for the case where no cell-particle contact was allowed (see Figure 518) confirming that particle endocytosis and/or particle cell contact was needed to trigger cell death. 5.5.4 Fully Oxidized Particles: Particle Loading Effect The evidence collected from the previous experiments suggested that internalization of the aluminum nanoparticles was involved in the toxicity mechanism of these particles. Even though reaction of the particles inside the cell was hypothesized to be the trigger for toxicity there was the possibility that just the uptake of enough solid part icles was causing cell death. In order to clarify the role of unreacted aluminum in the toxicity mechanism of these particles the following experiment was performed. Particles were stored in deionized water for over 3 months until they were fully oxidized to aluminum hydroxide. The microcalorimetry data shown in Chapter 4 confirmed the quick and total reaction in water of the different aluminum powders tested. Because only the smaller size powders and the high aspect ratio flakes showed significant toxicity the larger (micron) size aluminum powders were not tested. The cells were exposed as described in 5.3.1 to the oxidized particles. Results are summarized in Figure 5-19. The pre-reacted aluminum hydroxide nanoparticles showed no significant toxicity. A slight reduction in the quartz toxicity was also observed in agreement with the literature that reports quartz toxicity being dependent on surface characteristics (Schins et al., 2002). The diffusion of water and oxygen in explains its surface change (Doremus 1998) and therefore different toxicity levels. The findings from this set of experiments confirmed the hypothesis of the reaction of aluminum nanoparticles inside the cells being directly involved in the toxicity mechanism of these materials.

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117 5.5.5 Ph Enzymatic Activity During their normal cycle cells engulf different solid materials, molecules, and extracellular fluid by phagocytosis and pinocytosis. By digesting the contents of the membrane bounded compartments (endosomes) the cell extract the needed nutrients for its growth and reproduction (Alberts et al., 2002). The normal degradation process of endocytosed materials requires the transfer of the endosome contents to distinct intracellular compartments to ultimately end in the lysosomes (see Figure 5-20). This process is highly regulated by pH. Early endosomes of tubulovesicular morphology are reached within 1 to 2 min after uptak e. There, material to be recycled to the cell membrane is sorted from that destined to end in the lysosomes. Next endocytic material arrives in large multivesicular perinuclear late endosomes and is finally transferred to and degraded in lysosomes (Bayer et al., 1998). The vacuolar proton ATPase (v-ATPase) establishes an acidic pH in the lumen of endocytic organelles that gradually decreases from 6.2 in early endosomes to 5.5 in late endosomes and to 4.5 in lysosomes (Bayer et al ., 1998, Yamamoto et al., 1998). The hypothesis being investigated in this experiment was that by blocking the acidification of the endosomal compartments and ultimately thei r fusion with lysosomes containing active acidic enzymes would reduce or completely stop the toxicity caused by the reaction of aluminum nanoparticles in these digestive compartments. Bafilomycin A1 (baf A1) is an specific inhibitor of the vacuolar H+-ATPase isolated from Streptomyces griseus (Bayer et al., 1998, Ohta et al., 1998, Yamamoto et al., 1998). The use of such a specific chemical allowed investigating the potential influence of the acidic enzymatic activity on the degradation of aluminum nanoparticles inside the lysosomes and catalyzing their toxicity. The experimental protocol was the same that the one used in previous experiments (3.3.1 and 5.3.1). No reference about baf A1 used for the purpose of investigating toxicity of nanoparticles was found. The concentrations of antibiotic tested were chosen following other researchers work with this

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118 chemical for the purpose of characterizing internal cellular transport and protein sorting. The baf A1 concentrations of 100 nM, 10 nM and 1 nM were tested in this experiment. The LDH results presented in figures 5-21 and 5-22 are the average of two set of experiments, each containing four replicas, and the error bars represent the standard error of the replicate data points from their mean. The baf A1 treatment was successful in reducing the toxicity caused by the aluminum nanoparticles at a maximum time interval of 24 hrs. v-ATPases establish and maintain a luminal pH in endocytic and exocytic compartments. These ATPases are specifically inhibited by the drug bag A1 and secondary effects such as inhibition of receptor-ligand dissociation, altered trafficking of transmembrane proteins, inhibition of endosomal carrier vesicles budding, inhibition of late endosome-lysosome fusion and fragmentation of early endosomes have been reported (Bayer et al., 1998) Thereafter the antibiotic becomes toxic causing cell death by apoptosis (Ohta et al., 1998). It was found that very little concentration of baf A1 is needed to reduce the toxicity of aluminum nanoparticles in this system. Maximum values for toxicity reduction were achieved with baf A1 concentrations of 10 and 100 nM. However is importa nt to point out that according to the literature the effect of this substance in the cellular meta bolism is greatly dependent on the cell line (Bayer et al., 1998) The conclusion from this set of experiment is that the acidic pH and/or lysosomal fusion with the endosomes are needed to trigger aluminum nanoparticle toxicity. Because no reaction of aluminum was observed when particles were brought to an acidic pH after suspension in media (Chapter 4) the activity of hydrolytic enzymes seem to be a key factor in the toxicity of aluminum nanoparticles on A549 cells. However, it was also noted that the levels of toxicity from the aluminum flakes with baf A1 treatment were still significant. It is hypothesized that the higher cell death consistently caused by the aluminum flakes is a co mbination of the material effect (reaction of the aluminum inside the cells) and the irregular high aspect ratio shape that causes higher stress on the cells as the particles are phagocytosed and has the potential of rupturing the cell membrane. TEM

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119 micrographs were also examined and the phagocytosis of particles under the baf A1 treatment was confirmed as shown in Figure 5-23. Table 5-1. Differences between necrosis and apoptosis Necrosis Apoptosis Morphological Features Loss of membrane integrity Membrane blebbing, but not loss of integrity Begins with swelling of cytoplasm and mitochondria Aggregation of chromatin at the nuclear membrane Ends with total cell lysis Begins with shrinking of cytoplasm and nucleus condensation No vesicle formation, complete lysis Ends with fragmentation of cell into smaller bodies Disintegration (swelling) of organelles Formation of membrane bound vesicles (apoptotic bodies) Biochemical Features Loss of regulation of ion homeostasis Tightly regulated processes involving activation and enzymatic steps No energy requirement (passive process) Energy (ATP) dependent (active process) Random DNA digestion Prelytic DNA fragmentation Postlytic DNA fragmentation Activation of caspase cascade Physiological Significance Affects groups of contiguous cells Affects individual cells Evoked by non-physiological disturbances Induced by physiological stimuli Phagocytosis by macrophages Phagocytosis by adjacent cells or macrophages Significant inflammatory response No inflammatory response

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120 Figure 5-1. A549 cell in normal growth conditions under the light microscope at 63X magnification. A partial section of some neighboring cells can be observed. Figure 5-2. Experimental protocol used for the bi oassay test. Cells were seeded in the microtiter plates and allowed to reach equilibrium, and th en particles were added to the cultures and after the appropriate exposure time the different assays were processed.

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121 Released LDH: Time Course for 250 g/ml-5 0 5 10 15 20 25 4244872 Exposure Time (hrs)% Cytotoxicity NanoTek P25 Al Quartz Figure 5-3. Release of LDH from the time course expe riments. Values plotted represent the cell death rate compared to the control cells. LDH Released Concentrat ion Course at 48 hrs 0 5 10 15 20 25 31.2562.5125250500 Concentration g/ml% Cytotoxicity NanoTek P25 Al Quartz Figure 5-4. Release of LDH from the concentration course experiments. Values plotted represent the cell death rate compared to the control cells.

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122 Figure 5.5. Caspases 3/7 activity at 4 hours exposure in concentration course. Figure 5.6. Caspases 3/7 activity at 48 hours exposure in concentration course.

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123 A B Figure 5-7. Electron Microscope images of nanoparticle uptake by A549 cells. A) TiO2 nanoparticles endocytosized inside the cell (arrows) on a 48 hrs exposure. B) Higher magnification image showing the formation of a clathrin Figure 5-8. Representation of the major forms of cellular endocytosis (transport from the plasma membrane into the cell).

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124 A B Figure 5-9. Histopathology of A549 cells after nanoparticle uptake. A) Aluminum nanoparticles inside endosomes (arrows) after a 24 hrs exposure. Dilated mitochondria (m) indicating respiratory stress and potential necrotic death. B) Higher magnification showing the dilated mitochondria and the accumulation of aluminum nanoparticles inside the endosomes. Figure 5-10. Typical results of LDH release from a time course for A549 cells exposed to 250g/ml of different size aluminum particles.

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125 Figure 5-11. Typical results of LDH release from a concentration course after a 48 hrs exposure to different size aluminum particles. A B Figure 5-12. Electron Microscope images showing aluminum nanoparticle phagocytosis by A549 cells. A) Typical uptake of aluminum 30 nm particles after a 12 hr exposure. B) After 24 hr exposure higher degree of accumulation was observed generally for all the materials tested.

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126 A B Figure 5-13. Electron Microscope images showing aluminum nanoparticle accumulation inside A549 cells. A) Phagocytosis and accumulation of 80 nm aluminum particles inside A549 cells after 24 hr exposure. B) The aluminum nanoparticles were accumulated in larger compartments as they were internalized by the cells. Unidentified debris around the particles was observed inside the endosomes/lysosomes. A B Figure 5-14. Electron Microscope images of A549 cells after exposure to aluminum particles. A) Endosome containing residual material from a larger aluminum particle after 24 hr exposure. B) Higher magnification of the endosome/ lysosome content. It appears to be a network of bubbles hypothetically produced from the reaction of aluminum inside the cell.

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127 A B Figure 5-15. Endocytosis of aluminum flakes by A549 cells. A) A549 cell after a 12 hr exposure to aluminum flakes showing clearly phagocytosed particles. B) The vacuoles enclosing the particles seem to undergo swelling and internal sub membranes can be observed within the endosome. A B Figure 5-16. Electron Microscope images showing the uptake of quartz particles by A549 cells. A) A549 cell after a 24 hr exposure to quartz. Particles can be clearly distinguished inside the endosomes/lysosomes. The white areas around the particles are due to voids in the section caused by uneven cutting of the sections due to the hardness of the quartz particles. B) The quartz particles seem to organize in packed conformations inside the cell.

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128 Figure 5-17. Cross section of a cell culture well showing the filter insert used to isolate the nanoparticles from the cells surface. Figure 5-18. Release of LDH from cell exposure to the aluminum reaction products trough a filter insert.

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129 Figure 5-19. Release of LDH at after 48 hrs exposure to the unreacted and reacted particles. Figure 5-20. Transport mechanism of endocytosed materials from the extracellular fluid to the lysosomes inside the cell.

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130 Figure 5-21. Release of LDH for particle exposure with different Bafilomycin A1 treatments after 24 hrs exposure. Minimum toxicity reduction was achieved with 10 and 100 nM baf A1. Figure 5-22. Release of LDH for particle exposure with different Bafilomycin A1 treatments after 48 hrs exposure. The antibiotic causes cell death at extended periods of time.

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131 A B Figure 5-23. Transmission Electron Microscope images confirming endocytosis after baf A1 treatment. A) 50nm aluminum particles outside an A549 cell after 12 hrs exposure. A thick coat around the particles can be appreciated. B) Uptake of 50 nm aluminum particles after 24 hrs exposure and under 10nM baf A1.

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132 CHAPTER 6 CONCLUDING REMARKS 6.1 Summary The main objective of this investigation was to establish a correlation between nanoparticle characteristics (i.e. size and shape) and their potential toxicity to living organisms. With that purpose a comprehensive study of the in vitro toxicity of aluminum nanoparticles was designed and accomplished. The correlation between size, shape and nanoparticle toxicity to human lung A549 cells was established and a possible mechanism for that toxicity was hypothesized and tersted. Figure 6-1 represents the proposed mechanism of toxicity. After cell exposure to different nanoparticle suspensions the aluminum nanoparticles studied were found to end inside the endosomal/lysosomal compartments of the cell causing different levels of cell death depending on their size and shape. The hypothesis was that the nanoparticles were passivated by the oxide/hydroxide layer plus other substances adsorb from the physiological media (i.e. proteins and phosphates). This would allow the particle to reach the acidic compartment of the cell unreacted and then react there due to the caustic environment provided by acidic pH and the catalytic activity of the enzymes contained in these compartments. Thorough characterization of the materials was performed and the reactivity of the nanoparticles in the different media relevant to th is investigation was assessed. These experiments confirmed the possibility of aluminum nanoparticles reaching intracellular compartments unreacted. The different scenarios of cell/particle interaction were simulated and the toxicity measured corroborating that the cell death observed was caused by the reaction of the aluminum nanoparticles inside the cells. Lastly a very specific antibiotic meant to block the proton pumps that acidify the endosomal compartments was used and significant reduction of toxicity, especially for the spherical particles, was achieved. The results from these last experiments verified that the acidic environment and enzymatic activity are critical factors on aluminum nanoparticle toxicity. The aluminum flakes

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133 exhibit a significantly different behavior and higher levels of toxicity which could very likely be a result of their high aspect ratio. The toxicity observed is hypothesized to be a combination of the unique nanoaluminum chemistry and the physical damage caused by the flakes after being endocytosized. This last supposition, howeve r, is to be further investigated. The toxicity mechanisms hypothesized during this investigation was therefore proven to hold true. 6.2 Conclusions From the knowledge and results collected from my research the following conclusions are highlighted: As emphasized in the most recent literature available in the field of nanotoxicology, a thorough characterization of the materials tested in the di fferent environments relevant to physiological conditions was found to be fundamental for the interpretation of the results from the biological endpoints. The toxicity of nanoparticles is closely related to their behavior in physiological environments. In depth knowledge of the chemistry and surf ace properties of the materials tested in the conditions relevant to the route of exposure is essential to better understanding the outcome of biological assays. The toxicity of aluminum nanoparticles was found to be closely related to their size and shape which ultimately define their reactivity. This potentially could be extrapolated to other nanomaterials which properties are similar to those of nanoscale aluminum once verified comparable behaviors in physiological environments. Understanding the mechanism of nanoparticle/cell interactions would ultimately allow a more predictive risk assessment of nanomaterials. Common properties, like reactivity for metals, would be a reasonable framework to classify nanomaterials according to their potential toxicities. However, the results from isolated in vitro studies are to be taken with caution and direct extrapolations to in vivo scenarios are not trivial. Communication between different scientific disciplines can be challenging but is essential when approaching multidisciplinary research like the embodied by nanotoxicology. 6.3 Recommendations For Future Work The research presented in my dissertation establishes an appropriate baseline for further investigation of more subtle interactions of aluminum nanoparticles with cells. It would be

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134 interesting to analyze their effect on different cell lines like pulmonary endothelial cells and macrophages that potentially could also be exposed to nanoparticles after inhalation and will be involved in the clearance of these particles from the respiratory system. A more detailed intracellular chemistry investigation would be also beneficial. The effect on neighboring cells after aluminum toxicity could potentially open the door to increased toxicity or even medical application of these particles. A controlled delivery of these particles to targeted cells could potentially be used for therapeutic purposes if the reaction of aluminum inside one cell can be controlled without damaging the surrounding tissue. In any case cculture investigation of several cell lines are recommended as well as whole animal studies that will elucidate the ultimate fate of these particles when exposed to more complex organisms. From the experiences collected during my study it seems noteworthy to point out to future researches in this field a list of issues to take into account when investigating toxicity of nanoparticles: Acquisition of enough material of each sample to last for the entire duration of the investigation should be attempted at the beginning of the study. Batch to batch differences in nanoparticles can be enough to introduce significant variability in the experiments Storage of nanomaterials in inert environments is also important to avoid unwanted aging effects that are not controllable in many instances and hard to quantify. Classical bioassays are design, so far, to inves tigate effects of soluble toxins. When dealing with nanoparticles special attention to the protoc ols used is important to avoid artifacts caused by the nanoparticles on the measurements. Characterization of the nanoparticles should be carried out in situ or as close to the relevant conditions as possible for every experiment performed. When trying to understand new materials and their effects on physiological environments it is recommended to expand the search for references to as many fields as possible. Sometimes the answer to a question is far from where the question was asked.

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135 Figure 6-1.Sketch of the in vitro toxicity mechanism for aluminum nanoparticles established in this research

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PAGE 153

153 BIOGRAPHICAL SKETCH Maria Palazuelos Jorganes was born in Santander (Spain) in 1977. After completing her basic academic education she followed the chemical engineering program at the University of Cantabria. She got her degree from the Technical School of Industrial Engineers in 2001. Before that she had completed a nine-month research experience abroad at the Delft University of Technology in The Netherlands where she first discovered particle science and technology. From there she came to the Particle Engineering Research Center in Gainesville, Florida, and joined the chemical engineering department at the University of Florida as a PhD student on the Summer of 2002. During this time Maria has had the privilege to travel around the world presenting her work and to learn from renowned experts in her field. After her graduation on May 2007 Maria wants to pursue a scientific career in industry where she can use her skills to be nefit society and grow into a better professional and ultimately a better person.


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IN VITRO TOXICITY ANALYSIS OF NANOSCALE ALUMINUM:
PARTICLE SIZE AND SHAPE EFFECTS














By

MARIA PALAZUELOS JORGANES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2007











































O 2007 Maria Palazuelos Jorganes










































To my husband Scott,
my sister Amalia and my parents Pepa and Luis.









ACKNOWLEDGMENTS

I thank my mentor Dr. Kevin W. Powers for his unconditional support and guidance

throughout what it has been an intense journey in science and personal growth. It was thank to him

that I expanded my research experiences and that I practiced the "Philosophy" component of my

doctoral degree in our endless conversations about very different topics. To my advisor Dr. Richard

B. Dickinson goes my sincere gratitude for chairing my advisory committee and always being there

when I needed him. He has taught me to be rigorous about my work and I am truly appreciative for

that. My research was possible thanks to the generous sponsorship of Dr. Brij M. Moudgil and the

NSF. I consider myself very fortunate for the diverse and comprehensive formation that I have

received from the Particle Engineering Research Center. I also thank the rest of my advisory

committee members for their support, Dr. Spyros Svoronos and Dr. Yiider Tseng.

I want to thank the whole PERC "family" for their help, encouragement and friendship.

Special thanks are due to Gill Brubaker, Gary Scheiffele, Kathryn Finton, Jacqueline Gesner and

Vanessa Kuder. My gratitude also goes to the administrative personnel for their helpful assistance.

I am grateful to Dr. David Moraga for being a true catalyst for my research; he introduced me

to tissue culture and biotechnology techniques needed in my investigation. I could have not asked for

a better tutor. I specially thank Kerry Siebein for her wiliness to help me with HRTEM anytime.

Thanks also go to Dr. Greg Erdos and Dr. Sharon Matthews for their help with EM microscopy in

biological samples. Dr. Steve Roberts and the rest of the "Nanotox" group have been an important

contribution to the common goals of our group and I sincerely thank each of them.

My time in Gainesville has provided me with great friends and lots of memories that I will

cherish forever. Thanks go to my friends and colleagues, Vijay Krishna, Rhye Hamey, Stephen

Tedeschi, Anna Fuller, Marco Verwij s, Dauntel Specht, Milorad Djomlija and all the other with

whom I shared this time with, for making this journey so much better.










I owe this life-changing experience to Professor Brian Scarlett who was my mentor during

some of the most important transitions in my professional and personal life. He became a dear friend

who was an excellent example while providing guidance and security. I deeply miss him.

I give "gracias" to my parents, Luis and Pepa. Their love and sacrifices for our family have

been my biggest gift in life. I also thank my sister Amalia for always showing me the bright side of

life.

Last but certainly not least I thank my husband Scott; I have come this far thanks to him. He is

my haven, keeping me sane and loving me every step of the way.











TABLE OF CONTENTS


page

ACKNOWLEDGMENTS ............ ..... ._ .............. 4.....

LIST OF TABLES ............ ..... ._ ...............8....


LIST OF FIGURES ............ ..... ._ ...............9....


ABSTRACT................ ............... 12

CHAPTER


1 INTRODUCTION................ ............. 14

1.1 M otivation and Research Outline .........._..... .........._.. ............... 14.
1.2 Nanotechnology: Definitions and Historical Context ............... .................... 15
1.3 Nanotoxicology: A Discipline on Its Own ........._. ......._._. ...............18
1.4 Toxicity of Ultrafine and Nanomaterials: Literature Review. ................. ....................... 20
1.4. 1 History of Particle Toxicology: Ultrafine Particles ......... ................. ............... 20
1.4.2 Guidelines to Risk Assessment ................. ................. 21............
1.4.3 Nanoparticle Interaction with the Lung ............... .................... 22
1.4.4 Toxicity Review for Quartz and Titania .....___.....__.___ .......____ ..........2

2 NANOSIZED ALUMINUTM....................... ........30

2. 1 What is Aluminum?.............. ................ 3 0
2.2 Aluminum Nanoparticles................ ............ 3 1
2.2. 1 Economical and Social Impact. ................. ...............31...............
2.2.2 Synthesis of Nanoaluminum ................. ......... ........ ................. 32...
2.2.3 Regarding the Oxide Layer on Aluminum Nanoparticles .................... ............... 36
2.3 Toxicology Profile of Aluminum ................. ...............41........... ...
2.3.1 Sources of Aluminum .................. .. ........... ................. 41..
2.3.2 Aluminum Assimilation into the Body ................. ................. 43......... ..
2.3.3 Aluminum Distribution in the Body ............... .................... 43
2.3.4 Aluminum Distribution in the Cells ................. ................. 44...........
2.3.5 Systemic Effects Induced by Aluminum ............... .................... 44
2.3.6 Open Questions and Knowledge Gaps................ ................. 48

3 CHARACTERIZATION OF NANOPARTICulate SYstems ................. .......... ............... 51


3.1 Particulate Systems of Interest for this Research ....._.__._ ..... ..___.. .....__... ........5
3.2 Before Dosage: "As Received" .............. .................... 52
3.2. 1 Sampling ................. .. .......... .... ............... 52...
3.2.2 Density, Surface Area, and Porosity ............... .................... 55
3.2.3 Size and Shape ................. ................. 58......... ...
3.2.3.1 Imaging techniques................ .............. 59
3.2.3.2 Light scattering techniques ................. ...............61........... ..
3.2.4 Surface and Bulk Chemical Composition ................. ................. 64......... ..












3.2.4. 1 FTIR ........._...... ....._ ... ....._._.... ...........6
3.2.4.2 X-ray photoelectron spectroscopy (XPS) ................. .. ............... .......... 66
3.2.4.3 Energy dispersive spectrometry (EDS) ................. ................. ......... 66
3.2. 5 Zeta potential: "Surface" Charge ............ .....___ ...............68.
3.2.6 Crystalline Phase................ .................70
3.2.7 Solubility ................... ... .......... ...............71......
3.3 In Physiological Media: "As Dosed" ............. .....................72
3.3.1 Sampling ................... ................. 72......... ...
3.3.2 Particle Size Distribution ........................... ........73
3.3.3 Surface Chemistry,............... ...............73
3.3.3.1 Zeta potential in media ................. ............. ...............74.
3.3.3.2 Protein adsorption on aluminum nanoparticles ............... ..... ............... 74
3.3.4 Solubility ICP in media................ .. ............... 76
3.4 In Physiological Environment: "After Dosage" ................ ...............77........... ..

4 REACTIVITY MEASUREMENTS ................. ...............95................


4. 1 Isothermal Heat-Conduction Microcalorimetry Technique ............... .................... 95
4.2 Aluminum Reaction in Aqueous Media: Size and Shape Effects .................... ............... 96
4.3 Aluminum Reactivity in Physiological Media ................. ........... ..... .......... ..... 9
4.4 Aluminum Reactivity in Acidic Physiological Environments ............... .................... 100

5 IN VITRO BIOAS SAY S ............... .................... 105


5.1 Cell Line: A549 ................... ........ .... ... ....... .. ... .. .............10
5.2 Issues When Dealing with Nanoparticles and Classical In vitro Bioassays ................... ...... 106
5.3 Bioassays: Cell Death and Possible Mechanisms ............... .................... 106
5.3.1 Cytotoxicity Detection (LDH) ................................... 108
5.3.2 Apo-ONE Homogeneous Caspase-3/7. ....___......_____ ........___ ...........10
5.4 Transmission Electron Microscopy: Sample Preparation ............... ................ .... 109
5.5 Experimental Approach................ ......... .............. 110
5.5.1 Toxicity Framework for Aluminum Nanoparticles ............... .................... 110
5.5.2 Size and Shape Effect ................. ................. 113........ ...
5.5.3 Particle-Cell Contact Effect .................. ............... ................. 115.
5.5.4 Fully Oxidized Particles: Particle Loading Effect ............... ................. ... 116
5.5.5 Ph Enzymatic Activity ........._..._... ................ 117....... .

6 CONCLUDING REMARKS ............... .................... 132


6. 1 Summary ............... .................... 132
6.2 Conclusions ............... ..... .... ................... 133
6.3 Recommendations For Future Work ............... .................... 133


LIST OF REFERENCES ........._..._... ...............136....... ......


BIOGRAPHICAL SKETCH ........._..._... ............... 153....... ....










LIST OF TABLES


Table page

3-1 Common errors associated with powder sampling ............... ....................79

3-2 Absolute density measurements of the powders investigated ................. ................. ...._ 79

3-3 Specific surface area and calculated mean particle diameter. ................. ................. ...._ 79

3-4 Size range and shape of the particles from image analysis ................. ......... ................ 80

3-5 Elemental surface composition from XPS analysis. .............. .................... 80

3-6 Isoelectric points and zeta potentials (Q) in different environments. .............. ..................... 80

3-7 Crystalline phase identified experimentally by XRD ................. ................. 80......... ..

3-8 Particle solubility of aluminum nanoparticles incubated in cell culture media (ppm) during
two different time intervals ................. ................. 81.............

3-9 Mass spectrometry results: most abundant proteins found adsorbed to the surface of Al 2
particles ................. ................. 8......... 1.....

4-1 Heat of reaction in water for the different aluminum powders investigated. ................... ........ 102

5-1 Differences between necrosis and apoptosis............... ............... 119











LIST OF FIGURES


Figure page

1-1 Length scale for obj ects in the nano and micron worlds. ................. .. ............... 27.........

1-2 Possible mechanisms by which nanomaterials interact with biological tissue.. ........................ 28

1-3 Biokinetics of nano-sized particles. ................. ................. 29......... ..

2-1 "Top-down" and "bottom-up" approaches to nano-synthesis in the context of man-made
processes as well as in the physiological environment ................. ...............49..............

2-2 Atomic model of the face centered cube Al lattice and the adsorption of an oxygen
molecule on the surface. .............. .................... 49

2-3 HRTEM of an aluminum nanoparticle. ................. ................. 50............

3-1 Particle size distributions of Al 3 measured by light laser diffraction ................. ................. 82

3-2 High Resolution TEM images of NanoTek TiO2~ ................. ................. 82...........

3-3 High Resolution TEM images of P25 TiO2. .......... ................ .. ............... 83.

3-4 Scanning Electron Microscope images of Min-U-Sil 5 quartz ................. ....................... 83

3-5 High Resolution TEM images of Al 1.. ............. ..................... 84

3-6 High Resolution TEM images of Al 2.. ............. ..................... 84

3-7 Scanning Electron Microscope images of Al 3. ................................... 85

3-8 Scanning Electron Microscope images of Al 4. ................. ................. 85...........

3-9 Electron Microscope images of Al 5 ................. ................. 86......... ..

3-10 Particle size distributions of the TiO2 and quartz powders "as received" by laser diffraction. 86

3-11 Particle size distributions of the different aluminum powders "as received" measured by
laser diffraction. .............. .................... 87

3-12 Particle size distributions of the aluminum and quartz powders "as received" measured by
dynamic light scattering. ................................... 87

3-13 Infrared (IR) reflection-absorption spectra of the different aluminum samples.............._..._. .... 88

3-14 Typical EDS spectrum from the TiO2 particles. ................. ................. 88...........

3-15 Typical EDS spectrum from the quartz particles. ......_.__._ ..... .___ ......._..........8

3-16 Typical EDS spectra obtained form the different aluminum powders. ..........._.. ................ 89











3-17 Distribution of hydrolysis products (x, y) ................. ...............90........... ..

3-18 Particle size distributions as % number for Al 1 and Al 2 in water and in media .........._.........91

3-19 2D-gel showing a typical gel electrophoresis result from the exposure of Al 80 nm particles
to culture media in under different experimental conditions. .............. .................... 92

3-20 A549 cell exposed to Al 2 (80 nm) for 24 hrs. ................. ......... ............... 93

3-21 Nanoparticles Al 2 outside an A549 cell in a 24 hrs exposure. ................. ....................... 93

3-22 Image from TEM of some Al 1 nanoparticles outside the cells after a 12 hrs exposure. .......... 94

4-1 Thermal Hazard Technology CLRC calorimeter design .........__ ....... __ ............... 103

4-2 Heat output from the reaction in water of the different aluminum powders tested .............. 103

4-3 Heat output from the reaction in culture media (RPMI 1640) of the different aluminum
powders tested................ ................ 104

4-4 Heat flow from the reaction of 50 nm aluminum in different media ................... ............... 104

5-1 A549 cell in normal growth conditions under the light microscope at 63X magnification. A
partial section of some neighboring cells can be observed. .................. ................ 120

5-2 Experimental protocol used for the bioassay test. ................. ................. 120...........

5-3 Release of LDH from the time course experiments ................. .. ............... 121...........

5-4 Release of LDH from the concentration course experiments.. ................ ....................... 121

5.5 Caspases 3/7 activity at 4 hours exposure in concentration course. .............. .................... 122

5.6 Caspases 3/7 activity at 48 hours exposure in concentration course. .............. ................... 122

5-7 Electron Microscope images of nanoparticle uptake by A549 cells ................. ........._..._... 123

5-8 Representation of the major forms of cellular endocytosis. ......... ................. ............... 123

5-9 Histopathology of A549 cells after nanoparticle uptake.. ...................... ............. 124

5-10 Typical results of LDH release from a time course for A549 cells exposed to 250Cpg/ml of
different size aluminum particles. ................................... 124

5-11 Typical results of LDH release from a concentration course after a 48 hrs exposure to
different size aluminum particles. ................................... 125

5-12 Electron Microscope images showing aluminum nanoparticle phagocytosis by A549 cells.. 125

5-13 Electron Microscope images showing aluminum nanoparticle accumulation inside A549
cells. .............. .................... 126











5-14 Electron Microscope images of A549 cells after exposure to aluminum particles. ........._...... 126

5-15 Endocytosis of aluminum flakes by A549 cells. ....._.__._ .... ... .__. ......._.........12

5-16 Electron Microscope images showing the uptake of quartz particles by A549 cells. ............. 127

5-17 Cross section of a cell culture well showing the filter insert used to isolate the nanoparticles
from the cells surface. ................................... 128

5-18 Release of LDH from cell exposure to the aluminum reaction products trough a filter insert. 128

5-19 Release of LDH at after 48 hrs exposure to the unreacted and reacted particles. ................... 129

5-20 Transport mechanism of endocytosed materials from the extracellular fluid to the lysosomes
inside the cell. .............. .................... 129

5-21 Release of LDH for particle exposure with different Bafilomycin Al treatments after 24 hrs
exposure.. .............. .. ................. 13......... 0....

5-22 Release of LDH for particle exposure with different Bafilomycin Al treatments after 48 hrs
exposure.. .............. .. ................. 13......... 0....

5-23 Transmission Electron Microscope images confirming endocytosis after baf Al treatment.. 131

6-1 Sketch of the in vitro toxicity mechanism for aluminum nanoparticles established in this
research ................ ................ 135........ .....









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

IN VITRO TOXICITY ANALYSIS OF NANO SIZED ALUMINUM:
PARTICLE SIZE AND SHAPE EFFECTS

By

Maria Palazuelos Jorganes

May 2007
Chair: Richard B. Dickinson
Major: Chemical Engineering

Nanostructured materials promise to revolutionize many key areas of science and technology.

As our ability to manipulate matter at the nanoscale increases, there is a need to assess the effects of

these materials on human health and the environment. Materials at the nanoscale are interesting and

useful because they possess properties that are different from the equivalent bulk or molecular scale.

These same properties can make toxicological profiles very different from those of the same

materials on a different scale. There is a rising consensus that toxicity analysis of nanomaterials

should start from a thorough physicochemical characterization of the materials under investigation in

order to be able to establish a proper correlation between the nanoparticles characteristics and their

effects and behavior in physiological environments. This research is a clear example of the necessity

of comprehensive studies when investigating the toxicity of nanomaterials.

Aluminum nanoparticles are being extensively used for their very unique energetic properties.

These materials offer a very promising market that is fostering many startup companies which are

expected to consolidate on strong technological positions. Aluminum is generally recognized as a

non-toxic material to humans and it is widely used for applications which imply direct human

contact. The effect of aluminum nanoparticles in human health is still an unknown.

My research consisted of an in vitro toxicity screening of aluminum materials from nano to

micron size, including spherical irregularly shaped particles. Several issues relating to size, shape,










detection and characterization of nanoparticles in the different environments relevant to in vitro

toxicity analysis were addressed and suitable protocols were developed. Lung human epithelial cells

were exposed to different concentrations of these materials and the effects were analyzed by means

of various toxicity tests. Some of the materials investigated caused elevated in vitro toxicity. Cells

endocytosed the particles and a clear correlation between the particle size, shape and the effects

observed was established. The hypothesized toxicity mechanism was explored using different

analytical techniques. The detected toxicity of aluminum nanoparticles was demonstrated to be a

direct effect of their reactivity inside the cells.










CHAPTER 1
INTRODUCTION

1.1 Motivation and Research Outline

The very rapid growth of nanotechnology in the last few years promises great technological

advances and application of nanomaterials in very diverse fields. Almost every scientific field and

industrial sector is currently looking to the potential of materials at the nanoscale (Roco, 2005; The

Royal Society & The Royal Academy of Engineering, 2004). With the implementation of new

nanosize products the uncertainties about their effect on human health and the environment greatly

justify the research efforts in this field (Dreher, 2004; Hoet et al., 2004; The Royal Society & The

Royal Academy of Engineering, 2004; Thomas and Sayre, 2005). The research here presented was

motivated by the lack of information about the interaction and potential toxicities of nanoparticles at

the cellular level. A comprehensive study was designed to establish the correlation between particle

characteristics and their potential toxicity on an in vitro model.

* The rest of Chapter 1 includes a review of the current state and trends in nanotechnology and
the field of nanotoxicology. Some of the more important findings regarding the toxic effects of
nanomaterials are covered by an extensive literature review with a specific focus on inhalation
and lung interactions relevant to this investigation.

* Interest in aluminum was stimulated by the Air Force which is exploring the use of nanoscale
aluminum powders for energetic applications. Chapter 2 describes in detail the special
properties of these materials, the different processes used for their production as well as what
is known about the toxicity of aluminum.

* Knowledge of the materials tested is a prerequisite to fully understand their behavior in
physiological environments. Chapter 3 illustrates the diverse techniques and methods used for
the characterization of the different aluminum powders studied during this research.

* Aluminum is a reactive element passivated by an oxide coating that is less protective as size
decreases into the nano range. However, once introduced in physiological media the particles
are further protected (proteins and phosphate adsorbed onto the particle surface) and stay
unreacted.

* When testing for in vitro toxicity it was found that aluminum nanoparticles were penetrating
the cells and ending in cellular compartments (endosomes and lysosomes) meant for transport
and digestion inside the cell. A correlation between particle size, shape and toxicity was
established and it was hypothesized that the unreacted aluminum nanoparticles were being up
taken by the cells and exposed to the acidic and catalytic activity of the enzymes contained in










the lysosomes triggering the aluminum reaction inside the cells. This would ultimately be the
cause for the observed cell death.

* To test this hypothesis microcalorimetry was conducted to assess the reaction of aluminum in
water vs. media (simulating extracellular environment) vs. acidic media (simulating the
lysosome). Chapter 4 explains how these experiments were performed and the results
collected.

* To further test on the hypothesis for in vitro toxicity the cell metabolism was modified to avoid
the acidifieation of the endosomes and ultimately the enzymatic activity. Chapter 5 describes
how the different biological experiments were designed and performed and the results
obtained.

* Cell toxicity was reduced by suppressing the acidifieation of the endosomes and the enzymatic
reactions despite the fact that aluminum nanoparticles were still penetrating the cell. Proof of
hypothesis Q.E.D. Chapter 6 summarizes the findings of this research as well as suggestions
for further investigations in this topic.

1.2 Nanotechnology: Definitions and Historical Context

Nanotechnology definitions are as abundant as references to this topic. The National

Nanotechnology Initiative (NNI) defines nanotechnology as the understanding and control of matter

at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel applications.

Encompassing nanoscale science, engineering and technology, nanotechnology involves imaging,

measuring, modeling, and manipulating matter at this length scale (National Nanotechnology

Initiative, 2007). The Royal Society and The Royal Academy of Engineering in the UK distinguish

between nanoscience and nanotechnologies in their report "Nanoscience and Nanotechnologies:

Opportunities and Uncertainties" published in July of 2004. In this report, nanoscience is defined as

the study of phenomena and manipulation of materials at atomic, molecular and macromolecular

scales, where properties differ significantly from those at a larger scale. The definition for

nanotechnologies states that they are the design, characterization, production and application of

structures, devices and systems by controlling shape and size at the nanometer scale.

The word nanometer has its origin on two Greek expressions, "nanos" (dwarf) and "metron" (a

measure). It describes the length unit equal to one billionth of a meter (Inm = 10-9m). Nanoscale










things are naturally present in our environment and as artificial products of nanotechnology (see

Figure 1-1.)

Matter in this size range exhibits physical, chemical, and biological properties that differ from

those shown by individual atoms, molecules or bulk material. Properties of materials can be different

at the nanoscale for two main reasons: larger relative surface area than the same mass of the same

material in a larger form, and the surfacing of quantum effects at the lower end of the nanoscale.

These two phenomena can cause nanomaterials to be more chemically reactive and potentially affect

their optical, magnetic and electrical behavior. The proportion of atoms present at the surface

compared to those in the bulk is much larger in nanomaterials giving them unique properties.

Nanotechnology research and development are directed toward understanding and creating improved

materials, devices, and systems that exploit these new properties.

The first historical mention of nanoparticles and their use can be found more than 2000 years

ago when ancient Chinese and Egyptians made carbon black as a byproduct of combustion and used

it for its colorant properties. The definition of nanomaterials includes biomolecules like DNA and

some other biopolymers; therefore, life itself is related to nanotechnology. However, it was not until

1959 when the concept of nanotechnologies was first laid out by the physicist Richard Feynman, in

his lecture "There Is Plenty of Room at the Bottom" (Feynman, 1959). Feynman foresaw the

possibility of manipulating and controlling things on a small scale. The term "nanotechnology" was

not used until 1974 by Norio Taniguchi who described it as the ability to process, separate,

consolidate, and deform materials by one atom or one molecule (The Royal Society & The Royal

Academy of Engineering, 2004). Later on, in 1986, Drexler outlined the long-term potential for

nanotechnology and the possible implications for humanity. From a bottom-up approach, the author

builds "universal assemblers" from a proper arrangement of atoms in his book "Engines of Creation"

(Drexler, 1986).










Since then, the evolution of nanotechnology has been predicted to follow certain trends and

several phases of development can be distinguished (Hood, 2004; Roco, 2005; The Royal Society &

The Royal Academy of Engineering, 2004). At this time the most widespread use of these materials

is in the form of "passive" nanostructures or simple particles designed to perform one task.

Nanomaterials are being applied today in cosmetics, paints, clays, cutting tools and polymer

composites to give a few examples. The second one entails "active" nanostructure prototypes being

use for drug delivery, sensors, transistors and other special devices. This phase is being implemented

currently and should fully develop in the next Hyve to ten years. In the longer term more advanced

devices that will respond to their environment with thousands of interacting components are

envisioned in diagnosis and therapeutic tools, implants, nano-engineered filters and military

equipment. The potential benefits that nanotechnologies could have to humankind are being

considered and revised by leading experts in industry, academia and government and though

concerns about the safety of engineered nanomaterials have been raised, several fundamental

innovations are expected from nanotechnology (Roco et al., 2005d):

* Interdisciplinary teams of scientists, engineers and social experts are being fostered under the
umbrella of nanotechnology research facilitating interchange of knowledge and findings.

* Nanotechnology could enable increased social connectivity by providing improvements in
computing, sensing, communications, data storage, and display capabilities.

* Energy independence for major industrial nations caused by a host of efficiencies facilitated by
nanotechnology is a feasible possibility.

* The trend in nanotechnology indicates that affordable nanoscale medical diagnostic and
treatment devices will be available as well as advanced biomedical solutions to chronic
diseases, and visualization of biological processes within the human body.

* Protection equipment for hazardous environments will benefit from nanosensors incorporation
enabling them to adjust to vital signs as well as to exposure levels of toxic agents.

Nanotechnology will greatly contribute to general economic growth. It is already a

multimillionaire industry that is only expected to grow. Some nanomaterials used on information and










biotechnologies could be worth $1 million/ton. Annual production of these products could reach 10-

10,000 tons a year (The Royal Society & The Royal Academy of Engineering, 2004).

1.3 Nanotoxicology: A Discipline on Its Own

With the rapid advancement in the field of nanotechnology, there has been increased concern

regarding the potential risks associated with the widespread use of engineered nanomaterials (Dreher,

2004; Hoet et al., 2004; The Royal Society & The Royal Academy of Engineering, 2004; Thomas

and Sayre, 2005). Because of the increasing number of nanomaterials and the wide range of

applications, research associated with the potential risk of nanoparticles to biological organisms has

become a discipline on its own referred as "nanotoxicology" (Barnard et al., 2006; Donaldson et al.,

2004; Hoet et al., 2004; Holsapple et al., 2005; Nel et al., 2006; Oberdorster et al., 2005b).

Conducting reproducible and reliable toxicological studies with nanostructures is complicated by the

behavior of particulate matter in biological settings and the difficulty in making in situ measurements

of properties such as size, shape and surface chemistry. Because of this complexity, risk assessment

of nanomaterials requires the close collaboration of experts in different fields like, toxicology,

materials science, chemistry, medicine and molecular biology. Due to the wide variety of

nanomaterials available, any extrapolation of the attributes of a particulate system to "similar"

materials for the same or different organisms has to be made with caution. There are few attempts at

constructing general principles that help the researcher approach these issues (Moghimi et al. 2005;

Powers et al., 2006; Roberts et al., 2004; Zhi et al., 2006,). The unusual physicochemical properties

of nanomaterials are linked to their small size, chemical composition, surface structure, solubility,

shape and aggregation. It is reasonable to believe that these unique properties will cause unidentified

effects in biological systems. Figure 1-2 depicts the possible mechanisms of interaction between

nanoscale materials and biological organisms and how different materials properties, like surface

chemistry or solubility, can affect those interactions (Nel et al., 2006). In fact, investigation in this










Hield is splitting in two directions, possible toxic effects as well as therapeutic and diagnostic

applications of nanomaterials (Hoet et al., 2004; Moghimi et al., 2005).

Particles in the nanosize range can enter the human body via several routes: respiratory system,

gastrointestinal tract (GIT), skin and the circulatory systems. Contact with nanomaterials can occur in

occupational exposures, by parenteral administration in medical applications and from ambient water

and the food chain depending on the final fate of these materials in the environment. Nanotoxicology

is also concerned with the possible translocation of these materials once they enter the organism.

Because of their small size these nanoparticles and nanostructures are very likely to interact with

cells, body fluids and proteins that can help in their migration throughout the human body (see Figure

1-3) to Einally accumulate in target organs or to be eliminated through the normal excretion pathways

(Donaldson et al., 2006; Oberdorster et al., 2005a). Until now there has been little clinically relevant

evidence of engineered nanomaterials causing toxicity and the abundant studies found in the

literature are still too scattered to draw meaningful conclusions. It is still to be determined if the

distinctive properties of nanomaterials will introduce new mechanisms of injury and whether these

will result in new pathologies. Biological systems respond to multiple pathways of injury in a limited

number of pathological outcomes, such as inflammation, apoptosis, necrosis, fibrosis, hypertrophy,

metaplasia and carcinogenesis (Holsapple et al., 2005; Nel et al., 2006). Even in the case scenario

that nanomaterials will not cause new pathologies, they could introduce new mechanisms of inj ury

that will require special tools, assays and approaches to assess their toxicity. While new tools and

methods are being developed as the Hield matures, the investigation of potential toxicities of

nanomaterials should not be delayed. It could start by applying the conventional study methods

traditionally used to assess chemical toxicity.

The community involved in nanoscience and nanotechnology has come to a consensus about

the need of understanding and addressing the interaction of nanomaterials with biological systems.

So far, the range of approaches and methods used to reach conclusion regarding the effects of










manufactured nanomaterials and ultrafine particles has led to different results. This inconsistency

indicates the urgent need for standardized tests in order to get comparable results in screening

nanomaterials for potential adverse effects (Oberdorster et al., 2005a).

Nanotoxicology should establish the principles and procedures that will ensure the safety of

this technology for workers, consumers and the environment. Considering the large number of

different nanomaterials produced and tested and the trend that this industry is following,

toxicological studies should be predictive and pragmatic. The goal of nanotoxicity research should be

to develop a series of tests and assays that could predict the possible outcomes of interaction of a new

material with biological systems. Ideally, a sounded database of information about the new materials

tested will be collected and will allow a much faster and economic classification of nanomaterials as

safe or as possible hazards. In terms of regulation of nanomaterials it is recommended (Balshaw et

al., 2005; Nel et al., 2006) that decisions will be taken based on scientific evidence of toxicity, which

should consider specific products or product lines and the likelihood of an exposure risk.

1.4 Toxicity of Ultrafine and Nanomaterials: Literature Review

Although the term nanotoxicology and its use is relatively new, particle toxicology is a mature

science that has investigated the effects of particulate matter on environmental and human health for

the last 25 years (Donaldson et al., 2000). It is almost an impossible task to summarize the abundant

literature existent about particle toxicity. Nonetheless, in this section a historical overview of the

topic is given with a center of attention on the inhalation route of exposure and the in vitro cell

interactions that ultimately relate to the investigation presented in this dissertation.

1.4.1 History of Particle Toxicology: Ultrafine Particles

The first meeting presenting research in this Hield was held in Cardiff in 1979. The research

done at that time mainly involved in vitro cytotoxicity (toxic effect on cells) analysis focusing on

asbestos and other mineral dusts as well as epidemiological studies of the affected population.

Exposure to high concentrations of particulate matter in those years was mostly in mines and metal










industries employees where workers developed respiratory diseases like metal fume fever, asbestosis

and silicosis (Wagner et al., 1982). Since then, the interest in particle toxicology has focused more on

the effect that ultrafine particles (aerodynamic diameter <0. 1 Ctm) have on human health. The

definition of "ultrafine particles" roughly overlaps with that of "nanoparticles." The first expression,

ultrafine particles, refers specifically to the aerodynamic behavior of the particulates and it is mostly

used for nanoparticles generated in an uncontrolled fashion. Meanwhile, the second term,

nanoparticles, entails a true physical dimension of the particles and it generally refers to engineered

nanomaterials.

Air pollution is generated from natural sources like volcanic activity and forest fires and

manmade sources from activities like heating, cooking, industrial manufacturing and the use of

internal combustion engines. (The Royal Society & The Royal Academy of Engineering, 2004).

Ultrafine particles are a component of urban environmental air pollution. Epidemiological studies of

the effect of air pollution show a link between morbidity and mortality and the amount of particulate

matter. Laboratory-based studies through in vivo animal exposures and in vitro cell studies report

findings of increased pulmonary inflammation, cytokine and chemokines release, production of white

blood cells, oxygen-free radical production in the lungs, endotoxin mediated cellular and tissue

responses, stimulation of irritant receptors and modification of key cellular enzymes in response to

ultrafine particles exposure (Nel, 2005; Oberdorster et al., 2005a).

1.4.2 Guidelines to Risk Assessment

One of the qualities of nanomaterials is that they can be synthesized in highly homogenous

forms with desired sizes, shapes and surfaces properties. What is an advantage in terms of application

is a challenge in terms of their risk assessment. Risk assessment of any material traditionally is an

evaluation of the toxicity inherent to that material, the probability of exposure, and the dose-response

data available for that material (Balshaw et al., 2005; Environmental Protection Agency, 1993).

Dealing with the unique properties of nanomaterials means an unfeasible prediction of their possible










effects in biological systems based on the knowledge available for classical bulk materials. Limited

research has been done up to this day in terms of correlating biological effects of engineered

nanomaterials with properties like size, shape or surface charge. Smaller particles result in a larger

surface area and number of particles per unit mass, increasing their potential for biological

interactions (Hoet et al., 2004; Oberdojrster et al., 2005a). In terms of probability of exposure of

nanoparticles, it is important to define measures for exposure that describes their hazards (Donaldson

et al., 2001). Donaldson et al., provide possible candidates for measures of exposure to ambient

particulate matter (PMlo) considering inhalation as the route of exposure:

* Physical measures
Total airborne mass concentration (Cpg/m )
Particle number concentration (number of particles/m )
Specific surface area (m2/m3)
* Chemical measures
Polycyclic aromatic hydrocarbon (PAH) concentration (Cpg/m )
Sulphate concentration (Cpg/m )
* Factors to consider for risk management
How does PMlo exert its harmful effects on the lungs?
How practical is to carry out the measurement routinely?
What health endpoint is of interest?

In the case of nanomaterials, other factors like state of dispersion, surface charge, ability to

deliver transition metals, solubility, particle-cell interactions, and possibility of migration throughout

the body, to mention a few examples, are being examined within the disciplines of nanotoxicology

and nanomedicine. A better understanding of the biological interactions of nanoparticles will allow a

more "predictive" risk assessment of these materials instead of a recompilation of epidemiological

data from harmful exposures that could had been avoided otherwise.

1.4.3 Nanoparticle Interaction with the Lung

One of the possible routes of exposure to nanoparticles for humans is inhalation, lungs are the

largest surface-area organ the in the organism. The lungs consist of two different parts: airways

(transporting the air in and out the lungs), and alveoli (where the gas exchange occurs). Human lungs










contain about 2300 km of airway and 300 million alveoli. The surface area of the lungs is about 140

m2 in adult humans.

The airways are an active epithelium protected with a viscous layer of mucus and behave like a

robust barrier. The air in the alveoli, on the contrary, is only separated from the blood stream by a

500 nm layer of epithelial cells and extracellular matrix. Spherical solids can be inhaled when their

aerodynamic diameter is less than 10 Ctm (PMlo). The smaller the particles, the deeper they can travel

into the lungs. When particles are smaller than 2.5 Ctm (PM2.5), they can reach the alveoli.

Nanoparticles are deposited mainly in the alveolar region. Fibers are defined as solid materials with a

length to diameter radio of at least 3:1. Their penetration into the lung depends on their aerodynamic

properties. Fibers with a small diameter will penetrate deeper into the lungs, while very long fibers

(>>20 Ctm) are predominantly stuck in the higher airways (Hoet et al., 2004).

The likelihood of retention or clearance of inhaled particles is dependent on several factors: (1)

the site(s) of particle deposition; (2) the quantity of particles deposited; (3) the physicochemical

characteristics of the particles; (4) and the particle-cell interactions. Particles deposited in the upper

airway will be more rapidly cleared than those accumulated in the alveoli (Tran et al., 1999).

Clearance mechanisms are different for the different sections of the respiratory airway. The different

mechanisms are due to two processes: (1) physical clearance (mucocilary movement, macrophage

and epithelial phagocytosis, interstitial translocation, lymphatic drainage, blood circulation and

sensory neurons); (2) and chemical processes (dissolution, leaching and protein binding)

(Oberdorster et al., 2005a).

The phagocytosis of fibers and particles, happening in the deep alveolar region, results in the

activation of macrophages and induces the release of cytokines, chemokines, reactive oxygen species

(ROS), and other mediators that can result in sustained inflammation and eventually fibrotic changes.

The physical and chemical properties of the solid materials reaching the alveoli can affect the

phagocytosis efficiency, thus increasing the retention half time of those materials and allowing










interaction with the pulmonary epithelial cells (Hoet et al., 2004). Research regarding the interaction

of nanoparticles with lung tissue is being actively pursued.

1.4.4 Toxicity Review for Quartz and Titania

In the context of the research concerning this dissertation, two materials other than nanosize

aluminum were used as reference controls. Quartz and titania are commonly used as positive and

negative controls respectively for toxicity studies. In this section, a literature review about the

biological effects found for these two materials is presented.

Quartz: Silica (SiO2) Can Occur in non crystalline (amorphous) or in crystalline forms.

Crystalline silica may be found in more than one form being alpha form the most abundant. This

form is so abundant that the term quartz is often used in place of the general term crystalline silica.

Quartz is a component of nearly every mineral deposit. Exposure to silica has been reported from

many different industries and activities like, agriculture, mining, milling, construction, glass, cement,

abrasives, ceramics, foundries, machinery, rubber and plastics, paint, etc. The exposure to respirable

crystalline silica is associated with silicosis (a type of nodular pulmonary fibrosis) and other silica

related diseases such as pulmonary tuberculosis, lung cancer, and chronic obstructive pulmonary

disease (National Institute for Occupational Safety and Health, 2002). The Intemnational Agency for

Research on Cancer (IARC) and the National Toxicology Program (NTP) have both listed respirable

crystalline silica as a carcinogen to humans. The NIOSH exposure limit for respirable crystalline

silica is 0.05 mg/m Numerous epidemiological studies are available that relate cumulative

crystalline silica exposure data to the incidence of silicosis and other silica-related diseases. For

example, from these studies (National Institute for Occupational Safety and Health, 2002), 1 to 7

silicosis cases are predicted per 100 workers to occur at concentrations of 0.025 mg/m3 over a 45-

year working lifetime. Despite the evidences of a correlation between respirable crystalline silica and

the higher rate of respiratory diseases associated with it the exact mechanisms of the quartz toxicity

are still unknown. Castranova (2000) reviewed in vitro and in vivo studies that reported possible










mechanisms involved in the development of lung diseases associated with long term exposure to coal

mine dust and crystalline silica. The results found supported four basic mechanisms of toxicity:

* Direct cytotoxicity of coal dust or silica, resulting in cell damage, release of lipases and
proteases, and eventual lung scarring.

* Activation of oxidant production by pulmonary phagocytes, such as alveolar macrophages.
When oxidation production exceeds antioxidant defenses, lipid peroxidation and protein
nitrosation occur, resulting in tissue injury and consequent scarring.

* Activation of mediator release from alveolar macrophages and alveolar epithelial cells.
Chemokines recruit leukocytes and macrophages from the pulmonary capillaries into the air
space. Once there, these leukocytes are activated by proinflammatory cytokines to produce
reactive species increasing oxidant injury and lung scarring.

* Secretion of growth factors from alveolar macrophages and epithelial cells that stimulate
fibroblast proliferation and induces fibrosis.

The surface reactivity and characteristics of the quartz particle has been linked to the toxic

effects observed (Borm et al., 2001). The use of freshly fractured quartz Eine sized particles, like Min

U Sil, as a positive control for in vitro cytotoxicity studies is a widely accepted practice (Tsuji et al.,

2005).

Titania: Titanium dioxide (TiO2), 81So named titania, is a noncombustible, white, solid,

crystalline, odorless powder. TiO2 Can Occur in different crystalline forms, rutile and anatase are two

of the most common ones. TiO2 does not absorb visible light but strongly absorbs ultraviolet (UV)

radiation. Titania is used mainly in paints, varnishes, lacquer, paper, plastic, ceramics, rubber, and

printing. It is widely used as white pigment because of its high refractive index. The occupational

exposure to TiO2 iS regulated by OSHA (Occupational Safety and Health Administration) under the

permissible exposure limit (PEL) of 15 mg/m3 as total dust (over and 8 hour period) and 5 mg/m3 as

respirable dust (National Institute for Occupational Safety and Health, 2005). In summary, few

TiO2-related health effects have been identified in case reports. Lung particle analysis indicated that

workers exposed to respirable TiO2 can accumulate particles in their lungs that may persist for year

after the exposure has ended. Titania deposited in the lungs was often contaminated with other










agents, most commonly silica, at much lower concentration than TiO2 particles. The chronic tissue

reaction to lung deposited titania is distinct from chronic silicosis. Most cases presented a local

macrophage response with associated fibrosis that was generally mild, but of variable severity, at the

site of deposition. Overall, the available epidemiological studies of TiO2-exposed workers present a

range of environments, from industry to population based. In general, these studies provide no clear

evidence of elevated risk of lung cancer mortality or morbidity among those workers exposed to

titania dust (National Institute for Occupational Safety and Health, 2005). On the contrary some

animal studies have shown very different results in term of lung overload, inflammation and

cytotoxicity depending on the animal model used (Bermudez et al., 2004). The lung clearance rate

and nano P25 TiO2 toxicity in rodent species seems to follow an inverse relationship. Faster

pulmonary clearance was correlated to less lung toxicity. Multiple studies suggest tumor response in

rats exposed to ultrafine TiO2. The mechanism associated seems to be a secondary genotoxicity

involving chronic inflammation and cell proliferation rather than direct genotoxic effect of the TiO2

particles (National Institute for Occupational Safety and Health, 2005). Lung clearance of particles in

humans is greater than in rats. The origin of the lung cancer in rats indicates particle lung overloads

not relevant for humans though the possibility of migration of the nanoparticles to other organs is a

greater concern for humans (Borm et al., 2004, Oberderster et al., 2005). The species differences

become critical when extrapolating results from animal models to humans.

There are studies indicating the greater inflammatory response in animal studies to ultrafine

than to fine titania particles (Baggs et al., 1997, Ferin et al., 1992, Oberderster et al., 2005).

Nevertheless, TiO2 particles are found to be rather benign in terms of cell death ad metabolic activity

and are commonly used as a positive control for in vitro toxicity (Oberderster et al., 2005).
























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including the contribution of surface species and coatings and interactions with other
environmental factors (e.g., UV activation). Reproduced with permission of AAAS from
Nel, A., Xia, T., Madler, L., and Li, N. (2006). Toxic potential of materials at the
nanolevel. Science 311, 624.













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CHAPTER 2
NANOSIZED ALUMINUM

The main purpose of the research presented in this dissertation is to investigate the correlation

between size and shape of aluminum nanoparticles and their effects on toxicity in vitro in a human

lung cell line. In this chapter, nanosize aluminum relevance is reviewed in depth, from the different

methods of manufacturing and possible applications to the potential toxicities of this metal.

2.1 What is Aluminum?

Aluminum is the most abundant metal and the third most abundant element, after oxygen and

silicon, in the earth's crust. It is element 13 on the periodic table (second row of Group III) and it has

a molecular weight of 26.98 g/mol, which makes it a very light metal. It is widely distributed and

constitutes approximately 8% of the earth's surface layer. Aluminum however is a very reactive

element and it is never found as free metal in nature. It is found combined with other elements, most

commonly with oxygen, silicon, and fluorine. These chemical compounds are normally found in soil,

minerals (e.g., sapphires, rubies, and turquoise), rocks (especially igneous rocks), and clays. These

are the natural forms of aluminum rather than the silvery metal. The metal is obtained from

aluminum containing minerals, primarily bauxite. Small amounts of aluminum are also found in

water in dissolved or ionic form. The most commonly found ionic forms of aluminum are complexes

with hydroxyl ions (ATSDR, 1999).

Aluminum has low density, high electric and thermal conductivities, high reflectivity and high

corrosion resistance. Many of the common applications of aluminum rely on the durability of the

material after the aluminum atoms on the surface of the metal quickly combine with oxygen in the air

to form a thin, strong and protective coating of aluminum oxide or alumina. Because aluminum is

very soft it is usually mixed with small amounts of other metals to form aluminum alloys, which are

stronger and harder. This allows the use of aluminum in beverage cans, pots and pans, airplanes,

siding and roofing, and foil. Aluminum compounds are found in many consumer products such as










antacids, astringents, buffered aspirin, food additives, and antiperspirants. Aluminum metal powders

were often used in explosives and fireworks (ATSDR, 1999) and more currently they are a main

component of rocket propellants, munitions and composites with unique properties (Lowe, 2002).

2.2 Aluminum Nanoparticles

2.2.1 Economical and Social Impact

There are three big subcategories in the nanomaterials business; nanoclays or nanocomposites,

nanoscale metals and oxides, and carbon nanomaterials as nanotubes (Wood et al., 2002). Most of

the optimistic predictions for the nanotechnology revolution can already be seen in the current

successes and trends of nanometals. Metals are the world's oldest technological materials and due to

their wide applications and enhanced properties when in nanosize form, they were among the earliest

nanomaterials to be commercialized. The nanometals industry includes nanoparticles, nanolayers and

thin Eilms, nanofibers, and bulk nanostructured metals and alloys. The development of metal

nanoparticles had been decelerated by the fact that their high surface energy and reactivity makes

them dangerous to produce and handle. Only recent advances in the technology allow for the

production and safe handling of these materials in significant quantities (Lowe, 2002). The market is

dominated by simple metal oxides, such as silica, alumina and titania, and nanoscale metals such as

aluminum. These particles are about 83% of the world nanoparticle market with an estimated value

of around $900 millions (Wood et al., 2002).

Aluminum powders and granules (diameters < 1mm) worldwide annual sales are estimated to

be 200K tons per year. The major businesses involved in this market are the metallurgical, chemical,

and paint and pigment industries. The value of aluminum powders is so dependant on size that the

difference in price between the bulk material and the nanoparticles is over two orders of magnitude

from roughly $1/1b to $700/1b respectively (Keams, 2004). These prices are expected to drop as

demand increases and production processes are scaled up. The promising market and applications of










these materials is fostering many new startup companies, which are expected to consolidate under

bigger firms with a strong technology position (Wood. et al, 2002).

One of the unique properties of nanoaluminum is the vast quantity of energy stored in this

material. According to the Director of the Weapons and Materials Research at the US Army

Research Laboratory, energetic materials and ingredients that are produced on the nanoscale have the

promise of increased performance in a variety of ways including, sensitivity, stability, energy release

and mechanical properties (Miziolek, 2002). Thus, the areas of greatest interest for the application of

nanoaluminum are fuels for space and naval vehicles and propellants for the military. Aluminum

powder is used today in solid rocket boosters and there is an ongoing drive to reduce launching costs

and increase payload. The increase in bum rate is between 2 and 10 times, when using nano versus

regular aluminum powder according to the claim from Argonide that their nanoaluminum 'Alex'

doubles the burning rate and increases maneuverability and thrust compared to standard 20 Ctm sized

spherical aluminum powder (Keams, 2004; Rai et al., 2004). In the military context, there is interest

in the potential of the turbulent reaction of finely divided aluminum in contact with water to propel

super-cavitating naval vehicles and weapons and in the super-thermitic reactions for pyrotechnics,

primers and detonators (Lowe, 2002; Keams, 2004).

2.2.2 Synthesis of Nanoaluminum

There are several industrial methods for manufacturing of nanomaterials that can be

subdivided in two categories: "top-down", and "bottom-up". The first one refers to the size reduction

of larger size material fragments and/or devices by means or methods such as grinding or etching.

Nano thin aluminum is an important component of computer chips and new nanodevices like nano-

electro-mechanical system (NEMS). The second one involves the synthesis of larger structures from

chemical precursors or assembly of molecules. The manufacturing of nanoparticles usually requires a

bottom-up approach (The Royal Society and The Royal Academy of Engineering, 2004). The current

technology used for manufacturing is mostly a combination of both approaches in which bulk










materials are reduced to the atomic level to then "grow" the nanoparticles and/or nanostructures from

those atomic units by condensation, agglomeration, crystallization, etc. This evolution is illustrated in

Figure 2-1 which also depicts the presence of nanomaterials in biological environments and the man-

made manufacturing being ":inspired" by the smaller molecules in living organisms.

The chemical synthesis is generally used to produce nanoparticles, the process can occur in

two different phases: gas, or liquid. The focus materials of this research consist of aluminum, which

is normally transformed into nanoparticles using gas-phase techniques. Due to its very high reactivity

any liquid phase method results in an aluminum oxide/hydroxide layer around the particles. Particle

synthesis by reaction in the gas phase starts with seed generation, usually by a burst of homogeneous

nucleation when reactions produce condensable products or when volatile products are quenched.

This may be physical nucleation of a single species, but often involves complex chemical reactions.

Once the seed particles are present, they may grow by condensation or physical vapor deposition,

chemical vapor condensation, or coagulation. Coagulation dominates when the number concentration

of particles in the nucleation burst is large, which is normally the case in industrial powder synthesis

reactors (Masuda et al., 2006).

The production market for nanomaterials is highly competitive and has originated many

companies and patents around specific manufacturing processes. In the case of nanoparticles there is

also a very dynamic demand on these products that can rapidly change. Until now, the scale-up in

production of nanoparticles is limited for several reasons. Any new process or technology must be

able to exceed (in terms of economic value) what is already in place, and it must be of value (or

perceived value) to the consumer. The technology used in current industrial processes is already

generally very advanced, and so nanotechnologies will only be used where the benefits are high.

There are also technical barriers that start with the difficulty of scaling a process up from the

laboratory to an industrial setting. These barriers include inadequate characterization and measuring

tools and capabilities to enable on-line and in-line monitoring and processing control based on










nanoscale features. Along side the technical barriers there are those related to regulation such as

classification and standardization of nanomaterials and processes, and the management of any health,

safety and environmental risks that may emerge (The Royal Society and The Royal Academy of

Engineering, 2004).

Different companies have manufactured the different size aluminum nanoparticles used for this

research. The specific details about the production steps used by each company are proprietary but

general outlines of their processes are provided as follows:

QSI-NanoTAI PrOcess by Quantum Sphere Inc.: This company uses an adaptation of the gas

phase condensation method. Metal is vaporized using resistance heating at a temperature above the

boiling point of the material, until a sufficient rate of vaporization is achieved. By computer control

of the metal flux, chamber pressure, temperature and gas flow, nanopowders having the desired size

and particle distribution can be easily made at production rate desired. According to the company the

resistance heated vapor condensation method provides the best quality powder having the lowest

level of agglomeration and fewest impurities (Quantum Sphere 2006).

Pulsed Plasma Process by Nanotechnologies Inc.: Aluminum rods, such as alloy 1350 or

greater purity, are the starting material. These electrodes are placed inside a sealed vessel end-to-end

with a small gap between the ends. The sealed vessel is filled with carefully control atmosphere of

inert gases such as helium or argon. A very high electrical current (> 5 kV and >50 kA) is pulsed

through the electrodes for about a millisecond. The ends of the electrodes ablate into aluminum

plasma with a +2 or more energy state, which expands rapidly into the gasses. As the plasma ball

expands it cools and falls back to natural valence state, reforming aluminum nanoparticles. The

aluminum nanoparticles are collected while they are still in the inert atmosphere, because the

particles at this point are extremely pyrophoric. Small amounts of oxygen are then added very slowly

to create a passivation layer of aluminum oxide on the surface of the particles. This layer allows the

production workers to handle the particles in normal air, though still with caution. If the oxygen is










added to quickly, the resulting exothermic oxidation causes the particle to burn. Adding the oxygen

slowly allows the particle oxidation rate to be diffusion-limited (Nanoscale, 2006).

Inert Gas Atomization by Valimet Inc. and Toyal America Inc.: Both companies use

variations of the same method to produce micron size high purity aluminum particles. Atomization is

the process used commercially to produce the largest tonnage of metal powders. The raw material is

melted and then the liquid metal is broken into individual particles. To accomplish this, the melt

stock, in the form of elemental, multi-element metallic alloys, and/or high quality scrap, is melted in

and induction, arc, or other type of furnace. After the bath is molten homogenous, it is transferred to

a tundish, which is a reservoir used to supply a constant, controlled flow of metal into the atomizing

chamber. As the metal stream exits the tundish, it is struck by a high velocity stream of the atomizing

medium (water, air or inert gas as in the case of aluminum). The molten metal stream is disintegrated

into fine droplets that solidify during their fall through the atomizing tank. For aluminum atomization

a small amount (+ 3%) of oxygen is added to the atomizing gas in order to produce a passivated

surface on the powder being produced (Antony et al., 2003, Masuda et al., 2006).

Vacuum Deposition Technology by Sigma Technologies International Inc.: High aspect

ratio aluminum flakes, nano size in height, are produced with this technology. The vacuum

deposition involves simultaneous deposition of polymer and various metal and ceramic coatings, on a

rotating drum. Thousands of layers are deposited at high speed forming a multilayer nanocomposite

material. The bulk nanocomposite is removed from the drum and it is reduced into a fine

polymer/metal or polymer/ceramic powder, which for some applications may be further reduced to a

nanoflake pigment. The polymer is designed to dissolve in a solvent, leading to the production of

metal pigment (usually aluminum), composed of individual flakes, that have a cross sectional

diameter of 5-20 tpm and a thickness of about 20-30 nm (Sigma Technologies Inc., 2006).










2.2.3 Regarding the Oxide Layer on Aluminum Nanoparticles

When reducing particle size to the nanometer range the ratio of surface area to volume for the

same mass basis is significantly increased. Thus, surface physical and chemical properties are

decisive in understanding the behavior of nanomaterials. Aluminum is a very reactive element, with a

high heat of combustion (= -31 kJ/g), and some of its reactions occur with explosive violence. On the

other hand, the rapid formation of a thin oxide layer prevents the further attack by oxygen and retards

chemical reactions of the aluminum such as with acids (Ramaswamy et al., 2005). Many researches

interested in the combustion and energetic applications of nanoaluminum agree that the

characteristics and integrity of this oxide layer defines most of the thermo chemical behavior of this

material (Aunmann et al., 1995; Gromov et al., 2006; Ramaswamy et al., 2004, 2005; Schultze et al.,

200). The thickness, composition, and structure of the oxide layer on the particles are responsible for

the main differences in powder performance due to heterogeneous oxidation. The thermo chemical

activity of the powders is related to the method used for powder production, the storage time and

conditions, the particle size distribution, the specific surface area, the oxide layer thickness on the

particles and the total metal content in the powder (Gromov et al., 2006). Generally the oxide layer is

considered to be nonporous, very different from iron metal, which forms a porous oxide layer or rust,

easily penetrable by water and allowing corrosion beneath the superficial layer of rust (Ramaswamy

et al., 2005). Aluminum metal oxidizes very easily due to the large free energy of formation for the

oxide (= -378 kJ/mol of Al203) (Askeland, 1989). The type of oxide film determines the rate at which

oxidation occurs and weather the oxide causes the metal to be passive. The relative volumes of the

oxide and the metal define three different types of behavior. This ratio is described by the Pilling-

Bedworth equation for the following oxidation reaction:

nM +m O, a M, O,,, (2-1)










oxide volume per metal atom (a~)Psrl
P B ratio = (2-2)
metal volume per atom nZ (M2serl)(Poxlde)

Where M~ is the atomic or molecular mass, p is the density, and n is the number of metal atoms

in the oxide as defined in equation 2-1. If the Pilling-Bedworth (P-B) ratio is less than one the oxide

occupies a smaller volume than the metal from which is formed. Tensile stresses develop in the oxide

film, causing the film to crack and become porous. Oxidation can then continue rapidly. If the ratio is

equal to one, the volumes of the oxide and the metal are equal and the filmed formed is considered to

be adherent, nonporous and protective. In most cases, the oxide film tends to be protective until the

P-B ratio exceeds about two. If the P-B ratio is greater than two, the oxide volume is greater than that

of the metal and as the thickness of the film increases, high compressive stresses develop in the

oxide. The oxide may flake from the surface, exposing fresh metal, which continues to oxidize

(Askeland, 1989).

In the case of the aluminum, the P-B ratio depends on the density of the possible oxides forms

around the metal core. Considering the following values for molecular masses and densities in

equation (2-2): Moxide = 101.963 g/mol, Mmeta l= 26.982 g/mol, pmetal = 2.7 g/cm and the density of

aluminum oxide varying from 3.0 g/cm3 for low density amorphous Al203, to 3.98 g/cm3 for the

crystalline oc phase of Al203 (Gutierrez et al., 2002), the P-B ratio values range from 1.7 to 1.3.

According to the P-B ratio equation (2-2) then, the oxide layer around aluminum will be of more

protection in the crystalline form than in the amorphous phase.

For application, passive films should be stable. Nonetheless, a typical feature is their

variability under various conditions. According to Schultze et al. (2000), the most important

processes on metal oxides are given by:

* Growth (transfer of oxygen from the electrolyte or surrounding gas into the oxide)

* Corrosion (transfer of metal ions from the oxide into the electrolyte)

* Reduction (possible at very negative potentials or in reductive environments)










* Chemical dissolution (when the growth by oxidation equals the degradation by corrosion)

* Modification (intended and controlled, or not controlled as a result of changing conditions)

* Electron transfer reactions (hydrogen or oxygen evolution)

* Capacitive charging

In the context of toxic effects of nanoparticles in biological environments as humans, animals

or ecosystems it is important to consider the possibility that the "passivation" from further reaction

for metals like aluminum might not be adequate once the nanoparticles are exposed to the unique

conditions of physiological fluids.

Modern and more advanced analytical techniques have allowed a more detailed observation of

the atomic layer around the aluminum nanoparticles and a deeper understanding of the mechanisms

involved in its formation, growth and implications in the reactivity of these materials. Following, a

summary of a model proposed by Ramaswamy et al. in 2005 is included to illustrate the phenomena

occurring at the atomic level on the surface of aluminum nanoparticles as oxidation occurs.

Aluminum has a face-centered cubic lattice with the unit cell parameter a of 4.05 A. According

to this model oxygen adatoms adsorbedd atoms) are able to "move" through the aluminum

interatomic space to fill the lattice until electric charge equilibrium is obtained and no further atoms

can penetrate. The face-centered cubic unit cell of crystalline aluminum consists of single Al atoms

located at the eight corners of the cube plus another atom centered in each face of the unit cubic cell.

This structure leaves available octahedral sites (1.20 A in diameter) with enough space for an oxygen

molecule (2.4 A in total length, or 4 oxygen atomic radiuses (0.6 A)) to enter the surface on a

vertical orientation. When a clean aluminum metallic surface (free from oxygen and oxide) is

exposed to air or oxygen, oxygen molecules will attach themselves by physical adsorption (van der

Waals forces) almost instantaneously (Figure 2-2). The oxygen molecule becomes "clamped" and

permits the second phase of the reaction, a combined chemisorption and dissociation of the oxygen










molecule, to take place. The oxygen thus combines with the metallic basis by sharing electrons. The

high temperatures typical in the nanoparticle production processes provide the energy required by the

oxygen to undergo this step. These same temperatures also excite the aluminum atoms to a state that

let them shift in the lateral direction enough to allow the oxygen atom to move into the lattice. The

oxygen adatom can fit snuggly into the next octahedral interstitial site and when a new oxygen

molecules deposits on the surface it can "push" it down. Furthermore the presence of oxygen atoms

seems to have an autocatalytic effect for further oxidation exciting the surrounding aluminum atoms

by combination reactions. As the oxygen atoms fill a unit cell, further oxygen molecules deposited on

the surface can "push" the oxygen atoms farther down until a 2.5 nm oxide is created. This is the

equivalent to the saturation of 6 unit cells. No further oxidation occurs when the interatomic space is

so filled up with oxygen atoms that there is not extra space for any additional ones to move into after

electric charge equilibrium has been reached.

This model justifies the difficulty of forming single crystalline oxide on the surface of

nanoparticles, but rather an overall amorphous or part-crystalline structure is created. The single

molecular sheets produced following this model agree with high resolution TEM micrographs that

show molecular layers appearing to superimpose at the surface of the nanoparticles with some

"crystalline" mismatch. Figure 2-3 shows an example of the TEM images taken for the materials

used in this research in which the layered structure of the oxide layer can be observed.

The crystalline areas of the surface oxide are believed to be a-aluminum oxide. The main

phase composition of the oxide, forming upon aluminum combustion in oxygen, depends on the

conditions of its formation and under usual combustion conditions it is a-alumina, the most stable

phase of alumina. The a phase of alumina has a rhombohedral lattice that can grow into crystals with

a platelet morphology, which may explain the observed laminar layers on the nanoaluminum surface.










Another important observation made by Ramaswamy et al. (2004, 2005) is the presence of

water molecules on the surface of the aluminum nanoparticles. This was confirmed by Prompt

Gamma Neutron Activation Analysis (PGAA), which detected hydrogen before and after drying of

the samples. A large amount of hydrogen was found in the nanoparticles after the adsorbed water was

released under vacuum. This means that the nanoaluminum coating is porous and some of the water,

which is deposited into the coating, reacts to form aluminum hydroxide. The inner surface layer is

thus considered to consist ofoc-aluminum oxy-hydroxide Al-O (OH) and the hypothesis is confirmed

with the results from PGAA for hydrogen content and from the X-ray diffraction data.

This model explains the formation and some of the properties of the oxide-hydroxide layer that

is found in aluminum nanoparticles. It also elucidates how the "passivation" layer might in fact fail to

protect the nanoparticle core from further reaction under extreme environments such as high

temperatures or extreme pH environment in which the integrity of the coating can be compromised

(Kolics et al., 2001). When a metal is oxidized at high temperature and then allowed to cool the

difference in thermal expansion coefficients between the metal and the oxide is another factor that

can affect its stability. Typically the oxide film has a lower expansion coefficient, when the particles

cool down the metal contracts a greater amount than the oxide and the compressive stresses imposed

on the oxide may cause it to fail, specially is the P-B ratio is already high (Askeland, 1989)

Another possible reason for the failure of the oxide layer in aluminum nanoparticles to protect

them from further reaction is that the decreasing radius of curvature, for smaller size particles, could

affect the mechanical stability of the oxide layer making it more porous and fragile (Schultze et al.,

2000, Rat'ko et al., 2004 a, 2004 b). In a comparison between aluminum nanoparticles and flakes

Ramaswamy et al., (2004, 2005) found the coating of the nanoparticles to be much more porous than

the one on the flakes surface. Thus water adsorbed onto the surface can penetrate deeper on the

nanoparticles than on the flakes.










2.3 Toxicology Profile of Aluminum

In this section a thorough review of the literature available on aluminum toxicity is presented.

Even though the investigation of possible toxic effects from aluminum in diverse forms is well

established there is very little information about the consequences that exposure to aluminum

nanoparticles have on biological organisms.

Aluminum is ubiquitous element extensively used in modern life. It is a nonessential metal that

was previously considered virtually innocuous to humans (Sorenson et al., 1974). In the last two

decades several studies suggest that aluminum has some toxicity towards plants, some aquatic

animals and humans (Ganrot, 1986; Nayak et al., 2002; Pineros et al., 2001; Yokel, 2001).

2.3.1 Sources of Aluminum

Environmental Exposure: Natural aluminum occurs in the soil and makes about 8% of the

earth's surface (Sposito, 1996). Higher concentrations may be found in soil surrounding waste sites

associated with certain industries such as coal combustion and aluminum mining and smelting

(ATSDR, 1999). The largest source of particle-borne aluminum is the dust from ores and rock

materials. Dust particles are released onto the environment by both natural processes (weathering of

aluminosilicate crystal material) and human activities (mining and agriculture) (Nayak, 2002). In the

atmosphere aluminum is usually found as aluminosilicates associated with particulate matter and the

background levels of aluminum generally range from 0.005 to 0. 18 mg/m3 (Sorenson et al., 1974).

The presence of aluminum in natural water is normally small. Acid rain leaches the abundant

aluminum from the soil and contributes significantly to the environmental inputs (Harris et al., 1996).

Dietary Exposure: Many natural foods contain aluminum that has been transferred from the

soil to the plant roots when the pH is lower than 4.5-5.0 (Matsumoto, 2000). Aluminum is also

present in many manufactured foods and is added to drinking water for purification purposes.

Aluminum containing additives are common in processed cheese, baking powders, cake mixes,

frozen dough, pancake mixes, etc. (Nayak, 2002). Leaching of aluminum from beverage cans and










cookware can also occur. It has been estimated that about 20% of daily aluminum intake comes from

cooking utensils made of aluminum (Greger et al., 1985; Lin et al., 1997). The daily average of

aluminum in the daily diet is approximately 3.5 mg in Japan (Matsuda et al., 2001) and 3.4 mg in the

United Kingdom (Ysart et al., 2000)

iatrogenic Exposure: Some medications commonly used contain aluminum and in some cases

they are a direct source of aluminum into the blood stream. Acute aluminum intoxication cases in

clinical practice are uncommon but have been found to occur, with a fatal outcome in some instances

(Nakamura et al., 2000). There are several nonprescription drugs containing aluminum that are

commonly used and increase aluminum exposure in a large population. Examples of these drugs are

some antacids, buffered aspirins, antidiarrheal products, douches and hemorrhoidal medications

(Nayak, 2002). Some patients consume as much as 5 g of aluminum daily in antacids and buffered

aspirins (Lione, 1983; Flaten, 2001). Another source of aluminum introduced directly onto the

bloodstream is the use of aluminum adjuvants in vaccine products. The FDA limit of aluminum in

each vaccine dose is 0.85 mg.

Occupational Exposure: Certain occupational groups are among the highest aluminum

exposed populations. They are workers of aluminum refining and metal industries, people employed

in printing and publishing, in automotive dealerships and service stations, and individuals involved in

fabricating metal products (U.S. Public Health Service, 1999). Several studies have reported

cognitive changes, possible impairments, and other occupational hazards in relation to exposure to

aluminum dusts and fumes (Bast-Patterson et al., 1994; McLachlan, 1995; Rifat et al., 1990; White

et al., 1992). A significant finding is the accumulation and long-term retention of aluminum within

the respiratory tract of individuals repeatedly exposed in occupational settings (Schlesinger et al.,

2000)










2.3.2 Aluminum Assimilation into the Body

Aluminum absorption seems to be very low, but many factors can enhance its assimilation in

animals and humans (Deng et al., 1998).

Even though intestinal absorption of aluminum is very poor there are many organic dietary

components that are potential chelators of aluminum and may enhance its absorption (Deng et al.,

2000; Venturini-Soriano et al., 2001, Dayde et al., 2003). Examples of these compounds present in

normal diets are: tartaric acid, glutamic acid, malic acid, and succinic acid. They are common acids

in fruits, in industrial foods and beverages.

Miners, smelters and other metal workers are mainly exposed to aluminum through dusts and

aerosols. It has been suggested the accumulation of aluminum in the brain via absorption through the

olfactory system (Exley et al., 1996; McLachlan et al., 1980; Roberts, 1986) or systemized through

the lung epithelia (Gitelman et al., 1995), and through the gastrointestinal (GI) tract when

particulates are swallowed (Rollin et al., 1993). Aluminum absorption appears to be more efficient in

the respiratory system than in the GI tract (Yokel et al., 2001).

Dermal applications of aluminum compounds in cosmetic, antiperspirant, and health care

products generally do not induce harmful effects on skin or other organs (Flaten et al., 1996;

Sorenson et al., 1974).

Aluminum absorption from in vivo animal studies is very low (1%) and seems to be sensitive

to the amount of aluminum intake (Greger, 1983). To enter the body aluminum has to cross a layer of

epithelial barrier. Despite of the abundant information collected over the years about the health

effects of exposure to aluminum the interactions of GI, olfactory, pulmonary, and dermal epithelia

with aluminum are not well understood (Nayak, 2002).

2.3.3 Aluminum Distribution in the Body

The total aluminum content in healthy humans is approximately 30-50 mg (ATSDR, 1999).

The total body aluminum is a flux between different systemic compartments (Exley et al., 1996).










Unequal aluminum distribution throughout the various tissues has been reported in normal,

aluminum-exposed humans and in aluminum-treated experimental animals (Yokel et al., 2001).

From the total body aluminum about one half is in the skeleton and about one fourth is in the lungs

(Ganrot, 1986). The brain is an important accumulation organ regardless of the route of exposure

(McLachlan et al., 1980; ATSDR, 1999). The route, dose, and duration of the exposure characterize

the distribution of the metal among the different target organs (Ding et al., 1997). In terms of the

temporal pattern of pulmonary clearance of aluminum compounds from the lungs or the potential

translocation to other organs little is known (Schelesinger et al., 2000). With increasing age, the

aluminum loadings in lungs, liver, kidneys and brain have been found to be increased (ATSDR,

1999), which is an indication of the possible accumulation of aluminum overtime.

2.3.4 Aluminum Distribution in the Cells

The same manner that aluminum distributes unevenly in the body accumulating in different

target organs, aluminum distributes within the cells. Aluminum ions accumulate in the lysosomes,

cell nucleus, and chromatin (Karlik et al., 1980). A correlation between the intranuclear aluminum

accumulation and aluminum neurotoxicity has been suggested (Uemura, 1984). It has also been

reported that aluminum present in the lysosome may be associated with dementia (van Rensberg et

al., 1997). Aluminum has been found in the cytosolic, mytochindrial, lysosomal, and nuclear

components. Accumulation of aluminum in the different cellular compartments has been found to be

specific to the cell lines investigated (Exley et al., 1996; Julka et al., 1996).

2.3.5 Systemic Effects Induced by Aluminum

Toxic effects of aluminum in the brain, liver, skeletal, muscles, heart and bone marrow are

well established but the mechanisms of action are poorly understood (Flaten et al., 1996). There is

also a lack of information on its cellular sites of action (Levesque et al., 2000).

Neural System: Despite off the little information available regarding the molecular

cytotoxicity of aluminum there are evidences in literature of its neurotoxicity (Suwalsky et al., 1999).










In vivo studies in rats have found higher levels of aluminum in the brain of young aluminum-exposed

rats than in those of older aluminum-exposed rats (Gomez et al., 1997) and prenatal and postnatal

development inhibition of the brain (Yumoto et al., 2001). In humans there are several neurological

manifestations attributed to aluminum intoxication. They include the following: memory loss,

tremor, jerking movements, impaired coordination, sluggish motor movement, loss of curiosity,

ataxia, myoclonic jerks and generalized convulsions (Crapper et al., 1980; Zatta, 1994). Some of the

neuro-pathological conditions associated with elevated aluminum levels in the brain are Alzheimer's

type senile and presenile dementia, Down syndrome with manifested Alzheimer' s disease,

amyotrophic lateral sclerosis affecting spinal cord and brain, Parkinson's dementia with

neurofibrillary degeneration, dialysis encephalopathy, striatonigral syndrome alcohol dementia with

patchy demyelination senile plaques of Alzheimer' s disease and aged brain (Crapper et al., 1980;

Zatta, 1994). However the relationship between these pathological disorders and aluminum is still

controversial.

In terms of acute exposure to aluminum in adults some of the symptoms reported are agitation,

confusion, myoclonic jerk, grand mal seizures, obtundation, coma and death (Bakir et al., 1986).

Musculoskeletal System: From patients with renal dysfunction under dialysis treatment there

are studies of chronic aluminum poisoning, in these cases the skeletal is the main target (Kerr et al.,

1992). In animal studies, tracer analysis shows that the liver, kidney, muscle, and heart also

accumulate aluminum (Walker et al., 1994). Rodriguez et al. (1990) showed a decreased osteoblast

surface, increased osteoid accumulation, and cessation of bone formation after aluminum

administration to rats. In their study, aluminum was toxic to osteoblasts and inhibited mineralization

even when osteoblasts were not decreased in number.

For humans osteomalcia, bone pain, pathological fractures, proximal myopathy and failure to

respond to vitamin D3 therapy are the common outcomes of aluminum induced musculoskeletal

toxicity (Alfrey, 1984).










Respiratory System: In the case of inhalation exposure the effects of aluminum are mainly

localized on the respiratory system. Workers in the aluminum industry develop symptoms like

asthma, cough, lung fibrosis, or decreased pulmonary function but whether these effects are only due

to aluminum exposure is questionable. The working conditions and external factors other than

aluminum affecting these workers complicate the isolation of the cause for these conditions

(ATSDR, 1999).

In animal studies aluminum exposure causes macrophages proliferation in broncho-alveolar

lavage fluid and granulomatous reactions. Granulomatous reactions were characterized by giant

vacuolated macrophages, which were associated with pneumonia in some cases (ATSDR, 1999).

Cardiovascular System: Accumulation of aluminum in the heart is another outcome from

aluminum exposure observed in the literature. Hemodialysis patients develop cardiac hypertrophy

that in some cases has been related to aluminum accumulation (Zatta, 1994). Myocardial cells can

accumulate aluminum in lysosomes (Gallet, 1987) and aluminum buildup in the myocardium has

been associated with cardiomyopathy. Hemodialysis patients show a higher prevalence of arrhythmia

and sudden death than other populations with normal renal function (Zatta, 1994).

Hepatobiliary System: Evidences of the adverse effect of aluminum on the liver have been

documented for animal studies in the literature (Stein et al., 1987). Despite high accumulation of

aluminum, liver function is seldom affected due to biliary excretion. Abnormalities in hepatic

function associated with aluminum include increased serum bile acid concentration and glucuronyl

transferase activity and reduced mixed-function oxidase level and bile flow. In addition, decreased

taurine conjugation with bile acids which may be associated with cholestasis (blocked bile flow)

have been reported (Klein et al., 1989). It has also been observed that acute administration of

aluminum adversely affects hepatic drug metabolism and protein synthesis (Cherroret et al., 1995;

Jeffrey et al., 1987).










Endocrine System: Autopsy and histological tissue studies from patients of encephalopathy

associated with dialysis have shown numerous intracytoplasmic black colored fine granular

inclusions in endocrine tissues (pituitary, parathyroid, and adrenal) suggesting accumulation of

aluminum in these organs (Reusche et al., 1994). Aluminum has also been reported to concentrate in

lysosome-like structures of parathyroid gland without alteration of the cellular ultra structure (Galle,

1987). High levels of parathyroid hormone are suggested to be associated with the preferential

deposition of aluminum in the brain, bone, and parathyroid gland (Burnatowska-Hledin et al., 1983).

Parathyroid hormone levels are disrupted by aluminum in animals and humans (Fernandez et al.,

2000; Jeffrey et al., 1996). It has been speculated that aluminum induced neurological disorders,

bone disease, and anemia may indirectly cause in many dialysis patients, parathyroid hormone

toxicity (Mayor et al., 1983). The role of aluminum in the malfunctioning of the parathyroid gland is

not clear but hypertrophy is often associated with aluminum poisoning (Galle, 1987).

Urinary System: Urological dysfunction can both cause and result from aluminum

accumulation though impaired renal function is not a prerequisite for increased tissue aluminum

burden (Mayor et al., 1986). Because of their fixnction the kidneys can rapidly concentrate aluminum

but they can also get rid of it by excreting through the urine (Flaten et al., 1996). Nevertheless there

are animal studies that have found significant lysosomal damage in response to aluminum (Stein et

al., 1987 .

Blood and Hemopoietic System: There are several indicators of the toxicity of aluminum to

the hemopoietic system like: microcytic, hypochromic anemia, or decreased numbers of red blood

cells (O'Hare et al., 1982). Even in the absence of signs of anemia, ingested aluminum may depress

hematopoiesis by affecting red blood cell production and cell destruction (Garbossa et al., 1996).

Despite of the literature available suggesting a connection between aluminum toxicity to the blood

the exact mechanism of aluminum-induced anemia is still debatable.










Reproductive System: In animal studies, the testes have been found a target organ for

accumulation of aluminum for long-term exposed rats (Gomez et al., 1997). In the same way,

numerous intracytoplasmic black-colored fine granular inclusions were found in testes tissue

collected from dialysis patients (Reusche et al., 1994).

2.3.6 Open Questions and Knowledge Gaps

Despite the abundant available knowledge about the possible effects and diseases caused by

aluminum exposure there are still several questions unresolved (Nayak 2002):

* What are the organ-specific variations in aluminum toxicity?

* What is the role of differential aluminum kinetics in different organs?

* How does aluminum enter the body by different routes?

* What are the molecular mechanisms) of aluminum toxicity that may characterize features of
aluminum toxicity common to all target organs?

The studies reported here were conducted on forms of aluminum other than nanoparticles of

pure metal. During the time invested in this investigation no studies were found with regard to the

biological effects of aluminum nanoparticles.

The research presented in my dissertation addresses some of the possible toxicities and

mechanisms of aluminum nanoparticles at the cellular level in a lung epithelial cell model that could

ultimately be affected by aluminum nanoparticles after inhalation.














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Figure 2-1. "Top-down" and "bottom-up" approaches to nano-synthesis in the context of man-made
processes as well as in the physiological environment. Currently both approaches overlap
for most of the nanomaterials being produced.


Figure 2-2. Atomic model of the face centered cube Al lattice and the adsorption of an oxygen
molecule on the surface. Generated with CAChe software from Fujitsu.




























Figure 2-3. HRTEM of an aluminum nanoparticle. (A) A multilayer oxide/hydroxide layer is
observed around the aluminum nanoparticle. (B) Higher magnification shows mismatch
in the molecular layers of the surface oxide that could j ustify their tendency to easily
exfoli ate.










CHAPTER 3
CHARACTERIZATION OF NANOPARTICULATE SYSTEMS

In order to establish any correlation between nanoparticle characteristics like size and shape,

and their biological effects, a thorough characterization of the particles is essential. In this chapter,

the techniques used for the physicochemical characterization of these materials and the results

obtained for the systems of interest, are described. The chapter follows a logical sequence used in the

characterization of nanomaterials for biological applications: as received, in physiological

environment, and after dosage.

The importance of 'knowing', to the best of the available capabilities for analysis, the material

to be tested for toxicity has been widely recognized in the research community working in this field

and several recommendations can be found in the literature (Bucher et al., 2004, Oberdorster et al.,

2005a, 2005b, Powers et al., 2006). Complete characterization of nanoparticles includes such

measurements as density, size, size distribution, shape and other morphological features as,

crystallinity, porosity and surface roughness, chemistry of the material, solubility, surface area, state

of dispersion surface chemistry, and other physicochemical properties (Powers et al., 2006).

Some of those nanoparticle properties like shape, density, and porosity for example are not

expected to change when in a biological environment. At the other hand, others like particle size

distribution and surface chemistry are very likely to change in physiological media due to the

adsorption of biomolecules onto the surface and the different states of dispersion that the

physiological conditions will promote. Thus, the importance of taking into account the very likely

changes by characterizing the materials as close as possible to the conditions of before, during, and

after their interaction with biological organisms. An exhaustive material characterization is time

consuming, expensive and complex. The extent to which it should be carried on depends on the

objectives of the study.










The main obj ective of this investigation is to analyze the effect that size and shape of certain

commercially available aluminum nanopowders have on pulmonary in vitro toxicity. The limitations

of the available techniques and the complexity of the physiological environments are here discussed,

as well as the protocols that have been developed during this research.

3.1 Particulate Systems of Interest for this Research

The material of main focus of this investigation is aluminum in different sizes and shapes,

ranging from tens of nanometer to tens of micron, and from spherical particles to irregular flake like

morphologies. As mentioned in chapter 1 (1.4.4) other materials other than aluminum were tested in

order to establish a toxicity reference frame. The particulate materials used were crystalline quartz

and titanium dioxide. For reference purposes the names used hereafter in this chapter for the different

samples investigated are listed bellow:

* NanoTek: TiO2 fTOm Nanophase Technologies Corp.
* P25: TiO2 frOm Degussa
* Quartz: quartz Min-U-Sil 5 from US Silica
* Al 1: QSI-Nano aluminum from Quantum Sphere Inc.
* Al 2: 80 nm aluminum from Nanotechnologies Inc.
* Al 3: H1-2 aluminum from Valimet Inc.
* Al 4: x-81 aluminum from Toyal Inc.
* Al 5: aluminum flakes from Sigma Technologies International Inc.
* Al 6: AlCl3 x 6H20 from Fisher Chemical, Fisher Scientific.

3.2 Before Dosage: "As Received"

All the particulate materials used in this research were acquired from the different

manufacturers that make them as powder systems commercially available. They were received in dry

state and the total amount available varied depending on the powder, from few micrograms to the

order of kilograms. Upon arrival powders were processed in several steps for characterization.

3.2.1 Sampling

Reliable powder sampling constitutes the first step of most powder characterization and

processing procedures. Sampling particulate matter entails collecting a small amount of powder from










the bulk, such that this smaller fraction best represents the physical and chemical characteristics of

the entire bulk (Holdich, 2002; Jillavenkatesa et al, 2001).

Samples were prepared taking into account the issues listed by the National Institute of

Standards and Technology (Jillavenkatesa et al, 2001):

* Quantity of powder from which samples are being obtained

* Amount of sample required

* Powder characteristics, including but not limited to flow characteristics of the powder, shape
and size of the particles, tendency to segregate, surface chemistry that may cause the powder to
be hygroscopic, etc.

* Mechanical strength of the powder, i.e., are the particles friable and thus, likely to fracture
during transport or during sampling

* Mode by which powders are transported

* Possibility of powder contamination, and acceptable limits of contamination

* Duration of time needed to conduct the sampling procedure

As general guidelines for powder sampling one can refer to the "golden rules of sampling"

(Allen, 1981):

1. A powder should be sampled in motion

2. The whole stream of powder should be taken for many short intervals of time in preference to
part of the stream being taken for the whole of time.

Powder batches can vary from several tons to a few grams, which is the case for many

nanomaterials. There are several techniques and devices that have been developed to aid in

representative sampling of powders. Their design incorporates the "golden rules" to the greatest

possible extent (Jillavenkatesa et al, 2001).

Several of the powders used in this investigation were received in bulk quantities in the order

of kilograms or hundreds of grams. This was the case for NanoTek, P25, Al 2, Al 3, and Al 4. For

easier handling and consistent sampling in further analysis, the sample sizes were reduced to the

order of tens of grams and stored in hermetically sealed containers throughout the length of this










research. For sample sub-division of the bulk amounts scoop sampling was used. The nanopowders

investigated exhibited, with exception of the P25 and the aluminum SIGMA flakes, very poor flow

characteristics and strong adhesive forces that did not allow the use of more reliable sampling

devices like the spinning riffler. The scoop sampling technique is a widely used, simple method for

sample division. An operator, using a scoop, extracts the laboratory samples from some portion of

the bulk sample. This technique is only appropriate for materials that are homogenous and exhibit

poor flow characteristics, which is the case of the nanopowders used for this research. One of the

drawbacks of this technique is that all the bulk material does not go through the sampling process.

The other drawback is the dependency on the operator to decide where to scoop the material and

what quantity of the sample to extract. This sampling technique is largely influenced by segregation.

In order to minimize this problem samples were not taken from the surface of the bulk and the

material was shaken in the container before scooping the subsample (Jillavenkatesa et al, 2001).

Despite off all the precautions mentioned above, there are several sources of error in the

process of sampling both systematic and random. While these errors cannot be eliminated, standard

protocols were implemented to minimize their influence in the final results. Some of the common

errors associated with sample preparation, as well as the protocols implemented during this research

to minimize those errors are summarized in Table 3-1.

One of the powders, Al3, was noticed to be more susceptible to sampling variability than the

others due to its very polydisperse particle size distribution and the cohesiveness of its particles. As it

can be observed in Figure 3-1 different subsamples of this powder can result in large differences, up

to one order of magnitude, in the mean particle size. As expected, the particle size distribution based

on particle numbers was more sensitive to this phenomenon than the particle size distribution based

on volume. This is due to the fact that a number size distribution is more skewed towards the size

range which the more abundant number of particles on it whereas the volume distribution will be

skewed towards the size range with the largest particles (and or agglomerates). Thus whenever more










nanoparticles were subsampled the mean particle size for the number size distribution was closer to

that of the smaller particles found in the powder Al 3. From the SEM images taken of this powder

sample the presence of particles in the nanosize range around the bigger 2-3 Ctm particles was

observed and found to be frequent (Figure 3-7).

For the materials of interest to this research the subsamples were collected from different

regions of the bulk and mixed together in smaller containers (stocks) used to hermitically store the

powders. Some of the characterization techniques required dry samples. In those cases appropriate

amounts of powder were collected from the stock specimens prepared for this research. For the

characterization techniques requiring dispersion of the powders standard protocols were developed

and will be detailed in following sections.

3.2.2 Density, Surface Area, and Porosity

Density is simply the mass of a quantity of matter divided by the volume of that same quantity.

There are three densities associated with powders. The absolute density, also referred as the true or

skeletal density, which excludes both the pores that may be in the particles and their interparticle

spaces; the envelope or apparent density, includes the pores but excludes interparticle spaces; and the

bulk density which includes both pores and interparticle spaces (Webb et al., 1997). For powders the

bulk density depends on vibration and applied forces. It is not an intrinsic property of the material

and it is not relevant for in vitro toxicity studies.

The absolute and apparent densities are identical for a nonporous object. Absolute density by

definition excludes all the open pores, i.e., the pores that have access to the outside. The apparent

density includes pore spaces up to the plane of surface.

In this research the absolute density of all powders was measured using a Quantachrome

Ultrapyc 1000 gas pycnometer. A known amount of dry powder is placed in a sealed chamber of

known volume and exposed to a series of elevated and then released gas pressures to flush away

atmospheric gases and vapors. Then the gas is released into another chamber of known volume. The










pressures of both chambers are measured before and after the gas expansion, which allows

calculating the sample volume. The gas used by this instrument is helium that with an atomic radius

of 31 pm penetrates and fills all open spaces including pores open to the surface. The result of

dividing the known mass by this measured volume is the absolute density.

Results of the absolute density measurements are detailed in Table 3-2. The density values

measured for the titania and quartz powders matched the reference values found in the literature. In

the case of the aluminum nanoparticles the values measured were closer to that of pure aluminum for

the larger size particles of samples Al 3 and Al 4 than for the smaller particles. As has been explained

in chapter 2 the aluminum particles exhibit an oxide layer due to the very high reactivity of

aluminum. Considering the total density of a 'coated' particle as:

Vcore surface laver
Ptotal = Pcore + surface layer(31
Total Vtotal

Where p are the densities and V are the volumes of each component. According to equation 3-

1, the density of the smaller nanoparticles will be more influenced by the density of the surface layer

than the density of the larger particles for which the ratio of surface layer to total volume is much

smaller. This explains higher density values for the smaller sizes of aluminum nanoparticles and the

aluminum flakes which higher surface areas are expected to be covered by some form of aluminum

oxide/hydroxide.

Surface Area is a measurement of the "exposed" surface of a material. There is an inverse

relationship between particle size and surface area. For the same mass of materials the smaller the

particles the larger is the surface area. The large surface area is one of the main features that make

nanoparticles so desirable for many different applications. Actual particles of whatever size if

examined at the molecular scale, display planar regions, but also are likely to include lattice

distortions, dislocations, and cracks. This means that the actual exposed surface of particles is greater

than would be calculated assuming any one geometric shape (Webb et al., 1997).









Surface area is usually measured by gas adsorption. In this research the instrument used to

measure specific surface area was the Quantachrome NOVA 1200. Dry sample is evacuated of all

gas and cooled to the temperature of liquid nitrogen, 77 oK. At this temperature, inert gases such as

nitrogen, argon and krypton will physically adsorb on the surface of the sample exposed to the gas.

An adsorption isotherm (one temperature) is usually recorded as volume of gas adsorbed versus

relative pressure (i.e., sample pressure / saturation vapor pressure). One or more data points of the

adsorption isotherm must be measured and the BET (after Brunauer, Emmett and Teller) equation is

used to give specific surface area from this data. The BET equation is used to calculate the volume of

gas needed to form a monolayer on the surface of the sample. The actual surface area can be

calculated from knowledge of the size and number of the adsorbed gas molecules.

Nitrogen was used measure BET surface for all the samples except for the quartz. The surface

area of this powder is very low, and krypton was used instead of nitrogen for a more sensitive

measurement because of its lower saturation vapor pressures at liquid nitrogen temperature.

BET surface area measurements can also be used to derive the mean diameter of the particles

forming a powder (Holdich, 2002).


X = (3-2)
SSA -p

Where X is the mean particle diameter in Ctm, SSA is the specific surface area in m2/g and p is

the absolute density of the powder in g/cm3. This technique is a reliable way to estimate a mean

particle size of the particles in a powder, especially for nanoparticles for which the surface area is so

high.

Results from the BET measurements are summarized in Table 3-3 for every powder. Because

not enough sample of Al 1 was available to experimentally measure the density with the gas

pycnometer the reference density value of pure aluminum was taken to calculate the mean particle

diameter from BET. The mean diameter values calculated from the BET data match the values given










by the manufacturing companies as mean particle sizes for the nanometer samples (NanoTek, P25,

Al 1 and Al 2). This is expected as SSA measurements by BET is the technique mostly used by

manufacturing companies to calculate and report the mean particle size of their products

Porosity can be estimated by analysis of EM images (SEM and TEM) at a high enough

magnification to reveal particle surface morphology (see Figures 3-2, 10). This technique is only

valid to detect pores open to the particle surface, unless ultra sectioning of the particles is performed

to obtain cross section of the particles that would show their core topography. In the case of toxicity

research one should be more concern about open pores that could affect the surface properties of the

nanomaterials in terms of accessible surface, surface roughness and adsorption of molecules onto the

surface. As specified by the manufactures all the nanomaterials used in this research were found to be

non porous.

3.2.3 Size and Shape

Knowledge of particle size, shape, and their distributions, of a powder is a prerequisite for a

more accurate interpretation of any biological interaction observed for nanopowders. Particle size

and particle size distribution (PSD) have a very significant effect in properties of powders and

finished obj ects like: bulk density, mechanical strength, optical, electrical, and thermal properties. As

discussed in previous chapters, many researchers have investigated the possible correlation between

size and shape of nanoparticles and their effects in biological systems. Nonetheless, in most cases

conclusions were based in mean particle diameters reported by manufacturers that do not totally

describe the complexity of real particulate systems. In order to conduct an accurate particle size

analysis, one ideally should have a representative sample, a well dispersed system, and a physical

measurement technique that is carefully selected to produce data relevant to the intended use. For

example, if a test material is administered as an aerosol, it would be logical to pick an aerosol

technique. One has to measure an adequate number of particles across the entire breadth of the size

distribution for statistical validity. For a monodisperse system this is relatively easy. However, as










polydispersity increases it may be necessary to measure a large number of particles to accurately

portray the size distribution (Masuda and Inoya 1971). This number becomes larger still if the

interest is focused on the "tails" of the distribution rather than on the mean or the median.

An ensemble method of measurement (such as laser diffraction, centrifugal sedimentation,

impaction, etc.) is normally preferred because the parameter measured (scattering pattern, mass) is

generated by large numbers of particles. Counting techniques (such as microscopy and image

analysis) should include enough particles to reach the desired statistical reliability. For dry "as-

received" powders, BET surface area is often used to estimate average size (based on a nonporous

spherical model) and has the added advantage of providing a direct measurement of specific surface

area (SSA). (Allen 2004, b) It should be noted, however, that this common method of reporting

particle size assumes a monodispersed spherical system and cannot be used to determine the breadth

or shape of the size distribution (see Figure 3-1).

3.2.3.1 Imaging techniques

Microscopy-based techniques for particle size characterization provide a powerful tool for

characterization of particle size, size distribution and morphology. They involve direct observation of

particles and the consequent determination of size based on a defined measure of diameter. If

compared with other available techniques for particle size analysis, a significant advantage of

microscope-based techniques is the ability to determine particle shape or morphology in addition to

make a direct measurement of size. Typically, the calculated sizes are expressed as the diameter of a

sphere that has the same proj ected area as the proj ected image of the particle. The calculated sizes or

size distribution can then be converted to, or expressed in, different measures (area, mass or volume

distributions) with suitable precautions (Jillavenkatesa et al, 2001).

Different types of microscopes can be used for size/shape analysis of powder materials by

image analysis. Optical and electronic microscopes (EM) like the scanning electron (SEM) and the

transmission electron (TEM) microscopes are the traditional choices depending on the powder










properties. Magnification and resolution limits of these instruments determine which one to use in

every case. In the case of nanomaterials EM are required as the lower resolution limit for an optical

microscope is about 1 Ctm. This implies higher cost in sample preparation and analysis time (Masuda

et al., 2006).

The common errors in imaging techniques are: (1) errors associated with sample/specimen

preparation; (2) errors associated with instrumentation/equipment and image analysis; (3) errors due

to human and other factors.

Another important consideration when dealing with imaging techniques for particle size

characterization is the number of particles to be analyzed in order to obtain a statically reliable result.

To estimate the number of particles to be analyzed for a specific powder, the National Institute of

Standards and Technology refers to the mathematical theory developed by Matsuda and linoya in

1971 (Jillavenkatesa et al, 2001). According to the equations developed in that theory, the total

number of particles to analyze depends on the standard deviation of the particle sizes, the shape of

the particle size distribution, the type of distribution (i.e., number, area, and volume) and the desired

range for error. In order to get a mass median diameter within 5% error with 95% probability for a

powder with a typical standard deviation of 1.60 about 61000 particles are required (Matsuda et al.,

1999). While this number would be dramatically reduced to about 15000 particles for a 10% error,

the elevated number of particles to be analyzed for a reliable particle size distribution measurement

demotes the use of EM imaging for that purpose. Nonetheless EM images are required for shape

assessment as well as to detect the presence of large particles, which are indistinguishable from

agglomerates when using ensemble techniques for particle sizing.

All the powders of interest for this research were examined under a JSM 6335 F SEM and a

2010 F scanning TEM, both from JEOL. Samples were prepared by dispersing a little amount of the

powders in pure ethanol. The dispersions were sonicated in a 275 CREST Ultrasonics bath for 30

seconds and a drop of the suspension was poured on a sample stub for SEM, or a carbon-cupper grid










for TEM samples. Samples were dried and in the case of the SEM samples coated with a nano thin

coat of gold to aid electron conductivity. During the collection of the pictures the samples were

manually scanned and representative images for particle size and shape assessment were recorded.

The different instruments used were properly calibrated. Some of the images collected for every

sample are shown (see figures 3-2, 10) and a summary of the size range and shape assessment is

listed in Table 3-4.

High Resolution TEM (HRTEM) also allows imaging the atomic crystalline lattice when the

sample conditions are proper. In some instances a clear difference in structure between the particles

core (crystal lattice) and surface can be appreciated in the image for the aluminum powders.

3.2.3.2 Light scattering techniques

As observed in the images collected for all the samples investigated in this research the reality

of most commercial powders is some degree of size polydispersity and particle shapes that can be

very different from an ideal sphere. Particle sizing methods that analyze a sample on its entirety and

rely on statistics to deconvolute the measured information into some sort of particle size distribution

are known as ensemble techniques. One of the most common phenomena used to measure particle

size is the interaction between light and matter (Jillavenkatesa et al, 2001). Light has properties that

can be used for determining particle size and particle size distributions. In the case of diffraction

instruments, the angle at which the light is diffracted depends upon the wavelength of the light and

the particle size. The angle of diffraction is measured to determine size. Another feature of light that

can be used for determining the particle size is the frequency. Frequency change or shift information

is used in Dynamic Light Scattering for particles that are very small compared with the wavelength

of the light.

The instruments used in this research differ on the type of light interaction and principle

measured. One of them uses laser diffraction and the other one dynamic light scattering. Samples

were prepared in the same manner for both analysis and when suitable measurements were taken in










parallel. A small quantity of the powder was dispersed in deionized water and sonicated for 30

minutes in a 275 CREST Ultrasonics bath with an ultrasonic frequency of 2 kHz and sonic power

average of 135 W. In order to determine an optimum ultrasonication time particle size distributions

were measured after each 5 minutes interval until a minimum size was achieved repeatedly. At that

point it was assumed that the particles had been properly dispersed. Because the size range,

sensitivity, configuration, principle measured and deconvolution algorithms of both instruments are

very different, results were taken as "complimentary" instead of comparable. By combining both

results a better idea of the whole size distributions is acquired for nanosize powders.

Beckman Coulter LS13 320 multi-wavelength laser diffraction particle size analyzer:

This laser diffraction instrument offers a rapid analysis with a relatively easy sample preparation,

measurements are relatively inexpensive, and it allows dry and liquid dispersion powder analysis.

This instrument is capable of measuring particle sizes from 40 nm to 2mm. A 750nm laser is used for

analysis in the range from 400 nm to 2mm and polarization intensity differential scattering (PIDS)

assembly sizes from 40 to 400 nm and improves resolution in the 400 to 800 nm range. Regarding its

limitations, the instrument performance is highly dependent on instrument design (e.g., laser sources

of different wavelengths, different number and position of the detectors), the state of dispersion, and

the knowledge of the sample optical properties.

Laser diffraction techniques are based on three basic assumptions (Jillavenkatesa et al, 2001):

(1) particles scattering light are spherical in nature; (2) there is little to no interaction of the light

scattered by different particles; (3) the scattering pattern collected on each detector is the sum of the

individual patterns generated by each particle in the sample diffracting the laser at that angle

Laser diffraction methods cannot distinguish between dispersed and agglomerated particles.

The previous image analysis provided a qualitative assessment of the larger particle size for each

powder. This evaluation is helpful to determine if the larger sizes observed in a particle size

distribution measured by a light scattering instrument could be single particles or the effect of










agglomeration. Results from this instrument for every powder "as received" are plotted as relative %

number and relative % volume distributions in Figures 3-10, 11. Several observations can be pointed

out from these measurements:

* The large difference in the mean sizes from the number and volume particle size distributions
for each powder. This is to be expected due to the wide particle size range observed in the
micrographs as well as for the agglomeration occurring that could not be totally avoided in the
experimental conditions of close to neutral pH and no dispersion aids used. Only a powder
made of perfectly dispersed spheres of the same size would have identical number and volume
size distributions.

* Both TiO2 pOwders were confirmed to have very narrow particle size distributions and very
small difference in size between the two different manufacturers.

* The quartz powder contained a considerable fraction of nanoparticles (100 nm or smaller) as
noticed in the particle size distribution by number and the images analyzed (see Figure 3-4
(B)).

* The aluminum powders were found to cover a very wide particle size range. Al 3 with a mean
particle size of about 2 Ctm according to the manufacturer was found to contain a very large
fraction by number of particles in the nanosize range and that observation was confirmed by
image analysis of the micrographs taken.

* The smaller size powders, Al 1 and Al 2 showed agglomeration after the dispersion protocol
by sonication. Some of the particles in these powders are expected to hardly agglomerate
during the manufacturing process and therefore remain aggregated after sonication. Better
dispersion was achieved for the Al 2 powder as indicated by the volume particle size
distribution.

* The particle size distribution of Al 5 should be only taken as a qualitative measurement of size.
This powder consist of flake like particles and as previously explained light scattering methods
for particle size measurements are based on the assumption that the particles are perfect
spheres, thus the validity of the data obtained has not been proven. In this case image analysis
was considered the primary method for particle size assessment.

Microtrac UPA 150 ultrafine particle analyzer: Dynamic light scattering (DLS) theory is a

well established technique for measuring particle size over the size range from a few nanometers to a

few microns. When a coherent source of light (such as a laser), having a known frequency is directed

at moving particles, undergoing random thermal (Brownian) motion, the light is scattered at a

different frequency. The shift is termed a Doppler shift or broadening, and the concept is the same for

light when it interacts with small moving particles. For the purposes of particle measurement, the










shift in light frequency is related to the size of the particles causing the shift. Due to their higher

average velocity, smaller particles cause a greater shift in the light frequency than larger particles. It

is this difference in the frequency of the scattered light among particles of different sizes that is used

to determine the sizes of the particles present (Jillavenkatesa et al, 2001).

The light source of this instrument is a 780 nm laser, it measures particles within a size range

of 3.2 nm to 6.4 tpm, and it requires a very small sample volume. These two properties are very

attractive for nanoparticles size measurements. At the other hand, the fact that the calculations for

size from the velocities measured are better suited for mono modal distributions can affect the

reliability of the results for the real powders analyzed.

As seen on the microscopic images and the PSD obtained from laser diffraction the powders

here investigated present wide particle size distributions and bimodality in some cases. To avoid the

effect of very large particles or agglomerates that fall outside the measurable range by this technique,

measurements were taken over long periods of time (180 seconds). This allowed for the big particles

and/or agglomerates to settle at the bottom of the instrument without being taken in the calculations.

Due to the valid size range from this instrument, it was not used to analyze sample Al 4. The typical

size distributions measured after dispersion by sonication for the different aluminum powders and the

quartz are depicted in Figure 3-12. This technique provides better size resolution in the smaller size

range of the particle size distribution allowing to better describe the lower size tails of the particle

size distributions obtained from laser diffraction. On the other hand the upper size limit for this

technique does not include the larger size particles and/or agglomerates that were detected by laser

diffraction and image analysis.

3.2.4 Surface and Bulk Chemical Composition

In order to understand the biological effects observed from nanoparticles it is essential to know

the chemical composition of the powder. Of special importance is the chemical composition of the









surface as this is the first and most dominant area of interaction for nanomaterials. Most of the

techniques used for this purpose are based in an energy analysis of the radiation coming from a

sample after excitation with a specific source. The photons or x-rays emitted from a particular

specimen are intimately related to the atomic and molecular composition of that sample.

3.2.4.1 FTIR

Infrared (IR) spectroscopy is a useful technique for characterizing materials and providing

information on the molecular structure, dynamics, and environment of a compound. When irradiated

with IR light (photons) a sample can transmit, scatter, or absorb the incident radiation. Absorbed IR

radiation usually excites molecules into higher-energy vibrational states. This can occur when the

frequency (energy) of the incident light matches the energy difference between two vibrational states

(or the frequency of the corresponding molecular vibration). Obtaining an IR spectrum requires

detection of intensity changes as a function of wavenumber or frequency. Fourier transform infrared

spectroscopy (FTIR) uses an interferometer to modulate the intensity of each wavelength of light at a

different audio frequency. The instrument used for this analysis is an FTIR-Raman/FTIR Microscope

Magna 760 from Thermo Electron that has a resolution of up to 0. 1 cm-l and it covers mid-IR (400-

4000 cm- ) as well as most of the NIR (1 1,700-2,000 cm- ) (ASM Handbook, 1986). It is a useful

technique to identify organic and inorganic materials and to identify molecular species adsorbed on

surfaces. The technique however provides very little elemental information, requires for the

molecules to be detected to exhibit a change in dipole moment in one of its vibrational modes upon

exposure to IR, and needs the matrix to hold the sample to be relatively transparent in the spectral

region of interest.

The spectra collected from the different aluminum samples are shown in Figure 3-13. The Al-

O-Al stretching and bending vibrations (< 1000 cm- ) were unsplit and broad. Significant absorption

bands can be observed in the ranges 700-1300 cm- (Al-OH bend), 1300-1800 cm-l (H-OH bend) and

3 000-3 800 cm-l (AlO-H stretch). These peaks confirm the presence of an oxide/hydroxide layer on










the surface of all the aluminum materials used in this research as well as the absence of foreign

contaminants on the surface. The shift of the peaks observed as well as their broadness for each

sample can be explained by the different rate of crystallinity at the surface (Geiculescu and Strange,

2003) depending on the manufacturing process and the aging process of the different aluminum

samples.

3.2.4.2 X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) spectra are obtained by irradiating a material with a

beam of x-rays while simultaneously measuring the kinetic energy and number of electrons that

escape from the top 1-10 nm at the surface of the materials being analyzed. XPS is a chemical

analysis technique that can be used to analyze the chemistry of the surface of a material. It is a valid

to detect all elements except hydrogen and helium. The detection limit for this technique is in the

parts per thousand (ppth) range (ASM Handbook, 1986). The results from the XPS analysis for all

the powders tested during this research are summarized in Table 3-5. The data confirmed the

elemental surface elemental composition expected for the powders. The presence of carbon in this

analysis is common due to adsorption on the particles of organic species containing carbon from the

environment as well as from possible contamination of the samples by residual carbon present in the

instrument during the measurement. Because of the nature of the analysis the quantitative results

obtained should be taken as approximate values more than as an accurate measurement of the

amounts present at the surface. Nonetheless the analysis confirmed the absence of foreign species

within the detection limits of this technique.

3.2.4.3 Energy dispersive spectrometry (EDS)

This technique is available in electron microscopes by using a dedicated detector

(spectrometer) for the collection of x-rays emitted from a sample after the interaction with the beam

of electrons used for imaging. These x-rays are characteristic of the atomic species with which the

electrons have interacted. This technique allows for detection of elements as light as carbon (Z > 6).










Even though quantitative analysis of the results can be performed, it is only valid for elements with

an atomic number > 11 and it is limited by the efficiency of the detector and its ability to resolve x-

rays of similar energy (ASM Handbook, 1986).

Energy dispersive spectrometry (EDS) spectra of all the samples characterized for this research

were acquired at the same time that TEM images were collected. Some typical examples of the

spectra collected are shown in Figures 3-14, 16. It is important to point out that the presence of

carbon in these spectra could come from different sources other than the sample. The particles are

supported by a copper grid with a carbon lacey that holds the particles in place for analysis. Both

elements, C and Cu can therefore appear on the spectrum depending on how close to the grid and

lacey the examined particles are and on the angle of the proj ected x-rays with respect to the detector.

Another possible source for C in the spectra is the deposition of organic contaminants present in the

microscope sample chamber at the time of analysis. Thus the C and Cu found for most of the samples

was not considered to be relevant for the analysis but a source of error. The spots for analysis were

selected in the neighborhood of the particle edge, so the contributions to the spectra would be mainly

due to the elements on the surface layer, as well as in areas in the middle of the particles, so the

results would mainly indicate the composition of the particle core. The presence of oxygen was

detected for all the particles analyzed. The only exception were the very large particles > 500 nm

were the aluminum peak was much more intense and no significant amount of oxygen was detected.

This is explained by the fact that the ratio of oxide layer to aluminum core for the larger particles is

much smaller for the big particles than for the smaller ones. The oxide layer around the particles acts

as a passivation coat more effective for the large particles so larger proportions of pure aluminum are

expected to be found in their core (Ramaswamy et al., 2004, 2005).










3.2.5 Zeta potential: "Surface" Charge

When nanoparticles are introduced in a liquid medium charge may arise at their surface due by

any of several possible mechanisms. The most important mechanisms for charge generation on

colloidal materials are: (1) surface dissociation; (2) ion adsorption from solution; and (3) crystal

lattice defects.

The surface charge of nanoparticles is important for two reasons: (1) it is a major factor in

determining the particle dispersion characteristics and (2) it will influence the adsorption of ions and

biomolecules onto the particle surfaces, which may change how the cells "see" and react to the

particles (Powers et al., 2006).

The development of a net charge at the particle surface affects the distribution of ions in the

neighboring interfacial region, resulting in an increased concentration of counter ions (ions of

opposite charge to that of the particle) close to the surface. Thus an electrical double layer is formed

in the region of the particle-liquid interface. The double layer consists of an inner region, which

includes ions bound relatively strongly to the surface (including specifically adsorbed ions), and an

outer, or diffuse, region in which the ion distribution is determined by a valance of electrostatic

forces and random thermal motion. The potential in this region, therefore, decays as the distance

from the surface increases until, at sufficient distance, it reaches the bulk solution value,

conventionally taken to be zero. The potential at the boundary (surface of shear) between the particle

with its most closely associated ions and the surrounding media is known as the zeta potential r. The

zeta potential is a fitnction of the surface charge of the particle, any adsorbed layer at the interface,

and the nature and composition of the surrounding medium in which the particle is suspended. It is

usually, but not necessarily, of the same sign as the actual potential at the particle surface but unlike

the surface potential, the zeta potential can be measured experimentally. It reflects the effective










charge on the particles and is therefore related to the electrostatic repulsion between them, which

makes it relevant to dispersion and stability control of colloids (Hunter, 2001).

The surface charge and potential of an insoluble metal oxide, which is the case of all the

materials used in this investigation (considering the Si as a metalloid), is determined in part by the

pH in the solution in which is immersed. For such systems the If and the Off ions are the potential-

determining ions, as a result of reactions like the one represented by equation 3-3.
H+ OH
M -OH; t M -OH 4 M O + H2 (3-3)
K K


The presence of hydroxyl groups on metal surfaces has been amply demonstrated by IR

spectroscopy. They can often be removed by heating to high temperatures but gradually return when

the surface is exposed to water, either liquid or vapor (Hunter, 2001).

An important value to consider when measuring the zeta potential is the point of zero charge

(p.z.c.). Because the p.z.c is not measurable experimentally, the isoelectric point (i.e.p.) is used as a

valid replacement. The i.e.p is defined as the pH at which the zeta potential at the shear plane is zero

( =0). The resultant zero charge means no electrostatic repulsion among the particles, which will

then come close enough for the van der Waals attractive forces to take over and cause particle

agglomeration.

In order to evaluate the electrical potential of the materials investigated as received when in a

liquid suspension the zeta potential of the powders was measured in water using two different

instruments: the AcoustoSizer II from Colloidal Dynamics, and the Zeta Reader Mark 21. The first

instrument applies a high frequency electric Hield to a colloidal dispersion and the electric Hield

induces and oscillation of the suspended particles. The oscillation of the particles generates sound

waves that correspond to a particle size distribution and zeta potential. The zeta potential is measured

as a function of pH as the suspension is automatically titrated to acidic or basic values using 0. 1N

HCI or 0. 1 N NaOH respectively. This instrument allows to easily finding an experimental value for










the isoelectric point of the particles suspended. The second instrument uses a microscope and digital

image capture to look at the particles streaming past the light source and displays the motion on the

computer monitor. A big advantage of this last instrument is that allows to qualitatively estimating a

zeta potential distribution because the particles measured are being visualized. It also allows

measurement in very dilute samples and small volumes which in the case of nanoparticles can be a

limiting factor for applying other techniques. On the other hand it only allows to measure single point

zeta potentials and titrations have to be made manually making more laborious to fully determine the

zeta potentials versus pH relationship for each material.

After the particles were suspended in deionized water, the electrolyte background was set at

0.01 N KCl and the pH was brought to 7.4 (physiological pH of cell culture media) by adding the

proper amount of either HCI or NaOH. The zeta potential was then measured and the values found

are listed in Table 3-6. In order to measure the i.e.p the same sample preparation was followed but

the pH was not adjusted. The suspensions were then titrated and the found i.e.p. values found are

summarized in Table 3-6.

3.2.6 Crystalline Phase

Several studies have recognized the higher toxicity of crystalline forms compared to the same

chemical composition of an amorphous form of materials like silica (Fenoglio et al., 2003, Fubini et

al., 2004, Warheit et al., 2003, Wierzbicki et al., 2003). A crystalline material is a three

dimensionally periodic arrangement of atoms in space. Describing a unit cell as the smallest fraction

of sample having all the fundamental properties of the crystal as a whole best depicts this

arrangement. The location of the atoms within the unit cell depends on the types of atoms, the nature

of their bonds, and their tendency to minimize the free energy by a high degree of organization

(ASM Handbook, 1986).

X-ray Powder Diffraction (XRD) is an efficient analysis technique to identify and characterize

unknown crystalline materials. Monochromatic x-rays are used to determine the interplanar spacings









of the unknown materials. X-ray diffraction peaks are produced by constructive interference of

monochromatic beam scattered from each set of lattice planes at specific angles. The x-ray

diffraction pattern is a Eingerprint of periodic atomic arrangements in a given material (ASM

Handbook, 1986).

Samples were prepared by attaching a small amount of the powder to a glass slide and

analyzed with a Philips XRD 3720 instrument that emits monochromatic Cu, x-rays. The results

obtained are in agreement with the specifications given by the manufacturers and the crystalline

structures of the different powders are summarized in Table 3-7. The phases listed describe the

crystalline structure of the bulk rather than the surface of the particles due to the operation conditions

of this instrument and its interaction with the samples.

3.2.7 Solubility

An important property of nanomaterials is the very high surface area compared to larger size

particles. When a material is partially or totally soluble the rate at which that solubilization occurs is

usually increased due to the larger contact area between solvent molecules and the surface of the

particles. When investigating toxicity of nanomaterials an important aspect is the possibility of

solubilization of the particles under the different physiological conditions of pH that the particles

might Eind throughout a biological organism. Aluminum hydroxide is practically non soluble at

around neutral pH (6.5-7) but it is also amphoteric which means its solubility increases at acidic and

basic pH' s (see Figure 3 -17). From the plotted distribution of hydrolysis products it can be observed

the very low solubility of Al ions. This phenomena is even more emphasized at pHs between 6 and 8

with a minimum solubility of about 10-6.5 m Al"3 at pH =: 7 (Baes and Mesmer, 1976) corresponding

to the close to neutral pH found in media and other physiological fluids.









3.3 In Physiological Media: "As Dosed"

By definition physiological media is any environment in which a specific organism lives and

thrives. In the case of tissue culture it refers to buffered solution of salts, amino acids, vitamins and

other molecules required for the normal metabolism of the cultured cells. Usually these buffered

solutions are supplemented with appropriate additives to optimize cell growth and preserve cell

culture development over extended periods of time. Hereafter "complete media" will refer to the

mixture of RPMI 1640 media + 5% Fetal Bovine Serum (FBS) and 1 % antibiotic-antimycotic.

3.3.1 Sampling

In order to dose the cells with the nanomaterials the particles were first suspended in liquid.

The sampling of nanomaterials from liquid suspensions is more reliable assuming that the particles

are properly dispersed and that sedimentation of bigger particles and/or agglomerates is avoided by

keeping the liquid under stirring conditions. However because all the materials investigated in this

research were received in dry state all the issues referring to sampling and detailed in section 3.2. 1

applied in this case. The particles were first suspended, from the dry state, in ethanol (200 proof) and

left overnight under the UV light of the incubator with the purpose of sterilizing the particles before

exposing the cells to them. This was done with the purpose of avoiding toxicity caused by potential

pathogens carried by the particles before dosing the cells. Next particles were resuspended in

deionized sterile water according to the following protocol:

1. Centrifuged particles out of the ethanol for 15 minutes at 21000 ref
2. Extracted the alcohol supernatantt) and added sterile deionized water
3. Vortex tubes for 30 seconds and repeated steps 1 and 2
4. Vortex again and repeated 1 and 2 for a 3rd time
5. Particles were left in the 3rd WaSh
6. Applied sonication in an ultrasound bath (2kHz) for 30 minutes


Final concentration of the stock water suspensions were measured by a gravimetric method.

The liquid of 1 ml of suspension was evaporated in a known weight plate and the residual dry










particles were weighted. The stock water suspensions were then diluted in complete media to a final

concentration of 500Cpg/ml.

3.3.2 Particle Size Distribution

Particle size distributions of the particles in media "as dosed" were measured using DLS.

Figure 3-18 shows and example of the typical particle size distributions obtained for Al 1 and Al 2

plotted as differential % number distributions. For comparison purposes the same plot includes the

particle size distribution of the same powders measured in water. Agglomeration was assessed

visually and quantified by an increasing mean of the particle size distributions measured in water and

media. A similar tendency for agglomeration was observed for all of the materials. In the case of the

titania the zeta potential of the particles at a pH of 7.4 is only about 20 mV, which indicates a low

electrostatic barrier to agglomeration. The IEP values of the titania samples are very close to the

neutral pH found in the in vitro growth media which explains their tendency to agglomeration in

neutral physiological fluids. The agglomeration in complete media could also be due to the

compression of the double electric layer caused by the high ionic strength of the physiological media

which would result in reduced electrical repulsion.

3.3.3 Surface Chemistry,

Nanoparticles have been found to adsorb proteins on the surface when suspended in

physiological fluids (Bousquet et al., 1999; Lundqvist et al., 2004; Surve et at, 2006). The

mechanism by which this phenomenon occurs is depending on the nanoparticle surface properties. In

some instances the interaction of nanoparticles with proteins has been also linked to their toxic

potential and final fate (Borm et al., 2006b; Hoet et al., 2004). In an attempt to characterize the

surface of the nanomaterials tested once they encounter the biomolecules and salts present in the

culture media two different parameters were quantified, zeta potential and protein adsorption.










3.3.3.1 Zeta potential in media

The nanomaterials were suspended in complete media and the zeta potential was measured by

electrophoresis in the Zeta Reader Mark 21. The values obtained are detailed in Table 3-6. As

expected due to the high ionic strength of the physiological media the values of zeta potential for the

different powders were very low, in many cases close to zero. This explains the tendency to

agglomeration observed when the nanoparticles were suspended in media.

3.3.3.2 Protein adsorption on aluminum nanoparticles

As a basic approach to a analyze proteins adsorbed to aluminum nanoparticles after exposure

to in vitro culture conditions the following experiment was performed.

Cells were cultured as described in Chapter 5 (5.1) and plated in 12 well pates. To minimize

erroneous results in the protein analysis from cellular debris around the particles after in vitro

exposure, plates with a filter insert (400 nm pore size) in each well were used. This plate design

separates the bottom of the well were the cells grow adherently and the culture media. Cells were

plated and incubated at 37 oC for 48 hours before nanoparticle exposure. This allows cells to fully

adhere to the plates and begin growing exponentially and reaching approximately 60-70 %

confluency. Al 2 nanoparticles were dosed to the cells at 500 tpg/mL in culture media and incubated

at 37 oC for 60 hr. The particles were dosed inside the filters so that they would not touch the cells

surface. Thus, particles inside the filter chamber were isolated from the cellular debris generated after

cell death but they were exposed to the metabolites produced by the cells as they grew on the bottom

of the well. Proper controls were run in parallel. After an exposure time of 60 hrs the particles were

recovered from the plates and the culture media was washed five times using phosphate buffer saline

by vacuum filtration through the filter inserts. Samples were stored at -80 oC for further analysis by

gel electrophoresis and mass spectrometry.

Gel Electrophoresis: Electrophoresis is the transport of charged molecules through a solvent

by an electrical field. Any charged ion or group will migrate when placed in an electric field. As










most biological polymers carry a net charge at any pH other than their isoelectric point, they will

migrate at a rate proportional to their charge density. The mobility of a molecule through the electric

field will depend on the strength of the field, the net charge, the size and shape of the molecule, and

on the ionic strength, viscosity, and temperature of the medium in which the molecules are moving.

In gel electrophoresis proteins are run through a polymeric matrix (the gel) driven by an

electromotive force and they separated based on their charge and velocity traveling through the gel

and compared to a known marker for several proteins. Gel electrophoresis was performed using the

Invitrogen NuPAGE Bis-Tris Electrophoresis System. Samples were prepared with 1X sample

buffer, which desorbs the proteins, and 1X reducing agent, which further denatures the protein. The

samples were then micro centrifuged at 5,000 g for 5 min and the supernatant was used for analysis.

Samples without nanoparticles were prepared accordingly diluting at 1:10. All samples were heated

at 70 oC for 10 min before use. Electrophoresis was performed at 200 V for 55 min. The gel was

stained with the Invitrogen Colloidal Blue Staining Kit as directed. In order to have an in-gel

digestion for mass spectrometry, a gel was run for only 5 min as stated, but was stained with 100 ml

of Coomassie Blue + 20% methyl alcohol for 4 hr and then de-stained using 100 ml of deionized

H20 + 25% methyl alcohol overnight with a sponge pad. A typical gel collected after this type of

experiments is shown in Figure 3-19. The particles were found to adsorb a very wide range of

proteins directly from the complete media, fourth column on the gel from the left, as well as form the

exposure to cell activity, second and third columns on the gel from the left. This observation suggests

that the adsorption of proteins onto the particles is a dynamic process in which the surface does not

get permanently saturated of serum proteins but an interchange with the proteins present in the media

at any time happens.

Mass Spectrometry: Mass spectrometry is a technique that measures the mass of individual

molecules that have been converted into ions (i.e., molecules that have been electrically charged).

The mass unit of measurement is the Dalton (Da) and is defined as 1 Da = 1/12 Of the mass of a single










atom of the isotope of carbon-12 (12C). This follows the accepted convention of defining the 12C

isotope as having exactly 12 mass units.

A mass spectrometer does not actually measure the molecular mass directly, but rather the

mass-to-charge ratio of the ions formed from the molecules. Thus the charge of an ion is denoted by

the integer number z of the fundamental unit of charge, and the mass-to-charge ratio m/z therefore

represents Daltons per fundamental unit of charge. In many cases, the ions encountered in mass

spectrometry have just one charge (z = 1) so the m/z value is numerically equal to the molecular

(ionic) mass in Da.

For this analysis, one dimensional (lD) high performance liquid chromatography (HPLC)

interfaced to electro spray ionization (ESI) quadra pole time-of-flight (TOF) tandem mass

spectrometry (MS and MS/MS) was utilized. The proteins underwent tryptic digestion before

analysis and the results were analyzed using the MASCOT database from Matrix Science. Results

can be seen in Table 3-9. Conclusion from this experiment were that the 80 nm aluminum

nanoparticles adsorb mainly the more abundant serum proteins (albumin, hemoglobin and other

serum proteins) after exposure to complete media in what seems to be a non-specifie adsorption.

3.3.4 Solubility ICP in media

In order analyze the potential solubility of aluminum nanoparticles in physiological

environments particles of every aluminum powder were suspended in complete growth media, RPMI

1640 + 5% (FBS), and incubated in the same 5% CO2, 37 C environment that the cells are cultured

in. After 24 hrs and 48 hrs incubation times the particles were filtered out of the suspensions through

0.2 Ctm syringe filters and the remaining supernatants were analyzed. The concentration of the

relevant Al +3 iOns found in solution was measured by using Induced Coupled Plasma Spectroscopy

(ICP). The ICP operates In the principle of atomic emission by atoms ionized in the argon plasma.

Light of specific wavelengths is emitted as electron return to the ground state of the ionized elements,










quantitatively identifying the species present. The detection limit for this technique is less than 1

ppm.

The results are summarized in Table 3-8. The starting concentration of the particle suspensions

was 500 Ccg/ml and the found concentrations of aluminum ions were below 1 ppm so it is concluded

that solubilization of the nanoparticles investigated in the physiological media is insignificant. The

results obtained from Al 6 ("soluble aluminum") also proof the very low solubility of Al in neutral

pH.

3.4 In Physiological Environment: "After Dosage"

Imaging Techniques: Once the nanoparticles have entered the in vitro system the array of

tools available for their characterization is very much reduced to imaging techniques. Tissue cultures

are complex systems in which the living cells are fed with enriched salt solutions supplemented with

vitamins, proteins and other essential molecules. The in situ characterization of nanoparticles in this

environment requires the use of an involved sample preparation process and TEM to capture the

images. Observations from the images collected are valid to "locate" the nanoparticles inside the

cells and appreciate the state of dispersion in vitro. Nonetheless, as can be appreciated in Figures 3-

20, 21 a more detailed characterization of the nanoparticles themselves becomes very difficult in this

type of images. In some instances particles appeared to be coated with a foreign substance that could

be proteins adsorbed to their surface, as proteins had been confirmed to adsorb onto the surface

(3.3.3.2). The sample processing involves protein Eixation that could theoretically preserve these

structures around the particle. In some instances the particles around the cells were captured in a well

dispersed stated allowing a qualitative characterization of their morphology in the physiological

environment around the A549 cells. Figure 3-22 shows a group of Al 1 particles outside the cell after

a 12 hrs exposure. The particles appear to have conserved an external coating around them that is

hypothesized to keep the aluminum core non-reacted up to that point. However, the very limited

amount of pictures that is feasible to acquire with this technique does not allow making a reliable










assessment about the nanoparticles after dosage. More TEM images of the tissue sections as well as

further discussion about the endocytosis phenomena observed will be discuss in Chapter 5.














Use of specially designed instruments like spinning
Non-representative sampling / Improper riffling when possible
dispersion during sample preparation If scooping, mix sample before and take several
subsamples across the bulk

If possible sample from a liquid suspension
Agglomeration of primary particles during
Use of dispersant aids when measuring the
sample preparation
primaryv" particle size"
Contamination or introduction of artifacts Use of clean instrumentation and containers
during sampling / storage Store in properly sealed containers
Store in inert gas and avoid extreme temperatures,
pressures and light exposure
Specimen degradation during storage
Check stability of properties over time until sure
that powder is stable
Only used to determine the primary particle distribution. It should not be used for toxicity testing unless it is
a biocompatible surfactant present along the exposure route.

Table 3-2. Absolute density measurements of the powders investigated
Samples Density (g/cc) Reference Value' (g/cc)
NanoTek 4.06 Anatase TiO? 3.84
P25t 3.95 Rutile TiO? 4.26
Quartz 2.66 Crystalline SiOz 2.64-2.66
Al 1 ----- Aluminum 2.70
Al 2 3.04 a-aluminaf A12O3 3.97
Al 3 2.77 y-aluminaf AlaO3 3.5-3.9
Al4 2.74 Monohydratef AlaO3.HzO 3.01
Al 5 3.91
"From CRC handbook of Chemistry and Physics, 1970-1971. Both TiOz powders used are approximately
80% anatase and 20% rutile. The reference value calculated for this ratio is 3.924. Not enough powder
available of this material to measure density with a gas pycnometer.f Density of several aluminum compounds
likely to be on the surface layer of the aluminum nanoparticles is listed for reference purposes.

Table 3-3. Specific surface area and calculated mean particle diameter.
Samples SSA (m /g) Calculated Mean Particle Diameter' (pmn)
NanoTek 36.69 0.041
P25 45.41 0.038
Quartz 5.75 0.401
Al 1 70.75 0.031t
Al 2 27.26 0.072
Al 3 8.08 0.268
Al4 2.25 0.973
Al 5 47.07 0.033
'According to equation 3-2, using the density values reported in table 3-1. Using the reference density value
for aluminum metal of 2.7g/cc


Table 3-1. Common errors associated with powder sampling


Error in powder sampling


Protocols for improvement










Table 3-4. Size range and shape of the particles from image analysis.
Samples Size range Shap
NanoTek 10-500 nm Spherical
P25 10-50 nm Rounded/ irregular
Quartz 100 nm-12 ptm Angular/ irregular
Al 1 20-50 nm Spherical
Al 2 30-125 nm Spherical
Al 3 100 nm-5 ptm Spherical
Al4 2.5- 60 ptm Spherical
Al 5' 100 nm-5 ptm/ 10-20 nm Flakes (high aspect ratio)
Diameter of the smallest and largest particles observed in the analysis.
tGeneric shape descriptor based on the "dominant" morphology of the particles.
Flake like particles: cross sectional diameter/ thickness

Table 3-5. Elemental surface composition from XPS analysis.
Samples O 1s (%) C 1s () Ti 2p' (%) Al 2p (%) Si 2(%)
NanoTek 47.31 29.64 23.05
P25 47.74 25.32 28.94
Quartz 43.69 19.66 36.65
Al 1 43.54 21.69 34.77
Al 2 43.84 22.81 33.35
Al 3 40.09 27.13 32.78
Al4 37.32 36.02 26.66
Al 5 37.11 33.74 29.15

Table 3-6. Isoelectric pints and zeta ptentials ()in different environments.
Samples IEP' (mV) inwaterat pH =7.4 mV in media at pH =7.4
NanoTek 6.3 -20.2 -0.8/-1.3
P25 6.6 -25.1 -0.2/-1.1
Quartz 1.3 -28.5 -8.4/-10.7
All1 +15.7/+23.6 -12.1/-16.7
Al 2 9.3-9.5 +18.2/+20.3 -1.1/-4.3
Al 3 +10.2/+12.7 -7.9/-10.3
Al4 +21.3/+23.5 -2. 1/-3.4
Al 5 +34.2/+3 8.5 -0.5/-3.7
*Isoelectric point: pH at which i = 0

Table 3-7. Crystalline phase identified experimentally by XRD
Samples Crystalline Phase
NanoTek Anatase/rutile 80/20 (tetragonal)
P25 Anatase/rutile 80/20 (tetragonal)
Quartz a-quartz (hexagonal)
Al 1 FCC (face centered cubic) aluminum
Al 2 FCC (face centered cubic) aluminum
Al 3 FCC (face centered cubic) aluminum
Al4 FCC (face centered cubic) aluminum
Al 5 FCC (face centered cubic) aluminum










Table 3-8. Particle solubility of aluminum nanoparticles incubated in cell culture media (ppm) during
two different time intervals
Samples Initial Concentration 24 hrs 48 hrs
Al 1 500 0.98 0.26
Al 2 500 0.16 0.15
Al 3 500 0.38 0.13
Al4 500 0.36 0.31
Al 5 500 0.14 0.16
Al 6 250 0.29 0.90



Table 3-9. Mass spectrometry results: most abundant proteins found adsorbed to the surface of Al 2
aricles
Mascot Hit MW' Score Protein name
1 71244 889 Albumin [bos taurus]
2 68083 645 Albumin
3 59720 639 Keratin 10, type I, cvtoskeletal human
Keratin, type II cytoskeletal 1 (cytokeratin 1) (Kl) (CK 1)
4 66149 580
(67 kDa cytokeratin) (hair alpha protein)
5 133442 495 Thrombospondin 1 precursor
6 133321 486 Thrombospondin 1 precursor
7 66110 426 Epidermal cytokeratin 2 [homo sapiens]
8 54986 416 Keratin, 54K type I cytoskeletal bovine
9 62320 398 Cytokeratin 9 [homo sapiens]
10 133555 300 Thrombospondin [mus musculus]
11 70611 274 Serum albumin precursor (allergen Fel d 2)
12 6509 245 similar to Keratin, type II cytoskeletal 1 (cytokeratin 1) [rattus
norregicus]
13 516677 248 Apolipoprotein B-100 precursor human
14 36015 173 Apolipoprotein E [Bos taurus]
15 39193 210 Alpha-2-HS-glycoprotein precursor (fetain-A)
16 67857 201 Albumin [canis familiaris]
17 47249 201 Apolipoprotein B 100 alcess alces]
18 29888 196 Keratin B1 [xenopus laevis]
19 51010 189 Keratin type 16
20 7147 147 Keratin complex 2, basic, gene 17: keratin complex 2, gene
17 [mus musculus]
'MW = molecular weight in Da













Al (3): Aluminum Valimet H2


-Volume A
- - --- --- N um ber A -

-Volumie B
-Number B


0.01


O


10900


10000


Diamleter (mnicronls)



Figure 3-1. Particle size distributions of Al 3 measured by light laser diffraction. The curves
correspond to two different subsamples of the same powder and represent the effect that
polydispersity can have in sampling for cohesive powders.


Figure 3-2. High Resolution TEM images of NanoTek TiO2. A) 25kX area showing the size
polydispersity characteristic of this powder. B) 800kX magnification showing the
crystalline lattice of the particle, a very thin surface layer of about Inm is distinguished
around the particles.





SA $ sg8Mky~~ B

Figure 3-3. High Resolution TEM images of P25 TiO2. A) 50kX area showing a smaller size range
than for the NanoTek powder, and a more irregular shape of the particles. B) 200kX
magnification, a polyhedral shape with some angular corners can be observed.


A w rowrn ra B


Figure 3-4. Scanning Electron Microscope images of Min-U-Sil 5 quartz. A) 3.5kX magnification, a
very wide size range can be observed. B) 11IkX magnification showing the very sharp and
angular geometry of these particles.


























I r A

Figure 3-5. High Resolution TEM images of Al 1. A) 50kX area showing the spherical shape of the
particles and the relatively narrow particle size range. B) 200kX are where the oxide
coating of about ~2.5-3 nm in thickness around the particles is clearly observed.
















50 nm
A -e B

Figure 3-6. High Resolution TEM images of Al 2. A) 50kX representative example of the size and
shape of the particles in this powder. B) 100kX magnification showing the very uniform
oxide layer around the particles of about 3-5 nm in thickness.




























Figure 3-7. Scanning Electron Microscope images of Al 3. A) 5kX image showing the spherical
shape of the particles and the size polydispersity of this powder. B) Detail at 13kX
showing the presence of nanoparticles around and in between the larger micron size
particles.


Figure 3-8. Scanning Electron Microscope images of Al 4. A) 430X area where the very wide
particle size distribution of this powder can be appreciated. B) 700X image that shows
the rounded shape of the particles that despite not being perfect spheres proj ect a close to
circular area.



































Figure 3-9. Electron Microscope images of Al 5. A) 5,5kX area showing the wide range of size in
cross section of the flakes and the thickness estimated to be ~75 nm. B) 200kX
magnification showing the oxide layer around the flakes.





Particle Size Distribution by Laser Diffraction
10

Quarrz Number
1 6 - - -
- Quartz Volume
14 TIO2 Nanotek Number

- TiO2 Nanotek Volume
1 2 - - -
TIO2 P 25 Number

10 -----CU --------I ------ --- TiO2 25 Volume













0.01 0.1 1 10 100 1000 1000
Diameter (microns)


Figure 3-10. Particle size distributions of the TiO2 and quartz powders "as received" by laser
diffraction.


A '


0














Particle Size Distribution by Laser Diffraction


18-


16-


14 -


12-


10-


8-

-


4-


2-


0-
0.01


0.1 1 10 100 1000
Diameter (microns)


10000


Figure 3-11. Particle size distributions of the different aluminum powders "as received" measured by
laser diffraction.



Particle Size Distribution by Dynamic Light Scattering

60

-Al l Number
--Al l Volume
50 --
-A 2 Number
-A 2 Volume i
-A 3 Number IR:
40 --
-A 3 Volume
---A 5 Number
8~~ 30- -AIS5Volume
Quadtz Number
- Quadtz Volume

20


0.0010


0.0100 0.1000 1.09000

Diameter (microns)


10.90000


Figure 3-12. Particle size distributions of the aluminum and quartz powders "as received" measured
by dynamic light scattering.













AlO-H stretch
AIl 1


H-OH bend


Al OH bend



















a- rlEA tz etch


AI


All 3


Al
8 i

Al
9 i-
il


.,~ ... -- t
3500 0000


Wavenumbers (cm-l


Figure 3-13. Infrared (IR) reflection-absorption spectra of the different aluminum samples. The
regions of the spectra where the bond vibrations expected for these materials are
indicated in the plot by vertical lines.


Figure 3-14. Typical EDS spectrum from the TiO2 particles. The picture shows the particle and
location of the beam when the spectrum was collected
















O 1 2 3 4 5 6 7 910 1
ull Scale 117 dts Cursor 6.828 keV (0 cts) *r Eamrruwrapl


Figure 3-15. Typical EDS spectrum from the quartz particles. The picture shows the particle and
location of the beam when the spectrum was collected.








88


r..r





















01234
Skull Scale 118 cts Cursor~ 2 415 keV (0 cts)


ull Scale 771 cts C~ursor~ 2 415 WeV I2 cts) p*n Irmry w ,B


Figure 3-16. Typical EDS spectra obtained form the different aluminum powders. A) spectrum taken
from the smaller size particles, the presence of oxygen was detected when directing the
beam to the surface as well as towards the center of the particle. B) for larger particles the
presence of oxygen was not as evident indicating a smaller ratio of oxide with respect to
aluminum for the same interaction volume.















Io) Olm C~I(III)

i

lo1;13, 32
r I
II I L
(I (I
I I
r
I 1 T




I ,
,, ~I
I ,
,
,

1 1
2! I


2468


1012


2 4 6 8 10 12


L
I

I
I
prl
---4
I
o




o


2 3 4 5 6 7 8 9 10 11 12 13


Figure 3-17. Distribution of hydrolysis products (x, y) at I = 1 m and 250 in (a) 0. 1 m Al (III), (b) 105

m Al (III) and (c) saturated solutions of a-Al(OH)3. The dashed curves in a and b denote

supersaturated regions with respect to a-Al(OH)3; the heavy line in c is the total

concentration of Al (III). Reproduced from Baes and Mesmer 1976 with permission of

John Wiley and Sons Inc.





























---- -



---- --



---- ----


Particle Size Distributions: AI NP in water and media


I I I I

0.01 0.10 1.00100


10


20-



15



10-






0

0.0


-Al l1 water


- AI 1 media


-AI 2w~ater


AI 2 media


---------



---- --



----


-- -------



---- ------



-----------






'


Diameter (microns)




Figure 3-18. Particle size distributions as % number for Al 1 and Al 2 in water and in media showing

the typical agglomeration observed when these nanomaterials were resuspended in

complete media.































[~n cs in n I I.. I A nsuinB hi

Figur 3-19 2D-el showing typcal el elctrohoress reult fom te expsureof Al80 n
pa~lhrtlesr tocluemdai ne ifrn xeietlcniin.Prilsepsdt
wit hiherabudane o albumi anterlgtr wihprtnslike insln

































Figure 3-20. A549 cell exposed to Al 2 (80 nm) for 24 hrs. Particles can
what appears to be enlarged endosomes.


be seen inside the cells in


Figure 3-21. Nanoparticles Al 2 outside an A549 cell in a 24 hrs exposure. The particles appear
agglomerated and seem to be "coated" by what could be proteins adsorbed to the surface.

































Figure 3-22. Image from TEM of some Al 1 nanoparticles outside the cells after a 12 hrs exposure. A
coating around the particles can be observed.










CHAPTER 4
REACTIVITY MEASUREMENTS

4.1 Isothermal Heat-Conduction Microcalorimetry Technique

Calorimetry is a well known and widely used technique for measuring heat resulting from

reactions occurring in a given system. In the case of systems in which the heat rate is very small, e.g.

case of in vitro metabolic reactions, heat-conduction calorimetry is recognized as the best option to

analyze the system (Kemp, 2000; Lewis et al., 2003). Isothermal heat-conduction microcalorimetry

(IHCMC) is a type of heat-conduction calorimetry in which the detection sensitivity is very high (of

the order of + 0. 1 CtW and the test sample has a small mass (typically 1-3 g) or a small volume (20-30

ml). IHCMC consists on differential measurement of the temporal changes in the enthalpy (in J)

between a test material-test medium system and a reference material-reference medium system. The

system is kept at a constant temperature by different means of cooling-heating. The instrument used

for this research was a CtRC calorimeter from Thermal Hazard Technology. The design of this

instrument allows heat measurements in very small volume samples, 1.5 ml or less and it provides a

very low baseline noise in the order of 0. 5 tW. The test sample and the reference material are

contained in two separate identical ampoules and are kept at a constant temperature in separate,

identically constructed wells of the calorimeter (Figure 4-1). The heat flow of the sample material [Q

(in W)] is measured over time and the integral of this curve is directly proportional to the exothermic

or endothermic heat resulting from the processes occurring in the sample vial.

Another advantage of IHCMC is the possibility of collecting heat information of biological

processes from tissue cultured in vitro (Bottcher et al., 1997; Kemp, 2000, 2001; Lisowska et al.,

2004). It is important to point out that despite the simple experimental implementation of this

technique for in vitro metabolism measurements, the complexity of the natural reactions occurring in

living cells and their dependence on environmental factors make the interpretation of isolated

microcalorimetry experiments rather difficult.









One of the hypothesized toxicity mechanisms for aluminum nanoparticles in this research is

that the reaction of aluminum nanoparticles around or inside the cells would disturb their normal

cycle enough to cause cell death in a process that could be consider necrotic. In order to prove this

hypothesis the chemical reactivity of the different aluminum powders was evaluated in several

environments relevant to the in vitro cellular environment.

4.2 Aluminum Reaction in Aqueous Media: Size and Shape Effects

Despite the complicated formulation of complete media it is an aqueous solution the bulk of

which is water. Thus when investigating the possible reactions of aluminum powders in the

physiological environment the first logical step is to study their reactivity in water. Based on a great

body of literature and their own experimentation, Rat'ko et al (2004a), proposed the overall process

of the aluminum water reaction to be as follows:

Al203 + xH20 ++ Al203 xH20 where x = 2, 3


Al203 xH20 + (7 x)H20 + 20H- ++ 2[Al(OH4)(H20)]-

The hydration of the oxide film affects it homogeneity and this favors the access of water to

the metal surface and allows hydrolytic chemical reactions which are characterized by an increase in

pH.

2Al + 6H20 4 2Al(OH)3 + 3H2

Al (OH) 3 + 6 H 20 4 [ Al(H 20) 6 73 + 3 0H
Al203 + 9H20 4 [Al(OH)3(H20)3/

[Al(OH)3(H20)3] + 3H20 4 [Al(H2)O673 + 30H

When this reactions occur at low temperatures <100 C as is the case for the in vitro

experiments (37 C) relevant to this investigation the final product will be an amorphous Al (OH1)3. In

order to test the influence of particle size on the rate of reaction and the importance of the oxide layer

on delaying and/or totally avoiding the reaction of the particles in water the following experiment










was performed. A known amount of powder, 250Cpg/ml, was suspended in deionized sterile water and

gently dispersed by 30 seconds of bath ultrasonication. A reference cell was prepared by adding 1 ml

of the same water to an identical glass vial, both cells were carefully placed in the microcalorimeter

and the temperature was equilibrated at 37 C. After 45 minutes required for the instrument to reach

equilibrium state the heat output was measured and collected over extended periods of time, of up to

72 hours, in order to quantify the heat involved in the reaction of the particles. Experiments were

repeated 2 times and the measurements were found to have very good repeatability. Results from

these experiments are plotted in Figure 5-2.

From the data collected several qualitative observations can be made:

* The rate of reaction is directly linked to the mean size of the particles. The smaller particles
react more rapidly. The larger particles react later and over longer periods of time and for the
largest micron size particles no reaction is observed over the 72 hrs the samples were under
observation.

* Shape has also and influence in the rate of reaction. The aluminum flakes react at about the
same time that the 80 nm aluminum powder. Secondary heat pulses were observed in the case
of the flakes after the first exothermic reaction had happened.

* The amount of heat released is also directly related to the size of the particles.

* No reaction was observed for the quartz, which (being crystalline Sio2) is not expected to react
mn water.

All the above observations are consistent with the studies about the oxide layer that have been

reported in the literature (Schultze et al., 2000, Ramaswamy et al. 2004, 2005, Rat'ko et al., 2004 a,

2004 b). The efficacy of the oxide layer in protecting the aluminum core is reduced for the smaller

flakes and the irregular shapes as discussed in Chapter 2 (2.2.3). The sensitivity of this technique

allows for more quantitative analysis of the aluminum reactivity.


Al(s) + 3H20(1) 4 Al(OH)3(s) + 2 H2(g) (4-1)


Equation 4-1 describes the chemical reaction of the particle core, made of aluminum metal,

with water. The standard heat of this reaction (Sposito, 1996) can be calculated as:










AH y(A1(OHh)3 = -1288.25 kJ ol


AH,(Al) = OW/,,,


AH,(H,O) = -285.83 w;o; AH,(H,) = 09;o;


AHR = [ products reagents = (-1288.25) -[3(-285.83)1 = -430.76 m~ol

For 250 Ccg of pure aluminum the heat of reaction would be equal to:


250x16gx (-430.76 kmol) = 3.99 J (4-2)
26.98 8
Smol

As described in Chapter 3 all the aluminum particles used for this research were coated by and

oxide/hydroxide layer of about 2.5 nm in thickness. The aluminum contained in that layer will not

react to produce a hydroxide thus it should not be taken into account in the calculation of heat

produced by the reaction of the particles. A 2 nm thick layer around a, for example, 50 nm particle in

diameter means a reduction of total reactive aluminum mass of about 20 % according to the

following calculation:


y22 232~= 0.778 (4-3)


Therefore the total heat output of 250 Ccg of aluminum powder of 50 nm primary mean size is

expected to be a 20% less of that of 250 Ccg of pure aluminum. From equation 4-2 the heat

experimentally measured from the reaction of this sample should be about 3.99 x 0.778 = 3.11 J.

From Figure 4-2 the integral of the area under the peak for the reaction of aluminum 50 nm was

calculated as being 3.54 J + 0.05, which confirms the total reaction of the aluminum powder during

the first 5 hours after dispersion in water. The difference from the theoretical value could be due to

several reasons: (1) polydispersity of the original powder particle size distribution affects the

calculation of oxide to particle mass ratio; (2) thickness of the oxide layer taken as an average of 2. 5










for every particle when in reality thickness can vary among particles of the same sample; (3) particle

aging during storage can cause a thickening of the oxide layer around the particles.

A summary of the heat of reaction for the different size aluminum powders is presented in

Table 4-1 with a comparison of the theoretical heat to be expected out of a hypothetical

monodisperse sample of the same primary mean size as the tested sample with a 2 nm non reactive

layer around the particles. The ratio of aluminum metal in the core to total mass of the particle is

calculated using equation 4-3.

The results from this experiment verified the direct connection between particle size and shape

with the rate of reaction of aluminum nanomaterials in water.

4.3 Aluminum Reactivity in Physiological Media

When investigating cell interactions in vitro, the aluminum particles encounter a complex

environment rich in salts, vitamins, proteins and other biomolecules that could potentially modify

their behavior and reactivity. For this reason several experiments in different fluids were design to

simulate the possible different environments that the particle is exposed to in a cell culture. The first

experimental approach taken was to measure the rate of reaction of aluminum nanoparticles in the

culture media RPMI 1640 used to culture the lung cells investigated in the context of this research.

Particles were prepared the same way as the experiment in water. However cell culture media was

used instead of water. The pH of the suspensions in this buffered media was pH 7.4 (physiological).

Samples were kept in the microcalorimeter to constantly monitor the heat flow.

The results are shown in Figure 4-3. No significant reaction was observed from the aluminum

spherical particles when the plots where compared to the heat flow output of the reaction in water.

However the aluminum flakes did exhibit a unique pattern of incremental exothermic steps. The non

reactivity of aluminum nanoparticles in physiological media was postulated to be due to the

formation of an extra passivation layer around the native aluminum oxide/hydroxide consisting of

phosphate bound to the surface. The rapid and effective adsorption of phosphates species onto










aluminum hydroxide and oxides surfaces has been widely reported in research fields like

microelectronics (Scandurra et al., 2001) pharmaceutics (Tang et al., 1997) and environmental

chemistry (Tanada et al., 2003). In fact a chemical dipping into an organic bath containing

phosphating agents is been demonstrated to provide a very efficient passivation of aluminum surfaces

by the formation of one or two monolayers of ortho- and polyphosphates directly grafted onto the

alumina surface (Scandurra et al., 2001).

These findings would suggest the hypothesis of aluminum nanoparticles gaining and extra

layer of passivating material around their native oxide/hydroxide coating that would inhibit the

reaction of the aluminum in physiological media (with a concentration of phosphates of 0. 8 g/1) In

the case of the flakes the step like progression of the reaction in media could be explained by the

exfoliation of the passivating layers in areas of uneven coating due to the irregular surface

topography of these particles shown in Chapter 3 (Figure 3.9).

4.4 Aluminum Reactivity in Acidic Physiological Environments

From the lack of reaction over extended periods of time in physiological media the potential

for aluminum nanoparticles to maintain an unreacted aluminum metal core when dosed to the cells

was established. The pH in culture media, cell surroundings and the cytoplasm is known to be a

constant physiological pH of around 7.2 (Alberts et al., 2002). Despite the fact that pH is highly

buffered and controlled in physiological environments, it is not a unique value across the whole

organism. The normal metabolic cycle of a cell requires the presence of acidic compartments known

as late endosomes and lysosomes in which potentially particles could be found after interaction with

living cells (Albert et al., 2002). Due to the complexity in chemical composition and activity of these

acidic intracellular compartments a simple experimental approach was taken in order to characterize

the reactivity of aluminum nanoparticles within those compartments. The pH of the particle in media

suspensions prepared for the previous experiment was adjusting to pH 4 by addition of 1N HC1. 1 ml

of this acidic particle suspension was placed in the sample holder of the microcalorimeter and the