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IN VITRO TOXICITY ANALYSIS OF NANOSCALE ALUMINUM:
PARTICLE SIZE AND SHAPE EFFECTS
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
O 2007 Maria Palazuelos Jorganes
To my husband Scott,
my sister Amalia and my parents Pepa and Luis.
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
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
ACKNOWLEDGMENTS ............ ..... ._ .............. 4.....
LIST OF TABLES ............ ..... ._ ...............8....
LIST OF FIGURES ............ ..... ._ ...............9....
ABSTRACT................ ............... 12
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......... ...
220.127.116.11 Imaging techniques................ .............. 59
18.104.22.168 Light scattering techniques ................. ...............61........... ..
3.2.4 Surface and Bulk Chemical Composition ................. ................. 64......... ..
3.2.4. 1 FTIR ........._...... ....._ ... ....._._.... ...........6
22.214.171.124 X-ray photoelectron spectroscopy (XPS) ................. .. ............... .......... 66
126.96.36.199 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
188.8.131.52 Zeta potential in media ................. ............. ...............74.
184.108.40.206 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
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
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
Maria Palazuelos Jorganes
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.
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
* 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
* 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
* 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
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"
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
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.,
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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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/scal eof things.html. Last accessed March 4, 2007.
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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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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
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
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.,
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
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
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.
- -- -- -- -- '
Biotechnology (DNA, macromolecules, cells, etc) "LNano"~
Dime nsio n
1 mm -
N 1 l
O 10 nm -
0.1 nm -
C rLI 1.
1980 2000 2010
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
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.
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"
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
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
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)
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
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
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).
220.127.116.11 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.
18.104.22.168 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
* 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
* 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
* 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.
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
22.214.171.124 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.
126.96.36.199 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
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.
M -OH; t M -OH 4 M O + H2 (3-3)
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
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
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.
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.
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.
188.8.131.52 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.
184.108.40.206 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
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
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
(220.127.116.11). 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
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
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
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
- - --- --- N um ber A -
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.
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
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
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
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
Figure 3-10. Particle size distributions of the TiO2 and quartz powders "as received" by laser
Particle Size Distribution by Laser Diffraction
0.1 1 10 100 1000
Figure 3-11. Particle size distributions of the different aluminum powders "as received" measured by
Particle Size Distribution by Dynamic Light Scattering
-Al l Number
--Al l Volume
-A 2 Number
-A 2 Volume i
-A 3 Number IR:
-A 3 Volume
---A 5 Number
8~~ 30- -AIS5Volume
- Quadtz Volume
0.0100 0.1000 1.09000
Figure 3-12. Particle size distributions of the aluminum and quartz powders "as received" measured
by dynamic light scattering.
Al OH bend
a- rlEA tz etch
.,~ ... -- t
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.
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)
II I L
I 1 T
2 4 6 8 10 12
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
-Al l1 water
- AI 1 media
AI 2 media
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
[~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.
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
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
* 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
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)
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
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