Chapter 25
Chapter 25 .............................................................................................................. 1
Nanotoxicity ........................................................................................................... 2
1 Nanotoxicology and Nanoparticles ................................................................. 2
2 Potential Routes of Exposure .......................................................................... 5
2.1
Portals of Entry ................................................................................ 7
3 Historical Perspective ..................................................................................... 8
4 Nanoparticle Toxicity ................................................................................... 10
4.1
Mechanisms of Nanoparticle Toxicity ........................................... 12
4.1.1 Oxidative stress .............................................................................. 13
4.1.2 Calcium flux .................................................................................. 14
4.1.3 Inflammatory response................................................................... 14
5 Nanomedicines for Pancreatic Disease ......................................................... 15
6 Summary ....................................................................................................... 17
2
Nanotoxicity
G. R. Hutchison and E. M. Malone
Abstract Nanotechnology, including the field of nanomedicine, promises to revolutionise/improve the way in which we live our lives. This atomic, molecular and
macromolecular technology will introduce nanoparticles into our environment and
our daily routine. In response to this exciting technology, toxicologists have responded with the development of a specialised subcategory of toxicology, nanotoxicology. This chapter will introduce nanotechnology and nanoparticles and examine the origins of nanotoxicology, drawing on epidemiology and respirable
particle toxicology studies. These studies provide the foundation of what we today use to understand the toxicology of engineered nanoparticles. The focus of the
chapter will be on human and mammalian nanotoxicology and will summarise the
current understanding of the mechanisms of nanoparticle toxicity whilst describing the methodologies utilised to further knowledge in the area. In the context of
the book, this chapter will briefly examine the impact of nanotechnology and the
development of nanomedicines in relation to the pancreas. Nanomedicine relies
heavily on nano-specific toxicological concepts and findings to provide safe medical applications. Success in this area requires a collaborative approach involving
physicians, material scientists and toxicologists.
Exposure Routes, Hazard assessment, Mechanistic Nanotoxicology, Inflammation,
Experimental Models.
1 Nanotoxicology and Nanoparticles
Nanotoxicology, as proposed by Donaldson et al. (2004), is a subcategory of toxicology focused on the effects of materials in the nanoscale on living organisms and the potential, or likely, problems
caused by these materials. This new area of toxicology has developed because substances ordinarily innocuous can have adverse effects in the nanoscale (Service 2004; Lee, Qu and Copeland 2005).
The definitions for the terminology related to nanotoxicology have
evolved over time and there is some conflict in the literature so, at
this stage, it is important, for clarity, to discuss some of the current
terminology related to nanotoxicology. The British Standards Insti-
3
tution (BSI) has defined nanoscale as a, “size range of approximately 1nm to 100nm” (PAS 136; British Standards Institution 2007).
The National Nanotechnology Initiative (NNI) also uses the term
nanoscale to describe dimensions between approximately 1 and
100nm (http://www.nano.gov/html/facts/WhatIsNano.html accessed
on the 8th December 2009). Nanoparticles are defined by the BSI as
discrete pieces of material with all three external dimensions in the
nanoscale (PAS 136; British Standards Institution 2007). This definition is generally accepted and will be the definition followed in
this chapter. However, the BSI had previously defined nanoparticles
as “discrete pieces of material with one or more external dimensions
in the nanoscale” (PAS 71; British Standards Institution 2005). The
term now being used to describe this material is nano-object (PAS
136; British Standards Institution 2007), for example, nanotubes. In
addition, the term ultrafine particle has been in use for some time to
denote nanoparticles. The term nanoparticle is a relatively new term.
It is also important to be aware that nanomedicine can relate to nanometre size complexes from 1nm to 100s of nm in size (European
Scientific Foundation, 2005). Nanoparticles do not generally exist as
discrete uniform entities they can form agglomerates and aggregates
through chemical and physical interactions. Furthermore, they come
in a variety of different shapes and sizes. For example, High Aspect
Ratio Nanomaterials (HARN) which have a diameter in the nm
range, can range in length from nm to hundreds of microns. Thus the
material exists as long, thin particles however not all high aspect ratio nanomaterials are nanoparticles. Compositions can also vary,
from individual elements, for example nano gold, to complex engineered structures such as quantum dots with can have core made
from one element and outer shells made of other elements. Nanoparticles can also be engineered to be functionalised for a desired purpose. Therefore it is naive to assume that all nanoparticles will be
similar and behave in a generic manner.
Throughout evolution humans have been exposed to naturally occurring nanoparticles, for example, viruses and nanoparticles generated by active volcanoes. In addition to naturally occurring nanoparticles, there are anthropogenic nanoparticles, those generated
unintentionally, for example, by internal combustion engines, power
plants, incinerators, automobile and jet engines and those generated
4
intentionally including, metals, metal oxides and carbon (Oberdorster, Oberdorster and Oberdorster 2005). Historically, particle toxicology has been connected to industrial materials, for example coal
and asbestos (Donaldson and Borm 2000) and research has focused
on anthropogenic nanoparticles generated unintentionally, for example, the ultrafine (diameter less than 100nm) respirable air pollution
particles of PM10, particulate air pollution with aerodynamic diameter of less than 10µm, reviewed by Stone et al. (2007).
The number of nanoparticles generated intentionally, or engineered, has increased as a result of the rapidly developing field of
nanotechnology, an important research endeavour of the 21st century, which has been described as molecular manufacturing, one atom
or molecule at a time, and includes research and development technology of nanoparticles (McNeil 2005; Borm and Donaldson 2007).
The Project on Emerging Nanotechnologies (PEN) provides an extensive and diverse list of nanotechnology-based products currently
available to consumers. Those on the list include, cosmetics, sunscreens, sports clothing, electrical appliances and food packaging
products. (http://www.nanotechproject.org/inventories/consumer accessed on the 8th December 2009). Nanoparticles are also being
used for drug delivery, in vivo imaging and in vitro diagnostics as
nanoparticles operateing at the biomolecular scale; human cells are
10,000 – 20,000nm in diameter, a hundred to two hundred times the
size of nanoparticles. Their size, in combination with their surface
tailorability, solubility and multifunctionality, offer biologists the
opportunity to target, diagnose and treat diseases. However, the benefit of nanoparticles must be balanced with the potential health risks
associated with the manufacture, distribution and use of nanoparticles (McNeil 2005). The focus of the chapter will be on human and
mammalian nanotoxicology
The first rule of toxicology is that all substances produce an effect, but it is the dose that decides whether the effects are adverse or
beneficial (Paracelsus). Before exposure routes and toxicity can be
discussed it is important to understand the relationship between hazard and risk. There are three factors that require consideration when
making hazard/risk assessment; hazard, risk and exposure. A hazard
exists when a substance, object or situation has an intrinsic ability to
cause an adverse effect. Risk, on the other hand, is the chance that
5
such effects will occur: the risk can be high or negligible. These
alone do not allow a complete assessment. For harm to occur in
practice, in other words, for there to be a risk - there must be both
the hazard and the exposure to that hazard. Without hazard and exposure there is no risk. Regulatory, public health organizations and
industries all recognize the value of the toxicological triage process
that accompanies hazard classification and risk assessment. In the
field of Nanotechnology, nanotoxicologists are tasked with collating
the necessary hazard information for the new nanoparticles that are
currently being produced by the nanotechnology industry. In order
to advance our understanding of acute and chronic nanoparticle toxicity and nanoparticle translocation, biodegradation and elimination
from the body, we must first balance and prioritise efforts to understand how likely nanoparticles are to gain access to the human body
(Donaldson 2006).
2 Potential Routes of Exposure
Human exposure to nanoparticles can be described as incidental,
accidental or deliberate. For example, human exposure to engineered
nanoparticles, manufactured for a specific purpose, for example nanomedicines, can be intentional or deliberate. Exposure to engineered nanoparticles can also be incidental or accidental, for example nanoparticle release during nanoparticle manufacture, or use, can
result in occupational, consumer and/or environmental exposure.
Exposure can result from natural sources for example nanoparticle
release from forest fires, or anthropogenic nanoparticles generated
unintentionally (Borm et al. 2006). Figure 1 provides a summary of
the possible routes of human exposure to nanoparticles, potential interactions, and the possible clearance routes of nanoparticles from
the body. For example, it is well documented that inhaled nanoparticles deposit within the alveoli (Ferin et al., 1992 and Li et al., 1999)
and can subsequently be cleared by alveolar macrophages via the
mucocillary escalator and expelled by the nose or swallowed. Alveolar macrophage transport of nanoparticle from the lung could potentially lead to translocation of nanoparticle to other organs within the
6
body such as the gastrointestinal (GI) tract, and or lymphatic system
via macrophage migration to the local draining lymph nodes.
Nanoparticle exposure is not only a human health concern as they
equally effect the environment (Fernandes et al. 2007). Nanoparticles have also been utilised in environmental remediation/waste
treatment; free nanoparticles are added to contaminated environments in an attempt to clean up soils and/or groundwaters from organic and inorganic pollutants.
** Insert Figure 1 near here**
Fig. 1. Potential routes of human exposure to nanoparticles either via deliberate, incidental or accidental exposure. Arrows depict potential routes of travel through the body with nanoparticle
clearance via the lung, mucocillary escalator, gastrointestinal (GI) tract and lymph. As underlined, excretion can occur by route of the kidney or liver. Solid arrows depict known routes of
exposure. Dashed lines between target organs indicate that there is potential for translocation between primary target organs. Dotted lines depict the possible secondary target organs that could
be exposed to nanoparticles if translocation occurs. * denotes that deliberate injection can be
subcutaneous, intramuscular and not just intravenous.
Nanoparticles have many potential diverse applications and therefore there are a number of expected exposure routes associated with
nanoparticle utilisation and production; specifically inhalation, intravenous injection, ingestion and dermal application (Oberdorster,
et al. 2008).
Time is also an important parameter when considering exposure
in a relevant population. It is important to understand whether a
population is exposed over a long period of time i.e. chronic exposure, or whether a population comes into contact with the material
for short periods of time i.e. acute exposure. It is expected that
workers in the nanotechnology industry, generating nanoparticles or
using nanoparticles, will be exposed to higher concentrations than
consumers of, or recipients of, nanotechnology-based products. Inhalation and dermal routes of exposure are potentially greater for
workers in the nanotechnology industry. Consideration of the dose,
route of exposure and exposure period has a significant bearing on
the hazard/risk assessment of material in the nanoscale. For example, an employee in a factory may be exposed to high levels of a relatively inert nanoparticle, via inhalation and dermal routes of expo-
7
sure, over a prolonged period of time. Therefore, the concentration
of nanoparticles entering the body could increase over time and persist in the body resulting in a pathogenic effect. This pathogenic effect may not materialise in a consumer population if consumers are
exposed, by dermal routes only, to low levels of the relatively inert
nanoparticle over a short period of time.
2.1 Portals of Entry
Potential routes of nanoparticle exposure in the human population
include the respiratory tract, skin, GI tract, and in the case of biomedical applications possibly injection into the body, for example,
intravenous, subcutaneous or intramuscular injections. Any potential
adverse effects resulting from nanoparticle exposure may occur at
the various portals of entry, the primary target sites or organs, such
as the lungs and skin; however, it is possible that the adverse effects
may occur at distant sites, or secondary target organs such as the
kidney or liver (after nanoparticle translocation from the primary organ). For prediction of systemic toxicity, following nanoparticle exposure, systemic dose is another important parameter to consider.
The systemic dose is dependent upon both the barrier function and
clearance mechanisms at the portals of entry. It is postulated that
respirable particulate air pollution, or induced mediators from the
lung, are able to cross into the circulation, via the alveolar epithelium, and induce fibrogenic plaques in the cardiovascular system. Epidemiological studies support these findings as hospital admissions
increase during episodes of high air pollution. Predominantly these
adverse health effects are manifested in susceptible individuals who
had pre-existing pulmonary or cardiovascular disease (Pope et al.
1991; Dockery et al. 1993; Schwartz 1994). Studies addressing the
systemic translocation of nanoparticles from primary sites of deposition are beginning to unravel the dynamics of nanoparticle–organism
interaction, and provide the means to relate exposure to hazard data.
Nanoparticle translocation is a challenge for nanotoxicologists as
current techniques are not sensitive enough to track all nanoparticles
in vivo and to detect and measure nanoparticle concentrations ex vivo. Current techniques need to be adapted, or new techniques devel-
8
oped, to allow nanoparticle translocation to be assessed for all nanoparticles.
It is also important to mention that not all nanoparticles that enter
the body will deposit and accumulate within primary and secondary
organs. Some nanoparticles can be degraded by the bodies defence
mechanisms e.g. alveolar macrophages can readily clear inhaled particulate from the lung which is subsequently cleared via the mucocillary escalator. Additionally, a number of studies have shown that
some nanomaterials are readily excreted via the kidneys or GI tract.
From a nanotoxicologist’s perspective, removal of nanoparticles
from the body, by the body’s defence mechanisms, may be an ideal
outcome; however, a nano-engineer, producing a nanomedicine designed to bio persist, may consider this less than ideal.
3 Historical Perspective
Historically, as mentioned previously, nanoparticle research has
focused on the ultrafine component of PM10. PM10 is the commonly
applied international standard given to environmental particulate air
pollution that measures the mass of particles collected, with a 50%
efficiency for particles with an aerodynamic diameter of 10µm
(Maynard and Waller 1996). PM10 can be divided into three categories based on size; coarse (PM10 - PM2.5), fine (PM2.5 - PM0.1) and ultrafine (nano) (<PM0.1) (Donaldson and MacNee 2001). PM10 is
made up of a number of different components including fine and nanoparticles from combustion sources, transition metals, sulphates
and nitrates, as well as biological components, for instance, pollen,
endotoxin and wind blown dusts such as clays (Stone 2000) and
these components can vary between different PM10 samples. The association between adverse health effects and increased levels of particulate air pollution have been well documented, with more than
100 epidemiological studies published since 1992 (Folinsbee 1992;
Pope and Dockery 1999; Englert 2004). Although air pollution levels have gradually decreased since the late 1950’s due to the introduction of ‘Clean Air Acts’, as the international community has
moved away from solid fuels, which release high levels of sulphur
dioxide, to oil and petroleum resulting in a progressive increase in
9
levels of ozone, hydrocarbons, oxides of nitrogen and PM10 (Devalia
et al. 1996). Since publication of the WHO Air Quality Guideline
for Europe in 1987 and subsequent updates (Copenhagen, WHO Regional Office for Europe 2005) many epidemiology studies have
shown that PM10 levels below the set guidelines have caused adverse
health effects (Brunekreef et al. 1995). These effects are reviewed in
Pope et al. 1995, and demonstrate a clear relationship between increased levels of PM10 and exacerbations of asthma and chronic
pulmonary disease (COPD). Deaths, not only from respiratory causes but also from vascular causes, i.e. myocardial infarction and cerebrovascular accidents, are also related to levels of PM10 (Utell &
Frampton 2000). Air pollution is increasingly recognized as an important and modifiable determinant of cardiovascular disease in urban communities. Acute exposure has been linked to a range of adverse cardiovascular events including hospital admissions with
angina, myocardial infarction, and heart failure. Long-term exposure
increases an individual's lifetime risk of death from coronary heart
disease. The main arbiter of these adverse health effects seems to be
combustion-derived nanoparticles that incorporate reactive organic
and transition metal components. Inhalation of this particulate matter
leads to pulmonary inflammation with secondary systemic effects or,
after possible translocation from the lung into the circulation, to direct toxic cardiovascular effects. The Utah study by Pope and colleagues (1992) was ground breaking in linking air pollution, particularly PM10 with adverse health effects. The Utah scenario was a
landmark study as it is unusual for an environmental intervention
study to take place where the source of the pollution is switched off
and switched on again, allowing researchers to examine clearly the
effects of air pollution, providing a unique opportunity to examine
the health effects of PM10 in humans. The temporary closing of the
steel mill provided researchers with the unique opportunity to
demonstrate a correlation between exposure to PM10 and observed
health outcomes. Schwartz (2001), posed the question whether studies associating air pollution with increased hospitalisation and mortality rates, could in fact be confounded by influenza or other illness
and thus suggested there may be a ‘harvesting effect’ when it came
to data collection. Schwartz’s study concluded that deaths increased
only for patients outside the hospital which were exposed to increase
10
levels of air pollution, while death rates for patients not exposed to
the outside air remained consistent. Toxicity studies have provided
evidence of the toxicity of many nanoparticles, but epidemiological
proof for health effects of nanoparticles is limited. As such there is
currently no data available to make concentration-response functions
for nanoparticles that can be used in health impact assessment (Hoek
et al. 2010).
4 Nanoparticle Toxicity
The assessment of nanoparticle toxicity can include in vivo studies
or in vitro testing with nanoparticle dose being an important consideration. Artificially high doses are often used in initial, proof-ofprinciple, in vivo studies to induce a toxic effect, so that potential
toxicity can be ascertained, while limiting the number of animals
used. High doses can also be used in in vitro studies to induce a toxic effect, for example, 100µg nanoparticles/ml of cell culture supernatant. These studies should be followed up with realistic in vivo
doses (Oberdörster, Oberdörster and Oberdörster 2005). However,
not all nanoparticles are toxic and some are used successfully as
drug delivery vehicles (McNeil 2005) but toxicity testing is still required for the majority of nanoparticles.
The delivery of nanoparticles to animals in in vivo studies can involve a variety of methods. The method of exposure is usually selected based on the proposed or suspected route of entry, albeit incidental, accidental or deliberate, into the body. Traditionally particle
studies involving animals have focused on the lung as the primary
exposure site and can involve inhalation studies and/or instillation
studies. Inhalation studies involve inhalation of small doses, delivered daily, over various timescales (ranging from days to years)
while instillation studies involve the instant exposure of the animal
to a relatively high dose of the particle instilled into the lung (Donaldson et al. 2007). However, due to the possibility of nanoparticle
accumulation in the food chain and/or possible nanoparticle ingestion, nanoparticle toxicity testing can include intragastric dosing
where particles are introduced into the stomach (Folkmann et al.
2009). Nanoparticles are also being used in formulations for topical
11
use and therefore dermal toxicity studies are being utilised to assess
their safety (Jain et al. 2009). Additionally, particles utilised in nanomedicine may be deliberately injected into the bloodstream and
therefore this route of entry should be considered in nanoparticle
toxicity testing, when appropriate. Ultimately, the in vivo studies are
carried out to ascertain the adverse effects of nanoparticle exposure
on, not collectively or exclusively, the immune system, cardiovascular system, nervous system and digestive system.
In general, nanoparticle toxicity testing uses in vitro methods.
There are several advantages for in vitro toxicity testing and they include their relative low cost compared to in vivo studies and the
speed at which the testing can be completed and from an ethical perspective, the reduction of animal testing. It is important to state that
there are limitations and challenges with using in vitro assays to perform nanoparticle risk assessments as it has become apparent that
nanoparticles can interfere with the current in vitro testing methods,
reviewed by Kroll et al. (2009). Additionally, in vitro systems cannot fully replace the complex interactions that occur in vivo within
and between organs and cannot be used for true pharmacokinetic or
toxicokinetic studies. However, it has been estimated that hundreds
of thousands of animals would be required to ascertain the potential
hazard of each nanoparticle under development or production; therefore, there is a need for toxicologists to develop effective in vitro assays for the assessment of nanoparticle toxicity (Stone, Johnston and
Schins 2009).
Primary cells isolated from animals or humans can be used for in
vitro tests. They correspond closely to the cells in vivo but they have
a limited life span in vitro. Rodent cells are used in most instances as
human cells can be difficult to obtain. Cell lines are more commonly
used because they have an unlimited life span in vitro, under the appropriate conditions. However, they are transformed cells and therefore do not correspond as closely to the cells found in vivo as primary cells.
The first step of an in vitro study should be to determine the cytotoxic potential and LC50 (lethal concentration, 50%) of the nanoparticle of interest using toxicity or viability assays, of which there are a
wide variety (Stone, Johnston and Schins 2009). When sublethal
concentrations have been determined these can be used in subse-
12
quent tests analysing cell function, for example. In vitro toxicity
testing can also involve the assessment of reactive oxygen species
(ROS) production by nanoparticles or by cells exposed to nanoparticles. ROS are oxygen containing molecules that have unpaired electrons, and therefore are highly reactive. They can damage DNA, proteins and lipids and production by cells can be indicative of a stress
response (see Section 4.1.1). Additionally, as inflammation is central
to any potential adverse particle effects in vivo, many steps in the
process of inflammation can be analysed in response to nanoparticles in vitro. For example, calcium flux, non-specific stimulation of
receptors and pro-inflammatory gene expression. Furthermore, cell
death and oxidative stress are both stimulators of inflammation
(Donaldson et al. 2007).
Currently, to effectively ascertain the potential hazards of nanoparticles, it is necessary to complete a combination of in vitro and in
vivo studies (Oberdörster, Oberdörster and Oberdörster 2005; Donaldson et al. 2009). However, it is envisaged that scientists will develop a battery of in vitro tests to be used as alternatives to animal
testing (Stone, Johnston and Schins 2009).
4.1 Mechanisms of Nanoparticle Toxicity
Much of the research into the mechanism of nanotoxicity has been
conducted in the rodent lung. The small physical size of a nanoparticle means that there is an extremely large number of particles per
mass unit. The inhalation of such small particles can result in large
numbers being deposited in the lower alveoli. As a result, efficient
phagocytosis and clearance via the macrophage system can be prevented due to high burden (rodent phenomenon) (Oberdőrster 1992;
Salvi and Holgate 1999). Once in the lungs, nanoparticles can interact with epithelial cells, macrophages and neutrophils, whose activation results in the initiation, progression or prolongation of intracellular and extracellular signalling mechanisms that control
inflammation. In the extracellular environment, inflammation is activated and controlled by a wide range of molecules such as cytokines (Kelley 1990), prostaglandins (Park et al. 2006) and leukotrienes (Grayson et al. 2003). Pro-inflammatory cytokine expression
13
has been a major focus when examining the impact and effects of
pathogenic particles (Brown et al. 2004). Ongoing research strives to
ascertain whether any of the additional extracellular signalling
pathways may play a role in the potential toxic effects of nanoparticles. The role of the intracellular signalling pathways, in response to
nanoparticle exposure, is generally to control the production of the
extracellular pro-inflammatory mediators described above. At this
point it should be noted that nanoparticles, when in contact with biological systems, can be altered by adsorption of protein and other biological materials onto the surface of the particle, Deng et al.
(2009). This point is important for nanotoxicologists as it means that
nanoparticles can change upon introduction into biological conditions. Therefore there is the need for full characterisation of nanoparticles and to understand that these characteristics may change,
and in doing so may alter the behaviour of the raw material. A brief
summary follows describing the current mechanistic knowledge of
how nanoparticles can elicit a toxic response.
4.1.1 Oxidative stress
Oxidative stress occurs when there is an imbalance between oxidants
and antioxidants that favours oxidant presence, due to the excessive
production of oxidants or depletion of antioxidants. Although nanoparticles are small in size they present a large surface area for the
generation of free radicals as a result of redox cycling at the particle
surface. Brown et al. (2000, 2001) confirmed that particles of different composition, including carbon and polystyrene, generate ROS
and were more inflammogenic than larger particles of the same
chemical composition when added to lung cells. ROS are transferred to the interior of the cell where they can result in oxidative
stress, activating transcription factors for pro-inflammatory mediators (Stone et al. 1998; Donaldson et al. 1998; Brown et al. 2001 and
Donaldson and Tran 2002). Additionally, ROS can react with macromolecules such as lipids, proteins and DNA to compromise normal cell function, which can occur to such an extent that cell death
results (Li and Karin 1999).
14
4.1.2 Calcium flux
Nanoparticle carbon black, but not larger carbon black particles,
has been shown to induce calcium influx in a monocytic cell line
(Wilson et al. 2002) and in primary macrophages (Cathcart et al
1983). Furthermore, polystryrene nanoparticles have the ability to
induce calcium signalling in macrophages (Powel and Kanarek
2006). Calcium signalling induced by carbon black nanoparticle was
shown to be important in activating the transcription factor NFB
and production of the pro-inflammatory cytokine TNF (Brown et
al. 2007). There is substantial evidence that ROS and oxidative
stress can alter calcium signalling in cells. Cytosolic calcium is a
key intracellular signalling molecule that controls a variety of cellular processes (Hutchison et al 2005). There is also substantial evidence from the literature that calcium signalling can control inflammation (Ferin et al. 1992). Taken together this evidence would
suggest that ROS can modulate calcium signalling leading to the activation of an intracellular signalling cascade that results in transcription factor activation and the production of pro-inflammatory
cytokines.
4.1.3 Inflammatory response
The mechanism by which particles induce a pro-inflammatory response within the lungs is thought to be a consequence of the increased transcription of pro-inflammatory mediators regulated by increases in intracellular calcium and oxidative stress (Stone et al.
2000). In the lung, epithelial cells play an important role in the defence mechanisms of the body against foreign particles. They can
generate chemotactic factors that recruit phagocytic and inflammatory cells, such as macrophages, when exposed to damaging or infective agents. Attention has focused on the pro-inflammatory effects
induced by nanoparticles, due to the characteristic neutrophil driven
inflammatory response observed within the lungs of treated rats (Li
et al. 1999) and the associated increase in pro-inflammatory cytokine production, such as TNF alpha (TNFα), interleukin (IL)-8, and
IL-1. Some nanoparticles have been shown to stimulate epithelial
cells in vitro to produce the chemoattractant IL-8 (Montellier et al.
15
2007) and nanoparticle carbon black induces glutathione depletion,
in a human lung epithelial cell line, which is indicative of oxidative
stress (Stone et al. 1998).
It is important to note that a majority of the in vivo and in vitro
nanoparticle studies published over the last decade have used particles that were aggregated and/or agglomerated. In many of these
studies, despite a reduction in the exposed particle surface area, their
remains a clear relationship between particle surface area, inflammation and biological responses.
5 Nanomedicines for Pancreatic Disease
Nanotoxicology offers the potential to allow the safe development
of new nanotechnologies and applications including those related to
nanomedicine. Nanomedicine is still in its infancy; however, it has
major potential applications in a range of diseases and target organs
including diabetes and the pancreas: For example, glucose monitoring, insulin delivery, and diagnostic and imaging applications. New
nano-encapsulation technologies, such as layer-by-layer biocompatible nanofilms, involving the implantation of stable electrodes offer
the possibility of continuous glucose monitoring. Nanomedicines are
also being developed to solve current therapeutic problems such as
enhanced insulin delivery in diabetes by both improved islet encapsulation and oral insulin formulations. An artificial nanopancreas
could be an alternative closed-loop insulin delivery system where by
animal islets or insulin-producing cell lines are in a permanent selective membrane that facilitates glucose-dependent insulin release and
nutrient access across the membrane, while excluding the large proteins and cells of the immune system. As discussed in Chapter 10,
nanoparticles in the form of quantum dots or gold can be used in vivo to enhance targeted imaging. This technology could be used to
monitor tissue complications and single-molecule detection systems
could be employed to the study of molecular diversity in diabetes
pathology (Pickup et al. 2008). The technology is being used to aid
visualization of transplanted pancreatic islets, in doing so, advancing
the use of islet transplantation for treatment of type 1 diabetes by
helping to confirm technical success and to diagnose rejection. The
16
literature highlights a crucial need for non-invasive assessment tools
to monitor the success of cell transplantation. Several studies have
investigated whether an improved magnetic resonance imaging
(MRI) strategy, currently a non-invasive experimental cell-based
monitor of therapy, could be clinically applied to islet transplantation. The current strategies available use positron emission tomography and MRI to image islets. Advancements to improve these
strategies have involved the use of superparamagnetic iron oxide nanoparticles, as discussed in greater detail in Chapter 8, to enhance
labelling of cells and to visualise via MRI. Several studies have
demonstrated that islet visualization using superparamagnetic iron
oxide (SPIO) is eminently applicable to islet transplantation even in
humans (Tosco et al. 2008; Huang et al. 2009). Feridex, a representative dextran-coated SPIO nanoparticle with a diameter of 70–
140nm was extensively used for this purpose (Evenon et al. 2006). A
new SPIO agent, Resovist, currently approved for clinical use as a
liver imaging agent may also act as a labelling agent for islet transplantation. Resovist is composed of carboxydextran-coated iron core
nanoparticles comprising multiple crystals, with an overall hydrodynamic diameter of 62 nm. It was tested in vitro, and shown to enhance MRI imaging on islet viability, insulin secretion, and gene expression. This suggests that the technology can image islets with no
deleterious effect on islet function or gene expression (Kim et al.
2009). Nanomedicines are also being developed to improve the diagnosis and treatment of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the
United States (Kelly et al. 2008). The average survival of six months
is due, in part, to limitations in the diagnostic procedures; therefore
the disease can elude detection during its formative stages. Nanoparticle contrast agents have been developed to enhance the visualization of very small pancreatic tumours. Although many good nanoparticle-based contrast agents have been developed, most of them
work by targeting molecules expressed on the surface of tumour
cells. Unfortunately, normal (noncancerous) pancreas tissue express
many of these same molecules, making it difficult to distinguish tumours from healthy tissue (Kelly et al. 2008). Montet et al. 2006
devised an inverse targeting approach for imaging pancreatic cancer
by designing nanoparticles which bind to the peptide bombesin, a
17
ligand that will bind to receptor proteins only found on normal, as
opposed to cancerous, pancreatic cells. Therefore the target, on the
surface of normal pancreas cells, will be absent on PDAC. (Montet
et al. 2006). Montet’s studies in mice, injected with the nanoparticles
conjugated to bombesin peptide revealed that magnetic resonance
images displayed tumours because they did not bind to the nanoparticles.
Effective drug delivery in pancreatic cancer therapy remains a major
challenge. High resistance of tumors to chemo and radiation therapy
results in extremely low survival rates for pancreatic cancer. Recent
advances using nanoparticles to deliver cancer drugs and other therapeutic agents such as siRNA, suicide gene, oncolytic virus, small
molecule inhibitor, has been a success in recent preclinical trials (Yu
et al. 2009).
6 Summary
This chapter provides a summary of both historical and ‘state of the
art’ regarding studies of human and mammalian nanotoxicology. As
yet there is no reason to think that all nanomaterials will induce toxic or harmful effects. Previous experiences with particle air pollution
have provided a foundation for nanotoxicologists; however, it is now
evident that applying standard toxicological tests can only provide a
limited assessment of the potential hazards these new materials may
illicit. The literature available describes the current methodologies
and understanding of nanoparticle toxicity mechanisms. To advance
the science, new or improved methodologies must be developed to
allow a better understanding of how nanoparticles behave in situ.
The lack of information and research on nanomaterial toxicity is important to consider when assessing the potential health and safety issues. A hazard/risk assessment will have to be completed, for every
new nanoparticle, as is done for any new pharmaceuticals, diagnostics or medical materials. If done correctly it should allow a balanced approach to nanomaterial/medicine design and implementation.
18
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