# On the Toxicity of Therapeutically Used Nanoparticles: An Overview

Authors: A. El-Ansary; S. Al-Daihan
Journal: Journal of Toxicology
 (2009)

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## Abstract

Human beings have been exposed to airborne nanosized particles throughout their evolutionary stages, and such exposures have increased dramatically over the last century. The rapidly developing field of nanotechnology will result in new sources of this exposure, through inhalation, ingestion, and injection. Although nanomaterials are currently being widely used in modern technology, there is a serious lack of information concerning the human health and environmental implications of manufactured nanomaterials. Since these are relatively new particles, it is necessary to investigate their toxicological behavior. The objective of this review was to trace the cellular response to nanosized particle exposure. Therapeutic application of selected nanoparticles together with their range of toxic doses was also reviewed. Effect of therapeutically used nanoparticles on cell membrane, mitochondrial function, prooxidant/antioxidant status, enzyme leakage, DNA, and other biochemical endpoints was elucidated. This paper highlights the need for caution during the use and disposal of such manufactured nanomaterials to prevent unintended environmental impacts.


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## Body

## 1. Introduction

Nanotechnology  involves  the creation
and manipulation  of materials at nanoscale levels (1–100 nm) to create
products that exhibit novel properties [1]. The application of nanotechnology to
medicine, known as nanomedicine, concerns the use of precisely engineered
materials at this length scale to develop novel therapeutic and diagnostic
modalities [2]. Nanomaterials have unique physicochemical
properties, such as ultra small size, large surface area to mass ratio, and
high reactivity, which are different from bulk materials (in microscale) of the
same composition. These properties can be used to overcome some of the
limitations found in traditional therapeutic and diagnostic agents.
Nanotoxicology is emerging as an important subdiscipline of nanotechnology.
Nanotoxicology refers to the study of the interactions of nanostructures with
biological systems with an emphasis on elucidating the relationship between the
physical and chemical properties (e.g., size, shape, surface chemistry,
composition, and aggregation) of nanostructures with induction of toxic
biological responses [3, 4].Many people can get exposed to
nanostructures in a variety of manners such as researchers manufacturing the
nanostructures, patients injected with nanostructures, or people using products
containing nanostructures. In all cases, there will be unique routes of
exposure that will dictate the specific fate of nanostructures. The overall
behavior of nanostructures could be summed as follows: (1) nanostructures can enter the body via
six principle routes: intra venous, dermal, subcutaneous, inhalation,
intraperitoneal, and oral [5]; (2) absorption can occur where the
nanostructures first interact with biological components (i.e., proteins, cells);
(3) afterward, they can distribute to various organs in the body and may remain
the same structurally, be modified, or metabolized
[6]; (4) they
enter the cells of the organ and reside in the cells for an unknown amount of
time before leaving to move to other organs or to be excreted.Recently, many studies focus on the
safety issue of manufactured nanomaterials to minimize or eliminate their
nanotoxicity even before they are widely used
[3, 7–9]. This paper
seeks to provide a comprehensive review of all articles published on toxicity
of therapeutically used nanoparticles together with trials for modification of
these products aiming to improve their biocompatibility and minimize their
toxicity.


## 2. Nanomedicine

Nanomedicine has been defined as the
monitoring, repair, construction, and control of human biological systems at
the molecular level, using engineered nanodevices and nanostructures
[10].
Our body is constructed from nanoscale building blocks such
as DNA and proteins, which have long been targeted by the pharmaceutical
industry long before the emergence of nanotechnology
[11, 12].
This category of drugs includes aspirin, cisplatin, and other anticancer agents
as well as much more complex molecules like beta-blockers and anti-inflammatory
agents
[13]. The difference between nanomedicine and conventional
drugs is that nanomedicine is entirely based on small molecule chemistry. It
not only covers the therapeutic agents themselves, but also promise to combine
the abilities to deliver those agents to specific regions or tissues in the
body, to specific cells, perhaps to a specific location within a cell, and also
to make release of the therapeutic responsive to a physiological condition and
perform specific task
[14]. The increased biological activity of
nanoparticles can be either positive or desirable (e.g., antioxidant activity,
carrier capacity for therapeutic penetration of blood-brain barrier, and the
stomach wall or tumor pores), dispersed throughout the whole body including
entering the central nervous system, or negative and undesirable (e.g.,
toxicity, induction of oxidative stress, or cellular dysfunction) or a mix of
both
[3].Nanoparticles have been found to be
distributed to the colon, lungs, bone marrow, liver, spleen, and the lymphatics
after intravenous injection
[15]. Distribution is followed by rapid
clearance from the systemic circulation, predominantly by action of the liver
and splenic macrophages
[16]. Clearance and opsonization, the process
that prepares foreign materials to be more efficiently engulfed by macrophages,
occur under certain conditions for nanoparticles depending on size and surface
characteristics
[16].When inhaled, nanoparticles are found to be distributed to the lungs,
liver, heart, spleen, and brain
[15]. Nanoparticles are cleared in
the alveolar region via phagocytosis by macrophages facilitated by chemotactic
attraction of alveolar macrophages to the deposition site
[17, 18]. The
average half-life (t 1/2)
for nanoparticles in the respiratory tract is  ≈700 days in humans
[3].After intraperitoneal injection, nanoparticles have been found to cross
the transplacental membrane or cross the peritoneal cavity into uterus. This
affected the embryos cranial development and even caused embryo death
[19].After oral exposure, nanoparticles distribute to the kidneys, liver,
spleen, lungs, brain, and the gastrointestinal (GI) tract
[15]. Some nanoparticles can pass through
the GI tract and are rapidly eliminated in feces and in urine, indicating that
they can be absorbed across the GI barrier and into the systemic circulation
[15]. However, some nanoparticle systems can
accumulate in the liver during the first-pass metabolism
[3].Contact with nanoparticles through the skin can occur due to
occupational exposure during the manufacturing of solvents, pesticides, or
pharmaceuticals. Skin exposure to nanoparticles can also occur during
nonoccupational situations from the use of cosmetics and in the intentional
application of topical creams and other drug treatments
[3].


## 2.1. Nanosized Drug

Many approaches have been
developed to use nanoparticles in the area of biomedical imaging and drug
delivery.Quantum dots (QDs), one of the most well studied, are
nanocrystals that fluoresce in different colors depending on their sizes (e.g.,
cadmium selenide) [20]. They typically
have a core made of inorganic element, but  are generally coated with organic
materials such as polyethylene glycol to enhance their biocompatibility or
attach them to specific target molecule like proteins or DNA strands
[21].
If the target molecule is an indicator of disease, detection of that molecule
may indicate a higher propensity for disease. An example is to use
nanoparticles to bind to blood clots and to helpmake clots more visible
by ultrasound
[22].Nanoshells, another nanodelivery
system that is composed of copolymers, are used in combination with specific
wavelengths of light and heat technology for cancer therapy. Gold nanoshells (GNSs) are particularly
suitable for use in the surgical arena as their outer shell is composed of a
commonly used reduced inert gold. When activated by near infrared light, GNS
can raise surrounding temperatures to levels sufficient
for cellular ablation. This  strategy
was recently used by Stern and Cadeddu [23], for the therapeutic ablation
of urologic malignancies.Incorporation of cancer-killer genes
into nanocapsules is being tried out. One of the genes being investigated is
the gene elaborate tumor necrosis factor, a protein that is toxic not only to
cancer but also to healthy cells when injected in large doses. To avoid damage
to normal tissue, the nanocapsule is coated with sensors that target only on
tumor cells.Delivery of drugs to the brain has
always been a challenge. The use of nanoparticles to deliver drug to the brain
using the pathfinder technology is being investigated. This technology uses
nanoparticulate drug carriers in combination with the novel targeting principle
of “differential protein adsorption” to cross the blood-brain barrier
[24]. As the nanoparticles are not efficiently scavenged by
macrophages, the resulting increase
in blood circulation time and hence bioavailability are expected to extend the
duration of controlled system drug delivery or to improve the prospects for
nanoparticles to reach target sites by extravasation
[25].The delivery of magnetic
nanoparticles (MNPs) to or into various cell types has become an area of
increasing interest in the biomedical sciences
[26, 27]. Targeted
delivery is used to deliver drugs or genes by attaching them to MNPs and
locally concentrating the resulting complexes in vivo to the desired locale
[28].
Similarly, magnetic hyperthermia, the local concentration of MNPs and
subsequent heating via magnetic fields, has shown promise as a potentially
viable cancer therapy [29].Nanosized
calcium fluoride (CaF2) that could be used as F reservoir for more
effective F regimens was recently prepared
[30]. The nano-CaF2 can be used as an effective anticaries agent in increasing the labile F
concentration in oral fluid and thus enhances the tooth remineralization. It can also be very useful
in the treatment for the reduction of dentin permeability.Nanomedical
research could result in an array of new medical devices. Interesting research
projects include use of nanoelectromechanical device or nanowire field-effect
transistor to detect insect baculovirus and single influenza viruses,
respectively, were conducted
[31]. It is hoped that development of
these nanodevices can help physician to locate the problem areas in the body more
precisely. Other research works involve the use of biochips and microfluidic
devices to screen tissues for genetic differences and to design genetically targeted drugs
[32].


## 3. Nanotoxicology

While the small size of
particles is what makes nanotechnology so useful in medicine and industry, it
is also one of the main factors that might make them potentially dangerous to
human health. Research is now showing that harmless bulk materials opinion is
that the smaller the particles are the more reactive and toxic are their
effects.It is because any intrinsic properties of particles will likely
be emphasized with the increase in surface area per unit mass
[33, 34].The potential risks inherent to any new
technology are recognized. However, the special concern with nanotechnology is
the unique type
of toxicity due to surface modification. Enhanced endocytosis including
a potential for inflammatory and prooxidant activity are shown to be largely
dependent on nanoparticles' surface chemistry (coating) and in vivo surface
modifications
[3, 33]. Increase in pulmonary toxicity (e.g.,
inflammation, granuloma formation) of carbon nanotubes when compared with that
of the carbon black and carbonyl iron particles was seen in mice and rats
[7, 34].Oxidative stress caused by free
radicals generated by the interaction of particles with cells may result in
cell death. Evidence of mitochondrial distribution and oxidative stress
response after endocytosis of nanoparticles was noted. It was suggested that
nanoparticles, because of their small sizes, could act like haptens to modify
protein structures, either by altering their function or rendering them
antigenic, thus raising their potential for autoimmune effects
[35].There is an initial finding that
indicated that gold nanoparticles might move through a mother's placenta to the
fetus
[36]. Efficient uptake of nanoparticles via the
gastrointestinal tract has also been well documented in oral feeding studies
and gavage studies [37]. All these findings indicate that nanoparticles may
potentially present problems with body burdens and it is hypothesized that
nanoparticles, because of their long retention in the body tissues, might
repeat their highly catalytic activity with the host in cascade
[38].Though nanosilver-based dressing and
surgical sutures have received approval for clinical application, and good
control of wound infection is achieved, their dermal toxicity is still a topic
of concern. Despite laboratory and clinical studies confirming the dermal
biocompatibility of nanosilver-based dressings, several other researchers have
demonstrated the cytotoxicity of these materials [39–44].
Paddle-Ledinek et al. [45] exposed cultured keratinocytes to extracts of
several types of silver containing dressings. The results showed that extracts
of nanocrystalline coated dressings are among those, which are the most
cytotoxic. Keratinocyte proliferation was significantly inhibited, and cell
morphology was affected [45].Recently, the identification of
cytotoxicity of nanoparticles toward mammalian germline stem cells has aroused
great concern over the biosafety of nanomaterials [46]. In their
study, they used a cell line with spermatogonial stem cell characteristics to
test in vitro toxicity of several types of nanoparticles. The results showed
that of all the tested materials (Ag, MoO3, and Al), silver
nanoparticles were the most toxic with manifestations like drastic reduction of
mitochondrial function, increased membrane leakage, necrosis, and induction of
apoptosis. The findings are of significant practical implications because
silver nanoparticles are now able to access human sperms via a variety of
commercialized products like contraceptive devices and maternal hygiene items.
Based on this, fertility problems may occur. In addition, as a fair
extrapolation, another question emerged: what they will do to egg cells?Liver appears to be a major
accumulation site of circulatory silver nanoparticles
[47]. Like
germ line stem cells, similar patterns of cytotoxicity of silver nanoparticles
(decrease of mitochondrial function, LDH leakage, and abnormal cell
morphologies) were observed with in vitro BRL 3A rat liver cells, but to a
lesser extent [1]. In another study by the same researchers, a
neuroendocrine cell line (PC-12 cells) was exposed to silver nanoparticles as a
control against Mn nanoparticles and Mn2+
[48].
Experimental results showed that silver nanoparticles were toxic to
mitochondria than to Mn and Mn2+.All of these findings are of importance because considerable amount of
silver could be detected in rat brain following inhalation of silver
nanoparticles [47]. The  neurological toxicity of silver is not clinically
ascertained, however, several seizures cases have been related to exposure to
silver or silver compounds [49]. Thus, mitochondria seem to be
sensitive targets of cytotoxicity of silver nanoparticles. However, the
mechanism of silver nanoparticles action on mitochondria is yet to be
elucidated. In the study, with BRL 3A liver cell line, depletion of glutathione
(GSH) level and increased ROS were found in association with mitochondrial perturbation,
suggesting that oxidative stress might mediate the cytotoxicity of silver
nanoparticles. Based on these findings, a preliminary impression can be formed
that silver nanoparticles may interact with proteins and enzymes with thiol
groups within mammalian cells. These proteins and enzymes like glutathione, thioredoxin,
SOD, and thioredoxin peroxidase are key components of the cell's antioxidant
defense mechanism which is responsible to neutralize the oxidative stress of
ROS largely generated by mitochondrial energy  metabolism. As these effects of
Ag+ could be completely blocked by sulfhydryl reagents as GSH, the
surface  modification of silver nanoparticles by phosphoryl disulfides was effective in improving
silver biocompatibility and intracellular uptake
[50]. They prepared
the phospholipid derivatives containing disulfide groups to modify silver
nanoparticle surfaces. By adding sodium borohydride to reduce both disulfide
bonds of the derivatives and silver ion simultaneously, the generated thiol
group can be reacted with newborn silver atoms immediately to generate
nanoclusters. The assemblies consisted of either phosphorylcholine (PC) or
Phosphorylethanolamine (PE) head groups, which made the silver clusters
biocompatibile. In cell culture tests, the surfaces modified nanoparticles were internalized
into platelet and fibroblast cells in a short period of incubation without
harming the cells.The study of Pisanic II et al.
[51] indicates that even temporary exposure to Fe2O3 magnetic
nanoparticles (MNPs) results in a dose-dependent reduced ability rat
pheochromocytoma growing neuron cell line PC12 to respond to nerve growth
factor (NGF). PC12 cells exposed to different doses of Fe2O3 MNPs show reduced viabilities, increased cytoskeletal disruption, decreased
intracellular contact, and diminished ability to form mature neuritis in
response to NGF exposure as compared to control cells
(Figure 1). This may have
significant implication for in vivo phenotypic dependent in vitro uses of Fe2O3 in general.
Wiwanitkit et al.
[52] demonstrated in a preliminary
small study that the motility of spermatozoa was affected by the presence of
gold nanoparticles.
They noted that gold particles can penetrate sperm cells, which could result in
fragmentation. The possible spermatotoxicity of gold in industrial use has been
reported elsewhere as a cause of male sterility
[53].(a) Number of intracellular contacts
and (b)  formation of neuritis  in nano-Fe2O3 treated PC12 nerve cells.
(a)(b)


### 3.1. Ultrahigh Reactivity Provokes Nanotoxicity

Because the nanosize/surface area of
the nanosubstance is directly correlated to many essential characteristics like
surface properties, chemical reactivity, physical absorption ability, and so
forth, all these factors strongly dominate nanotoxicological behavior in vivo
[54]. The study of Chen et al.
[43] on
the acute oral toxicity of copper particles has shown a significant correlation
with its size distribution. With the particle size reducing from 17 μm (particle
number: 44 per μg; surface area 3.99×102cm2/g) to 23.5 nm
(particle number: 1.7×1010 per μg; surface area 2.95×102cm2/g), LD50 of copper
particle sharply increases from >5000 mg/kg (nontoxic) to 413 mg/kg (moderately toxic) based on the Hodge and Sterner
scale. For identical chemical composition, why do the nanocopper particles
possess unique biobehavior (nanotoxicity) in vivo comparing to those in bigger
size (in microscale)? Looking for answer for this question may provide an
insight into nanotoxicity reducing or elimination. They explained this on the
basis that nanocopper particles may not compromise the mice directly. The
nanocopper retained in gastric lumen can continuously react with the secreted
acid juice. The depletion of H+ results in metabolic alkalosis
because the HCO3  generated during production of gastric
acid will return to the circulation resulting in the formation of large
quantities of sodium bicarbonate  which increase the arterial blood pH
(Figure 2).Figure 2
Mechanism of
metabolic alkalosis induced by nanocopper ion.The elevation of blood pH motivates a set of
compensatory effects: (a) a respiratory compensation which is limited
[55],
(b) renal compensation starts relatively later but can sustain for a long time
(several days) [55, 56]. However, a series of abnormalities were
found in the pathological examination such as swollen glomerulus, dwindling in lumen
Bowman's capsules, and being signs of glomerulonephritis. The renal dysfunction
may largely weaken renal compensation in nanocopper group and deteriorate the
metabolic alkalosis.It is reported that copper ions
ingested are metabolized in liver and excreted via urine
[57]. If
the intake of copper exceeds the range of the tolerance, it would cause toxic
effects to hepatic and renal tissues, which is consistent with the finding of
Meng et al.
[58] that nanocopper possesses extremely high bioavailability, hence,
the original safety limit may be modified to much lower level. Based on these
finding, we can suggest that nano-and microcopper exhibit different biological
behaviors in
vivo via oral exposure routine. In terms of nanocopper particle, both copper
overload and metabolic alkalosis contribute to their grave toxicity.High chemical reactivity of Ag  nanoparticles was observed in the
reaction with hydrochloric acid: Ag (nanoparticles) + HCl→AgCl + H2;
the reaction product silver chloride was characterized by X-ray powder
diffraction to give a direct evidence for the reaction which has been proved
impossible for the bulk Ag [59].The microscale titanium dioxide (TiO2),
widely used in pharmaceutical and cosmetics industries, is considered as
biologically inert [60]. Such that, there was no obvious lung
toxicity in rats when a single instilled dose of TiO2 was 5 mg/rat or
50 mg/kg [61]. However, many studies have demonstrated that when TiO2 particles size decreased to nanoscale dimension, they could produce more
pulmonary toxicity than their bulk counterparts [62–65]. In a comparative study done by Li et al.
[66],
the acute pulmonary toxicity induced by 3 and 20 nm TiO2 was
investigated through measurement of selected biochemical parameters in
bronchoalveolar lavage fluid (BALF). At 3-day postexposure, the 3 nm TiO2 induced significant increase of albumin, alkaline phosphatase (ALP), and acid
phosphatase (ACP) concentrations in high-dose group (40 mg/kg) and also induced
significant increase of ALP and ACP concentrations in mid-dose group (4 mg/kg),
but did not induce significant
increase of total protein and LDH concentrations in any dose group. On the
other hand, 20 nm TiO2 induced significant increase of all
biochemical parameters in high- and mid-dose groups. At 3-day postexposure,
both TiO2 particles did not induce obvious pulmonary toxicity in
their low-dose (0.4 mg/kg) groups as evidence of no significant increase of all
biochemical parameters. The pH values of
3 nm TiO2 particles colloid were 5.38±0.12,  4.55±0.07, and 4.42±0.13
at concentrations of 0.1, 1, and 10 mg/mL, respectively, while at the
corresponding concentration, the pH values of 20 nm TiO2 particles
suspension were
5.5±0.19,  4.64±0.11, and 3.75±0.04, respectively. They reported that pH value
of TiO2 particles in medium, other than particle size, surface area,
and aggregation, plays important role in affecting TiO2 nanoparticles pulmonary toxicity.The
potentiated  toxicity of nanoscale vanadium oxide (V2O3)
compared to bulk  material is  demonstrated in human endo- and epithelial lung
cells and might be due to the higher catalytic surface of the particles
[60].
Reduction in cell viability is almost ten times stronger and starts with the lowest
concentrations of “nanoscaled” material (10 μg/mL). Vanadium oxide
leads to an induction of heme oxygenase 1 (HO-1) in a dose-dependent manner in
ECV304 cells, whereas a reduction in protein levels can be observed for the
epithelial cells (A549). Lipid peroxidation can be observed also for
“nanoscaled” vanadium oxide to a much stronger extent in macrophages
(RAW cells) than for bulk material. The observed effects cannot only be
explained by oxidation from V2O3 to V2O5 as there are significant differences between the novel nano-vanadium and all
used bulk materials (V2O3 and V2O5).
It appears rather to be a nanoeffect of a high surface reactivity, here coupled
with a yet unknown toxicity potentiating effect of a technically important
catalyst.


## 3.1. Ultrahigh Reactivity Provokes Nanotoxicity

Because the nanosize/surface area of
the nanosubstance is directly correlated to many essential characteristics like
surface properties, chemical reactivity, physical absorption ability, and so
forth, all these factors strongly dominate nanotoxicological behavior in vivo
[54]. The study of Chen et al.
[43] on
the acute oral toxicity of copper particles has shown a significant correlation
with its size distribution. With the particle size reducing from 17 μm (particle
number: 44 per μg; surface area 3.99×102cm2/g) to 23.5 nm
(particle number: 1.7×1010 per μg; surface area 2.95×102cm2/g), LD50 of copper
particle sharply increases from >5000 mg/kg (nontoxic) to 413 mg/kg (moderately toxic) based on the Hodge and Sterner
scale. For identical chemical composition, why do the nanocopper particles
possess unique biobehavior (nanotoxicity) in vivo comparing to those in bigger
size (in microscale)? Looking for answer for this question may provide an
insight into nanotoxicity reducing or elimination. They explained this on the
basis that nanocopper particles may not compromise the mice directly. The
nanocopper retained in gastric lumen can continuously react with the secreted
acid juice. The depletion of H+ results in metabolic alkalosis
because the HCO3  generated during production of gastric
acid will return to the circulation resulting in the formation of large
quantities of sodium bicarbonate  which increase the arterial blood pH
(Figure 2).Figure 2
Mechanism of
metabolic alkalosis induced by nanocopper ion.The elevation of blood pH motivates a set of
compensatory effects: (a) a respiratory compensation which is limited
[55],
(b) renal compensation starts relatively later but can sustain for a long time
(several days) [55, 56]. However, a series of abnormalities were
found in the pathological examination such as swollen glomerulus, dwindling in lumen
Bowman's capsules, and being signs of glomerulonephritis. The renal dysfunction
may largely weaken renal compensation in nanocopper group and deteriorate the
metabolic alkalosis.It is reported that copper ions
ingested are metabolized in liver and excreted via urine
[57]. If
the intake of copper exceeds the range of the tolerance, it would cause toxic
effects to hepatic and renal tissues, which is consistent with the finding of
Meng et al.
[58] that nanocopper possesses extremely high bioavailability, hence,
the original safety limit may be modified to much lower level. Based on these
finding, we can suggest that nano-and microcopper exhibit different biological
behaviors in
vivo via oral exposure routine. In terms of nanocopper particle, both copper
overload and metabolic alkalosis contribute to their grave toxicity.High chemical reactivity of Ag  nanoparticles was observed in the
reaction with hydrochloric acid: Ag (nanoparticles) + HCl→AgCl + H2;
the reaction product silver chloride was characterized by X-ray powder
diffraction to give a direct evidence for the reaction which has been proved
impossible for the bulk Ag [59].The microscale titanium dioxide (TiO2),
widely used in pharmaceutical and cosmetics industries, is considered as
biologically inert [60]. Such that, there was no obvious lung
toxicity in rats when a single instilled dose of TiO2 was 5 mg/rat or
50 mg/kg [61]. However, many studies have demonstrated that when TiO2 particles size decreased to nanoscale dimension, they could produce more
pulmonary toxicity than their bulk counterparts [62–65]. In a comparative study done by Li et al.
[66],
the acute pulmonary toxicity induced by 3 and 20 nm TiO2 was
investigated through measurement of selected biochemical parameters in
bronchoalveolar lavage fluid (BALF). At 3-day postexposure, the 3 nm TiO2 induced significant increase of albumin, alkaline phosphatase (ALP), and acid
phosphatase (ACP) concentrations in high-dose group (40 mg/kg) and also induced
significant increase of ALP and ACP concentrations in mid-dose group (4 mg/kg),
but did not induce significant
increase of total protein and LDH concentrations in any dose group. On the
other hand, 20 nm TiO2 induced significant increase of all
biochemical parameters in high- and mid-dose groups. At 3-day postexposure,
both TiO2 particles did not induce obvious pulmonary toxicity in
their low-dose (0.4 mg/kg) groups as evidence of no significant increase of all
biochemical parameters. The pH values of
3 nm TiO2 particles colloid were 5.38±0.12,  4.55±0.07, and 4.42±0.13
at concentrations of 0.1, 1, and 10 mg/mL, respectively, while at the
corresponding concentration, the pH values of 20 nm TiO2 particles
suspension were
5.5±0.19,  4.64±0.11, and 3.75±0.04, respectively. They reported that pH value
of TiO2 particles in medium, other than particle size, surface area,
and aggregation, plays important role in affecting TiO2 nanoparticles pulmonary toxicity.The
potentiated  toxicity of nanoscale vanadium oxide (V2O3)
compared to bulk  material is  demonstrated in human endo- and epithelial lung
cells and might be due to the higher catalytic surface of the particles
[60].
Reduction in cell viability is almost ten times stronger and starts with the lowest
concentrations of “nanoscaled” material (10 μg/mL). Vanadium oxide
leads to an induction of heme oxygenase 1 (HO-1) in a dose-dependent manner in
ECV304 cells, whereas a reduction in protein levels can be observed for the
epithelial cells (A549). Lipid peroxidation can be observed also for
“nanoscaled” vanadium oxide to a much stronger extent in macrophages
(RAW cells) than for bulk material. The observed effects cannot only be
explained by oxidation from V2O3 to V2O5 as there are significant differences between the novel nano-vanadium and all
used bulk materials (V2O3 and V2O5).
It appears rather to be a nanoeffect of a high surface reactivity, here coupled
with a yet unknown toxicity potentiating effect of a technically important
catalyst.


## 4. Protein-Nanoparticle Interactions

Within the medical device community, it is now well accepted that
material surfaces are modified by the adsorption of biomolecules such as
proteins in a biological environment
[67, 68], and there is
some consensus that cellular responses to materials in a biological medium
reflect the adsorbed biomolecule layer, rather than the material itself.
However, the importance of the adsorbed protein layer in mediating interactions
with living systems has been slower to emerge in the case of
nanoparticle-protein interactions. The key role of protein-nanoparticle interactions
in nanomedicine and nanotoxicity has begun to emerge recently with the
development of the idea of the nanoparticle-protein “corona”. This
dynamic layer of proteins (and other biomolecules) adsorbs to nanoparticle
surfaces immediately upon contact with living systems. The composition of the
protein corona at any given time will be determined by the concentrations of
the over 3700 proteins in plasma
[69] and the kinetic on and off rates (or
equilibrium binding constants) of each protein for the particular nanoparticle.
This corona may not immediately reach equilibrium when exposed to a biological
fluid. Proteins with high concentrations and high association rate constants
will initially occupy the nanoparticle surface, but may also dissociate quickly
to be replaced by proteins of lower concentration, slower exchange, and higher
affinity. Thus, the protein corona is the biological identity of a
nanoparticle, as it is what the cell “sees” and interacts with. Functional changes of proteins of
such complexes may be another mechanism by which particularly small
nanoparticle, with their large surface area as a binding interface, may induce
protein mal-functioning, which may lead to the pathogenesis and adverse health
effects [70]. Survey of the literature on nanoparticle-protein
binding shows that the vast majority of nanoparticle types studied, so far,
bind apolipoproteins [68]. At first sight, this is surprising result
and quite distinct from that for a flat surface. However, the fact that
apolipoproteins are known to be involved in lipoprotein complexes, which themselves have sizes on
the nanoscale ranging from 100 nm (chylomicron) to 10 nm (high-density
lipoprotein), may mean that there are specific size-dependent interactions that
drive the binding of apolipoprotein to nanoparticles. This is interesting from
the point of view of nanoparticle interaction with cells, as lipoprotein
complexes are involved in the general cellular processes of cholesterol
metabolism [2]. Thus, there are multiple receptors for apolipoprotein
complexes at cell surfaces that nanoparticles with surface-adsorbed
apolipoproteins can potentially exploit to enter cells [71]. If we
consider the issues of nanoparticle transport and fate in animals and humans,
then it is also relevant that apolipoprotein E has been found to associate to
some nanoparticles [67]. This has potentially significant
consequences for neurotoxicity and the development of neurotherapies, as
apolipoprotein E is known to be involved in trafficking to the brain [71].The first reports of the direct
biological influence of proteins adsorbed to nanoparticles are now emerging.
Single-walled carbon nanotubes (SWNTs) and 10 nm amorphous silica coated with
albumin have been shown to induce anti-inflammatory responses in macrophages,
measured as inhibited induction of cyclooxygenase-2- (Cox-2) by
lipopolysaccharide under serum-free conditions [72]. Blocking the adsorption of albumin by
precoating the nanoparticles with nonionic surfactant (Pluronic F127) also
inhibits the anti-inflammatory properties of the nanoparticles. These
observations suggest an important role for the adsorbed proteins in modulating
the uptake and toxicity of SWNTs and nanosized amorphous silica [72].
However, as these studies were conducted under serum-free conditions, it is unclear whether the
albumin would remain bound to nanoparticles under competitive binding
conditions, such as those occur
in plasma or in a cellular milieu. The interaction between human adult
hemoglobin (Hb) and bare CdS QDs dramatically alters the conformation of Hb and decreasing the
α-helix content of the secondary structure from 72.5% to 60.8%. Raman spectra
results indicate that the sulfur atoms of the cysteine residues form direct
bonds on the surface of the CdS QDs [73]. Functionalization of
nanoparticles surfaces with peptides is increasingly being used to control the
interaction of nanoparticles with proteins [69].


## 5. Discussion

Nanotoxicology
refers to the biokinetic evaluation of engineered nanostructures and
nanodevices. The need for this area of investigations became apparent after the
intensive expansion of nanotechnology, which in the last two decades has been
widely used in the pharmaceutical industry, medicine, and engineering
technology [74, 75]. Particle toxicology and the consequent adverse
health effects of asbestos fibers and coal dust serve as a historical reference
points to the development of nanotoxicological concepts.In
the area of medicine, nanomedicine has been defined as the monitoring, repair,
construction, and control of human biological systems at the molecular level,
using engineered nanodevices and nanostructures [10]. Macrophages as
specialized host defense cells, endothelium as thin specialized epithelial
cells that line the inner surface of lymph vessels and blood vessels serve as
gate keeper to control passage of materials together, and tumors are the most
common targets of nanoparticles. Within these biological targets, nanoparticles
favor the formation of prooxidants especially under exposure to light,
ultraviolet light, or transition metals; thereby destabilizing the balance
between the production of reactive oxygen species (ROS) and the biological
system's ability to detoxify or repair the system [74, 75]. ROS can
also be produced by the NADPH oxidase in phagocytic cells as target of
nanoparticle devices. Nanoparticles can modify mitochondrial function as well
as cellular redox signaling. Oxidative stress induced by nanoparticles is
reported to enhance inflammation through upregulation of redox-sensitive
transcription factors including nuclear factor kappa B (NFkB), activating
protein (AP-1), and extracellular signal regulator kinases (ERK) C-Jun,
N-terminal kinases JNK, and p38 mitogen-activated protein kinases pathways.
Figure 3 summarizes the most important recorded toxic effects of
therapeutically used nanoparticles reviewed in the present paper.Figure 3
A summary of the most important
recorded toxic effects of therapeutically used nanoparticles reviewed in the
present paper


## 6. Concluding Remarks

(1) Nanotechnology
is growing at an exponential rate and will undoubtedly have both beneficial and
toxicological impact and consequences on health and environment.(2) As a result of their properties,
nanomaterials differ substantially from those bulk materials of the same
composition,allowing them to perform exceptional feats of
conductivity,reactivity, and optical sensitivity. Possible
undesirable resultsof these capabilities are harmful interactions
with biologicalsystems with the potential to generatetoxicity.(3) Development of new techniques to show
accurate correlations between in vitro and in vivo studies is imperative to
accurately portray nanoparticle effects. Moreover, toxicity studies are
critical to establish the full in vivo potential of nanomedicine. Understanding
the physiochemical, molecular, and physiological processes of nanoparticles is
important for nanomedicine to become a reliable and sustainable treatment
modality.(4) In the future, nanoparticles could be
classified in terms of their biomolecule corona which mediates their
interaction with cellular machinery. This would represent a truly new paradigm in the
field of nanoscale toxicology and in the design of safe nanocarriers for
nanomedicine.(5) With this new opportunity to utilize the
unique properties of nanoparticles for research, industry, and medicine, there
is a responsibility  to test and optimize these new nanomaterials early during
the development process to eliminate or ameliorate identified toxic
characteristics.(6) The rapid commercialization of
nanoparticles requires focused environmental, health, and safety research, meaningful
and open discussion of broader societal impacts and urgent oversight.



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*Source: 754810-2009-01-25.xml*