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This journal is cThe Royal Society of Chemistry 2012 Chem. Soc. Rev.
Cite this: DOI: 10.1039/c2cs15355f
Intrinsic therapeutic applications of noble metal nanoparticles: past,
present and futurew
Rochelle R. Arvizo,
a
Sanjib Bhattacharyya,
a
Rachel A. Kudgus,
a
Karuna Giri,
a
Resham Bhattacharya
a
and Priyabrata Mukherjee*
abc
Received 23rd December 2011
DOI: 10.1039/c2cs15355f
Biomedical nanotechnology is an evolving field having enormous potential to positively impact
the health care system. Important biomedical applications of nanotechnology that may have
potential clinical applications include targeted drug delivery, detection/diagnosis and imaging.
Basic understanding of how nanomaterials, the building blocks of nanotechnology, interact with
the cells and their biological consequences are beginning to evolve. Noble metal nanoparticles
such as gold, silver and platinum are particularly interesting due to their size and shape
dependent unique optoelectronic properties. These noble metal nanoparticles, particularly of gold,
have elicited a lot of interest for important biomedical applications because of their ease of
synthesis, characterization and surface functionalization. Furthermore, recent investigations are
demonstrating another promising application of these nanomaterials as self-therapeutics. To
realize the potential promise of these unique inorganic nanomaterials for future clinical
translation, it is of utmost importance to understand a few critical parameters; (i) how these
nanomaterials interact with the cells at the molecular level; (ii) how their biodistribution and
pharmacokinetics influenced by their surface and routes of administration; (iii) mechanism of
their detoxification and clearance and (iv) their therapeutic efficacy in appropriate disease model.
Thus in this critical review, we will discuss the various clinical applications of gold, silver and
platinum nanoparticles with relevance to above parameters. We will also mention various routes
of synthesis of these noble metal nanoparticles. However, before we discuss present research, we
will also look into the past. We need to understand the discoveries made before us in order to
further our knowledge and technological development (318 references).
1. Historical perspective
Precious metals have a long and rich history of use harkening
back to the Egyptian First Dynasty. Gold in particular was a
much sought after metal and mined as early as 2900 BCE in
the deserts of Ethiopia and Nubia.
1
In Egypt, mineralogists
soon learned to purify this metal. Since then, there has been
evidence of objects made from gold from the Early Dynastic
period of Ur (2500 BCE) to Babylonia. Beni Hassan, a tomb
dating in the same era, has representative inscriptions of the
extraction process from quartz matrix and gold ores in addi-
tion to the weighing and melting processes.
2
2000 years later,
Darius, the king of Persia (558–486 BCE), was reported to
have received unrefined gold and gold dust as gifts from the
Ethiopians.
3
The early fascination with metals is further
illustrated by the keen interest of early alchemy philosophers
who believed in the spiritual connection between the seven
metals: gold, silver, mercury, lead, tin and iron, with the seven
heavenly bodies: the Sun, the Moon, Venus, Jupiter, Mercury,
Saturn and Mars.
2
It was believed that Earth housed the seeds
of metals and was under the influence of the heavenly bodies
(Fig. 1).
For alchemists, gold was greatly treasured as evidenced by
the common quest for the philosopher’s stone (lapis philoso-
phorum), an agent that would make possible transmutation of
base metals into gold.
4,5
Furthermore, gold was considered to
be indestructible and have immense medicinal value; hence
early alchemists set out to produce potable gold, ‘‘the elixir of
life’’.
5
In 8th century CE, an alchemist in Arabia, Jabir ibn
Hayyan, also known as ‘‘Geber’’ in Europe, succeeded in
dissolving gold in aqua regia, a mixture of nitric and hydro-
chloric acid.
1
By the 7th century, gold chloride had become
commonplace and in the early Renaissance, gold was
a
Department of Biochemistry and Molecular Biology, Mayo Clinic
College of Medicine, 200 First St SW, Rochester, MN 55905, USA.
E-mail: Mukherjee.priyabrata@mayo.edu
b
Department of Physiology and Biomedical Engineering, Mayo Clinic
College of Medicine, 200 First St SW, Rochester, MN 55905, USA
c
Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, 200
First St SW, Rochester, MN 55905, USA
wPart of the nanomedicine themed issue.
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recommended to purify blood and thought to have numerous
medical virtues.
2
The first use of gold in modern medicine was
in 1890 after the German bacteriologist Robert Koch discovered
that low concentrations of potassium gold cyanide, K[Au(CN)2]
had antibacterial properties against the tubercle bacillus.
6
In the
1920s gold therapy for tuberculosis was introduced
6
and in 1935
Jacque Forestier reported the use of gold to treat rheumatoid
arthritis.
7
Gold thiolates are still in use today to treat the disease.
In the United States, gold sodium thiomalate and gold
thioglucose are sold as Myochrysine and Solgonal respectively.
8
Auranofin (triethylphosphine(2,3,4,6-tetra-O-acetyl-b-1-D-thio-
pyranosato-S)gold(I)aresomeofthenewercompoundsinuseas
antiarthritic drugs (Fig. 2a–c). Other gold complexes have been
implicated in treatment of cancer and malaria (Fig. 2d and e).
Silver has been valued throughout history as a precious
metal and in ancient times it was considered to be more
valuable than gold. Believing it to be feminine in nature with
its white luster, silver was considered a symbol of purity.
Evidence of the use of silver to make ornaments and decorations
exists from as far back as 4000 BC.
9
Silver was oft referred to as
white gold and was known to the Greeks and inhabitants of the
region between the Indus and the Nile. A gold silver alloy was
used to make coins by 800 BC.
2
Alchemists associated silver
with the moon (oft referenced the element as luna), and hence
used it to cure ailments related to the brain (hence the term
lunatic). In Arabia, it was used to treat ‘‘falling-sickness’’ and
vertigo.
2
Hippocrates proclaimed that silver contained medicinal
Fig. 1 Alchemy symbol of (A) Gold, (B) Silver, (C) Platinum.
Rochelle R. Arvizo
Dr Rochelle R. Arvizo received
aBSinbiologyfromColorado
State University and performed
research under the supervision
of Professor Nancy Levinger,
Professor Deborah Crans and
Professor Dawn Rickey. She
did her graduate studies with
Professor Vincent M. Rotello
at the University of Massachu-
setts Amherst (20009). She is
currently a research fellow with
Professor Priyabrata Mukher-
jee at the Mayo Clinic in
Rochester, MN. Her research
interests include the molecular
mechanism of gold nanoparticles
as anti-angiogeic and anti-
tumorgenic agents.
Sanjib Bhattacharyya
Dr Sanjib Bhattacharyya
received his PhD from Univer-
sity of Missouri Columbia,
Missouri, in 2009 in Bio-organic
Chemistry. Currently, he is
working as a postdoctoral
research fellow in Department
of Biochemistry and Molecular
Biology and Biomedical Engi-
neering, College of Medicine,
Mayo Clinic, Rochester. His
research focus includes bioma-
terials, cancer nanotechnology,
targeted drug delivery and
Hydrogen sulfide signaling in
tumor genesis.
Rachel A. Kudgus
Dr Rachel A. Kudgus received
her B.S. in chemistry from
Binghamton University in New
York (2004) and performed
undergraduate research under
the supervision of Professor
Scott Handy. She later split
her graduate studies between
North Carolina State Univer-
sity and University of Colorado
at Boulder. She worked with
Professor Daniel Feldheim in
Colorado and with Professor
Christian Melander in North
Carolina and ultimately earned
her PhD in chemistry from
North Carolina State University (2010). She is currently a
research fellow with Professor Priyabrata Mukherjee at Mayo
Clinic, Rochester. Her research interests include the synthesis,
analysis and characterization of gold nanostructures for biomedical
applications.
Karuna Giri
Karuna Giri is a graduate
student with the Department of
Biochemistry and Molecular
Biology at the Mayo Graduate
School. She is from Kathmandu,
Nepal. She got her B.A in
Biology from Grinnell
College, Iowa in 2010. Her
current interest is studying
the interaction of engineered
gold nanoparticles with
biological macromolecules. In
her spare time she enjoys
travelling and the fine arts.
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properties and could cure multiple maladies.
10
Since silver was
touted to have antiseptic properties, Phoenicians used silver vials
for food storage to help prevent spoilage. Prior to the wide-
spread use of antibiotics, silver compounds were used to help
prevent infection during World War I.
11
In 1881, a physician
named Crede used silver compounds to help prevent gonorrhea
from being transmitted from the mother to new born babies.
12
Lunar caustic, silver nitrate amalgamated into sticks, was also
used in antiquity to cauterize wounds. Silver nitrate also has a
close relationship with photography. For silver, the history of
the art form begins in 1727 when John Herman Schulze, a
German professor first observed that silver salts turned black
when exposed to light.
13
Silver salts were further investigated
and even hundreds of years later continued to be the critical
component of photographic film.
Another noble metal, platinum was discovered on the
alluvial sands of the Pinto River in Columbia.
14
The first
reported use was by Egyptians and South Americans ca. 2000
years ago.
14
Archeologists uncovered an ancient Egyptian box
from B720 B.C. that contains hieroglyphic characters deco-
rated with platinum bands.
15
The Europeans did not know of
platinum until the 16th century when the Spaniards came
across the element following conquest of the lands of South
America. When mining for gold in Columbia, they found
lumps of platinum which they called platina del Pinto meaning
‘‘little silver of the Pinto river’’.
14
Some of the samples were
brought back to Europe in 1557 and studied by Italian-French
scientist Julius Caesar Scaliger who concluded that the metal
was not silver and in fact a new element, Platinum. Thus Julius
Caesar Scaliger is widely reported to have discovered the
metal. In 1735, a Spanish scientist Antonio de Ulloa redis-
covered platinum in South America and in 1783 French
chemist Francois Chabaneau successfully purified it thus
initiating the use of the metal for decorative purposes. A
famous object made from platinum in antiquity is a chalice
made in 1788 for Pope Pius VI. The popularity of the
metal rose in the following years and by the 19th century
platinum was in high demand for use in jewelry and industrial
purpose.
In 1965, Rosenberg et al. discovered that electrolysis using
platinum electrodes inhibited division of Escherichia coli.
16
The group subsequently reported that platinum salts,
[PtCl
6
]
!2
, generated via electrolysis, were responsible for the
Fig. 2 Structures of Gold complexes with antiarthritic, antitumor,
and antimalarial activity. (a–c) antiarthritic drugs; (d) gold (III) anti-
tumor agents; (e) antimalarial complex of chloroquine.
Resham Bhattacharya
Dr Resham Bhattacharya
graduated with a PhD. in
Molecular Microbioblogy
from the Bowling Green State
University, Ohio. Since the
beginning of the research
career her interest has been
in tumor angiogenesis and cell
signaling. Specifically study-
ing signaling, intervention-
therapy and chemoresistance
in ovarian cancer. She has
(a) elucidated the importance
of sonic hedgehog signaling in
ovarian cancer, (b) identified
microRNAs that regulate
ovarian cancer proliferation by targeting Bmi-1, and (c) func-
tionalized gold nanoconjugates to effectively target and kill
ovarian cancer cells by simultaneous conjugation with folic acid
and cisplatin.
Priyabrata Mukherjee
Dr Priyabrata Mukherjee has
a broad expertise in the area of
chemistry, materials science,
nanotechnology, cancer biology,
nanomedicine in general. Over
the past 17 years he has been
working on the biomedical
application of nanomaterials.
Presently, he serves as a Senior
Associate Consultant at Mayo
Clinic, Rochester. Dr Mukher-
jee’s laboratory is focused on
understanding the basic princi-
ples of cell-nanomaterial inter-
action as a way to discover new
molecular targets and signaling
events in various malignancies. This is a highly interdisciplinary field
where chemists, physicists, biologists, materials scientists and
clinicians work together to exploit the benefit of nanotechnology
in human health care.
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Chem. Soc. Rev. This journal is cThe Royal Society of Chemistry 2012
anti-proliferative action on the bacteria.
17
Thus began the
resurgence of investigations with cisplatin, which had
remained obscure since its first synthesis in 1845 by Michel
Peyrone.
18
In 1893, Alfred Werner had already elucidated the
structure of cisplatin but it wasn’t until Rosenberg that
the antitumor activity of cisplatin was studied and so began
the reign of the ‘‘Penicillin of cancer’’. After successful studies
in mice, the compound entered clinical trials in 1971. In 1977
cisplatin was licensed exclusively to Bristol-Myers Squibb and
by 1978 it was approved for use in the US by the Food and
Drug Administration.
18
In contrast to metals, metallic nanoparticles (NPs) and their
use may be considered a product of modern science since the
potential of nanotechnology was only realized in recent years.
Yet the synthesis and use of nanoparticles (NPs) dates back to
ancient times. The first evidence of metallic nanoparticles is
from 2000 years ago when gold NPs were a part of ancient
ayurvedic medicine in India.
19
56 nm sized colloidal gold NPs,
also called the swarna bhasmaI (gold ash) was mixed with
honey or cow ghee and given orally to patients to treat a wide
range of diseases including rheumatoid arthritis, bronchial
asthma, diabetes mellitus and other diseases of the nervous
system.
19,20
The aesthetic property of gold NPs was later
realized and exploited by the Romans. They used gold NPs
to color glass; an exemplary case in point is the Lycurgus cup
(Fig. 3). The colored glass and bronze cup is dated to the 4th
century Roman Empire and shows a ‘‘dichroic’’ effect i.e. the
cup appears pea green in reflected light but in transmitted light
it appears to be a deep wine red color.
19,21
Studies conducted
by the British Museum, which houses this work of art, report
that the cup composite is an alloy of 70nm NPs containing
70% silver and 30% gold.
21
Although one can only speculate
as to whether the use of NPs was purely accidental, artisans in
other cultures have shown deliberate exploitation of the
unique optical effect of NPs to create colorful church windows.
Silver NPs were used to stain glass a yellow color
22
while gold
NPs were used to produce a ruby red hue. In 9th century
Mesopotamia, silver and copper NPs were used to give pottery
gold like iridescent, metallic luster.
23
The Muslim culture forbids
the use of gold in artistic representation and so the artisans
devised a method to employ other metallic NPs to produce a
gold like result. Copper and silver salts and oxides were mixed
with vinegar, clay, and ochre and then applied to glazed pottery.
When ‘‘cooked’’ at high temperatures and a reducing environ-
ment, the metals ions would reduce and migrate to the outer
layer of the glaze forming a NP coat, thus producing a brilliant
gold-like end product.
But perhaps the first scientific study of metallic NPs in
colloidal systems and the first observation of the unique
optical properties of gold NPs was by English physicist and
chemist Michael Faraday in 1857.
24
Faraday was the first to
study and report the size dependent optical properties of gold
and silver colloids. Although it would be almost a century later
when the field of nanotechnology would take off, Faraday’s
observation that particles on the nanoscale behaved differently
from its bulk was critical and fundamental discovery. In 1908
Gustav Mie studied the mathematic correlation of NP size and
its optical manifestations.
25
In 1959, it was physicist Richard
P. Feynman who, almost a century after Faraday, memorably
championed the arrival of nanotechnology.
26
In that momentous
lecture to the American Physical Society at Caltech, he said,
‘‘There’s plenty of room at the bottom—an invitation to enter a
new field of physics’’ hinting at the potential for nanoscale design
to influence a wide range of fields such as optics and electronics.
The use of metal NPs has expanded in recent years
following significant developments in the synthesis process.
Metals like platinum and silver have long been used as
industrial catalysts. German chemist Johann Wolfgang
Dobereiner, who is also known as the founder of the study
of catalysis, was the first to discover the catalytic capability of
finely divided platinum.
27
In 1820, Edmund Davy an English
chemist had shown that chemically reduced platinum black
could promote alcohol oxidation. Dobereiner repeated this
experiment a year later and made the critical observation that
at the end of the conversion of alcohol to acetic acid, platinum
was unaltered and available to participate in another reaction.
He later went on to develop the Dobereiner lamp, which is
now appreciated as the first example to use a supported
catalyst, which involves a jet of hydrogen from zinc and
sulphuric acid that is spontaneously ignited in the presence
of platinum.
27
The nanoscale size of particles was later known
to enhance catalytic activity of a metal; thus metals in NP form
have been keenly studied as a way to cut down costs and
improve catalytic efficiency. It is interesting that gold was
historically considered to be catalytically inactive. But in 1985,
Graham Hutchings from the University of Cardiff, UK
reported that the gold ions could catalyze the hydrochlori-
nation reaction. Similarly, Masatake Haruta, from Tokyo
Metropolitan University in Japan, later observed that in NP
form gold could catalyze oxidation of carbon monoxide even
at low temperatures of !76 1C.
19
In recent years there has also
been increasing interest in the use of silver NPs as anti-
microbial agents. As mentioned earlier, this remarkable property
of the metal was known since antiquity to Greeks who used the
metal in their cooking and used it to for safe storage of water.
28
Modern application of nanoparticles extends even as far as
restoring centuries old works of art.
29
Thus NPs has been
Fig. 3 The Lycurgus Cup 1958,1202.1 in reflected (a) and transmitted
(b) light. Scene showing Lycurgus being enmeshed by Ambrosia, now
transformed into a vine-shoot. Department of Prehistory and Europe, The
British Museum. Height: 16.5 cm (with modern metal mounts), diameter:
13.2 cm. Reprinted with permission from rTrustees of the British Museum.
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involved in our life since time immemorial. Therefore in our next
section we will discuss their synthesis.
2. Modern synthesis of noble metal nanoparticles
Synthesis of noble metal nanoparticles (Au, Ag and Pt) has
exploded in the last few decades. The most popular techniques
are chemical reduction, physical processes and biological
methods. The physical properties of nanoparticles include size,
shape, structure and composition. Each of these aspects can be
altered or manipulated by varying either kinetic or thermo-
dynamic variables in the syntheses (Fig. 4). The ability to
control specific properties through minor alterations has led to
a major movement in research exploration of nanoparticles as
well as increased the potential for applications within the fields
of catalysis, electronics, therapeutics and diagnostics. Before
we discuss the therapeutic aspects of these nanoscale materials,
we must first have a look at their synthesis.
2.1 Chemical reduction
2.1.1 Gold nanoparticles. Collidial gold synthesis has been
intensively studied for centuries.
30,31
The most common
method of synthesis for gold nanoparticles (AuNP) is chemical
reduction. The simplest of these methods is the reduction of
gold salts in the presence of a reducing agent.
31,32
The first
documented study of the solution phase synthesis of gold
colloids was in 1857 when Michael Faraday reduced gold
chloride with phosphorous in an aqueous medium.
24
Turkevitch
et al. made the next critical discovery in 1951; he developed the
citrate reduction method.
33
This synthesis of citrate stabilized
AuNPs was based on a single-phase reduction of gold tetra-
choloauric acid by sodium citrate in an aqueous medium and
produced particles about 20 nm in size.
33
Frens et al. later
refined this method in 1973 in an effort to produce AuNPs of a
prechosen size.
34
Frens proposed modifying the ratio between
the tetrachloroaurate and the trisodium citrate, a method that is
still widely employed. Pursuing this strategy of simply modifying
reaction conditions such as ratios,
34
solution pH
35
and solvent
36
has allowed for better control of the gold nanoparticle size,
37
however the distribution was still variable.
In 1981, the Schmid cluster [Au
55
(PPh
3
)
12
Cl
6
] was reported
to have narrow dispersity (1.4 "0.4 nm). Unfortunately the
synthesis utilized diborane gas to reduce [Au(Ph
3
P)Cl] and
proved to be a delicate synthesis that was difficult to isolate in
a pure state.
38
Subsequently it was discovered by Mulvaney
and Giersig in 1993 that AuNPs could be stabilized using
alkanethiols of various chain lengths.
39
The last major contri-
bution to the field for AuNP synthesis was published in 1994
and is known today as the Brust-Schiffrin method.
40
This
method utilizes a two-phase synthesis that exploits thiol ligands
that strongly bind to gold due to the soft character of both
S and Au. Initially, a gold salt is transferred into an organic
solvent (toluene) with the help of a phase transfer agent such as
tetraoctylammonium bromide, and then an organic thiol is
added. Lastly an excess of a strong reducing agent, such as
sodium borohydride is added which produces thiolate protected
AuNPs.
40
The major advantages of this method are the ease of
synthesis, thermally stable NPs, reduced dispersity and control
of size.
41
Although the Brust synthesis was a significant step
forward and has proven extremely influential in the last 17 years
since its publication, it still didn’t provide the monodispersed
product that was so greatly sought. Nonetheless, efforts have
been made to narrow the size dispersity through purification,
42–44
ripening,
45
etching
42
and annealing.
46
Through modifications to
the Brust synthesis, such as variation in the pH, reactant concen-
tration, reduction time and aqueous methanol concentration,
Fig. 4 Representative chemical reduction schematics for nanoparticle synthesis. (A) Basic reduction of metal salts. (B) Reduction using sodium
citrate that is also the capping agent. (C) Reduction and synthesis using non-polar ligands: in this example, a nonpolar thiol. (D) Chemical ripening
using particle seeding and nucleation.
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Chem. Soc. Rev. This journal is cThe Royal Society of Chemistry 2012
monodispersed particles were ascertained.
47–56
In the last few years,
many groups have focused on producing monodispersed nano-
particles by exploring possible nanoparticle formation mechanisms
in order to control the size distribution. Natan et al. was an
innovator for the investigation of seeded growth of gold nano-
particles using modifications on the Frens synthesis.
57
Bastus et al.
have successfully synthesized monodispersed citrate stabilized
particles through kinetically controlled seed growth (Fig. 5).
37
Factors to consider for narrow size distribution are seed concen-
tration, number of steps and secondary nucleation events.
58
2.1.2 Silver nanoparticles. Silver nanoparticles (AgNPs),
though not as widely studied as gold nanoparticles, have made
a remarkable impact in the world of nanoscience. Consider-
able effort is being directed toward developing new appli-
cations and protocols for this promising and interesting noble
metallic nanoparticle. The simplest method to obtain silver
nanoparticles is through a reduction of silver nitrate (AgNO
3
)
in ethanol in the presence of a surfactant.
59
AgNPs are most
commonly synthesized via chemical reduction, which is based
on a two-step process.
60
The most widely used stabilizing
agents for silver nanoparticles are polyvinyl alcohol, poly
(vinylpyrrolidone), bovine serum albumin (BSA), citrate and
cellulose. By the use of these stabilizers, unwanted aggregation
of the particles is avoided.
The two most traditional solution phase synthesis routes are
the Lee-Meisel and the Creighton methods.
61
The Lee-Meisel
method was first published in 1982. It employed both AgNO
3
and Ag
2
SO
4
as metal precursors and was further reduced with
NaBH
4
, sodium citrate, and H
2
at various temperatures.
62
Unfortunately these procedures produced a variety of shapes
and sizes. The polyol method is an alternation on the Lee-Meisel
method. This method reduces silver salts with a diol solvent near
reflux temperatures in the presence of a polymeric stabilizing
agent. Size and shape control has been achieved with this type of
synthesis.
63
The Creighton method is the most common, produ-
cing particles with a fairly narrow size distribution through the
reduction of AgNO
3
with NaBH
4
.
64
Building on these pioneering
methods, the Yang group used sodium citrate under a range of
pH values (5.7–11.1) to control both the size and morphology of
silver nanoparticles.
65
It was found that high pH created both
rod and spherical particles due to a fast reduction rate silver
nitrate (Fig. 6). Triangle and other polygon shapes were observed
with lower pH values (5.7–11.1) primarily due to slow nucleation
and growth.
65
Thus, the morphology of spherical AgNPs can be
controlled via atwo-stepprocess:fastnucleationathighpH
followed by slowing down the growth phase by reducing the pH.
In some instances, the reducing and stabilizing agent is one
and the same. For example, polysaccharides can serve as both
the reducing and capping agent. In this novel method, starch
and b-D-glucose were gently heated in an aqueous solution
containing silver. Due to the weak surface binding of starch to
silver, the reaction is reversible at high temps, allowing for
variation. Starch alone can also create stable AgNPs (10–34 nm)
via an aqueous solution of silver nitrate autoclaved at 15 psi/
121 1C/5 min. In yet another example of this method, a heated
solution of AgNO
3
and heparin was used to produce AgNPs.
Through variation in heparin concentration, the AgNP size and
shape can be manipulated. In this case, heparin acts as the
reducer, stabilizer, and nucleating agent.
Like gold, a one step process is highly desirable for the
synthesis of silver nanoparticles.
66,67
Procedures to produce
AgNPs of uniform size and morphology are employing a
modified Tollens synthesis reaction:
Ag(NH
3
)
2+
(aq) + RCHO (aq) -Ag (s) + RCOOH (aq)
In the presence of ammonia (the solvent), silver ions are
reduced by polysaccharides, which yields AgNPs ranging from
50–200 nm.
68
The size of these particles is controlled by the (1)
concentration of ammonia and (2) the nature of the stabilizer
(etiher SDS, PVP, or Tween 80).
66,67
The mixture of glucose
with 5 mM ammonia produced 54 nm AgNPs.
66
Modifying
the pH of the solvent as well as the structure of the reducing
agent can further decrease size polydispersity. For example,
Fig. 5 Monodisperse citrate-stabilized gold nanoparticles with a uniform quasi-spherical shape of up to 200 nm and a narrow size distribution
were synthesized following a kinetically controlled seeded growth strategy via the reduction of HAuCl
4
by sodium citrate. The inhibition of any
secondary nucleation during homogeneous growth was controlled by adjusting the reaction conditions: temperature, gold precursor to seed
particle concentration, and pH. This method presents improved results regarding the traditional Frens method in several aspects: (i) it produces
particles of higher monodispersity; (ii) it allows better control of the gold nanoparticle size and size distribution; and (iii) it leads to higher
concentrations. Gold nanoparticles synthesized following this method can be further functionalized with a wide variety of molecules, hence this
method appears to be a promising candidate for application in the fields of biomedicine, photonics, and electronics, among others. Reprinted with
permission from ref. 37. Copyright r2011 American Chemical Society.
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This journal is cThe Royal Society of Chemistry 2012 Chem. Soc. Rev.
the narrowest size distribution of AgNPs (25 nm) was formed
by using maltose as the reducing agent and a pH of 11.5.
69
2.1.3 Platinum nanoparticles. Platinum complexes, such as
cisplatin, have been used for several decades to treat a number
of maladies. However, the use of platinum nanoparticles
(PtNPs) as therapeutics is still in its nascent state.
70
The most
common method for the synthesis of platinum nanoparticles is
by chemical reduction of metal salts, chief among these agents
are ethylene glycol and sodium borohydride.
71,72
For example,
Guo et al. formed PtNPs using borohydride as the reducing
agent and citric acid as a stabilizer.
73
By varying the ratio of
citric acid to the metal salt, they were able to form PtNPs
ranging in size.
73
The size and shape of PtNPs can be
controlled by the precursor reduction conditions while
employing supercritical fluid reactive deposition.
74
Herricks
and co-workers describe a scheme to generate PtNPs with
various morphologies.
71
In this method, polyethylene glycol
serves as the reducer and solvent. Further variation of struc-
ture was obtained by changing the NaNO
3
/Pt ratio.
71
Addi-
tionally, platinum nanoparticles exhibit size and shape
dependent catalytic properties.
75
Other capping agents such
as poly(N-vinyl-2-pyrrolidone) have been used in conjunction
with NaBH
4
reduction of H
2
PtCl
6
6H
2
O (Fig. 7).
76,77
Finally,
the size of PtNPs can be fabricated using chemical ripening.
78
The initial step of this multistep, multi-seed process begins
with small individual platinum seeds (B5 nm) in an aqueous
solution containing sodium citrate and L-ascorbic acid. The
final diameter of the PtNPs relies on the concentration of
chloroplatinic acid and the initial seed size.
78
Fig. 7 (a) TEM image and (b) size distribution of TTAB-stabilized cubic particles (average size: 12.3 "1.4 nm, 79% cubes, 3% triangles, and
18% irregular shapes). (c) TEM image and (d) size distribution of TTAB-stabilized cuboctahedral particles (average size: 13.5 "1.5 nm, 90%
cuboctahedra and 10% irregular shapes). Reprinted with permission from ref. 77. Copyright r2007 American Chemical Society.
Fig. 6 Growth of silver nanoparticles by the citrate reduction of silver nitrate under the range of pH from 5.7 to 11.1 was investigated
systematically and quantitatively. Reduction of the silver precursor (Ag+) was promoted with increased pH, attributed to the higher activity of the
citrate reductant under high pH value. Under high pH, the product was composed of both spherical and rod-like silver nanoparticles as a result of
the fast reduction rate of the precursor. Under low pH, the product was mainly dominated by triangle or polygon silver nanoparticles due to the
slow reduction rate of the precursor. The product that is dominated by spherical silver nanoparticles cannot be acquired by the one-step citrate
reduction method in the range of pH investigated, indicating the poor balance between the nucleation and growth processes in the reactions. On the
basis of the results of quantitative analyses, a stepwise reduction method, in which the nucleation and growth processes were carried out at high
and low pH, respectively, was proposed for the syntheses of spherical silver nanoparticles. Reprinted with permission from ref. 65. Copyright r2009
American Chemical Society.
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2.2 Physical methods
Noble metal nanoparticles can be successfully synthesized
using other procedures such as UV irradiation and micro-
waves, which may or may not employ reducing agents. For
instance, well defined (shape and size distribution) AgNPs
were formed from a direct laser irradiation of an aqueous
solution of silver nitrate and SDS.
79
In this instance, the
surfactant (SDS) also acts as the stabilizer, which further tunes
the size and shape of the nanoparticle. Additionally, the above
technique was also utilized employing benzophenone.
80
By
varying the time and laser power, the size of the AgNPs could
be controlled; short irradiation at a low power produced
B20 nm particles whereas 5 nm particles were generated with
longer irradiation times and a higher ionizing power.
80
Particle
size can also be controlled with the duration of photolysis.
81
In
this instance, the substrate initiates the reduction of Ag
+
to
form silver seeds (Ag
0
) upon excitation at 600 nm. A further
ripening process using high intensity laser excitation con-
trolled the growth rate of the silver seeds. Uniform water-
soluble silver nanoparticles (B26 nm) can also be formed via
microwave irradiation.
82
This method uses basic amino acids
(0.16 mmol) to reduce silver salts (20 mmol) in an aqueous
starch solution (0.4 mmol), which is heated for 10 s at 150 1C.
The rapid nucleation process, due to microwave irradiation, is
critical to the uniform size distribution of the nanoparticle.
Work done by Suzuki proposed a new method to fabricate
monodisperse spherical AgNPs ranging from 10 to 80 nm in
an aqueous solution.
83
This simple yet elegant method uses a
combination of seeding and laser treatments (Fig. 8). The
authors used ‘‘soft’’ irradiation: particles were heated and
melted by a single laser pulse. The final diameter (d
p
) of the
AgNPs can be calculated based on the assumption that the
reduction of Ag
+
is only at the surface of the seed particle
using the following equation: d
p
/d
p0
=(1+n
+
/n
s
)
1/3
.
Through changing the ratio of n
+
(silver ion) and n
s
(seed
particle), the average size of the particle can be controlled
through increasing the n
+
/n
s
ratio.
Photochemical reduction of gold salts has also been used to
form AuNPs.
84
This nanoformulation method employs a
continuous wave UV irradiation (250–400 nm), PVP as the
capping agent and ethylene glycol as the reducing agent. The
rate of formation of AuNPs with this method is dependent on
the glycol concentration as well as the viscosity of the solvent
mixture. This method was further improved upon by the
addition of Ag
+
to the solution, leading to an increase in
the production of Au
0
and the ensuing nanoparticles.
85
Fig. 8 TEM photographs for four colloids prepared in one-step syntheses with different n+/ns ratios: (a) n+/ns = 3.6; (b n+/ns = 18.9; (c) n+/
ns = 32.4; (d) n+/ns = 45. All photographs have the same magnification. The inserts are the histograms of particle size distribution calculated
only for spherical particles. Reprinted with permission from ref. 83. Copyright r2007 American Chemical Society.
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By using photochemically prepared seed particles, the size of
AuNPs can be further tailored.
86
In this preparation, spherical
particles (5 to 20 nm) were prepared with UV irradiation using
various ratios of surfactant (TX-100) to gold ion concen-
tration.
86
Larger particles were further formed by reducing
fresh Au(III) ions onto the surface of the seeds particles in
ascorbic acid. Through varying the [Au(III)]/[seed] ratio, the
surface size of the particle can be controlled.
Others have applied laser-based approaches to generate
spherical gold nanoparticles in the absence of a reducing
agent. In one method, thermal-free femtosecond laser radiation
was used to ablate gold in deionized water. Briefly, low laser
fluences (Fo400 J cm
!2
) produced 3–10 nm relatively
monodispersed gold particles.
87
Another group used laser
irradiation to elucidate the morphological changes induced
by ablation, comparing size dispersion between the second
(532 nm) or fourth harmonic (266 nm) of a pulsed laser.
88
It
was established that the mean size of the AuNPs could be
tuned by using the proper combination of laser ablation, laser
fluence and post-irradiation wavelength. Positively charged
nanoparticles can also be fabricated using pulsed laser light
without the incorporation of ligands or reducing agents.
89
Gold foil was pulsed with a femtosecond laser (delivering
120 fs laser pulses) at 800 nm in aqueous solution for a period
of 12 min. This method produces a surprisingly stable gold
nanoparticle in a variety of media primarily due to the partial
oxidation of the gold surface.
89
Irradiation and laser ablation techniques have also been
effectively used to create PtNPs. For instance, radiolytic
reduction of platinum complexes such as (Pt(NH
3
)
4
Cl
2
H
2
O)
can be stabilized with polyacrylic acid.
90
In another method,
irradiation was combined with ultrasonication to prepare
PtNPs.
91
In this process, H
2
PtCl
6
#6H
2
O was added to a
solution containing 10 mM polypyrole and SDS. By varying
the length and time of irradiation and ultrasonication, the
particle size is controlled.
91
Ablation of platinum targets in
aqueous solution using a nanosecond laser has also generated
PtNPs. This method, described by Cueto, used several laser
wavelengths and stabilizing agents to create a range of sizes
and shapes of PtNPs.
92
2.3 Biological methods
Numerous reducing agents have been studied, such as
hydroxylamine,
57,93
ascorbic acid
94,95
and biogenic methods
which utilizes an iodide-mediated reduction.
96
In addition to
aforementioned synthesis, some efforts have been put forth to
elucidate biological methods to produce nanoparticles. Plant
mediated particle synthesis has gained momentum due to
simplicity and eco-friendliness (See Table 1).
97
Biosynthesis
with plant extracts as well as Iodide mediated reductions of
AuCl
4
!
have been reported.
96,98
Blood compatibility also
makes green syntheses attractive.
98
For example, it has been
shown that Zingiber officinale extract can produce particles
ranging from 5–15 nm in diameter. The extract acts as a
reducing agent as well as a stabilizer and the biological benefits
are proven through physiological stability.
98
The use of micro-
organisms for synthesis has also emerged as an alternative to
chemical fabrication, yielding a strong area for investigation
into green syntheses. Photosynthetic bacteria,
99
prokaryotic
bacteria,
100–102
eukaryotic fungus
103,104
and plant extract
105–108
have all been employed for the reduction of aqueous metal ions
to produce metallic nanoparticles. Many biological methods
have a slow reaction rate and a wide distribution in particle
size.
109
However, a recent publication by Darroudi investi-
gated the role of sodium hydroxide as an accelerator to
generate AgNPs.
110
Briefly, silver nitrate, sodium hydroxide
and gelatin were mixed and then heated to 60 1C. After adding
glucose, the reaction was allowed to stir for an additional
15 min. It should be noted that the size of the formed AgNPs
was dependent on the volume of NaOH used. This method
yielded fairly monodispersed silver nanoparticles under 20 nm
in size.
In summary, optimizing nanoparticle synthesis is a prolific
area of research. Controlling size, shape, and distribution is an
elegant and arduous process. These reactions are ruled by
many variables such as reactant concentration, solubility,
reaction rate, reduction potential, temperature and time. All
of the parameters are intrinsically intertwined. Further investi-
gation is certain to be an ongoing area for increased tunability.
3. Therapeutic applications of noble metal
nanoparticles
Nanoparticle (NP) biotechnology is a burgeoning field with
immense potential for clinical and real world applications. To
realize this potential, especially in therapy it is necessary to
design and engineer NPs that can be targeted to tissues of
interest, as well as to produce specific, desired effect (with
minimal toxicity and environmental impact). Of particular
interest are NPs with a metallic core due to their purported
favorable safety profile in humans (indeed, colloidal metal has
seen medicinal use since ancient times), which has already
resulted in preclinical testing for imaging, diagnostic and
therapy. Defining the size of a nanoparticle is somewhat
obscure and debatable, with the colloquial designation being
less than 1 mm in diameter. Accordingly, the size of the
nanoparticles used in the field of bionanotechnology range
from 2 nm to 500 nm. Given that systems at these molecular
and atomic scales display essentially new properties due to
their small structure, innovative molecular design can be
precisely created along with a high degree of versatility. This
tailoring is largely due to ‘‘self-assembly’’ of the nanoscale
materials via charge compatibility and non-covalent
interactions.
As mentioned previously, nanoparticles, have proven to be
the most versatile and widely used constituents with broad
applications such as delivery vectors,
111
imaging agents,
112
synthetic inhibitors,
113
and sensors.
114
Thus these engineered
nanomaterials serve as unique multi-dimensional scaffolds that
vary from their bulk counterpart.
115
Inorganic nanomaterials
in particular are very attractive for various biomedical appli-
cations due their size and shape dependent optoelectronic
properties.
116,117
The use of nanoparticles in biology, takes
advantage of both the dimension and function of the inorganic
core, which in turn dictate certain physical properties.
116,118
For instance, the superparamagnetism of iron oxide and iron-
platinum nanoparticles and the size-dependent fluorescence of
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semi conductor nanoparticles are intrinsic to these particular
nanomaterials.
116–118
Furthermore, the size of these nano-
materials provides a large surface area to volume ratio; as
the particle size decreases, the amount (or number) of surface
atoms rapidly increase.
116,118
Through exploiting these extra-
ordinary properties, nanoparticle therapeutics can offer an
alternative platform for a wide variety of human maladies in
clinical settings.
116,119,120
3.1 Cellular uptake of noble metal nanoparticles
The cellular internalization of inorganic nanoparticles is an
area of intense research. Even though gold, silver and platinum
nanoparticles are noble metals, their mechanism of intra-
cellular uptake is not necessarily similar or well understood.
For instance there is contrasting evidence on the uptake
mechanisms of AuNPs. Geiser et al. used red blood cells to
elucidate intracellular uptake of gold nanoparticles.
121
Their
results support a diffusive mechanism of entry since AuNPs
were found in the cytosol free from membrane encapsulation
(i.e. endosomes). In comparison, it has been indicated that
cellular uptake of AuNPs is due to macropinocytosis
122
which
was later confirmed by further research in other groups.
123
Macrophages also easily internalized AgNPs, which were
found to localize to vacuoles within the cells, however the
authors were unable to discern the mechanism of internali-
zation.
124
In an analogous study by Yen et al., they showed
that AuNPs and AgNPs were confined in cytoplasmic vesicles
of the macrophages.
125
However the authors further specu-
lated that the protein corona formation influenced cellular
internalization of AuNPs, compared to AgNPs, thus compli-
cating the uptake process.
125,126
Original work from Taylor
and coworkers analyzed the cellular uptake of gold nano-
particles generated via laser ablation.
89
In this study, the
authors cultured bovine cells (GM7373) with the AuNPs
(15 nm, 50 mM) in a time course study. With the aide of
confocal microscopy, they were able to determine that the
AuNPs were passively taken up via diffusion through the
cellular membrane.
89
This is in contrast to gold nanoparticles
created through chemical means, which seem to prefer endo-
somal transport.
123,127,128
It may also be possible to tailor the
endocytotic uptake of nanoparticles. In a very recent study,
Bhattacharyya et al. shows that the endocytotic pathway can
be ‘‘switched’’ from a caveolar mechanism to pinocytosis.
129
The size and shape of nanoparticles also play a large role in
relation to cellular uptake in vitro. Spherical gold nano-
particles have higher cellular uptake than gold nanorods
owning to variable biophysical properties such receptor
diffusion kinetics.
130
The extent of nanoparticles exocytosis
is a function of nanoparticle surface size.
130
For instance,
Table 1 Examples of Plants used to synthesis metal nanoparticles. Adapted from ref. 97
Plant Origin Metal Size (nm)
Acalypha indica Silver 20–30
Apiin extracted from henna leaves Silver & Gold 39; 7.5–65 (respectively)
Avena sativa (oat) Gold 5–20 (pH 3–4) & 25–85 (pH 2)
Brassica juncea (mustard) Silver 2–35
Camellia sinensis (green tea) Gold 40
Carica papaya Silver 60–80
Citrus limon (lemon) Silver o50
Cochlospermum gossypium Silver 3
Coriandrum sativum Gold 6.75–57.91
Cymbopogon flexuosus (lemongrass) Gold 200–500
Cycas sp. (cycas) Silver 2–6
Datura metel Silver 16–40
Desmodium triflorum Silver 5–20
Eclipta sp. Silver 2–6
Enhydra fluctuans Silver 100–400
Eucalyptus camaldulensis (river red gum) Gold 1.25–17.5
Eucalyptus citriodora (neelagiri) Silver B20
Eucalyptus hybrida (safeda) Silver 50–150
Euphorbia hirta Silver 40–50
Ficus bengalensis (marri) Silver B20
Garcinia mangostana (mangosteen) Silver 35
Gliricidia sepium Silver 10–50
Honey Silver 4
Ipomoea aquatic Silver 100–400
Jatropha curcas (seed extract) Silver 15–50
Ludwigia adscendeous (ludwigia) Silver 100–400
Mentha piperita (peppermint) Silver & Gold 5–30, 90; 150
Moringa oleifera Silver 57
Murraya koenigii Silver & Gold 10; 20
Nelumbo nucifera (lotus) Silver 25–80
Ocimum sanctum (tulsi; root extract) Silver 10; 5
Ocimum sanctum (tulsi; leaf extract) Silver 10–20
Psidium guajava (guava) Gold 25–30
Scutellaria barbata D. Don (Barbated skullcup) Gold 5–30
Sesbania drummondii (leguminous) Gold 6–20
Syzygium aromaticum (clove) Gold 5–100
Syzygium cumini (jambul) Silver 29–92
Terminalia catappa (almond) Gold 10–35
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14 nm particles were rapidly cleared from cells twice as fast as
100 nm particles. Finally, cellular uptake of metallic nano-
particles has also been reported to be size specific, with
40–50 nm having the greatest effect on internalization into
cells.
131–133
Chan et al. has reported that 50 nm gold particles
can enter into cell at a faster rate with higher amount relative
to the other sizes.
130
In addition they also show that the
morphology of the particle also dictates the rate of cellular
uptake.
130
The size of monodispersed nanoparticles can also
be influenced by its surroundings, i.e. biological media. In a
new study by Albanse and Chan, they describe the interaction
of aggregated nanoparticles with three different cell lines
(HeLa, A549, and MDA-MB 435) in vitro.
134
The aggregation
of nanoparticles is primarily due to the amount of ions found
in biological medium.
135,136
Upon exposure, the electrostatic
nature of the nanoparticles is weakened and van der Waals
forces take over.
135
Furthermore, a more thermodynamically
favorable serum protein may displace the nanoparticle-
capping agent. It is primarily these destabilizing processes that
create nanoparticle aggregates in biological fluids (saliva, cell
culture medium, lung surfactants). This inevitable nanoparticle
aggregation creates a multitude of cellular responses.
134
The
results of this study showed the uptake into HeLa and A549
cells was decreased by 25% with aggregated nanoparticles
verses monodispersed particles. In contrast, the uptake of
aggregates into MDA-MB 435 cells demonstrated a two-fold
increase comparatively. Kudgus and coworkers also showed
size dependent nanoparticle uptake in vitro and in vivo in an
orthotopic model of pancreatic cancer. Pancreatic cell lines
(AsPC-1, PANC-1, and MiaPaca-2) were co-incubated with
gold nanoparticles with varying hydrodynamic radii (7 nm to
134 nm). Upon gold analysis, nanoparticles with a B20 nm
hydrodynamic radii exhibited the greatest uptake. Interestingly
enough, the outcome of their in vivo experiments mirrored
their in vitro results. Most importantly, Kudgus’ study eluci-
dated the design parameters for nanoparticle therapeutics.
32
Surface charge is also an important factor that moderates
cellular uptake of nanoparticles.
137,138
The functionality of the
nanoparticle surface further allows specific or nonspecific
interactions within the cellular lipid bilayer.
32
Since the cellu-
lar exterior is largely anionic, positively charged nanoparticles
can easily transverse the cellular membrane via electro-
statics.
137
Nonetheless, negatively charged nanoparticles have
also been observed in the cytosol.
138
This is most likely due to
the nanoparticles passively targeting lipophilic domains. In a
previous report, it is described that the structure and order of
capping agents on nanoparticles mitigates cellular uptake.
139
Cationic nanoparticles have also been shown to modulate
membrane potential of cells and their subsequent downstream
intracellular events. In the findings published by Arvizo et al.,
ovarian cancer and airway cells co-incubated with positive
nanoparticles depolarized the cell membrane and triggered the
release of intracellular calcium.
140
The effect was shown to
be dependent on the cell type. For instance, inhibition of
proliferation was observed in airway cells but the malignant cells
remained unchanged. Furthermore, there are other reports where
proteins in the serum help facilitate nanoparticle uptake into
cells.
141
In addition, the Rotello group has also reported that
zwitterionic nanoparticles (effective overall neutral surface
charge) can be highly efficient delivery system.
142,143
There have been a few publications that have investigated
the intracellular compartmentalization of nanoparticles. Silver
nanoparticles have been found in the cytoplasm and mito-
chondria of primary liver cells as well as the mitochondria and
nucleus of fibroblasts.
144,145
In keratinocytes, silver was found
to be localized to lysosomes, while lung cancer cells exposed to
AgNPs with different sugar coatings were found in the cyto-
plasm.
146
It was further noted that the rate of uptake was
dictated by the surface coating, with lactose having the
greatest effect on the rate of internalization in fibroblasts.
147
In addition, Lesniak et al. found silver within endocytic
vesicles.
148
AgNPs have also been purported to use macro-
pinocytosis and clathrin mediated uptake in NIH3T3 cells,
appearing in the cytoplasm as well as the nucleus.
145,149
It was
also further implied that AgNPs are directly toxic to the cancer
cells through DNA damage and increased production of
reactive oxygen species.
149
Breast cancer cells (MCF-7) treated
with colloidal silver lead to dose dependent apoptosis (LD
50
of
3.5 ng ml
!1
) and a significant increase in SOD activity but did
not affect the viability of normal PBMC cells.
150
Further
analysis of the intracellular uptake of AgNPs done by Greulich
and co-workers, using scanning electron microscopy, detected
nanoparticle aggregates in human mesenchymal stem cells.
151
Upon staining of the mesenchymal cellular structures, the AgNPs
agglomerates were located in endolysomal structures, but not in
the cellular nucleus or other cellular organelles.
151
Platinum
nanoparticles were also detected in intracellular vesicles in the
cytoplasm of HT29 cells using bright field electron microscopy.
74
Although there is evidence that Pt-NPs affects the integrity
DNA, the intracellular redox status of HT29 cells was not
altered.
133
3.2 Nanoparticles as anti-infective agents
The function of silver nanoparticles as antibacterial agents has
been well established and will not be covered further in this
review. However, little has been written on the role of nano-
particles as anti-virals. In one study, it is indicated that the
anti-viral properties of AgNPs biogenically formed are more
effective than chemically synthesized silver nanoparticles.
152
Likewise, Vero cells co-incubated with AgNPs were reported
to prevent plaque formation after being infected with the
Monkeypox virus.
153
Metallic nanoparticles have also been
described as a possible HIV preventative therapeutic.
154,155
In
a couple of studies, it is demonstrated that AgNPs prevented
the virus from binding to the host cells in vitro.
156,157
It was
further shown that silver acts directly on the virus as a
virucidal agent by binding to the glycoprotien gp120.
89
This
binding in turn prevents the CD4-dependent virion binding
which effectively decreases HIV-1’s infectivity.
158
Metallic
nanoparticles have also been effective antiviral agents against
herpes simplex virus,
159
influenza,
160
and respiratory syncytial
161
viruses.
3.3 Anti-angiogenic properties of metallic nanoparticles
It is well recognized that angiogenesis plays a central role in a
number of diseases such as cancer, rheumatoid arthritis, and
macular degeneration.
162–164
In normal conditions, angio-
genesis is tightly regulated between various anti-angiogenic
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(i.e. TSP-1, platelet factor 4) and pro-angiogenic growth
factors (i.e. VEGF, PDGF, TGF-b).
165
However, when the
balance is disrupted under pathological conditions, the angio-
genic switch is turned on.
165
This event induces highly abnormal
blood vessels which, become hyperpermeable to plasma proteins.
Some anti-angiogenic agents are being presently used in the
clinics, but a majority of them have been designed to only inhibit
VEGF
165
mediated signaling.
166
In addition, other reports
have indicated unexpected and serious toxicities of these conven-
tional agents including hypertension, thrombosis, and fatal
hemorrhage.
162,163
Furthermore, relevant clinical data indicates
that targeting a single pathway is not the most efficient or
effective mode of treatment.
167
3.3.1 Applications in tumor therapy. Owning to the above
concerns, noble metal nanoparticles might prove to be more
effective since they have been shown to target multiple path-
ways.
168
More importantly, the unusual toxicities associated
with conventional anti-angiogenic agents (as mentioned prior)
may be overcome if these nanoparticles alone can be efficacious
as an anti-angiogenic agent. In a landmark study, it was shown
that ‘‘naked’’ gold nanoparticles inhibited the activity of heparin-
binding proteins, such as VEGF
165
and bFGF in vitro and
VEGF induced angiogenesis in vivo.
169
However, non-heparin
binding proteins, (VEGF
121
and EGF) retained their intrinsic
activity. Further work in this area elucidated that heparin-
binding proteins are absorbed onto the surface of AuNPs
170
and were subsequently denatured.
171
The researchers also
showed that surface size, not surface charge, plays a large role
in the therapeutic effect of AuNPs.
171
In this study, Arvizo and
coworkers preincubated VEGF 165 with citrate reduced AuNPs
(d=5,10,and20nm)todeterminetheireffectondownstream
signaling in HUVEC cells (Fig. 9). The data demonstrated that
20 nm citrate reduced AuNPs had a dramatic effect on VEGF
signaling events such as receptor-2 phosphorylation, intracellular
calcium release, and proliferation comparatively. Mukherjee and
colleagues also tested the effect of gold nanoparticles on VEGF
mediated angiogenesis using a ‘‘mouse ear model’’ injected with
an adrenoviral vector of VEGF (Ad-VEGF—mimics the resulting
angiogenic response found in tumors).
169
Aweekafterthe
Ad-VEGF administration, mice treated with AuNPs developed
lesser edema than the sham treated mice.
Silver has also been shown to exhibit anti-angiogenic effects.
In a report by Eom and collegues, 40 nm silver nanoparticles
(AgNPs) were used to study their antiangiogenic properties in
bovine retinal epithelial cells (BREC) in vitro and a matrigel
plug assay in vivo.
172
The outcome of their experiment showed
that AgNPs inhibited cell proliferation and migration in
VEGF induced angiogensis in BRECs. Thus it is implied that
the PI3K/Akt signaling pathway is in some way targeted and
activated by AgNPs.
172
They went on to reveal the formation
of new blood vessels was suppressed by AgNPs in vivo. Further
work done by this group also described the anti-tumor effects
of 50 nm AgNPs in vitro and in vivo.
173
Dalton’s lymphoma
ascites (DLA) cell lines co-incubated with AgNPs displayed a
dose dependent toxicity through activation of caspase-3 and
inhibition of cellular proliferation. Furthermore, tumor bearing
mice injected with AgNPs demonstrated a reduction of ascites
production (65%) and tumor progression compared to the sham
treated mice.
173
Fig. 9 Binding of heparin-binding growth factors (HB-GFs) to gold nanoparticles leads to the inhibition of their function due to change in the
protein structure (A,B) Far UV-CD spectra was measured from 180 to 250 nm in a 1 cm cuvette. (A) 0.2 mg mL
!1
bFGF were incubated with and
without GNPs in 5 mM phosphate buffer. (B) 0.15 mg mL
!1
EGF were incubated with and without GNPs under similar conditions as listed above.
The blanks containing GNPs with same concentration in buffer were subtracted from each data set. (C) Cartoon representation of protein
denaturation on the surface of AuNPs. Modified with permission from ref. 171. Copyright r2011 Elsevier.
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3.3.2 Applications in multiple myeloma. The pathogenesis
and progression of multiple myeloma (MM) can also be
attributed to abnormal angiogenesis.
174,175
Even with intense
study, this malignancy of plasma cells remains fatal. Current
research proposes that the rise of angiogenic activity of the
myeloma cells is due to the increased expression of cytokines
including bFGF, VEGF, hepatocytes growth factor (HGF),
insulin like growth factor (IGF-1), and TGF-a
176
(treatment
strategies used in the clinics include a combination of chemo-
therapy either alone or with stem cell transplantation, gluco-
corticosteroids, thalimide, and proteome inhibitors (such as
Bortezomib).
175
Unfortunately, these treatments are not
restorative and the majority of patients go into relapse. As
mentioned above, work done in the Mukherjee group demon-
strated that gold nanoparticles inhibited the inherent function
of heparin binding growth factors.
169–171
These studies led to
the hypothesis that gold nanoparticles could also inhibit the
VEGF and bFGF dependent proliferation of MM cells. In all
three cell lines tested (OPM-1, RPMI-8266, and U-266), a dose
dependent inhibition of proliferation was observed in AuNP
treated samples (with no inhibition of normal cells at sub toxic
levels of AuNPs).
177
Further cell cycle analyses revealed an
arrest in the G1 phase of the cell cycle, with an up-regulation
of p21 and p27. This study is an important first step in show
casing the potential of AuNPs as a therapeutic moiety in the
treatment of multiple myeloma.
3.3.3 Applications in leukemia. B-chronic lymphocytic
leukemia (B-CLL) is the most widespread form of leukemia.
Primarily found in males, this disease affects B-lymphocytes
and causes infiltration of malignant cells into organs as well as
immune suppression. Not surprisingly, abnormal angiogenesis
was detected in the marrow of B-CLL patients with a
significant increase in the marrow vasculature.
178
Supporting
these observations, it was found that patients with this disease
also had a substantial amount of bFGF and VEGF in their
urine.
178
The biological component of this disease was further
confirmed with clonal cell studies of B cells from patients;
higher levels of bFGF resisted the apoptotic effects of the drug
fludarabine.
178
The anti-angiogenic properties of AuNPs stated
earlier, led to the possibility that the status of B-CLL cells could
also be modulated by AuNPs.
178
Indeed, B-CLL cells exposed to
gold nanoparticles exhibited an increase in apoptosis in a dose
dependent manner. The mechanism of apoptosis enhanced by
gold nanoparticles in B-CLL cells was further elucidated by a
clearly detectable PARP cleavageandadecreaseinanti-apoptotic
regulatory proteins such as caspase-3, Bcl-2 and Mcl-1.
178
3.3.4 Applications in rheumatoid arthritis. Angiogenesis
also plays a large role in the promotion and maintenance of
inflammatory diseases such as rheumatoid arthritis (RA).
Historically gold salts have been used to treat a multitude of
inflammatory diseases (see Fig. 3).
179
Clinicians first started
using gold complexes the early 1900s to help treat rheumatoid
arthritis.
180
However these gold(I) thiolates needed to be
injected and the response to treatment took several weeks to
months with patients incurring several adverse side effects.
181
In 1985, the oral drug Aurofin (Ridaurot) was introduced as a
safe and effective treatment for RA.
179,180
However, it was
later shown to be less effective than the original thiolates.
8
In a
current study, 13 nm gold nanoparticles were used to study
rats with collagen-induced arthritis.
182
Initial studies showed
that the nanoparticles bound to VEGF in the synovial fluid of
patients with RA affecting cellular proliferation and
migration. Subsequent histology from animal models showed
that TNF-aand IL-bwas considerably reduced after intra-
articularly administration of the nanoparticles. Moreover,
AuNP treatment resulted in further attenuation of arthritic
symptoms such as inflammation and reduced macrophage
infiltration. In a related study, gold beads were implanted
near the hip joints of dogs with hip dysplasia in a double blind
clinical trial.
183
After a 24-month period, 83% of the dogs in
the treatment group showed continuous pain relief from the
implantation compared to the placebo group.
183
3.4 Applications for anti-tumorigenesis
3.4.1 Hyperthermia/photothermal therapy. A combination
of surgery, chemotherapy, and radiation therapy constitutes
the conventional treatment regime for most cancers. Although
successful in many instances, these treatments are responsible
for significant damage to healthy tissue, with concomitant
health-related issues.
168
These issues arise in part from the
‘‘whole-body’’ approach of these therapies. To minimize the
damage of non-cancerous tissue, treatments could be applied
directly to the tumor, leaving neighboring tissue unaffected.
Two types of targeting can be used to enhance the efficiency of
tumor therapy. First, the tumor can be targeted spatially, with
the toxic effect of the therapeutic agent localized to the tumor
site. The second type of targeting is on the cellular level. By
targeting the treatment directly to tumor cells, other cells in
the vicinity of the tumor will not need to be sacrificed.
184
In
this regard, metallic nanoparticles have potential for non-
invasive tumor treatment.
185
Application of a magnetic field
will selectively heat the nanoparticles rapidly and efficiently,
allowing for selective destruction of tumor cells.
185
Current areas of research being actively pursued to localize
cancer treatment to affected regions of the body include photo-
dynamic therapy (PDT) and regional hyperthermia.
184,186–189
Targeting is primarily achieved by focusing the light source on
a region of the body. The wavelength of light that is readily
absorbed by tissue is 630–900 nm, otherwise known as the near
infrared region (NIR).
186
This spectral region minimizes the
light extinction by intrinsic chromophores in the healthy
tissue.
190
Another method of restricting healthy tissue damage
in tumor therapy is through regional hyperthermia.
187–189
In
general, hyperthermia is characterized by the damage of cells
from exposure to elevated temperatures.
189
Loss of membrane
integrity, DNA damage and biochemical pathway inhibition
have been implicated as causes of cellular death under these
conditions.
191
Amoderateriseinhomeostatictemperaturehas
been shown to induce apoptosis within a few hours. As tempera-
tures rise above 46 1C, necrosis is observed.
192
Although potentially
useful, this technology is limited by the difficulties of achieving a
localized, uniform heating of tissue (Fig. 10).
Recent innovations in nanotechnology have demonstrated
that metallic nanoparticles hold great promise as PDT and
hyperthermic agents. Research has shown the application of
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magnetic fields on metallic nanoparticles results in rapid
heating.
193
In this heating process, electrical currents
are produced in the gold particle by the oscillating magnetic
field, analogous to the current provided by an electrical
generator.
193,194
The resulting eddy currents create this rapid
heating which quickly dissipates from the nanoparticle into the
surrounding environment, incurring thermal ablation.
195
Pioneering work by Pitsillides illustrated that the surface
plasmon resonance (SPR) of nanoparticles is easily exploited
for PDT anti-cancer therapy.
196
Further work done in the
El-Sayed group demonstrated that AuNPs are effective PDT
agents and could ‘‘seek and destroy’’ cancerous cells.
187
Afour-
minute exposure at 25 W cm
!2
was lethal to malignant cells, a
two-fold decrease in comparison to normal cells (57 W cm
!2
). In
afairlyrecentreport,15nmcitratecappedAuNPswereusedto
treat A431 cells using photothermal therapy (PTT). In this study,
AuNPs were exposed to low laser light at different time intervals
and the morphology of the A431 cells was assessed along with
germane biological markers. After irradiation, it was shown that
AuNPs induced the eradication of the malignant cells through
ROS mediated apoptosis.
197
It is should also be noted that the
shape of the particle is also important in PDT therapy. In a
report by the Kanaras group, gold nanoparticles with different
morphologies were incubated with HUVEC cells and their
uptake after laser irradiation was investigated.
198
It was impli-
cated from their results that gold nanorods were taken up 100
times more that the other particles studied. However, they noted
that each particle was uniformly efficient in promoting cell death
when laser hyperthermia is employed.
Likewise, mice injected with goldnanoparticleshadasignificant
reduction of deep tissue tumors after a brief exposure to NIR.
199
Local laser induced hyperthermia has also been employed against
skin cancer.
200
In this study, AuNPs were injected into the tail vein
of mice for 5 days. Within 4–5 h, the nanoparticles accumulated
into skin tumors and showed complete inhibition.
200
Amore
efficient method for penetrating for deeper solid tumors is to use
radio waves, or radio frequency ablation (RFA).
201
At B14 MHz,
AuNPs were describe to thermally ablate cancer cells and
tumor tissue in vitro and in vivo.
202
Liver cancer cells (HepG2)
co-incubated with citrate reduced AuNPs (13 nm) demonstrated a
time-dependent cytotoxic effect upon exposure to the RF field.
The authors were able to further correlate their in vitro results
in vivo using a rat hepatoma model. Following RF exposure, rats
that were injected with nanoparticles revealed evidence of thermal
injury to the diseased tissue.
202
The frequency of the surface plasmon band (SPR) can be
tuned by changing the shape of nanoparticles to a rod.
203
One
of the advantages of gold nanorods is the duality of the
observed plasmon band that is tunable through its aspect
ratio. Second, the nanorods can be tailored further owing to
the distinctive surface chemistries along their crystal faces.
203
Moreover, the shift of the SPR allows for near-infrared (NIR)
absorption at the cross-sections, permitting a deeper penetration
into living tissues.
199
Utilizing these properties, von Maltzahn
et al. have developed nanorods that target and reduce tumors.
204
In this study, PEGcoated nanorods were injected into the tail
veins of tumor bearing mice. The treated mice were then exposed
to NIR and after 15 days showed a significant reduction in tumor
size. Expanding on their work, the authors tagged the nanorods
with SERS reporters and demonstrated their effectiveness at
imaging and ablating tumors in vivo.
199
3.4.2 Application in radiotherapy. Another common
treatment for patients with cancer is the use of ionizing radiation.
Although this method is effective for controlling the proliferation
rate of cancer cells, it can be invasive, side effects are numerous
and healthy tissue is often damaged. Metallic nanoparticles may
offer an advantage in this area by exploiting their excellent
optical properties, surface resonance, and wavelength tunability.
For example, upon X-ray irradiation, gold nanoparticles can
induce cellular apoptosis through the generation of radicals.
205
This treatment strategy has increased the killing of cancer cells
without harming the surrounding healthy tissue.
206,207
X-ray
irradiation of mice injected with AuNPs at 250 kV caused a
four fold decrease in tumor size and enhanced survival of the
animals.
208
Likewise, the intrinsic radioactive properties of
Au-198 (b
max
=0.96MeV;t/2 = 2.7 d) and Au-199 (b
max
=
0.46 MeV; t/2 = 3.14 d) nanoparticles makes them ideal
candidates for radiotherapy.
209
Furthermore, these particles have
revealing gamma emissions for dosimetry and pharmacokinetic
investigations.
209
Gannon et al. reported the destruction of
human cancer cells (Hep3B and Panc-1) via radiofrequency thermal
heating of non-targeted AuNPs (d=5nm).
210
Treatment with
67 mML
!1
of AuNPs and subsequent exposure to 13.56 MHz RF
field resulted in a 496% lethal injury to the cancerous cells.
Numerous studies have elucidated to the effects nano-
particles elicit upon cellular uptake under standard cell culture
conditions. For translational purposes of nanoparticles into
clinical trials, systematic studies are needed to assess the
nanoparticle-cell interaction after ionizing radiation (IR).
Fig. 10 Use of noble metal nanoparticles for therapy. Tumors can be
targeted with nanoparticles to induce hyperthermia and enhance
radiotherapy. However, non-specific targeting of the nanoparticles
may have severe toxic effects on healthy cells.
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In one report, researchers analyzed the uptake of nano-
particles (B5 nm) in mice colorectal adenocarcinoma tumor
cells (CT26).
211
Using transmission electron microscopy in
conjunction with confocal microscopy, it was revealed that
the absorption of the nanoparticles enhanced radiation
induced cellular damage.
211
These findings were later substan-
tiated by another group studying breast cancer.
207
Even
thought the intracellular uptake of nanoparticles was similar
for both the cancer cells (MCF-7) and normal cells (MCF-
10A), after irradiation cellular viability was significantly
reduced (40%).
207
These results confirmed 1) that irradiation
enhances the killing effect of nanoparticles and 2) low doses of
radiation can be used effectively thus decreasing localized
damage to the normal surrounding tissue.
207,211
In a more
recent study, biocompatible gold nanoparticles were used to
systematically study the survival rates of EMT-6 and CT26
cells after irradiation of 10 Gy from various sources: X-ray
emitter (from 6.5 keV to 6 MeV), synchrotrons, a laboratory
generator, an animal irradiator, an oncology linear accelerator
as well as a proton emitter widely used for therapy.
212
After
irradiation, the percentage of cell survival decreased in a dose
dependent manner, however these results were not significant.
212
The relative biological efficacy of proton beam therapy in
prostate cancer was increased by 20% with the internalized gold
nanoparticles.
213
Work done by Xu and coworkers demonstrated
dose and size dependent cytotoxicity of glioma cells when treated
with silver and gold nanoparticles, with 20 and 50 nm nano-
particles being the most effective at low radiation doses.
214
They
further theorized that the increased sensitivity to irradiation is
due to the release of Ag
+
from the nanoparticle. With its ability
to capture electrons, Ag
+
functions as an oxidizer thus increasing
production of intracellular reactive oxygen species.
214
Additional
work in this area has shown that the surface size of AgNPs
enhances the thermal sensitivity of glioma cells.
215
The size and
the amount of particle uptake into cells also affect its radio-
sensitization. This was elegantly demonstrated in a recent report
using nanoparticles ranging in size from 14–74 nm.
216
As antici-
pated, 50 nm AuNPs displayed the utmost enhancement factor
(REF) compared to 14 and 74 nm particles (1.43 vs. 1.20 and
1.25 @ 220 kVp respectively).
A novel experiment performed by Porcel et al. indicates that
platinum nanoparticles may have potential as an alternative
therapeutic for the treatment of cancer.
90
The combination of
Pt-NPs with hadron therapy resulted in enhanced DNA strand
breakage. The fast carbon ion irradiation of platinum led to
the production of OH
!
radicals thus amplifying the lethal
damage to DNA.
217
Human colon carcinoma cells (HT29)
showed a does and time dependent response when exposed to
platinum nanoparticles (Pt-NP).
133
It was further confirmed
that Pt
2+
ions are released from the Pt-NP (from cellular
endosomes) thus causing significant DNA damage and cellular
apoptosis.
74,218
Thus it is hypothesized that since Pt-NPs do
not directly interact with DNA, the soluble species of Pt forms
a complex with DNA similar to that of cisplatin.
74
The effects of radiotherapy has improved by exploiting the
enhanced permeability and retention effect (EPR) of interstitial
tumors.
219
The periphery of solid tumors is often the site of
angiogenesis, which in turn induces hyperpermeability. With
gaps up to 600 nm, nanoparticles could passively extravasate
into the interstitial space and potentially into the hypoxic center
of the tumor.
219
The landmark study by Paciotti was the driving
force in the field to augment the efficacy of radiotherapy through
metallic nanoparticles.
220
In a more recent study, mice bearing
A431 squamous tumors were intravenously injected with.
111
In-labeled pegylated 20 nm, 40 nm, or 80 nm AuNPs at a dose
of 150 mCi/mouse.
221
Upon analysis of the intra-tumoral
distribution, the 20 nm AuNPs showed higher tumor uptake
and extravasation from the tumor blood vessels than did the
40 and 80 nm AuNPs. Moreover, the smaller particles presented
the lowest uptake in the RES and had an increased circulation
residence time.
221
4. Pharmacokinetics, biodistribution, and
toxicology profiles
To realize their vast potential and clinical application, the
whole body effect of noble metallic nanoparticles need to be
assessed prior to clinical use. Studies evaluating the pharmaco-
kinetics, biodistribution and possible toxicities (in vitro and
in vivo) are needed to understand the broad spectrum of
tolerance and the possible side effects of nanomaterials,. The
size, shape and ligand formulation of these nanomaterials can
further alter their uptake and behavior in biological systems as
discussed above. Notwithstanding, the high surface to volume
ratio of nanoparticles, and unique physiochemical properties
may also play a role in nanoparticle toxicity. Hence this
section will discuss the effects that these nanoscale materials
have on biological systems as well as the impact experienced
by the surrounding environment.
4.1 In vitro studies
There are several discrepancies regarding the safety profile of
gold nanoparticles based on in vitro cellular assays. The
majority of these studies maintain that nanoparticles are fairly
non-toxic.
222,223
Incubation of citrate capped gold nano-
particles (10 nm) with immune cells (dendrites) did not alter
the immunocellular phenotypes, activation and cell death after
48 h treatment, even with a significant amount of internalization
of the nanoparticles. Though there was no apparent cytotoxicity,
the cytokine profile (IL-1, IL-6, IL-10 and IL-12) was considerably
changed after gold uptake implicating the modulation of the
immune response as a result of nanoparticle treatment.
222
The
Dechent group reported that 15 nm AuNP was fairly unreactive
(even at 6.3 mM) compared to 1.4 nm particles, stabilized with
triphenylphosphine.
223
The smaller (1.4 nm) AuNPs triggered
cellular necrosis (IC
50
=36mM) causing mitochondrial disruption
and ROS generation, whereas the other particles did not display
any notable toxicity. Addition of reducing compounds such as
glutathione and N-acetyl cysteine,amelioratedthecytotoxicityof
1.4 nm AuNP. Likewise gene array analysis revealed that stress
related genes were significantly upregulated upon 12 h treatment
with 1.4 nm particles, but not with 15 nm particles.
223
In contrast,
Ng et al. reported the effect of epigenetic modulation generated
by 20 nm gold nanoparticles in lung fibroblasts.
224
Gold treatment
caused a modification in gene expression levels with an up-
regulation of microRNA-155 and down regulation of the
PROS-1 gene. Albeit, AuNP treatment did not alter the DNA
methylation of the PROS-1 gene, rather chromatin condensation
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was observed in the nucleus by TEM analysis.
224
An analogous
study by Li and colleagues reportedthat20nmgoldnanoparticles
can trigger oxidative stress and autophagy in human lung fibro-
blasts.
225
The AuNP treatment also enhanced lipid peroxidation
levels as evidenced by malondialdehyde (MDA) adducts and
upregulation of many autophagy related gene (ATG-7). Further-
more, the authors observed autophagosome formation, specifi-
cally the increase of inflammatory enzyme cyclooxigenase-2
(COX-2) and PNK (polyneucleotide kinase 30-phosphatase) gene
production.
225
The Goering group investigated the biological
effect of 60 nm AuNPs in murine macrophage cells.
226
Though
TEM images demonstrated AuNPs inintracellularvesicles,the
cells did not elicit a pro-inflammatory response. In a report by
Schaeublin et al.,keratinocytes(HaCaT)wereexposedto1.5nm
AuNPs of varying charge (positive, negative, neutral).
227
Follow-
ing exposure to the three different AuNPs, there was a noticeable
disruption of cell morphology in a dose dependent manner.
226
However mitochondrial distress was only observed with the
charged AuNPs. While the charged AuNPs induced both an
increase in caspase-3 expression and nuclear localization of p53,
the neutral AuNP showed an increase in p53 localization (nucleus
and cytoplasm) only.
227
Thus the biological response is dependent
on the surface properties of AuNPs: the charged AuNPs induce
apoptosis and the neutral AuNP promote necrosis.
227
Likewise,
the Rotello group also discussed the cytoxicity of 2 nm cationic
AuNPs with various hydrophobicity in HeLa cells using mito-
chondrial, ROS, and comet assays (measures DNA damage).
228
The experimental outcome strongly suggests that the higher the
hydrophobicity, the greater the observed acute toxicity and
decreased DNA damage. Noticeably, these AuNPs can produce
considerable amounts of reactiveoxygenspecies(ROS)that
oxidatively damage DNA at doses that do not affect mito-
chondrial activity.
228
The above studies show that the surface size
as well as the charge of the nanoparticle plays a significant role in
cytotoxicty and genotoxicity. These are important factors to keep
in mind when designing nanomaterials for medicinal use.
In contrast, there are several studies that discuss the cyto-
toxic effects of silver nanoparticles. In an initial report by
Hussain and coworkers, the toxic effects of AgNPs (15, 100
nm; [5–50 ml] were evaluated in rat liver cells (BRL 3A).
229
After a 24 h exposure, there was a significant decrease in
mitochondrial function and LDH leakage. It was further
hypothesized that AgNP toxicity is mediated via oxidative
stress (increase in ROS, decrease in GSH, etc.)
229
A following
study by Park et al. used RAW264.7 cells to elucidate the
mechanism of AgNP toxicity.
230
The researchers exposed the
RAW264.7 (exposure time; 24, 48, 72, and 96 h to) to 69 nm
AgNPs with varying concentrations (0.2, 0.4, 0.8, and
1.6 ppm).
230
Their results showed that the viability of the
macrophages decreased in a dose and time dependent manner.
Further cellular analysis revealed a dramatic decrease in GSH
levels, a considerable increase in NO production and TNF-a
(2.8-fold) along with a complete arrest at the G
1
phase of the
cell cycle. Work by Piao et al. further clarified the mechanism
of cytotoxity of AgNPs (compared to AgNO
3
) using human
Chang liver cells.
231
As shown in the previous studies, GSH
levels were decreased while ROS levels increased in a time and
concentration dependent manner when the cells were incubated
with AgNPs. The researchers further elucidated that AgNPs
modulate the expression of Bax and Bcl-2 (mitochondrial depen-
dent apoptotic pathway) creating a loss of mitochondrial
membrane potential (MMP).
231
The subsequent release of cyto-
chrome C (due to decrease in MMP) resulted in activating
caspases-3 and -9 mediated by the JNK pathway. A more recent
report by Teodoro et al. not only corroborated the aforemen-
tioned deleterious effects of AgNPs; they also clearly demonstrate
that silver nanoparticles contribute to cellular damage.
232
In that
investigation the bioenergetics of rat liver mitochodria was
evaluated after acute exposure to AgNPs (40 and 80 nm). Both
sizes of AgNPs created an increase of the permeability of the
inner mitochondrial membrane and subsequently leading to
mitochondrial depolarization. This impairment of mitochondrial
function resulted in an uncoupling effect on the oxidative
phophorlyation system.
232
The molecular mechanism of AgNPs
cytotoxicty was further defined in a very recent report by the
Hyuan group.
233
Silver nanoparticles (ca. 40 nm) were incubated
with both liver cells (human Chang liver) and Chinese hamster
lung fibroblasts (V79-4) for up to 24 h with various concentra-
tions. Using flow cytometry and confocal microscopy, exposure
of AgNPs induced an overloading of mitochondrial Ca
2+
and
enhanced ER stress.
233
Further analysis via Western blotting
showed AgNPs increased the phosphorylation of PERK and
IRE1 along with an up-regulation of GRP78/Bip, which are
significant markers of ER stress. Altogether these results indicate
that AgNPs induce ER stress that eventually leads to cellular
apoptosis.
233
More importantly, this study also demonstrated
AgNP cytotoxity was not cell line dependent.
233
The biological
effects of AgNPs were also investigated using coronary endo-
thelial cells (CECs) and aortic rings isolated from rats.
234
It was
demonstrated that AgNPs induce NO-dependent proliferation in
adoseandsizedependentmannerinCECs.Interestingly,atlow
concentrations AgNPs induced vasoconstriction in rat aortic
rings but vasodilation at high concentrations. Thus the biological
responses mediated by AgNPs are selective and specifically
associated with the size and concentration of AgNPs.
The vast majority of toxicity studies performed is based on
determining the proper dosage of nanoparticles. Despite the
fact that ‘‘proper dosages’’ does not generate a ‘‘toxic response’’,
nanoparticles might modify cellular processes such as signal
transduction.
235
For example, silver nanoparticles where shown
to directly interact with Fyn kinase, thus creating signal inter-
ference in stem cells.
236
Likewise, cells treated with a low dose of
nanoparticles were shown to reduce the activity of caspases.
237
In
arecentexperiment,researchersstudiedthreeofthemostutilized
nanomaterials (silver, gold, and iron; d=10nm)todetermine
the downstream effect normal cellular processes.
238
Human
epithelial cell lines (A-431) were treated with 15 mgmL
!1
of
metallic nanoparticles (of similar size and morphology) and the
subsequent effect of the particles on EGF signal transduction was
evaluated. As predicted, silver nanoparticles caused a substantial
increase in ROS over either gold or iron particles. Gold nano-
particles had a considerable effect on EGF-dependent phos-
phorylation (20%), though the other particles also reduced
phosphorylation levels to varying degrees. Iron nanoparticles
had the greatest impact on EGF-dependent gene transcription;
minimal alterations were seen with either gold or silver particles.
The results of this in depth study demonstrate that metallic
nanoparticles can disrupt cellular functionality, with the
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composition of the core material uniquely affecting the signaling
response playing a significant role. The studies discussed above
imply that care is needed in the use of nanoparticles in medicine.
However with proper design, these studies also indicate the
potential utility of these systems as cytotoxic therapeutic agents
for cancer therapy.
4.2 Effect of mode of administration
It is essential to have a firm understanding of how nano-
particles interact in biological systems in vivo if they are to be
an effective pharmaceutical. Thus it is necessary to have
proper characterization of these nanomaterials using the
appropriate animal model alongside vigorous statistical analysis.
Depending upon the route of administration, the pharmaco-
kinetics, biodistribution, and toxicity profile of nanoparticles
vary (see Table 2). For instance, a few studies demonstrate that
intramuscular and intravenous injections of gold have a relatively
higher uptake versus oral administration.
239,240
As a case in
point, the toxicity and pharmacokinetics profile of Auranofint
(clinically approved gold complex drug—see Fig. 2) and gold
sodium thiomalate (also a gold complex—see Fig. 2c) are well
documented, nevertheless the mode of administration is still an
on going debate.
241
While, the bioavailability of injected gold
showed absorption maximum of only 20–25% after 2 h, inter-
mittent dosing of gold resulted inerraticconcentrationlevels
within patients.
239,242–244
Intravenous injection of gold complexes
can also lead to some accumulation in the dermis and deposition
in the cornea compared to oral administration.
245,246
In contrast,
orally administered gold complexes showed a higher half-life and
asteadybloodplasmaconcentrationofthroughoutthetreat-
ment. Furthermore, the majority (85–95%) of the gold complex
administered orally is excreted.However,theremainingamount
(5–15%) is passed through the urine.
244,247
The Brandau group
compared the biodistribution of AuNPs (d=1.4nmand18nm)
using two routes of administration:(i)intratrachealinstillation
into the lungs (IT); and (ii) tail vein injections (IV).
248
Analysis of
the results indicated the smaller nanoparticle translocated
through the respiratory tract after IT administration, whereas
the 18 nm particle remained in the lungs.
248
After IV adminis-
tration, both nanoparticles accumulated in the liver with the
18 nm AuNP showing 2-fold increase comparatively (18 nm =
93.6% ID/g). Work done in the Zhang group reported the
toxicological features of 13.5 nm AuNP is also dependent on the
route of administration.
249
Adosewindowof137.5–2200mgkg
!1
was administered orally, intraperitoneal (IP), or in the tail vein
injection of mice. The body weight, blood profile and other
phenotypical changes of the mice was observed and recorded.
249
Injections via tail vein had minimal toxic effects showing
minimal alterations to white blood cells and platelet counts.
Additionally, the change of hemoglobin concentration was not
statistically significant. However, intraperitoneal and oral
administration showed increased toxicity with a reduction in
red blood cell count.
249
A recently reported study compared the biodistribution of
silver salts (AgAc) to 14 nm PVP coated silver nanoparticles
orally administered to rats.
250
Despite the formulation, the
pattern of silver distribution into the organs was similar, with
the highest concentrations found in the small intestine,
stomach, kidneys, and liver though the uptake of silver in
the kidney, liver, lung and other organs were quite lower for
AgNP treatment with respect to AgAc treatment.
250
The
excretion of silver for AgNP treatment was relatively high in
fecal matter (63%) than in urine (0.005%). The amount of
silver present in bile fluid was 16 to 20 fold greater than in rat
plasma. Authometalliographic staining (AMG) showed silver
only on the surface of intestinal (ileum) villi but not in the
cellular cytoplasm. Renal papilla showed heavy staining of
AMG grains in the glomeruli and renal tubules; still, there was
no change in the staining pattern between animals exposed to
AgAc or AgNP.
250
Chrastina and Schnitzer reported the
biodistribution of PVP coated and radio I
125
labeled AgNP
(12 nm) in Balb/c mice injected intravenously.
251
The
CT-SPECT (single photon emission computerized tomography)
imaging revealed particles were mostly taken up by reticuloen-
dothelial system (spleen 41.5% and liver 24.5%) after 24 h; the
rest were distributed in all other organs in very low amount. This
indicates the particles relocate from the primary injection site and
then further distributed to a secondary location.
251
However
other reports indicate enhanced liver enzyme activity, higher
uptake by local macrophages, increased inflammatory response,
and liver damage.
251
This in depth analysis plainly demonstrated
nanosilver toxicity is contingent on the route of administration.
In addition, the toxic effects of silver nanoparticles are dose and
time dependent.
It is worth briefly discussing the effect inhalation of nano-
particles may have on biological systems. Evidence of silver
nanoparticle toxicity via inhalation was described in the in vivo
studies performed by Sung et al. and Kim et al. using Sprague-
Dawley rats.
252–256
In the initial studies, Sung et al. investigated
the possible biological effectsofprolongedexposureto18nm
AgNPs.
252
In this 90-day study, female and male rats were
exposed AgNPs for 6 h day
!1
,5days/week,for13weeksina
whole-body inhalation chamber. The end results of this
study show that AgNPs reduce lung function and produce
inflammatory lesions in the lungs in at a much lower mass
dose concentrations (2.9 $10
6
particles cm
!3
)comparedto
submicrometer particles. In their follow up study, bile-duct
hyperplasia in the liver increased dose dependently in both the
male and female rats under similar experimental conditions was
observed.
253
In their most recent report rats were exposed to
18 nm AgNPs for 4 h in a whole-body inhalation chamber and
then further observed for 2 weeks. After a full analysis of lung
function, the results demonstrated that acute inhalation exposure
to silver nanoparticles may not cause acute toxicity.
254
The results of the aforementioned studies was comparable
to investigations by Kim et al. using 60 nm AgNPs stabilized
with carboxymethylcellulose.
255
In this study, the effects of orally
dosed AgNP (60 nm; dosimetry 30 mg kg
!1
, 300 mg kg
!1
and 1000 mg kg
!1
) in both male and female rats was
extensively monitored for 28 days.
255
Over the course of AgNP
treatments, the rats did not show a significant change in body
mass index regardless of their sex or dosage. However, blood
chemistry analysis indicated elevated liver damage in the
group with the medium dosage (300 mg/Kg) indicated by
changes in ALP and cholesterol in both male and female
rats.
255
In a follow up study, 5 week old male and female rats
were administered 56 nm AgNPs via subchronic inhalation
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(30 mg kg
!1
, 125 mg kg
!1
, 500 mg kg
!1
) over a period of
90 days (13 weeks).
256
Numerous other parameters such as
metal ion (Mg
+2
, Na
+
,K
+
), total protein (albumin, gamma
glutamyl transpeptidase, alanine aminotransferase, etc.), reti-
culocyte, bilirubin, glucose, triglycerides, salt, calcium, blood
counts, histology and ICP-MS analysis were done 90 days post
treatment in both the male and female rats for all doses.
255
The
mice with the lowest dosage (30 mg kg
!1
) had no remarkable
toxicity whereas with the 125 mg kg
!1
dosages there was a
noticeable effect. The body mass index was significantly
decreased for male rats with the highest dosage (500 mg kg
!1
)
just after 4 weeks.
255
Histopathological analysis revealed con-
siderable liver damage, through bile-duct hyperplasia, in both
male and female rats and also a substantial change in cholesterol
and ALP (alkaline phosphatase concentration) with the
125 mg kg
!1
dosage.
256
The concentration of silver of all the
tissue samples collected showed a dose-dependent increase.
Furthermore female rats had a 2-fold higher concentration of
silver in the liver compared to the males. In conclusion, the above
studies demonstrate that reduction in lung function and inflam-
matory lesions appear after AgNPs enter the body. Thus accu-
mulation and, in some cases, damage tissues such as the liver,
lungs, and olfactory bulbs, or penetrate the blood–brain barrier
occurs.
4.3 Effect of particle size and morphology
The physiochemical parameters such as charge, size, and shape
of nanoparticles as well as the nature of the binding ligand
have to be considered during nanoformulation in order to
minimize toxicity and increase the therapeutic index. Initial
work by Hillyer demonstrated that the distribution of AuNPs
orally dosed was inversely proportional to the size of
the nanoparticles. An investigation by Geertsma exemplified
the wide dispersal of 10 nm gold nanoparticles throughout the
body, while the larger particles were only observed in the liver,
blood and spleen.
257
In another study by Pan et al., nano-
particles with a diameter of 1–2 nm are very toxic whereas
15 nm gold particles are relatively nontoxic in any cell type.
258
Furthermore, the authors showed that the cellular response is
size dependent, even with particles of similar size. Abdelhalim
et al. reported that Wistar-Kyoto rats infused with 10, 20 and
50 nm AuNP by a dosimetry of 3 or 7 days showed hepato-
toxicity and renal toxicity.
259
The smaller size particles had
more toxic effect than larger particles with ROS generation
leading to necrosis, renal tubular alterations, higher Kupffer
cell hyperplasia, and central veins intimae disruption.
259
In a
ground breaking study by Chen et al. reported on the in vivo
effect of naked particles ranging from 3 to 100 nm injected
IP.
260
BALB/C mice were injected with the variant gold
nanoparticles (8 mg kg
!1
/week dose). It was found that 8 to
37 nm particles caused acute toxicity including loss of appetite,
fur color change, sickness, reduced body weight, crooked spine
(after 14 days) in contrast to normal mice (Fig. 11). However,
particle core size of 3, 5, 50, 100 nm did not show any apparent
toxicity. Histopathological analysis evidenced an increased
population of Kupffer cell in hepatocytes and structural
deterioration of the lungs, liver and spleen. These observations
were linked to the presence of gold particles these sites, which
were detected by AuNP enhanced CARS signal (Coherence
anti-Stoke Raman Signal) (Fig. 12).
Table 2 A summary of in vivo toxicology profiles of nanoparticles. Adapted from ref. 249 and 311
NP Size Animal Mode Dose Period Side effects
AgNP 18 nm rat Inhalation 1.73 $10
4
/cm
3
6h day
!1
, 5days/week
for 4 weeks
Not much notable
changes in blood profiles
of the both male an
female rats
1.27 $10
5
/cm
3
1.32 $10
6
particles/cm
3
AgNP 18 nm rat Inhalation 1.73 $10
4
/cm
3
6h day
!1
, 5days/
week/12 weeks
Reduced alveolar
inflammation & bile
duct hyperplasia & liver
inflammation
1.27 $10
5
/cm
3
1.32 $10
6
particles/cm
3
AgNP 18nm rat Ingested 30 mg kg
!1
, 300 mg kg
!1
and 1000 mg kg
!1
AgNP mixed with diet
for 28 days
Changes in alkaline
phosphatase activity,
cholesterol changes &
liver damage
AgNP 13–15 rat Inhalation 1.73 $10
4
/cm
3
, 0.5 mgm
!3
6h day
!1
, 5days/
week/4 weeks
Number of goblet
containing neutral
mucins in liver increased
1.27 $10
5
/cm
3
3.5 mgm
!3
1.32 $10
6
particles/cm
3
61 mgm
!3
AgNP 22 nm mice Inhalation 1.97 $10
7
particles/cm
3
6h day
!1
, 5days/
week/2 weeks
Several gene expression
related to motor
neuronal disorders,
neurodegeneration
AuNP 4,10,28,58 nm Mice Oral 20 000 mg kg
!1
7 days None Observed
AuNP 10, 50 100 and 250 nm Mice Tail vein 77–120 mg kg
!1
24 h No adverse effects
were noted
AuNP 3,5,8,1217, 37 50 Mice IP 8000 mgg
!1
kg
!1
21 days 8–37 nm AuNP induced
severe sickness
AuNP 20 nm Mice IV 10 mg kg
!1
1 day, 1 week, 1 & 2
months
None noted
AuNP 12.5 nm Mice IP 40–400 mg kg
!1
day
!1
8 days None noted
AuNP 13.5 nm Mice (pregnant) IV,oral, IP 137.5–2200 mg kg
!1
14 & 28 days No gold found in the
fetuses’
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Ligands like mPEG, chitoan, dextran, polylacticcoglycolic
acid (PLGA) can be functionalized onto the nanoparticle
surface in order to improve blood retention time, decrease
deposition into the liver and reduce immunogenic reactions.
The pharmacokinetics and passive uptake of mPEG AuNPs of
various size, (from 20 nm to 100 nm), into tumors was studied
in tumor bearing CD1 mice.
261
The half-life of the smaller
particles (t/2 = 51 h) was 8-fold higher compared to the larger
particles (t/2 = 6.6 h). Interestingly, the larger nanoparticles
had a greater accumulation into the tumors, implying passive
uptake into the matrix is core size dependent. The pharmaco-
kinetics and biodistribution of PEG-AuNPs (d= 20, 40, and
80 nm) were also examined in nude mice.
221
This investigation
revealed that 20 nm AuNP had the longest half-life and
significantly higher tumor extravasation compared with the
40 and 80 nm AuNPs.
Park et al. reported the size dependent toxicity of the silver
nanoparticle when mice were exposed to 1 mg kg
!1
of AgNP
(22, 42, 71, 323 nm) by oral dosage for two weeks.
262
The
22 nm particles showed increased toxicity with higher immune
cell infiltration (B cell & higher CD8+ T cell subtypes) and
increased level of TGF-band cytokine production (specially
IL-10 and 12) whereas the larger size particles did not have an
adverse effect (Fig. 13). The organ weight was unaltered in any
of these treatments irrespective of size as compared to
controls.
262
The 22 nm particles count was higher in brain
tissue (possibly penetrating blood-brain barrier) than larger
particles, which were not well distributed in the brain. There
was also a dose dependent toxicity (0.25 mg kg
!1
, 0.5 mg kg
!1
and 1.0 mg kg
!1
) for 42 nm particles with the highest treat-
ment dosage showing an increase in ALP, AST (aspartate
transaminase), and ALT (alanine transaminase) levels. Histo-
pathology analysis indicated minor damage to the cortex of
the kidneys, but serious changes in morphology in the liver
and small intestines was not noted.
262
In a analogous study by
the De Jong group, the authors demonstrated a size dependent
(20, 80 and 110 nm) biodistribution of silver nanoparticles via
IV administration for 5 consecutive days and further monitored
for 16 days.
263
Nanoparticles were rapidly cleared from circul-
ation and dispersed to all the organs irrespective of size. Larger
particles mostly accumulated in spleen, followed by the liver and
Fig. 11 Average lifespan of mice receiving AuNPs with diameters
between 8 and 37 nm was shortened to different extents. The average
lifespan (L
50
) was defined as the time beyond which half of the mice
died. Mice injected with GNPs outside the lethal range behaved
normally. The break marks on the top of bars indicate no death
observed during the experimental period. Reprinted with permission
from ref. 260. Copyright r2009 Springer.
Fig. 12 H&E staining showed AuNP-induced abnormality in major
organs. (Top to bottom) HE staining for liver, lung, and spleen. The
left column shows tissues from 5 nm GNP-treated animals. The right
column shows tissues from 17 nm GNP-treated mice. Reprinted with
permission from ref. 260. Copyright r2009 Springer.
Fig. 13 The serum levels of cytokines and IgE after oral administration
of AgNPs (42 nm). Mice were treated with AgNPs with different doses of
0.25 mg kg
!1
,0.5mgkg
!1
,and1mgkg
!1
for 28 days. Mice were
sacrificed after treatment of 28 days and experiments were performed
using 3 samples. The concentration unit of cytokines is pg ml
!1
and that
of IgE is ng ml
!1
serum. Significantly different from control group, *Po
0.05, **Po0.01. Reprinted with permission from ref. 262. Copyright
2010 Elsevier.
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lung, while small particles (20 nm) were primarily deposited in
the liver, then subsequently distributed to the kidneys and
spleen.
263
Nanomaterials undergo aggregation upon contact with
biological fluid and media, thus changing their identity and
possible effects on living systems.
134
Gosens elegantly demon-
strated the effects of individual nanoparticles (50 and 250 nm)
and their aggregates by using a pulmonary inflammation animal
model in male rats.
264
The study of pulmonary inflammatory
markers (cytokines), TEM analysis, cell counts and cytotoxicity
analysis from bronchoalveolar lavage fluid (BALF) indicated that
citrate coated 50 nm and 250 nm (single particles) as well as their
aggregates did not cause severe toxicity, with the exception of mild
pulmonary inflammation. Additionalanalysisofbloodcellcounts
(lymphocytes, basophilis, neutrophils, eosinophils and macro-
phages), concentration of inflammatory cytokines (TNF-a,IL-6)
and other serum protein (fibrinogen, LDH, albumin) level were
unaltered in both single and agglomerated particles.
264
4.4 Effect of surface chemistry
The surface chemistry of nanoparticles is also an influential
factor in pharmacokinetics and biodistribution. This information
could provide design principles for optimizing delivery to tumors.
For instance, in one study mice were injected with one of five
types of gold nanoparticles (d=5–22nm)containingeithera
positive, negative or neutral surface charge.
265
Analysis of the
blood, excrement, and various tissues demonstrated that the
positive 5 nm AuNP was higher in the blood post injection
compared to the other charged nanoparticles. Additionally, the
positive nanoparticle was also observed to have the largest
accumulation in the kidneys (24% ID/g) and thusly ‘‘trapped’’.
The negative and neutral charged particles show statistically
significant accumulation in the liver comparatively.
265
Bio-
distribution studies in different mouse strains (immunodeficient
vs. immunocompetent) demonstrated that surface charge of gold
nanoparticles and their modes of systemic administration uniquely
alter their pharmacokinetics, organ distribution and tumor
uptake.
266
Neutral and zwitterionic particles provide high systemic
exposure and low clearance when administered through intrave-
nous administration. Intraperitoneal-administered nanoparticles
demonstrated substantially lower systemic exposure than the
IV-administered nanoparticles, suggesting inability of the particles
to cross the peritoneal barrier. Low plasma clearance for both
administration routes was reflected in the increased tumor uptake
of the neutral and zwitterionic nanoparticles in a subcutaneously
implanted xenograft model of ovarian cancer.
266
Furthermore,
AuNPs administered IV accumulated mainly in the liver followed
by the spleen and kidneys (positive AuNPs had the least accumul-
ation), however IP administered AuNPs were concentrated in the
pancreas, followed by the RES.
266
Particle size along with its
surface charge can also mediate its biodistribution. In a study by
Hirn and co-workers, rats were injected into the tail vein with
radio labeled gold nanoparticles ofvarioussizes(1.4–200nm)and
charges (positive or negative).
267
The biodistribution of the
negatively charged
198
AuNPs was shown to be size dependent
and had the greatest accumulation in the liver.
267
However the
accumulation of the positively charged particles had a varied
pattern. It was further hypothesized that protein binding and
exchange on the particles surface modulated the uptake of the
nanoparticles.
267
Finally, in a study by Zhu and coworkers, zebra
fish were exposed to AuNPs (HD = B10 nm) of various surface
charge (hydrophilic: positive, negative, neutral, and a hydrophobic
positive) over a range of times (24, 48 and 72 h).
268
Over time, it
was shown that the positive nanoparticles were taken up more
readily that the negative and neutral AuNPs. However, fish
exposed to the hydrophobic AuNPs expired within 24 h. The
positive, negative and neutral AuNPs mostly accumulated in the
intestine. Additionally, the charged AuNPs were excreted whilst
the neutral AuNP tended to stay in the body. In contrast, the
hydrophobic AuNP appeared to be more widely distributed with
the largest concentration seen in the gills, heart and dorsal fin.
These results are indicative of a strategic methodology: develop-
ment of hydrophilic nanoparticles decreased their toxic profile.
4.5 Accumulation of nanoparticles in the brain
Nanoparticle therapeutics is currently being developed to
combat brain disorders such as Parkinson and Alzheimer’s.
In order to treat these diseases, nanoparticles must be able to
cross the blood brain barrier (BBB) without harming the
integrity of the brain. One investigation found that 24 h after
exposure, AgNPs and copper nanoparticles injected intrave-
nously into rats resulted in edema localized to the proximal
frontal cortex and the ventral surface of the brain.
269
Analo-
gous findings were observed in mice that were systemically
exposed to AgNPs or through a direct injection into the brain
ventricular space.
270
In another investigation, inductively
coupled plasma mass spectrometry (ICP-MS) and transmission
electron microscopy was used to analyze the distribution of
AgNPs (50–100 nm) in the main organs of rats.
271
After sub-
cutaneous injection, the authors observed that AgNPs were
mainly dispersed in the kidneys, liver, spleen, lungs, and brain
as discrete nanoparticles.
271
These results were markedly different
from another publication using BSA coated AgNPs 2 nm in size
injected via IP.
272
Tissues were harvested from the rats after 24,
96, and 168 h and assessed for silver content using ICP-MS and
imaged using TEM. ICP analysis revealed significant accumul-
ation of silver in the liver and spleen after 96 and 168 h exposure
to AgNPs. Closer examination of brain tissue revealed evidence
of silver induced damage even though AgNPs were not observed
via TEM imaging.
272
It was further hypothesized by the authors
that Ag can cross the blood brain barrier, but not as a defined
nanoparticle.
272
In a report from the Soto group, 12.5 nm
AuNPs (40, 200, 400 mgkg
!1
day
!1
)wereadministeredtothe
tail vein of mice every day for 8 days. In all of the organs
examined, there was a proportional increase in gold accumul-
ation, including uptake into the brain. Their findings demon-
strate the possibility of targeting brain tissue using AuNPs
without generating noticeable toxicity.
273
In a contrasting report,
AgNPs of various sizes (25, 40, 80 nm) were used to investigate
their inflammatory effects on primary rat brain microvessel
endothelial cells (rBMEC).
274
Accumulation into the rBMECs
was demonstrated to be size dependent, with the 25 nm AgNP
showing the largest uptake. Furthermore the 25 nm AgNP
also showed to have significant effect on cellular viability, perme-
ability, cytotoxicity, morphology and inflammatory response as
opposed to cells exposed to the 40 and 80 nm AgNPs.
274
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The results of these studies indicate more work needs to be done
to produce a viable therapeutic nanoparticle for neurological
diseases.
4.6 Surface permeation of nanoparticles in vivo
The therapeutic applications of metallic nanoparticles are
fairly diverse. With its touted antimicrobial properties, silver
particles are found in a wide range of products ranging from
clothing to topical creams. Even so, the safety profile for silver
in these applications is still under investigation. Korani et al.
reported the dermal toxicity of silver nanoparticles in the
animal model of male guinea pigs.
275
For the subchronic
toxicity study, a concentration range of (100, 1000 and
10 000 mg mL
!1
) nanosilver was selected whereas for acute
measurement only two doses (1000 and 10 000 mg ml
!1
) were
chosen. For animals treated topically, a reduction in the
thickness of the epidermis and dermis was observed in the
low-dose group, with a slight increase of inflammatory
Langerhans cells. However collagen fibers were reported to
be normal. Skin toxicity was shown to be dose dependent; an
increase in the concentration of AgNP manifested skin
toxicity. During the course of the acute study, animals exposed
to 10 000 mg ml
!1
of nanosilver had disrupted collagen fibers
as well as a higher macrophage infiltration with acidophilic
cytoplasm.
275
It was also observed that damage to hepatocytes
(indicated with the higher amount of Kupffer cells) as well as
some necrosis is also dose dependent, but spleen toxicity was
not see with histopathological analysis.
275
Using a porcine
skin model, AgNPs was revealed to be in the upper stratum
corneum layers of the skin.
276
Treatment with AgNPs caused
localized inflammation (circa 14 days), whilst AgNPs cultured
with keratinocytes were observed to be enclosed in cyto-
plasmic vacuoles.
276
After application to damaged human
skin, transmission electron microscopy revealed silver pene-
tration in the outermost layer of the epidermis and deep
stratum corneum.
277
Particle size was also shown to be a factor for in vivo
permeation. Makino et al. used citrate capped gold nano-
particles with different core sizes (d= 15, 100 and 200 nm) and
determined their permeation coefficient.
278
They demonstrated
that the 15 nm AuNP had the greatest permeation, being
found in the deep regions of the skin. However the larger
particles remained on the surface of the skin.
278
In subsequent
experiments, mice were injected with gold nanoparticles (d=15,
50, 100 and 200 nm) to study theirensuingbiodistribution.
279
Analysis using inductively coupled plasma mass spectrometry
(ICP-MS) revealed that most of thegold,regardlessofsize,was
present in the liver, lung and spleen. The smallest nanoparticle
displayed the greatest biodistribution throughout the mouse.
Furthermore, both the 15 nm and 50 nm AuNPs crossed the
blood brain barrier. However, the presence of the 200 nm particle
was very insignificant in any of the tissues analyzed.
279
4.7 Effect on embryonic development
Beside mammals, other non-mammalian system such as Zebra
fish, Drosophila and C. elegans has also been used to evaluate
the toxicity of silver nanoparticles.
280
Gorth et al. studied the
effect silver nanoparticles had on the growth rate of
Drosophila, from egg to pupapte.
281
They found that smaller
sized particles (20–30 nm) had mild effect on the growth rate.
Eggs treated with larger particles showed significant toxicity
(500–1200 nm) with only 10% developing to pupate at a
100 ppm concentration. The toxicity occurred via Hsp-70
upregulation, oxidative damage, and lipid peroxidation in
the larvae.
281
The transport and biocompatibility of AgNPs
was investigated using real time. In this study, Kim et al. used
single AgNPs (5–46 nm) to determine the mechanism of
transport in developing embryos.
282
The authors observed
AgNPs accumulation throughout the development stage, with
abnormal development relating to the concentration of AgNPs
(Fig. 14).
282
Another study using Zebra fish models, a dose
dependent toxicity and phenotype changes were also observed.
Treatment with silver nanoparticles resulted in abnormal
phenotypic shapes such as twisted body axes and notochord,
as well as pericardial edema.
283
Beside that, hepatotoxicity and
changes in mRNA level for several detoxifying enzymes like
catalase, glutathione peroxidase were decreased whereas Bax,
p21, Noxa genes were upregulated.
255,283,284
A similar report
published by Wu et al. studied the early development of
Japanese Medeka (Oryzias latipes). The authors observed fin
fold malformations, oxidative DNA damage, and genetic
aberrations.
285
Silver nanoparticles suspended in water can
also exacerbate the hypoxic sensation of the Eurasian perch
fish (Perca fluviatilis)
286
as well as severely affecting embryonic
growth of oysters due to increased mRNA production of
metallothionein.
287
Additional genomic analysis of C. elegans
treated with AgNPs revealed the impairment of reproductive
budding due to up-regulation of SOD-3 (superoxide dismu-
tase) aberration in daf-12 genes.
280,288
However, in a study by
Browning et al., they observed that AuNPs (11.6 nm) are
much more biocompatible than AgNPs.
289
In their investi-
gation, they observed AuNPs were able to passively diffuse
through chorionic-pore-canals. Even though the amount of
AuNPs accumulation was directly related to its concentration,
the majority of the embryos (74%) developed into normal
fish.
289
4.8 Other potential side effects of nanoparticles
Argyria is one of the reported side effects in patients exposed
to colloidal silver. Over a long period of time, silver will
deposit in the skin thus giving the patient a blue hue
(via ingestion of 6.4 g of colloidal silver over a period of a
year).
290
Besides that, night vision problems, bowl pain,
respiratory trouble, reduced creatinine clearance, increase in
b-acetyl-B-Dglucoseaminidase are also associated with the
workers exposed to silver dust.
291,292
Some of the workers
also had tarnished corneas and conjunctiva due to inhalation.
293
Another report discusses a patient that ingested colloidal silver
three times a year over a two-year period resulted in hyper-
lipidemia, diabetics and hypertension along with blue-grey facial
color.
294
Other accounts of neurological disorders was recently
documented in a 75 year old man who self mediated with
colloidal silver.
295
Exposure to AgNPs via inhalation over a
14-day period can also stimulate the expression of 468 genes
in the cerebrum. Amongst these, several genes were linked to
numerous neurodegenerative disorders.
296
Rahman et al.
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demonstrated similar results of neurotoxicity from AgNPs.
Analysis of mouse brains exposed to nanosilver yielded increased
ROS generation, apoptosis and gene modulation.
297
The studies
presented here yielded different results seen of patients with
poisoning from silver inhalation.Thisclearlyillustratestheneed
for more in-depth studies on thesafetyprofilesofnanosilver
prior to commercial use.
As mentioned earlier, gold collids has been used for centuries
in medicine without any notable side effects. One contributing
factor is largely due to the fact that gold (0) is extremely inert.
298
Gold is also used in small quantities in dental prostheses, pastries,
chocolates and sometimes in alcoholic beverage.
181
Nonetheless,
enzymes from saliva can transform gold (0) to gold (I), which is
consequently engulfed by immune cells.
181,299
By and large the
observed toxicity for gold (I)compoundsisskinandmucous
hypersensitivity along with macular and papular rash, esosino-
philia, erythema nodosum and various other allergic reactions.
300
In some cases, oral administration of gold complexes have also
Fig. 14 Representative optical images of (a) normally developed and (b–g) deformed zebrafish: (a) the normal development of (i) finfold, (ii) tail/
spinal cord, (iii) cardiac, (iii–iv) yolk sac, cardiac, head and eye; and (b–g) deformed zebrafish: (b) finfold abnormality; (c) tail and spinal cord
flexure and truncation; (d) cardiac malformation; (e) yolk sac edema; (f) head edema: (i) head edema; (ii) head edema and eye abnormality; (g) eye
abnormality: (i) eye abnormality; (ii) eyeless. Scale bar = 500 mm. Reprinted with permission from ref. 282. Copyright r2007 American Chemical
Society.
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been reported to trigger diarrhea.
301
Very low incidence of
nephrotoxicity has been reported with minor proteinurea while
injected with gold complexes.
302,303
Gold complexes can also
generate some hematological disorders and is not recommended
for pregnant women due to its teratogenic properties.
304
Other
toxicological effects is due to the oxication of effects of gold (I)to
gold (III)byphagolysosomalenzymesandotherredoxproteins
such as myeloperoxidase.
181
The major cause of this toxicity has
been attributed to free radical formation, thereby causing oxidative
stress.
305
The slow clearance of these nanoparticles and their
increasing accumulation in the liver and spleen, kidneys and lungs
can also be the cause of increased damage due to oxidation.
181
It
has also been reported that nanoparticles trigger thrombosis,
hemolysis and other immunogenic reactions while being systemi-
cally transported.
306
In healthy conditions, the human body contains a basal level
of gold in the range of 0–0.001 ppm.
307
Gold is also found in
small amounts in skin (0.03 ug/g), hair (0.3 ug/g) and nails
(0.17 mgg
!1
).
239,245,247,308
Gold (I), relatively inert compared to
gold (III), is reasonably stabilized in aqueous solution via
capping agents. Most of the gold in circulation gets adsorbed
by albumin and/or globulin and remain bound to plasma for
months.
309
Depending on the patient, systemic gold uptake is
varied. For instance, with patients that are smokers, there is an
increased adherence of gold to red blood cells.
239,310
It is mostly
absorbed in the lymphatic and reticuloendothelial systems
(due to their metal absorbing capacity) whereas liver and bone
marrow account for 25% of the total uptake.
239,242,308
4.9 Toxic effect of other metallic nanoparticles
Some toxicological reports are also documented for other
metal nanoparticles beside gold and silver metal.
311
In
comparative study, Ashrarani et al. reported on the size dependent
toxicity of Au, Ag and platinum nanoparticles using zebrafish
model over 72 h period.
131
They found polyvinyl alcohol capped
(PVP) Pt particles (3–10 nm) delayed hatching, impaired the
mortality, crippled backbone, cardiac abnormality, along with
brain accumulation of platinum.Goldandsilveruptakewasquite
high compared to platinum as evidenced by ICP-OES. Yet, the
toxic profile of gold nanoparticles was trivial relative to silver
nanoparticles (highest toxic profile) and moderate for platinum
particles.
131
Hussain et al. reported that manganese (Mn-40 nm)
particles can trigger oxidative stress in neuroendocrine cells
(PC-12) and a dose dependent cleavage of dopamine (DA) and
its metabolite.
312
It is known that manganese has potential to
cause in vitro and in vivo toxic effects, however not much is
published on MnNP.
313
The experimental evidence with morpho-
logical assessment, MTT assay, and metabolite measurements of
DA indicates the neurotoxicity of the MnNP.
312,313
Another
report by Wang et al. demonstrated the neurotoxicity of manga-
nese and copper nanoparticles in PC-12 cell lines.
314
Copper
particles (90 nm) had similar effects as MnNPs such as DA
depletion and modulation of gene expression. Monoamine
oxidase A (maoa) was enhanced by Cu-90 nm treatment while
tyrosine hydroxylase (Th) was down regulated by Mn-40 nm
treatment.
314
There was no major effect observed for Ag-15
treated with MnNPs in relationship to dopamine regulation even
at a high dose. Many ROS responsive genes (gpx1, gss) and neuro
pathophysiological relevant genes (park2, a-synuclein) were also
altered by the Cu-90 nm and Mn-40 nm treatment.
314
This is
indicative of oxidative stress mediated by dopamine degradation.
Both Mn-40 nm and Cu-90 nm treatment caused upregulation of
Snca and Park2. Both proteins are often linked with many
neurodegenerative diseases such as Parkinson’s and Alzheimer
due to proteins being generated with an aberrant confirmation.
314
These studies implicate that CuNP and MnNP particle mediated
protein misfolding can lead to neurotoxicity and neurological
pathogenesis in domaminergic cells, having a susceptibility to
cross the blood brain barrier (BBB).
314
Prabhu et al. reported a
dose (10–100 mM) and size dependent toxicity of Cu-NP (40, 60,
80 nm) treated for 24 h in a dorsal root ganglion (DRG) cell
derived model from rats.
315
Cell viability study (MTT assay) and
light microscopic analysis indicated the formation of vacuoles and
neurons depletion and neurite network cleavage.
315
The Cu
particle showed maximum toxicity at highest dose and smaller
size. The effect of Cu-NP toxicity could occur due to disruption of
mitochondrial dehydrogenase activity in vitro and in vitro,thus
triggering ROS production. Chen et al. reported that oral dosage
(LD
50
413 mg kg
!1
)ofcoppernanoparticlesinmicemightcause
severe toxicity to many organs such as kidney, liver, spleen,
brain.
316
The in vitro analysis showed the generation of Cu
3+
ion is the cause of lethality, which could have a different in vivo
metabolism.
317
Furthermore copper nanoparticle can be smoothly
absorbed into body through skin and respiratory tract. Given the
increasing use and demand of nanomaterials in industry, health-
care, and cosmetics, safety measures are a necessity to protect
human health and the surrounding environment.
5. Concluding remarks and future outlook
In this review, we have discussed modern synthesis and some
recent applications of noble metal nanoparticles in medicine.
We have also described some possible toxic affect these
particles may elicit on living systems and their surrounding
environment. One of the major challenges in nanobiotechnology
is to improve the efficacy of nanoparticle therapeutic and the
reduction of any intrinsic side effect. With the rapid surge in the
development in nanomaterials, new treatment strategies are
being explored that has the potential to overcome existing
problems using noble metal nanoparticles. Even with their
fantastic promise, the impact on human health (positive and
negative) needs to be fully understood prior to wide spread use.
Furthermore, our experimental setups need to be well thought
out and carefully executed for proper interpretation of the
data presented. In a recent publication, it was eluded to that
the uptake and subsequent affects nanoparticles elicit on cells
might be arbitrary.
318
The cellular uptake of nanoparticles is
paramount to their therapeutic applications and possible toxicity.
Thus when designing in vitro experiments, numerous other
aspects must be considered. As mentioned earlier, nanoparticles
may aggregate in biological medium,
134
due to the high ionic
environment, and settle. This sedimentation might effect the
dynamic interactions between the nanoparticles and the cells.
Furthermore, the authors argue the probability that the uptake of
nanoparticles and subsequent cellular response is variable. Other
considerations also need to be made when conducting in vitro
experiments such as the protein corona. When nanoparticles are
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exposed to biological fluids, they may interact and be coated with
proteins in solution. This protein coating, or protein corona,
mediates the biological response and the intrinsic physical–chemical
properties of the nanoparticle.
126
These factors mentioned above
should be taken into careful consideration when designing the
experimental setup, executing the plan, and in data interpretation.
For a successful translation of nanoparticles to the clinics,
several features need to be considered. First of all, the attributes
and characteristics of nanoparticle therapeutics need to be strictly
and rigorously defined. Throughout the literature, it is apparent
that the biodistribution and pharmacokinetics is largely dependent
on the nanomaterial. Thus necessary measures need to be done to
examine possible toxic effects of each nanoparticle fabricated.
Even though there are several reports stating ‘‘naked’’ gold
nanoparticles are biologically inert (evident since its medicinal
use in antiquity), the capping agents may change the toxic profile
of the particle. Similarly, the hydrodynamic diameter and surface
charge may also affect the efficacy of the nanoparticle. The
increasing surface size as wellaschargemodulationaffectsthe
cell-nanoparticle dynamics.
32,140,170
Furthermore, the increase in
hydrophobicity of the nanoparticle surface is directly related to the
toxic effect in living systems.
268
The application of nanoparticles in medicine is an emerging
field with the potential to have a positive effect on human
healthcare. Although more research is necessary, nanotechnology
can play an intimate role in individualized medicine. Since they
show fundamentally new properties at the atomic and supra-
molecular scales (1–100 nm), novel molecular architectures can be
fabricated with a high degree of precision and flexibility. Owning
to these intrinsic properties, noble metal nanoparticles can be fine-
tuned to be effective therapeutics and diagnostic agents.
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