1. health and fitness
2. disease
3. cancer

APPLICATION OF GOLD NANOPARTICLES IN
DIAGNOSIS
AND TREATMENT OF CANCER CELLS
BY
CHUNG CHEE LOONG
SCHOOL OF ARTS AND SCIENCE
TUNKU ABDUL RAHMAN COLLEGE
KUALA LUMPUR
2010/2011
APPLICATION OF GOLD NANOPARTICLES IN DIAGNOSIS
AND TREATMENT OF CANCER CELLS
By
CHUNG CHEE LOONG
A project report handed to the School of Arts & Science in partial fulfilment of the
requirement for the Bachelor of Science, Campbell University, USA and Advanced
diploma in science, Tunku Abdul Rahman College, Kuala Lumpur
2010/2011
ACKNOWLEDGEMENTS
I hereby would like to thank my supervisor, Ms Chia Meow Lin for her help on my final
year project. She had been very generous and helpful in guiding me how to complete
my thesis. She had never put stress on me during this period to complete my thesis and
with the guidance I was able to do my thesis on time. Besides that, I would like to thank
my family for giving me moral support and my friends who had given ideas to me for
my thesis.
Without all who care and love, I would not able to complete this thesis smoothly. I
appreciate what they had done. Once again, thanks.
i
Contents
Page
ACKNOWLEDGEMENTS
Contents
List of Tables
List of Figures
List of Symbols and Abbreviations
Abstract
1.0 Introduction
I
II-III
IV
V-VII
VIII-IX
X-XI
1-4
2.0 Synthesis of gold nanoparticles
2.1 Synthesis of gold nanorods
6-7
2.2 Synthesis of gold nanoshells
7-8
2.3 Synthesis of gold nanospheres
2.4 Synthesis of gold nanocages
9
9-11
3.0 Applications of gold nanoparticles for cancer diagnosis
3.1In vitro assay
13-18
3.2 In vivo imaging
25-29
4.0 Implementations of gold nanoparticles in cancer therapies
4.1 Drug carriers and anchoring ligands
34-39
4.2 Photothermal therapy
43-46
5.0 Impact of gold nanoparticles on the diagnosis
and therapeutics in cancer
6.0 Future direction and Conclusion
49-52
57
ii
List of references
58-64
iii
List of Tables
Table
1 Description and advantages of QDs, AuNPs and
superparamagnetic nanoparticles
Page
20
2 P-values for average volume change in HSC-3 tumors following near-infrared PPTT
by 808 nm irradiation of pegylated gold nanorods
47
3 Bioaccumulation of GNPs with respect to the total injected does in different organs
( % ID/organ)
53
4 Biochemical parameters in the serum of mice treated with GNPs
53
5 Different parameters compared with the control and gold treated
55
iv
List of figures
Figure
An example of a cancer cell
1
2(a)
(b)
Page
5
Nanorods
Nanospheres
(c) Nanocages
(d) Surface Enhance Raman Scattering
3
5
Diagram illustration of HGNs for Surface enhanced-Raman scattering (SERS)
imaging of cancer cells.
4
A summary of some current researched applications of gold nanoparticles.
5
Transmission Electron Microscope images
12
19
(a)PEG-uPA-NR incubated with 400 nM uP
(b)PEG-uPA-NR incubated without uPA
21
6
The relative absorbance (%) is directly proportional to the time (min).
21
7
Accumulation of the PEG–peptide-modified gold nanorods in tumor.
22
8
The GNP-based assay for cancer diagnosis.
22
9
Applications of GNP assay for cancer diagnosis.
23
10
Diagram illustration of heparin-immobilized gold nanoparticles (AuNP-HHep) for
metastatic cancer cell detection.
23
11
Detection of metastatic states of tumor cells.
24
12
Fluorescence microscopic image of cancer cells.
30
13
Cancer cell diagnostics using dark field light scattering imaging of spherical gold
nanoparticles.
14
30
Cancer cell diagnostics using dark field light scattering imaging of
gold nanorods.
30
v
15
(a) Histological sections of silver stained tumor xenografts from mice intravenously
(tail) injected with 100µl of 10mM PBS following 24 hour circulation and
pegylated gold nanorods.
(b) 2 hour
(c) 6 hour
(d) 24 hour accumulation
16
The high and low resolution optical images of SiHa cells labeled with
anti-EGFR/gold conjugates.
17
19
The structure of platinum complexes.
20
Schematic illustration of in vivo targeting of the nanoconjugate
and its therapeutic efficacy.
22a
33
Transmittance and reflectance images of engineered tissue constructs
labeled with anti-EGFR/gold conjugates.
21
32
Laser scanning confocal reflectance and confocal fluorescence images
of precancerous cells labeled with anti-EGFR/gold conjugates.
18
31
Diagram illustration of the targeted apoptotic cancer cell death.
33
40
40
41
Cell proliferation of B16F10 and A549 was determined by trypan
blue exclusion assay after incubation with AuNP-Hep or
AuNP-Hep/PEG-RGD for 3 day.
22b
41
Diagram illustrating cellular caspase-3 activity of B16F10
(αvβ3 integrin positive cells) and A549 (αvβ3 integrin negative cells)
after treatment of AuNP-Hep lacking PEG-RGD or AuNP-Hep/PEG-RGD.
22c
Confocal microscopic images of B16F10 and A549 cells following incubation
with AuNP-Hep/PEG-RGD.
23
42
42
Average change in tumor volume for HSC-3 xenografts following near-infrared
PPTT treatment by control (♦), intravenous (■), and direct (●) injection
of pegylated gold nanorods.
47
vi
24
Laser photothermolysis (800 nm) of superficial rat tissue with intradermal
injection of gold nanoparticle.
25
48
Thermograms of rat’s skin surface with different depth injection of nanoshells
after laser irradiation during 30 s.
48
26
Histological analysis of various organs after GNPs treatment.
54
26
Toxicity studies of gold nanoparticles in mouse organs.
56
vii
List of symbols and abbreviations
AgCl
AgNO3
AuNPs
AuNP-HHep
BSA
CTAB
CoCl2
CV
C225
DDS
DHLA
DNA
EDC
EG
EGFR
Fe3O4
Gem
GF-AAS
GNPs
HaCat
HAuCl4
HER2
HeLa
HPLC
HGNs
HSC
HOC
ICP-MS
K2CO3
MCF-7
MNPs
mRNA
N2
NaBH4
Na2S
NaHS
NCR
NIR
NIH3T3
NH4OH
OD
PEG
PBS
PHNPs
PKC
PPTT
PS
Silver chloride
Silver nitrate
Gold nanoparticles
Heparin immobilized gold nanoparticles
Bovine serum albumin
Cetyltrimethylammonium bromide
Cobalt(II) chloride
Crystal violet
Cetuximab
Drug delivery system
Dihydrolipoic acid
Deoxyribonucleic acid
1-ethyl-3-(3-dimethylaminopropyl)
Ethylene glycol
Epidermal growth factor receptor
Iron(III) oxide
Gemcitabine
Graphite furnace atomic absorption spectrometry
Gold nanoparticles
Human keratinocyte
Chloroauric acid
Human epidermal growth factor receptor 2
High metastatic human epithelial carcinoma cell
High performance liquid chromatography
Hollow gold nanospheres
Hematopoietic stem cell
Human ovarian carcinoma
Inductively coupled plasma mass spectrometry
Potassium carbonate
Low metastatic human breast adenocarcinoma cell
Metal nanoparticles
Messenger ribonucleic acid
Nitrogen gas
Sodium borohydride
Sodium sulphide
Sodium hydrosulfide
National cancer registry
Near-infrared laser
Non cancerous cell
Ammonium hydroxide
Optical density
polyethylene glycol
Phosphate buffered saline
Porous hollow nanoparticles
Protein kinase C
plasmonic photothermal therapy
Polystyrene
viii
PVP
QDs
RT-PCR
RTK
RGD
SERS
SEM
SeV
SPR
THPC
TEM
TNF-α
TK
uPA
poly(vinyl pyrrolidone)
Quantum dots
Real time-polymerase chain reaction
Receptor tyrosine-kinase
Arginine-glycine-aspartic acid
Surface-enhanced Raman scattering
Scanning electron microscope
Sendai virus
Surface Plasmon resonance
tatrakis(hydroxymethyl)phosphonium chloride
Transmission electron microscopy
Tumor-necrosis factor-alpha
Tyrosine-kinase
Urokinase-plasminogen activator
ix
Abstract
Conventional strategies for cancer intervention include surgery, chemotherapy, and
radiation therapy. Recently, nanoparticles offer a great promise in biomedical
application which can be used as novel diagnostic and therapeutic method. The medical
application of nanoparticles takes advantage from the very small size which allows the
modified nanoparticles to be conjugated with different kinds of therapeutic drug to
penetrate the tumors with a high degree of specificity. A lot of advantages can be
achieved by using the nanoparticle-mediated targeted delivery of drugs. For example, it
can significantly reduce the dosage of the drugs, better specificity, better bioavailability
and low toxicities. In this thesis, the main aim is to introduce another new method for
cancer treatment by using the gold nanoparticles conjugated with therapeutics drug or
gold nanoparticle-based photothermal therapy to destroy the target cells instead of using
the conventional methods.
Gold nanoparticles can be used for molecular imaging (in vivo) where the gold
nanoparticles conjugated with anti-EGFR bind specifically to cancer cells and the
nanoparticles scatter light strongly in the visible region which can be distinguished from
the healthy cells. There can be no doubt that gold nanoparticles can be used for
molecular diagnosis. Besides, the gold nanoparticles conjugated with different kinds of
drug or protein such as polyethylene glycol, aptamer, heparin, protein kinase C-alpha
can deliver the ligands to target specifically to the cancer cells. Eventually, this will
induce some reactions to take place and thus enable easy and more efficient detection
and identification of cancer cells through transmission electron microscopy, scanning
electron microscopy or fluorescence image analyzer.
In addition, gold nanoparticles have been employed as drug delivery vehicles.
Combination of gold nanoparticles with anticancer drugs can reduce the dosage of the
x
anticancer drugs while providing better specificity, low toxicities and enhanced efficacy.
The ideal therapy is to deliver multiple drugs specifically to the tumor and induce the
apoptosis effect, blocking the receptor or reduce the growth of cancer cell. The most
common anticancer drugs are gemcitabine, cetuximab, heparin, uPA, trastuzumab and
so on. Besides, gold nanoparticles have unique properties to act as a photothermal agent
to destroy the cancer cell when the gold nanoparticles absorbed some light energy or
after laser radiation.
It was also suggested that the bioaccumulation and toxicity of gold nanoparticles
after repeated administration in animals did not show any increment of the dose and
toxic effects. It is certain that the gold nanoparticles can be safely used.
Ultimately, the gold nanoparticles will undoubtedly be one of the most critical
advancements to improve the biomedical application in the cancer diagnostics and
therapeutics. Hence, gold nanoparticles have high potentials and can be widely applied
in diverse areas such as anticancer drug delivery, photothermal agents, and agents for in
vivo imaging and in vitro assay.
xi
1.0 Introduction
In Malaysia, cancer is a foremost public health problem worldwide. According
to the Cancer Incidence in Peninsular Malaysia 2004-2006 report, published by the
National Cancer Registry (NCR), around 67700 new cases about cancer were
successfully diagnosed among 43.7% of males and 56.3% of females. The annual crude
rate of cancer cases for males found out is 100.2 per cent per 100,000 population, and
132.1 per cent per 100,000 for females. (Lim and Halimah., 2004-2006). Moreover,
cancer can be developed in any ages. According to the report that is stated, the median
age at the diagnostic stage for cancer in Malaysian can be divided into two groups. The
Malaysian males were 59 years and 53 years for Malaysian females. The top five
cancers that can be occurred in among the Malaysia children (0-14 years) were
leukemia, cancers of the brain, lymphoma, cancers of the connective tissue and kidney.
In the group of young adults (15-49 years old), the common cancers were nasopharynx,
leukemia, lymphoma, lung, colon and rectum in men, and cancers for the breast, cervix,
ovary, uterus, thyroid gland and leukemia in women. In older people (50 years old and
above), cancers of the lung, colon, rectum, nasopharynx, prostate and stomach were
predominant among men, while cancers of the breast, cervix, colon, uterus, lung and
rectum occurred commonly in women. Among these cancers, the top five most frequent
cancers occurred in Malaysian are breast cancer following by lung cancer, colon and
rectum cancer, cervical cancer and leukemia (Lim and Halimah., 2004). Therefore, new
treatment strategies are urgently required to combat cancers.
An example of a
cancerous cell is shown in Figure 1. Currently an active field in cancer research is
nanomedicine.
Nanotechnology is a multidisciplinary field that involves the biology, chemistry,
engineering and medicine of functional systems at the molecular scale (1-100nm or
1
even smaller). In our human cells, biological molecules such as amino acids, receptor,
antibodies and enzymes are typically larger than the nanoparticles which are several
hundred nanometers smaller in size. Due to this advantage, the nano-sized particles can
be used in a wide range of biomedical application which is detection, diagnosis of
cancer cell and treatment of cancers (Cai and Chen., 2007). Furthermore, based on the
interaction of nanoscale devices with molecular and cellular components, the cancer
nanotechnology is widely used in diagnosis and cancer therapy. The surface modified
nanoparticles conjugated with therapeutic drugs can make a way into the malignant
cells with high degree of specificity (Patra et al., 2008; Cuenca et al., 2006). The crucial
point in the cancer nanotechnology is targeted delivery in a localized way. Furthermore,
in cancer therapeutics, nanoparticles-mediated targeted delivery of drugs might
significantly decrease the quantity of the drugs with advanced specificity, less toxicities,
and improved bioavailability. In this review, we will focus on the biomedical
applications of gold nanoparticles (AuNPs) in the diagnosis and treatment for cancers.
Gold nanoparticles can be synthesized with well-controlled size distribution and with
stunning precision in various sizes, structures, composite and shapes such as nanoshells,
nanorods, nanocages and so on. There are many types of gold nanoparticles based on
the size, shape and physical properties. They can be divided into gold nanorods, gold
nanospheres, gold nanocages, gold nanoshells, and gold surface enhanced Raman
scattering (SERS) (Figure 2). Each type of gold nanoparticles has its own significant
function to diagnose and treat the malignancy cells in our body. Moreover, each of them
appeared in different sizes (example gold nanorod is around 20nm and gold nanosphere
has a diameter around 2nm).
Long time ago, gold nanoparticles were actually called colloids, emulsion, or
aerosols, and included many natural and man-made suspensions (Kreuter., 2007).
2
Colloidal radioactive gold or gold salts were used as therapeutic agents for intraarticular injection in people having a disease like rheumatoid arthritis and for treating
cancers nowadays. A result of interdisciplinary biomedical research, gold nanoparticles
can be applied in a wide range of functions including cancer therapy, molecular
diagnosis, molecular imaging, targeted therapy, and bioinformatics. There are quite a lot
of reasons for the use of gold nanoparticles in nanomedicine including the ease to
produce in many forms or shapes for example it can be in spheres, rods, shells, cubic
and so on by using templates and modifying the reaction conditions. In addition, it is
easy to synthesize gold nanoparticles by several safe, commercially cheap, easy and
reliable methods. Nanoparticles also can be synthesized in the sizes within the ranges of
2-100nm by changing the reaction parameters. Due to the presence of negative charge
on the surface of gold nanoparticles, they are highly reactive and this surface property
facilitates the synthetic advancement using numerous biomolecules. Moreover, the
surface of gold nanoparticles has a strong interaction with the amine-containing
molecules in human body like amino acid, enzyme, DNA, organic molecules and so on.
So the surface of gold nanoparticles can be easily modified (Bhattacharya et al., 2007).
Gold nanoparticles are well-known for being highly biocompatible and they are less
toxic to the human body when interacting with the tumor cell. Besides, they are good
biosensors because of the structure of their unique optical as well as of their electronic
behavior (Hainfiels and Slatkin., 2002). The synthesis of gold nanoparticles can be
referred to many types of the gold nanoparticles where it can be synthesized with wellcontrolled size distribution, or with stunning precision for example like nanoshells,
nanorods, nanocages and so on. Moreover, there is a way to synthesize each of the gold
nanoparticles in the particular form. The development in gold nanotechnology has the
advantages of enhancing conventional methods as well as promoting the development
3
of novel approaches for detection and treatment of tumour cells. For the diagnostic
purposes, methods can be divided into in vitro and in vivo. As an example of the in vitro
method, gold nanoparticles are conjugated to anti-epidermal growth factor receptor
(EGFR) for the early diagnosis of oral cancer cell (Kah et al., 2007). For the in vivo
method, for example, the surface enhance Raman scattering (SERS) nanoparticles can
target a tumor marker such as epidermal growth factor receptor (EGFR) on human
carcinoma cells and in tumor-xenograft mice.
Diagnosis is done with Raman
spectroscopy by scanning and the imaging (Qian et al., 2008). In this summary,
therapeutics for cancer cells is divided into two parts which is photothermal therapy for
the destruction of carcinoma tissue in particular organs and the use of gold nanoparticle
as drug delivery vehicles (Huang et al., 2010, Visaria et al., 2006). Photothermal
contrast agents are used in photothermal therapy in which the photon energy is
transmitted into heat sufficient to kill tumor tissue in the targeted organ via thermal
effects (Huang et al., 2010). Targeted therapy has been brought to the forefront of
cancer management. Targeted delivery system can be prepared using the polyethylene
glycol-coated (PEG) gold nanoparticles loaded with tumor-necrosis factor-alpha (TNFα to deal with the tumor tissue and reduce the systemic toxicity (Visaria et al., 2006).
The objective of this review is to learn how the gold nanoparticles become a
cutting edge technology for use in the diagnosis and therapeutics of cancer cells. Further
discussion will highlight the improvement afforded. Nanotechnology has offered a
faster and more efficient way for cellular imaging, detection, diagnosis and therapy in
modern biomedical research.
4
Figure 1: An example of a cancer cell.
Source: www.conspiracyplanet.com
Figure 2: (a) nanorods (www.physorg.com) ;(b) nanospheres
(www.seashelltech.com) ;(c) nanocages (bit.bme.jhu.edu) ;(d) SERS
(www.rsc.org/Publishing/Journals)
5
2.0 Synthesis of gold nanoparticles
Different types of method are used to prepare gold nanoparticles of various shape, size,
and physical properties. In this review, gold nanorods, gold nanoshells, gold nanocages
and gold nanospheres, associated with the surface-enhanced Raman scattering particles
(SERS) will be further discussed.
2.1 Synthesis of Gold nanorods
There are a lot of methods to synthesize gold nanorods. For example, synthesis of gold
nanorods using electrochemical synthesis by addition organic solvent has been done
(Huang et al., 2007) and seed-mediated growth was introduced by Nikoobakht and Elsayed (2003) and this is the most frequently used gold nanorod preparation. In the seedmediated growth metho, firstly, HAuCl4 was added to cetyltrimethylammonium
bromide (CTAB). Six hundred microlitres of ice-cold NaBH4 was added to the stirred
solution and allowed to react for several minutes, forming the pale brown gold seed
solution. Then, more HAuCl4 was added to CTAB and AgNO3. After that ascorbic acid
was added, followed by gentle mixing to form the transparent growth solution. The seed
solution was added to the unstirred growth solution and allowed to react for estimated
for a few hours. Nanorods synthesized by this method are approximately 12nm in width
and 50nm length, with a longitudinal Plasmon absorption maximum at 800nm. Gold
nanorod solutions were centrifuged twice for more than ten minutes and re-dispersed in
deionized water to remove excess CTAB molecules. The mPEG-SH was added to the
~1nM colloidal nanorod solution at a final concentration. Rods were sonicated
overnight and centrifuged for more than ten minutes and re-dispersed in deionized water
to remove non-specifically bound PEG molecules. The pegylated gold nanorods were
again centrifuged for a few minutes and re-dispersed in the phosphate-buffered saline to
6
the desired optical density at 800nm. Extinction spectra of the pegylated nanorod saline
suspensions showed no peak shift, broadening or reduction over a 1-week period prior
to injection (Dickerson et al., 2008).
Earlier, Jane et al. (2001) stated that gold
nanorods can be produced by seeds of gold salt which interact strongly with the
reducing agent like NaBH4 in this experiment. The nucleation sites for nanorods are
provided by the gold seeds and then added to the growth solution of gold salt to interact
with a weak reducing agent like CTAB. These seeds which served as the nucleation
sites for nanorods are then mixed with a growth solution of gold salt with a weak
reducing agent such as the ascorbic acid and hexadecyltrimethylammonium bromide
(Jane et al., 2001). In this process, the weak reducing agent is slightly different with the
method by Dickerson et al. (2008).
2.2 Synthesis of Gold nanoshells
The gold nanoshells have sort of different ways to synthesize it. Using the surface
plasmon resonance (SPR) peak, the gold nanoshells can be designed and fabricated by
changing the composition and dimensions of the layers (Huang et al., 2010). In this
review,
the
synthesis
of
the
gold
nanoshells
using
the
tetrakis(hydroxymethyl)phosphonium chloride (THPC)-gold-decorated polystyrene
particles which are obtained from the attachment of gold nanoparticles to functionalized
polystyrene particles will be discussed. The gold nanoshells can be synthesized by using
the THPC-gold-decorated polystyrene (PS) particles. The THPC-gold particles attached
to the PS spheres were used as nucleation sites for the further reduction of gold,
resulting in deposition of a thin layer of gold on the nanoparticle surface. The gold shell
can be grown on the nanoparticle surface after a solution of gold hydroxide was
prepared. Potassium carbonate (K2CO3) was dissolved in HPLC grade water in reaction
7
flasks. Then, the solution was stirred for a few minutes. Next, HAuCl4 was added to the
solution. Initially, the mixture will turn light yellow and slowly became colorless after
half an hour. This observation indicated that gold hydroxide was formed. The resulting
solution was aged for a day in the dark before it was used. The gold-decorated
polystyrene particles were mixed with gold hydroxide solution and then stirred gently.
To begin the reduction of gold on to the gold-decorated polystyrene particles, a small
amount of formaldehyde was added to the stirred mixture. This step was followed by
the addition of ammonium hydroxide to adjust the pH of the solution to be slightly basic.
This typically required a few drops of NH4OH solution. The solution was gently stirred
for approximately more than seven hours. During this time, the solution changed from
colorless to light blue, which is an indication of shell formation. The solution was then
allowed to stand for another one hour. By varying the volume ratio of gold hydroxide to
the polystyrene particle solutions, the thickness of gold nanoshell could be varied. To
investigate the effect of the reducing agent on the gold nanoshell morphology and
formation of free gold nanoparticles, other reducing agents such as sodium borohydride
and hydroxylamine hydrochloride were also used to deposit gold from solution onto the
THPC-gold-decorated polystyrene solutions (Yong et al., 2006). Another method with
gold nanoshells can be produced by a silica core around 100nm and a thin shell of gold
about few nanometers. The shell was formed by aging the gold clusters attached on the
silicon core. The red shift can explained as the results of the hybridization of the
plasmons of the inner sphere and outer cavity. The SPR wavelength of gold
nanoparticles can be controlled by changing the shell thickness (Huang et al., 2010).
Compared to the method that we discussed, the synthesis of gold nanoshells using the
above method is more complicated because it involves the reduction of HAuCl4 to form
gold nanoparticles before attaching to the polystyrene (PS) particles. The shell grows
8
when formaldehyde and gold hydroxides react simultaneously while the reduction
occurred to form gold nanoshells. However, this method is typically used to synthesize
the gold nanoshells.
2.3 Synthesis of Gold nanospheres
Hollow gold nanospheres (HGNs) can be prepared by cobalt nanoparticles synthesized
by reducing CoCl2 with NaBH4 under a N2 purging condition and used as templates of
HGNs. HAuCl4 was added a few times in small amount of aliquots. Here, gold atoms
were nucleated and grown up to small shells around the cobalt template. When the
solution was exposed to an ambient condition by stopping N2 purging, cobalt was
completely dissolved and a hollow interior was formed. At this stage, the color of the
solution changed from dark brown to deep blue. The wall thickness could be controlled
by changing the concentration of HAuCl4. Figure 3(a) shows a schematic illustration for
the synthetic production of HGNs whereby Figure 3(b) shows their transmission
electron microscopy (TEM) images. Lee and colleagues (2009) measured the diameter
of the HGNs and stated that it was relatively bigger than the wall thickness of the HGNs.
Antibody was conjugated with HGNs where the Raman reporter CV (crystal violet) was
adsorbed on the surface of HGN by electrostatic interaction. DHLA was used for
antibody conjugation, and mercaptoethanol was used to occupy the remaining
adsorption sites on the surface. The terminal –COOH group of DHLA was activated
using NHS and EDC for antibody conjugation (Figure 3(c)) (Lee et al., 2009).
2.4 Synthesis of Gold nanocages
To produce gold nanocages, silver nanostructures need to be synthesized first. The
silver nanoparticles of different shapes have been synthesized. Single-crystal nanocubes
9
have become the most exciting and useful structure particularly for the production of
gold nanocages. Gold nanocages have a lot of useful biomedical applications such as in
optical imaging contrast enhancement and photothermal treatment in cancer therapy
(Siekkinen et al., 2006, Chen et al., 2006).
In a typical synthesis of silver nanocubes, small amounts of ethylene glycol (EG) were
heated by stirring with a Tefloncoated magnetic stirring bar for about 1 hour in a glass
vial. During the EG was heated in the experiment, the EG solutions consist of AgNO 3
compound and poly (vinyl pyrrolidone) (PVP) were prepared. Solutions of Na2S or
NaHS in EG were also prepared for half an hour prior to injection. Brusquely after
putting the sulfide solution, a micro-pipettor was used. Both the AgNO3 and PVP
solutions were sequentially injected to the solution. A clear and colorless solution
immediately changed to purple-black, followed instantly by a transparent bright yellow
color after silver nitrate was added. The appearance of yellow color indicates the
formation of small silver particles. The solution changed to an orange–yellow color and
some silver nanoparticles were seen to deposit on the wall of the vial after a few
minutes into the reaction. If experiment still to be continued, the solution turned to a
lighter, whitish-brown color but remained opaque. The last step is the acetone was used
to dilute the final product and allowed undergoes centrifugation, washed with waster,
and then suspended in water for future use. Both Na2S and NaHS gave similar results
(Siekkinen et al., 2006). A fixed amount of the as-synthesized silver nanocubes was
dispersed in the solution (PVP and water) and undergoes magnetic stirring and then
heated to boil for ten minutes in a synthesis of gold nanocages. Using a syringe pump, a
specific amount of HAuCl4 in certain concentration was added to the flask under
magnetic stirring. The solution was heated for another ten minutes until the color of the
10
system was stable. The sample was centrifuged and washed with saturated NaCl
solution to remove excess AgCl and then washed again with water several times to
remove excess PVP and NaCl before characterization by SEM and TEM when the
solution was cooled down to room temperature, (Siekkinen et al., 2006). The silver
nanocubes have been demonstrated as a sacrificial template to generate gold nanocages
in this experiment. Some of the sulfur is straightly to adsorb on the surface of assynthesized cubes due to strong interaction between sulfur and silver. However, this
sulfur did not interfere with the galvanic replacement reaction between Ag and HAuCl4:
3Ag + HAuCl4 -Æ Au + HCl + 3AgCl
(Siekkinen et al., 2006).
11
Figure 3 Diagram illustration of HGNs for Surface enhanced-raman scattering (SERS)
imaging of cancer cells: (a) experimental procedure that generates HGNs by templating
against a cobalt nanoparticle; (b) TEM images of HGNs prepared using the template of
cobalt nanoparticles; (c) antibody conjugation onto HGNs which involved Raman
reporter CV, DHLA, and Mercaptoethanol (Lee et al, 2009).
Source: Lee et al., 2009
12
3.0 Application of gold nanoparticles for diagnostics in cancer
3.1 In vitro assay
These are thrilled times, with constant improvement in the recital of current diagnostic
methods for cancer or maybe other diseases. The use of nanoparticles provides the
advantages to help promote in vitro diagnostics to the medical breakthroughs. Quantum
dots (QDs), gold nanoparticles (AuNPs), and superparamagnetic nanoparticles are the
most promising nanoparticles for in vitro diagnostic applications. Oligonucleotide and
antibodies conjugated with nanoparticles have been report for protein detection. A lot of
studies have discussed about the advantages of QDs, AuNPs and supermagnetic
nanoparticles (Table 1). However, in this review, gold nanoparticles will be discussed
more. One of the unique optical properties of gold nanoparticles is a phenomenon
known as surface Plasmon resonance (SPR). According to Azzazy and Mansour (2009),
when an electromagnetic radiation of a certain wavelength much smaller than the
diameter of AuNPs, hit the particles and induces coherent, resonant oscillations of the
metal electrons across the nanoparticles SPR occurred and this SPR will result in strong
optical absorbance and scattering properties of the AuNPs. Furthermore, Azzazy and his
co-workers (2009), also mentioned that the AuNPs had a strong absorption which can
be used in colorimetric detection of analytes by measuring changes in the refractive
index of AuNPs environment caused by adsorption of target analytes (Azzazy et al.,
2009).
There are numerous examples (Figure 4) of the usage of gold nanoparticles to
diagnose the cancer cells conjugated with different drugs or proteins. The major drugs
and proteins that commonly used are polyethylene glycol, aptamer, protein kinase C and
heparinase. As suggested by Niidome and co-workers (2010), polyethylene glycol
13
(PEG)-peptide-modified gold nanorods which can bind with hydrophobic interaction to
tumor cells in vitro and the PEG chains that are released from the surface of the gold
can be detected by transmission electron microscopy (TEM) (Figures 5a and 5b). PEGpeptide-modified gold nanorods contained peptide substrate for urokinase-type
plasminogen activator (uPA), which is expressed on malignant tumors, and could form
aggregates in response to uPA activity. The uPA-expressing cells bind with the gold
nanorods in vitro and the binding was also mediated by the cleavage of the peptide
component. The presence of uPA in tumor homogenate could be detected using the
PEG-peptide-modified gold nanorods was investigated by Niidome et al. (2010). In the
study, Mouse T41 breast carcinoma cells, that are known to express a high level of uPA,
were subcutaneously inoculated into mice. After inoculation a homogenate was mixed
with gold nanorods and the decrease in absorption at 900nm was monitored (Figure 6).
Niidome et al. (2010) found a larger amount of the gold detected in the tumor as
compared with the control after intravenous injection of the gold nanorods (Figure 7).
However, the key factor is the density of the PEG-peptide on the surface of the gold
where it did not only maintain the stability of the gold nanorods under physiological
conditions, but also secured the accessibility of uPA to the peptide component covered
by the PEG layer. When the delivery system is applied to tumor imaging and
photothermal tumor therapy, the absorption of the gold nanorods at the near infrared
light region should be maintained. Lots of molecules such as serum and extracellular
matrix proteins in the tumor tissue non-specifically bind to the surface of the gold
nanorods immediately after the peptide cleavage. As a result, aggregation that decreases
the absorption of the nanorods would be hindered (Niidome et al., 2010).
14
A different approach was attempted earlier by Medley and coworkers in 2008.
Medley et al. (2008) reported the use of aptamer-conjugated gold nanoparticles. They
took advantage of the selectivity of conjugation chemistry and spectroscopic benefits of
gold nanoparticles to allow for sensitive detection of cancer cells. When indicating if
target cells were present, the samples would change color; while there is no change of
color for the non-target samples. In addition, different types of target and control cells
based on the aptamer used can be used and so widens the capability of the assay for
diseased cells detection. On the basis of these qualities, aptamer-conjugated gold
nanoparticles could become a powerful instrument for point of care diagnostics (Medley
et al., 2008). The aptamer-conjugated gold nanoparticles in vitro assay is different from
the method of PEG-peptide-modified gold nanorods which are expected to bind strongly
to the tumor cells. However, both in vitro assays are very useful for the diagnosis of the
cancer cells.
Kang et al. (2010) also developed a novel gold nanoparticle (GNP)-based
colorimetric assay for cancer diagnosis. There is a fundamental difference between the
GNP-based colorimetric assay compare with Niidome et al. (2010) method and Medley
et al. (2008) method. This system is based on the noncrosslinking aggregation
mechanism with a cationic protein kinase C (PKC) α-specific peptide substrate, which
is used as a coagulant of citrate-coated GNP with anionic surface changes (Figure 8).
After that, phosphorylation of the peptide substrate by the PKCα enzyme could change
the color of GNP dispersions. When the colors changed to red indicated that
phosphorylation of the peptide by PKCα enzyme on GNP aggregation was happened.
While for non-phosphorylation, the color of the GNP changed from the red to blue,
indicating the GNP aggregation had occurred with the addition of samples treated with
15
normal tissue lysates. Based on their results, the concentration of protein in the cell used
for the method showed no direct effect on the GNP dispersions. The examination of the
effect of bovine serum did not show any GNP aggregation. However, lysozyme induced
their aggregation in low concentration but not in the high concentration (1.0 or
<2.0µg/ml). Kang et al. (2010) used this system to the diagnosis of breast cancers.
Normal human breast and human breast cancer tissue were collected and applied with
GNP assay. They found that breast cancer tissue has higher levels of activated PKCα
than normal breast tissues. After reaction of the peptide substrate with lysate, the
solution was mixed with the GNP dispersions. As shown in Figure 9, a lower OD level
was identified from cancer tissue lysates compared with those in normal tissue lysates.
That is no any evidence of PKCα activity in normal breast tissue. Moreover, the system
also showed that clear red or purple colors from most of the cancer samples (nearly
67%), but failed to detect red purple from the other cancer samples. Based on these
results, the GNP assay developed here can be used for initial screening during cancer
diagnosis was suggested. This study is the first report on the application of the GNPbased colorimetric assay to diagnosis of cancer and the GNP assay for a combination of
protein kinases may provide a more detailed diagnosis (Kang et al., 2010).
Another approach suggests that the gold nanoparticles conjugated with another
drug are introduced by Lee and co-workers (2010) which are developed a new class of
theragnostic (therapy and diagnostic) nanomaterials which is heparin immobilized gold
nanoparticles (AuNP-HHep) for metastatic cancer cell imaging and apoptosis (Figure
10). The AuNP-HHep is the surface of gold nanoparticles personalized with fluorescent
dye labeled heparin molecules to detect a metastatic stage of cancer cells that overexpress heparin-degrading enzymes. As a result, AuNP-HHep showed a characteristic
16
of fluorescence quenching. With increasing haparinase unit, it can be seen that the
stronger fluorescence recovery can be gradually attained after incubation. The
fluorescence recovery was caused by heparinase-induced, specific cleavage of heparin
chains that were anchored onto the surface of AuNPs. Clear fluorescence signals were
emitted by the cleaved heparin fragments as the conjugated fluorescence dyes in the
released heparin chain were not quenched any more by AuNPs. The study proved that
the expression levels of heparinase could be easily visualized by the fluorescence image
analyzer. In the Lee and co-workers (2010) study, they incubated AuNP-HHep
nanoparticles with three different metastatic activities cancer cell which were divided
into high metastatic human epithelial carcinoma cells (HeLa), low metastatic human
breast adenocarcinoma cells (MCF-7), and non-cancerous cells (NIH3T3). Another test
was done to confirm that the heparinase activity of cancer cells corresponds with their
increased metastatic potential (Figure 11). High metastatic cells show greater and upregulated heparinase mRNA levels than low metastatic cells. The three cells were
evaluated by photoluminescence spectrophotometry to prove that AuNP-HHep
nanoparticles could detect the metastatic state of cancer cells by haparinase/heparinaseinduced fluorescence recovery (Figure 11). These three cells exhibited different levels
of fluorescence recovery (Lee et al., 2010).
In brief, the application of gold nanoparticles to diagnostics in cancer cells can
be done by several of gold nanoparticles conjugated with different of the drug or protein
to diagnose the cancer cell. It can be found out that using the PEG-peptide-modified
gold nanorods and AuNP-HHep nanoparticles is more constructive due to both of the
gold nanoparticles are valuable for detection of the cancer cell and destruction of tumor
17
tissue compare with others. However, the GNP-based colorimetric assay can provide a
more detailed diagnosis for cancer cells compared with the other methods.
18
Application of
gold nanoparticles
to diagnostics in
cancer (In vitro
assay)
Polyethlene glycol
(PEG)-peptidemodified gold
nanorods bind with
tumor cells
(Niidome et al.,
2010).
Aptamerconjugated gold
nanoparticles to
detect the cancer
cell (Medley et al.,
2008).
Protein kinase C
(PKC) α-specific
peptide substrate,
bind with citratecoated GNP (Kang
et al., 2010).
AuNP-HHep to
detect a cancer
cells (overexpressed heparindegrading
enzymes) (Lee et
al., 2010).
Results can be
detected by
transmission
electron
microscopy
(TEM)
Results can be
observed by the
color changes and
the absorbance
reading
The cancer cells
can be identified
by changes of the
color &OD
Results can be
visualized by the
fluorescence image
analyzer.
Figure 4: A summary of some current researched applications of gold nanoparticles.
19
Table 1
Description and advantages of QDs, AuNPs and superparamagnetic nanoparticles
Nanoparticles
Structure
Signal detection
methods
Benefits
References
QDs
-Nanocrystals
typically
composed of a
core
semiconductor
e.g. cadmium
selenide (CdSe)
which is
enclosed in a
shell of another
semiconductor
with a larger
spectral bandgap
e.g. zinc sulfide
(ZnS)
-A third silica
shell can be
added to the
nanocrystal for
water solubility.
Fluorescence
measurement using a
fluorometer,
fluorescence
microscopy,
or wide-field
epifluorescence
microscopy.
-Broad excitation
range
-Narrow
emission bands
-Photostability
-Optical
tunability
(control of
emission
wavelength
by size control)
-Multiplexing
Azzazy et al,
2006; Jain, 2007;
Alivisators et al,
2005; Goldman
et al, 2006
AuNPs
Either a
dielectric core
(normally gold
sulfide
or silica)
enclosed within
a thin gold shell
(known
as nano-shells)
or simply a
spherical gold
nanoparticle.
Several detection
methods can be used
such
as colorimetric,
scanometric, electronic
and
electrochemical,
scattered light, surfaceenhanced
Raman scattering.
Optical tunability
-Strong optical
signal.
Azzazy et al,
2007; Baptista et
al, 2008; Jain,
2007
Superparamagneti
c
nanoparticles
Composed of
magnetic metals
like iron, or
alloys of
different metals.
Magnetometer
-High sensitivity
due to detection
of subtle
modifications in
magnetic
character.
Jain , 2005 and
Jain, 2007
20
Figure 5a: TEM images of PEG-uPA-NR0.01 incubated with 400 nM uPA at 37 oC for
30 min. Figure 5b: TEM images of PEG-uPA-NR0.01 incubated without uPA at 37 oC
for 30 min. The PEG layer was stained with 1% phosphotungstic acid (Niidome et al.,
2010).
Source: Adapted from Niidome et al, 2010
Figure 6: Decreases in the light absorption of the PEG–peptide-modified gold nanorods
mediated by tumor homogenate. (Niidome et al., 2010).
Source: Adapted from Niidome et al, 2010
21
Figure 7: Accumulation of the PEG–peptide-modified gold nanorods in tumor. After
intravenous injection of PEG-uPA-NR0.1, PEG-uPAscr-NR0.1, PEG-uPA-NR0.01 and
PEG-uPAscr-NR0.01 into tumor-bearing mice, the mice were sacrificed at 72 h. The
tumors were collected and the amount of gold in the tumor was quantified using ICPMS. Closed bars and open bars indicate the amounts of PEG-uPA-NR and PEGuPAscrNR, respectively, in the tumor. Data represent mean values for n = 3 and the bars are
standard errors of the means. **P <0.05 (Niidome et al., 2010).
Source: Adapted from Niidome et al, 2010
Figure 8: The GNP-based assay for cancer diagnosis. This system is based on the
noncrosslinking aggregation mechanism and a cationic PKCα-specific substrate peptide
is used as a coagulant of citrate-coated GNPs with anionic surface charges (Kang et al.,
2010).
Source: Adapted from Kang et al, 2010
22
Figure 9: Application of the GNP assay for cancer diagnosis. After reaction of the
peptide substrate with normal human breast cancer tissue lysates for 1 hour, each
solution was mixed with GNP dispersions (Kang et al., 2010).
Source: Adapted from Kang et al, 2010
Figure 10: Diagram illustration of heparin-immobilized gold nanoparticles (AuNPHHep) for metastatic cancer cell detection (Lee et al., 2010).
Source: Adapted from Lee et al, 2010
23
Figure 11: Detection of metastatic state of tumor cells using high metastatic cancer cell
line (HeLa) with high expression of heparanase, low metastatic tumor cell line (MCF-7)
with low expression of heparanase, and non-tumor cell line (NIH3T3).
(Relative heparanase expression level (Hpa) of each cell line as determined by RT-PCR)
(Lee et al., 2010).
Source: Adapted from Lee et al, 2010
24
3.2 In vivo imaging
Specific and effective therapies are needed to reduce the mortality caused by many
common types of malignant tumours, sensitive diagnostic tools are urgently required to
improve the early detection of malignancies and the accuracy of tumour localization.
Another way of looking at the diagnosis of cancer cell is achieved through the
development of in vivo tumor binding, targeting and molecular imaging based on the
surface surface-enhanced Raman scattering effect of gold nanoparticles (Qian et al.,
2008). Zhang and his coworkers (2009) attempted to determine the effect of conjugation
and particle size on the stability and pharmacokinetics in mice. They injected the
tumour-bearing nude mice intravenously with pegylated gold nanparticles. Mice were
killed several hours after injection. After that, several steps were done to prepare for
staining and imaging under fluorescent microscope. Texas red and fluorescein
isothiocyanate filter sets were used to visualize CD31 and EGFR expression,
respectively, and a dark filed condenser was used to visualize AuNPs (Figure 12)
(Zhang et al., 2009). When the pegylated gold nanoparticles conjugated tumor-targeting
ligands in mice, the conjugated SERS nanoparticles were able to target tumor markers
such as the EGFR expressed on human cancer cell surface and in xenograft tumor
models (Zhang et al., 2009).
Gold nanoparticles can be excited by white light produced from a halogen lamp
for bright field imaging. For dark field microscopy, anti-EGFR conjugated AuNPs
bound specifically to the over-expressed EGFR on the cancer cell surfaces and this can
be used to distinguish them from the healthy cells (El-sayed et al., 2005) (Figure 13).
The nanoparticles scatter light strongly in the green-to-yellow region (El-sayed et al.,
2005).
Huang et al. (2006) also demonstrated that gold nanorods, via a poly
25
(stryenesulfonate) linker, can conjugate with the anti-EGFR antibodies (Figure 14). This
study successfully demonstrated that the gold nanorods can be a very useful imaging
contrast agent for cancer cell diagnosis. Gold nanorods have a similarity with the gold
nanospheres where the antibody-conjugated nanorods are specifically bound to the
cancer cells, whereas the distribution is random in the case of healthy cells (Huang et al.,
2006).
Huang and his co-workers (2007) specifically detected the human oral cancer
cells using gold nanorods conjugated to anti-EGFR antibodies. Gold nanorods
conjugated to anti-epidermal growth factor receptor (anti-EGFR) antibodies was
assembled and aligned on the human oral cancer cells. Immnoconjugated gold nanorods
and nanospheres were shown to exhibit strong Rayleigh (Mie) scattering useful for
imaging. Molecules near the nanorods on the cancer cells are found to give a Raman
spectrum that is greatly enhanced (due to the high surface plasmon field of the nanorod
assembly in which their extended surface plasmon fields overlap), sharp (due to a
homogeneous environment), and polarized (due to anisotropic alignments). These
observed properties can be used as diagnostic signatures for cancer cells (Huang et al.,
2007).
In another study, Dickerson et al. (2008) showed pegylated gold nanorod
accumulation by attenuation of near-infrared transmission using a special device assay
for in vivo imaging monitoring. The tumor-bearing mice were injected with gold
nanorods via the tail vein, and euthanized at specified time points. Following injection,
tumors were excised at varying time intervals, fixed, sectioned and stained with silver to
visualize the extent of particle loading. A positive control was used where nanorods
were directly-injected into tumors. Based on the study, the histological sections of silver
26
stained tumor xenografts from mice intravenously (tail) injected with 100µl of 10mM
PBS at the tail following 24 hour circulation were prepared as the control (Figure 15a).
The tumor sections following 2 and 6 hour of accumulation are shown in Figures 15b
and 15c. The results proved that there is no appreciable accumulation of particles
observed at these time points. However, a high particle loading was observed following
24 hour of circulation and it can be seen in figure 15d (Dickerson et al., 2008).
Dickerson et al, 2008 used the silver staining of the tissue sections for examination of
accumulation of gold nanoparticles to the tumor and the tissue also used attenuation of
near-infrared transmission using a special device assay (Dickerson et al., 2008).
In addition, Sokolov and co-workers (2003) demonstrated another class of
molecular specific contrast agents for vital optical imaging of precancers and cancers,
based on gold nanoparticles conjugated to probe molecules with high affinity for
cellular biomarkers. In their experiment, Sokolov and co-workers (2003) used the
monoclonal antibodies conjugated with gold nanoparticles against EGFR that is
overexpressed in epithelial precancers for molecular specific optical imaging. The size
of gold nanoparticles is ~12nm in diameter. This size is estimated to be the same as the
size of antibodies which are routinely used for molecular specific labeling and targeting.
Three biological models of cancer with increasing complexity demonstrated the
application of gold bioconjugated for vital reflectance imaging. Firstly, suspensions of
cervical cancer cells were explored; the SiHa cells are well-characterized cervical
epithelial cancer cells that overexpress EGFR. After that engineered tissue constructs,
three-dimensional cell cultures that mimic major features of epithelial tissue, were
explored. Next, the application of contrast agents in normal and neoplastic fresh
27
cervical biopsies was done, this is the model system that most closely resembles living
human epithelial tissue (Sokolov et al., 2003).
Based on the results, gold nanoparticles conjugated to anti-epidermal growth
factor receptor (anti-EGFR) antibodies via nonspecific adsorption to recognize the
EGFR proteins on the cervical carcinoma cells and tissues (Sokolov et al., 2003). The
cancer cells treated with bovine serum albumin (BSA)-adsorbed nanoparticles were
compared with the cancer cells treated with gold nanoparticles conjugated with EGFR.
Those incubated with the targeted particles scattered strongly due to the bound
nanoparticles on the membrane of the cancer cells (Figure 16). From the first sight, the
bound conjugateds was appeared as randomly distributed sharp and bright spot at the
upper of the cells and then bright rings can be observed in the optical cross-sections
through the middle of the cells through a series of focus confocal reflectance images of
labeled cells. It was pointed out that labeling prevail establish on the surface of surface
of the cellular cytoplasmic membrane when compared with the labeling pattern with
transmittance images of the cells. This is reliable with the truth that antibodies EGFR
have molecular specificity to the extracellular domain of EGFR. Unlabeled cells cannot
be resolved on the dark background due to the much higher intensity of light scattering
than the unlabelled cells. No labeling was observed when gold nanoconjugates with
BSA were added to the cells (Figures 16E and 16F).
Besides that, the reflectance and the fluorescence confocal images of the
abnormal biopsy ratify predominant binding of the anti-EGFR/gold conjugates to the
cytoplasmic membrane of the epithelial cells were compared (Figures 17A and 17B).
Yet, in vivo application of these contrast agents rely on the capability to distribute the
agents throughout the epithelium in the targeting organ site. For the new direction of
28
diagnostic method and the study of the molecular changes associated with cancer
progression, it is necessary to carry the gold nanoparticles throughout the whole
epithelium. The study of PVP was used to deliver the gold nanoparticles throughout the
epithelium (Figure 18). In the pure PBS buffer and PBS buffer with the presence of
PVP, the top of engineered tissue have been employed the anti-EGFR/gold conjugates.
The constructs were rinsed and bulky transverse sections were prepared after incubation,
and viewed under the transmittance and confocal reflectance microscopies. When the
presence of PVP has been applied by the conjugates, uniform labeling is attained
throughout the whole depth (Figure 18A and 18B). Only the surface layer of epithelial
cells in the engineered tissue constructs was labeled when gold conjugates were applied
in PBS (Figures 18C and 18D). The remarkable potential of the contrast agents
presented in the study indicated the ability of vital reflectance microscopies for in vivo
molecular imaging to be extended. When this great possible of contrast agents was used
where provided the ability to image the distribution of EGFR expression in living
neoplactisc cervical tissue and providing the possibility to assess molecular pathology in
vivo (Sokolov et al., 2003).
The expected improvements for in vivo and in vitro diagnostics include increases
in analytical sensitivity without sacrificing specificity. Furthermore, non-amplification
assays are cheap detection technologies which are fast and require small sample sizes.
With regard to in vivo imaging, enhancement of conventional imaging agents has
resulted in higher sensitivity and finer resolution of tumors. Moreover, the application
of nanotechnology to develop novel imaging agents has resulted in new roles for noninvasive imaging in the detection, staging and overall management of patients with
cancer (Fortina et al., 2007).
29
Figure 12: Fluorescence microscopic image of cancer with an original magnification,
40X of an A431 tumor removed from a mouse 2 days after intravenous injection of 20nm gold nanoparticles (GNPs) coated with thioctic acid-anchored polyethylene glycol
with a molecular weight of 5000. The slice was stained for platelet–endothelial cell
adhesion molecule-1 (CD31, red) and epidermal growth factor receptor (blue). GNPs
were visualized using a dark field condenser (pseudocolored green). (A) Some
nanoparticles remained in the blood vessel (arrows), while (B) others entered the
extravascular fluid space (arrow heads) (Zhang et al., 2009).
Source: Adapted from Zhang et al, 2009
Figure 13 (above) and Figure 14 (below)
Figure 13: Cancer cell diagnostics using dark field light scattering imaging of spherical
gold nanoparticles
FIugre 14: Cancer cell diagnostics using dark field light scattering imaging of gold
nanorods. The anti-EGFR-conjugated gold nanoparticles are bound to the cancer cells
assembled in an organized fashion, while they are randomly distributed around normal
cells, thus allowing for the optical differentiation and detection of the cancer cells (Elsayed et al., 2005).
Source: Adapted from El-sayed et al, 2005
30
Figure 15: Histological sections of silver stained tumor xenografts from mice
intravenously (tail) injected with (a) 100µl of 10mM PBS following 24 hour circulation
and pegylated gold nanorods following (b) 2 hour, (c) 6 hour, and (d) 24 hour
accumulation. Arrow indicates staining of red blood cells (observed in all tumors)
(Dickerson et al., 2008).
Source: Adapted from Dickerson et al, 2008
31
Figure 16: From A to D showed high resolution and from G to I showed low resolution
optical images of SiHa cells labeled with anti-EGFR/gold conjugates. For the E and F
showed the nonspecific labeling using gold conjugates with BSA. For the A, C and E
images used the laser scanning confocal reflectance whereas B, D and F images used
combined confocal reflectance/transmittance for the labeled SiHa cells and it can be
obtained with 40 X. The scattering from gold conjugates is false-colored in red
(Sokolov et al., 2003).
Source: Adapted from Sokolov et al, 2003
32
Figure 17A and 17B: Laser scanning confocal reflectance and confocal fluorescence
images of prencancerous cells labeled with anti-EGFR/gold conjugates (Sokolov et al.,
2003).
Source: Adapted from Sokolov et al, 2003
Figure 18: For A, C and E are Transmittance and B and D is reflectance images of
engineered tissue constructs labeled with anti-EGFR/gold conjugates. The densely
packed, multiple layers of cervical cancer (SiHa) cells are the structures of the tissue
constructs. The contrast agents were added on top of the tissue phantoms in 10% PVP
solution in PBS (A and B) or in pure PBS (C and D) (Sokolov et al., 2003).
Source: Adapted from Sokolov et al, 2003
33
4.0 Applications of gold nanoparticles to therapeutics for cancer cell
4.1 Anticancer drug
Gold nanoparticles can be used as therapeutics in cancer therapy where the anticancer
drugs are conjugated together with the gold nanoparticles to target the tumor cells.
Usually, cisplatin, transtuzumab, gemicitabine, and heparin are used as anticancer drugs.
All of these anticancer drugs have strong effect to kill the cancer cells. In 2009, Cheng
and coworkers explored the therapeutic approach of delivering cisplatin, a very
powerful therapeutic agent stored into porous hollow nanoparticles (PHNPs) of Fe3O4,
against the numerous solid tumors. Cisplatin functions by interacting with DNA to form
intrastrand cross-linked adducts and by interfering with cell transcription mechanisms.
However, Cheng et al. (2009) found that the potential of cisplatin is restricted by its
tendency to target both of the tumor cell and healthy cells, its chemical instability, its
poor water solubility and its low lipophilicity. To overcome these problems porous
hollow nanoparticles of Fe3O4were used. Besides that, the PHNPs were conjugated with
trastuzumab. The bioconjugated cisplatin-loaded hollow nanoparticles exhibited high
targeting efficiency and a remarkable cytotoxicity against the cancer cells (Cheng et al.,
2009). This ideology can be applied into gold nanoparticles (Au- Fe3O4) which are able
to act as target-specific nanocarriers to deliver different platin complex instead of
cisplatin into HER2-positive breast cancer cells with strong therapeutic effects (Figure
19). The function of Fe3O4 is replaced by optically active Au NPs. There are reasons
why gold nanoparticles rather than the iron oxide nanoparticles are used. The reasons
are the presence of Fe3O4 and Au surface facilitates the stepwise attachment of an
antibody and a platin complex, and the structure can serve as both a magnetic and an
34
optical probe for tracking the platin complex in cells and biological systems (Xu et al.,
2009).
However, cisplatin kills healthy cells together with the tumor cells. The problem
is minimized in another study in which urokinase-type plasminogen activator (uPA) was
used. Niidome et al. (2010) reported a gold nanorod targeted delivery system for tumors
by constructing a peptide substrate for uPA. This uPA is expressed specifically on
malignanat tumors. The uPA was inserted between the PEG-chain and the surface of the
gold nanorods. The PEG-peptide-modified gold nanorod constructed is a novel targeted
delivery system (Niidome et al., 2010). Another study from Kinoh et al. (2009) also
discussed about the relationship of uPA to the tumor cells. Kinoh et al. (2009)
developed one specific vector in which the substrate sequence (SGRS) of the uPA was
inserted into the tryptic cleavage site, which triggers membrane fusion activity in the
infected cells. The vector used is called the oncolytic Sendai virus (SeV) vector. The
uPA-responsive SeV showed syncytia formation in uPA-expressing in tumor cells.
Subsequently, extensive cell death through massive cell-to-cell spreading without
significant dissemination to the surrounding noncancerous tissue was observed. So, the
substrate peptide of uPA is also expected to be a promising candidate that can act as a
biosensor to activate drug at the malignant tumor site (Kinoh et al., 2009).
Niidome et al. (2010) used PEG-peptide-modified gold nanorods to deliver the
uPA to targeting site. Instead of using the uPA, recently Colombo et al. (2010)
discussed the use of trastuzumab together with nanoparticles in clinical therapy. Some
breast cancers produce protein biomarkers such as estrogen receptor, progesterone
receptor and human epidermal growth factor receptor 2 (HER2) which make therapeutic
choices specific. Trastuzumab is always used as an antibody that binds selectively to
35
the HER2 in the breast cancer cells. By binding to protein it causes the cells no longer
are able to divide uncontrollably. The use of nanoparticles will ultimately enable the
tailoring of specific anticancer treatment to an individual patient’s specific tumor
protein profile, and thus potentially leading to personalized medicine (Colombo et al.,
2010).
The use of antibody-conjugated of nanoplatforms has greater localization of
therapy to the diseased tissue or organ, while the introduction of significantly lower
doses of drug into the bodies greatly reduces the possibility of side effect. Colombo et
al, (2010) reported their study on the use of trastuzumab conjugated nanoparticles as
drug delivery agents as an advanced therapy of HER2-positive breast cancer and they
reported that the use of a combination of trastuzumab with another targeting agent was
shown to induce superior disease control. Furthermore, according to the study of Yang
et al, (2007) trastuzumab-conjugated magnetopolymeric nanohybrids incorporating
doxorubicin, a microbial-derived antharacycline drug acting as inhibitor of
topoisomerase II was developed and widely used for treatment of breast carcinoma cells
(Yang et al., 2007).
However, the trastuzumab is a very high cost treatment and it is also high in
toxicity. So, there is another study using an anticancer drug which is relatively low-cost
and medium toxicity than the trastuzumab (Patra et al., 2010). Patra and coworkers
(2010) developed a gold nanoparticles based targeted drug delivery system (DDS) for in
vitro and in vivo therapeutic application in pancreatic cancer. In this study gemcitabine
was the anti-cancer drug and cetuximab (C225) and anti-epidermal growth factor
receptor (EGFR) antibody was the targeting agent. The C225 is a common targeted
therapy. By binding to epidermal growth factor receptors (EGFR), C225 can be
36
classified as a monoclonal antibody and signal transduction inhibitor (Patra et al., 2010).
The anti-EGFR was used based on recent evidence from studies which stated that EGFR
is overexpressed in cancer cells (Zhang et al, 2009; Huang et al, 2010). According to
Patra and coworkers (2010), tyrosine kinase (TKs) is overexpressed in pancreatic cancer
cells and a rational approach to treat the cancer is by blocking the receptor tyrosine
kinase (RTKs). Gemcitabine was chosen because it is the more advanced chemotherapy
drug for pancreatic cancer and it can also be used for the treatment of breast, head and
neck as well as ovarian cancers. The results clearly showed that targeted delivery
system resulted in significant inhibition of proliferation in vitro and orthotopic
pancreatic tumor growth in vivo. Therapeutic efficacy was tested in human tumor
xenografts implanted subcutaneously in nude mice. Therefore, Patra and coworkers
(2010) believed that the orthotopic model is a better way of testing in vivo efficacy of
targeted delivery system. Recently, they had also demonstrated the generation of an
orthotopic human xenograft model of pancreatic cancer where tumor progression can be
monitored non-invasively by bioluminescence from the implanted cells. Biodistribution
studies as determined by inductively coupled plasma analysis demonstrated minimal
uptake in vital organs such as liver and kidney whereas significant accumulation of gold
was detected in the tumor (Figure 20a). Figures 20b and c correspond to the luciferase
imaging of the control group (C225 +Gem) and experimental group (Au-C225-Gem) at
the end of the study, respectively. Mice treated with Au-C225-Gem had a significant
tumor growth inhibition when compared with its non targeted counterpart. These results
were further confirmed by measuring the size and circumference of the tumor. Growth
inhibition in vivo was significant compared with all other nontargeted groups as shown
in Figure 20d (Patra et al., 2010).
37
In addition, another anticancer drug is heparin. Heparin is known to have a lot of
biological functions such as anti-coagulation, anti-inflammation, anti-tumor cell
proliferation and so on. For metastatic cancer cells, heparanase and haparinase are overexpressed for cell migration through heparin. Lee et al. (2010) also used intracellular
heparin delivery for targeted apoptotic cancer cell death. This was attempted using a
cell adhesive peptide, arginine-glycine-aspartic acid (RGD) conjugated onto the heparin
immobilized AuNPs using a poly (ethylene glycol) (PEG) as a spacer. The modified
heparin and RGD conjugated with prepared AuNPs were delivered into αvβ3 integrin
over-expressing cancer cells to induce apoptotic effect as illustrated in the Figure 21.
The AuNP-Hep/PEG-RGD was specifically taken up by αvβ3 integrin over-expressing
cells that can be found in the certain metastatic tumor cells via receptor –mediated
endocytosis through specific RGD- αvβ3 integrin interaction. After that, the free heparin
molecules will be released out from the endocytosed AuNP-Hep/PEG-RGD into the
cytoplasm by cleaving the gold-thiol linkage under the reductive intracellular
environment (Lee et al., 2010).
Lee et al, 2010 also examined two cancer cells for targeted cellular uptake,
intracellular release of free heparin, and subsequent apoptotic cell death. The two cancer
cells used were αvβ3 integrin over-expressing mouse melanoma cells (B16F10) and
moderately expressing lung carcinoma cells (A549). After three days incubation with
Au-NP-Hep/PEG-RGD, B16F10 cells exhibited far greater extent of cell growth
inhibition (which was approximately 78% cell growth inhibition) than A549 cells
showed which was only approximately 10% cell growth inhibition. The results clearly
revealed target cell specific growth inhibition effect of AuNP-Hep/PEG-RGD (Figure
22a). As a negative control, the AuNP-Hep without PEG-RGD was also used to treat
38
the two cells. Both αvβ3 integrin receptor positive/negative cells showed similar cell
proliferation profiles without any sign of growth inhibition. This suggests that Au-NPHep/PEG-RGD was transported within cells via a target-specific manner. In another
experiment, the enzymatic activity of caspase-3 within cells was measured to assess if
the cell growth inhibition effect was indeed caused by apoptotic cell death (Figure 22b).
The caspase-3 is a protease known to play a central role in triggering an apoptotic cell
death. Based on the results obtained by Lee et al. (2010), caspase-3 activity was slightly
increased for B16F10 cells, but not for A549 cells. It was also observed that AuNP-Hep
without RGD targeting moieties did not show any significant change in the caspase-3
activity for both of αvβ3 integrin positive/negative cells, suggesting cells specific
apoptosis-inducing effect of AuNP-Hep/PEG-RGD. Moreover, to visualize cell specific
cellular uptake for αvβ3 integrin positive/ negative cells, Lee et al. (2010) used
fluorescien-labeled AuNP-Hep/PEG-RGD in their experiment. The confocal images
showed strong green fluorescence signals recovered from the released heparin
molecules homogeneously distributed within the cytoplasm only for B15F10 cells,
whereas no fluorescence was observed for A549 cells. This indicated target cell specific
endocytosis and subsequently cytoplasmic release of free heparin. When the apoptotic
effect of released heparin within cells was examined by measuring the activity of
caspase that produce red fluorescence, only B16F10 cells showed red color, proving
target cell specific apoptotic cell death. The merged images also strongly support the
αvβ3 integrin targeted cellular uptake and apoptotic effect of AuNP-Hep/PEG-RGD
(Figure 22c) (Lee et al., 2010).
39
Figure 19 (A): Platinum complexes used for therapies; (B): dumbbell-like Au–Fe3O4
NPs conjugated with trastuzumab (Herceptin) and a platin complex (Xu et al., 2009).
Source: Adapted from Xu et al, 2009
Figure 20: Results illustrate in vivo targeting of the nanoconjugate and its therapeutic
efficacy. A, the quantification of the amount of gold taken up by the tumor, kidney, and
liver under different treatment groups (n=3). A comparative bioluminescence image
from the mice treated with a mixture of C225 and gemcitabine (C225+Gem; B) or Au–
C225–Gem (C) i.p. (n=10). D, effect of different treatment groups on tumor growth
inhibition in vivo (Patra et al., 2010).
Source: Adapted from Patra et al, 2010.
40
Figure 21: Diagram illustration of the targeted apoptotic cancer cell death for αvβ3
integrin positive cells upon treatment of heparin and PEG-RGD immobilized gold
nanoparticles (AuNP-Hep/PEG-RGD) (Lee et al., 2010).
Source: Adapted from Lee et al, 2010
Figure 22a: Axis-y-cell profileration (%) schematic illustrated targeted cell growth
inhibition effect of AuNP-Hep/PEG-RGD to αvβ3 integrin positive cells (B16F10). Cell
proliferation of B16F10 and A549 was determined by trypan blue exclusion assay after
incubation with AuNP-Hep or AuNP-Hep/PEG-RGD for 3 day (Lee et al., 2010).
Source: Adapted from Lee et al, 2010.
41
Figure 22b: Axis-y caspase-3 activity (relative pNA (pmole) / protein (ug)). Diagram
illustrated cellular caspase-3 activity of B16F10 (αvβ3 integrin positive cells) and A549
(αvβ3 integrin negative cells) after treatment of AuNP-Hep lacking PEG-RGD or AuNPHep/PEG-RGD. There was no statiscally significant change in caspase-3 activities for
blank and AuNP-Hep samples (Lee et al., 2010).
Source: Adapted from Lee et al, 2010.
Figure 22c: The result showed that confocal microscopic images of B16F10 and A549
cells following incubation with AuNP-Hep/PEG-RGD. Heparin was fluorescently
labeled with fluorescein (green) and apoptosis related-caspase was detected by magic
red assay (red). Cell nucleus was stained with DAPI (blue) (Lee et al., 2010).
Source: Adpated from Lee et al, 2010.
42
4.2 Photothermal therapy
Taking advantage of the plasmon-resonant nanoparticles unique properties, optimization
and amplification of laser heating based on light absorption have been studied. The use
of gold-nanoparticles-based photothermal therapy for the destruction of cancer cells or
tumor tissues, may be potentially important implication in the clinical testing as cancer
therapy. A dipole collective oscillation due to surface charge separation of the
conduction band electrons is induced when a nobel metal, such as gold, is exposed to
light. At specific frequency the amplitude of the oscillation of these free electrons
reaches the maximum which is known as surface plasmon resonance (SPR) and there is
a strong absorption of incident light which can be assayed using a UV-Visible
spectrophotometer (Huang et al., 2006).
In support of this theory or principle claimed by Huang et al. (2006), Dickerson
et al (2008) tested the oncological treatment based on the use of photon energy
selectively administered and converted into heat sufficient to induce cellular
hyperthermia called plasmonic photothermal therapy (PPTT). Their work demonstrates
the feasibility of in vivo PPTT treatment of HSC-3 tumor xenografts using easilyprepared plasmonic gold nanorods and a small, portable, inexpensive near-infrared
(NIR) laser. Using previously eastablished treatment conditions, the change in tumor
volume for HSC-3 xenografts was recorded over 13-day consecutively for control mice,
as well as those treated by intravenous and direct injection followed by near-infrared
PPTT treatment. Here, control mice were subjected with 15μl direct injection of 10mM
PBS to the tumor interstitium, with no NIR exposure. The average change in tumor
volume for each group was plotted (Figure 23) and statistical hypothesis testing for
differences in average tumor growth were performed (Table 2). More than 96%
43
decrease in average tumor growth for directly treated HSC-3 xenografts and more than
74% decrease in average tumor growth for intravenously-treated HSC-3 xenografts at
day 13 can be observed from figure 22. In addition, over the monitoring period, the
reabsorption of more than 57% of the directly-treated tumors and 25% of the
intravenously-treated tumors was observed (Dickerson et al., 2008).
Besides that, the average tumor growth at day 13 for directly and intravenouslytreated tumors was significantly less than that observed in untreated control groups.
Based on the results, there were gradually increasing differences in the observed
efficacy for direct and intravenous treatments during the experiment, reaching statistical
significance at the 8% level on day 13 in white rats (Dickerson et al., 2008).
In the study of Maksimova et al. (2007), the efficiency of the therapy was
demonstrated by the effective optical destruction of cancer cells by local injection of
plasmon-resonant gold nanoshells followed by continuous wave (CW) semiconductor
laser irradiation at certain wavelengths. The theory of this study was virtually the same
as that of Huang et al. (2006). The temperature characteristic of animal skin at
hypodermic and intramuscular introduction of gold nanoparticles in vivo has been
measured (Figure 24 and Figure 25). For the study, laboratory white rats were used. The
experimental animals were fixed at the horizontal position supine with hair shaved off.
Every animal was labeled by a symmetrical centerline of stomach as shown in Figure 24.
Three sections to the left from the centerline were used as control where heating under
laser radiation was done without nanoparticles and marked by asterisks (“●”, “●●”, and
“●●●”). Nanoshell suspension was injected in three sections to the right from the
centerline. In the section marked as “X”, “XX”, and “XXX”, the injections were
intradermal, subcutaneous, and intramuscular, respectively. The injection depth was
44
roughly 5mm, the laser power was 2.5W, and the distance between the fiber and skin
surface was about 15mm. After laser irradiation and temperature registration, the
histological examination of tissues from all sections was carried out and after 10s of
irradiation, the coagulation of tissue marked by X symbol also observed visually
(Maksimova et al, 2007).
Next, Maksimova and coworkers (2007) heated up the control section of the rat
tissue without nanoparticles to 46oC. This heating cannot lead to irreversible injuries of
tissues but can lead to cell damage if given sufficiently prolonged treatment. In the case
of the intramuscular irradiation (“XXX”), Maksimova et al. (2007) did not see any
significant changes in the color and structure of tissue surface during the irradiation
time even though the surface temperature was approximately 7 oC higher than in the
control measurements without nanoparticles. Thus, the temperature in the region of
nanoparticle localization could significantly exceed the surface temperature recorded by
the thermal imaging system. As a conclusion, it is found that laser thermolysis in the
region of nanoparticles localization did not damage the surface tissues. (Maksimova et
al., 2007).
The results were further confirmed by the histological examination. In Figure 25,
the local temperature can reach or almost exceeds 60oC with the hypodermic injection
of nanoparticles. Denaturation of proteins should occur rapidly at this temperature. The
degree and speed of such protein denaturation depends on the value and duration of
temperature excess, as well as on the protein nature. The critical denaturation
temperature for a majority of tissue components is about 57oC. After 15-20 second,
Maksimova and coworkers (2007) observed surface albication of skin followed by the
thermal burn of skin. With the intracutaneous introduction of nanoparticles, the visual
45
changes in the biological tissue were observed for irradiation times less than 10 seconds.
At a temperature near 70oC, noticeable dehydration of biological tissues was observed
(Maksimova et al., 2007). After the water withdrawal, the dried tissue is heated rapidly
to a temperature of 150oC, at which the carbonization process begins. The maximum
value of the local temperature in this case can exceed 180oC due to rapid expulsion of
water from the local section of biological tissue. In these experiments and in a number
of rare cases, Maksimova et al. (2007) observed the formation of nanoparticles
aggregates.
46
Figure 23: Average change in tumor volume for HSC-3 xenografts following nearinfrared PPTT treatment by control (♦), intravenous (■), and direct (●) injection of
pegylated gold nanorods. Errors for control (n = 10), direct injection (n = 8), and
intravenous injection (n = 7) groups reported as standard error of the means. Control
mice were treated by interstitial injection of 15 lL 10 mM PBS alone, while intravenous
PPTT treatments were performed by administration of 100 lL pegylated gold nanorods
(ODk=800 = 120, 24 h accumulation) followed by 10 min of 1.7–1.9 W/cm2 NIR laser
exposure. Direct PPTT treatments were performed by administration of 15 lL pegylated
gold nanorods (ODk=800 = 40, 2 min accumulation) followed by 10 min of 0.9–1.1
W/cm2 NIR laser exposure (Dickerson et al., 2008).
Source: Adapted from Dickerson et al, 2008
Table 2: P-values for average volume change in HSC-3 tumors following near-infrared
PPTT by 808 nm irradiation of pegylated gold nanorods.
Source: Adapted from Dickerson et al, 2008
47
Figure 24: Laser photothermolysis (800 nm) of superficial rat tissue with intradermal
injection of gold nanoparticles. Symbols ●,●●and ●●● stand for control (without
nanoparticles); symbols X, XX designate the hypodermic injection of 0.1 ml
nanoparticle solution; symbols XXX designate the intramuscular injection of 0.1 ml
nanoparticles to depth of about 5 mm (Maksimova et al., 2007).
Source: Adapted from Maksimova et al, 2007
Figure 25: Thermograms of rat’s skin surface with different depth injection of
nanoshells after laser irradiation during 30 s: (a) control without nanoparticles; (b)
intramuscular injection of 0.1 ml of silica/gold nanoshells (depth is about 5 mm); (c)
subcutaneous injection and (d) intradermal injection (Maksimova et al., 2007).
Source: Adapted from Maksimova et al, 2007
48
5.0 Safety issues of gold nanoparticle technology on the diagnostics and
therapeutics in cancer
Gold nanoparticles (GNPs) provide a great possibility for biomedicine field. However,
it is necessary to know the bioaccumulation and local or systemic toxicity associated
with the application of GNPs in therapy and drug delivery. Currently, there is no
evidence to prove the in vivo accumulation of gold nanoparticles after repeated
administration. Lasagna and coworkers did a study to assess the biosafety of gold
nanoparticles in 2010. Different doses of GNPs upon intraperitoneal administration in
mice were done continued for 8 days to understand the bioaccumulation and toxic
effects (Lasagna et al., 2010). After administration of GNPs, the mice were examined
daily for survival and evident behavioral changes. Besides that, mice were also weighed
everyday. To examine morphological changes, the brain, lungs, liver, spleen, heart and
kidneys were removed and weighed. Serum biochemical analysis, hematological
analysis, histopathological examination and statistical analysis were used to understand
toxicological studies.
Lasagna and coworkers (2010) employed two methods to
measure gold in various tissues, including brain, liver, kidney, spleen and lungs:
inductively coupled plasma-mass spectrometry (ICP-MS) and graphite furnace atomic
absorption spectrometry (GF-AAS). The results showed that the amounts of gold
detected in each tissue were similar. Moreover, the result also showed that in kidney,
liver, spleen, and lungs, there was significant increase in the amount of gold after
repeated injection of GNPs. The same results were obtained in the blood and brain. The
bioaccumulation profile of gold/g of tissue showed that spleen> liver=kidney>
lungs>brain (Table 3). Interestingly the percentage of gold accumulated decreased when
the GNPs dose increased, suggesting efficient clearance of GNPs from the body.
49
Furthermore, Lasagna and co-workers (2010) observed that accumulation of GNPs in
different organs after repeated administration did not produce any mortality or gross
behavioral changes in mice receiving GNPs at the doses studied. Tissue color, size and
morphologies also remain unchanged after treatment with GNPs. No results of atrophy,
congestion or inflammation were observed. To determine if GNPs produce renal
toxicity, they also determined the levels of urea nitrogen and creatinine in blood, which
are metabolites associated with the functionality of the kidney (Lasagna et al., 2010).
The levels of bilirubin and alkaline phosphatase in blood were tested as a measure of
hepatic and biliary functionality.
In addition, Lasagna and coworkers (2010) also
determined the levels of uric acid, since hypouricemia (a decrease of uric acid in the
blood) is a common sign of drug toxicity. A detailed analysis of all these metabolites at
different doses of GNPs as compared to controls showed no statistically significant
differences in any of the parameters tested (Table 4). Additional hematological studies
were done by Lasagna and co-workers (2010) to assess changes at the levels of red
blood cells, white blood cells, hemoglobin, hematocrit, mean corpuscular volume,
mean corpuscular hemoglobin concentration, red blood cell distribution width,
neutrophils, lymphocytes, monocytes, easinophils, basophils and platelets. None of
these parameters showed any statistical difference between the control and the
experimental mice groups. Finally, in order to search in more details for possible toxic
effects, histological examination GNPs showed no tissue damage in any of the sections
obtained from the kidney, liver, spleen, brain or lungs (Figure 26)(Lasagna et al., 2010).
The study of BarathManiKanth et al. (2010) pointed out about the mice were
injected with AuNPs at a dosage of small amount for more than ten days and daily
examined for any changes in the morphology and behaviour. As a result, the mice did
50
not show any significant side effect of toxicity like weight loss, lack of desire for food,
weak body, changes in fur colour, tiredness, less energetic and so on. All the nude mice
are survived throughout the experimental period without showing any abnormalities
was proved. Analysis of various hematological parameters in the gold treated and
control animals are compared each other. There was no significant amendment except
trivial differences in certain parameters (Table 5) (BarathManiKanth et al., 2010).
Furthermore, the study also showed that the gold nanoparticles treated organs
did not show any major morphological alteration in comparison to control. Normal
alveolar geometry and normal appearing alveolar septum was showed in the lung
histopathology (control groups) (Figure 27A). After the treatment of gold nanoparticles
at a concentration of certain amount day-1 was proved that the same histopathological
finding was seen (Figure 27B). Normal renal cortex and glomerular tufts was proved
control kidney is the healthy kidney and used for the kidney histological studies in this
study (Figure 27C). For the treatment of gold nanoparticles at a dosage of small amount
did not lead to any disorders in the histology. They showed normal glomerular tubules
and renal cortex (Figure 27D). In the Liver histopathology sections from the control
animals are showing normal hepatic portal triad and central vein (Figure 27E). The gold
nanoparticles treated liver also showed normal hepatocytes with clear central vein
showing no morphological changes significant in comparison to control (Figure 27F).
There were no any changes due to the treatment of gold nanoparticles at a dosage of
small amount per day according to the study over the spleen histology also revealed.
The control and gold treated spleens showed normal lymphoid follicles and sinuses
(Figure 27G-H) (BarathManiKanth et al., 2010). It is undoubtedly true that the
51
accumulation of gold nanoparticles in different organs after repeated administration did
not produce any mortality or any indication of toxicity (BarathManiKanth et al., 2010).
Moreover, Colombo et al. (2010) also stated that the GNPs are generally
considered relatively safe because gold has been demonstrated to be a biocompatible
metal in many cases. However, other kinds of NPs, including Metal Nanoparticles
(MNPs) and Quantom dots (QDs), which contain iron or hard metals, respectively, need
to be carefully evaluated for potential toxicity ( Colombo et al., 2010).
The cytotoxicity of gold nanoparticles has been studied quite extensively using
various in vitro model systems and it is found that cytotoxicity depends on the size of
the gold nanoparticles. Fadeel and Garcia-Bennett (2010) reviewed that gold
nanoparticles with a size of 13nm and above, commonly typified as colloids, may be
viewed as non-toxic. By contrast, gold particles below 2nm have shown an unexpected
degree of toxicity in different cell lines. A case in point, the gold clusters compound
Au55 with a distinct particle size of 1.4nm has been shown to interact in a unique
manner with the major grooves of DNA, which could account for the remarkable
toxicity of these nanostructures (Fadeel and Garcia-Bennett, 2010). However, gold
clusters with perfectly complete geometries could be envisioned for cancer treatment,
particularly in the case of metastatic melanoma, an aggressive cancer and notoriously
difficult to treat, if toxicity to normal cell types can be controlled (Fadeel and GarciaBennett, 2010).
52
Table 3 Bioaccumulation of GNPs with respect to the total injected does in different
organs( % ID/organ)
µg/kg/day
ICP-MS
40
200
GF-AAS
400
40
200
400
Brain
± 0.001
Lung
± 0.002
Spleen
± 0.034
Kidney
± 0.056
Liver
± 0.357
0.023 ± 0.006
0.006 ± 0.001
0.006 ± 0.001
0.022 ± 0.006
0.006 ± 0.001
0.006
0.037 ± 0.013
0.011 ± 0.001
0.006 ± 0.002
0.036 ± 0.013
0.011 ± 0.001
0.006
0.204 ± 0.199
0.291 ± 0.219
0.096 ± 0.034
0.206 ± 0.198
0.281 ± 0.203
0.096
0.398 ± 0.173
0.184 ± 0.046
0.139 ± 0.058
0.413 ± 0.176
0.186 ± 0.044
0.139
1.983 ± 0.566
1.828 ± 0.881
1.355 ± 0.361
2.009 ± 0.587
1.812 ± 0.867
1.320
Table 4 Biochemical parameters in the serum of mice treated with GNPs.
Groups
ALKP(U/L)
URIC(mg/dL)
Control
3.45 ± 0.8
89.25 ± 9.8
40 lg/kg/day 3.93 ± 0.6
± 10.9
200 lg/kg/day 4.02 ± 1.1
± 16.4
400 lg/kg/day 4.17 ± 0.8
76.66 ± 3.7
CREA(mg/dL)
UREA(mg/dL)
TBIL(mg/dL)
0.24 ± 0.08
26.25 ± 2.06
1.25 ± 0.53
0.27 ± 0.02
24.01 ± 2.44
0.90 ± 0.28
86.60
0.26 ± 0.06
24.02 ± 3.65
0.75 ± 0.31
91.01
0.24 ± 0.05
26.20 ± 2.58
0.52 ± 0.26
Source: Lasagna et al, 2010
53
Figure 26: Histological analysis of various organs after GNPs treatment. Tissues were
stained with hematoxilin/eosin as indicated in Materials to assess for potential effects of
GNPs treatment on the organ morphology and cellular damage. The size of the bar
corresponds to the following: lung, 130 µm; spleen, 35 µm; liver, 30 µm; kidney, 100
µm, brain 75 µm (Lasagna et al., 2010).
Source: Adapted from Lasagna et al, 2010
54
Table 5: Each value represents the mean ± S.D of n = 6 Hb, hemoglobin; cells; RBC,
red blood cells; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin content; HCT, hematocrit. Numerical values (±)
in the parenthesis are considered as 'Standard Deviation (SD)'. P values were calculated
using one way ANOVA followed by Students-'t' test by comparing between different
groups (control vs. treatment) and values are considered to be non-significant (P > 0.05)
(BarathManiKanth et al., 2010).
Source: Adapted from BarathManiKanth et al, 2010
55
Figure 27: Toxicity studies of gold nanoparticles in mouse organs. Histological
specimens of mice tissues (lung, kidney, liver and spleen) collected from mice
euthanized on day 15, stained with hematoxylin and eosin (H and E) showed normal
histology (BarathManiKanth et al., 2010).
Source: Adapted from BarathManiKanth et al, 2010
56
6.0 Future Research and Conclusion
The use of nanotechnology in cancer diagnosis and therapeutics offers exciting
impossible mission and it is a cutting edge technology in this era. The use of gold
nanoparticles or other nanoparticles conjugated to specific antibodies allows the
possibility of simultaneously detecting multiple molecular targets in small tumor
samples, on which treatment decisions can be made. Looking forward the future, there
are several steps of research directions and guidelines that are particularly important.
Firstly, the design and structure of nanoparticles with monofunctions, dual task, three
utility or multiple tasks will become a new directions. A more diverged and varied
targeted drug delivery system can be experimented with one drug or a combination of
two drugs or multiple drugs, with one targeting agent or multiple targeting agents along
with an imaging agent. Hence, the nanoparticles can be used for targeting, imaging,
sensing, therapy and detecting (Cai et al., 2008)
In the coming years, the application of nanotechnology-based diagnostics and
therapeutics in clinical management will be increased. Moreover, identification of new
molecular markers/targets that will only be observed on cancer cells would be ideal to
speed up the advancement of nanoparticle–based targeted therapy. This is particularly
promising in the field of cancer research because of cancer heterogeneity and the
development of drug resistance. A single targeted therapy may not be effective for
every population of patients. In fact, the use of nanotechnology could revolutionize not
only oncology, but also the entire discipline of medicine.
57
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