Artificial Cells, Nanomedicine, and Biotechnology

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Application of gold nanoparticles in biomedical

and drug delivery

Hadis Daraee, Ali Eatemadi, Elham Abbasi, Sedigheh Fekri Aval, Mohammad
Kouhi & Abolfazl Akbarzadeh

To cite this article: Hadis Daraee, Ali Eatemadi, Elham Abbasi, Sedigheh Fekri Aval,
Mohammad Kouhi & Abolfazl Akbarzadeh (2016) Application of gold nanoparticles in biomedical
and drug delivery, Artificial Cells, Nanomedicine, and Biotechnology, 44:1, 410-422, DOI:
10.3109/21691401.2014.955107

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Artificial Cells, Nanomedicine, and Biotechnology, 2016; 44: 410–422
Copyright © 2014 Informa Healthcare USA, Inc.
ISSN: 2169-1401 print / 2169-141X online
DOI: 10.3109/21691401.2014.955107

Application of gold nanoparticles in biomedical and drug delivery

Hadis Daraee2, Ali Eatemadi2, Elham Abbasi1, Sedigheh Fekri Aval1,2, Mohammad Kouhi3 &
Abolfazl Akbarzadeh1,2
1Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,

Iran, 2Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences,
Tabriz, Iran, and 3Department of Physics, College of Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran

Abstract quintessence—“quinta essentia auri,” which he detected by the

Nanoparticles are the simplest form of structures with sizes in
the nanometer (nm) range. In principle any collection of atoms
bonded together with a structural radius of  100 nm can
be considered nano particles. Nanotechnology offers unique
approaches to probe and control a variety of biological and
medical processes that occur at nanometer scales, and is expected
to have a revolutionary impact on biology and medicine. Among
the approaches for exploiting nanotechnology in medicine,
nanoparticles offer some unique advantages as sensing,
image enhancement, and delivery agents. Several varieties of
nanoparticles with biomedical relevance are available including,
polymeric nanoparticles, metal nanoparticles, liposomes,
micelles, quantum dots, dendrimers, and nanoassemblies. To
further the application of nanoparticles in disease diagnosis
and therapy, it is important that the systems are biocompatible
and capable of being functionalized for recognition of specific
target sites in the body after systemic administration. In this
review, we have explained some important applications of gold
nanoparticles.
Keywords: biocompatible, drug delivery, gold nanoparticle,
immunoassay, photothermal
Introduction
Gold is one of the first metals to have been discovered;
the history of its study and application spans at least
some thousand years. The initial information on colloi-
dal gold can be found in treatises by Arabian, Chinese,
and Indian scientists, who tried to attain colloidal gold as
early as in the fifth-fourth centuries BC. They utilized it for
medicinal (Indian “liquid gold,” Chinese “golden solu-
tion”) and other functions. In the Middle Age, in Europe,
colloidal gold was studied and used in alchemist laborato-
ries. Paracelsus wrote about the curative properties of gold
reduction of gold chloride by vegetable dig outs in alcohols or
oils. He used the “potable gold” for healing a number of mental
diseases and syphilis. His contemporary, Giovanni Andrea,
utilized “aurum potabile” as a therapy for patients with lep-
rosy, plague, epilepsy, and diarrhea. In 1583, the alchemist,
David de Planis-Campy, who served, when the doctor to
Louis XIII of France recommended, his “longevity elixir,”
a colloidal solution of gold in water. Nanoparticles based
on gold chemistry have attracted significant research and
practical consideration lately. They are flexible agents with
a selection of biomedical applications including use in
highly sensitive analytical assessments, ablation thermal
and radiotherapy development, as well as drug and gene
delivery (Chiu and Rana 2003, Soutschek et al. 2004, Jeynes
et al. 2013, Prigodich et al. 2009, Dhar et al. 2009, Horisberger
et al. 1975). For biomedical uses, external functionalization
of gold nanoparticles (GNPs) is necessary in order to target
them to specific disease areas and allow them to selectively
interact with cells or biomolecules. The resulting GNP have
unique properties, such as size- and figure-dependent visual
and electronic characteristics (Qian et  al. 2008, Niemeyer
et  al. 2003, Wheeler et  al. 1999, Javier et  al. 2008, Huang
et  al. 2009, Loo et  al. 2005), a high surface area to amount
ratio, and surfaces that can be readily modified with ligands
containing functional groups such as thiols, phosphines, and
amines, which display affinity for gold surfaces (Stamatoiu
et al. 2012).
By using these functional groups to fix the ligands,
additional moieties such as proteins oligonucleotides, and
antibodies can be used to impart even greater functional-
ity (Wang et al. 2010, Pavlov et al. 2004). The broad range of
application for GNP is based on their unique physical and
chemical properties. In particular, the visual properties of
GNP are determined by their plasmon resonance, which
is associated with the combined excitation of conduction

Correspondence: Dr. Abolfazl Akbarzadeh, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel/Fax:  984113355789.
E-mail: Akbarzadehab@tbzmed.ac.ir and Dr. Mohammad Kouhi, Department of Physics, College of Science, Tabriz Branch, Islamic Azad University, Tabriz,
Iran. Tel/Fax:  984113844395. E-mail: kouhi@iaut.ac.ir
(Received 31 July 2014; revised 8 August 2014; accepted 12 August 2014)

410

electrons and localized in the broad region, from the visible
to the infrared (IR) region, depending on the particle size,
shape, and structure (Thiruppathiraja et  al. 2011). Taking
into account the large volume of data published and the
high speed at which they are updated, our review aims
to take a broad view of the outcomes obtained over the
past several years in the most promising directions in the
use of GNP in modern medical and biological studies
(Horisberger et al. 1975) (Figure 1).
Application in biomedical
Biomolecule-directed nanoparticles organization:
Nanoparticles as biolabels
The dimensions of the metal nanoparticles are similar to
those of biomolecules such as proteins (enzymes, antigens,
antibodies) or DNA whose dimensions are in the range of
2–20 nm (Wang et al. 2010, Neely et al. 2009, Kamnev et al.
2002). Immobilization of biomolecules onto nanoparticles to
give new hybrid nanobiomolecules has been achieved by a
variety of techniques including electrostatic binding, physi-
cal adsorption, covalent coupling, and specific recognition.
Under appropriate conditions, non-covalent bonding is a
general strategy to bind colloidal gold and macromolecules,
with little or no change in the specific activity of the bound
macromolecule.
DNA-gold nanoparticles assemblies and sensors
Negatively charged DNA was found to substitute citrate
ions around gold nanoparticles to form a DNA-nanopar-
ticle probe, which was confirmed by electrophoresis and
fluorescence (Russier-Antoine et al. 2008). DNA function-
alized gold and semiconductor nanoparticles have been
prepared using the n-alkylthiolated DNA and also using
DNA containing several adenosylphosphothioate residues
at their ends (Neely et al. 2009). DNA is used as a template
to prepare nanocrystal chains consisting of two or three
1.4-nm particles on a single oligonucleotide strand. Con-
jugates of gold nanoparticles-oligonucleotides are of great
interest for detection of DNA hybridization because of its
application in the diagnosis of pathogenic and genetic
diseases. Most of the DNA hybridization techniques utilize
fluorescent, chemiluminescent, and radioactively labelled
probes that require special instrumentation or both
(Figure 2).
Figure 1. Near Infrared window.
Gold nanoparticles in biomedical and drug delivery 411
Figure 2. Gold nanoparticles in biosensor.
Protein-based recognition systems: Enhanced
immunosensing
Though, a large number of complementary binding pairs
are available, nucleic acid based conjugation might offer
advantages over protein based assembly (Dykman and
Khlebtsov 2011). These biomolecule based coupling sys-
tems were useful in various diagnostic applications and for
generating inorganic nanoparticle networks (Neely et  al.
2009). The conjugation of proteins on colloidal gold nano-
particles is achieved by the electrostatic interactions between
negatively charged citrate on surfaces of gold nanopar-
ticles and positively charged groups of the proteins (Zharov
et  al. 2003). The experimental gold nanoparticle-protein
conjugate architectures involve either direct binding of
antigen-gold nanoparticles bioconjugates to an antibody
modified surface or the exposure of an antibody derived
surface to free antigen and then to a secondary antibody-
gold nanoparticles conjugate. Biosensors for immunoas-
says in human serum have been developed (Huang et  al.
2008).
Drug delivery
Nanoparticles can easily enter cells although the
mechanism(s) involved are not well understood. The
nanoparticle influx occurs by endocytosis; the particles are
inserted and diffused through the lipid bilayer of the cell
membrane. Furthermore, these nanoparticles were shown
to be able to enter the cells even after linkage to proteins
such as antibodies. Nanoparticles conjugated with anti-
bodies against exclusive cancer cell surface receptors have
been used to specifically bind with cancerous cells; the
functionalized nanoparticles have also been used for
targeted entry into cells. Phthalocyanine-stabilized gold
nanoparticles have been shown to be a potential delivery
Figure 3. Colloidal gold nanoparticles.
412 H. Daraee et al.
vehicle for photodynamic therapy (Doubrovsky et al. 2010).
Gold nanoparticles with a size of 20 nm have been conju-
gated to various cellular targeting peptides to provide func-
tional nanoparticles that penetrate the biological membrane
and target the nucleus. Various nanoparticles have also
applied as targeted biomarkers and drug-delivery agents for
diagnosis and medical treatment of cancer.
Cytochemical labels and other applications
Colloidal gold nanoparticles prepared in sizes from 1 to
25 nm are electron dense due to the high atomic number
of the gold atoms, and this makes them ideal for electron
microscopy. Site-specific labeling of biological macro-
molecules finds use in histological applications. Colloidal
gold nanoparticles are used as cytochemical labels for the
study of macromolecules with transmission and scanning
electron microscopy, light microscopy and freeze-etch
electron microscopy and also to enhance the signals of both
surface enhanced Raman spectroscopy and surface Plas-
mon resonance. A further advantage of using the colloidal
gold marker is that the colloidal gold nanoparticles can be
easily counted and thus the cytochemical signal may be
evaluated quantitatively. Thus, gold nanoparticles serve as
large surface area platforms for organo-functional groups
that interact with the capillary surface, the analytes, or
both. The use of gold nanoparticles in conjunction with
chip-based capillary electrophoresis to improve the selec-
tivities between solutes and to increase the efficiency of the
separation has been reported (Akbarzadeh et al. 2012a). In
summary, gold nanoparticles that are functionalized with
proteins have long been used as tools in the biosciences.
Gold nanoparticles were utilized both as color reporting
agents and also as the signal amplification probes for the
detection of the antigen (Figure 3).
Photothermal cancer cell therapy in near
infrared region using Anti-epidermal growth
factor receptor antibody conjugated gold
nanorods
Reducing a material’s size to the nanometer length scale
(which is the length scale of the electronic motion that deter-
mines the material’s properties) makes it sensitive to further
reduction in size or a change in shape. In semiconductor
nanoparticles, the property change results from quantum
detention of the electronic shift. Recently, photothermal
therapy using the absorption properties of antibody conju-
gated gold nanoshells (Akbarzadeh et  al. 2012c) and solid
gold nanospheres have been demonstrated to selectively
kill cancer cells leaving the healthy cells unaffected. In
order to use long wavelength laser irradiation that pen-
etrates tissue optimally (can be over 10 cm in penetration
depth depending on tissue types) for in vivo photothermal
treatment (650–900 nm), (Akbarzadeh et  al. 2012c) the
absorption band of the nanoparticles has to be tuned by
adjusting the ratio of the thickness of the gold shell to the
diameter of the silica core (about 120 nm in diameter) and
thus enabling photothermal therapy in this region. Carbon
nanotubes absorb naturally in this region and have recently
been proposed as near-infrared therapy agents. It is impor-
tant to mention that a surface plasmon field is applied in
drug delivery and gene therapy (Akbarzadeh et al. 2012d).
Gold nanoparticles in biosensor applications
The basic principle involved in the design of a biosensor
based on gold nanoparticles is that the GNP are function-
alized or capped with a thiolated biomolecule which upon
identifying the complementary biomolecule causes change
in the optical absorption of GNP (Ebrahimnezhad et al. 2013).
GNP functionalized with antigen (antibody) aggregate when
matching antibody (antigen) binds causing a shift in the
plasmon absorption (Alimirzalu et  al. 2014, Hosseininasab
et al. 2014). Significant efforts have focused on the binding of
the oligonucleosides to metal surfaces and colloids for a vari-
ety of applications, including a multiplexed DNA detection
technology, rapid sequencers based on surface-enhanced
Raman spectroscopy or surface-enhanced Raman scatter-
ing (SERS) from single DNA bases (Alimirzalu et  al. 2014,
Hosseininasab et al. 2014), and the real-time DNA detection
methodology. In the present project, we have developed
a new SERS substrate based on gold nanoparticles in an
agarose matrix that provides better enhancement in the
Raman signal of DNA nucleosides than that with commer-
cially available gold nanoparticles.
Bioconjugation of GNP
Hybrid gold nanoparticles are produced by the interaction
of highly reactive nascent GNP with chemical functional-
ities present on specific molecules of biological interest
(including peptides and proteins). The conjugation proto-
cols that are applied for production of radiolabel led biocon-
jugates, traditionally used for cancer diagnosis and therapy
(Rezaei-Sadabady et al. 2013), can be extended to labeling
nanoparticles of gold and other metals with tumor specific
peptides. The biomolecule chosen for bioconjugation with
GNP is the seven amino acid truncated bombesin analogue
(BBN8–14) that is known to target gastrin releasing peptide
(GRP) receptors that are overexpressed in a variety of neo-
plasma including small cell lung, prostate, breast, gastric,
pancreatic, gastrointestinal carcinoid, and colon cancers
(Nejati-Koshki et al. 2013, Ghasemali et al. 2013). S-S group
undergo oxidative addition to GNP and the reaction is very
selective, even in the presence of thiol groups. Thioctic acid,
a biological antioxidant (Mollazade et al. 2013) believed to
exhibit metal chelating properties (Akbarzadeh et al. 2014)
contains disulfide and carboxylic acid groups to conjugate
Figure 4. Design strategy for bioconjugate/hybrid gold nanoparticles.
  
to peptide. S-S group acts as a chelating moiety to hold the
GNP and five carbon atoms act as a space between S-S and
the biomolecule. The thioctic acid modified bombesin is
used for GNP bioconjugation (Figure 4).
Application of gold nanoparticles for
immunosensors
Immunosensors are important analytical tools based on
the detection of the binding event between antibody and
antigen. Among types of immunosensors, electrochemical
immunosensors are attractive tools and have received con-
siderable attention because they are easy and economical
for mass production, are robust, and achieve excellent detec-
tion limits with small analyte volumes. A novel and sensitive
electrochemical immunoassay for immunoglobulin G (IgG)
is using a colloidal gold label via anodic stripping voltam-
metry technology. Furthermore, a novel electrochemical
immunoassay based on the precipitation of silver on
colloidal gold labels has been reported (Figure 5).
The enhancement in sensitivity for an electrochemical
immunoassay is achieved by the autocatalytic deposition
of Au3 onto GNP. After the interfacial competitive immuno­
reactions, the formed HRP labeled immunoconjugate
showed good enzymatic activity for the oxidation of
o-phenylene diamine by H2O2. The technique is mainly
based on the detection of a change in physical properties as
a result of antibody–antigen complex formation. The direct
determination of immunospecies by detecting the change
of impedance caused by immunoreactions has been dem-
onstrated. A simple and sensitive label free electrochemical
immunoassay electrode for detection of carcinoembryonic
antigen (CEA) is obtained as follows: CEA antibody (CEAAb)
is covalently attached on glutathione (GSH) monolayer-
modified GNP and the resulting CEAAb-GNP bioconjugates
were immobilized on Au electrode by electrocopolymeriza-
tion with o-aminophenol (OAP).
Citrate and transferrin
Citrate-functionalized gold nanoparticles can be prepared
on a relatively large scale. These methods allow for the
synthesis of citrate-capped spherical nanoparticles with
diameters ranging from 5 to 250 nm (Akbarzadeh et  al.
2013a, Ahmadi et  al. 2014). This well-established synthesis
and the ability to finely control size have contributed to
citrate-functionalized nanoconjugates forming the basis of
recent investigations of the uptake of gold nanoparticles by
Figure 5. Gold nanoparticles for immunosensors.
Gold nanoparticles in biomedical and drug delivery 413
cells (Alizadeh et  al. 2014). Their study demonstrates that,
in a HeLa cell model, the amount of time that the citrate
particles remain internalized is independent of the par-
ticle size when they have diameters between 14 and 74 nm.
However, the size does affect the overall number of nano-
particle conjugates internalized throughout the experi-
ment. By using inductively coupled plasma atomic emission
spectroscopy (ICP-AES) to determine the intracellular gold
content, these researchers determined that citrate-capped
gold nanoconjugates with diameters of 50 nm are most
readily internalized by HeLa cells (Figure 6). The mechanism
by which the citrate-capped gold nanoconjugates enter
cells were shown by recorded transmission electron micros-
copy images of internalized “bare” citrate nanoconjugates
and showed that the particles were mainly localized within
vesicles inside of the cells (Pourhassan-Moghaddam et  al.
2014).
They correlated cell uptake with the nonspecific
adsorption of proteins to the citrate-capped nanoparticle
surfaces. The negatively charged citrate surface presents
a suitable scaffold to attach positively charged proteins
such as transferrin, which is expected to make possible
and get better entry into cells. The images obtained sug-
gest vesicle formation at the cell surface and nanoconju-
gate internalization through endocytosis (Anganeh et  al.
2014, Davoudi et al. 2014). Many investigations in cells use
citrate-capped GNP as important precursors of covalent
conjugates with additional functionality, because further
derivatization has been shown to boost uptake capability
(Akbarzadeh et  al. 2012b), alter intracellular localization
(Valizadeh et al. 2012, Akbarzadeh et al. 2013b), or impart
functionality that can be used to affect a cellular response
(Pourhassan-Moghaddam et  al. 2013, Kouhi et  al. 2014).
Indeed, citrate-coated particles are generally not ideal
structures for investigations and internalization studies
on cells.
Amines
This method has been developed for production of gold
nanoparticles. The Brust–Schiffrin technique allows for
production of monodisperse gold nanoparticles ranging
from 1 to 3 nm in diameter (Russier-Antoine et  al. 2008).
The resultant nanoparticles are stabilized by a monolayer of
alkanethiolates. The composition of the monolayer can be
changed from the beginning to the end through a substitu-
tion reaction to consist of specific functionalities, depending
on the intended use of the nanoparticles (Eatemadi et  al.
2014). Accordingly, gold nanoconjugates functionalized with
a monolayer of amine-terminated alkanethiolates (hereafter
referred to as amine-functionalized) have been prepared for
various biological applications.
Gene transfection
The ability to induce control over biological systems at
the genetic level is a fundamental concept in experimental
biology, and embraces huge promise for developing novel
treatments of disease (Huang et al. 2009). The search for the
best method for controlling gene expression is ongoing.
414 H. Daraee et al.
Figure 6. Transmission electron microscopy imaging and measurements of gold nanoparticles in cells. A) Graph of number of gold nanoparticles
per vesicle diameter for various nanoparticle sizes. B)–F) TEM images of gold nanoparticles with sizes of 14, 30, 50, 74, and 100 nm, respectively,
trapped inside vesicles of a HeLa cell.
Amine surface groups are positively charged at physio-
logical pH values, and thus amine-functionalized nanocon-
jugates electrostatically act together with negatively charged
nucleic acids.
Actually, these nanoconjugates are talented to transfect
these cells with a superior efficiency than the universally used
cationic polymer transfection agent polyethylenimine (PEI,
60 kDa). Building on these observations, these researchers
have recently shown that gold nanoparticles functionalized
with lysine moieties are highly effective at delivering DNA
plasmids, and outperform a profitable vector by a factor of
28 (Abbasi et al. 2014a).
The concentration of PEI was used to control the size
of the functionalized nanoparticles from 2.3 to 4.1 nm
in diameter. The resultant nanoconjugates deliver
plasmid DNA to COS-7 cells more efficiently than PEI
alone.
Stability
In addition to providing functional groups, surface-bound
ligands also contribute to the constancy of the GNP. The
constancy of the nanoconjugates is an significant con-
sideration for their impending use as therapeutic means
because they must preserve their constancy under
inconsiderate conditions such as in the cell or in the
bloodstream. It was found that increasing the net positive
charge on the nanoparticle surface caused a more rapid
dislocation of ligands, whereas more negatively charged
nanoconjugates did not display measurable disloca-
tion of surface-bound ligands (Abbasi et  al. 2014a). This
result is consistent with studies by our research group on
the stability of 13 nm oligonucleotide/gold nanoparticle
conjugates which found that the negatively charged thio-
lated oligonucleotide ligands are not easily displaced in
intracellular environments or by small molecules such as
glutathione (Kouhi et al. 2011).
 
Oligonucleotides
Over the past decade, our research group and others have
synthesized, characterized, and applied polyvalent DNA
functionalized gold nanoconjugates (DNA-GNP). Indeed,
the optical, catalytic, and binding properties of DNA-
GNP have been used for a variety of colorimetric (Wang et al.
2010, Pavlov et  al. 2004, Thiruppathiraja et  al. 2011), elec-
tronic, scanometric (Kamnev et al. 2002), and Raman-based
detection strategies, some of which have recently been com-
mercialized and approved by the FDA.
Synthesis
Nanoconjugates densely functionalized with synthetic
oligonucleotides are prepared by mixing alkanethiol-
terminated oligonucleotides and citrate-capped GNP.
Oligonucleotide ligands dislocate the citrate from the
GNP through creation of a gold–thiol bond. Methods
have been optimized for functionalizing particles with
diameters ranging from 2 to 250 nm (Vahedi et al. 2013). This
polyvalent material has a number of emergent properties
that are unique compared to the properties of the oligonu-
cleotides or the GNP alone (Figure 7).
Properties
One unusual but now fairly well understood property of
DNA-GNP is their ability to bind complementary nucleic
acids with a high affinity. Additionally, the oligonucle-
otides on the GNP surface are close enough such that the
counterions associated with one oligonucleotide also act to
screen negative charges on adjacent oligonucleotides. In the
context of cellular applications, it was theorized and subse-
quently it makes obvious that the higher binding constant
of the DNA-GNP would cause better intracellular binding
of the target molecule, thus raising the helpfulness of anti-
sense gene regulation. Nucleic acids are often hampered
 Gold nanoparticles in biomedical and drug delivery 415

Figure 7. The synthesis of the oligonucleotide gold nanoconjugates: Alkanethiol-terminated oligonucleotides are added to citrate-stabilized
GNP, thereby displacing the capping citrate ligands through formation of a gold–thiol bond. Subsequent addition of a salt shields repulsion
between the strands, thus leading to a dense monolayer of oligonucleotides.

in biological investigations by enzymatic hydrolysis, which
directs to degradation and cause them inactive. Another
emergent property of DNA-GNP is resistance to degradation
by enzymes such as DNase I. Two explanations have been
proposed as the origin of this enhanced stability:
The local Na concentration is the dominant factor that
contributes to the improved constancy of DNA. The resis-
tance of DNA-GNP to enzymatic degradation is an important
property that renders these structures extremely promising
candidates for introducing nucleic acids into cells, where
oligonucleotide degradation has historically been a major
challenge.
Cellular uptake
Perhaps the most surprising property of DNA-GNP is
their ability to enter a wide variety of cell types. Indeed,
because of their high negative charge, most researchers at
the time would have predicted that the nanoparticles would
not enter cells.
At DNA surface loadings of greater than about 18 pmol
cm22, cellular uptake can exceed one million DNA-GNP
per cell. The importance of the polyvalent arrangement of
oligonucleotides to cellular uptake can be further empha-
sized when comparing DNA-GNP to other types of GNP.
For instance, HeLa cells internalize only a few thousand
citrate-coated gold particles, compared to over one million
DNA-GNP under nearly identical conditions. Importantly,
fluorescence spectroscopy studies reveal that the thiolated
oligonucleotides remain bound to the GNP after cellular
internalization (Figure 8). Interestingly, biophysical charac-
terization of DNA-GNP after exposure to serum-containing
media reveals changes in the charge and size of the nano-
conjugates. The interaction of polyvalent nanoparticle
conjugates with proteins provides a possible mechanism of
recognition and subsequent internalization of these highly
negatively charged particles, the details of which are still
under intensive investigation.
Applications in cells
Methods based on nucleic acids for detecting and controlling
gene expression have had a significant impact on fundamental
studies of gene pathways and functions. Methods for control-
ling gene expression include the use of antisense oligonucle-
otides and small interfering RNA (siRNA).

Figure 8. Fluorescent microscopy images of C166-EGFP cells incubated for 48 h with gold nanoconjugates functionalized with dual fluorophore-
labeled oligonucleotides (3′-Cy3 and 5′-Cy5.5) only reveal fluorescence from Cy5.5 (706–717 nm, upper left). Negligible fluorescence is observed
in the emission range of Cy3 (565–615 nm, upper right). Transmission and composite overlay images are shown in the lower left and lower right
quadrants, respectively. The arrows indicate the location of the cell.
416 H. Daraee et al.  

Table I. Cell types that internalize polyvalent DNA gold nanoconjugates. increased the gene silencing capability to greater than 75%
Cellular internalization was determined using mass spectrometry and (Figure 9). Indeed, GNP can be encoded with a suite of
cell-associated fluorescence measurements.
designer oligonucleotides that confer improved properties,
Cell type Designation or source
ranging from amplified target specificity to catalytically
Breast SKBR3, MDA-MB-321, AU-565
Brain U87, LN229 improved biological processing. GNP densely functionalized
Bladder HT-1376, 5637, T24 with LNA form remarkably stable duplexes (Russier-Antoine
Colon LS513 et  al. 2008) with complementary nucleic acids, and can be
Cervix HeLa SiHa
Skin C166, KB, MCF, 10 A easily manipulated and governed under biologically relevant
Kidney MDCK, 293T conditions. For function in cells, the use of LNA-modified
Blood Sup T1, Jurkat GNP enlarges the efficiency of gene knockdown compared
Leukemia K562
Liver HepG2 to analogous DNA-modified GNP.
Ovary CHO
Macrophage RAW 264.7 Intracellular detection and imaging
Oligonucleotide-based probes to visualize and detect
An ideal gene regulation system—from a search intracellular RNA, including those used for in situ staining,
standpoint—should feature high uptake efficiencies across molecular beacons, and fluorescence resonance energy
all cell types, strong binding affinity for target nucleic acids, transfer (FRET) probes are important biological tools to
high intracellular stability, and very low toxicity. Recently, measure and quantify biological activity in living systems.
DNA-GNP were used as agents to alleviate several of the Recently, our research group has developed novel intra-
challenges that are commonly associated with the applica- cellular detection probes termed “nanoflares” that take
tion of nucleic acids in cells (Table I). advantage of the properties of DNA-GNP (Lin et  al. 2008).
Nanoflares are oligonucleotide-functionalized gold nano-
particles that are hybridized to short, fluorophore labeled
Antisense gene control complements designed to provide an intracellular fluores-
As a demonstration of this concept, DNA-GNP were cence signal that correlates with the concentration of a spe-
extended, which target the mRNA sequences that code for cific nucleic acid or molecular goal. In the lack of a target,
enhanced green fluorescent protein (eGFP) expressed in the fluorophore is near to the nanoparticle surface, which
mouse endothelial cells. An antisense sequence comple- quenches its fluorescence. Target binding releases the fluo-
mentary to an internal coding region of the mRNA for eGFP rophore, thereby generating a signal that can be detected
was used in the proposal and creation of “antisense nano- inside a live cell. Nanoflares can distinguish between differ-
particles”. Initial experiments make obvious a silencing ent cell types on the basis of the expression profile, and give
of approximately 20%, but further optimization of the a semi-quantitative real-time readout of gene expression
experimental parameters and conjugate structure has in a living sample (Figure 10). Several problems commonly

Figure 9. A) Representative Western blots showing the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HeLa cells treated with
various concentrations and compositions of the gold nanoconjugates. GAPDH expression is reduced in a dose- and sequence-dependent manner.
a-Tubulin is shown as the loading control. B) Relative decrease in GAPDH expression in HeLa cells. a-Tubulin was used as a loading control and for
subsequent normalization of GAPDH knockdown. The error bars represent the standard deviation from at least three Western blots.
 Gold nanoparticles in biomedical and drug delivery 417

Figure 10. “Nanoflares” are gold nanoconjugates functionalized with oligonucleotide sequences complementary to a specific nucleic acid
target (messenger RNA) hybridized to short fluorescent sequences. In the absence of a target, the nanoflares are dark because of quenching by the
gold nanoparticle. In the presence of a target, binding displaces the short flare through the formation of a longer (more energetically favorable)
duplex. The result is a fluorescence signal inside the cell, which indicates the target has been detected. Scale bar: 20 mm.

associated with intracellular RNA detection, including toxic-
ity, intracellular instability, and the difficulty associated with
cell entry, are obviated as these nanoparticles are densely
functionalized with oligonucleotides.
RNA interference
Recently, we determined that RNA-GNP can be syn-
thesized and subsequently introduced into cells
without the use of transfection agents. Traditional
RNAi uses molecular RNAs, which have very short
half-lives as a consequence of the instability of ribo-
nucleotides to RNase-type enzymes, thus limiting
their efficacy (Chiu and Rana 2003, Soutschek et  al.
2004). In the case of RNA gold nanoconjugates, a
dense monolayer of surface-immobilized RNA increases
the protection from nonspecific degradation both in cell
culture media and in the intracellular environment.
Cellular detection
In addition to intracellular applications, a colorimetric
assay has been developed that uses DNA-GNP for the detec-
tion of cancer cells or tissues. In particular, GNP were func-
tionalized with a monolayer of aptamers selected to have a
high affinity for surface receptors expressed by a cancer cell
line (CCRF-CEM). The aptamer-functionalized nanocon-
jugates accumulate on the cell surfaces, which causes their
surface plasmon resonances to interact. This results in a red
shift in the loss spectra, thus providing a direct recite of tar-
get binding.
Peptides
The targeting portions of many proteins are short stretches
of oligopeptides. Peptide-based nuclear localization sig-
nals have been employed to increase efficacy of conjugated
biomolecules and modify the intracellular localization.
Peptide nanoconjugates
Conjugation of peptides to gold nanoparticles is achieved
through attachment to bovine serum albumin (BSA) and
subsequent electrostatic association. The resulting nano-
conjugates enter the nucleus of HepG2 cells in culture.
Fascinatingly, only nanoconjugates functionalized with
peptides containing both nuclear localization signal (NLS)
and a receptor-mediated endocytosis (RME) are able to
enter the nucleus of these cells (Figure 11).
Peptide/DNA-gold nanoparticle conjugates
We recently prepared gold nanoconjugates functionalized
with both antisense oligonucleotides and NLS or HIV Tat
peptides (Jeynes et  al. 2013). Our synthetic strategy uses
thiolated oligonucleotides and cysteine-terminated peptides
to functionalize the GNP external surface. As the oligonucle-

Figure 11. Images of nanoparticle–peptide complexes incubated with HepG2 cells for 2 h. Complexes were: A) nuclear localization peptide,
B) receptor-mediated endocytosis peptide, C) adenoviral fiber protein, and D) both nuclear localization and receptor-mediated endocytosis
peptides.
418 H. Daraee et al.
otides and oligopeptides are conflictingly charged, the addi-
tion of salt is necessitated to monitor conflictingly charged
biomolecules during synthesis.
Multifunctional and multi component DNA nano
conjugates
Recently, our research group has demonstrated that
nanoflares can be adapted for both intracellular mRNA
detection and gene knockdown (Prigodich et al. 2009). These
nanoflares enter cells and bind mRNA in a location suitable
for gene knockdown, so decreasing the relative wealth of
mRNA, whereas simultaneously releasing a fluorescent flare.
Here, the nanoflare gives a read-out of gene regulation within
the cell. In addition, one can, in principle, create all sorts of
cell-sorting genetic screening assays by using the nanoflare
approach. Other therapeutic nanoconstructs have been
designed to take advantage of the uptake of DNA-GNP by
cells. These constructs, similar to their canonical DNA coun-
terparts, deliver the drug payload effectively to cells (Dhar
et al. 2009). Future work in this area will examine regulating
gene expression to chemo sensitize the cells while deliver-
ing drugs and materials. Such multi-component conjugates
should reduce the total of chemotherapeutic agent required
for therapeutic usefulness while simultaneously reducing
systemic toxicity.
Antibodies
Antibody-labeled gold nanoconjugates have been used in
immunohistochemistry for almost 40 years (Horisberger
et  al. 1975). Synthetic methods to produce antibody-gold
nanoconjugates include adsorption (Horisberger et  al. 1975),
N-hydroxysuccinimide (NHS) ester chemistry (Horisberger
et  al. 1975, Qian et  al. 2008), and oligonucleotide-directed
immobilization (Niemeyer et  al. 2003). Antibodies can
adsorb onto GNP through hydrophobic and ionic inter-
actions, or through chemisorption of native thiol groups
present in their chemical structure (Wheeler et  al. 1999).
However, conjugates synthesized with this method have lim-
ited stability because the proteins are easily desorbed (Javier
et al. 2008). GNP functionalized with monolayers containing
NHS esters can be reacted with the primary amine groups of
the antibody to form more stable organizations. Alternately,
DNA GNP can be hybridized with antibodies that have been
conjugated to complementary oligonucleotides (Javier et al.
2008).
Imaging
GNP modified with antibodies specific to cancer associ-
ated proteins have been used to image cancerous cells.
Light microscopy experiments show that conjugates bind
to cancerous cells with six-times greater affinity than the
noncancerous controls, thus making this method poten-
tially helpful for the recognition of cancer cells (Huang
et al. 2009).
Photothermal therapy
Gold nanorods and nanoshells conjugated with antibod-
ies are being developed as photothermal therapy agents
that use antibody-coated surfaces to hone on cancerous
cells. These cells were then irradiated with near-IR light
at a frequency that is resonant with the surface plasmon
resonance of the nano-shell. Light absorption directs
to heating, which causes cell death (Loo et  al. 2005).
Nano-shells conjugated to control antibodies did not dem-
onstrate this influence because of the lack of nano-shell
binding on the cell exteriors. These conjugates are also
being expanded as materials that merge photothermal
therapy with near-IR imaging capabilities (Stamatoiu et  al.
2012).
Lipids
Recently, lipids have joined oligonucleotides, peptides,
and antibodies as biomolecules used to modify GNP. In
this synthesis, thiolated lipids or alkane thiols along with
apolipoprotein A1 (APOA1), a protein component of HDL,
are adsorbed onto the surface of GNP. Next, a second lipid
is adsorbed onto the GNP surface through hydrophobic
interactions between the thiolated species and lipid tails.
Trouble-free methods for synthesizing HDL with control
over the shape, composition, and size had not been revealed
prior to these studies. It is being increasingly realized that
shape, size, and chemistry of HDL has an impact on its
in vivo physiology, and these structures might confirm use-
fulness as therapeutics and imaging agents (Russier-Antoine
et al. 2008).
Therapeutics
Natural HDL is critical for transporting cholesterol from
macrophages in atherosclerotic plaques and from the body,
and increasing the HDL levels may give an improvement
to reversing or preventing atherosclerosis. To that end,
our research group synthesized HDL mimics called HDL-
GNP whose size as well as protein and lipid contents are
like those of natural HDL (Figure 12). To the best of our
knowledge, this is the first measured binding constant for
any form of HDL and a cholesterol derivative. This is
important as it presents a key data point from which
to assess future constructs and their capability to bind
cholesterol as well as their potential as novel therapeutic
candidates.
Imaging
In addition to cholesterol transport, HDL-GNP mimics have
been employed to image macrophage cells in vivo. Mac-
rophage density is indicative of high-risk atherosclerotic
plaque, thus making it a beautiful imaging target. Tomogra-
phy images of the mice aortas demonstrated a build-up of
HDL-GNP, thus indicating that the nanoparticles possibly
will be applied to atherosclerotic imaging.
Gold nanoparticles in diagnostics
Visualization and bioimaging
Gold nanoparticles have been in active use in the identi-
fication of chemical and biological agents The visualiza-
 Gold nanoparticles in biomedical and drug delivery 419

Figure 12. Templated synthesis of spherical HDL nanoparticles through use of thiol-terminated peptides and the protein (APOA1). Adapted
from Ref. [111], with permission from the American Chemical Society; Copyright 2009.

tion methods with the use of GNP and optical microscopy Raman scattering (Lin et  al. 2008), have been used to
(Wang et al. 2010), in particular, confocal laser microscopy, enhance the sensitivity of the analytical homophase reaction
have gained increasing popularity in medical and biologi- (Figure 13).
cal research. The methods for obtaining confocal images
include fluorescence detection (confocal fluorescence Dot immunoassay
microscopy) or resonance elastic or two-photon (multipho- At the early stages of the development of immunoassays,
ton) light scattering by plasmon nanoparticles (resonance preference was given to liquid phase methods, in which the
scattering confocal microscopy or two-photon luminescence unbound antigen or the bound antibodies deposited were
confocal microscopy). These methods are based on detect- removed using dextran-coated activated coal. The solid-
ing micro-objects using an optical microscope in which the phase methods have lately been widely used (first used in
object’s luminescence is excited because of the simultane- radioimmunoassay of proteins), since they supply the pos-
ous absorption of two (or more) photons; the energy of each sibility to considerably make simpler the analysis procedure
of them being inferior than that required for fluorescence and shrink the background signal. The mainly widespread
excitation. The main advantage of this technique is that solid-phase carriers are polystyrene plates and nitrocel-
the strong reduction in the background signal results in lulose membranes. Enzymes (peroxidase, alkaline phos-
the contrast being enhanced. phatase, etc.) and radioactive isotopes (125I, 14C, 3H) are
extensively used as a label in membrane tests (dot and blot
Analytic methods for diagnostics
Homophase methods
This method is based on two principles: 1) the color and
absorption spectrum of a sol vary little upon biopolymer
adsorption on individual particles (Thiruppathiraja et  al.
2011), 2) when particles approach a distance that is less
than one-tenth of their diameter, the sol’s red color alters
into purpuric; the absorption range broadens and shifts Figure 13. Sol-particle immunoassay: a scheme of conjugate aggregation
into the red region (Thiruppathiraja et  al. 2011). These caused by binding to target molecules (a) and corresponding changes
in the sol color and absorption spectra.
changes in the absorption spectrum can be easily detected
either spectrophotometrically or visually, this method
was subsequently used for performing immunoassay of
the antigens of schistosomes and rubella viruses and for
the quantitative determination of immunoglobulins, for
determining thrombin (using aptamers) and glucose, for
the direct detection of cancer cells and leptospira cells in
urine, and for determining markers of Alzheimer’s disease
(Neely et al. 2009) and protease activity (Neely et al. 2009).
The simultaneous use of conjugates of gold nanorods and
nanospheres with antibodies for detecting tumor anti-
gens was described. The data on the determination of the
hepatitis B virus in blood using gold nanorods conjugated
with specific antibodies were published. Various opti- Figure 14. Dot immunoassays of a normal rabbit serum (1 by using
cal methods, including different versions of IR Fourier 15-nm GNPs and silica/gold nanoshells (180-nm-core diameter and
(Kamnev et  al. 2002) and UV-vis beam absorption or 15-nm gold shell) conjugated to sheep’s antirabbit antibodies. The IgG
quantity equals 1 mg for the first (upper left) square and is decreased
deflection spectroscopy, hyper-Rayleigh, differential static by two-fold dilution (left to right). The bottom rows (2) correspond to a
and dynamic light scattering, as well as surface-enhanced negative control (10 mg BSA in each square).
420 H. Daraee et al.
analyses). In 1984, four studies were separately published in
which colloidal gold was employed as a label for solid-phase
immunoanalysis. The use of GNP conjugates in solid-phase
analysis is based on the fact that the strong red coloration
of a gold-containing marker permits one to determine visu-
ally the results of a reaction that was carried out on a solid
carrier. “Immuno-gold techniques” in a dot blot assay are
better than the other types of assays (e.g., immunoenzyme
assay) in sensitivity (Beckmann et al. 1994), cost, speed, and
simplicity (Figure 14).
Immunochromatography
Immunochromatographic assay (Dykman and Khlebtsov
2011) is based on eluent motion along the membrane
(lateral diffusion), resulting in the formation of specific
immune complexes that are detected as stained bands on
different membrane regions. Enzymes, stained latexes,
and quantum dots are used as labels in these systems; but,
in the overwhelming bulk of cases, gold nanoparticles are
employed. The sample under investigation migrates along
the test strip due to capillary forces. If an illustration contains
the desired compound or immunologically close ones when
the sample passes through the absorbing device, a reaction
with specific antibodies labelled with colloidal gold occurs,
accompanied by the formation of an antigen–antibody
composite. The colloidal preparation is involved in the reac-
tion of competitive binding with the antigen immobilized
in the test zone (haptene conjugated with a protein carrier
is usually used for immobilization in the detection of
low-molecular-weight compounds) (Figures 15 and 16).
Plasmon resonance biosensors
New unique technologies are currently being used for the
design of biosensor devices, including monolayer self-
assembly of metal particles, nano lithography, and vacuum
evaporation. These properties of metal clusters served as the
basis for the design of new promising plasmon resonance
biosensor systems (SPR-biosensors) based on the conver-
sion of biospecific interactions into an optical signal. GNP-
based biosensors are applied not only in immunoanalysis,
but also for the detection of nucleotide sequences. A record
sensitivity was achieved for these sensors in pioneer stud-
ies in the zepto-molar range based on the recording spectra
of resonant scattering from individual units. This unlocked
the gate for the registration of intermolecular interactions
at the stage of individual molecules.
Figure 15. Positive (1) and negative (2) results of an
immunochromatography assay.
 
Figure 16. Scheme for detection of target molecules with a BIA core™
device based on a total internal reflection prism covered by a thin gold
layer. Adapted from Ref.
Gold nanoparticles in therapy
Photothermal therapy using gold nanoparticles
In 2003, GNP were applied for the first time as agents
for photothermal therapy (Soutschek et  al. 2004, Zharov
et  al. 2003); it was latter proposed to refer to this kind of
therapy as plasmonic photo thermal therapy (PPTT). A
new method for selective damage of target cells, which is
based on the use of 20–30 nm gold nanospheres radiated by
20-ns laser pulses (532 nm) in order to create Tumor local
warming-up, has been described (Pitsillides et al. 2003). The
sandwich technology consisting of labeling T-lymphocytes
with GNP conjugates was used for the pulse photothermy in
the model experiment.
Photodynamic therapy using gold particles
The photodynamic method (Doubrovsky et  al. 2010,
Akbarzadeh et  al. 2012a, 2012c, 2012d, Ebrahimnezhad
et al. 2013) is applied in the therapy of oncological diseases,
certain dermal or infectious diseases, and is based on the
use of light-sensitive agents—photosensitizers (including
dyes) and, typically, visible light of a certain wavelength. It
is well-known that metal nanoparticles are efficient fluo-
rescence quenching agents. Thus, gold nanostructures with
plasmon resonance show promise for the selective PPTT of
oncological and other diseases.
The use of gold nanoparticles as therapeutic agents
Gold nanoparticles are increasingly actively employed not
only in cell photo thermolysis experiments and diagnostics,
but also for therapeutic reasons. Accumulation of GNP in
the tumor is attested by the modification in the color of the
tumor; the tumor obtains a bright red/purple color (the color
typical of colloidal gold and its aggregates), which coincides
with the maximum of tumor-specific activity of the TNF (Ali-
mirzalu et al. 2014, Hosseininasab et al. 2014, Rezaei-Sada-
bady et al. 2013, Nejati-Koshki et al. 2013, Ghasemali et al.
2013, Mollazade et al. 2013, Akbarzadeh et al. 2014, 2013a,
Ahmadi et al. 2014).
Immunologic properties of gold nanoparticles
A significant number of studies devoted to the use of GNP
in designing DNA vaccines with gene constructions encod-

ing proteins, to which antibodies had to be produced, have
been published, Gold nanoparticles can stimulate anti-
body synthesis in rabbits, rats, and mice if a lower dose of
the antigen is used in comparison with the amount that is
required when using a number of conventional adjuvants.
Gold nanoparticles used as antigen carriers were shown
to stimulate the phagocytic activity of macrophages and
affect the functioning of lymphocytes, which most likely
are accountable for their immune-modulating effect. The fact
is that gold nanoparticles act both as a carrier and an adjuvant.
We believe that the detection of adjuvant properties in GNP cre-
ates favorable conditions for the development of a new genera-
tion of vaccines (Alizadeh et al. 2014, Pourhassan-Moghaddam
et  al. 2013, 2014, Anganeh et  al. 2014, Davoudi et  al. 2014,
Akbarzadeh et al. 2012b, 2013b, Valizadeh et al. 2012).
Conclusions
It is now widely accepted that GNP conjugates are excellent
labels for solving the problems of bioimaging, which can
be implemented using various optical technologies, includ-
ing resonance scattering dark-field microscopy, confocal laser
microscopy, different variants of two-photon luminescence of
GNP, optical coherence tomography, acoustic tomography, etc.
GNP conjugates have found application in analytic studies that
can be based both on modern instrumental methods (surface-
enhanced Raman spectroscopy, LISNA, IR Fourier spectros-
copy, etc.) and the use of simple solid-phase or homo phase
procedures (dot analysis, immune chromatography). Finally,
there is a necessity to continue and broaden studies on the bio-
distribution and the toxicity of GNP. First of all, a coordinated
program is required, which would reveal the correlations
between particle parameters (size, shape, functionalization
with various molecular probes), experimental parameters
(model, doses, method, and administration scheme, observa-
tion duration; organs, cells, subcellular structures under study,
etc.), and the observed biological effects. Coordinated efforts
in the introduction of standards for the particles and methods
used for the testing of nanomaterial toxicity are also required
(Kouhi et al. 2014, Sadat Tabatabaei Mirakabad et al. 2014, Eat-
emadi et al. 2014, Abbasi et al. 2014a, 2014b, Kouhi et al. 2011,
Malekynia et al. 2010, Vahedi et al. 2013).
Authors’ contributions
AA conceived of the study and participated in its design and
coordination. MK participated in the sequence alignment
and drafted the manuscript. All authors read and approved
the final manuscript.­­
Acknowledgments
The authors thank Department of Medical Nanotechnology,
Faculty of Advanced Medical Science of Tabriz University
for all supports provided.
Declaration of interest
The authors report no declarations of interest. The autors
alone are responsible for the content and writing of the
paper.
Gold nanoparticles in biomedical and drug delivery 421
This work is funded by 2014 Drug Applied Research
Center Tabriz university of Medical Sciences Grant.
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