International Journal of

Molecular Sciences

Review
Gold Nanoparticles in Diagnostics and Therapeutics
for Human Cancer
Priyanka Singh 1 , Santosh Pandit 2 , V.R.S.S. Mokkapati 2 , Abhroop Garg 1 ID
,
Vaishnavi Ravikumar 1 and Ivan Mijakovic 1,2, *
1 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark,
2800 Kgs. Lyngby, Denmark; [email protected] (P.S.); [email protected] (A.G.);
[email protected] (V.R.)
2 Systems and Synthetic Biology Division, Department of Biology and Biological Engineering,
Chalmers University of Technology, 41296 Chalmers, Sweden; [email protected] (S.P.);
[email protected] (V.R.S.S.M.)
* Correspondence: [email protected]; Tel.: +46-070-982-8446




Received: 8 June 2018; Accepted: 3 July 2018; Published: 6 July 2018


Abstract: The application of nanotechnology for the treatment of cancer is mostly based on
early tumor detection and diagnosis by nanodevices capable of selective targeting and delivery
of chemotherapeutic drugs to the specific tumor site. Due to the remarkable properties of gold
nanoparticles, they have long been considered as a potential tool for diagnosis of various cancers
and for drug delivery applications. These properties include high surface area to volume ratio,
surface plasmon resonance, surface chemistry and multi-functionalization, facile synthesis, and stable
nature. Moreover, the non-toxic and non-immunogenic nature of gold nanoparticles and the high
permeability and retention effect provide additional benefits by enabling easy penetration and
accumulation of drugs at the tumor sites. Various innovative approaches with gold nanoparticles are
under development. In this review, we provide an overview of recent progress made in the application
of gold nanoparticles in the treatment of cancer by tumor detection, drug delivery, imaging,
photothermal and photodynamic therapy and their current limitations in terms of bioavailability and
the fate of the nanoparticles.

Keywords: gold nanoparticles; cancer; protein corona; biocompatibility; photoimaging; drug delivery;
photothermal therapy; photodynamic therapy; clinical trials; toxicology

1. Introduction
With recent advances in nanotechnology and medical science, numerous nanoparticles and
nanomaterials have emerged from different bulk elements such as gold, silver, iron, copper, cobalt,
platinum, etc., which are synthesized either biologically or physiochemically [1,2]. The ability to
manipulate nanoparticle features, such as their physical, chemical and biological properties, opens up
many possibilities to explore these nanoparticles in drug delivery as image contrast agents and for
diagnostic purposes [3]. Among various organic and inorganic nanoparticles, gold nanoparticles
possess unique optical and Surface Plasmon Resonance (SPR) properties, due to which it has become
the first choice for researchers, particularly in the biological and pharmaceutical field (Figure 1). Due to
the optical properties of gold nanoparticles, they are especially utilized in ultrasensitive detection
and imaging-based therapeutic techniques required for the treatment of lethal diseases, such as
cancer. Cancer is a disease state caused by abnormal cell growth and is the third leading cause of
mortality worldwide. According to the World Health Organization (WHO, www.who.int), cancer
caused 8.8 million deaths in 2015. Current cancer treatment is based on chemotherapeutic drugs,

Int. J. Mol. Sci. 2018, 19, 1979; doi:10.3390/ijms19071979 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 1979 2 of 16

usually involving chemo or radiation therapy, with the aim to kill the cancer cells [4,5]. However,
these treatments often result in several side effects due to the damage caused to the surrounding
healthy tissues. In addition, delays in diagnosis and a high incidence of relapse result in lower survival
rates. Treating cancer cells by utilizing a nanoparticle-based drug delivery approach plays a key role
in overcoming the limitations of conventional treatment methodologies by providing simultaneous
diagnosis and treatment [4]. Consequently, a considerable amount of research focusing on gold
nanoparticle-based nanocarrier development and their potential applications in cancer biology and
nanomedicine has been carried out [6]. In this review, we focus on providing further new insights for
exploring the gold nanoparticle applications as a tool in cancer diagnostics and treatment.

Figure 1. Important properties of gold nanoparticles.

Typically, gold nanoparticles of controlled size and shape have been synthesized by various
physical (microwave and ultraviolet (UV) irradiation, laser ablation), chemical, and biological ways.
Chemical synthesis generally utilizes chemicals and solvents, which are associated to environmental
and human health impacts. In addition, it demands extreme conditions (e.g., pH, temperature) which
are not optimal [7–9]. On the other hand, biological nanoparticle synthesis (plants and microorganisms
mediated) is a relatively new, eco-friendly, and promising area of research with a considerable
potential for expansion [10–12]. Numerous medicinal plants have shown potential to produce stable
gold nanoparticles within a few seconds [11,13–15]. Microorganisms are also equally capable of
adsorbing gold atoms and accumulating gold nanoparticles by secreting large amounts of enzymes,
which are involved in the enzymatic reduction of gold ions [16,17]. These biologically synthesized
gold nanoparticles have become an attractive and potential option to explore as a tool for biosensors,
immunoassays, targeted drug delivery, photoimaging, photothermal therapy (PTT), and photodynamic
therapy (PDT) (Figure 2). Interestingly, in human cancer and cell biology, various types of gold
nanoparticles, such as gold nanorods, nanocages, nanostars, nanocubes, and nanospheres, have become
effective tools. Their application in cancer diagnostics and therapeutic development is due to their
favorable optical and physical properties that provide a potential platform for developing cancer
theranostics. The optical properties of gold nanoparticles rely on SPR. In principal, SPR is a process
whereby the electrons of gold resonate in response to an incoming radiation, causing them to both
absorb and scatter light. In addition, some specifically shaped gold nanoparticles contribute to photon
capture cross sections that are four to five-fold greater than those of photothermal dyes. These attributes
Int. J. Mol. Sci. 2018, 19, 1979 3 of 16

are exploited to obtain localized heating either to destroy the cells or for drug release, underlying the
therapeutic applications. In addition, gold nanoparticles possess tunable properties, which allow for
the synthesis of nanoparticles of specific size and desired shape, resulting in a plasmonic resonance shift
from 520 to 800–1200 nm (complex shapes) [18]. Susie et al. showed the change in optical properties
and resonance of gold nanoparticles (ranging from 500 to 1200) by slightly changing the nanoparticles’
shape from nanospheres of 15–30 nm to nanorods of 2.5–7.5 Aspect ratio (AR.) [19] The range between
800 and 1200 is therapeutically useful because the body tissue is moderately transparent to Near
Infra-Red (NIR) light, thereby providing an opportunity for therapeutic effects in deep tissues by
photothermal and photoimaging approaches. Another important property is the available surface
area. It is well known that the surface area of nanoparticles is inversely proportional to their size,
which results in a large surface area to volume ratio. In other words, nanoparticle have a large surface
area available for drug loading, conjugation, or binding of any gene or biological moiety of choice,
thus increasing drug solubility, stability, and pharmacokinetic parameters [20]. The available surface
area also plays a critical role for the application of gold nanoparticles in cancer diagnostics, specifically
in photo-imaging and photothermal therapy. In photothermal therapy, smaller nanoparticles are
preferred as light is mainly adsorbed by the nanoparticles and thus efficiently converted to heat for
destruction of cell, whereas in photo-imaging, lager nanoparticles are preferred because of their higher
scattering efficiency. In addition, biological responses to nanoparticles tend to scale with surface area.
This means that when nanoparticles are exposed to a biological environment, such as serum or plasma,
more proteins from the surroundings bind to small nanoparticles with a larger surface area-to-volume
ratio than to those with a larger size and a smaller surface area-to-volume ratio. In parallel with
the above-mentioned properties, the tailored surface functionalization of gold nanoparticles has
also evinced considerable interest. The possibility to conjugate gold nanoparticles with a variety of
biologically active moieties, especially with amine and thiol groups, provides possibilities for important
biomedical applications ranging from diagnostics, targeting specific delivery of drugs/genes, imaging,
and sensing for electron microscopy markers [21,22].

Figure 2. Different approaches of gold nanoparticles in cancer diagnosis and treatment.

Despite all these benefits, biocompatibility of gold nanoparticles is a crucial factor to take into
account prior to clinical applications. Although the inert nature of gold nanoparticles makes them
relatively biocompatible, the cytotoxicity of the nanoparticles, which is more or less dependent on
Int. J. Mol. Sci. 2018, 19, 1979 4 of 16

their shape, size, surface properties, and chemical composition, has to be further evaluated. Once the
particles are internalized by the cells, the proteins present in the physiological environment form
a coating called a “corona” on the surface of the nanoparticles, resulting in a nanoparticle-protein
complex [23]. This protein corona is quite complex and variable in structure and plays a key role in the
biodistribution of nanoparticles throughout the body. Dobrovolskaia et al. reported that untreated
plasma usually contains approx. 3700 proteins, and the gold nanoparticles that come in contact with the
plasma form a “protein corona complex” containing fewer than 100 proteins [24]. Gold nanoparticles
inside the corona complex contain opsonins on their surface, which are recognized by the immune
cells (part of reticuloendothelial system (RES)). These proteins ultimately determine the route of
nanoparticle internalization and eventually affect the fate of the nanoparticles in the body (i.e., rate and
route of clearance from the bloodstream and body, volume of distribution, organ disposition, etc.)
(Figure 3) [25]. So far, various proteins have been reported to have been isolated from the corona
complex in the plasma, such as albumin, fibrinogen, Immunoglobulin G (IgG), Immunoglobulin M
(IgM), transferrin, etc. This corona complex can cause changes in particle size and charge, which in
turn affect the internalization process into the macrophages of the RES and the overall distribution
in the body. Certain proteins allow macrophages to easily recognize nanoparticles; for example,
IgG opsonins, and fibrinogens are reported to promote phagocytosis and nanoparticles removal from
the body [26], whereas dysopsonins such as albumins are reported to cause prolonged blood circulation
of nanoparticles [27]. To prevent the nanoparticles from immune recognition, scientists introduced
a process called “PEGylation”. In this approach, nanoparticles “hide” by masking their surface with
a poly-ethyleneglycol (PEG) layer. This saves them from immune recognition, in essence prolonging
their blood circulation. PEGylation can be done by covalent linking that entraps or adsorbs PEG
chains onto the surface of the nanoparticle. Once the nanoparticles are internalized, they can be
used in tumor imaging, PTT, and PDT. Although PEGylation can help in avoiding rapid recognition
by the RES, complete avoidance is rarely achieved as nanoparticles may still be recognized and
taken up by the RES system (Figure 4). Though a lot of work in this regard has been conducted,
many challenges remain and must be tackled before PEGylation can be put into practice. In this review,
we summarize the importance and advantages of gold nanoparticles and their utilization in several
aspects of cancer therapeutics.

Figure 3. Distribution of nanoparticles with varying coatings and bound proteins. PEG = poly-ethyleneglycol.
Int. J. Mol. Sci. 2018, 19, 1979 5 of 16

Figure 4. Systemic delivery of multifunctional gold nanoparticles for cancer bioimaging, photothermal
therapy (PPT), and photodynamic therapy (PDT). EPR = enhanced permeation and retention;
NIR = near infra-red; ROS = reactive oxygen species.

2. Gold Nanoparticles as Drug Carriers

Traditional drug delivery approaches for chemotherapeutic drugs, i.e., oral or intravenous
administration, result in the dissemination of the drug in the whole body, with only a fraction
of the drug reaching the tumor site. This, however, can have side effects on healthy tissues and
organs. This problem of side effects is circumvented by targeted drug delivery approaches, which can
be defined as a process in which a specific biologically active compound or drug is released at
a specific location in a controlled manner. The development of nanoparticles has opened up enormous
possibilities for drug delivery. Due to their small size, they can efficiently pass through the capillaries to
reach their target cells. The chemotherapeutic drugs can be loaded or attached to nanoparticles and can
be targeted either passively or actively to the tumor site. Tumor tissue generally has a leaky vasculature,
which allows the nanoparticles to accumulate easily. This is also known as the enhanced permeation
and retention (EPR) effect. This form of passive targeting utilizes the pathophysiological properties
of the tumor tissue. However, there are certain limitations to this approach, which include arbitrary
targeting and inefficient dispersion of drugs in tumor cells. Additionally, not all tumors exhibit the
EPR effect. In active targeting, the ligands of tumor specific biomarkers such as monoclonal antibodies,
aptamers, peptides, and vitamins are conjugated on to the nanoparticle surface. These ligands then
interact with their receptors on the tumor cells, allowing for endocytosis and subsequent release of
the drug. Thus, active targeting offers a higher probability of endocytosis as compared to the passive
targeting approach [28].
Gold nanoparticles have caught the attention of scientists for their use as drug carriers because of
their SPR, optical, and tunable properties. They can be prepared in a broad range of core sizes (1 to
150 nm), which makes it easier to control their dispersion. The presence of a negative charge on the
surface of gold nanoparticles makes them easily modifiable. This means that they can be functionalized
easily by the addition of various biomolecules such as drugs, targeting ligands, and genes. In addition,
the biocompatibility and non-toxic nature of gold nanoparticles makes them an excellent candidate for
Int. J. Mol. Sci. 2018, 19, 1979 6 of 16

their use as drug carriers [29,30]. For example, methotrexate (MTX), which has been used to treat cancer
for decades, upon conjugation with gold nanoparticles displayed higher cytotoxicity towards numerous
tumor cell lines as compared to that of free MTX. MTX was observed to accumulate in the tumor cells
at a faster rate and to a higher level when conjugated with gold nanoparticles [31]. Another drug,
doxorubicin (DOX), when bound to gold nanoparticles via an acid labile linker, showed enhanced
toxicity against the multi drug resistant MCF-7/ADR breast cancer cell line, thus overcoming the multi
drug resistance to some extent due to the enhanced uptake of the gold nanoparticle-tethered drug
followed by its responsive release within the cell [32]. In the past, peptide-drug-conjugates (PDCs) have
been investigated for their use as anticancer agents [33–36]. However, their stability in the blood, liver,
and kidneys pose a significant challenge to their successful use as an anticancer molecule. Recently,
it was shown that this difficulty can be by-passed by conjugating these PDCs to gold nanoparticles.
The authors reported an increase in the half-life of PDCs from 10.6–15.4 min (administered alone),
to 21.0–22.3 h (upon conjugation with gold nanoparticle), while retaining cytotoxicity [37]. Apart from
synthetic drugs, phytochemicals have also shown the potential of being used as anticancer drugs but,
similar to PDCs, they too have certain problems such as low specificity, short half-life, fast clearance
rate, and inefficient cell penetration. These problems in using phytochemicals can be by-passed by
conjugating them to gold nanoparticles. For example, kaempferol (a phytochemical) conjugated to
gold nanoparticles displayed both significantly higher apoptosis and inhibition of angiogenesis in
MCF-7 breast cancer cells as compared to kaempferol alone [38]. Table 1 lists some of the studies
performed to investigate the anti-tumor applications of gold nanoparticles in drug delivery.

Table 1. Anti-tumor applications of gold nanoparticles in drug delivery.

Nanoparticle
Nanoparticle Outcome Cell Lines Ref.
Size (nm)
Higher cytotoxicity towards numerous cell
lines as compared to free MTX. Lewis lung carcinoma
MTX-AuNP 8–80 [31]
Suppression of tumor growth with (LL2) cells
MTX-AuNP but not with free MTX.
Enhanced toxicity against multi drug
DOX-Hyd@AuNP 30 MCF-7/ADR cancer cells [32]
resistant cancer cells.
A549 lung epithelial
Platinum-tethering exhibited higher
cancer cell line, HCT116,
(Pt(R,R-dach))-AuNP 26.7 cytotoxicity as compared to free oxaliplatin [39]
HCT15, HT29, and RKO
that could enter the nucleus.
colon cancer cell lines
Cellular uptake of targeted particles was
Tfpep-AuNP conjugated with LN229 and U87 human
5.1 significantly higher than that of the [40]
photodynamic pro-drug Pc 4 glioma cancer lines
non-targeted ones.
Higher cell death as compared to
CPP-DOX-AuNP 25 HeLa cells and A549 cells [41]
previously tested 41 nm AuNP.
Enhanced drug accumulation and retention
HepG2-R, C0045C,
FA-Au-SMCC-DOX as compared to free DOX in multi drug [42]
and HDF
resistant cancer cells.
Increased efficacy of BHC against
FA-BHC-AuNP 20–60 Vero and HeLa [43]
cancer cells.
Enhanced cellular uptake and cytotoxicity 4T1 mouse mammary
Au-P(LA-DOX)-b-PEG-OH/FA NP 34 [44]
against cancer cells. carcinoma cell line
Induction of early and late apoptosis in
A549, H460, and H520
DOX@PVP-AuNP 12 lung cancer cells and upregulation of tumor [45]
human lung cancer cells
suppression genes.
Enhanced half-maximal effective drug
DOX-BLM-PEG-AuNP 10 concentration, providing rationale for HeLa cells [46]
chemotherapy using two drugs.
The biomimetic nanoparticle loaded with
4T1 mouse mammary
EpCam-RPAuN 48 PTX was used in combination treatment [47]
carcinoma cell line
(PTT and chemotherapy).

AuNP: Gold nanoparticle, AuN: Gold nanocage, BHC: Berberine hydrochloride, BLM: Bleomycin, CPP:
Cell penetrating peptides, DOX: Doxorubicin, EpCam: epithelial cell adhesion molecule, FA: Folic acid, Hyd:
Hydrazone, MTX: Methotrexate, PEG: Poly ethylene glycol, PLA: Poly L-aspartate, (Pt(R,R-dach)): Active ingredient
of oxaliplatin, PTT: Photothermal therapy, PTX: Paclitaxel, PVP: Polyvinylpyrrolidone, SMCC: Succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate, Tfpep: Transferrin peptide.
Int. J. Mol. Sci. 2018, 19, 1979 7 of 16

3. Gold Nanoparticles in Photothermal Therapy and Photoimaging

In this section, we discuss the photothermal and photoimaging applications of gold nanoparticles,
which are still under development. Photothermal therapy (PTT) is referred to as photon mediated
induction of the localized therapeutic temperature that can stimulate hyperthermic physiological
responses. In simple terms, it melts the tumor in molten gold. This therapy uses metal nanoparticles
(such as gold) that can exhibit SPR and efficiently convert light into heat [48]. The extent of heat
generation is directly proportional to the incident excitation power and the nanoparticle itself.
In addition, the wavelength of plasmonic absorption can be tuned by optimizing the surface properties,
shape, and size of the nanoparticles. The nanoparticles used in PTT are generally rod-shaped or
shell-shaped gold nanoparticles, and when they are being introduced in to a biological environment,
like any other foreign material, the cellular uptake can be limited. Several groups have reported
different methods of tagging, functionalization, etc. based on which method seems to largely improve
the uptake [49]. Near infra-red (NIR) light is chosen for PTT due to minimal absorption by the
tissues at these wavelengths (650–900 nm) [50], which is enough to induce cytotoxic damage [51].
There have also been other studies on PTT where gold nanoparticles were used at a maximum
absorption of 795 nm and also on branched gold nanoparticles functionalized with nanobodies that
can effectively kill cancer cells leaving healthy cells unharmed [52,53]. Lin et al. carried out the first
demonstration of gold nanoparticles in PTT where 30 nm gold nanospheres conjugated with IgG
antibodies were used [54]. This in vitro demonstration was specifically targeted towards CD8 (cluster
of differentiation 8) receptors on lymphocytes where 95% of the gold nanoparticle (AuNP)-IgG treated
cells were killed. The most successful gold nanoparticles that are approved by the Food and Drug
Administration (FDA) and are in ongoing human pilot studies are PEGylated gold nanoparticles.
They have shown enhanced accumulation and absorption at the tumor site in the NIR region [55].
According to the reported studies, more than 50% of the mice treated with a single administration of
PEGylated gold nanoparticles and a single 10 min laser exposure showed no recurrence of the disease
even after 2 weeks [55]. Further optimized PEGylated silica gold nanocore shells are being studied for
the head and neck tumors in humans (Nanospectra Biosciences INC NCT00848042).
Photodynamic therapy (PDT) is another form of cancer treatment that utilizes light,
photosensitizers and oxygen from the tissues. Unlike PTT, which is oxygen independent, PDT is
completely dependent on the availability of tissue oxygen. In the case of PDT, a photosensitizing
agent such as porphyrin is intravenously injected into the tissues and excited by specific wavelengths,
leading to the energy transfer that generates reactive oxygen species (ROS) and causes cell death by
apoptosis. PDT typically has a very low chance of causing mutations as the ROS do not accumulate
in the cell nuclei. Recently, PTT and PDT were applied simultaneously in melanoma-xenografted
mice. The lipid-coated gold nanocages were exposed to a 980 nm continuous wave (CW) laser,
which raised the temperature (~10 ◦ C) and generated the singlet oxygen, ultimately damaging
the tumor growth [56]. The biggest drawback with PDT is that the photosensitizers are not
generally soluble in the physiological environment, which inhibits their uptake by the diseased
tissues. Nevertheless, some studies do show that these photosensitizers can be conjugated to the
gold nanoparticles, thereby facilitating their uptake and delivery to the cancer tissues [57]. On the
other hand, PDT has its own advantages in terms of minimal invasiveness, no cumulative toxicity,
and reduced morbidity [58], which has proven effective for lung cancer [59], head and neck cancers [60],
and skin cancer [61]. The important and interesting aspect is that PDT and PTT can also be combined
to have an effective dual therapy where gold nanoparticles (nanorods and nanocages) were used
in conjugation [61–63]. One good example is the work by Seo et al., where methylene blue loaded
mesoporous silica coated gold nanorods were used for PTT/PDT dual therapy [64]. The photosensitizer
was physically adsorbed on the pores of the silica shell. Upon irradiation with NIR (near infra-red)
light at 780 nm, the viability of CT-26 cells (Murine colon carcinoma) was found to decrease by 31%
for the cells transfected with gold nanorods, while the methylene blue (MB) loaded nanocomposites
showed an 11% drop, which indicates a synergistic effect of dual therapy [64]. An important aspect that
Int. J. Mol. Sci. 2018, 19, 1979 8 of 16

has to be noted here is the effect of PTT therapy on PDT or vice versa. Liu et al. presented a detailed
analysis of this dual therapy where they clearly showed that the hypoxic environment induced by PDT
does not affect PTT as PTT is an oxygen-independent therapy.
Photoimaging is an advanced technique that can help detect early stage tumors and guide the
surgeons for precision treatment. One of the biggest challenges today for the surgeons is to have a clear
picture of where the tumor ends and the healthy tissue begins. During an operation, the surgeons
face a nearly impossible task of deciding to what extent the tumor has to be removed: being too
conservative can leave some tumor cells behind, and being too liberal can result in removing healthy
tissues that can be vital. Being too conservative is the reason that most of the tumors recur with time.
Magnetic Resonance Imaging (MRIs) and Computed Tomography (CT) scans are limited and can
only detect tumors above a size of several millimeters or approximately 10 million cells, meaning
that the tumors are detected only when they reach a certain threshold. Photoimaging is a novel
approach in cancer treatment where millions of functionalized gold nanoparticles are site specifically
injected into the tumor, where they specifically bind to the cancer cells and scatter (shine), making
it easier for the surgeons to identify the tumor and healthy cells. Gold nanoparticles (nanorods,
nanocages, and nanoshells) are known to be the best available photo imaging nanoparticles for cancer
therapeutics due to their bio inertness and their ability to provide increased spacial and temporal
resolution for imaging [65].

4. Recent Advance to Explore Gold Nanoparticles in Clinical Trials

Very few clinical trials are being actively carried out for the approval of gold nanoparticles for
cancer diagnostics and therapy (Table 2) [66,67]. According to the literature, the FDA has approved
few gold nanoparticle-based technologies for diagnostic and therapeutic purposes in medicine [55,68].
The cytotoxicity of gold nanoparticles is highly dependent on the size and morphology of the particles,
environmental scenario, and the method of production [66,69,70]. One of the clinical trials being carried
out by Astra Zeneca in partnership with Cytimmune mainly focuses on gold nanoparticle-based cancer
treatment (http://cytimmune.com/#pipeline). Their first phase of trials was successfully completed.
Aurimune (CYT-6091) was used as a vehicle to deliver the recombinant human tumor necrosis factor
alpha (rhTNF) into tumors, which disrupted the blood vessels, enabling chemotherapeutic drugs to
penetrate the tumor and damage the cancer cells. Safe delivery of highly effective doses of rhTNF
to tumor cells was observed [71]. The authors also found that the dose of rhTNF administered after
immobilization to gold nanoparticles could be three times higher than its usual dose without any toxic
effect [71]. The PEG layer also decreased the uptake of nanoparticles by the mononuclear phagocytic
system (MPS) and aided in their accumulation in the tumor masses via the EPR effect.
Due to the favorable ability of gold nanoparticles to absorb NIR-light, interest towards PTT
has increased of late. Researchers are mainly focusing on the photothermal conversion efficiencies,
selective targeting of cancer cells, enhanced cancer cell destruction, and in vivo bio-distribution of the
nanoparticles [72]. For example, Aurolase® , developed by Nanospectra, are silica-gold nanoshells
coated with (poly)ethylene glycol (PEG) and designed to thermally ablate the solid tumors following
stimulation with a NIR light source. The absorption of light leads to an increase in the local temperature,
which thermally dissolves the solid tumors [73]. In a recent clinical trial, AuroLase® particles were
used in a localized therapy for the treatment of primary or metastatic lung tumors (NCT01679470).
Another trial, last updated in September 2017 (NCT00848042), was reported as being completed
for the treatment of patients with refractory and/or recurrent tumors for head and neck cancer.
A currently active trial uses AuroLase® as an imaging technology during focal ablation of prostate
tissue using nanoparticle-directed laser irradiation. Since no active drug is used in this approach,
AuroLase® is activated externally at the target site, thus avoiding any toxicity towards the normal cells.
This is the only ultra-focal tumor ablation therapy designed and dedicated to maximize the treatment
efficacy while causing minimum side effects. In addition to this, several gold nanoparticle-based
theranostics have recently advanced to clinical trials. One such currently ongoing trial is aimed at
Int. J. Mol. Sci. 2018, 19, 1979 9 of 16

the safety evaluation of NU-0129, a spherical nucleic acid (SNA) formulation composed of small
interfering RNAs (siRNAs) targeting the Bcl-2-like protein 12 (BCL2L12) sequence and conjugated
to gold nanoparticles, that has potential antineoplastic activity (NCT03020017). Kharlamov et al.
studied the safety and feasibility of two delivery techniques for gold nanoparticles for the treatment of
atherosclerosis. The first is a PTT approach with silica-gold nanoparticles and the second is a magnetic
navigation approach with silica-gold iron bearing nanoparticles (NCT01270139) [74]. Results obtained
in this trial suggested that PTT using gold-silica nanoparticles is associated with a significant regression
of coronary atherosclerosis and an acceptable level of safety for clinical practice. CNM-Au8 is a gold
nanocrystal suspension drug developed by Clene Nanomedicine (Salt Lake City, UT, USA) for the
demyelinating disorder neuromyelitis optica (NMO) (NCT02755870). A randomized placebo controlled
trial is ongoing in healthy individuals to evaluate the safety, tolerability, and pharmacokinetics of
CNM-Au8. Another gold-nanoparticle based clinical trial is currently underway, with the aim to
evaluate the feasibility of a novel method in oncology involving breath analysis with a nanosensor
array for identifying gastric diseases (NCT01420588). It has been suggested that the nanosensor
array could provide the missing non-invasive screening tool to distinguish gastric cancer and related
precancerous lesions [75]. A clinical trial on another novel diagnostic approach (electronic nose sensor)
with gold nanoparticles is ongoing for the evaluation of its performance in the diagnosis of pulmonary
arterial hypertension (NCT02782026).

Table 2. Lists of clinical trials of gold nanoparticles.

Clinical trials.gov
Name Materials Application
Identifier
Laser responsive thermal ablation of solid
Silica-gold nanoshells coated NCT00848042,
AuroLase® tumors: head/neck cancer, primary and/or
with PEG NCT01679470
metastatic lung tumors
Prostate, head and neck, lung MRI/US fusion
Silica-gold nanoshells coated imaging and biopsy in combination with
AuroLase® NCT02680535
with PEG nanoparticle-directed focal therapy for ablation
of prostate tissue
A Spherical Nucleic Acid Targeting BCL2L12 in recurrent glioblastoma
NU-0129 NCT03020017
(SNA) Gold Nanoparticle multiforme or gliosarcoma patients
Silica-Gold Plasmonic photothermal therapy of
Silica-Gold Nanoparticles NCT01270139
Nanoparticles flow-limiting atherosclerotic lesions
Evaluation of safety, tolerability, and
CNM-Au8 gold nanocrystal pharmacokinetics of CNM-Au8 in healthy male NCT02755870
and female volunteers
Sensors functionalized with gold nanoparticles
Gold Nanoparticles Gold nanoparticles Organic functionalized gold nanoparticles NCT01420588
Detection of gastric lesions
Exhaled breath olfactory signature of
Gold Nanoparticles Gold nanoparticles NCT02782026
pulmonary arterial hypertension

5. Current Limitations
As described above, gold nanoparticles do show promise and potential to be used in cancer
diagnostics and therapeutics. Nevertheless, it is imperative to consider the other side of the coin,
i.e., unintended side effects on human health. A number of individual studies previously addressed
the cytotoxicity, effect of size on toxicity, efficacy, biodistribution, retention time, and physiological
response of nanoparticles. However, many of them are seen to contradict one another. Absence of
coherent information on the actual effect of nanoparticles could have delirious effects and a negative
impact on human health. While the discussed issues in general are applicable to any nanoparticle,
examples described below are specific to gold nanoparticles.
Toxicity: The toxicity of gold nanoparticles to biological systems has always been an issue of
concern. Properties of gold nanoparticles such as shape, size, surface chemistry, targeting ligand,
elasticity, and composition largely influence their toxicity. This, in combination with the complexity and
the heterogeneity that exists amongst human cells and tissues, makes it challenging to comprehensively
Int. J. Mol. Sci. 2018, 19, 1979 10 of 16

probe the effect and response of the biological system to the administration of gold nanoparticles.
Surface charge has been reported to influence toxicity of gold nanoparticles, wherein positively
charged particles were found to be more toxic than negative or neutral particles [76]. On the other
hand, other groups found no toxicity induced by positively charged gold nanoparticles [77] and no
toxicity of negatively charged particles [78]. This discrepancy arises due to the unique physiochemical
nature of nanoparticles, and no single standardized assay is currently available that could universally
be applied to test the toxicity effect of all nanoparticles. The lack of such robust standardized assays
leads to varying interpretations or assumptions that limit nanoparticle administration. Toxicity assay
using Caenorhabditis elegans (ISO 10872 method) is widely used to assess the effect of nanoparticles on
multicellular organisms. Hanna et al. recently reported an artifact caused while testing the toxicity of
positively charged gold nanoparticles using the C. elegans assay [79]. The authors initially observed
growth inhibition of the nematodes when fed with E. coli along with a suspension of positively
charged gold nanoparticles. However, they deduced that this observation was a false positive as
gold nanoparticles heteroagglomerate with E. coli cells, influencing the ability of the nematodes to
feed. On repeating the assay in the absence of E. coli, the authors observed a reduced toxicity effect,
illustrating unforeseen artifacts that could occur in such widely used toxicity assays. Ginzburg et al.
showed a synergistic toxicity effect induced by gold nanoparticles in the presence of additives such
as surfactants, while the individual components separately exhibited low toxicity [80], underlining
the importance of identifying strategies for selecting safe nanoparticles-additive/ligand/modifier
combinations. Gold nanoparticles were also shown to have species-specific differences with respect
to biodistribution, pathophysiologic response, and retention time. Bahamonde et al. observed that
gold nanoparticle-treated mice and rats responded differently, wherein a number of rats died on gold
nanoparticle administration while no fatality was seen amongst the mice [81]. Additionally, the authors
also noticed a relatively higher accumulation of the gold nanoparticle in rats as compared to the mice,
highlighting the fact of differential physiological response even amongst closely related groups.
Size and Biodistribution: Apart from toxicity assessment, size and biodistribution of nanoparticles
are also significant factors to take into consideration. Tang et al. reported increased cytotoxicity of
smaller gold nanoparticles (8 nm) coated with reduced glutathione when tested on a human hepatic cell
line as compared to that of the larger particles (37 nm) [82]. On the other hand, Rosli et al. recorded that
50 nm gold nanoparticles exhibited higher cytotoxicity in a breast cancer cell line as compared to their
13 and 70 nm counterparts [83]. Connor et al. studied the cytotoxicity of a series of gold nanoparticle
sizes ranging from 4 to 18 nm on human leukemia cells and found that none of the sizes were not
harmful to cellular function [84]. Liang et al. showed that PEG-coated gold nanoparticles of 4.8 nm had
the highest toxicity effect on Hela cells whereas the 12.1 and 27.3 nm counterparts showed low toxicity
and the 46.6 nm counterpart exhibited absolutely no toxicity [85]. Li et al., however, reported that
regardless of the size of the nanoparticles, the observed cytotoxicity was due to dose-dependency [86].
Sonavane et al. noticed a similar accumulation of gold nanoparticles in the liver while testing the
effect of nanoparticle size on biodistribution. The authors observed that gold nanoparticles of all
sizes mainly accumulated in organs like liver, lung, and spleen. The 15 nm particles accumulated in
tissues including blood, liver, lung, spleen, kidney, brain, heart, and stomach whereas much larger
particles (200 nm) showed a very minute presence in organs including blood, brain, stomach, and
pancreas [87]. With respect to biodistribution, Fraga et al. assessed the biodistribution of ~20 nm
citrate- and pentapeptide CALNN (cysteine–alanine–leucine–asparagine–asparagine) -coated gold
nanoparticles and found them to mainly accumulate in liver [88]. Cho et al. analyzed gold contents
of 13 nm PEG-coated gold nanoparticles and found them to accumulate in the liver and spleen [89].
Li et al. showed the accumulation of larger PEG-coated gold nanoparticles (42.5 and 61.2 nm) in the
liver. Additionally, the authors also observed a longer retention time (poor elimination rate) possibly
causing further safety issues [86]. Aside from those issues mentioned above, it is vital to take into
account the non-biodegradability and non-porous properties of gold nanoparticles which would likely
have an effect on the pharmacokinetics [90].
Int. J. Mol. Sci. 2018, 19, 1979 11 of 16

Thus, currently, there is controversy and inconsistency regarding the potential of gold
nanoparticles for clinical applications, and there is an inherent need for the development of universally
applicable methods to evaluate the biocompatibility of gold nanoparticles and to have a firm
understanding of their interaction with the living system. Lastly, it is also worthwhile to reflect on
whether the cost of synthesis would justify the underlying therapeutic capabilities of gold nanoparticles
(concept discussed in general in [91]).

6. Concluding Remarks and Future Perspectives

In this review, we highlighted the recent advances in the development and application of
gold nanoparticles in cancer diagnostics and treatment. Gold nanoparticles’ optical properties,
easy synthesis, possible control over size and shape, colloidal stability, and the ability to tune
the surface chemistry to achieve easy conjugation with biological moieties make them favorable
for biomedical applications. Importantly, surface and core properties of gold nanoparticles can
be optimized for individual and multifold applications including molecular recognition, chemical
sensing. and imaging. Thus, photoimaging and PTT are attractive approaches to treat the tumor
cells. Millions of functionalized gold nanoparticles can be released into the blood stream and bind
to specific cancer cells, and in turn either aid in treatment via PTT or enable photoimaging for the
successful removal of the tumor by operation. Despite these advances with gold nanoparticles, there is
still a requirement for more cost-effective gold nanoparticle-based systems, which will allow cancer
diagnosis and treatment at an early stage with a high level of specificity. The very first step for
developing such a nanosystem is to understand the nanoparticle properties described above and
the nature of the protein corona complex, which eventually influences its uptake and distribution
throughout the body. Gold nanoparticles are actively employed in drug delivery, PTT, and in imaging
techniques, despite these approaches having limitations owing to non-specific binding and the potential
activation of the normal host immune response. These issues have been tackled by using PEGylation
Mask technology thereby rendering the nanoparticle surfaces inert with respect to protein absorption,
thus reducing the probability of an immune response. Paradoxically, if a particle is so well coated that
it becomes “invisible” to the immune system, it will also probably lose its ability to bind to specific
receptors. To overcome these in vivo barriers, gold nanocomplexes are further modified with targeting
ligands and bioresponsive linkers. However, extensive modification may cause unwanted toxic effects.
In principle, active targeting of the nanoparticle is also possible, although further work on methods to
evade the immune system en route to the target is a prerequisite. Thus, there are a number of critical
issues that need to be addressed such as reproducible and reliable manufacturing methods/assays,
long-term health effects, stability, and cellular and immune responses. This calls for continued research
in the development of the techniques described above, particularly with respect to active targeting
and PTT. Moreover, nanoparticle fate is an equally important and substantial aspect to acknowledge
before exploiting it in the clinical trial applications. Hence, in this respect, a great deal of research will
be required to focus on internalization of the nanoparticles, their subsequent localization, relevant
immunological response, and, most importantly, their excretion from the human body.

Acknowledgments: We acknowledge the financial support from the H.C. Ørsted fellowship, co-funded by
Marie Skłodowska Curie, to P.S. and Novo Nordisk Foundation and VINNOVA to I.M.
Conflicts of Interest: The authors declare no conflict of interest.

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