Received 8 May 2016
Received in revised form 5 August
Accepted 8 August 2016
Available online xxx
This study validates the utility of Gum Arabic-conjugated gold nanoparticles (GA-AuNPs) and laser to induce photother-
mal inhibition of hepatocarcinogenesis, via employing a diethylnitrosamine (DEN)-mediated hepatocellular carcinoma
model. This work included both of in vitro and in vivo studies; to investigate the GA-AuNPs cytotoxicity and phototoxi-
city in hepatic cell line; to delineate the GA-AuNPs therapeutic efficiency in DEN-induced preneoplastic lesions (PNLs)
in the liver of Balb-C mice. The therapeutic effects of GA-AuNPs on the mediators of apoptosis, inflammation, and tu-
mor initiation, as well as the histopathological changes in preneoplastic liver have been investigated. Our results infer
that GA–AuNPs in combination with laser irradiation led to a significant reduction in the cell viability and in histone
deacetylase activity in hepatocarcinoma HepG2 cells. In chemically-induced PNLs mice model our results have demon-
strated that GA-AuNPs, with or without laser irradiation, induced cancer cell apoptosis through the activation of death
receptors DR5 and caspase-3 and inhibited both of the PNLs incidence and the initiation marker (placental glutathione
S-transferase; GST-P). The laser-stimulated GA–AuNPs significantly reduced the tumor necrosis factor-αlevels. In sum-
mary, GA–AuNPs with laser treatment inhibited liver PNLs via the induction of the extrinsic apoptosis pathway and the
inhibition of inflammation.
© 2016 Published by Elsevier Ltd.
Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com
Photothermal therapy mediated by gum Arabic-conjugated gold nanoparticles
suppresses liver preneoplastic lesions in mice
Amira M. Gamal-Eldeen a, b, ⁎, Dina Moustafa c, Sherien M. El-Daly a, d, Enas A. El-Hussieny e, Samira Saleh f,
Menka Khoobchandani g, h, Kathryn L. Bacon g, h, Sagar Gupta g, h, k, l, Kavita Katti g, h, Ravi Shukla i,
Kattesh V. Katti g, h, j, k, l, ⁎
aCancer Biology and Genetics Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt
bDepartment of Biochemistry, National Research Centre, Cairo, Egypt
cDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, October 6 University, 6 October City, Giza, Egypt
dDepartment of Medical Biochemistry, National Research Centre, Cairo, Egypt
eZoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt
fDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt
gDepartment of Radiology, University of Missouri, Columbia, MO 65212, USA
hInstitute of Green Nanotechnology, University of Missouri, Columbia, MO 65212, USA
iCentre for Advanced Materials and Industrial Chemistry; School of Applied Sciences; Health Innovation Research Institute; RMIT University, Australia
jResearch Reactor, University of Missouri, Columbia, MO 65212, USA
kDepartment of Physics, University of Missouri, Columbia, MO 65212, USA
lDepartment of Bioengineering, University of Missouri, Columbia, MO 65211, USA
Hepatocellular carcinoma (HCC) is reported affect 500,000 people
annually . Hepatocarcinogenesis is the multistep process character-
ized by several genetic variations causing aberrant growth and malig-
nant transformation of liver cells. Formation of preneoplastic lesions
(PNLs) is considered the early step in hepatocarcinogenesis, where
part of them developed through time into carcinomas . Thus target-
ing these lesions can be a successful tool to prevent the development
Photothermal therapy (PTT), an efficient minimally invasive ap-
proach, involves photothermal sensitizers' usage that can transform
photon energy into thermal energy, which can cause irreversible cel-
lular damage, and eventually cell death. For PTT applications, the ab-
sorption band for the nanoparticle (NP) sensitizers is preferred in the
⁎⁎ Corresponding authors.
Email address: firstname.lastname@example.org (A.M. Gamal-Eldeen )
near-infrared region (NIR > 700 nm) to enhance the light penetration
into tissues .
Nanotechnology is an uprising area of research that is intensively
applied nowadays in the medical field. Gold nanoparticles (AuNPs)
have been actively used in PTT , where in the presence of NIR ra-
diation, AuNPs produce what is known as plasmon resonance effect
(PR) where NPs can absorb and scatter light of wavelengths larger
than that of NPs and thus causing a localized heating. This local heat-
ing would cause tumor tissue damage and/or release of payload mole-
cules of therapeutic importance. The frequency of PR mainly depends
on the nanoparticle size and shape [5,7]. The efficiency of AuNPs
as PPT is mainly depending on the size, shape and physical proper-
ties of these nanoparticles. Nanospheres, nanorods, nanoshells, nanos-
tars, and nanocages have all been reported as subtypes of AuNPs .
AuNPs are carriers and enhancers of specific photoactive functional
groups to achieve effective penetration across cancer cell membranes.
The versatile surface chemistry and unique photophysical character-
istics of AuNPs providing a building block for multifunctional
1011-1344/© 2016 Published by Elsevier Ltd.
2 Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
nanoplatforms for PTT applications. AuNPs can be conjugated to bio-
logically active moieties for effective localized therapy at tumor sites
[7–9]. We have successfully developed a new generation of hybrid
AuNPs that demonstrated their cancer cell-targeting features for tu-
mors molecular imaging and therapy using tumor specific nanoprobes
We are currently interested in the utility of Gum Arabic (GA) en-
capsulated AuNPs (GA-AuNPs) as potential PTT probes, since the
highly branched polysaccharide structure of GA consisting of a com-
plex mixture of glycoproteins encompassing arabic acid, with residues
of rhamnose, glucuronic acid, galactose, and arabinose, which may
promote GA-AuNPs adhesion to the cell surface proteins and max-
imize its photothermal effects . GA had FDA-approval for hu-
man consumption . GA application to achieve in vitro and in
vivo stability for Au and iron oxide NPs were established for imag-
ing and therapy , however, PTT of GA-AuNPs has remained rel-
atively unexplored. We herein aim to delineate the photothermal ef-
ficiency of GA-AuNPs on the PNLs in the early stage. Our investi-
gations included both in vitro and in vivo studies; to investigate the
GA-AuNPs cytotoxicity and phototoxicity in hepatic cell line; to de-
lineate the GA-AuNPs therapeutic efficiency in diethylnitrosamine
(DEN)-induced liver PNLs in Balb-C.
2. Materials and Methods
2.1. Synthesis of Gum Arabic Encapsulated AuNPs (GA-AuNPs)
GA conjugated nanoparticles were synthesized and characterized
following our standard protocol [7c–e]. Briefly, 100 μL of 0.1 M
NaAuCl4aqueous solution was added to 6 ml of 12 mg GA aqueous
solution, stirred at 80 °C for 10 min. To this mixture, 40 μL of 0.1 M
aqueous solution of reducing agent, P(CH2NHCOOH)3(THP-Gly;
also referred to as ‘Katti’peptide)  was added. Immediately af-
ter, the color changed from yellow into a ruby red, which indicated
NPs formation. The metallic core size of GA stabilized AuNPs is
21 ±6 nm (Fig. 1a–c). By dynamic light scattering instrument (Ze-
tasizer Nano S90, Malvern Instruments Ltd., USA), the GA-AuNPs
hydrodynamic size was found to be 78 ±4 nm thus confirming the
GA encapsulation of AuNPs. GA-AuNPs has a negative zeta potential
(− 35.5 ±3 mV), an indication of significant in vitro stability . Sta-
bility measurement that.
involved dilutions in aqueous media, saline, PBS buffer and hu-
man serum albumin have confirmed that GA-AuNPs are robust under
in vitro profiles for potential biomedical applications (Fig. 1d).
2.2. In vitro Application
Human HCC cell line (HepG2; ATCC, Rockville, MD, USA) were
routinely cultured in RPMI 1640 medium containing 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/
ml streptomycin. Cells were cultured in a CO2incubator at 37 °C,
under humidified environment. Diode laser (Quanta System, Milan/
Italy) emitting continuous wave of light was utilized. All in vitro ex-
posures were performed in these conditions: wavelength 807 nm ,
average power 500 mW, beam diameter 3.0 cm and power density
50 mW/cm2. The time period of exposure was 10 min. The laser light
was coupled with monocore optical fiber, and the use of a biconvex
lens ensured homogeneous exposure of the 96-well plate.
2.3. Cell Viability Assay
To evaluate the phototoxicity as well as the cytotoxicity of GA
and GA-AuNP in the presence or absence of laser irradiation, 3,4,5-di-
methylthiazol-2,5-diphenyl tetrazolium bromide (MTT) cell viabil-
ity assay was applied. HepG2 cells were cultured in 96-well plate
(0.5 ×10  cells/well) for 24 h, and then treated for 24 h with in-
creasing concentrations of GA or GA-AuNP solutions, followed by a
10 min laser exposure. After 24 h, cells were submitted to MTT assay.
2.4. Estimation of Histone Deacetylase Activity
The activity of histone deacetylase (HDAC) was measured in
HepG2 cell lysate by colorimetric kit (BioVision, USA) according to
the manufacturer's instructions. HepG2 cells were treated with GA
(57.7 μg/ml) or GA-AuNP (8.6 μg/ml) with or without laser exposure.
3. In vivo Anti-Neoplastic Experiments
Animal experiments were performed adhering to the Ethical Com-
mittee guidelines of the National Research Centre, Egypt, and the Na-
tional Institutes of Health, USA, for the animal care and use. Male
wild-type Balb-c mice (18–20 g; 4 weeks old; Theodor Bilharz in-
stitute, Cairo, Egypt) were housed in a 12 h light-dark cycle, with
free access to sterilized standard chow and water ad libitum. Mice
were allowed to acclimate for 7 days prior to experiments. Hepatic
PNLs were developed in mice using DEN, as previously described
by Kushida et al. . A total of 224 mice were randomly subdi-
vided into two large groups: 1. Normal group (PNLs-free group, con-
trol) were subdivided into 6 subgroups (n= 16/group) including con-
trol untreated group, laser-, GA-, GA + laser-, GA-AuNPs,
GA-AuNPs + laser- treated groups. 2. PNLs-induced group
(PNLs-bearing group, n= 112 mice) received an intraperitoneal (IP)
injection of DEN (50 mg/kg b. wt.) every 2 weeks for a total of
12 weeks. At that point, three mice were randomly assigned for a
histopathological examination to check hepatic PNLs. After confirm-
ing PNLs occurrence, PNLs- bearing mice were classified into 6
subgroups (n= 16/group) including DEN (positive control),
DEN + Laser, DEN + GA, DEN + GA + Laser, DEN + GA-AuNPs,
and DEN + GA-AuNPs + Laser.
Mice received either an intravenous injection of GA (0.2 mg/
100 μl/mouse) or GA-AuNP (30 μg/100 μl/mouse) with or without
10 min laser exposure. The laser source used was the same one used
in the in vitro study but with an average power of 5 W. The laser
energy was delivered to the treatment site in a non-contact mode
from the skin surface, where the left region of the mouse abdomen
were irradiated by for a single 10 min session. Mice were gross-ob-
served for 4 weeks for any clinical signs. Individual body weights
were recorded in day 0 and every week. Weight loss > 20% was con-
sidered unacceptably toxic. After 4 weeks, mice were anesthetized;
blood was withdrawn and the plasma was separated. The livers were
excised, rinsed multiple times in ice cold PBS. A liver part was pre-
served in 4% paraformaldehyde/PBS for histopathological examina-
tion, and another liver part (40 mg) was homogenized by grinding in
liquid nitrogen and lysed in 1 ml ice-cold lysis buffer (50 mM Tris
pH 8, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sul-
phate, 0.5% Na-deoxycolate monhydrate and protease inhibitor cock-
tail tablet), and then the lysate was centrifuged at 13,000gat 4 °C for
Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx 3
Fig. 1. a. UV–visible absorption spectra of GA-AuNPs dissolved in serum-free RPMI 1640 media; b. Transmission electron microscopy image of GA-AuNPs; c. Histogram of par-
ticles distribution; d. In vitro stability of GA-AuNPs in various biological fluids as confirmed by UV–VIS absorption spectra. Cytotoxicity at different concentrations of GA and (e)
GA-AuNPs (f) against HepG2 liver cells in the presence (triangle-line) and absence (circle-line) of laser irradiation, using MTT assay. Data was expressed as mean ±S.E. of the
percentage of control cells (n= 6). g. Effect of GA and GA-AuNPs with and without laser irradiation on HDAC activity in HepG2; data expressed as μM deacetylated substrate/mg
15 min. The supernatant was stored at − 80 °C until used for biochem-
3.1. Histological, Histochemical and Immunohistochemical Analyses
Unbiased histopathological examination (blind to treatment) was
performed on the sections of Paraffin-embedded tissues, by hema-
toxylin and eosin (H & E) staining. To evaluate the cell death mode,
liver tissue sections were stained with a dual DNA dyes; acridine
orange (AO) and ethidium bromide (EB) mixture (100 μg/ml) .
Images were captured under fluorescent microscope (Axiostar plus,
Zeiss, Goettingen, Germany) and digital camera (PowerShot A20,
Canon, USA). PCNA is a valuable marker for evaluating the cel-
lular proliferative activity. PCNA and Placental form of glutathione
S-transferase (GST-P) were detected in liver sections by immunohisto
4 Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
chemical analysis, using fluorescence icothiocyanate (FITC) conju-
3.2. Evaluation of Inflammation and Apoptosis Mediators
Liver tissue level of tumor necrosis factor-α(TNF-α) was quanti-
fied using Sandwich ELISA technique . Levels of Cytochrome C
in tissue homogenates was measured using a commercial quantitative
ELISA kit (Abcam #154471, USA), using the manufacturer's proto-
col. The levels of caspase-3 and death receptors (DR4 and DR5) in the
tissue homogenate were measured by indirect ELISA .
3.3. Statistical Analysis
The data obtained from different experiments were analyzed by
(SPSS) program version 11, using one way ANOVA test/Tukey's
Post Hoc. Data were expressed as mean ±SD. Significant was se at
4.1. In Vitro Experiments
Analysis of HepG2 cell viability after being treated with GA or
GA–AuNPs revealed that, without laser irradiation, both GA
(9.0–57.7) μg/ml and GA-AuNPs (1.6–8.6 μg/ml) did not induce any
cytotoxicity. Treating cells with GA followed by laser session did not
induce cell death even at high concentration (57.7 μg/ml), while in
case of GA–AuNPs, laser irradiation induced cell death, where the vi-
ability decreased to 62% at concentration of 8.6 μg/ml. The IC50 of
GA–AuNPs/laser was calculated and found to be 11.7 μg/ml (Fig. 1e
and f). The results indicated that the only significant change in HDAC
activity was detected in HepG2 cells treated with GA–AuNPs/laser
(p< 0.05). The other treatment modalities showed no significant re-
duction in HDAC activity (Fig. 1g).
4.2. In Vivo Experiments
4.2.1. Histopathological Examination
Liver sections of the normal groups (control untreated, laser, GA,
and GA–AuNPs) showed a normal histopathology structure of hepatic
lobule. However normal mice treated with GA + laser showed slight
activation of kupffer cells. In mice treated with GA-AuNPs + Laser
showed slight cholangitis, as shown in (Fig. 2). In hepatic PNLs-in-
duced groups (DEN, DEN + Laser, DEN + GA and
DEN + GA-AuNPs) the histopathological examination showed kary-
omegaly of hepatocytic nuclei, cholangioma and oval cells, hyper-
plasia, as well as a significant kupffer cells proliferation with por-
tal infiltration. Hepatic PNLs were induced, in mice that were treated
with DEN + GA + laser, with hepatocytomegaly, karyomegaly and fo-
cal hepatic necrosis that was associated with inflammatory cells infil-
tration. DEN + GA-AuNPs-treated mice showed oval cells hyperpla-
sia and significant induction of apoptotic hepatocytes and inhibition of
PNLs (Fig. 2).
4.2.2. Apoptosis and Necrosis Analysis
Another morphologic evaluation was carried out by dual DNA
staining by AO/EB. All normal groups showed high predominance
of viable cells of organized structure green nuclei, however slight
morphological changes GA-AuNPs mice with/without laser were ob-
served, where few cells with bright green to orange staining were
detected indicating early to late apoptosis (Fig. 3). In PNLs-induced
groups, the important morphologic change observed was in
GA-AuNPs or GA-AuNPs + laser, where a high incidence of early
apoptosis (bright green cells) and late apoptosis (cells of condensed
and fragmented yellow to orange chromatin) was predominant. It is
also important to mention that in PNLs-induced mice treated with
GA-AuNPs + Laser, although the majority of cells were in the late
apoptotic state, we also observed few necrotic cells displaying orange
to red nuclei (Fig. 3).
4.2.3. PCNA Detection
PCNA is considered as an index of the cell turnover rate, as it is
associated with S phase and DNA replication in the cell cycle. The
PCNA positive cells and colonies appeared as bright green stained
solitary cells or regions. Undetectable PCNA-positivity was noticed in
all of the normal groups (Fig. 5). On the other hand, nuclear staining
of PCNA was highly detected in all PNLs-induced groups (p< 0.05),
an indication of the high proliferative activity of the cells. However,
in the two PNLs-induced groups that were treated with GA-AuNPs
with/without laser showed a significant suppression in PCNA activity
comparing to the DEN group (Fig. 5). These findings pointed out the
probability that GA-AuNPs alone can halt the cell proliferation even
without laser stimulation.
4.2.4. GST-P Positive Foci
GST-P is known as a carcinogen detoxification enzyme and it is
one of the most reliable markers for tumor initiation. In the present
study, all of the normal groups showed morphologically normal cells
with no positive staining of GST-P (Figs. 4, 6). On the contrary, the
expression of GST-P was markedly detectable in all PNLs-induced
mice and was obviously in strong GST-P positive foci. However a
significant reduction (p< 0.05) in the GST-P positive foci was de-
tected in the PNLs–induced groups received GA-AuNPs with or with-
out laser exposure (Figs. 4, 6).
4.2.5. Estimation of TNF-
A significant elevation of TNF-αconcentration that was observed
in all of PNLs-induced groups comparing with normal groups. In the
PNLs groups treated with GA-AuNPs with/without laser exposure, a
noticeable reduction in TNF-αconcentration was found compared to
DEN group (Fig. 6).
4.2.6. Apoptosis Mediators
The level of Cytochrome-c, a responsible trigger of intrinsic apop-
tosis, showed no significant change in all groups with different treat-
ment modalities (data not shown). However in the case of other apop-
totic mediators (caspase-3, DR4 and DR5), the significant changes
(p< 0.05) were detected in caspase-3 and DR5 levels in all of
GA-AuNPs-treated groups (healthy and PNLs-induced groups with/
without laser), which is another indication that treatment with
GA-AuNPs alone have an apoptotic effect on cells (Fig. 7).
4.2.7. Discussion and Conclusion
GA is regarded as an alternative biopolymer for the coating and
stabilization of nanostructures including iron oxide, magnetic NPs
[16,17,18], AuNPs [10,18d] and as a PT agent . In the present
study aimed to validate the hypotheses that AuNPs conjugated with
GA matrix are effective probes for the use in PTT, we have used
GA-AuNPs produced by the reduction of Au salt with a water soluble
trimeric glycine-phosphine (P(CH2NHCOOH)3). This novel reducing
agent was discovered in our laboratory . Detailed procedures for
the synthesis of a large NPs variety have been described using simi-
lar family of NPs initiators . These GA-AuNPs have optimum sizes
Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx 5
Fig. 2. Representative photos for H&E stained liver sections from different mice groups (×400).
(Fig. 1c) and favorable in vitro stabilities for potential applications un-
der in vivo profiles (Fig. 1d) . The negative zeta potential value of
GA-AuNPs provides the necessary repulsive forces resisting particles
agglomeration and therefore stay stable in different solutions and ex-
pected to have long-term in vitro and in vivo stability of the nano-par-
Since rod-shaped AuNPs, among other structures, revealed a
higher PTT than sphere-shaped AuNPs, we planned to use GA to
prepare rod-shaped AuNPs in the future studies. In the present study
we investigated the PPT for the successfully prepared sphere-shaped
GA-AuNPs. It is known that the spheres size of is more efficiently
taken up by cells than the nanorods shape, moreover spheroid AuNPs
are characterized by the ease and fast method of preparation compar-
ing to other nanostructures . In previous report, NIR at 540 nm 
and at 800 nm  were used to stimulate PTT in spheroid AuNPs.
Huang et al. , who prepared spheroid AuNPs of similar diame-
ter size and similar surface plasmon absorption intensity (540 nm) and
reported that NIR at 800 nm enhanced the second harmonic genera-
tion and the two photon absorption. They also found that the use of
800 nm to irradiate AuNPs spheres had increased the photothermal
destruction effect of AuNPs on the cells. Accordingly, in this study,
6 Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
Fig. 3. Representative images for the analysis of cell death type in liver sections from different mice groups, as stained by AO/EB and captured under fluorescence microscope
(×200), showing living cells (green), early apoptotic cells (bright green nucleus with condensed/fragmented chromatin), late apoptotic and necrotic cells (condensed/fragmented
chromatin; dark orange). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
we preferably selected NIR laser 800 nm to enhance the PTT effi-
ciency of AuNPs.
In order to prove the GA-AuNPs stability at appropriate concen-
trations for cellular dilutions, we have performed dilution studies
and estimated stability by monitoring the plasmon resonance wave-
length after every successive addition of 0.1 ml in doubly ionized
water through additions to 1 ml of GA-AuNPs solutions. Our results
have confirmed that absorption intensity of AuNPs (540 nm) did not
change even at very dilute conditions. It is, thus, conceivable that the
encapsulation of AuNPs by GA phytoconstruct is aiding the stabi-
lization in aqueous media, involving saline, PBS buffer, serum albu-
min solutions and different other buffers, as a biologically relevant
medium and at concentrations optimum for biomedical applications
(Fig. 1D). Our next goal of this study was to evaluate the cytotoxicity
of GA-AuNPs with or without laser exposure on a hepatic cell line (in
vitro study), and then to study GA-AuNPs potential anti-preneoplastic
effect in hepatic preneoplastic-induced mice model.
Our studies have revealed that various concentrations of both GA
and GA-AuNPs (without laser exposure) were safe and did not in-
duce any cytotoxicity in HepG2 cells. This finding is in accordance
with previous reports, which found that using gold nanospheres (4,
12, and 18 nm in diameter) with stabilizing capping agents (cysteine,
glucose, citrate, or biotin) for the treatment of human leukemia cells
caused no cell death [18d,20,21]. However, our investigations involv-
ing the treatment of HepG2 cells with GA-AuNPs followed by laser
irradiation resulted in a remarkable phototoxicity due to the collective
Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx 7
Fig. 4. Representative photos for GST-P expression in liver sections from different mice groups (×400), as stained by FITC-labeled antibodies.
PT effect resulting from GA-AuNPs nanoparticulate assembled struc-
tures. Our findings corroborate results observed by Liu et al. ,
and Richardson et al.,  who have suggested that the PT effect of
AuNPs are due to their multiple mobile electrons, which are strongly
enhanced by absorption of incident photons of laser beam that matches
collective plasmon resonance, thus resulting in efficient conversion
of photon energy into heat energy. This heat energy in close prox-
imity to tumor cells and surrounding tumor matrix would result in
effective tumor therapy through the excess heat energy transferred.
The phototoxic effect of GA-AuNPs on HepG2 cells was also medi-
ated by a significant reduction in the HDAC activity, suggesting that
these NPs, in addition to their repressor proliferation effect, can also
show a post-translational modification of DNA histone proteins. This
is in accordance with other studies, where Sule et al. , suggested
that AuNPs can bind to the sulfhydryl groups on the surface of his-
tone deacetylase and thus repress its activity. Mazumder et al. , re-
ported that AuNPs considered as epigenetic modulator by modifying
chromatin connections with lamin proteins and core histones.
As part of our extensive evaluation of PT effect of GA-AuNPs
on tumor initiation, we have induced hepatic preneoplastic lesions in
mice using the DNA alkylating agent DEN . The histopathologi-
cal examination of liver tissues harvested after the different treatment
modalities revealed that the administration of GA or GA-AuNPs or
exposure to laser separately resulted in no histopathological change,
8 Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
Fig. 5. Representative photos for PCNA expression in liver sections from different mice groups (×400), as stained by FITC-labeled antibodies.
thus confirming the safety of both. Al-Kenanny et al.,  had re-
ported the safety of GA when used in liver injury induced by gen-
tamycin. El-Sayed et al. , had reported the safety of NIR laser us-
age and observed that the viability of cancer cells was not affected
when exposed to different laser power density (19, 25, 38, 50, 64,
or 76 W/cm2) for 4 min using CW argon laser. Both studies [27,28]
are confirming the safety of each treatment modality when using sep-
arately, accordingly the therapeutic effect was due to laser-stimu-
lated AuNPs. Administration of GA-AuNP and subsequent exposure
to laser irradiation resulted in massive apoptotic cancer cell death
as noted from the histopathological examination. This was also sup-
ported from AO/EB staining (Fig. 3), which demonstrated that treat-
ment with GA-AuNPs induced apoptosis to some extent without laser
exposure but when it is combined with irradiation, apoptosis was
predominant with few necrotic cells. Activation of apoptosis is the
sole determinant of most of the effect anti-tumor strategies. Apopto-
sis involves two distinct pathways, the extrinsic (death receptor) path-
way, which begins with the binding of an appropriate ligand to a
subset of death receptors (such as Fas, TNF, DR3, DR4, DR5) 
and the intrinsic (mitochondria mediated) pathway, which is initiated
when cells are exposed to severe oxidative stress and hypoxia result-
ing in an elevated mitochondrial permeability with consequent release
of pro-apoptotic molecules such as cytochrome-c into the cytoplasm
which in turn initiate apoptosis . In the extrinsic pathway, activa-
tion of death receptors is followed by the formation of a death-induc-
ing signaling complex (DISC). Binding of the initiator caspase 8 to
Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx 9
Fig. 6. (a) Number of GST-P positive cells in liver sections from different groups, as stained by the corresponding FITC-labeled antibody. Sections were imaged and analyzed by
image analyzer. (b) TNF-αconcentration in liver homogenate of different groups, as measured by sandwich ELISA.
the DISC resulted in an activation of the downstream effector cas-
pase-3 and initiate the extrinsic apoptotic program . This mech-
anism was in accordance with our findings, where the detected ele-
vation of apoptosis after GA-AuNPs treatment was also accompanied
by a significant increase in the levels of DR5 and caspase-3. Liu et
al.  reported that Au-AuNPs conjugated with GA may strongly
interact with asialoglycoprotein receptors of hepatocytes due to the
presence of arabinogalactan in GA. Asialoglycoprotein receptors are
known to mediate recognition of apoptotic hepatocytes by their viable
neighbors . Therefore, we infer that GA-AuNPs, in our present in-
vestigation, may interact with the asialoglycoprotein receptors of he-
patocytes, thus in turn enhanced the DR-5 up-regulation and activate
caspase 3, resulting in apoptosis enhancement.
In our endeavor to delineate the inhibitory effects of GA-AuNPs
on the neoplastic process in the liver, we evaluated the positive stain-
ing of GST-P foci. The protein content of GST-P in preneoplastic cells
is exceedingly high, comprising > 1% of the soluble cytosolic pro-
teins and therefore has permitted identification and evaluation of ini-
tiation stage of carcinogenesis . Our investigations have inferred
that hepatocytes of normal groups showed no GST-P positive foci,
which is in accordance with other studies . However, mice sub-
jected to DEN administration showed hyperplastic liver nodules with
strong positive staining of GST-P indicating the presence of hepato-
cellular foci, this finding further corroborate earlier observations of
Ledda-Columbano et al. , and Perra et al. , who also reported
that DEN administration increased the number of induced GST-P pos-
itive cells. These hyperplastic nodules can easily progress into HCC,
indicating that the high GST-P + expressing hyperplastic liver nod-
ules serve as cancer precursors . The observed significant attenu-
ation in the growth of hepatic pre-neoplastic foci positive for GST-P,
as shown in Figs. 4 and 6a, upon treatment with GA-AuNPs with or
without laser exposure, provide a solid rationale that these NPs have
an inhibitory effect on tumor initiation.
Neoplastic process is often associated with chronic inflammation
, therefore we have considered the effects of GA-AuNPs on in
flammatory mediators. In the present study, the chronic inflammation
state in all PNLs-induced mice was proved by the enhanced TNF-α, as
a result of DEN administration , which was significantly reduced
upon treatment with GA-AuNPs. Functionalized AuNPs have been
reported in several studies to cause significant reduction in the ex-
pression of different inflammatory mediators with consequent attenu-
ation in the local macrophages production of TNF-αand IL-6 [39,40].
Meanwhile, it is also reported that GA protein alone has anti-inflam-
matory effects due to suppression of leucocytes infiltration, which is
considered to be a source of inflammatory mediators . In tradi-
tional medicine GA is used as a treatment for the intestinal mucosa
inflammation . Taken together the previous reports and our own
findings, it appears that the anti-inflammatory effects of GA-AuNPs
(even in the absence of laser irradiation) may presumably be due to
the combined effects of AuNPs and GA. The in vitro and the in vivo
findings from our current investigations corroborate an important con-
clusion that the functionalized GA–AuNPs are efficient PTT agents as
they have demonstrated efficacy in inhibiting liver PNLs and inflam-
mation via induction of the extrinsic pathway of apoptosis.
Conflict of Interest Statement
The authors declare no conflict of interest.
We are grateful to laser Research Unit, NRC, Egypt, in particu-
lar Prof. Dr. Ali Shabaka for their efforts. This work was financially
supported by October 6 University and the National Research Centre,
Cairo, Egypt. We thank the University of Missouri for intramural sup-
port through University of Missouri Interdisciplinary Intercampus Re-
search Program (IDIC), funded by UM System and the MU campus.
Ravi Shukla acknowledges the generous VC research fellowship sup-
port from RMIT University.
10 Journal of Photochemistry & Photobiology, B: Biology xxx (2016) xxx-xxx
Fig. 7. (a) Caspase-3, (b) DR4 (black bars) and DR5 (light gray bars) were measured in
liver homogenate of different groups by ELISA. Data was expressed as the mean ±SE
of absorbance readings. Statistical analysis was carried out. The bar labels; a: p< 0.05
comparing with untreated group, and b: p < 0.05 comparing with DEN-treated group.
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