Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39

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Colloids and Surfaces B: Biointerfaces

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PST-Gold nanoparticle as an effective anticancer agent with immunomodulatory

properties
Manu M. Joseph a , S.R. Aravind a , Sheeja Varghese a , S. Mini b , T.T. Sreelekha a,∗
a
Laboratory of Biopharmaceuticals, Division of Cancer Research, Regional Cancer Centre (RCC), Trivandrum 11, Kerala, India
b
Department of Biochemistry, University of Kerala, Trivandrum, Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: Polysaccharide PST001, which is isolated from the seed kernels of Tamarindus indica (Ti), is an antitumor
Received 28 August 2012 and immunomodulatory compound. Gold nanoparticles have been used for various applications in cancer.
Received in revised form 5 November 2012 In the present report, a novel strategy for the synthesis and stabilization of gold nanoparticles using anti-
Accepted 29 November 2012
cancer polysaccharide PST001 was employed and the nanoparticles’ antitumor activity was evaluated.
Available online 17 December 2012
PST-Gold nanoparticles were prepared such that PST001 acted both as a reducing agent and as a cap-
ping agent. PST-Gold nanoparticles showed high stability, no obvious aggregation for months and a wide
Keywords:
range of pH tolerance. PST-Gold nanoparticles not only retained the antitumor effect of PST001 but also
Polysaccharide
Tamarindus indica
showed an enhanced effect even at a low concentration. It was also found that the nanoparticles exerted
PST-Gold nanoparticles their antitumor effects through the induction of apoptosis. In vivo assays on BALB/c mice revealed that
Immunomodulation PST-Gold nanoparticles exhibited immunomodulatory effects. Evaluation of biochemical, hematological
Cancer and histopathological features of mice revealed that PST-Gold nanoparticles could be administered safely
without toxicity. Using the polysaccharide PST001 for the reduction and stabilization of gold nanopar-
ticles does not introduce any environmental toxicity or biological hazards, and these particles are more
effective than the parent polysaccharide. Further studies should be employed to exploit these particles
as anticancer agents with imaging properties.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction They have been used as anticancer, immunomodulatory, antivi-

Despite advancement in the prevention, diagnosis and treat-
ment of cancer, it is still the leading cause of mortality worldwide.
Although chemotherapy regimens represent a major treatment
modality, they are accompanied by undesirable side effects due
to the severely toxic nature of these chemicals. Therefore, novel
pharmaceutical agents with improved specificity and efficacy that
are also nontoxic to normal cells would be of immense clinical
value. Polysaccharides are a group of widely occurring natural
biological macromolecules with tremendous structural diversity,
and their biological activities have attracted much attention
in medicine [1,2]. Antitumor, immunomodulatory, antimicrobial,
antiulcer, antioxidant and several other pharmacological activities
from various polysaccharides have been reported [3–5].
Tamarindus indica (Ti), a tree from the family of Leguminosae,
is widely grown in India and other Asian countries, and its com-
ponents are utilized in daily human life. Polysaccharide isolated
from the seed kernels of Ti mainly consists of xyloglucans, which
have been shown to possess various pharmacological properties.
∗ Corresponding author. Tel.: +91 4712522378; fax: +91 4712447454.
E-mail address: lekhasree64@yahoo.co.in (T.T. Sreelekha).
ral, antioxidant and even artificial tear agents in modern medicine
[6–11]. Ti polysaccharide possess various properties, including
biocompatibility, biodegradability, high viscosity, high thermal sta-
bility, broad range of pH tolerance and adhesiveness, that facilitate
their broad usages as stabilizers, thickeners, gelling agents, binders
and additives in food and pharmaceutical industries; they are also
used for controlling drug release in pharmaceutical applications [9].
Gold nanoparticles (AuNPs) have recently emerged as an attrac-
tive candidate for targeted delivery of various therapeutic agents
[12,13] due to their distinctive features, including reasonably low
cytotoxicity, tunable surface characteristics and stability under
in vivo conditions. AuNPs selectively accumulate in tumor cells,
showing bright-light scattering, and hence can serve as specialized
microscopic probes to study cancer cells [14]. The conventional
methods for the synthesis of AuNPs involve toxic chemicals
[15], but the use of natural materials, including plants, algae
and microbes, have also been reported [16–19]. Recently, certain
polysaccharides, such as sucrose, cellulose and chitosan, have also
been used for the synthesis and stabilization of AuNPs [20,21].
These approaches would not introduce environmental toxicity or
biological hazards.
The polysaccharide PST001, which is extracted from the seed
kernels of T. indica, was previously isolated and characterized by our

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.11.046
 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39
laboratory; it was found to be an anticancer agent with excellent
in vitro and in vivo immunomodulatory properties and no toxicity
to normal cells [6–8]. The aim of the present study was to com-
bine effective delivery of the antitumor and immunomodulatory
activities of PST001 by generating AuNPs using the polysaccharide.
2. Materials and methods
2.1. Isolation and purification of polysaccharide PST001 from
seed kernels of Tamarindus indica
Ripened seeds of Ti were obtained from the Thiruvanantha-
puram District of Kerala. The seeds were dried and powdered,
and then the polysaccharide PST001 was isolated as previously
reported [6,22]. The isolated polysaccharide was purified with gel
filtration chromatography using Sephadex G-200; 0.001 M phos-
phate buffered saline (PBS) was used as the eluent, and the purified
product was lyophilized. The total carbohydrate content was deter-
mined by Dubois’s method [23] using d-glucose as the standard.
2.2. Preparation and characterization of PST-Gold nanoparticles
After the isolation of polysaccharide, the nanoparticles were
prepared. The preparation of AuNPs using PST001 was performed
based on earlier reports [20,21,24,25], with modifications. All glass-
ware used was cleaned with freshly prepared aqua regia solution
(HCl:HNO3 3:1) and rinsed thoroughly with distilled water prior to
use. To 1 mM solution of HAuCl4 (1 ml), 3 ml of 10 mg/ml solution
of PST001 was added drop-wise with constant stirring on a mag-
netic stirrer plus hotplate heated to 70 ◦ C; the process continued
for 2–3 h until an intense red-colored solution was obtained. The
particles thus formed were named PST-Gold nanoparticles. Cap-
ping agents can be used to stabilize the nanoparticles and prevent
aggregation, but no external capping agents were added in this case.
The PST-Gold nanoparticles were initially characterized with
UV–vis spectroscopy (Bio Spec-1601, Shimadzu), transmission
electron microscopy at an accelerated voltage of 80 kV (Hitachi
TEM system) and finally with dynamic light scattering (Malvern
DLS instrument V2.0).
2.3. Stability assay of PST-Gold nanoparticles
Once the nanoparticles were prepared, their stability was eval-
uated by spectroscopy over the parameters of time and pH at
ambient temperature. In the pH stability study, the pH of PST-Gold
nanoparticles was adjusted using 0.1 N hydrochloric acid (pHs 1,
2, 3, 5 and 6) and 0.1 M sodium hydroxide (pHs 12 and 14) on a
calibrated pH meter (Cyber Scan 510).
2.4. Cell cultures
The human cancer cell lines MCF-7 (breast cancer), K562
(leukemia) and A549 (adenocarcinoma) were obtained from the
National Centre for Cell Science, Pune, India. A375 (melanoma),
HepG2 (hepatocellular carcinoma) and HCT116 (colon cancer) cells
were kindly provided by RGCB (Rajiv Gandhi Centre for Biotech-
nology), Thiruvananthapuram, India. The cells were maintained in
DMEM media with 10% fetal bovine serum at 37 ◦ C and 5% CO2 in
an incubator (Heraeus BB 15).
2.5. Cytotoxicity assay
The growth inhibition capacity of PST-Gold nanoparticles was
evaluated on cancer cell lines and isolated normal lymphocytes by
the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium)
assay as previously reported [3,6]. This assay’s function is based on
33
the cleavage of tetrazolium salt by mitochondrial dehydrogenases
in viable cells. The absorbance was measured at 570 nm using a
microplate spectrophotometer (BioTek Power Wave XS).
The proliferation rate and inhibitory rate of the cells were cal-
culated with the following formulas:
Proliferation rate (PR) % = [Abs sample/Abs control] × 100;
Inhibitory rare (IR) % = 100 − PR.
MTT assays were performed on cancer cell lines and lympho-
cytes containing PST-Gold nanoparticles, PST001 and standard
citrate-capped AuNPs (Sigma G1652) that are of the same size as
PST-Gold nanoparticles.
2.6. Acridine orange–ethidium bromide staining assay
Acridine orange–ethidium bromide dual staining is the most
commonly used method to detect apoptosis based on the differ-
ential uptake of two fluorescent DNA binding dyes by viable and
nonviable cells [26]. The experiment was performed as described
previously [7]. The cells were observed under an inverted fluores-
cent microscope under an FITC filter (Olympus 1X51).
2.7. Flow cytometric evaluation of Annexin V–FITC staining
Annexin V–FITC staining assay was performed with FITC
Annexin V Apoptosis Detection Kit (BD Pharmingen #556547, BD
Biosciences, San Jose, CA) as per the manufacturer’s instructions
and as previously described [7]. FITC-conjugated Annexin V, which
binds to phosphatidylserine, was detected using a FACS Calibur flow
cytometer (BD) and analyzed with the CellQuest Pro software.
2.8. In vivo toxicity studies
Evaluation of compound toxicity in a biological system requires
special attention. The effect of PST-Gold nanoparticles on in vivo
systems was evaluated by acute and subacute toxicity assays on
5- to 6-week-old male BALB/c mice as previously described [6].
Briefly, for acute toxicity studies, the compound was administered
intraperitoneally (ip) up to a concentration of 2000 mg/kg body
weight. For subacute toxicity assays doses of the nanoparticles cor-
responding to various fractions of the LD50 (1/5, 1/10 and 1/20)
were prepared and administered intraperitoneally to each group
of mice for 14 consecutive days, whereas the control group was
treated with the vehicle (PBS) only. On the 15th day, the animals
were sacrificed by cervical dislocation; blood, femur bones and
internal organs were collected and used for further evaluation [27].
All animal studies were performed in accordance with the Institu-
tional Animal Ethical Committee’s (IAEC) approval.
2.9. In vivo immunomodulation studies
An immunomodulator is a substance that affects the immune
system. Lymphocytes were isolated from the blood 14 days after
administration of the compound. Its proliferation status was eval-
uated with the MTT assay as described before, and the status of
CD3+, CD4+ and CD8+ cells was determined by flow cytometry and
CellQuest Pro [6]. Various biochemical and hematological param-
eters were also evaluated. Femur bones were collected and cut at
the level of epiphyseal plates of the proximal and distal ends of the
bone. Bone marrow cells were aspirated after flushing with RPMI
media, and cell counts were calculated as described previously [6].
34 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39

2.10. Statistical analysis IC50 values of 70.3 ± 1.2 ␮g/ml and 48.9 ± 1.8 ␮g/ml, respectively,
after 48 h of incubation with the nanoparticles, whereas the native
Data were expressed as the mean ± standard deviation (SD) of polysaccharide failed to inhibit 50% of cell growth even at a higher
three replicates and were analyzed using GraphPad PRISM soft- concentration and with a longer incubation period (Fig. 2a and
ware version 5.0 (GraphPad Software, USA). One-way analysis of b, and Supplementary Fig. 1a and b) in both cell lines. The cyto-
variance was used for repeated measurements, and the differences toxic potential of the nanoparticles increased in a dose-dependent
were considered to be statistically significant if P < 0.05. The IC50 manner up to 100 ␮g/ml in both cell lines. PST-Gold nanoparticles
values were calculated using the Easy Plot software. also exhibited excellent cytotoxic potential against A549 (adeno-
carcinoma), A375 (melanoma), HepG2 (hepatocellular carcinoma)
and HCT116 (colon cancer) cells. IC50 values of the nanoparti-
3. Results and discussion
cles were 53.4 ± 0.9 ␮g/ml and 33.8 ± 1.1 ␮g/ml at 48 h for A549
and A375 cells, respectively, whereas the IC50s for PST001 in the
3.1. PST-Gold nanoparticles were prepared and are stable
two cell lines were 82.03 ± 1.6 ␮g/ml and 61 ± 1.7 ␮g/ml, respec-
tively, after 72 h of incubation. PST-Gold nanoparticles exhibited a
The polysaccharide PST001 isolated from the seed kernels of Ti
dose-dependent increase in cytotoxicity in A375 cells but reached
was found to be pH neutral, and the total sugar content was 98%,
optimum cytotoxicity at 100 ␮g/ml in A549 cells (Fig. 2c and d, and
as determined by the phenol–sulfuric acid method. PST0001 was
Supplementary Fig. 1c and d). The cytotoxic potential increased in
purified by gel filtration chromatography, lyophilized and stored.
a dose-dependent fashion up to 10 ␮g/ml for HepG2 cells, and an
We found that PST001 successfully reduced HAuCl4 to form gold
IC50 value of 6.5 ± 0.5 ␮g/ml was obtained after 48 h of incubation.
nanoparticles with a deep red color (Fig. 1a). PST001 not only acted
In HepG2 cells, the IC50 dose of PST001 was 33.7 ± 1.3 ␮g/ml after
as a reducing agent for the production of gold nanoparticles but
72 h of incubation (Fig. 2e, and Supplementary Fig. 1e). In HCT116
also served as a capping agent, imparting stability to the PST-Gold
cells, PST-Gold nanoparticles also exhibited a dose-dependent
nanoparticles and preventing their re-aggregation without the use
increase in cytotoxicity, with an IC50 of 50.07 ± 1.5 ␮g/ml at 48 h,
of any other capping agent. The successful production of PST-Gold
whereas the IC50 of PST001 was 82 ± 1.2 ␮g/ml at 72 h (Fig. 2f, and
nanoparticles was evaluated using UV–vis spectroscopy, which
Supplementary Fig. 1f). Standard citrate-capped gold nanoparti-
produced the characteristic peak of gold nanoparticles approxi-
cles of 20 nm in size and 1 mM HAuCl4 were found to be nontoxic
mately 550 nm (Fig. 1b); PST001 and HAuCl4 failed to produce any
in all of these cell lines. To evaluate the cytotoxic potential of
characteristic peak from 350 to 750 nm. At an accelerated voltage
PST001 in normal cells, an MTT assay was performed on iso-
of 80 kV, TEM evaluation of the nanoparticle size clearly showed
lated normal lymphocytes. PST-Gold nanoparticles were found not
that the majority of the particles were circular in shape with an
only to be nontoxic to the cells across all concentrations but also
average size of 15–20 nm (Fig. 1c). This result was confirmed using
strongly immunostimulatory. A proliferative index (PI) of 1.19 was
DLS, which also indicated that the particles have an average size of
obtained at a concentration of 0.1 ␮g/ml. Standard gold nanoparti-
20 nm (Fig. 1d). Although PST-Gold nanoparticles do not produce
cles showed little toxicity at higher concentrations but exhibited
any visible aggregation, their stability was evaluated for various
no immunostimulatory effects (Fig. 2g). Earlier we showed that
time intervals (Fig. 1e) and they remained stable even up to 12
PST001 alone was much less cytotoxic to cancer cells than PST-
months. Evaluation of nanoparticle stability over a wide range of
Gold nanoparticles. Because PST001 was found to be nontoxic to
pH using UV–vis spectroscopy clearly demonstrated a high toler-
normal cells, the nanoparticles were also checked for cytotoxic-
ance for pH change (Fig. 1f) with no significant shift in the position
ity on isolated normal lymphocytes. We found that, in addition to
of the peak over a pH range of 1–14. The present study investigated
being nontoxic, PST-Gold nanoparticles also exerted immunostim-
the preparation of gold nanoparticles using the water soluble, anti-
ulatory effects with a PI of up to 1.19 on the lymphocytes. Various
cancer polysaccharide PST001 isolated from the seed kernels of T.
polysaccharides were previously reported to have both anticancer
indica and further evaluated their biological potential as a cyto-
and immunomodulatory effects [1,3,6]; however, the present study
toxic agent specific to cancer cells. Water soluble polysaccharides
revealed that PST-Gold nanoparticles are more potential than other
were found to be therapeutically applicable for various pharmaco-
compounds.
logical applications, including the use as drug carriers [28]. Gold
nanoparticles have a long history of use in various pharmacologi-
3.3. PST-Gold nanoparticles exert anticancer effects through
cal applications [29–31]. The production of PST-Gold nanoparticles
induction of apoptosis
using PST001 does not include the use of any hazardous chemi-
cals and environmental toxins and hence is a “green” nanoparticle
Because PST-Gold nanoparticles exhibited significant cytotox-
synthesis process [32]. This process also does not require the use
icity specifically against cancer cells, their mode of cell death
of a capping agent because PST001 itself acts as a capping agent
induction was evaluated using various apoptotic assays. Mem-
in addition to its role as a reducing agent. PST-Gold nanoparti-
brane blebbing, which is a hallmark of apoptosis, refers to irregular
cles have an average size of 20 nm and are stable for up to 1 year
bulges in the plasma membrane of a cell caused by localized
with a wide range of pH tolerance. Therefore, they are more stable
decoupling of the cytoskeleton from the plasma membrane. Mor-
than commercially available borohydrate- or citrate-reduced gold
phological evaluation of the cell lines treated with PST-Gold
nanoparticles [15].
nanoparticles at a concentration of 10 ␮g/ml for 48 h using phase-
contrast microscopy revealed a decrease in the number of cells
3.2. Anticancer effect of PST-Gold nanoparticles exhibiting morphological features of apoptosis, such as distorted
shape and membrane blebbing in comparison with the control
Because PST001 is reported to be an anticancer agent with group (Supplementary Fig. 2). Using acridine orange–ethidium bro-
immunomodulatory properties, the PST-Gold nanoparticles were mide staining, cells treated with PST-Gold nanoparticles showed
also evaluated for their anticancer potential on various cancer cell a change in color from green to yellow/red with associated
lines. This potential was found to be highly significant (P < 0.001) apoptotic features such as membrane blebbing, nuclear conden-
in all cell lines examined. Cytotoxicity of PST-Gold nanoparticles sation and presence of apoptotic bodies compared to the control
was also evaluated in the cancer cell lines. Breast cancer cell line (Fig. 3, insert). PST-Gold-induced apoptosis was confirmed by flow
MCF7 and leukemia cell line K562 were growth-arrested with cytometry analysis of Annexin V staining. There was a significant
 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39 35

Fig. 1. Preparation and stability assay of PST-Gold nanoparticles. (a) Photographic image of freshly prepared PST-Gold nanoparticles. (b) UV–vis spectra of PST-Gold nanoparti-
cles, HAuCl4 and PST001. (c) TEM images of PST-Gold nanoparticles, magnified 20,000×. (d) DLS spectra of PST-Gold nanoparticles. (e) Stability assay of PST-Gold nanoparticles
in various time intervals and (f) in various pH conditions. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)

(P < 0.001) increase in the Annexin V positive cells in all of the
cell lines treated with 10 ␮g/ml PST-Gold nanoparticles for 48 h
(Supplementary Table 1). In MCF 7 cells, the controls exhibited
2 ± 0.07% Annexin V positive cells, whereas compound treated cells
were 41.4 ± 1.5% positive (Fig. 3a and b). A similar pattern was
observed for K562, A549 and A375 cell lines, where the control
cells were 4.5 ± 0.9%, 11 ± 1.2% and 16 ± 1.1% Annexin V positive
and PST-Gold nanoparticle-treated cells were 8.3 ± 0.9%, 35 ± 1.6%
and 51 ± 1.9% positive, respectively (Fig. 3c–h). The percentages
of apoptotic HepG2 and HCT116 cells significantly increased from
3.5 ± 0.6% and 9.9 ± 1.3% in the controls to 47 ± 2.1% and 32.5 ± 2.2%
in nanoparticle-treated cells, respectively (Fig. 3i–l). The enhanced
activity exhibited by the nanoparticles compared with the parent
polysaccharide might be due to the increased uptake of the par-
ticles via endocytosis because of their smaller size and increased
surface to volume ratio [33].
3.4. In vivo evaluation of PST-Gold nanoparticles
Even though PST-Gold nanoparticles exhibited significant cyto-
toxicity against cancer cells and immunomodulatory effects in vitro,
their activity in biological systems requires further evaluation.
In vivo toxicity of PST-Gold nanoparticles was assessed on 5- to
6-week-old male BALB/c mice. There were no behavioral changes
or visible toxicity symptoms upon ip administration of PST-Gold
up to a concentration of 2000 mg/kg; hence, the LD50 was taken
to be 2000 mg/kg. After the administration of the compound at
different concentrations for 14 consecutive days, animals were
sacrificed by cervical dislocation and various parameters were ana-
lyzed. None of the PST-Gold nanoparticle-treated animals from any
of the groups exhibited weight loss throughout the duration of the
experiment. Various biochemical and hematological parameters of
the sacrificed animals were assessed for any toxicity induced by
the nanoparticles (Supplementary Table 2). There was an increase
in the RBC and WBC count in the groups administered with the
compound, regardless of the concentration, which again reinforced
the immunostimulatory effects of PST-Gold nanoparticles. There
was no significant variation in any of the common biochemical
parameters for the compound-treated mice compared with the
control; moreover, no toxicity was observed to be associated with
the administration of the compound. Hematoxylin–eosin staining
was performed on the livers, kidneys, lungs, hearts and spleens
of the sacrificed animals to evaluate any pathological changes
associated with compound administration at the organ level. No
significant pathological changes were observed with the heart,
lung and spleen in any of the PST-Gold-treated mice compared
with the control group (Supplementary Fig. 3). PST-Gold nanopar-
ticles administered at a concentration of 100 mg/kg appeared to
have no effect on the liver and kidney (Fig. 4c and d), and mice
treated with 200 and 400 mg/kg compound exhibited slight cyto-
logical abnormalities. The hepatocytes appeared to be slightly
swollen in morphology with enlarged cytoplasm and normal nuclei
in the mice given 200 mg/kg compound (Fig. 4f); the shape of
the cells appeared to be irregular at the higher concentration
(Fig. 4h), but the nuclei remained normal. Although the glomeruli
appeared normal for the mice given 200 mg/kg PST-Gold nanopar-
ticles, the cells lining the renal tubules showed slight changes
with a swollen morphology (Fig. 4e). Similar to the changes in the
glomeruli, the tubular cells also appeared larger with a smaller
lumen (Fig. 4g).
36 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39

Fig. 2. MTT assay on cancer cell lines and lymphocytes. (a) Cytotoxicity profile on 48-h incubation of PST-Gold nanoparticles, PST001 and standard citrate capped gold
nanoparticles on MCF-7 cells, (b) on K562 cells, (c) on A549 cells, (d) on A375 cells, (e) on HepG2 cells, (f) on HCT116 cells and (g) on isolated normal lymphocytes at 72 h of
incubation. Results are expressed as the mean ± SD.

Compound-treated mice exhibited a significant (P < 0.001)
increase in the lymphocyte proliferation status both at the time
of sacrifice (0 h) and at 72 h after in vitro incubation in comparison
with the control group (Fig. 5a). Although all concentrations of the
nanoparticles showed significant immunomodulatory activity, the
mice given with 100 mg/kg PST-Gold nanoparticles showed maxi-
mum lymphocyte proliferation with PIs of 1.8 ± 0.01 and 1.6 ± 0.08
at 0 h and 72 h, respectively. Immunophenotyping of lymphocyte
subsets with CD markers (CD3, CD4 and CD8) showed an increase
in the proportion of the T lymphocyte population in the PST-
Gold nanoparticle-treated mice compared with the control group
(Fig. 5b). Mice given 100 mg/kg compound exhibited a significant
increase in all of the T lymphocyte subsets, with PIs of 48.7 ± 4.5%,
35.7 ± 4% and 11.2 ± 2% for CD3, CD4 and CD8 subtypes, respec-
tively. In comparison, the PIs in the control group were 22.8 ± 3.1%,
24.5 ± 3% and 3.3 ± 0.3% for CD3, CD4 and CD8 subtypes, respec-
tively. The elevated T cell population serves as an index for the
immunostimulatory property of PST-Gold nanoparticles. The bone
marrow cellularity was determined by counting the number of
bone marrow cells from the femur bones of the sacrificed animals.
The group administered with 100 mg/kg PST-Gold showed the
maximum bone marrow cell count at 8 × 106 cells/femur, whereas
 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39 37

Fig. 3. Apoptotic evaluation of cell lines treated with 10 ␮g/ml PST-Gold nanoparticles for 48 h by Annexin V–FITC staining assay. The insert figure shows acridine
orange–ethidium bromide staining images of the same on corresponding cell lines. MCF 7 cells (a) control and (b) PST-Gold. K562 cells (c) control and (d) PST-Gold.
A549 cells (e) control and (f) PST-Gold. A375 cells (g) control and (h) PST-Gold. HepG2 cells (i) control and (j) PST-Gold. HCT116 cells (k) control and (l) PST-Gold. (For
interpretation of the references to color in text, the reader is referred to the web version of this article.)

the control group produced 4.8 × 106 cells/femur. Notably, the bone
marrow cell counts were significantly higher (P < 0.001) in the
PST-Gold treated mice than in the control group across all concen-
trations (Fig. 5c).
Acute toxicity assay performed on nanoparticle-treated BALB/c
mice did not show any lethal effect, and the LD50 was taken
to be 2000 mg/kg. Histopathological evaluation of various organs
after hematoxylin–eosin staining demonstrated no pathological
symptoms for the heart, lung and spleen, but slight changes were
observed with the liver and kidney in mice administered with
200 and 400 mg/kg nanoparticles. However, analysis of various
biochemical parameters revealed no abnormality compared with

Fig. 4. Light microscopic images of H&E staining of various organs of BALB/c mice after 14 days administration of PST-Gold nanoparticles and normal saline. Control (a)
kidney and (b) liver. 100 mg/kg PST-Gold (c) kidney and (d) liver. 200 mg/kg PST-Gold (e) kidney and (f) liver. 400 mg/kg PST-Gold (g) kidney and (h) liver.
38 M.M. Joseph et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39

Fig. 5. Immunomodulatory effects of PST-Gold nanoparticles on BALB/c mice after 14 days of administration of the compound. (a) Lymphocyte proliferation status of the
sacrificed animal at the hour of sacrifice (0 h) and after 72 h. (b) Immunophenotyping for the status of lymphocyte subsets. (c) Bone marrow cellularity from the bone marrow
cells of femur bones. Results are expressed as the mean ± SD. Statistically significant differences are at **P < 0.01, ***P < 0.001, ns is the non-significant, as compared with the
control group.

the control mice. The slight pathological abnormalities observed
with the liver and kidney at higher concentrations of nanoparticles
were expected to be reversible. The in vivo immunomodulatory
effects of PST-Gold nanoparticles were further demonstrated by
significant increases in the lymphocyte proliferation status, bone
marrow cell count and T lymphocyte population in mice treated
with the nanoparticle for 14 days relative to the controls. Con-
ventional chemotherapeutic agents often suppress the immune
system of the host [34], which warrants the search for anticancer
agents that are nontoxic to normal cells. Gold nanoparticles have
been evaluated in diverse applications, including in vitro assays,
in vitro and in vivo imaging, cancer therapy and drug delivery.
Tumor-targeting technologies that exploit gold’s inherent bio-
compatibility are being developed to deliver drugs directly into
cancerous tumors. Additionally, simple, cost-effective and sensi-
tive diagnostic tests are being developed for the early detection
of prostate and other cancers. Furthermore, application of gold
nanoparticles in radiotherapy has been reported recently. Roa et al.
reported that gold nanoparticle sensitizes prostate cancer cells to
radiation by regulating the cell cycle [35]. Gold nanoparticle could
also provide advantages in terms of radiation dose enhancement
[36], as reported by Wan et al. Therefore, PST-Gold nanoparticle
needs to be evaluated for its various applications in cancer treat-
ment and management.
pathological abnormalities were observed based on H&E staining
at higher concentrations of the nanoparticle, the groups given lower
doses of the nanoparticle were found to be unaffected. Immunos-
timulatory effects of PST-Gold nanoparticles on BALB/c mice were
more pronounced 14 days after administration of the compound, as
demonstrated by a significant increase in bone marrow cell count
and CD3/4/8 counts. The data presented here suggest that PST-Gold
nanoparticle may be used as an effective anticancer agent with
strong immunomodulatory potential.
Acknowledgements
We greatly acknowledge the Council of Scientific and Industrial
Research (CSIR), Govt. of India, for the research fellowship; Kerala
State Council for Science, Technology and Environment (KSCSTE),
Govt. of Kerala, for the financial support; and the National Institute
for Interdisciplinary Science and Technology (NIIST), Thiruvanan-
thapuram, for the DLS analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.
2012.11.046.

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