Research Article

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Mitochondria protection with ginkgolide B-loaded

polymeric nanocapsules prevents diethylnitrosamine-
induced hepatocarcinoma in rats
Aim: Hepatocellular carcinoma (HCC) has no successful pharmacotherapeutic remedy. The aim of this study
was to ascertain whether ginkgolide B (GB)-loaded polymeric nanocapsules can prevent diethylnitrosamine
(DEN)-induced HCC in rats. Materials & methods: GB was fabricated in two types of nanocapsules of which
one was polyethylene glycol coated (N1GB) and the other was uncoated (N2GB). These nanocapsules were
orally gavaged during DEN-induced HCC development in rats. Results: Nanocapsulation of GB enabled
aqueous suspension and slow time-dependent release of the compound. Anticarcinogenic potential of
N2GB was reflected by its ability in the management of DEN-induced reactive oxygen species generation,
mitochondrial dysfunction, p53, NF-kB, inducible nitric oxide synthase, COX-2 and VEGF expressions, and
induction of apoptosis in cancer cells in the rat liver. Conclusion: Positive zeta-potential on N2GB surface
might have offered higher hepatic accumulation of GB, especially at the electron-dense organelle
mitochondria. Mitochondria protection against DEN-induced oxidative damage ensured HCC prevention.
Original submitted 27 June 2012; Revised submitted 4 December 2012

KEYWORDS: COX-2 n diethylnitrosamine n ginkgolide B n hepatocellular carcinoma Swarupa Ghosh*1,

n inducible NOS n mitochondria n nanocapsule n NF-kB n ROS n VEGF
Hepatocellular carcinoma (HCC) is a deadly
disease contributing a third of cancer-related
deaths worldwide [1] . Past efforts have brought
very limited success in treating HCC patients
chemotherapeutically. Currently, liver trans-
plantation is the most effective way to prolong
the life of HCC patients [2] . With the present
drugs being mostly palliative in function, target-­
based new drugs are being generated and inves-
tigated but with inadequate clinical efficacy.
This necessitates a novel approach to confer pro-
tection against development of a lethal disease
such as liver cancer. Research to find alternative
‘leads’ for cancer prevention and therapeutics
focuses on natural sources. However, few of
these compounds have come out of clinical trials
successfully, facing vital challenges of systemic
toxicity and poor bioavailability.
Ginkgo biloba extract prepared from leaves
of the living fossil tree G. biloba is used in tra-
ditional Chinese and in occidental medicine.
It is one of the most commonly prescribed
medications in Germany and France and one
of the most widely used herbal dietary supple-
ments in the USA [3,4] . Ginkgolide B (GB), a
diterpene and the major unique component
isolated from G. biloba extract is reported to
competitively inhibit the binding of platelet-
activating factor (PAF) to its specific receptors
in various tissues [5] . Recent studies revealed
Sandhya Rekha
Dungdung1, Somsubhra
that GB showed antineoplastic effects in a Thakur Choudhury2,
breast cancer cell line through its anti­oxidant, Somsuta Chakraborty2
antiangiogenic and gene-­regulatory actions [6] . & Nirmalendu Das2
Although G. biloba extract was found to 1
Cell Biology & Physiology Division,
Indian Institute of Chemical Biology,
signifi­cantly suppress proliferation and increase 4, Raja SC Mullick Road,
cytotoxicity in hepatoma cells in vitro [7] , the Kolkata-700032, India
2
Drug Development, Diagnostics &
beneficial effect of GB against development of Biotechnology Division, Indian Institute
HCC in vivo is a fairly uninvestigated avenue. of Chemical Biology,
Nanocapsules offer an interesting alternative 4, Raja SC Mullick Road,
Kolkata-700032, India
form of drug-delivery tool attributable to the *Author for correspondence:
feasibility of incorporating hydrophobic as well Tel.: +91 33 2499 5771
Fax: +91 33 2473 0284
as hydrophilic molecules, long shelf life and ghosh.swarupa1@gmail.com
better cellular uptake [8,9] . The biodegradable
polymer poly(d,l-lactide-co-glycolide) (PLGA)
is the most frequently used biomaterial owing
to its excellent biocompatibility, and it is
already commer­cialized for a variety of drug-
delivery systems, such as blends, films, matri-
ces, microspheres, nanoparticles and pellets,
among others [10,11] . Nanoparticles of this poly-
mer have been shown to significantly enhance
the chemo­preventive action of naturally occur-
ring compounds, such as quercetin and cur-
cumin, among others [12,13] . Since no systematic
work on the effect of GB on liver carcinoma
development in animals has been carried out,
it was thought worthwhile to investigate the
efficacy of GB and its nano­c apsulated forms
in combating diethylnitrosamine (DEN)-
induced carcinogenesis in rat livers. DEN is part of

doi:10.2217/NNM.13.56 © 2013 Future Medicine Ltd Nanomedicine (Epub ahead of print) ISSN 1743-5889
Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das
the most intensively studied model of chemi-
cally induced liver cancer develop­ment both in
rats and mice. DEN treatment has elucidated
important molecular and cellular pathways
involved in HCC development (e.g., STAT-3P
and IL-6) and can be used to identify new tar-
gets in order to block chemically induced liver
carcino­genesis in humans [14,15] . Encouraging
results of nanocapsulated GB on DEN-induced
HCC prompted us to study the possible anti-
carcinogenic mechanisms of this compound.
Hence we investigated its effect on some of the
key players in the development of HCC, such
as reactive oxygen species (ROS) production,
mitochondrial dysfunction, p53, inflammation
and angiogenesis in the liver.
Materials & methods
„„ Chemicals
DEN, bovine serum albumin, PLGA (molecu-
lar weight: 50,000–75,000), GB, 2,6-dichloro-
indophenol, phenazine methosulfate, succinic
acid and didodecyldimethylammoniumbromide
(DMAB) were purchased from Sigma Aldrich
(MO, USA). Ethyl acetate (analytical reagent
grade), HPLC-grade chloroform and methanol
were purchased from E Merck (Mumbai, India).
All other reagents were of analytical grade.
„„ Nanocapsulated GB preparation
A modified emulsion–diffusion–evaporation
method was used to make the GB nanocapsules.
A total of 5 mg GB was dissolved in 1 ml ethyl
acetate. The organic solution of PLGA (25 mg
in 2.5 ml in ethyl acetate) and drug was emulsi-
fied with 5 ml of an aqueous phase containing
DMAB. The resulting oil in water emulsion
was stirred at room temperature for 3 h before
homo­genizing at 15,000 rpm for 5 min. The
organic solvent was removed by constant stir-
ring in a water bath set at 40°C. The suspen-
sion was ultracentrifuged at 105,000 ×  g for
1 h [8] . The pellet was washed with phosphate-
buffered saline twice and resuspended in 2 ml
phosphate-buffered saline.
Table 1. Amount of ginkgolide B present in liver homogenates of
experimental rats subjected to diethylnitrosamine-induced
hepatocellular carcinoma.
Parameter DEN + N1GB treated DEN + N2GB treated
GB in liver homogenate 34.32 ± 3.16 83.4 ± 4.52*
(µg/g tissue)
Results are expressed as mean ± standard error; n = 5 animals. The DEN + N1GB-treated group was
compared with the DEN + N2GB-treated group.
*p < 0.001.
DEN: Diethylnitrosamine; GB: Ginkgolide B; N1GB: Polyethylene-glycol coated ginkgolide B;
N2GB: Uncoated ginkgolide B.
doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print)
Polyethylene glycol (PEG)-incorporated
nanocapsules were prepared using PEG (molec-
ular weight: 4000) as a component material
taken along with PLGA and drug in ethyl ace-
tate in a PEG:PLGA material ratio of 1:1. PEG
was also added as an emulsifier in the aqueous
phase. PLGA and drug concentrations were the
same as that of the formulation without PEG.
PEG-containing nanocapsules will be referred
to as N1GB and those without PEG as N2GB
henceforward.
„„ Characterization of nanocapsules
To estimate the intercalated drug in nano­
capsules, the pellets were dissolved in 2 ml ethyl
acetate and kept for 3 days at 4°C. The opti-
cal density was measured at lambda maximum
(GB) 220 nm, and the percentage incorporation
of drug in nanocapsules was calculated against a
standard plot of GB. Percent encapsulation was
calculated. The size and shape were measured
by atomic force micro­scopy using the Agilent
Technologies 5500 PicoPlus™ atomic force
microscopy system (Agilent Technologies, CA,
USA). Images were taken in the acoustic mode
with cantilevers having a resonance frequency of
146–236 kHz, a tip height of 10–15 µm and a
tip length of 225 µm. Freshly cleaved clean mica
sheet was chosen as a solid substrate. Both probe
and cantilever were immersed completely in the
water solution during the experiment. Images
were captured and analyzed using PicoScan™
5.33 software from Molecular Imaging Corpo-
ration (AZ, USA). Nanocapsules were further
verified for size and shape under transmission
electron microscope (Tecnai™ G2 BioTWIN
Spirit, 120 kV; Fei Company, OR, USA) using
uranyl acetate stain [16] . Zeta-potential and
polydispersity index were measured using a
zetasizer (Zetasizer Nano ZS; Malvern Instru-
ments, Malvern, UK). The in vitro release study
was performed following the method of Win
and Feng [17] .
„„ Animals & experimental design
Adult male Swiss Albino rats, each weighing
approximately 100–120 g, were acclimatized
to laboratory conditions (26–28°C; 60–80%
relative humidity with 12-h light/dark cycle)
for 7 days prior to commencement of treat-
ment, during which time they received food
and drinking water. All rats used in this study
received proper care and handling in compli-
ance with the Animal Ethics Committee, India,
and experimental procedures were carried out
only after approval by the institutional animal
future science group
 Ginkgolide B nanocapsules against hepatocarcinoma
ethics committee. Animals were randomly
selected for groups, and the carcinogen and drug
were administered as per their individual body-
weights. Rats were divided into six groups with
five animals in each group. Rats in the normal
group (Group A) were kept as untreated controls
and injected intra­peritoneally with three doses of
olive oil (0.5 ml) with 15-day intervals between
doses. All other rats were injected intraperito-
neally with three doses of DEN (200 mg/kg
of bodyweight in 0.5 ml olive oil) with 15-day
intervals between doses. Group B animals were
kept as a DEN-­administered control group. Ani-
mals in Group C, D, E and F were treated orally
by gavage with GB (100 mg/kg of bodyweight
of GB suspended in neutral oil), GB (5 mg/kg
of bodyweight of GB suspended in neutral oil),
N1GB containing 5 mg/kg of bodyweight of GB
and N2GB containing 5 mg/kg of bodyweight
of GB, respectively, once per week from the day
of first DEN administration up to 16 weeks. All
animals were provided with a normal diet and
drinking water without any treatment for the
next 2 weeks and sacrificed thereafter. At the
end of 18 weeks, final bodyweight was docu-
mented and blood collected from the heart of
each animal. Serum aspartate trans­a minase,
serum alkaline phosphatase and serum alanine
transaminases were determined using a standard
kit manufactured by Coral Clinical Systems
(Goa, India). Serum a-­fetoprotein was mea-
sured by ELISA using a-fetoprotein antibody
from Santa Cruz Biotechnology (TX, USA)
and Roche Elecsys® 2010 Immuno­a ssay Ana-
lyzer (Roche Diagnostics GmbH, Mannheim,
Research Article
injected onto a HPLC C18 Symmetry ® column
(Waters Corp­oration, PA, USA). The mobile
phase consisted of methanol and water (60:40,
v/v) at a flow rate of 0.3 ml/min [19] .
„„ Histological examination of liver of
experimental animals
Formalin-fixed liver portions of experimental
animals were embedded in paraffin blocks, and
5-μm thin sections were cut on a microtome. The
sections were stained with hematoxylin–eosin
using standard staining protocol and examined
under a light microscope.
„„ Liver mitochondria &
submitochondrial particle preparations
Liver mitochondria were isolated from the rat tis-
sue using a differential centrifugation technique
following the method of Navarro and Boveris [20] .
„„ Mitochondrial ROS measurement
Intracellular ROS level was measured in liver
mitochondria [8] . Fluorescence was measured
spectrofluorometrically (LS-3B; Perkin Elmer,
MA, USA) at 499 nm excitation and 520 nm
emission wavelengths. The data were normalized
to normal values, and the normal was expressed
as a value of 100%.
12
*** NS Liver weight
10
Relative liver weight
*
8 **
***
Weight (g)

Germany). After blood collection, all rats were

6 ***
dissected and their livers were isolated promptly NS
and washed with cold physio­logical saline. Final *
**
liver weights of all animals were noted. A part of 4 ***
the organ was immediately used for mitochon-
dria preparation and another part was fixed in 2
10% formaldehyde and processed for histologi-
cal examination. The remaining part was kept 0
at -80°C [18] .
Normal DEN DEN + GB DEN + GB N1GB N2GB
(5 mg/kg) (100 mg/kg)
„„ Quantitation of GB in liver
Liver tissues were homogenized in five vol- Figure 1. Liver weight (in grams) and relative liver weight of experimental
umes of 25 mM 4-(2-hydroxyethyl)-1-piper- rats after 18 weeks of first diethylnitrosamine administration. Relative liver
azineethanesulfonic acid. Homogenates from weight = liver weight (in grams)/final bodyweight (in grams) × 100. DEN-treated
experimental rats were deproteinized with 1% control groups were compared with normal animals and drug-treated groups were
phosphoric acid, vortexed and centrifuged. compared with the DEN control group. Values are mean ± standard error of the
mean for five rats.
The supernatant was mixed with three volumes *p < 0.05.
of ethyl acetate, vortexed and centrifuged at **p < 0.01.
1000 rpm for 5 min. The ethyl acetate layer was ***p < 0.001.
removed under nitrogen and dried. The residue DEN: Diethylnitrosamine; G: Ginkgolide B; N1GB: Polyethylene glycol-coated
was dissolved in 120 μl methanol, 50 μl was ginkgolide B; N2GB: Uncoated ginkgolide B; NS: Not significant.

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Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das

Table 2. Effect of nanocapsulated ginkgolide B on hepatic toxicity in diethylnitrosamine-induced hepatocellular

carcinoma.
Parameter Normal DEN treated DEN + GB DEN + GB DEN + N1GB DEN + N2GB
(5 mg/kg) (100 mg/kg) treated treated
treated treated
Alkaline 37.82 ± 4.15 79.52 ± 7.06*** 73.11 ± 6.92NS 65.74 ± 5.34* 56.59 ± 5.12** 47.38 ± 4.63***
phosphatase
(Ka)
Serum 121.36 ± 9.02 238.59 ± 14.68*** 215.42 ± 11.63NS 182.38 ± 9.55* 157.18 ± 7.13** 139.73 ± 6.85***
aspartate
transaminase
(IU/l)
Serum alanine 32.54 ± 3.06 114.23 ± 5.19*** 107.06 ± 4.58NS 95.82 ± 3.92* 54.93 ± 3.56** 41.76 ± 3.28***
transaminase
(IU/l)
a-fetoprotein 2.42 ± 0.25 8.67 ± 0.71*** 8.43 ± 0.81NS 5.37 ± 0.78* 5.25 ± 0.46** 3.98 ± 0.27***
(ng/ml)
Results are expressed as mean ± standard error; n = 5 animals. The DEN-treated group was compared with normal, and the experimental groups were compared with
the DEN-treated group.
*p < 0.05.
**p < 0.01.
***p < 0.001.
DEN: Diethylnitrosamine; GB: Ginkgolide B; N1GB: Polyethylene glycol-coated ginkgolide B; N2GB: Uncoated ginkgolide B; NS: Nonsignificant.

„„ Lipid peroxidation, mitochondrial number of stained cells in ten randomly selected

membrane fluidity, reduced
glutathione succinate dehydrogenase,
nicotinamide adenine dinucleotide
oxidase & mito­chondrial membrane
potential assays
Lipid peroxidation in the mitochondrial
membrane was determined by measuring the
amount of conjugated diene formed therein.
Fluorescence depolarization, associated with
hydrophobic f luorescence probe diphenyl
hexa­triene, was used to monitor the changes
in the fluidity of the lipid matrix accompany-
ing the gel-to-liquid crystalline phase transi-
tion. A part of the tissue homogenate was used
to estimate glutathione (GSH) level spectro-
photometrically [8] . Succinate dehydrogenase
(SDH) and nicotinamide adenine dinucleotide
(NADH) oxidase activity in liver mitochondria
were assayed using the method of Ghosh et al.
[8] . Mitochondrial membrane potential was
measured by rhodamine 123 fluorescence [8] .
„„ In situ DNA fragmentation assay
Formalin-fixed, paraffin-embedded, glass slide-
mounted liver sections of experimental rats were
stained with anti-5-bromo-2’-deoxyuridine
(BrdU); fluorescein isothiocyanate antibody
using the Apo-BrdU In Situ DNA Fragmen-
tation Assay Kit (#K401-60; Biovision, CA,
USA) following the manufacturer’s protocol.
BrdU-positive cells were counted per field at 10×
magnification using a Glasgow cell-counting
graticule (Datasights, Enfield, UK). The mean
fields in each slide was expressed as the number
of BrdU-positive cells.
„„ Immunofluorescence
Deparaffinized, rehydrated liver sections were
subjected to standard immunostaining tech-
niques followed by incubation with primary
antibody (nitric oxide synthase [NOS] 2 and
VEGF, Santa Cruz Biotechnology) solution and
then incubation with Texas Red® (Santa Cruz
Biotechnology) and fluor­escein isothiocynate-­
conjugated secondary antibody (Santa Cruz
Biotechnology) solutions separately. The images
were observed under a fluorescence microscope
[21] . VEGF and inducible NOS (iNOS)-positive
cells were counted per field at 10× magnification
using a Glasgow cell-counting graticule. The
mean number of stained cells in ten randomly
selected fields in each slide was expressed as the
number of VEGF and iNOS-positive cells.
„„ p53, p21, NF-kB & COX-2
immunoblotting
Liver tissues were subjected to standard SDS-
PAGE procedures, followed by electrophoretic
transfer to polyvinylidene fluoride membrane
(Millipore, MA, USA) at 15 V for 20 min
by using Trans-Blot ® SD Semi-Dry Blotting
Aparatus (Bio-Rad, CA, USA), blocked with
bovine serum albumin in phosphate-buffered
saline followed by incubation with the primary
p53, p21, COX-2 and NF-kB antibodies sepa-
rately for 3 h and then in secondary alkaline

doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print) future science group

 Ginkgolide B nanocapsules against hepatocarcinoma Research Article

A B

C D E

F G

Figure 2. Representative photomicrographs of hematoxylin–eosin-stained 10% formalin-fixed

paraffin-embedded liver sections of experimental animals (under 10× magnification) after
18 weeks of first diethylnitrosamine administration. Rats received three intraperitoneal injections
of diethylnitrosamine (DEN; 200 mg/kg) with 15-day intervals between doses. Experimental rats except
normal and DEN-treated control groups were orally administered different doses and formulations of
ginkgolide B (GB) on a weekly basis from the day of first DEN administration up to 16 weeks.
(A) Showing liver section of normal rats having proper arrangement of hepatic cords. (B) Showing liver
sections of DEN-exposed rats having hyperplastic nodule (inset) with cells having higher nuclear-to-
cytoplasmic ratio and atypical nuclei. (C) DEN-treated rat liver showing loss of architecture and large
fluid-filled spaces, cells not arranged in a single plane. (D) Liver sections of DEN-treated rats showing
formation of septa and large accumulation of inflammatory infiltrates indicated by the black arrow.
(E) GB (100 mg) + DEN-exposed rat liver sections show accumulation of fibrous tissue and septa
formation indicated by the dashed arrow. (F) Polyethylene glycol-coated ginkgolide B + DEN-treated rat
liver sections show interportal bridging fibrosis. (G) Uncoated ginkgolide B + DEN-treated rat liver
sections show near-normal portal tract with regular hepatic cellular arrangement.

phosphatase-conjugated goat anti-rabbit IgG (see online at www.futuremedicine.com/doi/

antibody (1:1000) for 1 h 30 min. Bands were
visualized by color development using Sigma
Aldrich premixed BCIP®/NBT substrate solu-
tion. Their pixel density was analyzed with
ImageJ software system (NIH, MD, USA).
„„ Statistical analysis
Statistical analysis was performed with one-way
analysis of variance with post hoc Tukey’s test
using SPSS® software version 15.0 (SPSS Inc.,
IL, USA). In all cases, p < 0.05 was taken as the
minimum level of significance.
Results
„„ Physicochemical characterization of
nanocapsules
The physicochemical characteristics, such as
particle size, zeta-potential, polydispersity index
and drug encapsulation efficiency of N1GB and
N2GB are summarized in Supplementary Figure  1
suppl/10.2217/NNM.13.56). The images
revealed that nanocapsules have regular spheri-
cal shape with average size distribution and
smooth surface. No apparent aggregation was
detected. Use of DMAB in the aqueous phase
decreased particle size. Zeta-potential values
indicated stable suspensions of both N1GB
and N2GB. In vitro drug release demonstrated
a biphasic pattern with an initial burst during
the first 24 h (Supplementary Figure 1), followed by
a slow, sustained release for both types of parti-
cle. N1GB showed a comparatively faster release
than N2GB. After 5 days, the cumulative drug
release was 38 and 31%, respectively.
„„ GB level in liver following N1GB &
N2GB feeding to rats exposed to DEN
Table 1 indicates the amount of GB level in rat
liver at the end of the experimental regimen
– that is, 18 weeks from the commencement

future science group www.futuremedicine.com doi:10.2217/NNM.13.56

Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das

Table 3. Effect of nanocapsulated ginkgolide B on mitochondrial reactive oxygen

species, lipohydroperoxide content, reduced glutathione and glutathione-S-
transferase in diethylnitrosamine-induced hepatocellular carcinoma in rats.
Experimental groups Lipohydroperoxide Reduced glutathione GST (U/mg
content (μmol/mg (μg of protein)
protein) glutathione/g tissue)
Normal 0.49 ± 0.03 18.32 ± 1.14 0.92 ± 0.07
DEN treated (A) 4.54 ± 0.48*** 39.57 ± 1.32*** 3.17 ± 0.27***
A + GB (5 mg/kg) treated 4.27 ± 0.53NS 36.12 ± 1.27NS 2.93 ± 0.21NS
A + GB (100 mg/kg) treated 3.18 ± 0.37* 33.57 ± 1.48* 2.31 ± 0.17*
A + N1GB treated 2.53 ± 0.06** 31.23 ± 1.57** 1.78 ± 0.14**
A + N2GB treated 0.95 ± 0.03*** 25.41 ± 1.08*** 1.18 ± 0.08***
Results are expressed as mean ± standard error; n = 5 animals. The DEN-treated group was compared with normal, and
the experimental groups were compared with the DEN-treated group.
*p < 0.05.
**p < 0.01.
***p < 0.001.
DEN: Diethylnitrosamine; GB: Ginkgolide B; GST: Glutathione-S-transferase; N1GB: Polyethylene glycol-coated
ginkgolide B; N2GB: Uncoated ginkgolide B; NS: Nonsignificant.

of the study. GB was detected by HPLC only
in nanocapsule-treated groups and not in the
‘free’ drug-treated groups. A significantly higher
amount of GB was found to be present in DEN
plus N2GB-treated rat liver than in DEN plus
N1GB-treated rat liver.
„„ Body growth, liver weight, liver
morphology & serum markers
DEN (three intraperitoneal doses of 200 mg/kg
at 15-day intervals) treatment caused an increase
in liver weight with a decrease in body growth
(Figure 1) . The liver exhibited small grayish-white
nodules on the surface of the cirrhotic liver. The
marker enzymes of liver function were found
to be significantly altered from their nor-
mal counter­parts. a-fetoprotein expression, a
marker of HCC development, was found to be
high in DEN-exposed rats (Table 2) . Oral treat-
ment of GB (5 mg) was found to be completely
ineffective; GB (100 mg) could significantly
inhibit DEN-induced micronodule formation
and hepatic function alteration. Nanocapsule
formulations (N1GB and N2GB) containing
5 mg GB were found to be more effective than
GB (100 mg). However, the maximum protec-
tion was achieved through N2GB oral treat-
ment (p < 0.001) against DEN-induced HCC
incidence.
„„ Pathomorphology of liver sections
Hematoxylin–eosin-stained liver sections of nor-
mal rat exhibited cords of normal hepatocytes,
central veins and normal-looking sinusoids lined
by Kupffer cells, and the overall histology was
within normal limits (Figure 2A). DEN-exposed
rats showed hyperplastic nodule (Figure 2b inset),
the cells having higher nuclear-to-­c ytoplasmic
ratio with atypical nuclei, loss of architecture
and large fluid-filled spaces, cells not arranged
in a single plane (Figure 2C) , and formation of
septa and large accumulation of inflammatory
infiltrates (Figure 2D). GB (100 mg) plus DEN-
exposed rat liver sections showed no signs of
HCC, but prominent cirrhotic damage char-
acterized by accumulation of fibrous tissue and
septa formation (Figure 2E) . N1GB plus DEN-
treated rat liver sections showed interportal
bridging fibrosis (Figure 2F), whereas N2GB plus
DEN-treated rat liver sections showed near-
normal portal tract with regular hepatic cellular
arrangement (Figure 2G) .
„„ Lipid peroxidation & antioxidant
status in liver
Conjugated diene generated as a result of lipid
peroxidation caused by DEN-induced oxidative
stress was studied. DEN-exposed control ani-
mals showed a significant increase (p < 0.001)
in diene level in the liver compared with normal
animals. GB (100 mg) could decrease diene levels
to 3.18 µmol/mg protein (p < 0.05) and N1GB
could arrest the level to 3.53 µmol/mg protein
(p < 0.01) in the liver. However, maximal protec-
tion was achieved through N2GB oral treatment
post-DEN exposure, depicting the anti­oxidative
behavior of the formulation (Table 3) . GSH, the
most important intracellular endogenous antiox-
idant, and glutathione-S-transferase (GST), the
free radical detoxifying enzymes, were studied.
Rats exposed to DEN showed a marked increase
in liver GSH levels from 18.32 to 39.57 µg/mg
tissue (Table  3) . No significant protection was
observed in the case of rats treated with free
GB (5 mg). However, GB (100 mg) and N1GB
treatment could retain the GSH levels compared

doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print) future science group

 Ginkgolide B nanocapsules against hepatocarcinoma Research Article
Table 4. Effect of nanocapsulated ginkgolide B on reactive oxygen species
generation, SDH and nicotinamide adenine dinucleotide oxidase activity in rat
liver mitochondria by the induction of diethylnitrosamine-induced hepatocellular
carcinoma.
Groups H2DCF NADH oxidase level SDH activity (µl DCIP
fluorescence (nmol of oxidised reduced/min/mg
(% of normal) NADH/min/mg protein) protein)
Normal 100 ± 6.5 5.89 ± 0.18 1.52 ± 0.09
DEN treated (A) 247 ± 15.8*** 10.77 ± 0.38*** 0.42 ± 0.04***
A + GB (5 mg/kg) 238 ± 6.2NS 10.69 ± 0.41NS 0.40 ± 0.02NS
treated
A + GB (100 mg/kg) 192 ± 11.5* 8.83 ± 0.54* 0.68 ± 0.08*
treated
A + N1GB treated 164 ± 12.7** 7.98 ± 0.48** 0.73 ± 0.07**
A + N2GB treated 127 ± 10.6*** 6.27 ± 0.39*** 0.93 ± 0.06***
Results are expressed as mean ± S.E; n = 5 animals. The DEN-treated group was compared with normal, and the
experimental groups were compared with the DEN-treated group.
*p < 0.05.
**p < 0.01.
***p < 0.001.
DCIP: 2,6-dichlorophenol indophenol; DEN: Diethylnitrosamine; GB: Ginkgolide B; H2DCF: 2’,7’-dichlorofluorescein;
NADH: Nicotinamide adenine dinucleotide; N1GB: Polyethylene glycol-coated ginkgolide B; N2GB: Uncoated ginkgolide B;
NS: Nonsignificant; SDH: Succinate dehydrogenase.

with DEN-treated animals, but maximal reten-
tion towards normal values at the higher level
of significance (p < 0.001) was shown in rats
treated with N2GB. A similar pattern was seen
in GST activities where N2GB most proficiently
showed less GST activity than DEN-induced
control animals (Table 3) .
„„ ROS level in rat liver mitochondria,
mitochondrial respiratory complex
enzyme activities & mitochondrial
membrane potential dissipation
The f luorescence intensity produced by
2’,7’-dichlorofluorescein diacetate on oxida-
tion to 2’,7’-dichlorofluorescein is proportional
to the amount of ROS produced by the mito­
chondrion. DEN exposure of experimental
animals caused a 2.5-fold increase in mito-
chondrial ROS generation with concomitant
increased NADH oxidase activity and reduced
SDH activity in the rat liver. Rats treated with
GB (100 mg) and N1GB showed a signifi-
cant decrease in mitochondrial ROS level and
NADH oxidase activity, and elevated activity
of SDH (p < 0.05 and p < 0.01, respectively).
N2GB offered maximum benefit (p < 0.001)
against DEN-induced ROS generation in
mitochondria and respiratory complex enzyme
dysfunction (Table  4) . Due to DEN exposure,
potential-driven rhodamine uptake was found
to be lowered from 1.93 to 0.67 nM rhodamine
incorporated/mg protein using succinate as a
substrate, at the end of 18 weeks from com-
mencement of the study, indicating a decrease
in mitochondrial membrane potential (Figure 3) .
Considerable repression in membrane dissipa-
tion (p < 0.05) was achieved through oral treat-
ment with GB (100 mg) and N1GB in liver
mito­c hondria. On the other hand, animals
treated orally with N2GB along with DEN
administration showed a significant increase in
mitochondrial membrane potential (p < 0.001).
„„ p53, p21 & apoptosis in liver
p53 expression in the liver cytosol of DEN-
exposed rats was significantly lower than
normal (Figure 4A) . Very strong p53 expression
was observed in DEN-exposed rats treated
with GB (100 mg). Basal level of p53 expres-
sion was observed in N2GB-treated animals.
p53-induced p21 expression was observed in
DEN plus GB (100 mg)-treated rats. Liver
sections, when studied for apoptosis and DNA
fragmentation, showed no more than normal
fluorescence in DEN-exposed control rats.
BrdU fluorescence indicates DNA fragmen-
tation and apoptosis. The maximum number
of fluorescent cells was observed in DEN plus
GB (100 mg)-treated rats, indicating induc-
tion of apoptosis in the initiated cancer cells.
However, an improved histology along with a
lower number of fluorescent cells was observed
in N2GB-treated rats (Figure 4B) .
„„Inflammation & iNOS, COX-2 &
NF-kB expression in liver
iNOS, COX-2 and NF-kB are some of the
key players involved in inflammation. Since

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Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das

A
fluorescent signal was recorded in DEN-treated
2.5
control rats (Figure 5A,ii) . Considerable fluores-
incorportated/mg protein (nM)

*** cence was observed in DEN plus GB (100 mg)-

2
** treated rats (Figure 5A,iii) with a gradual decrease
* in fluorescent signal in DEN plus N1GB-treated
Rhodamine

1.5 rats (Figure  5a,iv), while a near-normal level of

fluo­rescence was observed in DEN plus N2GB-
1
treated rats (Figure 5A,v) . Western blot analysis of
*** NS COX-2 expression in the cytosolic fraction of
experimental rat liver showed a significantly
0.5 increased expression in DEN-exposed rats. A
gradual decrease in expression was observed
0 with different formulations of GB, with N2GB
Normal

(5 mg/kg)

(100 mg/kg)

N1GB + DEN

N2GB + DEN
DEN

offering maximum reduction in expression.

DEN + GB

DEN + GB

NF-kB (p65) protein expression was studied in

the cytosolic fraction of the liver. A sharp dis-
appearance of NF-kB protein from the cytosol
was observed in hepatocarcinogenic conditions
induced by DEN exposure. DEN plus N2GB-
B 0.9
treated animals showed almost normal NF-kB
0.8 expression.
***
Membrane microviscosity

0.7
0.6
„„ VEGF expression & neoangiogenesis
**
([r*/r - 1] - 1)

* in rat liver
0.5 VEGF expression is a marker of neoangiogenic
0.4 signal. Effect of GB nanocapsules on hepatic
*** NS
0.3 VEGF expression due to DEN-induced hepato­
carcinogenesis in rats was studied. VEGF
0.2
immuno­fluorescent localization was visualized
0.1 in liver sections from animals sacrificed 18 weeks
0 after the commencement of the study. Green flu-
Normal

orescence indicates VEGF expression. Normal

(5 mg/kg)

(100 mg/kg)

N1GB

N2GB
DEN

DEN + GB

DEN + GB

rats showed no fluorescence (Figure 6A,i) , while

strong intense fluorescent signal was recorded in
DEN-treated control rats (Figure 6a,ii) . A signifi-
cantly lower level of fluorescence was observed
in DEN plus N1GB-treated rats (Figure 6a,iii) and
Figure 3. Effect of ginkgolide B nanocapsules on diethylnitrosamine- a near-normal minimal level of fluorescence in
induced mitochondrial membrane potential and microviscosity in hepatic DEN plus N2GB-treated rats (Figure 6a,iv) .
cells of rats sacrificed 18 weeks after the commencement of the study.
(A) Membrane potential of liver mitochondria and (B) liver cell membrane
microviscosity of experimental rats 18 weeks after first DEN administration. Discussion
DEN-treated control groups were compared with normal animals and drug-treated In this study, the authors formulated GB in two
groups were compared with the DEN control group. Values are mean ± standard different nanocapsules and demonstrated that
error of the mean for five rats. a concurrent administration of hepatocarcino-
*p < 0.05. gen DEN along with oral feeding of GB-loaded
**p < 0.01.
***p < 0.001. polymeric nanocapsules can prevent develop-
DEN: Diethylnitrosamine; GB: Ginkgolide B; N1GB: Polyethylene glycol-coated ment of hepato­c arcinogenesis in rats. N2GB
ginkgolide B; N2GB: Uncoated ginkgolide B; NS: Nonsignificant. offers a significantly higher level of GB payload
to the liver and is most effective in preventing
hepato­c arcinogenesis is inherently associated DEN-induced carcino­genesis. In relation to can-
with an inflamed liver, the authors studied the cer development, GB may be a useful chemo­
expression of these proteins. Immunofluorescent prevention tool but the difficulty remains to
staining of liver sections with anti-iNOS and resolve the effective dose, time of administration
Texas Red-conjugated anti-rabbit IgG anti­ and to steer the compound to the target site.
bodies showed normal rat liver with minimal PLGA nanocapsules equipped with sustained
red fluorescence (Figure 5A,i), while strong intense drug-releasing ability offer a unique nontoxic,

doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print) future science group

 Ginkgolide B nanocapsules against hepatocarcinoma Research Article
A i ii 60
DEN + GB DEN DEN p53
Normal DEN (100 mg/kg) + N1GB + N2GB 50 *** p21

Pixel density (AU)

40 ***
***
p53 30 ***
20
10
p21 0
Normal DEN DEN + GB DEN + DEN +
(100 mg/kg) N1GB N2GB

B
FITC PI Merged
vi

i a i b i c 900

800 *

700

BrdU-positive cells/field
ii a ii b ii c 600

500 **

400

iii a iii b iii c 300

200 **
***
100

iv a iv b iv c 0
i ii iii iv v

v a v b v c

Figure 4. Effect of ginkgolide B nanocapsules on diethylnitrosamine-induced p53 and p21 expression and in situ DNA
fragmentation in hepatic cells of rats sacrificed 18 weeks after commencement of the study. (A,i) Western blot analysis of p53
and p21 protein expression in cytosolic fraction of experimental rat liver. (A,ii) Histogram showing representative pixel intensities
(arbitrary units of densitometric analysis using ImageJ software [NIH, MD, USA]) of the immunoblot performed with different individual
rats. (B) Representative photomicrographs of BrdU-positive cells observed by double staining with BrdU (under FITC filter) and PI staining
of liver sections. (B,i,a–B,i,c) Liver sections from normal rats (10×). (B,ii,a–B,ii,c) Liver sections from DEN-treated control rats (10×).
(B,iii,a–B,iii,c) Sections from DEN + GB 100 mg/kg-treated rats (10×). (B,iv,a–B,iv,c) Sections from DEN + N1GB-treated animals (10×).
(B,v,a–B,v,c) Liver sections from DEN + N2GB-treated rats (20×). (B,vi) BrdU-positive cells per field of liver sections of experimental rats.
BrdU-positive cells were counted per field at 10× magnification using a Glasgow cell-counting graticule (Datasights, Enfield, UK).
The mean number of stained cells in ten randomly selected fields in each slide was expressed as the number of BrdU-positive cells.
*p < 0.05.
**p < 0.01.
***p < 0.001.
BrdU: Anti-5-bromo-2’-deoxyuridine; DEN: Diethylnitrosamine; FITC: Fluorescein isothiocyanate; GB: Ginkgolide B; N1GB: Polyethylene
glycol-coated ginkgolide B; N2GB: Uncoated ginkgolide B; PI: Propidium iodide.

biodegradable, nonimmunogenic solution of
delivering drugs, peptides, aptamers and other
bioactive molecules in biological systems. The
size and size distribution of nanoparticles are
important in determining their stability for
drug release and cellular uptake efficiency [22] .
Small-sized part­icles have the property of get-
ting opsonized and cleared out of systemic cir-
culation by the reticulo­endothelial system. PEG
functionalization has been a major approach

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Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das

A vi 800
***

iNOS-positive fluorescent
700
600
*
500

cells/field
i ii iii iv v
400
300 ***
200
100
i a ii a iii a iv a v a
0
i ii iii iv v

B i ii 200
DEN + GB DEN DEN
180 *** COX-2 ***
Normal DEN (100 mg/kg) + N1GB + N2GB
160 NF-κB
140
COX-2
120 * **
100
80 ***
NF-κB ***
60
40
20
β-actin 0
Normal DEN DEN + GB DEN + DEN +
(100 mg/kg) N1GB N2GB

Figure 5. Effect of ginkgolide B nanocapsules on diethylnitrosamine-induced expression of inflammation modulators in

hepatic cells of rats sacrificed 18 weeks after the commencement of the study. (A) Representative photomicrographs of rat liver
sections showing iNOS localization visualized by immunofluorescent staining of sections with anti-iNOS and Texas® Red-conjugated anti-
rabbit IgG antibodies using standard procedures. Red fluorescence indicates iNOS activity. (A,i) Normal rats show minimal red
fluorescence, while (A,ii) a dark intense fluorescent signal was recorded in DEN-treated control rats, (A,iii) considerable fluorescence
was observed in DEN + GB (100 mg/kg)-treated rats, (A,iv) with a gradual decrease in fluorescent signal in DEN + N1GB-treated rats,
and (A,v) a near-normal level of fluorescence seen in DEN + N2GB-treated rats. (A,i,a–A,v,a) Phase contrast images of their
corresponding fluorescence images. (A,vi) iNOS-positive cells were counted per field at 10× magnification using a Glasgow cell-counting
graticule (Datasights, Enfield, UK). The mean number of stained cells in ten randomly selected fields in each slide was expressed as the
number of iNOS-positive cells. (B,i) Western blot analysis of COX-2 and NF-kB (p65) protein expression in the cytosolic fraction of
experimental rat liver. (B,ii) Histogram showing representative pixel intensities (arbitrary units of densitometric analysis using ImageJ
software [NIH, MD, USA]) of the immunoblot performed with different individual rats.
*p < 0.05.
**p < 0.01.
***p < 0.001.
DEN: Diethylnitrosamine; GB: Ginkgolide B; iNOS: Inducible nitric oxide synthase; N1GB: Polyethylene glycol-coated ginkgolide B;
N2GB: Uncoated ginkgolide B.

to modify nanocarriers, reduces the chance of
getting opsonized and subsequent macrophage
uptake, thereby reducing phagocytosis [22] . Use
of PEG as a component material as well as in
the aqueous phase might have contributed to
the higher particle size compared with N2GB.
PEG incorporation imparted a negative zeta-
potential to the particles (Supplementary Figure 1) .
Usually, via the enhanced permeability and
retention effect, cancer cells have the ability to
concentrate a drug 20-times more when deliv-
ered through nanoparticulated system [23] . Poly-
meric nanocapsules with a positive or neutral
charge enter most of the cell lines studied, while
those with a negative charge internalize mostly
in the cancer cell lines. The positive potential
residing on N2GB surface was a result of the
positive cationic surfactant DMAB used in the
formulation. The in vitro drug-release patterns
indicate N2GB particles containing DMAB
have slower drug release. N2GB nanocapsules
offer higher uniformity than N1GB nano­
capsules, as observed from polydispersity index
values (Supplementary Figure  1) . HPLC data con-
firmed that the nanocapsulated formulations
were able to maintain the drug in its active form
for quite an extended period compared with the
free doses (Table 1) . However, higher hepatic GB
level in N2GB-fed animals indicate a slower
and extended release of the drug at the desired
organ. Bigger size of the particles along with an
initial burst of drug as observed in the in vitro
release study caused a faster release of drug from
N1GB compared with only DMAB-stabilized

doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print) future science group

 Ginkgolide B nanocapsules against hepatocarcinoma Research Article
A B 100

***
90

80
i i a
70

VEGF-positive cells/field
60

50
ii ii a
40

30 **

iii iii a 20
***
10

0
iv iv a i ii iii iv

Figure 6. Effect of ginkgolide B nanocapsules on hepatic VEGF expression due to

diethylnitrosamine-induced hepatocarcinogenesis in rats. (A) Representative
photomicrographs of rat liver sections following sacrifice 18 weeks after the commencement of the
study. VEGF immunofluorescent localization visualized by tagging sections with anti-VEGF and
fluorescein isothiocyanate-conjugated IgG antibodies using standard procedures. Green fluorescence
indicates VEGF expression. (A,i) Normal rats show no fluorescence, (A,ii) strong intense fluorescent
signal recorded in diethylnitrosamine-treated control rats, (A,iii) significantly lower level of
fluorescence in diethylnitrosamine + N1GB-treated rats and (A,iv) a near-normal minimal level of
fluorescence seen in diethylnitrosamine + N2GB-treated rats. (A,i,a–A,iv,a) Micrographs are phase
contrast images of their corresponding fluorescence images. (B) VEGF-positive cells were counted per
field at 10× magnification using a Glasgow cell-counting graticule (Datasights, Enfield, UK). The
mean number of stained cells in ten randomly selected fields in each slide was expressed as the
number of VEGF-positive cells.
*p < 0.05.
**p < 0.01.
**p < 0.001.

uncoated particles (i.e., N2GB). A faster release
from N1GB may have been responsible for GB
clearance from the organ.
Carcinogenesis is a long-term, multistep
process involving a spectrum of changes in
the biological system. Serum a-fetoprotein is
a useful marker to screen HCC. a-fetoprotein
serum levels usually fall after childbirth, but
HCC patients are diagnosed with a very high
level of this protein in serum [24] . We found
that N2GB treatment was able to arrest DEN-
induced HCC incidence reflected from body
growth, liver weight and hyperplastic nodule
formation, serum a-fetoprotein level, liver func-
tion enzyme activities, liver inflammation and
neoangiogenesis.
There may be several mechanisms by which
GB could exert its anticarcinogenic potential.
Upon metabolism, DEN produces toxic free
radicals, a major factor involved in all steps of
carcinogenesis – that is, initiation, promotion
and progression [25] . High dichlorodihydro­
fluorescein diacetate fluorescence was found to
be generated in mitochondria of DEN-adminis-
tered rats. DEN-induced elevated ROS produc-
tion disrupts cellular redox balance and mito-
chondrial output (Table  4) . Our findings have
shown a significantly higher (p < 0.001) conju-
gated diene level in liver mitochondria of DEN-
exposed rats (Table  3) . DEN-induced decrease
in membrane microviscosity of hepatic cells
(Figure 3) might also be attributed to the accu-
mulation of lipids and protein oxidative damage
due to cellular ROS buildup as well as ROS gen-
erated from defective mitochondria created dur-
ing the carcinogenic process. Lipid peroxidation
products may play an important role in medi-
ating the decreased membrane micro­viscosity
causing the hyper­permeable membrane, thus
allowing serum alanine trans­aminases and

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Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das
aspartate transaminase that originally resides
within the cytoplasm to be released into the
bloodstream.
GST as well as GSH content and other
glutathione-­metabolizing enzymes are over­
expressed in actively proliferating cells
(Table 2) [26] . Therefore the elevated level of GSH
and GST might be due to an overexpression of
these antioxidants as a consequence of enhanced
proliferation and rapid sequesteration of anti­
oxidants by tumor cells (Table 3) . Warburg, in his
pioneering work, mentioned about the ‘injury’
created in mitochondrial respiratory machinery
as a result of carcinogenesis with a subsequent
‘compensatory’ glycolytic ATP production [27] .
An increased NADH oxidase activity suggests a
higher ROS production. The impaired electron
transport system and oxidative phosphorylation
in mito­chondria is reflected from the decreased
SDH activity (Table 4), suggesting lower proton
accumulation and a dissipation of mitochondrial
membrane potential (Figure 3) . The administra-
tion of N1GB and N2GB could inhibit DEN-
induced mitochondrial ROS generation that
drives hepatic cells towards carcinogenic dam-
age, with N2GB offering highest protection.
Lower ROS levels in N2GB-treated animals
explain the lesser amount of conjugated diene
in the cell membrane as well as higher mem-
brane microviscosity. Lowering of GSH and
GST in N1GB- and N2GB-treated animals
reflects a state of lower ROS and fewer divid-
ing cells in the liver, giving a positive indication
of the reduction of oxidative burden generated
in the liver due to DEN administration. Mito­
chondrial respiratory complex enzymes were
also protected from ROS-induced damage in
the N2GB-treated group.
The role of mitochondria in tumor sup-
pression has been recently elucidated [28,29] .
In tumors associated with mutations in com-
plex II subunits, accumulation of succinate is
suggested to be the underlying cause of carcino­
genesis because it encourages hypoxic acclima-
tization  [30,31] . SDH, the complex II enzyme,
is the only electron transport chain complex
enzyme that is entirely encoded by the mito-
chondrial DNA. Thus decreased SDH activity
observed in DEN-exposed rats gives an indi-
cation of any DNA damage occurring in the
nuclear genome due to the application of the
carcinogen. Impaired p53 signaling is the most
common genetic modification found in cancer
and it plays a decisive role in apoptosis [32] . p53 is
involved in controlling cellular metabolism and
regulates glycolysis and oxidative metabolism
doi:10.2217/NNM.13.56 Nanomedicine (Epub ahead of print)
in divergent directions [33–35] and is therefore
considered as one of the molecular moderators
steering cells away from oxidative metabolism.
In the present study, downregulated p53 expres-
sion together with lower SDH activity in DEN-
exposed rats reflects the fact that an impair-
ment of p53 expression and function might be
due to mitochondrial dysfunction (Figure 4) . A
quite unexpected, strong p53 expression in GB
(100 mg/kg)-treated cells indicate p53 accumu-
lation due to the stress generated by DEN, but
not its inactivation as found in DEN-treated
control animals. DNA damage causes p53
protein levels to increase and induce the syn-
thesis of p21, an inhibitor of cyclin-­dependent
kinases, leading to arrest of the cell cycle. In
our study, the DEN-exposed group showed
low levels of p21 and a high level in the GB
(100 mg/kg)-treated group. p53 inactivation in
DEN-exposed animals might have caused p21
not to be expressed in response to stress, but
oral administration of GB (100 mg/kg) actu-
ally stimulated the DEN-induced increase in
p53 and p21 in rats. Upstream regulation of
ROS and mitochondrial dysfunction might
have prevented DNA damage and subsequent
upregulation of p53–p21 signaling in N2GB-
treated animals. Liver sections of DEN-exposed
rats showed that GB was able to induce DNA
fragmentation in hyperplastic nodules contain-
ing cancerous cells in DEN-exposed animals
(Figure 4) . This is consistent with our p53 results.
The lower number of apoptotic cells in N2GB-
treated animals indicate the lower number of
initiated cancer cells in that group, a clear reflec-
tion of the anticarcinogenic activity of GB. Thus
by protecting the mitochondria, N2GB might
have prevented p53 inactivation and inhibited
the process of carcinogenesis.
Histological evidence indicates that HCC
development occurs in the backdrop of chronic
inflammation [36] . NF-kB is the central media-
tor of inf lammation [37] , regulating other
inflammatory molecules either upstream or
downstream. Increased expression of COX-2
and iNOS observed in several human HCCs
and in chemically induced animal tumors [38,39]
were also found to be strongly expressed in our
DEN-administered animals (Figure 5) . Involve-
ment of PAF in the tumor microenvironment
has been implicated in numerous pathophysi-
ologic processes [40] . PAF effects are mediated
by a G-protein-linked transmembrane receptor
[41] , and binding on its receptor leads to tumor-
induced angio­genesis and metastasis. In addi-
tion, PAF can induce the expression of several
future science group
 Ginkgolide B nanocapsules against hepatocarcinoma
angiogenic factors such as TNF-a, IL-1b, FGF
and VEGF in endothelial cells through the tran-
scription of NF-kB [42] . N2GB was most success-
ful in controlling DEN-induced COX-2, iNOS,
NF-kB and VEGF expression (Figures 5 & 6) . GB,
the PAF antagonist, might have played the
most crucial role in reducing the DEN-induced
inflammatory and neoangiogenic signals.
Nanocapsulation of GB in biodegradable
polymer PLGA stabilized with cationic surfac-
tant DMAB was found to be the most effec-
tive in preventing DEN-induced HCC in the
rat liver. Nanoparticles upon entry into a cell
are localized to different subcellular parts
(e.g., cytoplasm and lysosomes) depending on
the nanoparticle’s surface charge [43,44] . The pos-
itive surface charge possessed by N2GB might
have been the key factor controlling hepatic cel-
lular internalization of nano­capsules followed by
their rapid accumulation at the electron-dense
mitochondria. A higher mitochondrial accu-
mulation might have led to a buildup of GB
concentration in the mitochondria, capable of
protecting the organelle from dysfunctioning
that subsequently leads to HCC development.
Dysfunctioning mitochondria arising out of
DEN administration appears to be the key fac-
tor in controlling redox status, inflammation,
cell proliferation and neoangiogenesis in the
liver. Easy accessibility of GB at hepatic mito-
chondria facilitated by N2GB could prevent
DEN-induced mitochondrial oxidative injury-
associated pathophysiologic changes in rat liver.
Protecting the mitochondria thus plays the most
decisive role in inhibiting the DEN-induced
hepatocarcinogenesis process.
Research Article
Future perspective
Human beings are constantly exposed to vari-
ous carcinogens and hence are threatened with
the chances of developing cancer during their
lifetime. This experiment of using a nontoxic
compound of natural origin in polymeric
nanocapsules stabilized with cationic surfac-
tant indicates the clinical prospect of using
such nanofabrications against the development
of a deadly killer disease such as liver cancer.
Financial & competing interests
disclosure
S Ghosh (Council for Scientific and Industrial Research
Senior Research Fellow) acknowledges the Council of
Scientific and Industrial Research, Government of India
for financial support. The authors acknowledge AK Das,
Associate Professor at the Department of Pathology,
Calcutta National Medical College and Hospital,
Kolkata (India) for histopathological interpretation,
AK Ghosh and S Lahiri for HPLC operation, and
T Muruganandan (Technical Officer, Indian Institute
of Chemical Biology, Kolkata) for atomic force microscope
operation. The authors have no other relevant affiliations
or financial involvement with any organization or entity
with a financial interest in or financial conflict with the
subject matter or materials discussed in the manuscript
apart from those disclosed.
No writing assistance was utilized in the production
of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate
insti­t utional review board approval or have followed the
princi­ples outlined in the Declaration of Helsinki for all
human or animal experimental investigations.

Executive summary
Nanocapsulation of ginkgolide B
ƒƒ Nanocapsulation of ginkgolide B (GB) enabled aqueous suspension, oral delivery and slow, time-dependent release of the compound.
GB level in liver
ƒƒ Uncoated GB (N2GB) offered higher GB accumulation in hepatic tissue.
Effect of zeta-potential of nanocapsulated GB on intracellular uptake
ƒƒ Positive zeta-potential residing on N2GB surface might have been the key factor controlling accumulation at the electron-dense
mitochondria.
Nanocapsulated GB on diethylnitrosamine-induced hepatocellular carcinoma development in rats
ƒƒ N2GB was most the potent in bringing down diethylnitrosamine-induced experimental hepatocellular carcinoma incidence in rats.
Nanocapsulated GB on mitochondria
ƒƒ Diethylnitrosamine-induced mitochondrial oxidative injury was prevented by N2GB application.
Nanocapsulated GB on inflammation in liver during carcinogenesis
ƒƒ N2GB treatment was able to bring down diethylnitrosamine-induced inducible nitric oxide synthase, COX-2, NF-kb and VEGF expression
in the rat liver.
Nanocapsulated GB on apoptosis
ƒƒ N2GB could induce apoptosis to cancer cells while protecting the overall physiology of the experimental animals.
Nanocapsulated GB on chemoprevention against hepatocellular carcinoma
ƒƒ N2GB is a potential chemopreventive formulation against hepatocellular carcinoma.

future science group www.futuremedicine.com doi:10.2217/NNM.13.56

Research Article Ghosh, Dungdung, Choudhury, Chakraborty & Das
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