Environmental Toxicology and Pharmacology 43 (2016) 27–37

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Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Nanopharmaceutical approach using pelargonidin towards

enhancement of efficacy for prevention of alloxan-induced DNA
damage in L6 cells via activation of PARP and p53
Asmita Samadder a,∗ , Suresh K. Abraham a , Anisur Rahman Khuda-Bukhsh b
a
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
b
Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India

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

Article history: Alloxan is an environmental food contaminant that causes DNA damage in living cells and induces hyper-
Received 9 August 2015 glycemia. Pelargonidin (PG), an active ingredient found in extract of various fruits and vegetables, has
Received in revised form 9 February 2016 been nanoencapsulated (NPG) with poly-lactide-co-glycolide (PLGA) and tested for efficacy in preven-
Accepted 10 February 2016
tion of alloxan (ALX)-induced DNA damage in L6 cells in vitro. Glucose uptake, reactive oxygen species
Available online 12 February 2016
(ROS) generation, glucose transporter 4, glucokinase levels and mechanism of activation of DNA repair
proteins (PARP and p53) have been studied in ALX-induced L6 cells. Drug–DNA interaction has been
Keywords:
analyzed using calf thymus DNA as target through circular dichroism and melting temperature profile.
PLGA encapsulation
Pelargonidin
NPGs were physico-chemically characterized by standard protocols using dynamic light scattering and
Alloxan transmission electron microscopy. Pre-treatment with both PG and/or NPG was effective in reducing
DNA stability curve ALX-induced oxidative stress and showed favourable effects for protection against DNA damage by acti-
L6 cell line vating DNA repair cascades. Results suggested ∼10-fold increase in efficacy of NPG than PG in prevention
DNA repair cascade of alloxan-induced oxidative stress and DNA damage.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction antioxidant, anti-inflammatory, anticarcinogenic and antigeno-

Diabetes has emerged as a common metabolic disorder affecting
a large section of the human population (Samadder et al., 2011). At
present, many new antidiabetic drugs with minimum harmful side
effects are being developed. In spite of that, herbal medicines with
relatively less cytotoxic effects are becoming popular for treating
diabetic patients. Hence, research on a wide variety of phytochem-
ical combinations for treating diabetes (Samadder et al., 2011;
Samadder and Khuda-Bukhsh, 2014; Roy et al., 2008) is gaining
importance. Alloxan, an environmental food contaminant, which
is consumed as a food item made up of flour (a staple food in a
large part of the human population), is implicated to induce a con-
dition of hyperglycemia in animal models in vivo as well as in vitro
cultured cells including L6 cells (Lenzen and Panten, 1988).
Anthocyanidins are non-toxic plant pigments found in orange,
red, blue and purple coloured fruits and vegetables (Khandelwal
and Abraham, 2014a). Anthocyanidins have been reported to have
∗ Corresponding author. Present address: Department of Zoology, Dumdum Moti-
jheel College, Kolkata 700074, India. Tel.: +91 9874548900
E-mail address: asmita.samadder@gmail.com (A. Samadder).
toxic activities (Khandelwal and Abraham, 2014b). Pelargonidin
(PG) is one among the six most abundantly available antho-
cyanidins and is known to exert antidiabetic effects in animal
models (Roy et al., 2008; Mirshekar et al., 2010). These reports
prompted us to examine if the antidiabetic efficacy of PG could be
enhanced through its nanoencapsulation to form nano-PG (NPG) by
a biodegradable environ-friendly non-toxic polymer, poly-lactide-
co-glycolide (PLGA).
Currently, nanoformulations of several antidiabetic drugs are
used in therapeutic applications, as they offer non-toxic and effi-
cient carrier system for targeted drug delivery. These nanoforms
render enhanced drug bioavailability within the cells/tissues, or
both (Samadder et al., 2012, 2013a,b). Information on the cura-
tive properties of some phytochemicals and their nanoforms are
now available. However, studies for understanding their preven-
tive potential are scanty. Therefore, there is a need to evaluate the
relative protective potential of nanoencapsulated phytochemicals
against induced stress in suitable biological in vitro models.
Interaction of phytochemicals with proteins, deoxyribonucleic
acid (DNA) and other biological targets often results in modulation
of key activities of cellular processes such as immune responses,
signal transduction, mitosis, DNA repair mechanism, apoptosis

http://dx.doi.org/10.1016/j.etap.2016.02.010
1382-6689/© 2016 Elsevier B.V. All rights reserved.
28 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37
and oxidative stress (Saha and Khuda-Bukhsh, 2013). Identifica-
tion of cellular targets and active sites of the nanoparticles is a
prerequisite for ascertaining therapeutic potentials and efficacy of
biologically active molecules. DNA is thought to be the cellular tar-
get of many therapeutic molecules (Samadder et al., 2011, 2013a;
Bhadra and Kumar, 2010). Interaction of DNA with small natural
products and their derivatives, or synthetic products having poten-
tial drug value, has therefore attracted special attention. Several
techniques like circular dichroism spectroscopy and the method
used for determination of melting temperature profile provide sig-
nificant information on the nature, specificity of the interactions
and DNA stability. Similarly, assessment of DNA structure/damage,
induction of cytotoxicity and generation of reactive oxygen species
(ROS) by ALX are important for this investigation.
Thus, the main objectives of the present work were to: (i)
produce PLGA-loaded pelargonidin (NPG); (ii) assess relative pro-
tective effects of NPG vis-a-vis PG on ALX-induced oxidative stress
and DNA damage in L6 cell line; (iii) examine relative binding abil-
ity of PG and NPG with calf thymus DNA (CT-DNA), and cellular
DNA, and (iv) determine the possible signalling pathway involved
in DNA damage and generation of ROS as a sequel to treatment of
either PG or NPG.
2. Materials and methods
2.1. Chemicals
All the chemicals used for our study were of analytical grade and
procured from Sigma Aldrich. St. Louis, MO, USA.
2.2. Cell culture
Skeletal muscle cells, L6, obtained from National Centre for
Cell Science (Pune, India) were grown in 5% carbon dioxide atmo-
sphere at 37 ◦ C in Dulbecco’s modified Eagle’s Medium (DMEM)
supplemented with 10% foetal bovine serum and 1% antibiotic. For
experimental studies, the cells were allowed to grow to 80–90%
confluence, after which they were harvested in ice-cold buffer
saline (PBS), plated at desired density and re-equilibrated for 24 h
before any treatment (Samadder et al., 2013b).
2.3. Preparation of nanoencapsulated pelargonidin (NPG)
Pelargonidin (PG), purchased as Pelargonidin Chloride (Sigma),
was encapsulated in a biodegradable polymer poly-lactide-co-
glycolide (PLGA) to form nanoencapsulated pelargonidin (NPG)
by following the solvent displacement technique (Fessi et al.,
1989) under optimal conditions. Briefly, 10 mg of PG and 50 mg
PLGA were dissolved in 3 ml acetone. This organic phase mix-
ture was added dropwise to 10 ml of aqueous solution containing
100 mg (F68; w/v) (1% polyoxyethylene–polyoxypropylene) stabi-
lizer and stirred in a magnetic stirrer at room temperature until
the organic solvent evaporated completely. The redundant sta-
bilizer was removed from the nanoparticles by centrifugation at
25,000 g at 4 ◦ C for 30 min. After completion of the reaction process,
the pellets of nanoparticles thus obtained were re-suspended in
Milli-Q water, washed and weighed before storing at 4 ◦ C for further
use. Thus from the initial amount of 10 mg PG, we obtained about
100 mg of NPG as the end product, accounting for about a 10-fold
increase in the yield from PG to NPG. We used equal amount PG or
NPG to comprise a dose, and thus the dose of NPG became almost
10 times reduced as compared to that of PG. Blank nanoparticles,
which served as control, were also prepared in the same way except
that addition of PG was excluded from the process. However, when
the nanoparticles of PLGA alone, without PG or NPG, were tested
against alloxan-treated L6 cells for any possible protective efficacy
against hyperglycemia, they did not show any. Therefore, the use
of blank nanoparticles as one arm of the control was later aban-
doned. Further studies were conducted for evaluating comparative
efficacy of PG and NPG.
2.4. Transmission electron microscopic (TEM) study
The aqueous suspension of NPG was prepared for TEM by pla-
cing a drop of the suspension on a copper grid, allowing the water
to evaporate. Observations were made under TEM (JEOL-2100 F)
operated at an accelerating voltage of 200 kV.
2.5. Zeta potential measurement
The zeta potential of the nanoparticles was determined by in
Nano-ZS instrument (Malvern Instruments, Southborough, UK) fol-
lowing the standard technique (Samadder et al., 2013a).
2.6. Determination of NPG loading and encapsulation efficiency
NPG loading was estimated by following the standard pro-
tocol (Samadder et al., 2013b) with slight modification. PG
concentrations were determined by a UV/VIS spectrophotometer
(SHIMADZU-UV-1700) at 293 nm where the characteristic absorp-
tion peak of PG appeared. A standard plot of PG (0–50 ␮M) was
prepared under identical conditions. The PG-loading content and
encapsulation efficiency (EE) were calculated according to the fol-
lowing formula: EE (%) = [(drug fed − drug loss)/(drug fed)] × 100.
2.7. Treatment of L6 cells with alloxan (ALX)
ALX was diluted in sterilized Milli-Q water at a concentration of
1 M and was stored at 4 ◦ C. Cells were exposed to freshly prepared
dilution of ALX, 1 mM in DMEM media for 2 h (Li et al., 2002). The
concentration of ALX and duration of treatment used in the study
were decided on the basis of results from preliminary range-finding
trials.
2.8. Dose selection of PG or NPG to protect against ALX-induced
damage in L6 cells
PG was dissolved in DMEM media at a concentration of 10, 20
and 30 ␮M, respectively. L6 cells were incubated with all the con-
centrations of PG separately for different time periods and further
exposed to standardized dose of ALX (1 mM). Differences in intra-
cellular glucose concentration were measured and 30 ␮M dose of
PG was found to give the best result at 2 h pre-incubation time
(Kamenickova et al., 2013). Therefore, 30 ␮M dose of PG (corre-
sponding to ∼9.0 ␮g/ml of PG) for 2 h was selected for further
study as the standard dose. Doses of NPG were likewise prepared
at 4.5 ␮g/ml and 9.0 ␮g/ml and cells were exposed to freshly pre-
pared doses of NPGs in DMEM media for 2 h after optimizing them
in a similar range-finding trial.
2.9. Assessment of cytotoxicity of NPG
To assess the cytotoxicity of NPG, MTT [3(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium-bromide] assay (Samadder et al.,
2012) was performed in normal L6 cells.
2.10. In vitro release of PG from NPG
In vitro release of PG from NPG capsule was estimated from the
PG standard curve, after administration of NPG to L6 cells. The cells
were then lysed in PBS and centrifuged at 2000 g. The absorbance
 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37
was taken to calculate the amount of PG in it at 293 nm (max of
PG).
2.11. Circular dichroism measurements
Circular dichroic spectra were monitored at 25 ◦ C to detect any
change in the structure of CT-DNA in the region (200–650 nm)
using 1 cm path length rectangular quartz cuvette (Jasco spectropo-
larimeter; J-720).
2.12. Melting temperature profile and assessment of stability of
CT DNA
Melting temperature profile data of CT-DNA, CT-DNA + PG and
CT-DNA + NPG were obtained with the aid of a SHIMADZU-UV-
1700 spectrophotometer fitted with a temperature programme.
The recording chart read the temperature and absorbance differ-
ences between the reference cuvettes (which were the same as the
sample except without DNA) and the sample cuvettes at 260 nm.
From the melting temperature curve, the changes in Gibbs free
energy were determined for CT-DNA alone, CT-DNA + PG and CT-
DNA + NPG deploying the following series of equations: y = mx + c;
where m = slope which is equivalent to the equilibrium constant
K. From this K, changes in Gibbs free energy were calculated from
the equation ıG = −RT ln K, where R = gas constant (8.31 J/K/mol),
T = change in temperature; here T (Kelvin) = 373 [100 ◦ C (for all
the series) + 273]. The stability and changes in the temperature
for denaturation of DNA were then ascertained from the equation
ıG = ıH − TıS, ıH = enthalpy (which was constant for all sets) and
ıS = entropy.
2.13. Evaluation of glucose uptake in L6 cells
Glucose uptake in control and experimental L6 cells was esti-
mated following a standard procedure (Samadder et al., 2013b).
2.14. Estimation of DNA damage by fragmentation
The extent of DNA damage was determined by extracting DNA
from L6 cells by standardized phenol–chloroform method. There-
after, DNA gel electrophoresis was performed in 1% agarose gel
(Samadder et al., 2011).
2.15. Morphological changes
Microscopic observations were also made to identify the mor-
phological differences in control and experimental L6 cells (Nikon
Eclipse TiE).
2.16. Determination of reactive oxygen species (ROS)
ROS generation in L6 cells was assessed under fluorescent
microscope (Nikon Eclipse TiE) and fluorescence activated cell
sorter [FACS (BD Science Calibur)] using a cell permeable fluores-
cent probe, 2 7 -dichloro dihydrofluorescein diacetate (H2DCF-DA)
(Samadder et al., 2011). Cells were counterstained with Hoechst
dye to determine the ROS-induced nuclear condensation. Micro-
scopic fluorescence intensity was quantified by using the analysis
software (NIS Elements AR, Version-4.00).
2.17. Detection of glucose transporter 4 (GLUT4) and
poly(ADP-ribose) polymerase [PARP] proteins
Fluorescein isothiocyanate (FITC)-tagged secondary antibody
was used for localization of GLUT4 and PARP proteins in L6 cells
by following the standard procedure of immunofluorescence assay
29
(Samadder et al., 2013b). The images were photographed under
Andor Spinning Disk Confocal Microscope. The fluorescence inten-
sity of the proteins was quantified by Analysis software, NIS
Elements AR, Version-4.00.
2.18. Analysis of PARP and p53 proteins by indirect ELISA
Expressions of PARP and p53 proteins were analyzed by using
anti-p53 and anti-PARP primary antibodies (Santa Cruz Biotech-
nology, Inc., USA) and alkaline phosphatase-conjugated secondary
antibodies (Sigma Aldrich, USA) following a standard protocol
(Samadder et al., 2013a). The absorbance was measured at 405 nm
in an ELISA reader (Thermo Electron Corporation Multi Scan EX).
2.19. Reverse transcriptase polymerase chain reaction (RT-PCR)
analysis
Extraction of cellular RNA was made using Trizol reagent fol-
lowing the manufacturer’s instructions (Samadder et al., 2013a).
Synthetic oligonucleotide was purchased from Chromus Biotech
Pvt., Ltd., Bangalore, India. The forward and reverse primer
sequences [Bioserve Biotechnologies (India) Pvt., Ltd., Hyderabad,
India] used for specific amplifications are: G3PDH(F:5 -
ATGGGGAAGGTGAA-GGTCGG-3 and R:5 -GGATGCTAAGCAGTT
GGT-3 ), GLUT4(F:5 -AAGATGGCCACGGAGA-GAG-3 and R:5 -
GTGGGTTGTGGCAGTGAGTC-3 ) and Glucokinase(F:5 -CACCCAA
CTGCGAA-ATCACC-3 and R:5 -CATTTGTGGGGTGTGGAGTC-3 ).
After PCR amplification, the DNA bands were photographed and
densitometrically analyzed by Image-J software.
2.20. Statistical analysis
All the data were presented as mean values of three indepen-
dent experiments after analyzing them statistically to determine
the level of significance of the differences between the mean values
by Student’s t-test and one-way ANOVA followed by LSD post hoc
test using SPSS 14.0 software; **p < 0.05 was considered significant.
3. Results
3.1. Characterization of NPGs
NPGs were ∼12 nm in size with fairly uniform shape and
morphology as observed under TEM imaging captured in lower
magnification at 40,000× magnification (Fig. 1a) and higher mag-
nification at HRTEM: 800,000× magnification (Fig. 1b). The spectral
band for Fast Fourier Transformation (FFT) and the 2D power
spectral density (Fig. 1c) showed uniform spatial frequency of topo-
graphic planar signal of the formulated NPGs. The zeta potential
value was −17 mV (Fig. 1d).
3.2. Assessment of PG loading, encapsulation efficacy, burst
release and in vitro release kinetics of NPG
PG loading and encapsulation efficacy of PLGA to form NPG were
calculated to be 87.57% (Supplementary Table 1a). The loss of PG
during NPG formulation (PG burst release) was also found to be
minimal (Supplementary Table 1b). Intracellular release of PG from
NPG commenced immediately after administration of NPG in L6
cells (0–2 h) (Supplementary Fig. #), which accounts for the ease in
biodegradability of PLGA to release PG from NPG capsule.
3.3. Assessment of cellular cytotoxicity of NPG
Administration of NPGs did not show any change in percent-
age viability of L6 cells as compared to that of untreated normal
30 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37

Fig. 1. Characterization of PLGA-encapsulated nanopelargonidin (NPG). (a) Transmission electron microscopic (TEM) images of NPG (lower magnification). (b) High-resolution
TEM (HRTEM) image of NPG. (c) Fast Fourier Transformation (FFT) spectral density. (d) Zeta potential of the NPG.
 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37 31

Fig. 2. Interaction of PG and NPG with CT-DNA. (a) Circular dichroic spectra of CT-DNA incubated with PG and NPG. (b) The melting curves of CT-DNA in the presence of PG
and NPG. (c) Stability curve for Gibbs free energy from temperature for denaturation of CT-DNA in the presence and absence of PG and NPG.

control (normal: 97.7 ± 1.9, NPG at the highest dose and the longest
treatment interval: 95.09 ± 3.54) (Supplementary Fig. ##).
3.4. Analysis of CD spectral band
The CD spectrum of CT-DNA showed a positive band at 273 nm
due to base stacking and a negative CD band at 245 nm due to a
change in helicity, which are characteristic features of a typical B-
form DNA. When they were free in the solution, PG and NPG alone
did not have a CD spectrum. Introduction of PG and NPG with CT-
DNA (separately) showed interaction, which led to an increase in CD
molar ellipticity; NPG showed a greater shift in the spectral band
thereby suggesting strong binding of the NPG with CT-DNA than
that of PG alone (Fig. 2a).
32 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37

Fig. 3. Alloxan-induced stress assessment. (a) Glucose uptake quantification. (b) DNA damage estimation (Ln1:Control; Ln2:ALX-treated cells; Ln3:PG + ALX-treated cells;
Ln4:NPG(I) + ALX-treated cells; Ln5:NPG(II) + ALX-treated cells), **p < 0.5 vs. ALX-treated set of cells. (c) Flow cytometric quantification of ROS using DCFDA. A – Control, B –
ALX-treated cells, C – PG + ALX-treated cells, D – NPG(I) + ALX-treated cells, E – NPG(II) + ALX-treated cells.

3.5. Evaluation of melting temperature profile and stability curve
of CT-DNA
The melting temperature (Tm ) profile of CT-DNA alone (in the
absence of PG or NPG) was observed to be at 82.5 ± 0.5 ◦ C. Introduc-
tion of PG or NPG (separately) with CT-DNA led to an increase in Tm
value to 87.5 ± 0.5 ◦ C and 90.0 ± 0.5 ◦ C, respectively (Fig. 2b). The ıG
values were obtained from the data of the stability curve, which are
as follows: −16,013.17 for CT-DNA, −16,264.99 for CT-DNA + PG,
and −16,949.10 for CT-DNA + NPG (Fig. 2c). The proportionality val-
ues were found to indicate that NPG interacted with the DNA to
make it the most stable form because of the highest negative ıG
value among the other two, i.e., CT-DNA alone and CT-DNA + PG. The
increasing order for temperature of denaturation of the CT-DNA is
as follows: CT-DNA < CT DNA + PG < CT-DNA + NPG, i.e., CT-DNA will
denature first, followed by CT-DNA + PG and CT-DNA + NPG.
3.6. Quantification of glucose uptake in L6 cells
There was a decrease in the level of glucose uptake in
ALX-treated cells as compared to normal control. On the
contrary, glucose uptake was inhibited in the cells incu-
bated with either PG + ALX or NPG + ALX (Fig. 3a, Supplemen-
tary Table 1c and d); NPG showed the maximum inhibiting
effect.
3.7. Assessment of DNA damage
There was a marked increase in smearing of the DNA in
ALX-treated cells as compared to that of normal cells. On
the contrary, the occurrence of DNA smear was highly dimin-
ished in cells treated with PG + ALX or NPG + ALX; noticeably,
the NPG pre-treated group of higher dose showed better
 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37 33

Fig. 4. Analysis of morphological changes, reactive oxygen species (ROS) generation using DCFDA and nuclear condensation by counterstaining with Hoechst in different
sets of L6 cells. Arrowhead denotes ROS generated disruption in morphology in ALX-treated cells. In NPG(I) + ALX-treated cells arrowhead shows lesser quantum of ROS from
disrupted cells.

result when compared with that of the PG pre-treated group
(Fig. 3b).
3.8. Evaluation of ROS generation leading to morphological
anomaly
Generation of ROS as estimated through FACS and from the
intensity profile of the microscopic images revealed that there was
an elevation of ROS as observed from quadrant shift in FACS and
modulation in fluorescence intensity in ALX-treated group, when
compared with control set (Figs. 3c, 4, 5a). Interestingly, in PG + ALX
and NPG + ALX treatment sets, this phenomenon was not signifi-
cant. Damage in the cellular morphology was also prevented to a
greater extent in the same manner, possibly reflecting the above
consequences.
3.9. PCR analysis for expression of GLUT4 and glucokinase
When compared with ALX-treated cells, a significant difference
in the expression of GLUT4 and glucokinase mRNA was observed in
experimental cells treated with PG + ALX or NPG + ALX. NPG pre-
incubated cells gave better response as compared to PG alone
(Fig. 5b and c, Supplementary Table 1i–l).
3.10. GLUT4 and PARP protein localization
Results of intensity curves revealed that there was a sharp
decline in fluorescence intensity in ALX-stressed L6 cells when
compared to untreated control in case of GLUT4, whereas in case of
PARP, it was just the reverse. Moreover, cells treated with PG + ALX
or NPG + ALX, showed higher GLUT4 (Fig. 6) and PARP (Fig. 7a)
34 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37

Fig. 5. Quantification of ROS and expression of mRNA. (a) Intensity profile of this figure images. A – Control, B – ALX-treated cells, C – PG + ALX-treated cells, D – NPG(I) + ALX-
treated cells, E – NPG(II) + ALX-treated cells. (b, c) mRNA level expression and band intensity in L6 cells: RT-PCR analysis for mRNA expression and the band intensity of
G3PDH (housekeeping gene), GLUT4, and glucokinase in different cell sets: Ln1:Control; Ln2:ALX-treated cells; Ln3:PG + ALX-treated cells; Ln4:NPG(I) + ALX-treated cells;
Ln5:NPG(II) + ALX-treated cells, ***p < 0.001 vs. ALX-treated set of cells.

protein localized in them (green fluorescence for positive localiza- control. The increase was more prominent in NPG-treated set of
tion) as compared to ALX-stressed cells. This difference was more cells (Fig. 7b, Supplementary Table 1e–h).
striking in NPG pre-treated cells than that of the PG pre-treated
cells. 4. Discussion and conclusion

3.11. Assessment of p53 protein by ELISA
Cells treated with PG + ALX or NPG + ALX showed a gradual
increase in p53 concentration when compared to that of untreated
The overall results of our present study show that ALX treat-
ment has increased ROS level in L6 cells when compared to the
untreated control. However, in PG + ALX or NPG + ALX treated L6
cells, the quantum of ROS generated was clearly less, with NPG
showing greater degree of protection. The same trend was also
 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37 35

Fig. 6. Confocal microscopic analysis of GLUT4 protein. Localization and quantification of GLUT4 protein in control and experimental cell sets.

noted when DNA damage was taken into consideration. Here, the
quantum of DNA damage was reduced, more by the treatment of
NPG than by PG. In fact, an equivalent dose of NPG produced a
∼10-fold greater protective effect as compared to that of PG. This
was primarily because of the greater cell-penetrating ability of the
NPG, being smaller in size and having negative electrical charges,
higher entrapment efficiency and by possible protection and/or
improved activation of DNA repair genes PARP and p53. Biodegrad-
able property of the nanocarrier is another important factor; it not
only helps in suspended drug release, but also spares the cells of
any toxic effect of by-products that may accumulate in the cells.
PLGA polymers were not only reported to be non-toxic in nature
but also considered as an efficient carrier system for intracellular
drug delivery due to their biodegradable nature (Weissenböck et al.,
2004; Bhattacharyya et al., 2011). Thus, NPG has greater potential
of being used strategically in future antidiabetic drug formulation.
L6 skeletal muscle cells are known to play a key role in glu-
cose homeostasis through the kinetics of glucose transporter. As
part of the peripheral tissue, they can effectively portray initial
changes indicative of diabetes. Thus, L6 cells are considered suitable
36 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37

Fig. 7. Expression of different proteins in L6 cells. (a) Localization and quantification of PARP protein in control and experimental cell sets counterstained with Hoechst. (b)
Quantification of PARP and p53 proteins by indirect ELISA techniques, ***p < 0.001 vs. ALX-treated set of cells.

material for studying mechanism of action of various antidiabetic
drugs (Samadder et al., 2013b).
The binding ability of a drug to DNA is considered as a desirable
quality to effectively intervene at the DNA level. CD spectral data
in this study clearly indicated that the drug could bind on specific
sites of DNA, with modulating helicity of DNA leading to certain
conformational changes that in turn could probably be attributed
to the ability of drugs to block some specific active sites of DNA,
preventing DNA damage and influencing cell cycle progression and
change in transcriptomes. Further, CD data revealed that NPG had
greater effects than PG and thereby caused greater alteration in
base stacking. DNA denaturation curve also revealed higher ele-
vation in melting temperature (Tm ) of CT-DNA by NPG than by
PG treatment alone. This was further confirmed by analyzing the
 A. Samadder et al. / Environmental Toxicology and Pharmacology 43 (2016) 27–37
changes in the Gibbs free energy and stability curve, which indi-
cated greater ability of binding of NPG to CT-DNA, making it more
stable. Further, results of our study revealed the ability of NPG to
activate the DNA repair pathway. Up-regulation in the expression
in PARP and p53 proteins provided evidence that NPGs also pos-
sessed the potential to initiate and enhance the progress of DNA
repair signalling cascades.
ALX is known to specifically inhibit uptake of intracellular glu-
cose by blocking glucokinase activity and induce generation of
reactive oxygen species (ROS), thereby damaging the DNA (Lenzen
and Panten, 1988). Introduction of NPG in L6 cells, prior to admin-
istration of ALX, could suppress the damage of glucose transporter
4 (GLUT4) and reduce the generation of ROS, better than that of PG.
NPG could also lower the inhibition of glucokinase activity, bet-
ter than PG, thereby affecting the level of glucose uptake when
compared with ALX-treated cells. DNA damage associated with ALX
treatment was also inhibited in NPG-treated cells. Thus, NPG could
act better on cellular protection to reduce free radical generation,
which is thought to be another benefit of using the nanoparticu-
lated drug having ability of greater penetrance and bioavailability
due to their PLGA coating.
Phytochemicals are now being quite frequently used as alter-
native medicines against myriads of diseases. In order to facilitate
their effective and safe use, emphasis is being increasingly given
to gather more precise information related to ease of deliv-
ery, targeted action, greater bioavailability, and lesser dose with
maximum effect. In recent years, therefore, nanoformulations of
several phytochemicals, both of natural and synthetic origin, have
been attempted and their efficacy against several diseases tested
(Samadder et al., 2012, 2013a; Das et al., 2012; Paul et al., 2013).
Since most antidiabetic drugs are for post-diabetic treatment,
results of this study point to the possibility of formulating a strategy
of introducing non-toxic drugs for preventing/delaying diabetes
to develop in the first place. This could open up a new area of
research if non-toxic and biodegradable nanopolymers can be fruit-
fully exploited with the right kind of phytochemicals for bringing
up new hope in the control and management of diabetes.
Conflict of interest
None to declare.
Transparency document
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found in the online version.
Acknowledgements
AS is grateful to DST Nanoscience & Technology (JNCASR) for
financial grant. ARK-B is thankful to UGC, New Delhi, for Emeritus
Fellowship.
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.etap.2016.02.010.
37
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