Environmental Toxicology and Pharmacology 46 (2016) 147–157

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Environmental Toxicology and Pharmacology

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

Nano-encapsulated chlorophyllin significantly delays progression of

lung cancer both in in vitro and in vivo models through activation of
mitochondrial signaling cascades and drug-DNA interaction
Jayeeta Das a , Asmita Samadder b,c , Jesmin Mondal a , Suresh K. Abraham b ,
Anisur Rahman Khuda-Bukhsh a,∗
a
Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India
b
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
c
Department of Zoology, Dum Dum Motijheel College, Kolkata-700074, India

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

Article history: Chlorophyllin (CHL), a sodium-copper-salt derived from chlorophyll, has been widely used as a food-
Received 27 March 2016 dye, also reportedly having some anti-cancer effect. We tested if PLGA-loaded CHL (NCHL) could have
Received in revised form 12 July 2016 additional protective abilities through its faster and targeted drug delivery in cancer cells. Physico-
Accepted 15 July 2016
chemical characterization of NCHL was done through atomic-force microscopy and UV-spectroscopy.
Available online 18 July 2016
NCHL demonstrated greater ability of drug uptake and strong anti-cancer potentials in non-small cell
lung cancer cells, A549, as revealed from data of% cell viability, generation of reactive-oxygen-species
Keywords:
and expression of bax, bcl2, caspase3, p53 and cytochrome c proteins. Circular dichroic spectral data
Chlorophyllin
Poly (lactic-co-glycolic) acid
indicated strong binding of NCHL with calf-thymus-DNA, causing a conformational/structural change in
A549 cells DNA. Further, NCHL could cross the blood-brain-barrier in mice and showed greater efficacy in recov-
Mice ery process of tissue damage, reduction in chromosomal aberrations and% of micronuclei in co-mutagens
Mitochondrial signaling (Sodiumarsenite + Benzo[a]Pyrene)-treated mice at a much reduced dose, indicating its use in therapeutic
Anti-cancer efficacy oncology.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Several naturally occurring phytochemicals have been reported

Lung cancer is the leading cancer killer in both men and women.
In one statistical survey (Centers for Disease Control and Preven-
tion, National Center for Health Statistics) approximately 402,326
have ever been diagnosed with lung cancer in the United States,
and an estimated 221,200 new cases of lung cancer are expected
to be diagnosed in 2015, which represents about 13% of all can-
cer diagnoses (American Cancer Society. Cancer Facts and Figures,
2015). Benzo[a]Pyrene (BaP), a major component of tobacco smok-
ing, is a source of polycyclic aromatic hydrocarbons (PAH), which
is attributed to cause nearly 90% of lung cancer cases. Arsenic is
another environmental carcinogen that also leads to lung cancer
(Das et al., 2012). Therefore, these two, when act together con-
tribute to a great extent towards initiation of lung cancer rapidly
in animal models and sometimes used conjointly to induce lung
cancer (Das et al., 2012).
∗ Corresponding author.
E-mail addresses: prof arkb@yahoo.co.in, khudabukhsh 48@rediffmail.com
(A.R. Khuda-Bukhsh).
to have strong anti-cancer effects (Egner et al., 2001; Das et al.,
2012; Abraham et al., 2012; Das et al., 2013a,b; Paul et al.,
2013). Chlorophyllin (CHL), a water-soluble sodium copper salt
derived from chlorophyll, is one such compound, which has
been used for decades as a food dye and wound-healing accel-
erant (Tumolo and Lanfer-Marquez, 2012) without any known
human toxicity. Recently Chlorophyllin has been shown to possess
anti-carcinogenic activity in very few cases (Gradecka-Meesters
et al., 2011; Vesenick et al., 2012; Nagini et al., 2015). Chloro-
phyll does not normally dissolve in water, and therefore food
resources of chlorophyll have less chance to bind to mutagenic
substances. On the other hand, Chlorophyllin, being water-soluble,
can significantly bind to environmental mutagens, mainly with
Benzo[a]pyrene (BaP) (Keshava et al., 2009). In fact, chlorophyllin
binds to mutagens twenty times better than resveratrol and about
thousand times better than xanthenes (Osowski et al., 2010).
Nanoparticles have been widely used for various applications
including drug delivery (Duncan and Izzo, 2005; Samadder et al.,
2013; Das et al., 2013a,b; Paul et al., 2013). Their physiochemical
properties including their small size and large surface area have
led to these advances. In drug delivery, they have been reported

http://dx.doi.org/10.1016/j.etap.2016.07.006
1382-6689/© 2016 Elsevier B.V. All rights reserved.
148 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157

Fig. 1. Characterization of nano-chlorophyllin (NCHL) by atomic force microscopy (AFM).

(a) 2D image of NCHL (b) Graphical representation of mean diameter of NCHL (c) 3D image of NCHL (d) FFT spectra of NCHL (e) in vitro release kinetics of NCHL.

to significantly improve the bioavailability of drugs and minimise
drug toxicity (Bawarski et al., 2008; Farokhzad and Langer, 2006),
thus leading to more efficient therapies.
Therapeutic prospective and efficiency of biologically active
molecules are dependent on the identification of their bio-target
and active sites on one hand and elucidation of the strength and
specificity of binding to the other (Samadder et al., 2011). Function-
ally, deoxyribonucleic acid (DNA) serves as the carrier of the genetic
information of the cell, and therefore it is thought to be the cellu-
lar target of many therapeutic molecules. Thus, the interaction of
DNA with small natural or synthetic products having potential drug
value has been under rigorous investigation since the discovery of
the double helical structure of DNA.
Therefore, the main objectives of the present study were to
test the hypotheses whether: (i) chlorophyllin can be effectively
nano-encapsulated with PLGA and if possible, whether (ii) the
encapsulated chlorophyllin (NCHL) would show better potentials
of delaying/interfering with the process of progression of cancer
development in sodium arsenite + BaP induced lung cancer in mice
at a reduced dose than un-encapsulated chlorophyllin, (iii) to deter-
mine if NCHL has the ability to cross the blood–brain barrier, and
(iv) to address the possible molecular mechanism of action and the
signaling pathways involved.
2. Materials and method
2.1. Reagents
All the reagents used in our study were of analytical grade were
purchased from Sigma, USA.
2.2. Formation of blank and drug loaded nanoparticles
The standard technique of solvent displacement method
(Samadder et al., 2016) was followed to encapsulate Chlorophyllin
(CHL) in poly(lactide-co-glycolide) [PLGA] to form nano-CHL
(NCHL) under optimal conditions. The same technique was used for
 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157
Fig. 2. Evaluation of structural integrity by UV spectroscopy and demonstration of antioxidant property of CHL and NCHL.
(a) UV spectra of CHL (b) UV spectra of NCHL (c) Graphical representations of DPPH radical scavenging activity of CHL and N CHL at different concentrations.
blank nano-particles formulation except for addition of CHL during
the process of preparation.
2.3. Atomic force microscopic (AFM) studies of NCHL
NCHL samples for AFM imaging were prepared following
the standard process (Samadder et al., 2013) and observed and
recorded through AFM (Veeco di CP-11) imaging in amplitude and
tapping modes.
2.4. Assessment of structural integrity of NCHL by UV
spectroscopy
UV spectral analysis of CHL and NCHL were undertaken in
JascoV-550 UV–vis spectrophotometer.
2.5. Determination of loading and encapsulation efficiency of
NHCL
The loading capacity and encapsulation efficiency (EE) of NHCL
was estimated by standard protocol (Samadder et al., 2013)
in a UV/vis spectrophotometer (SHIMADZUUV-1700) and calcu-
lated according by using the formula: EE (%) = [(drug fed − drug
loss)/(drug fed)] × 100
2.6. Circular dichroism (CD) spectroscopic study
Analysis of circular dichroic spectra was done by Jasco spec-
tropolarimeter; J-720 monitored at 25 ◦ C. Structural changes in
the CT-DNA, CT-DNA + CHL and CT-DNA + NCHL in the region of
149
200–650 nm were detected using 1 cm path length rectangular
quartz cuvette.
2.7. Melting temperature profile of CT-DNA
Differences in the melting temperature profile data of CT-DNA
alone, CT-DNA + CHL and CT-DNA + NCHL were obtained with the
aid of a SHIMADZU-UV-1700 spectrophotometer fitted with a tem-
perature program.
2.8. 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging activity assay of CHL and NCHL
The standard DPPH radical-scavenging method as described in
Samadder et al. (2011) was used for determination of free radical-
scavenging activity of both CHL and NCHL.
2.9. Cell culture procedure
A549, human non small lung carcinoma cells and WI-38,human
normal lung cells were procured from NCCS, Pune, India were cul-
tured in DMEM supplemented with 10% fetal bovine serum and 1%
PSN antibiotic at 37 ◦ C, 5% CO2 for experimental purpose.
2.10. Selection of dose of NCHL in cell culture% cell cytotoxicity
assay
A549 cells were cultured and grown at 105 cells/mL conflu-
ency in 96-well flat bottom microtiter plate and were treated
with different concentrations of CHL and NCHL. The anti-
proliferative efficacy of the drugs were determined using MTT
150 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157

(3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyltetrazolium bromide)
following standard technique (Das et al., 2013a). PLGA blank
nanoparticles served as control. Similar experiments were also per-
formed in peripheral blood mononuclear cells (PBMC) and normal
lung cells (WI-38) to verify the cytotoxic effect, if any, of CHL and
NCHL in normal cells.

2.11. Assessment of the intracellular entry of NCHL by

fluorescence microscopy

To assess the relative cellular uptake of CHL and NCHL, FITC was
tagged to CHL and FITC-tagged-PLGA was used to encapsulate CHL
to formulate FITC-NCHL (Samadder et al., 2012). A549 cells were
plated at 107 /plate and incubated with 2.76 ␮M of FITC-CHL and
similar dose of FITC-NHCL at different time intervals (0, 1, 4, 8, 12
and 24 h) to check intracellular entry through confocal microscopy
by using the standard protocol (Paul et al., 2013).

2.12. Assessment of generation of reactive oxygen species (ROS)

in A549 cells

Generation of ROS in A549 cells were assessed by undertaking

the standard practiced protocol of Das et al. (2013a,b) using 2 7 -
dichloro dihydrofluorescein diacetate acetyl ester (H2DCF-DA) and
observed under fluorescence microscope (Leica, Germany).

2.13. Immunoblot localization of different proteins in A549 cells

Immunoblot localization was conducted by using anti-caspase-

3, anti-p53, anti-bax and anti-bcl2 antibodies (Santa Cruz
Biotechnology, USA) and alkaline phosphatase conjugated sec-
ondary antibody (Sigma, USA) by following standard technique
(Samadder et al., 2011).

2.14. Intracellular localization of cytochrome c by confocal
microscopy
A549 cells were prepared for the immunofluorescence analysis
using MitoTracker Red (50 nM) along with incubation in secondary
fluorescent FITC antibody following the method reported in Das
et al. (2013a) and observed and photographed under Andor Spin-
ning Disk Confocal Microscope.
2.15. Isolation of mitochondria from A549 cells
Fig. 3. Assessment of drug-DNA interaction by circular dichroism (CD) and melting
temperature profile (Tm).
(a) CD spectral analysis CD spectrum of calf thymus DNA incubated with CHL and
NCHL (b) Melting temperature profile study (Tm). The melting curves of calf thymus
DNA in presence and absence of CHL and NCHL.
following the standard protocol (Das et al., 2013a) and pho-
tographed under Andor Spinning Disk Confocal Microscope.
2.18. Quantification of mitochondrial membrane potential
(MMP) in A549 cells

Perfused alveolar cells were suspended in mitochondria

isolation buffer and homogenization buffer for separation of mito-
chondrial fraction and cytosol fraction respectively as per standard
practice and kept at 80 ◦ C for further use (Das et al., 2013a).
2.16. Assessment of cytochrome c in mitochondria and in cytosol
by ELISA technique
Expressions of cytochrome c protein in mitochondrial and
cytosolic fraction were quantified through indirect ELISA fol-
lowing the standard technique (Samadder et al., 2011) using
anti-cytochrome c, primary antibody (Santa Cruz Biotechnology,
Inc., USA) and alkaline phosphatase-conjugated secondary anti-
body (Sigma Aldrich, USA). The absorbance was measured at
405 nm in an ELISA reader (Thermo Electron Corporation Multi Scan
EX).
2.17. Qualitative analysis of generation of intracellular ROS by
confocal microscopy
Intracellular ROS generation of was examined using 2 7 -
dichloro dihydrofluorescein diacetate acetyl ester (H2DCF-DA)
Modulations in mitochondrial membrane potential (MMP) were
assessed in alveolar cells isolated from different groups of mice,
using Rhodamine 123 (1 mM), a fluorescent probe and analyzed
them through flow cytometer (FACS Calibur ARIA, BD Bioscience)
(Samadder et al., 2013).
2.19. Assessment of apoptosis in A549 cells by annexin V FITC and
PI staining
To evaluate the phenomenon of apoptosis and necrosis,
AnnexinV-FITC-PI (Propidium iodide) dual staining of A549 cells
were performed as per standard practice (Das et al., 2013a).
2.20. Animals
Healthy inbred strain of Balb/c mice (Mus musculus) (6/8 weeks:
∼25 g) were housed for 14 days at a temperature of 24–26 ◦ C ± 2 ◦ C;
humidity of 55 ± 5% and 12-h light/dark cycle with access to food
and water ad libitum. The Animal Ethical and Welfare Committee,
University of Kalyani approved guidelines were followed for our
present study.
 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157 151

Fig. 4. Determination of IC50 dose and cellular uptake of CHL and NCHL.
(a) Graphical representation of MTT assay to determine IC50 values of CHL and NCHL.
(b) Graphical representation of MTT assay to test cytotoxic effects of CHL and NCHL on PBMC.
(c) Analysis of intracellular uptake and cellular cytotoxicity of CHL and NCHL in A549, PBMC and WI-38 cells.

2.21. Experimental design in different sets of mice 2.22. Isolation of alveolar (lung) cells

Around thirty healthy mice were randomly selected for our
experimental purpose. They were then randomly subdivided into
five groups, each group comprising six mice.
Group 1: Negative control: Normal diet and water ad libitum
were administered in six mice which served as normal control.
Group 2: Co-mutagenic group: Benzo[a]pyrene (BaP) at the rate
25 mg/kg b.w and sodium arsenite (SA) at the rate 10 mg/kg b.w,
were force-fed to a subgroup of twenty four mice, twice a week,
successively for 4 weeks to induce lung cancer at 16th week (Tran
et al., 2002).
Group 3: Drug-treated subgroup: A subgroup of six mice from
the co-mutagenic group was taken and force-fed with CHL 5 g/kg
b.w./day for 30 days (Kumar et al., 2012).
Group 4 and 5: Nano-drug treated subgroup (dose 1 and dose
2): A subgroup of twelve mice was randomly selected from the
co-mutagenic group. They were force-fed with 2.5 g/kg b.w/day of
NCHL(I) and 5 g/kg b.w/day of NCHL(II) for 30 days (six mice in each
group) through gavage.
Lung tissues of normal and different experimental mice groups
were removed aseptically and perfused in DMEM medium (Das
et al., 2012)
2.23. Range finding trial
The dose of CHL, NCHL(I) and NCHL(II) in mice group were
standardized in a range finding trial by feeding 2.5 g/kg b.w/day,
5 g/kg b.w/day and 10 g/kg b.w/day of CHL and/or NCHL to co-
mutagenic control group of mice. 5 g/kg b.w/day dose of CHL and
2.5 g/kg b.w/day and 5 g/kg b.w doses of NCHL were selected based
on histopathological deformities and their amelioration in drug
treated series of lung tissues for further studies to make economy
on cost and on mice lives.
2.24. Cytogenetical parameters
Assessment of chromosome aberrations (CAs) and micronuclei
(MN) formation to determine and analyze the changes in morphol-
152 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157

Fig. 5. Evaluation of generation of reactive oxygen species (ROS) and flow cyometric analysis of mitochondrial membrane potential (MMP) in A549 cells.
(a) Fluorescence microscopic images of ROS generation A = control A549 cells, B = CHL, C = NCHL(I) and D = NCHL(II).
(b) Flow cyometric analysis of mitochondrial membrane potential (MMP) in A549 cells A = control A549 cells, B = CHL, C = NCHL(I) and D = NCHL(II).

ogy of chromosome in different control and experimental sets of 3. Results

mice were undertaken by following standard protocol (Das et al.,
2012). 3.1. Characterization of CHL loaded nanoparticles (NCHL) and
their structural integrity
2.25. Histolopathological assessment
From AFM image, the PLGA-loaded CHL nanoparticles (NCHL)
Control and experimental sets of mice lung tissues were dis- were found to have a mean diameter of 22.0 ± 0.05 nm; these were
sected out and fixed in 10% normal buffered formalin following spherical in shape and had a smooth surface (Fig. 1a–c). The Fast
standard protocol and prepared for histopathological observation Fourier Transformation (FFT) image (2D) in AFM image also showed
under phase contrast microscope (Samadder et al., 2011). a uniform spatial frequency of topographic planar signal (Fig. 1d)
of the formulated NHCL. UV–vis spectral data revealed that NCHL
2.26. Detection of NHCL in brain tissues by crossing the
produced similar characteristic band peaks as that of CHL, i.e., one
blood-brain-barrier (BBB)
peak at 628 nm and the other at 402 nm (Fig. 2a–b).
To check whether NHCL could cross the BBB, NHCL were orally
3.1.1. Antioxidant property of CHL and NCHL
fed to five mice 5 g/kg b.w twice per day for 7 days and then they
The antioxidant property was evaluated by DPPH scavenging
were sacrificed via cervical dislocation. The brain tissues were pre-
method and showed in Fig. 2c. The percentage of DPPH scavenging
pared and observed under TEM microscopy as per standard practice
of CHL and NCHL elevated with the increase in their concentrations.
(Samadder et al., 2013).
3.2. Assessment of drug loading and encapsulation efficiency of
2.27. Statistical analysis
NCHL
Students’ t-test and ANOVA were used to determine the level
The encapsulation efficiency of CHL inside PLGA to form nano-
of significance of all the data obtained from three independent
encapsulated-chlorophyllin was calculated as 86.2%.
experiments based on the differences between their mean values.
 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157 153

Fig. 6. Assessment of cytochrome c release in A549 cells by using confocal microscopy and indirect ELISA.
(a) Confocal microscopic images A = control A549 cells, B = CHL, C = NCHL(I) and D = NCHL(II).
(b) Graphical representation of cytochrome C release by indirect ELISA.

Fig. 1e illustrated the in vitro release kinetics of NCHL. The
drug release was persistent and only 58.33% of the total drug was
released from nanoparticles at the 24th hour.
3.3. Circular dichroic spectral analysis
The spectrum data for circular dichroism (CD) of CT-DNA
showed a positive band at 275 nm and a negative band at 247 nm,
i.e., the characteristics features of right-handed B form DNA. Addi-
tionally, an induced extrinsic CD band was also observed in the
region 375–475 nm. CHL and NCHL both are a chiral molecule and
therefore, were not CD active by itself when free in solution. Signif-
icant increase in the spectral band intensities in both positive and
negative bands was observed in CT-DNA + CHL and CT-DNA + NCHL
when compared to CT-DNA alone; CT-DNA + NCHL showed greater
increase than the other two, indicating a stronger binding and inter-
action NCHL with CT-DNA than that of un-encapsulated from, CHL
(Fig. 3a).
154 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157

Fig. 7. Evaluation of expression of different proteins in A549 cells and apoptotic assay by using Annexin V FITC-PI in flow cytometer.
(a) Western blot of p53, bax, bcl2, caspase-3 and GAPDH as housekeeping gene Ln1 = control A549 cells, Ln2 = CHL, Ln3 = NCHL(I) and Ln4 = NCHL(II).
(b) Flow cytometric analysis of apoptosis = control A549 cells, B = CHL, C = NCHL(I) and D = NCHL(II).

3.4. Melting temperature profile assay
The melting temperature (Tm) for CT-DNA alone = 82.5 ± 0.5, CT-
DNA + CHL = 90.0 ± 0.5 and CT-DNA + NCHL = 95.0 ± 0.5 revealed an
increase in Tm value on addition of NCHL when compared to CT-
DNA alone or in combination with CHL (Fig. 3b).
3.5.2. Relative intracellular uptake of CHL and NCHL in A549 cells
Results of confocal microscopy indicate that the intracellular
uptake of NCHL commenced from 1 h interval onwards whereas
that of CHL begins at 4 h interval onwards. Moreover, the rate of
entry of NCHL was greater than that of CHL signifying the capacity
of more rapid entry and greater intracellular bioavailability of NCHL
than that of CHL (Fig. 4c).

3.5. In vitro assessment

3.5.1. Cytotoxicity assessment in A549 cells
The percentage of cytotoxicity of A549 cells varied in a dose
dependant manner as observed from the CHL and NCHL treated
series. The IC50 value for both CHL and NCHL was found to be
2.76 ␮M. Therefore, A549 cells were treated with 2.76 ␮M of CHL
and 2.76 ␮M and 5.52 ␮M of NCHL for further studies (Fig. 4a). In
our further experiments with peripheral blood mononuclear cells
(PBMC) and WI-38, human normal lung cells there was no signifi-
cant observable change in the percentage cell viability in the CHL,
NCHL(I) and NCHL(II) group when compared to untreated control
(Fig. 4b).
3.5.3. Evaluation of generation of reactive oxygen species (ROS)
in A549 cells
Results clearly revealed that ROS generation in A549 cells
treated with NCHL(II) was greater than that of CHL and/or NCHL(I)
when compared to untreated control A549 cells (Fig. 5a).
3.5.4. Quantitative analysis of mitochondrial membrane potential
(MMP) in A549 cells
When compared to untreated control set of A549 cells, drug
treated cells showed a decrease in MMP; the NHCL groups showing
greater decrease in MMP (Fig. 5b).
 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157 155

Fig. 8. Assessment of histopathology of lung tissue, cytogenetical parameters of bone marrow cells in mice and transmission electron microscopic (TEM) images to assess
ability of the nano-particles to cross the blood-brain-barrier (BBB).
(a) Histopathological analysis of lung tissues A = control mice, B = SA + BaP, C = SA + BaP + CHL, D = SA + BaP + NCHLI, E = SA + BaP + NCHLII (b and c) Cytogenetical assessment
of chromosomal aberration and (CA and MN) A = control mice, B = SA + BaP, C = SA + BaP + CHL, D = SA + BaP + NCHLI, E = SA + BaP + NCHLII (d) Bar graphs showing% on MN in
different groups (e) TEM images of brain tissue of mice A = control, B = NCHLII.

Table 1
Showing% of aberrated chromosomes, isolated from bone marrow cells in different groups of mice.

Treatment No. of Metaphase plates (n) % of aberrated metaphase plate

Rings Gaps Premature Chromosome Condensation (PCC) Breaks

Control 200 0 0 0 0
SA + BaP 200 2 ± 0.001 5 ± 0.007 19 ± 0.03 6 ± 0.02
CHL 200 2 ± 0.04 4 ± 0.02 14 ± 0.03* 5 ± 0.01*
NCHL-1 200 1 ± 0.02 4 ± 0.05 12 ± 0.07 ** 5 ± 0.09*
NCHL-2 200 1 ± 0.03* 3 ± 0.02* 11 ± 0.04** 5 ± 0.07*
*
p < 0.05.** p < 0.01 vs SA + BaP treated groups.
156 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157
3.5.5. Cytochrome c release in different drug-treated A549 cell
sets
Data from both confocal microscopy (Fig. 6a) and that of indirect
ELISA (Fig. 6b) revealed that there was an increase in Cytochrome
c expression in the cytosolic fraction in the drug-treated series
whereas a decrease in the expression of Cytochrome c was observed
in the mitochondrial fraction when compared to untreated control
A549 cells.
3.5.6. Expression of different protein localization in A549 cells
Up-regulation of p53, bax and caspase-3 protein and down-
regulation of bcl2 protein were clearly observed in CHL and NCHL
treated A549 cells when compared to that of untreated control;
NCHL(II) treated cells showed better result in every series (Fig. 7a).
The increase or decrease of each protein’s band intensities in each
series of western blot was done by taking GAPDH as internal stan-
dard in every case to show proper neutralization. The process of
internal standardization was repeated thrice for proper neutraliza-
tion of each group of mice. The band intensities of GAPDH remained
the same in each set of experiments at the time of internal standard-
ization.
3.5.7. Apoptosis assay by annexin V FITC-PI
Flow cytometric assessment of apoptosis in A549 cells was per-
formed using Annexin V-FITC-PI. Although percentage of necrotic
cells was negligible in all the treatment series, but both early
and late apoptotic cells significantly increased in a concentration-
dependant manner (Fig. 7b).
3.6. In vivo assessment
3.6.1. Histopathological analysis of lung tissues
When co-mutagen treated mice were administered furthered
with CHL and NCHL an amelioration in the damaged and degraded
and architectural tissue loss, NCHL administration showed better
results (Fig. 8a).
3.6.2. Cytogenetical assessment
Major and minor types of chromosomal aberrations (CA) and
micronuclei (MN) were observed in co-mutagenic group. However,
this particular trend of incidence were found to decrease in CHL and
NCHL treated series; NCHL(II) showed better results (Fig. 8b and c;
Table 1).
3.6.3. Transmission electron microscopic (TEM) images to assess
the presence of NCHL in mice brain
Results of TEM images suggested that NCHL could effectively
cross the blood-brain-barrier (BBB) (Fig. 8e).
4. Discussion
Results of the present study would suggest that NCHL had
a very small particle size of 22 nm, good entrapment efficiency,
smooth surface area, negative zeta potential, stability and sus-
tained intra-cellular release- all qualities of an ideal drug-carrier.
The nano-encapsulated drug thus showed a better efficacy even at
a ∼10 fold reduced dose because of faster entry into the cells and a
suspended release due to shorter diffusion path and larger surface
area. Further, analysis of UV spectral data of both NCHL and CHL
revealed two similar spectral peaks characteristic of CHL, one at
628 nm and the other at 402 nm. These peaks remained unaltered
even when the drugs were stored for a long period of time, sug-
gesting thereby their strong structural integrity and high storage
stability.
Results of CD spectra and Tm profile data clearly suggested that
NCHL interacted with CT-DNA at a greater scale than that of CHL.
Enhancement of the intensities of both the positive and negative
bands of DNA clearly suggests strong intercalation and stabiliza-
tion of the right-handed B conformation of DNA (Kong et al., 2008).
Therefore, it is possible that because of greater bioavailability of
NCHL, it could bind and interact with CT-DNA more effectively and
strongly than CHL, thereby interfering with the divisional activities
of the cells that could delay the process of cancer progression; NCHL
could also initiate more DNA damage in the process which could
have an impact on further accelerating the process of apoptosis,
preventing the cell from undergoing a successful division.
NCHL inhibited the growth of A549 cells in a dose-dependent
manner. The growth inhibiting effect of CHL, on the other hand, was
not as effective in inducing apoptosis in A549 cells, possibly because
of their slower entry into cells. Earlier studies implicated that
CHL increased oxidative stress to induce apoptosis via increased
ROS accumulation (Wang et al., 2011). Our results were in agree-
ment with the previous studies, albeit we observed a significantly
higher rate of ROS accumulation in NCHL treated cells than that in
CHL. Therefore, NCHL acted in different ways towards accomplish-
ing cancer intervention to prevent further progression through a
cascade of events: by intercalating with DNA to cause disarray
in divisional activity, to induce DNA damage contributing further
towards apoptosis, and also by increasing the level of intracellular
ROS that might cause further extensive DNA damage.
One earlier study (Roos and Kaina, 2013) also suggested that
drugs having capability of binding with the DNA could trigger apo-
ptosis more effectively in a cancer cell, than those which do not
have this capability. This may be due to modulatory effect in several
downstream molecules involved in the initiation and progression
of the apoptotic process. Further, it has also been reported that
increase in generation of ROS may lead to depolarization of mito-
chondrial membrane potential permitting leakage of cytochrome c
(cyt c) from mitochondria to cytosol, that also contributes to apo-
ptosis induction (Fulda and Kroemer, 2011). In our present findings,
the percentage of viable cells was decreased by NCHL adminis-
tration, presumably through initiation of activation of sequential
process of mitochondrial signaling pathway; the overall process
was further accentuated by the simultaneous accumulation of ROS,
loss of MMP, cyt c release into the cytosol, elevation in bax, caspase
3 and p53 proteins and depletion in bcl2 protein.
Redistribution of phosphatidyl serine (PS) to the outer side of
the plasma membrane is considered a significant step in the pro-
cess of programmed cell death (apoptosis) whereas it is confined
to the inner side in the viable cells; this phenomenon can thus act
as a marker for apoptotic event. Results of Annexin V-FITC/PI dual
staining reveal the relocation of PS to the cell membrane by signif-
icant increase in early and late apoptotic cells’ percentages, NCHL
acting more effectively than CHL implying that NCHL was superior
to CHL as an antitumor and anti-invasive agent.
To test if NCHL could have possible human application, we tested
if it had the desired ability to cross the blood-brain-barrier. Results
suggested clear presence of NCHL in brain tissue of mice imply-
ing its potential role for possible human application. Further, our
results showed that NCHL enhances drug bio-availability and has a
substantially longer half-life than CHL in mice in vivo, and showed
stronger protective effects on various types of chromosome aber-
rations, micronuclei and associated DNA damage, amelioration of
lung tissue damage and inhibition of tumor growth in SA plus BaP
treated mice. These effects were observed in animal model at a ∼10
fold reduced dose of NCHL than that of CHL.
Therefore, the overall results suggested that NCHL showed a
greater potential for the upstream event mediated mitochondrial
apoptosis than its un-encapsulated counterpart. However, further
studies on other higher animal models can be recommended, before
 J. Das et al. / Environmental Toxicology and Pharmacology 46 (2016) 147–157
testing in human model, for effective use in therapeutic oncology
in future.
Conflict of interest
None to declare.
Acknowledgements
This work was supported by DST nanoscience and technol-
ogy (AS), UGC-Resource Networking (SKA), DST-PURSE (JD), UGC’s
Maulana Azad National fellowship (JM) and UGC’s Emeritus fellow-
ship (ARKB).
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