Carbohydrate Polymers 160 (2017) 184–193

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

␬-Carrageenan: An effective drug carrier to deliver curcumin in

cancer cells and to induce apoptosis
Malairaj Sathuvan a,1 , Ramar Thangam b,1 , Mani Gajendiran c , Raju Vivek d ,
Sengottuvelan Balasubramanian c , Subramani Nagaraj a , Palani Gunasekaran e ,
Balaraman Madhan b,∗ , Ramasamy Rengasamy a,∗
a
Centre for Advanced Studies in Botany, University of Madras, Chennai 600 025, Tamilnadu, India
b
CHORD, CSIR-Central Leather Research Institute, Adayar, Chennai 600 020, Tamilnadu, India
c
Department of Inorganic Chemistry, University of Madras, Chennai 600 025, Tamilnadu, India
d
Department of Zoology, Bharathiar University, Coimabatore 641 046, Tamilnadu, India
e
King Institute of Preventive Medicine & Research, Chennai 600 032, Tamilnadu, India

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

Article history: The current study is to develop a natural drug carrier with seaweed derived polymers namely ␬-
Received 22 October 2016 Carrageenan (␬-Car) for drug delivery applications. ␬-Car is a natural polysaccharide which derived from
Received in revised form edible red seaweeds, they are easily available, non-toxic, cost effective, biodegradable and biocompatible
15 December 2016
nature. Curcumin (Cur) is a yellow-orange polyphenol existing in turmeric, which is predominantly used
Accepted 18 December 2016
as spice and food coloring agent. The ultimate use of polymeric composites, especially those composed
Available online 23 December 2016
of natural polymers, has become a very interesting approach in recent drug delivery applications, due
to their non-toxicity and biological origin. In this study the primary approach which depends on the
Keywords:
␬-Carrageenan
loading of Curcumin into ␬-Carrageenan was accomplished, and which (␬-Car-Cur) an active drug car-
Curcumin rier was developed for drug delivery against selected lung cancer cells (A549). Thus, the ␬-Car-Cur was
Drug-carrier synthesized by solvent evaporation method followed by freeze drying, and it was further characterized.
Cancer cells From this study, it has been reported that the high encapsulation efficiency, good stability, and successful
ROS generation release of Cur from the carrier (␬-Car) was achieved. The drug release was more active at acidic pH 5.0
Cellular apoptosis with the cumulative release of 78%, which is the favorable condition present in tumor microenviron-
ments. The in vitro cellular applications studies of ␬-Car-Cur demonstrated that, ␬-Car-Cur composites
induced higher cytotoxicity against selected cancer cells than free Cur and effectively involved to trigger
cellular apoptosis in A549 cancer cells. Further, it was also possessed that inhibition of cell growth and
changes in metabolic activity of cancer cells are the unique characteristic features of cellular apoptosis,
through reactive oxygen species (ROS) generation. It also observed that there was a decrease in mito-
chondrial membrane potential ( m m) which leads to a cellular apoptosis during treatment with
␬-Car-Cur. Hence, the study outcomes may provide the potential outline for the use of ␬-Car-Cur as a
promising tool to deliver drugs at intracellular level.
© 2016 Published by Elsevier Ltd.

1. Introduction
Polysaccharides are being widely utilized for drug delivery
related applications in recent years. Marine organisms such as
bacteria, microalgae, and seaweeds, have represented a largely
untapped reservoir of valuable materials (Ahmed, Adel, Karimi,
∗ Corresponding authors.
E-mail addresses: madhanclri@gmail.com (B. Madhan),
profrrengasamy@yahoo.com (R. Rengasamy).
1
These authors contributed equally to this work.
& Peidayesh, 2014). Among them, seaweeds are the most abun-
dant source of polysaccharides such as Carrageenan (iota, kappa,
and lambda), alginate, and agar. Carrageenan, a naturally occur-
ring anionic sulfated linear polysaccharide molecule extracted from
certain red seaweeds of Rhodophyceae family particularly from
Chondrus cripus, Euchema gigartina, Stellate iridaea, Hypnea, Solieria
agardhiella and Sarconema (Gómez-Ordóñez, Jiménez-Escrig, &
Rupérez, 2010). The major constituent of such algae is Carrageenan,
polysaccharides with the linear backbone built up by ␤-d-galactose
and 3,6-anhydro-␣-d-galactose with variable density in the sul-
fated group (Gómez-Ordóñez, Jiménez-Escrig, & Rupérez, 2014).

http://dx.doi.org/10.1016/j.carbpol.2016.12.049
0144-8617/© 2016 Published by Elsevier Ltd.
 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
Certain algae such as Gigartina stellata, Chondrus crispus, Eucheuma
spp., Iridaea spp., and Kappaphycus spp., are the major raw materi-
als source which is used for Carrageenan extraction (Pereira, 2013).
Carrageenan have shown several potential pharmaceutical appli-
cations including anti-coagulant, anti-cancer, anti-hyperlipidemic,
and immuno-modulatory activities (Wijesekara, Pangestuti, & Kim,
2010). In food industry, Carrageenans are widely utilized as
gelling, thickening, emulsifying and stabilizing agents, and has been
employed to improve the texture of cottage cheese, puddings dairy
desserts, binders and stabilizers in the meat processing industry
(Campo, Kawano, Braz, Silva, & Carvalho, 2009).
The hydrogels formed from ␬-Carrageenan are a suitable source
for drug delivery systems (Li, Ni, Shao, & Mao, 2014). It has been
reported that ␬-Car is potentially used as pharmaceutical material
due to its good biocompatibility and low toxicity, in spite of that
they also having easy gelling, thermo-reversibility of the gel net-
work and appropriate visco-elastic properties that enable them to
undergo harsh conditions (Varghese, Chellappa, & Fathima, 2014).
Hydrogels of Carrageenans bears a capable carrier to encapsulate
macromolecular drugs in oral drug delivery (Matricardi, Di Meo,
Coviello, Hennink, & Alhaique, 2013). Although ␬-Car is capable of
encapsulating drugs to prevent premature release and degradation
in the localized environment, to overcome these, there is a need of
an efficient drug carrier, as this will ensure the released drug into
the intestinal region (Liu, Zhan, Wan, Wang, & Wang, 2015; Necas
& Bartosikova, 2013; Paleos, Tsiourvas, Sideratou, & Pantos, 2013).
According to the physicochemical properties, ␬-Car is considered
to exhibit the suitable rheological and sustained release behavior
with potential use for an oral drug delivery vehicle (Leong et al.,
2011).
Curcumin (Cur) is a hydrophobic polyphenol pigment which is
predominantly available in turmeric. The curcumin powder derived
from rhizome of Curcuma longa that has a wide spectrum of biolog-
ical and pharmacological activities (Kim, Choi, & Lee, 2003; Singh,
Sidhu, Deepa, & Maheshwari, 1996). Recently it has been evidenced
that, curcumin is able to interact physically with its diverse range
of cellular targets including transcription factors, growth factor
receptors, cytokines, enzymes and genes regulating cell prolifera-
tion and apoptosis (Aggarwal, Kumar, & Bharti, 2003). Curcumin has
shown to be more effective against many cancer cells such as breast,
prostate, bone, head and neck, lung and (Kunnumakkara, Anand, &
Aggarwal, 2008; Wilken, Veena, Wang, & Srivatsan, 2011). Despite
all these promising characteristics, a major problem encountered
with Cur is that their solubility, they are extremely poor soluble
in aqueous solutions leading to low gastrointestinal absorption,
low bioavailability which limits its clinical efficacy (Ha et al., 2012;
López-Lázaro, 2008). Thus, we proposed in this study, the ␬-Car
based carrier-mediated Cur delivery might be the potential way
to overcome these problems without affecting the efficacy of cur-
cumin. In these aspects there are several recent researches which
are concentrated on delivering curcumin using different carrier
molecules, such as polymeric micelles, nanoparticles, liposomes,
lipid-based nanoparticles, hydrophilic polymers or hydrogels etc.,
(Gou et al., 2011; Maeda, Bharate, & Daruwalla, 2009; Naksuriya,
Okonogi, Schiffelers, & Hennink, 2014; Scarano, De Souza, & Stenzel,
2015; Song et al., 2011; Sun et al., 2012; Teong et al., 2015; Yallapu,
Jaggi, & Chauhan, 2012) Lung cancer, which is also known as pul-
monary carcinoma or lung carcinoma, is a type of malignant tumor
in lung tissues. Tobacco smoking, which induces majority of these
cancer (80–90%), thus is considered to be dangerous tumor that
threatens human health and life with high morbidity and mortal-
ity (Lieber, 1976; Sun, Schiller, & Gazdar, 2007). The incidence and
mortality of lung cancer have significantly increased worldwide. In
many cases, the malignancy of tumors is detected only at advanced
stages when chemotherapeutic drugs become increasingly toxic to
healthy cells. To improve this condition, both targeted drug deliv-
185
ery and early detection of cancer cells will be more supportive (Bae
& Park, 2011; Munoz et al., 2006). Chemotherapy is the primary
treatment for cancer and is applied in most cases because this dis-
ease is usually diagnosed at an advanced stage (Youlden, Cramb,
& Baade, 2008) including lung cancer. Therefore, there is a need
and development of novel formulation that depends on the drug
delivery system to deliver drugs with increased solubility, stabil-
ity, and pharmacological activities, resulting in the improvement
of therapeutic effects especially curcumin based nano-drugs.
In this present study, it is aimed to exploit a ␬-Carrageenan as
an effective carrier to load curcumin and to achieve drug release
behavior in two different pH (5.0 and 7.4) conditions. It is confirmed
that, the Cur was effectively loaded (incorporated) on ␬-Car com-
posites, which was confirmed using FT-IR and scanning electron
microscopy (SEM). The controlled release of Cur from ␬-Car-Cur is
higher at acidic pH 5.0, followed by pH 7.4. The in vitro anti-cancer
studies showed that ␬-Car-Cur potentially inhibited cell growth
of A549 cells in a time-dependent manner. Finally, the involved
anti-cancer mechanisms of ␬-Car-Cur have been studied using flu-
orescence microscopy analysis. The study results showed that the
␬-Car was effectively involved in delivering the loaded drug (Cur)
at the cellular environment through controlled release behavior to
the possible induction of cellular apoptosis.
2. Materials and methods
2.1. Materials
Curcumin (95.0% purity), ␬-Carrageenan, Dulbecco’s mini-
mal essential medium (DMEM), Penicillin, Streptomycin, 2 ,7 -
dichlorfluorescein-di-acetate, DAPI were purchased from Sigma-
Aldrich, USA. MTT was purchased from Hi-Media, laboratories,
India. Fetal bovine serum was purchased from Gibco, Inc, USA. All
other chemicals were of analytic grade. All the solutions used in the
experiments were prepared using ultrapure Milli-Q water using a
Millipore water purification system (Millipore, Milford, MA, USA).
2.2. Preparation of curcumin loaded -Carrageenan beads
␬-Car-Cur was prepared as follows, 0.1 g of ␬-Car was added
into 50 mL of distilled water and the temperature was adjusted to
80 ◦ C, the prepared contents were stirred using magnetic stirrer
until ␬-Car was dissolved completely. A stock solution of curcumin
was prepared in ethanol at a concentration of 1 mg mL−1 . In par-
allel 50 mg of Cur was dissolved in 5 mL of ethanol and added to
the above stirred solution. The solution was again re-stirred for
1 h at 80 ◦ C to obtain a clear, viscous and homogeneous solution
without bubble. Finally, the bead production process was carried
out using the interphase technique (López, Lázaro, & Marqués,
1997). The above-prepared mixture containing Carrageenan and
curcumin were dropped through a syringe into the beaker contain-
ing sunflower oil in aqueous phase (5% KCl). Beads were passed
through the oil layer and it was collected in the aqueous phase;
resulting in spherical beads left behind in the aqueous phase for
an hour. Ice was placed in the beaker to assist in the formation of
beads (Scheme 1). The solutions were maintained at 10 ◦ C for 5 h to
let the beads harden. After gelation process, the beads were filtered
and rinsed with water to remove excess potassium ion, then freeze
dried. The suspension was centrifuged at 12,000g for about 30 min.
and the residues were re-suspended in water until the pH reaches
7.4.
2.3. Characterization techniques
The FT-IR spectra of prepared samples (␬-Carrageenan, Cur-
cumin and ␬-Carrageenan loaded Curcumin) were recorded using
186 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
Scheme 1. (a) Steps involved in the preparation of Curcumin loaded ␬-Carrageenan through interphase method. (b) Schematic representation of cross-linking phenomenon
of developed ␬-Car-Cur materials.
a Fourier Transform Infrared Spectrometer (Nicolet 670 FTIR, USA)
with 16 scans per sample in the region of 400–4000 cm−1 using
KBr pellets. The drug release behaviors were studied at different
pH (5.0 and 7.4) conditions using UV–vis spectrophotometer in an
in vitro medium and the release conditions were analyzed using
UV–vis spectrophotometer (Hitachi U2900). The SEM analysis of ␬-
Carrageenan and ␬-Car-Cur were analyzed using Hitachi Scanning
Electron Microscope SU3500 Japan.
2.4. Determination of drug loading efficiency
The ␬-Car-Cur (5 mg) was ultra-sonicated and then 1.5 mg were
placed in 1.5 mL of 0.01 mg mL−1 drug solution in PBS and allowed
to equilibrate for 48 h at room temperature. The beads were then
removed from the loading solutions by centrifugation, at 12,000g
for 30 min. and were separated the supernatant, rinsed with PBS
and freeze dried. The drug content in the beads were determined
by UV–vis spectroscopy measurements at 428 nm of the solution
before and after encapsulation (Syed, Liew, Loh, & Peh, 2015). The
loading capacity and encapsulation efficiency of the hydrophobic
curcumin to beads were calculated using Eqs. (1) and (2), respec-
tively:
weightof the drug in Beads
Drug loading content(%) = × 100 (1)
weight of the beads
weight of the drug in Beads
Encapsulation efficiency(%) = × 100
weight of the feeding drug
(2)
2.5. In vitro drug release study
The ␬-Car-Cur (10 mg) were dispersed in phosphate buffer solu-
tion (pH 5.0 and 7.4) and incubated at 37 ◦ C on a rotary shaker.
The dispersions were centrifuged at different time intervals and the
amount of Cur released at different time interval were determined
using spectro-photometrically (Kadam, Bhingare, Nikam, & Pawar,
2013). Free curcumin is completely insoluble in water; therefore,
at predetermined time intervals, the solution was centrifuged at
6000 g for 7 min. to separate the released curcumin from the loaded
beads. The released curcumin
was re-dissolved in 3 mL ethanol to assay spectro-
photometrically at 428 nm. The concentration of released drug was
then calculated using the standard curve of curcumin in ethanol.
The percentage of curcumin released was determined from the
following equation,
Released curcumin from Beads-Beads
Release (%) = × 100
Total amount of curcumin in Beads-Beads
2.6. Cell viability assay
Human lung adenocarcinoma (A549) cells were cultured in
DMEM containing 10% fetal bovine serum, and 100 IU/mL of peni-
cillin and streptomycin (pH 7.4) at 37 ◦ C in humidified incubator
containing 5% CO2 . The cell viability was analyzed by Methylth-
iazol Tetrazolium salt (MTT) reduction assay was used to assess
the cell viability as described by Mosmann (1983). Briefly, the cul-
tured cells of A549 (1 × 104 cells/mL) were seeded in a 96- well
plate to the final volume of 100 ␮L/well, after 48 h, the cultured
 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
Fig. 1. SEM micrographs of freeze-dried samples of (a) Cur loaded ␬-Car bead (b) magnified surface view (c) Internal view of beads.
cells were treated with a different ranges of concentration of Cur,
␬-Car and ␬-Car-Cur for 24, 48 and 72 h respectively. After that,
100 ␮L of MTT (5 mg mL−1 ) solution was added to the treated cells,
and then the plates were incubated at 37 ◦ C for 4 h. The super-
natant was aspirated and 100 ␮L of DMSO was added to each well
to dissolve the formazan crystals. Then the absorbance was mea-
sured at 620 nm using microplate reader (THERMO MULTISKAN,
USA), and the inhibitory concentrations (IC50 ) were determined for
treated materials accordingly. The percentage of cell viability was
calculated using the following formula:
Mean experimental absorbance
Cell viability (%) = × 100
Mean control absorbance
The control wells were not treated with study materials.
2.7. Fluorescence microscopy analysis
A549 cells were cultured in a 6-well plate and maintained at
37 ◦ C with 5% CO2 for 48 h. Subsequently, the cells were exposed
with ␬-Car-Cur for 7, 24, and 48 h respectively with IC50 concen-
trations. Then, the cells were washed twice with PBS, fixed in
4% para-formaldehyde for 20 min, re-washed with 75% methanol,
and stained with DAPI (10 mg mL−1 ) at 37 ◦ C for 20 min in dark.
Subsequently, the stained cells were treated with Rhodamine-123
(Rh-123) (10 mg mL−1 ) stain for 30 min at 37 ◦ C in dark to ana-
lyze the mitochondrial membrane potentials (␺m). The cells were
then washed with methanol and analyzed for changes in  m with
appropriate wavelengths using fluorescent microscopy.
2.8. Measurement of reactive oxygen species (ROS) generation
Reactive oxygen species (ROS) are the one which generated
from cells during aerobic metabolism. They are highly reactive
187
molecules and excessive amounts of ROS may lead to protein and
DNA oxidation, protein cross-linking, and cellular death. The cell
culture models provide an identical tool in understanding these
mechanisms which ultimately lead to cell death (apoptosis). Thus,
the accumulation of ROS within cells and their release is stud-
ied using 2 -7 -di-chlorofluorescein di-acetate (DCF-DA) staining
analysis. The ability to estimate ROS levels is an important step in
understanding the mechanisms of cellular apoptosis. After seed-
ing 5 × 105 cells/well to a 6-well plate, the cells were treated with
compounds of ␬-Car-Cur for 7, 24, and 48 h respectively in a time
dependant manner. Subsequently, the treated cells were washed
with ice-cold PBS and incubated with DCFH-DA (50 ␮M in a final
concentration) at 37 ◦ C for 30 min in dark. Then, the cells were
washed twice and maintained in 1 mL of PBS. The ROS generation
was assessed using fluorescence microscope using an excitation
and emission wavelengths of 488 nm and 530 nm, respectively and,
the release of ROS from cells were measured by mean fluorescence
intensity (MFI) of 2 ,7 -dichlorofluorescein (DCF). The fluorescence
intensity of DCF is directly proportional to the amount of ROS
produced by the cells. The Cur, ␬-Car and ␬-Car-Cur treatment
and generation of ROS in the cells showed the de-acetylation by
intracellular esterase to non- fluorescent DCFH, which is oxidized
by ROS resulting in the formation of the fluorescent compound
DCF.
2.9. Statistical analysis
The experiments were performed in triplicates (n = 3) and the
data were expressed as mean ± standard error of the mean. Statis-
tical analysis was performed using Graph Pad Prism 6 (Graph-Pad,
San Diego, CA). The results were considered statistically significant
if *p ≤ 0.05.
188 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193

3. Results and discussion

3.1. Preparation of -Car-Cur

The gelation of ␬-Car consists of two-step mechanism which

involves, formation of rod-shaped ␬-Car double helical confor-
mation with an average length of the same order of magnitude
than followed by parallel aggregation of these helices (Daniel-da-
Silva, Ferreira, Gil, & Trindade, 2011; Spagnuolo, Dalgleish, Goff,
& Morris, 2005). An important feature that provides ␬-Car ver-
satile capabilities and its overall negative charge. Because of the
abundant sulphate groups on the sugar chain, ␬-Car can com-
bine with positively charged molecules (Dalgleish & Morris, 1988)
interact electro-statically with ␬-Car and drugs increases the drug
solubility (Pinheiro et al., 2012). The drug with possible complex
with ␬-Car is illustrated in (Scheme 1). The complex structure
may increase the solubility of the free compound by 15–30 times,
as these compounds becomes amorphous in the complex (Dai,
Dong, & Song, 2007). The purpose of conjugation of Cur to the
hydrophilic polysaccharide or encapsulation (like ␬-Carrageenan)
of Cur in the composite is mainly to improve its aqueous solubil-
ity. In the previous studies, it is reported that the polymer-drug
conjugate strategies could possibly improve the solubility and sta-
bility of Cur (Dey & Sreenivasan, 2014; Yallapu, Jaggi, & Chauhan,
2010) using different polymers. Hence, in this study the solubility
of Cur was so far achieved in an aqueous medium by loading with
␬-Carrageenan.
The freeze-dried beads (␬-Car-Cur) were spongy, more rigid and
it was found to be sensitive to the ambient pH conditions (acidic),
thereby suggesting that a matrix made from ␬-Car may be a promis- Fig. 2. FTIR spectra of (a) ␬-Car, (b) Cur, and (c) Cur loaded ␬-Car.
ing candidate for pH dependant controlled release. The possible
fabrication process and the preparation of Cur loaded Car com-
at 1642 cm−1 corresponds to the C O stretching frequency, a peak
posites are shown in Scheme 1. It was clearly observed that the
at 1072 cm−1 is due to the C O stretching frequency and a peak at
developed Cur loaded ␬-Car had fine dispersion and appeared to
1184 cm−1 includes the S-O stretching of sulphate ester salt (Fig. 2a)
be more soluble, unlike that of Cur which was completely insolu-
(Gómez-Ordóñez & Rupérez, 2011). The FT-IR spectrum of Cur in
ble in water solution due to the very low solubility. The Cur loaded
Fig. 2b shows the characteristic bands at 3514 cm−1 (phenolic O H
␬-Car forms a mesh or network-like structure, like the structure
stretching), 1597 cm−1 (C O stretching), 1642 cm−1 (aromatic C C
of the ␬-Car gelation, which suggests that the suitability of this
stretching), 2937 cm−1 (C H stretching), 1254 cm−1 (C H bending)
biopolymer for biomedical applications, including as a delivery
and 1279 cm−1 (C O stretching) (Fig. 2b) (Anand, Kunnumakkara,
vehicle for various drug molecules. Results from this study showed
Newman, & Aggarwal, 2007; Shelma & Sharma, 2013). The pres-
high loading efficiency of Cur to ␬-Car which could be explained
ence of both Cur and ␬-Car functional peaks in the FT-IR analysis
on the basis of chemical interactions (Scheme 1). The use of this
polymeric composite, especially those composed of natural poly-
mers, may be an interesting approach in drug delivery applications,
mainly because of the non-toxic and biological origin (Guo, Skinner,
Harcum, & Barnum, 1998). Overall, the incorporation of Cur into the
␬-Car matrix and the encapsulation efficiency still remains high,
and this matrix proved to be a successful material for drug delivery
applications.

3.2. Scanning electron microscopy analysis

The SEM micrograph of ␬-Car-Cur exhibited bead like morphol-

ogy and the bead structures were useful to load higher amount of
drug molecules (Fig. 1a). The Cur loaded ␬-Car SEM micrograph
also exhibited roughness on the surface of the beads, which were
accredited due to the presence of Cur (Fig. 1b). The figure revealed
the loading of Cur molecules and their dispersion on the ␬-Car
matrix (Fig. 1c).

3.3. Interaction of drug–polymer: FTIR analysis

The interactions of Curcumin with ␬-Carrageenan matrices were

analyzed using FT-IR (Fig. 2), the analysis exhibited a broad band
Fig. 3. Cumulative in vitro drug release profile of Curcumin from Cur loaded ␬-Car
centered at 3405 cm−1 corresponding to the polyhydroxy group, beads at pH – 5.0 and pH – 7.4. Data are expressed as the mean ± standard deviation
a peak at 2952 cm−1 is due to the C H stretching, a strong peak of three independent experiments. (* p ≤0.05).
 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
Fig. 4. The in vitro cytotoxicity effect of (a) ␬-Car (b) Cur and (c) Cur loaded ␬-Car
against selected A549 cells, the viability cells were studied and determined by an
MTT assay after 24, 48 and 72 h. Data are expressed as the mean ± standard error of
three independent experiments. (*p ≤ 0.05).
189
Fig. 5. (a). Analysis of ROS generation from treated cells using DCFH–DA Staining.
The cells were treated with Cur loaded ␬-Car for 7, 24 and 48 h and the treated cells
were stained for the production of intracellular ROS level, which is a characteristic
features for apoptosis induction. The DCFH-DA oxidized and the elevated DCF sig-
nal in green color was visualized under fluorescence microscopy. (b). The relative
release% of ROS from treated cells were expressed statistically as compared with
control cells in accordance with the mean fluorescence intensity. (* p ≤ 0.05). (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
indicates the conjugated composite materials of ␬-Car-Cur (Fig. 2c).
The analysis confirms the various chemical interaction between Cur
with ␬-Car into the polymer matrix. The natural polymer based
Cur activated composite materials have been successfully formu-
lated. Thus FT-IR results confirmed that the ␬-Car-Cur composite
were formulated via rapid mixing which was followed by solvent
evaporation and freeze drying techniques (Fig. 2).
3.4. Drug loading and drug release efficiency
The drug loading efficiency values of the Cur loaded ␬-Car was
determined and compared with different pH (5.0 and 7.4). The drug
loading efficiency of ␬-Car relatively depends on pH. The drug load-
ing content of 8.4% was estimated according to the procedure given
in materials and methods section and encapsulation efficiency was
73.6%, respectively. The pH-induced drug targeting properties of
Cur loaded ␬-Car have been investigated by dispersing Cur loaded
␬-Car in PBS solution at two different pH (5.0 and 7.4) conditions.
The in vitro cumulative drug release profiles of Cur loaded ␬-Car are
shown in Fig. 3. The in vitro cumulative drug release profiles indi-
cates that the presence of ␬-Car exhibits a rapid release which were
190 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193

more specific at lower pH (acidic) (Teong et al., 2015), and varies
with the pH in the following order pH 5.0 > pH 7.4, (Ye et al., 2012)
following that the rate of drug release increases with decreasing
pH (acidic). The released level of curcumin was calculated, and the
release of curcumin from this study indicates that the presence of
␬-Car may strongly influence the rate of Cur release in an acidic
environment. The Cur loaded ␬-Car system exhibits the controlled
drug release of about 78% and 43% at pH 5.0 and pH 7.4 respectively.
This reveals that the ␬-Car may act as a potential candidate to carry
drug molecules specifically on lower pH conditions of cancer cells
and their microenvironments. The Cur health benefits are strongly
limited by its reduced aqueous solubility and low oral bioavailabil-
ity (Anand et al., 2008; Bisht & Maitra, 2009). Also, the results of this
study state that the presence of ␬-Car has good cytocompatibility,
the Cur loaded ␬-Car shows a high drug loading capacity and sta-
bility which confirms that the ␬-Car might act as a potential drug
carrier to deliver anticancer drugs.
3.5. Cytotoxicity of Cur, -Car and -Car-Cur
The cell viability was observed in A549 cells with increas-
ing concentration of prepared materials. The dose response
study were performed with a different range of concentra-
tions at 50–500 ␮g mL−1 , 10–100 ␮g mL−1 , 10–100 ␮g mL−1 of
␬-Carrrageenan, Curcumin and ␬-Car-Cur for 24, 48 and 72 h
respectively. The reduction in cell viability was observed in cells
incubated with ␬-Car-Cur when compared that of free Cur, the
observed cytotoxicity for free Cur was about IC50 90, 80 and
60 ␮g mL−1 for 24, 48, 72 h respectively (Fig. 4b). As compared
to control, the cytotoxicity was induced by ␬-Car-Cur increased
with extending incubation time. The dose response effects of cells
treated with Cur loaded ␬-Car after incubation of 24, 48 and
72 h exhibited a significant IC50 values of 65, 50 and 40 ␮g mL−1
respectively, for 24, 48, 72 h (Fig. 4c). There were no significant
cytotoxicity observed even at higher concentrations treated with

Fig. 6. DAPI and Rh-123 staining analysis of A549 cells treated with Cur loaded ␬-Car at dose dependant concentrations for 7, 24 and 48 h and visualized under fluorescence
microscopy for the induction of apoptosis by changing the mitochondrial membrane potentials ( m) and nuclear integrity. The fluorescence images of green (Rh-123)
depicts the changes in  m which leads to the loss of membrane potential in A549 cells; The DAPI staining (blue color) shows the fragmented nuclear bodies and condensed
nuclear materials in treated cells compared with that of untreated (control) cells, which leading to the proposed apoptosis inductions. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
pure ␬-Car dose-dependent manner and its cytotoxicity IC50 value
were 350, 250 and 150 ␮g mL−1 24, 48, 72 h respectively (Fig. 4a).
The cytotoxicity study confirmed that the Cur did not lose its ther-
apeutic efficacy even after its conjugation with ␬-Car as it showed
a significant cell toxicity for cancer cells in 24, 48, 72 h respectively,
instead, the therapeutic values have been increased due to its effi-
cient delivery to the cells. These results indicated that Cur loaded
␬-Car have good cancer cell targeting and penetrating ability for
drug delivery in in vitro models.
3.6. Reactive oxygen species (ROS) generation
ROS have been reported to play a key role in early stage of
apoptosis detection in evidence of high level of intracellular ROS
production in apoptotic induced cells (Fulda, Gorman, Hori, &
Samali, 2010). Cur has an importance in the induction of cellu-
lar apoptosis through intracellular ROS generation (Thayyullathil,
Chathoth, Hago, Patel, & Galadari, 2008). In order to measure
this capacity of Cur loaded ␬-Car for intracellular oxidation, we
have analyzed the reactive oxygen metabolic by product H2 O2 ,
which would indicate the levels of intracellular ROS produced by
treated cells. Hence, the 2 ,7 -dichlorofluorescein-diacetate (DCF-
DA) assay was performed to study the generation of intracellular
ROS production during treatment with developed materials. The
cell permeable DCF-DA is commonly used as intracellular ROS
marker, upon passive diffusion into the cells is retained in intracel-
lular level on getting cleaved by the intracellular esterase (Sarkar
et al., 2015). Thus, it is observed a rapid accumulation of ROS
following with Cur treatment in cancer cells. When A549 cells
were treated with Cur loaded ␬-Car, the cells exhibited significant
production of ROS at 7, 24 and 48 h as compared to cell control
(untreated) (Fig. 5a and b). The results from this study are consis-
tent with previous reports of anticancer drugs which demonstrated
to induce ROS production, the report indicated that these ROS are
Scheme 2. Proposed anticancer activity and involvement of ␬-Car-Cur in apoptosis activation in lung cancer cells (A549). The released curcumin in cellular environment
targeting the cancer cell surfaces using ionic channels and it allow them to enter and induce mitochondrial membrane potential loss ( m) that finally to may lead to
activation of cellular apoptosis signaling for cell death.
191
generated as by-products of oxidative metabolism that frequently
damage cellular macromolecules such as DNA, lipids and proteins
(Schulze & Harris, 2012). Thus here it is concluded that Cur when
loaded with ␬-Car induced cell apoptosis through intracellular ROS
generation, and also revealed that this may lead to the activation of
both caspase-dependent and caspase-independent apoptotic sig-
naling pathways. Thus, the study outcomes confirm that the Cur
loaded ␬-Car has a remarkably enhanced the ability to induce
apoptosis in A549 cells as compared with that of free Cur, dur-
ing prolonged treatment, hence, the bioavailability of Cur may be
greatly improved with drug carriers for chemotherapeutic func-
tions.
3.7. Loss of mitochondrial membrane (␺m) and nuclear
damage potential analysis
The mitochondrial membrane potential ( m), fragmented
nuclear and their membrane integrity of apoptotic cells were
examined using Rh-123 and DAPI staining through fluorescence
microscopy. Upon staining with Rh-123, the loss of  m, in cancer
cells (A549) were observed in accordance to their morphological
changes after treating with Cur loaded ␬-Car at dose dependent
concentrations (IC50 ) for 7, 24 and 48 h respectively. There was
no positive staining found in the case of untreated (control) A549
cells with DAPI which was evident with no visible chromatin con-
densation (Fig. 6). However, the cells treated with IC50 of Cur
loaded ␬-Car, showed a chromatin condensation which appeared to
increase in number with the increase in time, the Cur loaded ␬-Car
treatment remarkably increased the fluorescence intensity which
was identified distinctly; this indicates the loss of mitochondrial
membrane as well as nuclear damage of the A549 cells. The mito-
chondrial membrane of treated cells showed abnormal structures,
which revealing the shredded cristae with disrupted outer mem-
brane integrity. From this, it was evidenced that the decrease/loss
192 M. Sathuvan et al. / Carbohydrate Polymers 160 (2017) 184–193
of mitochondrial membrane potential could promptly induce the
cells to release cytochrome c (Christensen, Jansen, Sanchez, &
Waterhouse, 2013). In addition to that, there have been several
studies in this area reporting that the abonormality in mitochon-
drial structures and disruption of nuclear integrity are actively
involved in the activation of cellular apoptosis-related functions
in cancer cells when exposed to recognized cancer therapeutic
agents (Hoeller & Dikic, 2009). Thus, our results give an evidence
for the inhibition of cancer cell growth by inducing apoptosis upon
treated with Cur loaded ␬-Car by means of typical morphological
characteristics such as cell shrinkage, aggregation, and spherical
micro-nucleation-condensation of chromatin, in the formation of
apoptotic bodies and nuclear fragmentations. This changes/damage
accord to clear signs of apoptosis induction in A549 cells.
4. Conclusion
To conclude this study, the transport and careful delivery of
chemotherapeutic drug to cancer cells become an important aspect
in drug delivery and development. Hence, the natural polymer like,
␬-Car has shown an enhanced ability to efficiently load and release
Cur in a well-defined medium for controlled release, which pro-
moted the loaded molecules to reach the desired cellular sites.
The controlled release of Cur was accomplished at pH 5.0 through
in vitro studies. The cytotoxicity of the Cur loaded ␬-Car had a sig-
nificantly high apoptotic activity in selected lung cancer cells of
A549 (Scheme 2). On the whole, from the investigation it is sug-
gested that the Cur loaded ␬-Car could be a potential candidate
for developing polymer based natural delivery vehicles with an
improved therapeutic function for pharmaceutical applications. In
fact, all these observations of this study revealed that the ␬-Car
might be an innovative and adequate alternate molecule for the
development of novel drug carrier systems to encapsulate drugs or
other bioactive agents into them against multiple health disorders.
The further research on this molecule will definitely explore more
valuable functions of this polymer in biomedical applications.
Acknowledgment
The author (R.T) acknowledges DST-SERB, Government
of India for the Postdoctoral Research Fellowship (Ref. No.
PDF/2016/000086).
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