Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

SYNTHESIS
AND
CHARACTERIZATION
OF
PECTIN
CAPPED
SILVER
NANOPARTICLES AND EXPLORATION OF ITS ANTICANCER POTENTIALS IN
EXPERIMENTAL CARCINOGENESIS IN VITRO
Baisakhi Moharana1, Preetha SP1, Selvasubramanian S1, Malathi S2 and Balasubramanian S2
1

Department of Veterinary Pharmacology & Toxicology, Madras Veterinary College, Chennai.

Department of Inorganic Chemistry, Guindy Campus, University of Madras, Chennai.

ARTICLE INFO
Article history
Received 27/11/2014
Available online
03/12/2014

Keywords
Fractionated Pectin Powder
(FPP),
Silver Nanoparticles
(Ag NPs),
FPP Capped Ag NPs,
Ehrlich Ascites Carcinoma
(EAC).

ABSTRACT
Nanotechnology holds promise for superior pharmacological effects in terms of targeted
delivery and small size but still many aspects of its pharmacological effects still has to be
uncovered for maximum utilization of this technology. Co-chemotherapy using
nanotechnology can be a better option to fully exploit the advantages of drugs in clinical
cases of cancer patients. Green synthesis of silver nanoparticle was carried out using chitosan
as capping agent with ascorbic acid as the reducing agent. Fractionated pectin powder (FPP) a
well known antimetastatic agent and biologically synthesized silver nanoparticles (Ag NPs)
were used in this study as a nanocomposite i.e., FPP capped Ag NPs. FPP capped Ag NPs
were characterized by TEM, FTIR, XRD, DLS and zeta potential to determine their size and
stability. TEM analysis revealed that FPP capped Ag NPs had size ranged between 35-45nm
with zeta potential -47 mV. The present study explored the positive role of FPP capped Ag
NPs as an antitumor agent using Ehrlich ascites carcinoma (EAC) cell line in vitro. FPP
capped Ag NPs caused a significant (1.8 fold) reduction in the dose of the nanocomposite in
attaining the IC50 value. Hence FPP capped Ag NPs proved to be a better candidate for cancer
chemotherapy.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Baisakhi Moharana et al. Synthesis and characterization of pectin capped silver nanoparticles
and exploration of its anticancer potentials in experimental carcinogenesis In vitro. Indo American Journal of Pharm
Research.2014:4(11).

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Corresponding author
Baisakhi Moharana
Department of Veterinary Pharmacology & Toxicology,
Madras Veterinary College, Vepery,
Chennai,Tamilnadu, India.
Pin-600007
+91-9498035048
baisakhimoharana@gmail.com

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INTRODUCTION
Non-selective binding, poor pharmacokinetic properties and side effects are the major limitations of conventional
chemotherapy. Nanotechnology holds promise for medication and nutrition because materials at nano meter dimension exhibit
properties different from those of both isolated atom and bulk material [1]. Cancer cells survive and multiply due to the decreased
intracellular concentration of conventional anti-cancer drugs [2]. Nanoparticles if properly designed, can cross physiologic barriers
due to their small size, delivering drugs in normally inaccessible sites with classical means [3]. Recently silver nanoparticle has
attracted the attention of scientists across the globe for its potent antimicrobial as well as anticancer properties. Green synthesis of
silver nanoparticles is considered as one of the cost effective method for synthesis of silver nanoparticles. There are several reports
regarding the green synthesis of silver nanoparticle from plant extracts [4], microbial sources [5] and biopolymers like chitosan as
reducing and stabilizing agents [6]. Silver nanoparticles have proven cytotoxic effects against HeLa cells [7] and MCF-7 cells [8].
Natural products have shown great promise in mitigating carcinogenesis and associated cellular aberrations, with substantial
numbers of anticancer agents being derived from natural sources [9]. Pectin is a natural plant polysaccharide with mounting reports
documenting its anti-tumour efficacy. Due to the limited bioavailability and non-degradable nature of plant-pectin, scientists have
developed modified citrus pectin (MCP) and fractionated pectin powder (FPP) with better anti-cancer efficacy. FPP was reported to
induce significant apoptotic activity than its counterpart MCP with its antitumour activities being mediated by the inhibition of Gal-3
and Gal-3 mediated interactions [10]. Tehranian et al. [11] reported cytotoxic effects of PectaSol in DU145 cells and LNCaP cells.
Jackson et al. [12] reported that FPP induced significant apoptosis compared to other pectin-like compounds in LNCaP cells.
Co-chemotherapy has attracted the focus of research not only due to its high potency and reduced toxicity [13], but it can
address simultaneously several receptors involved in the complicated carcinogenesis machinery. As the world battles with the growing
incidence of carcinogenesis, the search for novel chemo preventive agents to selectively target the tumour cells with negligible
toxicity to the host cells constitute an urgent priority.
The synthesis of Ag NPs and its encapsulation with fractionated pectin powder (FPP) has been reported in the present study.
The formulation has undergone specific characterization protocols to confirm its nano-nature. This study is designed to explore the
possible synergistic potential of the nano-composite i.e., FPP capped Ag NPs and formulation and its effect on the cancer cells.
MATERIALS AND METHODS
Chemicals and materials
Fractionated pectin powder (FPP) was obtained from Thorne Research, Dover, U.S.A. Silver nitrate, chitosan, ascorbic acid,
PBS were obtained from Sigma-Aldrich. MTT powder was obtained from Himedia. All other chemicals and solvents used were of
analytical grade.
Preparation of pectin capped nanoparticle
Green Synthesis of silver nanoparticles (Ag NPs)
Silver nitrate (AgNO3 - 10 mg) was added separately to previously prepared chitosan solution (1mg/ml) in 1% acetic acid.
The mixture was stirred at room temperature for one hour to make it homogenous. Then the temperature was increased to 90 C for 5
minutes, followed by addition of ascorbic acid (1mg/ml) in drops to the solution. This led to the immediate formation of Ag NPs
visualized as yellowish-red color solution. Then the solution was purified by repeated washing with deionized water. Concentrations
of Ag NPs were determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) (IIT, Madras). Then Ag NPs
were immediately characterized by UV-Visible spectroscopy.
Preparation of FPP capped Ag NPs
To 10 ml of the aqueous solution containing 5 mg Ag NPs, 50 mg FPP was added and sonicated for 5 minutes. Then the
mixture was stirred vigorously for 1 hour at room temperature. The product obtained was purified by centrifugation followed by
repeated washing with deionized water and then freeze dried.
Characterization of nanoparticles
The size, morphology and stability of the synthesized Ag NPs and FPP capped Ag NPs were characterized using the
following techniques.

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Transmission electron microscopy (TEM)

Approximately 15-20l of Ag NPs and FPP capped Ag NPs suspension were loaded on carbon-coated copper grids and
solvent was allowed to evaporate by incubation at 37C for 30 min in an incubator. The size and morphology of the Ag NPs were
estimated by TEM (Tecnai-10, Phillips-Neitherland).

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UV Visible Spectroscopy
For the preliminary determination of Ag NPs, UV visible spectroscopy was performed to measure the Surface Plasmon
Resonance (SPR) for the wave length ranging from 300-600 nm.

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Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of the freeze dried samples were recorded in KBr pellets using an FTIR spectrophotometer in the range of 4004000 cm-1 to identify the possible molecules responsible for the reduction of silver ions and to confirm FPP capped Ag NPs.
X-Ray diffraction (XRD)
XRD study of Ag NPs and FPP capped Ag NPs were done to determine the structural characterization of the nanoparticles by
using X-ray diffractometer. The intensity of the diffracted X- rays was measured as a function of the diffracted angle 2 and the
specimens crystalline phases and orientation.
Zeta potential measurement
The suspension of Ag NPs and FPP capped Ag NPs were diluted and the zeta potential was measured in Zetasizer Nano ZS.
Dynamic light scattering measurement (DLS)
DLS (Dynamic light scattering) analyzes the velocity distribution of particle movement by measuring dynamic fluctuations
of light-scattering intensity caused by the brownian motion of the particle and this technique yields a hydrodynamic radius or
diameter, which is calculated using the Stokes-Einstein equation. Dynamic light-scattering measurements (DLS) were performed for
analyzing the size groups of nanoparticles using a Nano ZS apparatus in aqueous solution.
Entrapment efficiency (EE)
The EE was determined by analyzing the nano particles spectrophotometrically. The amount of FPP capped Ag NPs was
estimated by dispersing a known amount of nanoparticles in 10 ml deionized water and stirring the sample vigorously. The absorbance
of the solution was measured at 230 nm and amount of FPP present was calculated from a previously drawn calibration curve of
concentration vs absorbance, with different known concentrations of FPP. The entrapment efficiency was calculated using the formula
given below

% EE

Amount of FPP present in FPP capped Ag NPs

------------------------------------------------------Amount of FPP used

x 100

IN VITRO STUDY
Cell line
Ehrlich Ascites Carcinoma (EAC) cell line was obtained from Amala Cancer Research Institute, Thrissur, Kerala,
India.
In vitro maintenance of EAC
The aseptically collected ascitic cells were washed with PBS and were incubated in Dulbecco's modified eagle's medium
(DMEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 g/ml) at 37C and 5% CO 2 atmosphere.

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RESULTS AND DISCUSSION

Characterization of nanoparticles
UV-Visible spectroscopy analysis
Optical properties of Ag NPs were determined by the excitation of plasmon resonances. Figure 1 illustrates the typical UVVis spectrum of Ag NPs. The UV-Vis absorption spectrum showed an intense absorption peak at 430 nm, originating from the surface
plasmon absorption of nano-sized silver particles.The absorption maxima of Ag NPs were reported in the range of 400500 nm [14].
max of Ag NPs was found to be 430 nm which is in accordance with a previous report by Zonooz and Salouti [15].

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MTT Assay
The 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide dye reduction assay was performed to determine the
cytotoxic effect of the individual drugs at 10 different concentrations. The basic principle is based on the reduction of MTT by
mitochondrial dehydrogenase, an enzyme present in the mitochondria of viable cells, to form a blue formazan product. Briefly, the
EAC cells were freshly harvested from EAC-bearing mice and the cell concentration was adjusted to 1x10 5 cells/ml and plated onto 96
well flat bottom culture plates with various concentration of FPP, Ag NPs and FPP capped Ag NPs. All cultures were incubated for 24
hours at 37 c in a humdified CO2 incubator. After 24 hours of incubation (37 c, 5% CO2 in a humid atmosphere), 20l of MTT
(5mg/ml in PBS) was added to each well and the plate was incubated for a further 4 hours at 37 c. The resulting formazan was
dissolved using dimethyl sulfoxide and absorbance of the solution was read at 570nm using a microplate reader. All determination was
carried out in duplicate. Concentrations of drugs showing 50% reduction in cell viability (i.e., IC 50 values i.e., 50% inhibitory
concentration) was calculated.

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FIG. 1 UV -Visible spectrum of silver nanoparticle

Transmission Electron Microscopy (TEM)
TEM images of FPP, Ag NPs and FPP capped Ag NPs are displayed in Figure 2 (A, B, C respectively). The size of FPP
capped Ag NPs ranges from 35-45 nm, whereas FPP size ranges from 15-20 nm and Ag NPs size ranges from 20-25 nm. The surface
morphology of FPP, Ag NPs, FPP capped Ag NPs are almost spherical in shape and well dispersed without any agglomeration. FPP
capped Ag NPs had comparatively larger size (35-45nm) than the individual components (FPP size ranges from 15-20 nm and Ag
NPs size ranges from 20-25 nm).

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Fourier Transform Infrared Spectroscopy (FTIR)

Figure 3 shows the FTIR spectra of FPP, Ag NPs and FPP capped Ag NPs. FPP has characteristic absorption bands at 3436
cm-1, 1602 cm-1, 1404 cm-1, 1104 cm-1, 1009 cm-1, 777 cm-1 and 640 cm-1. Ag NPs has absorption bands at 3436 cm-1, 2325 cm-1, 1629
cm-1, 1383 cm-1, 1063 cm-1 and 599 cm-1. FPP capped Ag NPs has absorption bands at 3423 cm-1, 2175 cm-1 , 1602 cm-1, 1411 cm-1,
1022 cm-1, 1097 cm-1, 780 cm-1 and 626 cm-1. The IR spectrum of FPP showed characteristic absorption bands at wave number 3436
cm-1 that belongs to OH stretching of pectin. Absorption bands at 1602 cm-1 indicates carbonyl stretching frequency, 1404 cm-1 is that
of C-O-H stretching frequency, 1104 cm-1 denotes C-C bond stretching frequency, absorption band at 1004 cm-1 is that of COOH
dimer stretching frequency, band at 770 cm-1 is associated with carboxylic acid C-OH stretching frequency and absorption band at 640
cm-1 belongs to low frequency vibration of pyranoid ring. Ag NPs do not have IR spectra and the spectral peaks that were obtained
belonged to the capping agent that had absorption bands at 3436 cm-1 indicating OH stretching frequency of the capping agent i.e.
chitosan, band at 2325 cm-1 is associated with CH stretching frequency of chitosan, carbonyl group of capping agent is represented at
1629 cm-1, absorption at 1383 cm-1 corresponds to C-H bending and 1063 cm-1 indicates C-O stretching of the capping agent,
absorption at 599 cm-1 is assigned to C-O-C bending frequency of the capping agent. FPP capped Ag NPs had absorption bands at
3423cm-1, 2175cm-1, 1602 cm-1, 1411 cm-1, 1022 cm-1, 1097 cm-1, 780 cm-1 and 626 cm-1. The spectra showed that majority of the
peaks belong to the pectin organic groups. The spectral results clearly demonstrate that there were shifts in the original peaks of FPP

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FIG. 2 Typical TEM images of the FPP, Ag NPs and FPP capped Ag NPs

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indicating the presence of biomolecules other than FPP in its structure. Hence, it was obvious that the Ag NPs with its capping agent
attributed to the altered IR-spectrum in FPP capped Ag NPs as against FPP alone.

FIG. 3 FTIR spectrum of FPP, Ag NPs, and FPP capped Ag NPs

X-Ray Diffraction (XRD)
The crystal structure of Ag NPs and FPP capped Ag NPs was further characterized by XRD analysis. The XRD patterns for
the synthesized Ag NPs and FPP capped Ag NPs are reproduced in the Figure 4. The four peaks appear at 2 = 37.97, 44.11, 64.35
and 77.27 that corresponds to (111), (200), (220) and (311) planes, respectively, of the face centered cubic Ag NPs. The XRD
patterns for the synthesized nanoparticles are in good agreement with reference of FCC structure from joint committee of powder
diffraction standard (JCPDS) - Card No-087-0720. In FPP capped Ag NPs, XRD pattern showed the presence of the peak associated
with Ag NPs along with peaks for FPP indicating maintenance of integrity of the crystalline structure of nanoparticles after formation
of nanocomposite.

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Zeta Potential
The zeta potential of Ag NPs, FPP and FPP capped Ag NPs were found to be -23 mV, - 40 mV and -47 mV, respectively. As
far as zeta potential is concern, the particles in suspension have a large negative or positive zeta potential, then they will tend to repel
each other and there will be fewer tendencies for the particles to come together and aggregate. The particles in suspension with zeta
potentials more positive than +30mV or more negative than -30mV are considered stable [16]. However, if the particles in suspension
have low zeta potential values, then there will be no force to prevent the particles coming together and aggregating [17]. Kumar et al.
[18] reported that the zeta potential value of Ag NPs was -26.2 mV, which was found to be highly stable. In the present study, the zeta
potential of Ag NPs seems to be fairly stable. The zeta potential of FPP capped Ag NPs indicates the highly stable nature of the

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FIG. 4 XRD pattern for the synthesized Ag NPs, and FPP capped Ag NPs

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capped formulation. The higher negative value is indicative of greater repulsion among particles which prevents aggregation in the
solution.
Dynamic light scattering measurement (DLS)
The particle size of the synthesized Ag NPs and FPP capped Ag NPs were determined using DLS technique. DLS size range
of Ag NPs and FPP capped Ag NPs were found to be 85 5 nm and 97 3 nm, respectively. DLS size ranges of Ag NPs and FPP
capped Ag NPs were found to be greater than TEM size. This might be due to the fact that DLS measures hydrodynamic diameter of
nanoparticles, where the amphiphilic nanoparticles were surrounded by water molecules [19]. Moreover, high swelling properties of
pectin in aqueous medium [20] may be attributed to be the cause of large size of capped formulation.
Entrapment efficiency (EE)
The calibration curve (Figure 5) shows concentration versus absorbance curve for FPP. Based on the calibration curve, EE of
FPP capped Ag NPs was found to be 86.4% (FPP: Ag NPs :: 10:1) which is used for further studies. Entrapment efficiency refers to
amount of drug binding to form the nanocomposite. Higher the efficiency, higher will be the binding of the drugs with less wastage in
the supernatant while preparation of the nanocomposite. The entrapment efficiency, in this study was found to be 86.4% which
indicate a better drug loading.

FIG. 5 Calibration curve for FPP concentration.

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In the present study IC50 values against EAC cell line was found to be 1600 g/ml, 260 g/ml and 890 g/ml by FPP, Ag
NPs, FPP capped Ag NPs respectively. Recently, Manivasagan [7] reported that the IC50 value of biosynthesized Ag NPs against
HeLa cells was at 200 g/mL concentration. Ranjitham et al. [8] showed that Ag NPs with an IC50 value of 190.501g/ml
significantly inhibited MCF-7 cells proliferation. Tehranian et al., [11] reported IC 50 value of PectaSol of 3mg/ml and 4 mg/ml in
DU145 cells and LNCaP cells respectively. In a recent report by Wang et al. [21] the inhibition rate of apple pomace pectin (APP)
was lower than citrus peel pectin (CPP) and the IC50 values of APP was higher than 10 mg/mL. The treatment of LNCaP cells with
increased concentrations of FPP showed that 3 mg/mL FPP induced maximum apoptosis. Lower concentrations of 0.01 mg/mL and
0.10 mg/mL of FPP did not affect LNCaP cells, whereas 1.0 mg/mL induced significant apoptosis [12]. Combination effect of
PectaSol and Doxorubicin showed that the IC50 values decreased 1.5-fold and 1.3-fold in the DU-145 and LNCaP cells respectively
[11]. In accordance with the earlier reports, FPP capped Ag NPs caused a significant (1.8 fold) reduction in the dose of the
nanocomposite in attaining the IC50 value, which shows that the nanocomposite has better efficacy than its individual counterparts.
The mechanism behind the potentiated anti-tumour effect of FPP capped Ag NPs may be attributed to the synergistic effect of FPP
and Ag NPs on the carcinoma cells. The nano-size of the formulation might have enhanced its effective permeability in to the cancer
cells. The enhanced cytotoxic effect of the capped formulation may be attributed to the destruction of the mitochondrial membrane
and the release of apoptogenic molecules. Further studies are needed to evaluate the exact underlying mechanism of the anticancer
properties of nano-composite formulation.

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IN VITRO CYTOTOXICITY ASSAY

The preliminary tumour cytotoxic activities of FPP, Ag NPs and FPP capped Ag NPs were evaluated against EAC cells at
different concentrations using MTT assay. The treatments demonstrated a considerable cytotoxicity against the EAC cells. The results
showed that EAC cells proliferation was significantly inhibited by FPP, Ag NPs and FPP capped Ag NPs with IC50 values mentioned
below.
Treatments
Concentration (IC 50)
FPP
1600 g/ml
Ag NPs
260 g/ml
FPP capped Ag NPs
890 g/ml

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CONCLUSION
From the present study, the beneficial role of co-chemotherapy and nanopharmacology in carcinogenesis is established. FPP
capped Ag NPs prove to be a valuable candidate in cancer chemotherapy at an optimal, cost effective dose. Further rigorous studies
are needed to elucidate the concrete mechanisms of antitumour activities of Ag-FPP nano formulation underlying carcinogenesis
which will be a useful contribution to the discipline of Nanopharmacology.
ACKNOWLEDGEMENT
The first author sincerely acknowledges the expertise rendered by Dr.S.Ramesh, Associate Professor and Head, Centralised
Instrumentation Laboratory, Madras Veterinary College, Chennai in conducting TEM analysis of the nanoparticles.
Competing Interests
The authors declare no conflict of interests.

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