Carbohydrate Polymers 148 (2016) 206215

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

Non toxic, antibacterial, biodegradable hydrogels with pH-stimuli

sensitivity: Investigation of swelling parameters
S. Sudarsan a,b , D.S. Franklin b , M. Sakthivel c , S. Guhanathan d,
a

Department of Chemistry, Periyar University, Salem 636011, Tamilnadu, India

Department of Chemistry, C. Abdul Hakeem College of Engineering and Technology, Melvisharam 632509, Tamilnadu, India
Research and Development centre, Bharathiar University, Coimbatore 641046, Tamilnadu, India
d
PG & Research Department of Chemistry, Muthurangam Government Arts College (Autonomous), Vellore 632002, Tamilnadu, India
b
c

a r t i c l e

i n f o

Article history:
Received 23 January 2016
Received in revised form 12 April 2016
Accepted 13 April 2016
Available online 3 May 2016
Keywords:
Biodegradation
Swelling studies
Sodium alginate
Cytotoxicity
pH-sensitive

a b s t r a c t
In this work, a series of pH-sensitive hydrogels were synthesized from Sodium alginate (SA), Ethylene
glycol (EG) and Acrylic acid (AA). Biodegradability of hydrogel was tested against soil burial test for
35 days and in vitro phosphate buffer solution test for 10 days respectively. Degradation of the sample
might be due to the breakdown of ester linkage and hydrophilic pendant functionality present in hydrogel.
The progression of biodegradation was examined by Scanning electron microscopy (SEM) and Fourier
transform infrared spectroscopy (FT-IR). Detailed swelling parameters such as swelling equilibrium Seq
(%) at various pH, biological uids (distilled water (DW), physiological saline 0.89% NaCl (PS), iso-osmotic
phosphate buffer at pH 7.4 (PB)) and equilibrium water content (EWC) have also been investigated, which
revealed that dynamic compassion of hydrogels. The hydrogel has shown strong antibacterial activity
against Escherichia coli (gram negative) and Staphylococcus aureus (gram positive) bacterias. Cytotoxic
assays, using MTT Assay in 3T3 broblast Cell line was performed. At 10 g/ml, cell viability was in
the range of 9294%. However, the cell viability (%) decreases with increasing concentration of sample.
The synergistic effect of biodegradable hydrogels possessing excellent swelling properties, high water
content, biocompatibility and wound healing tendency using in vivo test can be made as suitable candidate
for biomedical applications. In vivo wound healing studies conducted on a Wister albino rat model of
incision wound performed for 9 days. The results revealed that more accelerated wound healing have
been observed even in shorter duration. Thus, the synthesized hydrogel with great pH-responsiveness
and excellent drug delivery may have a great opening for biomedical applications.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Hydrogels are three dimensional macromolecular hydrophilic
cross-linked networks and imbibing large amount of water or
biological uids (Franklin & Guhanathan, 2014a, 2015a, 2015b).
Hydrogel consists of ionizable acidic or basic pendant groups such
as carboxylic acid, sulfonic acid or amine groups are responsible for
water retaining ability (Kevadiya, Joshi, Mody, & Bajaj, 2011). pHsensitive hydrogels are either accept or release proton as a result
of changing the external pH caused the phenomenon is known as
swelling (Qiu & Park, 2001; Lin, Zhou, Yingde, & Gunasekaran, 2009;
Sakthivel, Franklin, & Guhanathan, 2015). The swelling parameter
is a unique property for hydrogel. Hydrogel is a suitable material

Corresponding author.
E-mail addresses: srsudarsan29@gmail.com (S. Sudarsan),
sai gugan@yahoo.com (S. Guhanathan).
http://dx.doi.org/10.1016/j.carbpol.2016.04.060
0144-8617/ 2016 Elsevier Ltd. All rights reserved.

for biomedical applications due to its low toxicity, biodegradability and biocompatibility properties (Venkatesan, Bhatnagar,
Panchanathan, Kang, & Kipn, 2015; Venkatesan, Babour et al., 2015;
Lee & Mooney, 2011). The diffusion and swelling behavior of hydrogel was affected by the nature of hydrophilic group present in
hydrogel (Peppas, Bures, Leobandung, & Ichikawa, 2000; Franklin
& Guhanathan, 2013; Omidian & Ki, 2008).
Sodium alginate based biopolymeric hydrogels have been used
extensively in medical, environmental and biological applications due to their renewable origin, non toxicity, biocompatibility,
biodegradability and pH-sensitivity (Yoo, Song, Chang, & Lee, 2006;
Saether, Holme, Maurstad, Smidsrd, & Stokke, 2008; Yao, Ni,
Xiong, Zhu, & Huay, 2010; Kumar Gri et al., 2012; Venkatesan,
Babouret al., 2015; Horkay, Tasaki, & Basser, 2000; Venkatesan,
Babouret al., 2015; Cai, Ni, & Zhang, 2012; Sudarsan, Franklin, &
Guhanathan, 2015a; Sudarsan, Franklin, & Guhanathan, 2015b).
Ethylene glycol (EG) was chosen as a di-functional monomer to

S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

improve the properties of hydrogels because of its exibility and

biocompatibility (Savas & Guven, 2001; Chaturvedi et al., 2013;
Franklin & Guhanathan, 2014e; Risbud & Bhonde, 2000). Acrylic
acid (AA) based polymeric hydrogels were also used to fabricate
pH sensitive hydrogels (Nazar, Jahanzeb, & Sajid, 2011; Franklin &
Guhanathan, 2014f, 2014c, 2014d, 2015b, 2015a). Acrylic acid has
a tendency to make interactions with solvent and enhanced the
swelling behavior of hydrogels, particularly in drug delivery applications (Theil & Maurer, 1999; Nazar & Umrreen, 2014). The usage
of organic hazardous cross linker and organic solvents in the synthesis of hydrogels causes serious problems to the human being and
environment. (Franklin & Guhanathan, 2015). Therefore, hydrogels
with crosslinkers has limited its applications towards biomedical
eld.
Wound healing is the gradual restoration of injured tissues,
which reimbursement the dermis of the repaired tissue (Catanzano
et al., 2015). It is an injury in which the outer surface of tissue is torn,
broken or cut. However, the synthetic wound dressing materials
have acquired some drawback such as insufcient swelling ability,
water retention capacity etc. To overcome such setbacks, hydrogels
have chosen as an effective biomaterial due to higher bioadhesive
and wound healing properties. Hydrogels based wound dressing
would be helpful to provide a better healing effect on many types
of wounds (Jin et al., 2016).
Based on the careful analysis of literature, the efforts have been
taken towards the synthesis of sodium alginate based pH sensitive
biodegradable hydrogel with non-toxic nature to human being as
well as to the environment. In addition, healing efcacy on wound
using in vivo incision wound type have also been observed on Wister
albino rats model with morphological changes.
2. Experimental
2.1. Materials
Sodium
alginate
(SA)
(Mannuroate/Guluronate
ratio = 1.56,Viscous average M.Wt: 200,000) was purchased
from Sigma-Aldrich Company (Bangalore, India). Ethylene glycol
(EG) and Acrylic acid (AA), Na2 CO3 and NaHCO3 were purchased
from Merck, Pvt. Ltd., India.
2.2. Methods
2.2.1. Synthesis of polyelectrolyte SEA biopolymeric hydrogels
Synthesis of biopolymeric SEA hydrogel was the same as
described in our earlier communication (Sudarsan, Franklin, &
Guhanathan, 2015a; Sudarsan, Franklin, & Guhanathan, 2015b). In
brief, two stage polymerization has been done through condensation followed by free radical polymerization as follows,
Stage-I: synthesis of pre-polymer via condensation polymerization.
The pre-polymer was synthesized by condensation polymerization
among sodium alginate (SA) and ethylene glycol (EG) in nitrogen
atmosphere at 80 C for one hour with constant stirring resulted in
a glassy like pre-polymer chain.
2.2.1.2. Stage-II: synthesis of hydrogels via free radical polymerization. Further, acrylic acid (AA) and stoichiometric amount of
K2 S2 O8 initiator were also added to co-polymer at 80 C with constant stirring for 3 h in an inert nitrogen atmosphere. The formation
of yellowish glassy solid revealed the sodium alginate-ethylene
glycol-acrylic acid (SEA) hydrogel was obtained. The hydrogel was
allowed in distilled water for 2 days to expel out the unreacted
monomers. The hydrogel was dried at room temperature and stored

207

in sealed containers for further use. The general mechanism of the

formation of biopolymeric hydrogel have depicted in Fig. 1.
2.3. FT-IR spectroscopy analysis
The FT-IR spectra of SEA hydrogel was recorded on a FT-IR spectrophotometer 8400 S. Shimadzu spectrophotometer. The spectra
were recorded between 4000 and 400 cm1 at resolution of 2 cm1 .
2.4. Scanning electron microscopy (SEM) analysis
The SEM photographs were executed with Hitachi SU6600
variable pressure Field Emission Scanning Electron Microscope
(FESEM). The samples were mounted on the base plate and gold
amalgam-sputter coating to render them electrically conductive.
The scanning was synchronized with a microscopic beam to preserve the triing size over a large distance relative to the specimen.
2.5. Swelling studies
2.5.1. In vitro swelling studies of hydrogels in physiological body
uids
In vitro swelling studies conducted in simulated physiological
body uids viz., distilled water (DW), physiological saline 0.89%
NaCl (PS), iso osmotic phosphate buffer at pH 7.4 (PB), articial
urine (UR) and 1% glucose (GL) (CRC Hand book). To ensure the
complete equilibrium of the hydrogel was allowed to swell for
24 h. The equilibrium swelling (Seq %) of sample was calculated
by gravimetrically at different physiological medium using Eq. (1)
(Saradyin, 2002; Franklin & Guhanathan, 2014b; Sakthivel,Franklin,
& Guhanathan, 2015).
Seq % =

Weq Wd
100
Wd

(1)

where Weq is the mass of the swollen gel at equilibrium and Wd is

the mass of the dry gel.
2.5.2. Equilibrium water content
Higher water content of hydrogel at equilibrium is one of the
noteworthy properties due to increasing permeability and biocompatibility (Chen &Tan, 2006; Lokesh, Krishna Rao, Mallikarjuna
Reddy, Chowdoji Rao, Srinivasa Rao, 2008; Wang, Zhang, & Wang,
2009; Saraydn & Yasemin., 2010). The water absorbed by the gel
is quantitatively represented by the equilibrium water content
(EWC).
EWC% =

Meq Md
100
Meq

(2)

where Md is the dry weight of the hydrogel and Meq is the hydrated
weight of the hydrogel
2.6. Biodegradation studies
2.6.1. Soil burial test
The biodegradability test was observed in agricultural soil at
normal condition. The hydrogel (1 g by weight) was buried in soil at
a depth of 3 cm. The sample was removed from the soil at a regular
interval of 7 days. The sample was washed with distilled water and
dried in vacuum oven at 40 C until a constant weight was reached.
The degradation percentage was calculated using the following Eq.
(3) (Kasma et al., 2014).
Biodegradation (%) =

Wi Wf
100
Wi

(3)

where Wi is the initial weight of the hydrogel and Wf nal weight

after 7 days of soil burial.

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S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

Fig. 1. General formation and degradation of biopolymeric hydrogel.

2.6.2. In vitro phosphate buffer solution test

The in vitro degradation of the hydrogels was performed in PBS
solutions of acidic, neutral and alkaline pH media (pH 4.0, pH 7.4
and pH 10.0) as per the reported procedure (Yuan-Mei, Ping, YuMin, & Ai-qin, 2014; Zhao, Li, Guo, & Ma, 2015).
2.7. Antibacterial assessment
The bacterial cultures were sub-cultured periodically and maintained on nutrient agar (NA) medium at room temperature

(30 2 C) for further experiments. The antibacterial activity has

found modied agar well diffusion method. MullerHinton agars
were sterilized, poured on petriplates and allowed to solidify under
laminar airow. About 100 l [108 CFU/ml (colony forming units)]
of each bacterial culture was spread on the agar surface using sterile glass spreader. Wells of 10 mm diameter was made using sterile
cork borer on the agar media. About 100 l (0.0250.25 mg) of the
study material were added to the well and they were kept in refrigerator for 20 min for diffusion. Then, the plates were incubated for

S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

209

Fig. 2. Degradation of SEA (a) percentage weight loss (%) of SEA hydrogels in soil biodegradation from 7, 14, 21, and 30 days. (b) Weight loss (%) of SEA hydrogels in phosphate
buffer solution at pH 4.0, pH 7.4, and pH 10.0. (c) Comparative FT-IR spectra of SEA and their biodegradation at 7, 14, 21 and 30 days (soil burial test) (d) comparative FT-IR
spectra of SEA with degradation at pH 4.0, pH 7.4, pH 10 phosphate buffer solution test.

24 h at 37 C. Ethanol was used as a control. The antibacterial activity was evaluated by measuring the diameter of zone of inhibition
against the test organisms.

2.8. Minimum inhibitory concentration (MIC)

Hydrogel sample was dissolved in 10% DMSO. The initial concentration of hydrogel was 1 mg ml1 and end with 0.06 mg/ml

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S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

Fig. 3. SEM images of (a and b) SEA hydrogels (c and d) SEA hydrogels after soil burial test (e and f) in vitro degradation at pH 4.0 of SEA hydrogels (g and h) in vitro degradation
at pH 7.4 of SEA hydrogels.

concentration. The initial test concentration was serially diluted by

two-fold. Each well was inoculated with 5 l of suspension containing 108 colony-forming units CFU ml1 of bacteria. The antibacterial
agents were incubated for 24 h at 37 C for bacteria. The MIC of
hydrogel was determined as the lowest concentration of the hydrogel inhibiting the visual growth of the test cultures. After 24 h
incubation at 37 C, the microbial zone of inhibition was measured
using optical density (OD).
2.9. In vivo wound healing studies
Incision method have adopted for wound healing effects with
Wister albino rats of 150180 g of either sex. They were housed
individually in standard laboratory environment for 9 days of
period, fed with commercial pellets and water ad libitum. For inci-

sion wound model, animals were divided into three groups in

each model consisting of three animals as follows: (Group I-Only
wound, Group II-Silver sulfadiazine (0.01%) cream was used as standard, Group III-2% hydrogel Ointment Base). In incision wound
model, 6 cm long paravertebral incision were made through the
full thickness of the skin on either side of the vertebral column
of the rats, after all the animals of each group were anesthetized
under light ether anesthesia (Ehrlich & Hunt, 1968). No local or
systemic antimicrobials were used throughout the experiment.
The both edges kept together and stitched with Black silk surgical
thread and a curved needle was used for stitching. The continuous
threads on both wound edges were tightened for good closure of
the wound. After stitching, wound was left undressed then without ointment base, standard ointment and extracts ointment (SEA
hydrogels) were applied daily until 9 days; when the wounds cured,

S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

211

Fig. 4. (a) Equilibrium uid content (EFC%) of SEA hydrogels in DW, IP, UR, PS and GL. (b) Equilibrium water content (EWC%) of hydrogels at different pH.

the sutures were removed on the day 9 (Hemalata, Subramanian,

Ravichandran, & Chinnaswamy, 2001; Kuwano et al., 1994).
2.10. Cytotoxicity studies
The
MTT
(3-(4,
5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) tetrazolium reduction assay
was the rst homogeneous cell viability assay developed for a
96-well format that was suitable for high throughput screening
(HTS). The MTT substrate is prepared in a physiologically balanced
solution, added to cells in culture, usually at a nal concentration
of 0.20.5 mg/ml, and incubated for 14 h. The quantity of formazan (presumably directly proportional to the number of viable
cells) is measured by recording changes in absorbance at 570 nm
using a plate reading spectrophotometer. Viable cells with active

metabolism convert MTT into a purple colored formazan product

with an absorbance maximum near 570 nm. When cells die, they
lose the ability to convert MTT into formazan, thus color formation
serves as a useful and convenient marker towards the viable cells.
The exact cellular mechanism of MTT reduction into formazan
is not well understood, but likely involves reaction with NADH
or similar reducing molecules that transfer electrons to MTT.
Speculation in the early literature involving specic mitochondrial
enzymes has led to the assumption mentioned in numerous
publications that MTT is measuring mitochondrial activity. The
formazan product of the MTT tetrazolium accumulates as an
insoluble precipitate inside cells as well as being deposited near
the cell surface and in the culture medium. The formazan must be
solubilized prior to recording absorbance readings.

Fig. 5. (a) MIC studies of SEA hydrogels with E. coli and S. aureus (b) zone of inhibition of SEA hydrogels (c) cell viability of hydrogel with respect to different concentration
(d) MTT assays of SEA hydrogel.

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S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

3. Results and discussion

3.1. Biodegradation studies
The biodegradation studies of synthesized hydrogels have carried out by following methods viz., (i) soil burial test and (ii)
phosphate buffer solution (PBS) test.
3.1.1. Soil burial test
The changes in physical appearance and color of hydrogels after
soil burial test have an idea on biodegradation of the hydrogel. It
has been believed that the presence of ester bonds and active pendant groups in the hydrogels are responsible for biodegradation
(Zheng, He, Huynh, & Lee, 2010). The biodegradation of hydrogels are also depends on several factors such as pH, temperature,
mineral, nutrients and humidity in the soil. The degradation is
gradually increased on every week, and the hydrogel sample fully
degraded after 5 weeks. In addition, active functionalities enhances
the growth of micro-organism. Initially, the weight of the hydrogel was increased because of its water holding capacity from the
soil followed by reduction of weight, which has strongly been supported the biodegradation of hydrogel.
3.1.2. Phosphate buffer solution (PBS) test
In vitro degradability tests were carried out in PBS buffer (pH 4,
pH 7.4 and pH 10) at room temperature. The weight loss of the sample was monitored by every 3 h. This experiment was conducted for
10 days. The degradation percentages have been calculated using
Eq. (3) and depicted in Fig. 2(b) Initially swelling causes increase
of the weight of hydrogels and decrease of ller loading of saline
solution. At pH values greater than the hydrogel pKa, the COOH
groups of hydrogel dissociate to COO , increasing the number of
xed ionized groups within the biopolymeric hydrogel structure.
It produced electrostatic repulsion forces between the adjoining
ionized groups in the hydrophilic network. 100% of degradation
of hydrogel was observed in PBS method, due to the hydrophilic
network of the hydrogel.
3.2. Comparative FT-IR spectroscopy analysis
Fig. 2(c) illustrated the FT-IR spectral investigation on the
biodegradation of SEA hydrogels by soil burial test and PBS
test. There are four different stages of biodegradation have been
observed between 7, 14, 21 and 30 days. A characteristic strong
broad absorption band was observed at 3291.11 and 3310.05 cm1
might be due to a hydrogen bonded O H groups (Mandal, 2012).
The sharp band was found at 1738.55 cm1 indicated the ester
(C O) stretching (Chahatri et al., 2011). The C O- stretching were
also observed at 1016.64, 1157.22 cm1 (Shi, Wanga, & Wanga,
2011) which have conrmed the participation of monomers in
the polymerization. The peaks were recorded at 1593.84 and
1414.12 cm1 might be due to the asymmetric and symmetric
stretching vibration of carboxylate (COO ) ion (Zhang, Wang, &
Wang, 2007; Franklin & Guhanathan, 2014e; Karadag & Saraydin,
2002). After 7 days, the important stretching bands viz., OH
(3291.11 cm1 ), C O (1157.22 cm1 asym), (1414.12 cm1 sym),
C H (2922.42 and 2926.93 cm1 ) were completely vanished. The
unusual vanishing of peak might be due to the microbial disintegration on the hydrogels their by sacricing their functionality
Fig. 2(d). Almost all important peaks were diminished in the spectra
soon after 14, 21 and 30 days respectively. Similar to our observation Saruchi et al., 2015 have also identied for their hydrogel.
Fig. 2(c) and (d) summarized the enhanced degradation among
ascending the week on the weight loss of the SEA hydrogel using
soil burial test have been presented in Table 1.

Table 1
Biodegradation of SEA hydrogels in soil burial test.
Days

7 days

14 days

21 days

30 days

Weight loss%

20%

39%

63%

98%

Similarly, the biodegradation studies have also been carried out

for SEA hydrogels using PBS for 10 days immersed at three different
pH viz acidic (pH 4), neutral (pH 7.4) and basic (pH 10) at room
temperature. The results of FTIR assay on SEA hydrogels have also
been studied and compared for day one to 10 days. According to the
results on the degradation, the major stretching frequencies have
disappeared at basic pH. However, the results were less pronounced
in both acidic and neutral pH than basic pH. The reason for such
degradation in basic medium might be due to the anionic nature of
SEA hydrogels. Further, the deprotonation was high at more basic
medium. Hence, the enhanced degradation (Morais et al., 2013;
Papageorgiou et al., 2010), was observed in pH 10 (Fig. 2bd).
3.3. Comparative SEM morphologies studies
The morphological change of degraded hydrogels was scrutinized under SEM analysis (Fig. 3ah). The SEA parent hydrogel has
a cavities and soft smooth structure. The inner part of the hydrogel
SEA have shown in Fig 3a and b has randomly distributed aperture with an open structure (Karadag, Saraydn, & Guven, 2001;
Pathak, Yun, Lee, & Paeng, 2012). This observation was revealed
that these cracks are convenient for enhancement of water absorption. The morphology of SEA hydrogel in soil burial test after 30 days
was depicted in Fig. 3c and d. The sizably voluminous cracks were
formed because of looser hollowness/cavities became larger in
hydrogel. SEMs results clearly evidenced that the SEA hydrogel has
biodegradable in nature.
The SEM observation of biodegraded SEA hydrogel in PBS solution (pH = 4.0 and pH = 7.4) after 10 days have shown in Fig. 3e and
f. On comparison with parent SEA, at pH 4.0 showed an uneven distributed cavities and smooth morphology, whereas hollowness and
cavities have found for SEA hydrogel in pH 7.4. In basic pH the SEA
samples were completely disappeared. Hence, SEM images for SEA
hydrogel at pH 10 may not be possible to record. Biodegradability of the synthesized hydrogel was higher in the phosphate buffer
saline solution method than soil burial method. The reason for such
degradation might be due to the PBS saline environment enhances
ease of disintegradation of SEA hydrogels into small fragment than
the presence of microorganism in soil based disintegradation.
The basic relationship exists between the swelling of hydrogel
and nature of solvent. The signicance of in vitro swelling studies
found to have considerable attention in biomedical elds. Equilibrium swelling of the SEA hydrogels in various physiological body
uids (physiological saline, urea, glucose and distilled water) have
been studied and represented in Fig. 4(a). The inuencing factors of swelling equilibrium were ionic character of SA, pH and
ionic strength of the solutions. The presence of solutes in swelling
medium suppresses the swelling ratio due to decrease in osmotic
pressure of external solution.
3.4. In vitro swelling studies in biological uids
It has been found that, SEA and its series of hydrogels in the
simulated body uids were swollen in the following order as follows distilled water (DW) > iso osmotic phosphate buffer in pH 7.4
(PB) > urine (urea) (UR) > physiological saline (0.89% NaCl solution)
(PS) > glucose (GL) respectively. Seq % values of the hydrogels were
greater than the percentage of water present in the human body
(60%). Thus, the synthesized hydrogels have shown similarity to
living tissues.

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213

Fig. 6. Wound healing behavior of SEA hydrogels in comparison with standard Silver sulfadiazine (in vivo).

3.5. Equilibrium water content studies

Equilibrium Water Content (EWC) was observed for SEA hydrogels in different physiological body uids have shown in Fig. 4(c).
The EWC of SEA biopolymeric hydrogel at various pH viz., pH 4.0,
6.0, 7.4, 8.0 and 10.0 found to have 66.6, 97.4, 97.2, 95.7 and 98.1%
respectively. When the pH value greater than 7.4, the number of
COO groups increases, leading to charges repulsion among these
functional groups, instead of hydrogen bonding. So, the maximum
water content values reach at neutral and alkaline medium (Leal,
Borggraeve, Encinas, Matsuhiro, & Muller, 2013; Tianeng, Wang, &
Wang, 2011). Thus, the synthesized SEA biopolymeric hydrogels
and its series exhibit identical values with respect to uptake of
distilled water (97.7, 88.5, 89.9, 98.4, 94.1, 91.3, 95.7, 91.9, 91.6
and 98.4%). From Fig. 4 values, it has been clearly indicated that
the SEA biopolymeric hydrogel imbibed greater percent of water
content (Mandal & Ray, 2013). All EWC% values of the hydrogels
were greater than the percentage of water content present in our
body which is about 60% (Abdel Wahab, El-Naggar, Safaa, Abd Alla,
& Hossam, 2006; Rosiak et al., 1988). It could be concluded from

the above discussion that the synthesized hydrogels hold a greater

water content than human body.
3.6. Antibacterial assessment
The antibacterial activity is the most valuable property in
the eld of biomedical applications. Fig. 5(a) showed the plot
of optical density (OD) versus culture time for the SEA hydrogel
against two different bacterias viz., gram positive (Staphylococcus aureus-MTCC96) and gram negative (Escherichia coli-MTCC443)
respectively. The optical density might be a criteria of measuring the activity of the sample. The smaller OD of the medium,
the higher the values of antibacterial activity of sample. It has
been noticed that the biodegradable hydrogel exhibited the significant antibacterial activity towards both gram positive and gram
negative bacterias. The in vitro antibacterial properties of SEA
hydrogel was tested against Gram-negative E. coli and Grampositive S. aureus bacteria by modied agar well diffusion method.
The zone of inhibition around the tested samples have visualized in Fig. 5(b). The improved interfacial interaction among the
surface charges between the SEA hydrogels and bacterial sur-

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S. Sudarsan et al. / Carbohydrate Polymers 148 (2016) 206215

face might be the reason for improved antibacterial activity of

SEA hydrogel (Park & Barbul, 2004). It has been clearly visible
and more pronounced that the decrease of microbes after 24 h of
contact was observed. In the present investigation SEA hydrogel
has shown the higher zone of inhibition on gram negative bacteria (E. coli) than gram positive (S. aureus) bacteria. The results
of zone of inhibition of gram negative (E. coli) bacteria found to
have at (25 g/well = 11 mm, 50 g = 12 mm, 100 g = 13 mm and
250 g = 17 mm). However, the SEA hydrogel effectively inhibit
gram positive (S. aureus) bacteria at 250 g with 15 mm only.
According to the Standard Antibacterial test SNV 195920-1992,
specimens showing more than 1 mm microbial zone inhibition can
be considered as good antibacterial agents (Pollini Russo Licciulli
Sannino, & Maffezzoli, 2009). Based on the results, it has been
concluded that the SEA shown better antibacterial towards gram
negative bacteria than gram positive bacteria.
3.7. In vivo evaluation of the wound healing process
Hydrogels are presently used in several biomedical applications, where they acted as a three-dimensional scaffold for tissue
engineering applications. The results of wound healing experiment have illustrated in Fig. 6. After 5, and 9 days, in vivo wound
healing was performed with Wister rats and compared with standard and wound. The applied hydrogels have shown very good
accelerated wound healing performance against created wound,
compared with silver sulfadiazine (0.01%) cream as a standard. On
day 5, a signicant morphological change was observed on Wister
rat. Further, the complete wound healing with regular hair growth
has also been noticed for 9 days. An interesting observation was
made during the entire study of wound healing that, the hydrogel
showed faster and better epithelisation and granulation of wounds
after the 8 days than that of standard silver sulfadiazine cream.
3.8. In vitro cytotoxicity assays
Cytotoxic assays, using MTT Assay in 3T3 broblast Cell line
was performed to evaluate the toxicity of the samples at different
concentrations viz., (10 g/ml, 25 g/ml, 50 g/ml and 100 g/ml)
have represented in Fig. 5(c and d). The percentage of cell viability
was determined by interacting ability of the cells and the hydrogel.
At 10 g/ml, cell viability was in the range of 9294%. However,
at 100 g/ml was recorded in the range 6366% respectively. The
cell viability (%) decreases with increasing of sample concentration.
At low concentrations better cell viability was recorded, perhaps
at higher concentrations declining of viability and cell proliferation compared to the controls. Similar to our observation Michiko,
Kazuhiro, Kazuhiro, and Ryoji, (2000) have also observed for their
polymeric systems (RaeeMehr, 2011; Michiko et al., 2000). The
cell viability test was conducted for thrice to get high accuracy values for 24 h. According to GB/T 16886.5-2003 (ISO 10993-5:1999),
samples with cell viability larger than 75% can be considered as
showing no cytotoxicity (Yang, Yu, & Chen, 2009). Based on the
above results, SEA hydrogel is considered as a promising material with improved physical properties such as swelling, rate of
degradation with satisfying in vitro cytotoxicity. Hence, the synthesized hydrogel can be considered as non-toxic in nature and thus
compatible with living tissues.
4. Conclusion
In this study, synthesis of sodium alginate based biodegradable pH-sensitive biopolymeric hydrogel by solventless-greener
route. The entire experiments were carried out in the absence
of cross-linker and hazardous organic solvents. The biodegradation of the hydrogels have been carried out by soli burial test

and in vitro PBS buffer solution. The complete degradability was

observed in PBS buffer after 10 days. The degradation was gradually
increased in soil burial test on every week, and the hydrogel sample fully degraded after 5 weeks. The structural and morphological
changes of the biodegraded sample were examined by FT-IR and
SEM analysis respectively. The important stretching frequencies
have been vanished in biodegraded hydrogels while comparison
with parent hydrogels. Surface morphological changes with different environment caused improvement of cavities and hollowness
of hydrogels. Swelling parameters such as equilibrium swelling,
equilibrium water content at various pH from acidic to alkaline
has been investigated. In addition, swelling studies have also been
carried out in synthetic physiological body uids viz urine, physiological saline and iso-osmotic phosphate buffer. From the results,
it can be concluded that a signicant variation exists in the in vitro
release pattern of physiological simulated body uids from the
tested formulations. Especially, it has been observed that the agreeable amount of antibacterial activity against E. coli and S. aureus.
From the in vivo examination, it has been concluded that the SEA
hydrogels have an excellent wound healing capacity than that of
standard silver sulfadiazine. In addition, the imbibed physiological
body uids onto biopolymeric SEA hydrogels may be recommended
for applications such as biomaterials, biomedicine, biotechnology,
environment, sorption, separation, purication, immobilization
and many environment species makes the SEA hydrogel for more
popular in modern medical technology.

Acknowledgments
One of the authors, Mr. S.Sudarsan, gratefully acknowledges the
authorities of C. Abdul Hakeem College of Engineering and Technology, Melvisharam, Tamil Nadu, India for providing laboratory
facilities. The authors wish to thank Kovai Medical Centre Hospital (KMCH)- Pharmacy College, Coimbatore, Tamilnadu India for
technical help with in vivo examination.

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