Journal of Industrial and Engineering Chemistry 39 (2016) 127–135

Contents lists available at ScienceDirect

Journal of Industrial and Engineering Chemistry

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

Sulfanilamide and silver nanoparticles-loaded polyvinyl

alcohol-chitosan composite electrospun nanofibers:
Synthesis and evaluation on synergism in wound healing
Mani Ganesh a,b,*, Abidov Sch Aziz a, Udumansha Ubaidulla c, Pushparaj Hemalatha d,
Arthanari Saravanakumar a, Rramaswamy Ravikumar a, Mei Mei Peng a, Eun Young Choi e,
Hyun Tae Jang a,*
a
Department of Chemical Engineering, Hanseo University, 360 Daegok-ri, Seosan-si 356 706, Chungcheongnam-do, South Korea
b
Hillside College of Pharmacy, Bengaluru, Karnataka 560062, India
c
C.L. Baid Metha College of Pharmacy, Chennai 600097, India
d
Department of Chemistry, Anna University, Chennai 600025, India
e
Department of Cosmetology, Hanseo University, 360 Daegok-ri, Seosan-si 356 706, Chungcheongnam-do, South Korea

A R T I C L E I N F O A B S T R A C T

Article history: Novel silver nanoparticles-decorated chitosan (CS)-polyvinyl alcohol (PVA) composite electrospun
Received 4 March 2016 nanofibers, loaded with sulfanilamide for enhanced wound healing have been developed. Herein, formic
Received in revised form 19 May 2016 acid was used as a reducing agent to produce in situ colloidal silver nanoparticles (AgNPs) in the
Accepted 20 May 2016
composite polymeric solution with the active agent sulfanilamide. The prepared electrospun fibers
Available online 30 May 2016
were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM),
X-ray diffraction, ultraviolet spectroscopy, and thermal gravimetric analysis (TG). Further, in vitro
Keywords:
release, antimicrobial properties and in vivo wound healing activity were evaluated. The results revealed
Nanofiber
Sulfanilamide
that the composite fibers displayed a synergistic antibacterial and wound healing activities.
AgNP’s ß 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
Wound healing reserved.
Antimicrobial

Introduction
Ultra-fine structures including microspheres and microporous
and mesoporous nanostructures such as nanoparticles, nano-
spheres, and nanomachines, have been shown to play pivotal roles
in the fields of gas sorption, catalysis, and environmental and
medical science. Most importantly, mesoporous nanoparticles
including those made from silica and hydroxyapatite, and
microporous nanostructures such as bio-MOF have found applica-
tions in medical imaging, drug delivery, and bone and organ
replacements [1–8]. Over the last two decades, their tunable high
surface area, surface to volume ratio, softness, surface coverage,
high porosity, and high drug loading and delivery capacity to their
* Corresponding authors at: Department of Chemical Engineering, Hanseo
University, 360 Daegok-ri, Seosan-si 356 706, Chungcheongnam-do, South
Korea. Tel.: +82 41 660 1423; fax: +82 41 688 4143.
E-mail addresses: chemgans@gmail.com (M. Ganesh), htjang@hanseo.ac.kr
(H.T. Jang).
target tissues, has made them attractive in drug delivery research.
Nanofibers are among the ultra-fine nanostructures that are
produced using the principles of drawing [9], template synthesis
[10], phase separation [11], self-assembly [12], and electrospin-
ning [13]. A detailed discussion regarding these procedures can
found in the review by Huang et al. [14].
Electrospinning is the most versatile among the techniques
listed above, and can produce nanofibers of different diameters
with well-ordered surface morphologies. Herein, the nanofibers
were produced by applying a high electrical voltage to polymers
spun over the target electrode. The era of electrospinning began in
1934, when a series of patents were released describing the
technique [15–17]. Due its reliability, simplicity, versatility, and
potential uses in diverse fields, electrospinning has gained
increasing applications including fiber reinforcement [18], filtra-
tion [19,20], protective clothing [21,22], nanosensors [23], and
cosmetics [24].
In addition to the above exciting applications by their high
encapsulation efficiency, high loading capacity, simultaneous
delivery of diverse therapeutics, ease of preparation, and low

http://dx.doi.org/10.1016/j.jiec.2016.05.021
1226-086X/ß 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
128 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
cost, polymeric electrospun nanofibers using synthetic and
biological polymers have found applications in drug-loaded wound
dressing preparation, drug [25–30] and protein delivery [31], and
in tissue engineering scaffolds [32,33]. Electrospun nanofibers
have been reported to possess great potential for wound dressings
as a result of special characteristics such as the high porosity of the
nanofibrous membrane effectively contributing to air permeabili-
ty, providing the required oxygen for cell respiration, and having a
relatively small pore size that can preserve the wound from
bacterial infections. Moreover, the fibrous surface structure
displays strong adhesiveness to mucous layers since the nanopor-
ous structure instantly absorbs moisture into the voids.
Chitosan [poly[(1,4)-2-amino-2-deoxy-D-glucose], is an amino
polysaccharide obtained from chitin through the deacetylation
process. Chitin is the principal structural polysaccharide in
arthropods (crabs and insects), and the second most abundant
natural polysaccharide next to cellulose. It possesses antimicrobial
properties that are beneficial for wound dressing development.
Many authors have reported that the electrospinning of the CS
polymer has major disadvantages due to the use of solvents such as
trifluoroacetic acid (TFA), which is environmentally harmful, and
very toxic and corrosive, which has led to limited use in biomedical
applications. However, chitosan is not electrospinnable when
other solvents, such as acetic acid, are used. A common approach to
improve the electrospinnability of CS is to blend it with other
easily-electrospinnable polymers such as polyethylene oxide
(PEO), polyvinyl alcohol (PVA), and polylactic acid (PLA) [34].
The present study shows the preparation of in situ synthesized
silver nanoparticles-decorated composite nanofibers of CS and
PVA, embedded with sulfanilamide (SN) as a small molecule to
improve the antimicrobial and wound healing properties. The
effect of weight ratio and process parameters on the morphology of
the blended nanofibers were investigated using SEM. Nanofibers
were characterized using FTIR and DSC, and the release rate of
sulfanilamide and silver from nanofibers were also determined.
Experimental
Materials and methods
Polymers such as high molecular weight polyvinyl alcohol (PVA,
Molviol) low molecular weight chitosan (CS), and silver nitrate
(AgNO3), were obtained from Sigma–Aldrich (GmbH, Germany).
The model drug sulfanilamide and commercial grade solvents such
as acetic acid (99%), formic acid (85%), and isopropyl alcohol (IPA)
were purchased from Daejung Chemicals and Metals (Korea). DD
water used in the studies was obtained from an in-house
deionizing and distillation system. All other chemicals were of
commercial grade unless otherwise specified.
Preparation of the polymer solution
12% w/v PVA was prepared in the solvent (A) system consisting
of DD water and IPA (4:1). The mixture was heated to 90 8C for 1 h
to obtain a transparent homogenous solution. 3% CS (w/v) was
prepared in the solvent (B) system consisting of 60 ml 2% acetic
acid and 40 ml 85% formic acid (reducing agent for AgNO3) [35].
Preparation of the electrospun nanofibers
Various weight ratios (9:1, 8:2, 7:3, 6:4, and 5:5) of the above
polymer solutions (A and B) were mixed thoroughly for 1 h to
obtain a homogeneous solution. A nanospinning system (Nano-NC,
Korea), equipped with a high voltage power supply unit, was used
to provide high voltages in the range of 0–30 kV. Spinning
solutions were carefully loaded in a 10-ml syringe with a stainless
steel 21-gauge blunt needle, taking care to avoid the entrapment of
air bubbles. The positive electrode with a high voltage power was
connected to the needle tip. Aluminum foil wrapped around the
collector drum acted as a negative electrode as well as fiber
collector. The electrospinning process was carried out at ambient
temperature with a relative humidity of 45  5%. A fixed electrical
potential of 15 kV was applied, and the distance between the collector
and needle tip was kept at 15 cm. The solution feed rate was
controlled at 0.5 ml/min using a single syringe piston pump. The
collected nanofibers were dried overnight at 40 8C to remove residual
solvent. The spun nanofibrous material was carefully peeled from the
aluminum foil and cut in to specified dimension (1 cm  1 cm) for
further pharmaceutical evaluation. The polymer composition that
produced fine and smooth nanofibers was selected for drug and
AgNP’s loading and for pharmacological evaluation.
Drug and AgNP’s loading
The one-pot procedure was followed for the in situ drug and
AgNP’s (AgNP’s) loading. Herein the sulfanilamide (50 w/w% to
that of the total polymer) and silver nitrate (10 w/w% Ag to that of
the total polymer) were dissolved in 8 ml 4:1 DD water:IPA
mixture prior to PVA addition. The respective quantity of PVA to
obtain a 12% w/v solution was added to the mixture, and heated at
90 8C to obtain a homogenous PVA drug solution. Subsequently,
2 ml 3% CS solution in solvent (B) was added, and the solutions
mixed thoroughly until a homogeneous solution was obtained. The
solution was drawn into the syringe and fibers were formed using
the above-described electrospinning procedure (note: due to the
formation of AgNP’s, the color of the solution changes from
colorless to dark brown to black while mixing solvent B mixture to
PVA mixture).
Characterization
The surface morphology of the electrospun nanofibers was
studied using a JEOL-JSM 5600 scanning electron microscope (JEOL
Ltd., Japan). The samples were sputtered with gold plasma via a
sputter-coater (Cressington, Sputter Coater-108 auto) before
visualization under the SEM. The diameters of the electrospun
nanofiber were measured using the image visualization software,
Image-J (National Institutes of Health, USA). The average fiber
diameters were determined by measuring 30 fibers at random
from the SEM micrographs. The morphological characteristics and
particle size of the AgNP’s were observed using a Hitachi tunneling
electron microscope (Hitachi-800 TEM) with the solution of
nanoparticles in the solvent with the polymer. The solution was
placed over a copper grid and allowed to dry at room temperature,
and then placed in the sample holder of the TEM instrument and
analyzed. Fibers loaded with AgNP’s were also examined TEM. To
determine the crystal structural changes of the polymer and AgNP
and drug loaded polymer XRD was recorded using Rigaku Miniflex
diffractometer using CuKa radiation (l = 1.54 Å). The diffraction
data were recorded in the 2u range of 10–808 with a 0.18 step size
and a 1-s step time. SCINCO DSC N 650 differential scanning
calorimeter was used to record the DSC traces of the pure
sulfanilamide, PVA–CS and PVA–CS-sulfanilamide nanofiber mats
with a heating rate of 10 8C/min under helium atmosphere (40 ml/
min) to get the information on drug and polymer crystalline
changes after fabrication. Further to confirm the formation of the
AgNPs, the polymeric solution was subjected to UV–vis spectral
scanning using a Shimadzu UV mini-1240 UV–visible spectropho-
tometer with 1-cm quartz cells. A solution without reducing
agents, formic acid, or CS was used as a blank. Fourier-transform
infrared (FTIR) spectra were obtained on a Nicolet 6700 FT-IR
spectrometer at room temperature following the formation of a
 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
transparent pellet with KBr. 20 scans were taken at 4 cm1
resolution over the range of 4000–400 cm1. To determine the
mechanical stability of the nanofibers, thermogravimetric analysis
(TGA) was carried out using a SCINCO N-1000 thermogravimetric
analyzer in an N2 atmosphere, by heating approximately 10 mg
fiber sample in a platinum pan from 25 to 800 8C in increments of
10 8C/min.
Pharmaceutical evaluation
Swelling index
The swelling index was measured by immersing 100 mg fiber
material into phosphate buffer (pH 7.4) and weighing the material
at various time intervals. Following the immersion the material
was removed at specified time intervals, held over filter paper for a
few seconds to remove the surface water, and then weighed. The
increase in weight gives the swelling character of the fiber
material. The initial volume (V0) was noted, and the change in
physical volume was observed (Vt) at predetermined time intervals
of 1, 2, 4, 6, 10, and 12 h. The percentage swelling index was
calculated using the following formula (1) [36,37]:
V t V 0
Swelling indexð%Þ ¼ 100
V0
where V0 and Vt are the initial volume and final volume of the
nanofiber material, respectively.
In vitro release
Electrospun nanofibrous membranes were carefully peeled
from the aluminum foil and weighed exactly using a digital
balance. Each sample, weighing 100 mg (1 cm  1 cm) was
immersed in 100 ml phosphate buffer (pH 7.4, biomimetic pH),
with the temperature maintained at 37  1 8C, and the buffer was
magnetically stirred at 100 rpm. 1-ml samples were taken from the
buffer solution after 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 h. Following
sampling, the volume was replaced with fresh buffer. The concentra-
tion of sulfanilamide in the sample was determined using a UV–vis
spectrometer (Shimadzu Mini-1240, Japan) at 258 nm. Likewise the
Ag concentration in the release media and total concentration of
loaded AgNP’s in the respective quantity of nanofiber mat was
evaluated by total immersion on 50% HNO3 (as digestion media for
silver) using an atomic absorption spectrophotometer (Z5000,
Hitachi) [27].
Analysis of the release data
The release data obtained via the above procedure were
subjected to the Ritger and Peppas model in order to devise the
release mechanism [38,39]. The initial 60% cumulative release data
were used to estimate the diffusion exponent ‘n’ using the
following Eq. (2):
Mt
¼ Ktn
M1
where Mt is the amount of drug released at time t, M1 is the
nominal total amount of drug released at K the kinetic constant,
and n is the diffusion exponent that is used to characterize the
release mechanism. For spheres, a value of n  0.43 indicates
Fickian release and an ‘n’ value between 0.43 and 0.85 is an
indication of non-Fickian release (both diffusion-controlled and
swelling-controlled drug release). An ‘n’ value 0.85 indicates
case-II transport, which involves polymer dissolution and poly-
meric chain enlargement or relaxation.
In vitro antibacterial activity
The antibacterial activity of the hot DD water solution of
nanofibers was evaluated using the agar well diffusion method
129
since the synthesized nanofibers were rigid and the moisture in the
agar plates was not high enough for penetration. The antibacterial
activity was evaluated against Gram negative () Escherichia coli
(NCIM 2931) and Pseudomonas aeruginosa (NCIM 5029) as well as
Gram positive (+) Staphylococcus aureus (NCIM 2079) [27,40]. Nu-
trient agar medium was prepared, and 30–35 ml was transferred
into sterile petri dishes, upon which 1 ml working stock culture
(1  106 cells/ml) of the respective bacteria were placed (pour
plate technique) and mixed well to get a uniform mixture.
Following solidification of the medium, 4 wells were made in the
plates with a stainless steel cork borer. A fixed volume (50 ml) of
PVA fiber solution made in hot water (control in well 1), PVA–CS
sulfanilamide composite fiber solution (well 2), AgNP’s loaded
PVA–CS composite fiber solution (well 3), and sulfanilamide and
AgNP’s-loaded PVA–CS composite fiber solution (well 4) were
added to the wells and placed in a refrigerator for 30 min to allow
for diffusion. The plates were then incubated at 37 8C for 24 h, and
the zones of inhibition were measured. Note; all the formulation
were diluted to get 30 mg ml1 of sulfanilamide in final solution.
In vivo wound healing activity
In vivo wound healing activity was assessed in rats. Male Wistar
(1) rats (200–250 g) were obtained from the Central Animal House,
C.L. Baid Metha College of Pharmacy, Chennai. They were housed in
elevated wire cages, four animals per cage, with ad libitum access to
food (Lipton, Mumbai, India) and water. The study protocol was
approved by the Institutional Animal Ethics Committee, C.L. Baid
Metha College of Pharmacy, Chennai (Approval No. IAEC/XLII/04/
CLBMCP/2015).
The animals were randomized into four groups consisting of
three animals in each group. The animals were anesthetized with
an intramuscular injection of ketamine (75 mg/kg). Following
anaesthetization, the dorsal skin region of the animals was shaved,
and a 2 cm2 0.5 cm full-thickness incision was made on the dorsal
skin using a sterilized surgical blade [41].
The rats were divided into three groups (n = 6) and treated as
follows:
Group I: Control
Group II: Marketed formulation (sulfanilamide cream)
Group III: Sulfanilamide and AGNP’s-loaded nanofibers
The degree of wound healing is expressed as the wound
contraction ratio (WCR) as follows (Eq. (3))
A0 At
WCR ð%Þ ¼ 100 (3)
A0
where A0 and At indicate the initial wound area and the wound area
at time t, respectively. The wound area was measured using a slide
caliper.
(2) Statistical analysis
All the drug release experiments were conducted at least three
times, and all values are reported as the mean with standard
deviation (mean  SD). One-way analysis of variance (ANOVA) was
employed for the statistical analysis of the data. The fitting values
were obtained using commercially available software (Origin 8.5)
purchased from Origin Lab Inc. (Northampton, MA).
Results and discussion
Scanning electron microscopy analysis
The SEM images of the preformulated nanofibers with various
combinations of 10% PVA and 3% CS are depicted in Fig. 1. Since CS
is polycationic in nature, it cannot be electrospun alone into
130 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
Fig. 1. SEM images of PVA:CS nanofibers with different combination of polymers.
nanofibers without the use of highly concentrated acid, hence it
was mixed with the polymer PVA. As can be seen from Fig. 1, the
fibers formed from plain PVA and PVA:CS (8:2 and 7:3) are smooth
and continuous, with good structural integrity. However, increas-
ing the concentration of CS beyond 3% leads to adhesion of the
nanofibers, as seen with 6:4 PVA:CS composite, or a decrease in the
structural integrity by the formation of defective nanofibers (such
as balloons and spray drops). This may be due to the fact that the
ionic conductivity of the polymeric mixtures increases as the
amount of CS increases, as a result of the –NH3+ that forms upon
the addition of acetic and formic acids during the generation of the
solution. Thus, the formed –NH3+ will increase the repulsive forces
between them in the polymeric network, which will further induce
the formation of defective and non-continuous fibers under the
applied high voltage. The average diameter of the formed
nanofibers was approximately 200, 150, and 95 nm for neat
PVA, and 8:2 and 7:3 PVA:CS, respectively (data not shown). A
similar decreasing pattern in the diameter was also observed by
and Abdelgawad et al. [27], therefore, it was decided that 8:2
formulations would be used.
Fig. 2 shows the SEM images of nanofibers loaded with
sulfanilamide and those loaded with sulfanilamide and AgNP’s.
Both fibers are smooth, continuous, and sufficient for the loading of
active molecules for wound healing. The viscosity of the PVA/CS-
AgNPs mixed solution was lower than that of PVA/CS solutions that
improved the electrospinning ability of the solutions and fiber
diameter. The solution viscosity made an impact on fiber
morphology. In general the solution with high viscosity have
longer stress behavior or long relaxation time, which could impede
the jet formation during electrospinning while in the case low
viscous solution impeding in spinner jet formation get reduced,
this resulted in fiber with increased diameter. Further addition of
AgNPs/drug to CTS/PVA solution cause variation in its electrical
charge and conductivity that causes the changes in shape and size
of the droplet that might formed from the tip that leads to changes
in electrospinning process. The change in electrical charges is
resulted in fiber with varying diameter [27,42]. To observe the
AgNP’s formed during the in situ preparation, the solution was
diluted, spread over the TEM grid, dried well, and subjected to TEM
analysis. Fig. 3(a) shows that the AgNP’s are small and spherical,
with good textural properties to be loaded onto nanofibers. The
average particle size of the AgNPs thus formed was calculated as
18.5 nm (average from the diameter of 200 nanoparticles that were
taken into account). And the fibers decorated with particles were
also analyzed in the same way and presented in Fig. 3(b) and (c).
The in situ formation of the AgNP’s was further confirmed using UV
spectral analysis, which is discussed below.
UV spectral analysis of silver nanoparticles
The colorless AgNO3 solution turned a deep brown color upon
addition to the solution of polymers containing formic acid,
 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
Fig. 2. SEM images of sulfanilamide and AgNP’s sulfanilamide loaded nanoparticles.
Fig. 3. TEM images of (a) AgNP’s and (b) & (c) AgNP’s, sulfanilamide loaded nanofiber.
indicating the formation of AgNPs. The synthesized AgNPs in the
polymer solution were subjected to UV–visible spectral scanning
in order to record their surface plasmon resonance (SPR)
properties. In general, the spherical AgNPs showed the character-
istic SPR in the wavelength region of 400–450 nm. The results of
the spectral scanning are shown in Fig. 4, demonstrating the
presence of SPR of the formed AgNPs as a sharp peak at 420 nm.
This indicates the successful in situ formation of spherical AgNPs
[29–31] with the aid of formic acid and CS as reducing agents [35].
XRD and DSC analysis
As can be seen in Fig. 5, four major diffraction patterns
representing the formation of AgNPs are indexed at 38.166, 46.291,
Fig. 4. UV-spectral data of AgNP’s.
131
64.467, and 77.671 of the 111, 200, 220, and 311 lattice planes of
face-centered cubic (FCC) crystals, the results of which is coincided
with JCPDS card No. 04-0783 for FCC crystals [43,44]. The broad
diffraction peak near 20 (degree 2u) represents the semi crystalline
nature of the PVA polymer and the crystalline peaks responsible for
sulfanilamide were not observed in the XRD which depicts that the
drug was distribute finely into the polymer in amorphous form (as
it is present in low % while compared to polymers used). A similar
type loss in crystalline behavior of drug also reported by Ahed et al.,
where they studied with sodium salicylate PVA composite
[45]. Same was further supported by the DSC traces (Supplemen-
tary Fig. S2), where there is a melting endothermic peak was shown
at 164.5 8C for pure sulfanilamide but the same was not observed
in drug loaded nanofibers. The above results evidenced the
distribution drug in amorphous form not in crystalline form this
due to that the drug concentration in final nanofabrics was less
hence it was finely distributed as amorphous. In case of pure PVA–
CS fiber the melting peak for PVA was observed at around 230 8C
the same was little shifted higher temperature in case drug and
AgNPs loaded nanofiber this may be due to the cross linking on
polymer caused by the hydrogen bonded interaction of drug and
AgNPs.
FTIR analysis
Fig. 6 shows the FTIR spectrum of plain PVA and PVA–CS
polymers. In the plain PVA spectrum the broad band observed at
3180–3600 cm1 was assigned to O–H stretching vibration mode
arising from the inter- and intra-molecular hydrogen bonds of PVA.
The bands at 2900–3000 cm1 are due the alkyl group stretching of
PVA. The vibrational bands resulting from residual C5
5O and C–O of
PVA appear between 1700–1680 cm1. The –CH2 bending vibra-
tion was observed at 1750 cm1 [40,46]. In the case of the FTIR
132 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
Fig. 5. X-ray data of AgNP’s decorated nanofiber.
spectrum of the composite nanofibers, all the major peaks for PVA
and CS were observed. Due to the hydrogen bonding between the –
NH2 group of CS and the –OH group of PVA, individual peaks were
not observed in the composite, instead, a broad peak was seen at
3000–3700 cm1. The peak responsible for amide –C5 5O–NH2 was
observed at around 1650 cm1.
The FTIR spectrum of sulfanilamide-loaded composite nano-
fibers and sulfanilamide and AgNP’s loaded nanofibers were
shown in Fig. 7. FTIR spectrum of pure sulfanilamide was shown in
Supplementary Fig. S1, in that the high intensity bands appearing
between 3500–3400 cm1 and 3390–3337 cm1 were assigned to
the asymmetric and symmetrical stretching vibrations of –NH.
These bands may overlap with the symmetrical stretching
vibrations of the –NH2 of sulfonamide. Aromatic –CH stretching
vibrations of the aromatic rings were observed from the band
near 1370–1320 cm1. The bands at 1215–1175, 1125–1080, and
Fig. 6. FTIR-spectrum of nanofibers synthesized using different combination of PVA–CS.
860–810 cm1 are due to planar and extra-planar deformational
vibrations of two adjacent hydrogen atoms of the para-substitut-
ed benzene ring. The band responsible for S–N stretching
vibrations of sulfonamide appears between 940 and
890 cm1. The C–S stretching vibration was observed near
800 cm1 [47]. In the case of sulfanilamide-loaded composite
nanofibers, the peak responsible for the –NH and –NH2 of
sulfanilamide overlapped with the peaks of the –OH of PVA and CS,
and the –NH group of CS. Further amino groups (–SO2NH2 and
primary –NH2) of sulfanilamide formed hydrogen bonding with
the –OH of the polymers. Hence, together they gave a high
intensity broad band between 3100 and 3600 cm1. The bands due
to S–N and C–S stretching were observed in the vibrational ranges
of 940–890 and 800 cm1. Aromatic –CH stretching was observed
near 1370–1320 cm1.
TGA
Pure PVA showed three-step degradation weight loss behaviors
(Fig. 8). The first degradation weight loss appeared below 150 8C,
which is due to the removal of physisorbed and chemisorbed
water. The second degradation pattern that started around 300 8C
is associated with the degradation of the PVA backbone. The third
phase of degradation occurred at 600 8C as a result of the
degradation of the residual vinyl acetate groups of PVA
[36,48]. In the case of the CS composite of PVA, a similar type of
weight loss pattern was observed, however, their thermal stability
was slightly improved compared with pure PVA. Nevertheless,
there was an increase in final weight of the residues, which may be
due to an increase in total carbonaceous matters in the composite.
The weight increase in the AgNP’s nanofiber composite is still
greater than that of the remaining residues (around 8–12 wt%),
which may be due to the metal loaded in the composite (final char
weight 14.3 wt%).
 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
Fig. 7. FTIR-spectrum of (a) sulfanilamide loaded (b) sulfanilamide and AgNP’s
loaded nanofibers.
Pharmaceutical evaluation
Swelling index
The swelling index of the electrospun nanofiber materials was
determined using a gravimetric method. The swelling index of the
nanofibers plays an important role in the loading and release
behavior of a drug. Fig. 9 shows the degree of swelling of drug-
loaded and unloaded nanofiber patches at different time intervals.
The degree of swelling of PVA–CS patches in PBS pH 7.4 was
109.2  3.96, 120  5.5%, 150  4.3%, 178.56  4.68, 201.20  5.2%,
183  2.7, and 160  3% for the time intervals of 1, 2, 4, 6, 8, 10, and
12 h, respectively. In the case of drug-loaded PVA–CS patches, the
degree of swelling decreased due to the water-soluble sulfanilamide
can interacts with the polymeric chain through hydrogen bonded
crosslinks that restricting further entry of water to swell
[37,49]. However, after maximum swelling at 8 h, further increases
in time show a decrease in the degree of swelling in all cases. This
decrease in swelling is likely due to the degradation of polymers by
dissolution, hence there is a leaching of polymer from the nanofiber
patch and a decrease in its ability to swell.
Fig. 8. TGA traces of synthesized nanofibers.
133
Fig. 9. Swelling index of plain and drug loaded nanofibers.
The novel nanofiber system has a high degree of swelling (200%)
in case of the PVA–CS nanofiber patch compared with the PVA
nanofiber patch and the PVA–CS drug-loaded patch. This is due to
the presence of CS there was an increase in buffer solution intake. It
can be seen that after the drug-loading, the degree of swelling in
both the PVA and PVA–CS patches decreased. This demonstrates
that sulfanilamide/AgNP can crosslink with the polymer by
hydrogen bonding [37,47] that restricting the further entry of
water and the swelling of the drug-loaded patch, nevertheless,
after 8 h the degree of swelling of mats decreases in all cases. This is
due to the slow dissolution of the polymer through layer by layer
over a time due to erosion. Earlier study also supports the
possibility of polymer erosion [49].
In vitro release behavior
In vitro sulfanilamide release and AgNPs from PVA–CS
nanofibers as a function of time is shown in Fig. 10. The release
of the drug as well as AgNPs were fast during the initial hours and
became relatively slow during the later period. The in vitro release
profile specifies that the mean percentage cumulative release was
found to be 99% in the formulations after 24 h. However, a
Fig. 10. Sulfanilamide and AgNPs in vitro release behavior.
134 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
significant variation in the release pattern was observed in the first
hour, superseded by the PVA nanofibers (32.6%) followed by the
composite nanofibers (PVA–CS), where only 25% drug was released
after 1 h. The rate of drug release was higher in the case of the PVA
nanofibers compared with the PVA–CS nanofibers. In the case of
the PVA patch, almost 75% drug release was seen within 3 h, while
in the PVA–CS only around 50% drug release was seen. The
maximum (98.6%) drug release was seen within 5 h in the case of
the PVA nanofibers, with only 50% drug release seen in the same
time frame with the PVA–CS nanofibers. A controlled release
pattern was observed only in the PVA–CS nanofibers. The higher
cumulative drug release during the first hour in the case of PVA
nanofibers is due to the initial small burst release, which may be a
result of the hydrophilic nature of the polymer and a certain
amount of drug on the surface of the nanofibers. Maximum drug
release was observed up to 8 h due to the degradation of the water
soluble PVA in the nanofibers (erosion of the polymer). While in
PVA–CS drug-AgNP loaded nanofibers, the rate of release was slow,
since CS provides stability to the fiber structure as a result of the
intermolecular hydrogen bonding between PVA, CS, and sulfanil-
amide. The in vitro studies demonstrate that drug release takes
place within 6–8 h, which is good for acute injuries. This quick and
prolong release of sulfa and Ag from the nanofibers could provide
more rapid and constant antimicrobial activity at the wound site.
These novel nanofibers have a high surface area-to-volume
ratio, which increases the contact area of the wounded surface.
Compared with conventional sulphanilamide cream, these nano-
fiber materials are dry, which increases the stability of the drug.
Release kinetic study
Dissolution data of the PVA–CS nanofibers was fitted to various
kinetic models (zero order, first order, and Higuchi and Kersey-
meres-Pappas) in order to describe the drug and AgNP release
profile. The plot of the cumulative percentage drug/AgNP release
(Supplementary Fig. S3) as a function of square root of time
indicates the formulation follows Higuchi kinetics. In both case the
line of best fit obtained with R2 = 0.991 and 0.988 for the drug and
AgNPs respectively. The nanofiber shows a diffusion exponent
value of n = 0.619 and 0.716 of sulphanilamide and AgNP
respectively. Results indicating that these nanofibers follow a
non-Fickian anomalous release mechanism. This shows that the
drug is released in a diffusion-controlled mechanism coupled with
swelling and erosion mechanisms.
In vitro antimicrobial activity
Wounds are contaminated by a polymicrobial population of
aerobic and anaerobic bacteria. Common aerobic or facultative
pathogens are Staphylococcus aureus, Pseudomonas aeruginosa, and
Fig. 11. In vivo wound healing activity (a) control for PVA–CS nanofiber; (b) 7 days
after the application of AgNPs and sulfanilamide PVA–CS nanofiber; (c) 14 days
after the application of AgNPs and sulfanilamide PVA–CS nanofiber loaded PVA–CS
nanofiber; (d) control for conventional cream containing sulfanilamide; (e) 7 days
after the application of sulfanilamide cream and (f) 20 days after the application of
sulfanilamide cream.
Table 1
In-vitro antibacterial activity.
S. No. Formulation Zone of inhibition (in mm)
Gram (+) Gram ()
S. aureus E. coli P. aeruginosa
1. PVA 0 0 0
2. PVA + CS + SULFA 7.91  0.1 9.82  0.21 10.0  0.21
3. PVA + CS + AgNP’s 8.25  0.51 11.78  0.42 9.78  0.32
4. PVA + CS + SULFA + AgNP’s* 11.58  0.68 15.21  0.36 13.45  0.12
*(p < 0.05) is significant of combination of Sulfa and Ag Nanofibers compared to
individual nanofiber.
E. coli. These bacteria are present abundantly on the skin, and when
the skin gets injured the bacteria can badly affect the wound.
Table 1 shows the significantly (p < 005) increased zone of
inhibition of PVA–CS nanofibers loaded with sulfanilamide and
AgNP’s in all the dishes when compared to individual nanofiber.
The results are attributed to the fact that the antibacterial ability is
enhanced in the PVA–CS nanofibers. The combination of sulpha-
nilamide and AgNP’s loaded in the nanofibers amplified the
antibacterial effect.
In vivo wound healing activity
Wound healing is the process of regeneration of dermal and
epidermal tissues. In the present study, the process of wound
healing in rats was observed over time. Approximately 2 cm2 and
0.5 cm-deep wounds were generated in rats by making an incision
in the skin at time zero, as shown by Fig. 11. The control sample
showed very slow healing even after 20 days. The AgNPs and
sulfanilamide loaded PVA–CS nanofibers showed a faster rate of
healing than the control. The wound construction ratio (WCR) of
the PVA–CS nanofibrous matrices increased gradually in a similar
manner, and reached 90.76  4.3% after 7 days, whereas the control
wound reached only 55.26  3.5% after 20 days. The rate of wound
healing (reduction in wound area) was found to be maximum for the
AgNPs and sulfanilamide loaded PVA–CS composite nanofibers (7
days). However, the conventional formulation took more than 20 days
to recover the wound. The enhanced cell attachment of the nanofibers
was also attributed to the high wettability and positive surface charge
of the polymeric system.
In the control group after 20 days, the wound surface was
covered with fibrous debris, and a dense infiltration of polymor-
phonuclear leukocytes and proliferation of fibroblasts were
observed below the surface layer. However, the tissue debris
was not present on the wound surface in the drug-loaded PVA–CS
nanofiber groups after 5 days, and there was prominent
proliferation of young capillaries and fibroblasts. In the nanofiber
groups, epithelialization of the wound was complete after 7 days.
However, there was no significant difference observed in the
pattern of wound healing between any experimental formulations.
The combination of sulphanilamide and AgNP’s in the PVA–CS
nanofibers was able to significantly improve wound healing. This
accelerated healing is likely due to the high surface area and
microporous structure of the nanofibrous matrices, which are
advantageous in the promotion of cell attachment and prolifera-
tion, and release the drug to the wounds over a prolonged period.
Further to this sulphanilamide alone is incapable to enter in to the
cell wall lipopolysaccharide (LPS) of microbes efficiently but
AgNP’s have the capability to invade into the cell walls efficiently
through peptidoglycon part of cell wall by the destruction of LPS.
While the AgNPs complexes with sulphanilamide, the complexed
drug is easily pass thorough the cell wall membrane and leads to
synergistic antimicrobial and wound healing. The same type of
synergism was also reported with cephalexin along with AgNPs
[50,51].
 M. Ganesh et al. / Journal of Industrial and Engineering Chemistry 39 (2016) 127–135
Conclusions
One-pot fabrication of AgNP’s decorated with sulfanilamide-
loaded PVA–CS nanofiber material was developed and evaluated
for its enhanced wound healing activity. The developed composite
nanofibers have an average diameter of 150 nm, with a narrow size
distribution. Herein, formic acid was used as a reducing agent to
produce AgNP’s loaded fibers using an in situ preparation
procedure, which reduced the preparation time compared with
other procedures. The results of the TEM and XRD experiments
confirm the presence of AgNPs on the surface of the nanofiber
material. The presence of sulfanilamide was confirmed by FTIR
analysis. The results of the antimicrobial and in vivo wound healing
evaluation demonstrate the superior and synergistic activity as a
result of AgNP’s and sulfanilamide loading. It can be concluded that
the AgNP’s-decorated sulfanilamide-loaded PVA–CS nanofibers are
a better choice for enhanced wound healing activity than
conventional dosage form.
Acknowledgements
The authors gratefully acknowledge the financial support
received from Hanseo University Intramural Research Grant
2014, and we extend our thanks to the National Research
Foundation of Korea (NRF), funded by the Ministry of Science,
ICT, and Future Planning (Grant No. 2014-004694), for their partial
financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jiec.2016.05.021.
References
[1] R. Tania, L. Ignacio, P. Gonzalo, S. Beatriz, M. Hozanna, C. Avelino, K. Freek, X.L.I.X.
Francesc, G. Jorge, Nat. Mater. 14 (2015) 48.
[2] R. Ghorbani-Vaghei, S.M. Malaekehpoor, Tetrahedron Lett. 53 (2012) 4751.
[3] K. Rad-Moghadam, S.C. Azimi, J. Mol. Catal. A 363–364 (2012) 465.
[4] E.L. Sean, C.L. Sarah, Environ. Sci.: Nano 1 (2014) 200.
[5] M. Miguel, V.-R. Marı́a, J. Mater. Chem. 20 (2010) 5593.
[6] C.B. Marı́a, F.-J. David, P. Marcelo, J.R. Antonio, Q.S. Randall, J. Mater. Chem. B 2
(2014) 766.
[7] C.H. Rachel, D.R. Joseph, L. Wenbin, Curr. Opin. Chem. Biol. 14 (2010) 262.
[8] Z.W. Agnieszka, N. Katarzyna, H.M. Karolina, C. Halina, Pharmacol. Rep. 64 (2012)
1020.
[9] T. Ondarcuhu, C. Joachim, Euro. Phys. Lett. 42 (1998) 215.
[10] L. Feng, S. Li, H. Li, J. Zhai, Y. Song, L. Jiang, Angew. Chem. Int. Ed. 41 (2002) 1221.
[11] P.X. Ma, R. Zhang, J. Biomed. Mater. Res. 46 (1999) 60.
[12] G.J. Liu, J.F. Ding, L.J. Qiao, A. Guo, B.P. Dymov, J.T. Gleeson, T. Hashimoto, K. Saijo,
Chemistry – Eur. J. 5 (1999) 2740.
135
[13] H. Fong, D.H. Reneker, in: D.R. Salem (Ed.), Structure Formation in Polymeric
Fibers, Hanser, Munich, 2001, p. 225.
[14] Z.-M. Huang, Y.-Z. Zhang, M. Kotaki, S. Ramakrishna, Sci. Technol. 63 (2003) 2223.
[15] A. Formhals, US patent 1,975,504, 1934.
[16] A. Formhals, US patent, 2,323,025, 1943.
[17] A. Formhals, US patent, 2,349,950, 1944.
[18] Y.A. Dzenis, Y.K. Wen, Mater. Res. Soc. Symp. Proc. 702 (2002) 173.
[19] G. Renuga, K. Satinderpal, M. Zuwei, C. Casey, R. Seeram, M. Takeshi, J. Membr. Sci.
281 (2006) 581.
[20] D. Aussawasathien, C. Teerawattananon, A. Vongachariya, J. Membr. Sci. 315
(2008) 11.
[21] R.D. Nattanmai, K. Gopalu, S. Rangaraj, M. Palanisamy, R. Venkatachalam, Nano-
Micro Lett. 6 (2014) 46.
[22] M. Gorji, A.A.J. Ali, A.A. Gharehaghaji, J. Appl. Polym. Sci. 125 (2012) 4135.
[23] L. Zhang, J. Lou, L. Tong, Micro/Nanofiber Opt. Sens. Photon. Sens. 1 (2011) 31.
[24] F.-A. Anahita, Q. Lin, W.C. Yew, Y.C. Sui, AAPS PharmSciTech 11 (2010) 1164.
[25] R.U. Afeesh, A.M.B. Nasser, P.B. Tirupathi Pichiah, G. Gopalsamy, R. Nirmala,
Y.-S. Cha, C.-H. Junge, El.-N. Mohamed, H.Y. Kim, Carbohydr. Polym. 90 (2012)
1786.
[26] C. Florence, A. Ganka, P. Yves, J. Christine, Adv. Health Mater. 3 (2014) 2032.
[27] M.A. Abdelrahman, M.H. Samuel, J.R. Orlando, Carbohydr. Polym. 100 (2014) 166.
[28] A.T. Hang, B. Tae, J.S. Park, Carbohydr. Polym. 82 (2010) 472.
[29] Z. Yinghui, Z. Ying, W. Xiaomian, W. Lu, X. Ling, W. Shicheng, Appl. Surf. Sci. 258
(2012) 8867.
[30] H. Xiuli, L. Shi, Z. Guangyuan, H. Yubin, X. Zhigang, J. Xiabin, J. Control. Release 185
(2014) 12.
[31] W. Huaimin, W. Youzhi, Z. Xiaoli, H. Yawen, Y. Xiaoyong, M. Linsha, Z. Hao, L. Jiafu,
L. Qian, Y. Zhimou, Chem. Commun. 51 (2015) 14239.
[32] N. Ashammakhi, A. Ndreu, Y. Yang, H. Ylikauppila, L. Nikkola, Eur. J. Plast. Surg. 35
(2012) 135.
[33] J. Venugopal, S. Low, A.T. Choon, S. Ramakrishna, J. Biomed. Mater. Res. B: Appl.
Biomater. (2007) 34.
[34] Z.E. Maher, F.N. Hala, E.M. Rania, Mater. Sci. Eng. C 32 (2012) 1711.
[35] L. Jeong, W.H. Park, Int. J. Mol. Sci. 15 (2014) 6857.
[36] K.O. Kim, Y. Akada, W. Kai, B.-S. Kim, I.-S. Kim, J. Biomater. Nanobiotechnol. 2
(2011) 353.
[37] S.N. Alhosseini, F. Moztarzadeh, M. Mozafari, S. Asgari, M. Dodel, A. Samadiku-
chaksaraei, S. Kargozar, N. Jalali, Int. J. Nanomed. 7 (2012) 25.
[38] P.L. Ritger, N.A. Peppas, J. Control. Release 5 (1987) 23.
[39] N.A. Peppas, Pharm. Acta Helv. 60 (1985) 110.
[40] Y. Shigemasa, H. Matsuura, H. Sashiwa, H. Saimoto, Int. J. Biol. Macromol. 18
(1996) 237.
[41] S.K. Kulkarni, Handbook of Experimental Pharmacology, 3rd ed., Vallabh
Prakashan, New Delhi, 1997p. 49.
[42] D.H. Reneker, A.L. Yarin, Polymer 49 (2008) 2387.
[43] A. Saravanakumar, M. Ganesh, J. Jayaprakash, H.T. Jang, J. Ind. Eng. Chem. 28
(2015) 277.
[44] K. Jeeva, M. Thiyagarajan, V. Elangovan, N. Geetha, P. Venkatachalam, Ind. Crops
Prod. 52 (2014) 714.
[45] N. Ahad, E. Saion, E. Gharibshahi, J. Nanomater. (2012) 8, http://dx.doi.org/
10.1155/2012/857569.
[46] H.S. Mansur, C.M. Sadahira, A.N. Souza, A.A.P. Mansur, Mater. Sci. Eng. C 28 (2008)
539.
[47] A. Alsughayer1, A.-Z.A. Elassar, S. Mustafa, F. Al Sagheer, J. Biomater. Nanobio-
technol. 2 (2011) 144.
[48] C. Santos, C.J. Silvaa, Z. Büttel, R. Guimarães, S.B. Pereira, P. Tamagnini, A. Zillev,
Carbohydr. Polym. 99 (2014) 584.
[49] K. Kataria, A. Gupta, G. Rath, R.B. Mathur, S.R. Dhakate, Int. J. Pharm. 469 (2014)
102.
[50] P. Singh, R. Balaji Raja, Biores. Bull. 4 (2012) 171.
[51] S. Shrivastava, Nanotechnology 18 (2007) 225103.