International Journal of Pharmaceutics 564 (2019) 350–358

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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

A comparative study of wound dressings loaded with silver sulfadiazine and T

silver nanoparticles: In vitro and in vivo evaluation
⁎
Mina Mohsenia, Amir Shamlooa, , Zahra Aghababaieb, Homa Afjoulc, Shabnam Abdid,
⁎
Hamideh Moravveje, , Manouchehr Vossoughib
a
Center of Excellence in Energy Conversion (CEEC), School of Mechanical Engineering, Sharif University of Technology, P.O. Box 11155-9567, Tehran, Iran
b
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
c
Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
d
Anatomy Department, Tarbiat Modares University, Tehran, Iran
e
Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: In the current study, two series of antimicrobial dressings conjugated with silver sulfadiazine (SSD) and silver
Wound dressing nanoparticles (AgNPs) were developed and evaluated for chronic wound healing. Highly porous poly-
Silver nanoparticles caprolactone (PCL)/polyvinyl alcohol (PVA) nanofibers were loaded with different concentrations of SSD or
Silver sulfadiazine AgNPs and compared comprehensively in vitro and in vivo. SSD and AgNPs indicated a strong and equal anti-
Antimicrobial activity
microbial activity against S. aureus. However, SSD had more toxicity against fibroblast cells over one week in
Electrospinning
vitro culture. An in vivo model of wound healing on male Wistar rats was developed with a full thickness wound.
All the wound dressings indicated enough flexibility and hydrophilicity, which resulted an adequate adhesion
into the wound closure. After 30 days, the control group without any treatment indicated 31% wound closure
while the group treated with PCL/PVA (without antimicrobial components) indicated 44% wound closure.
Presence of antimicrobial components in the PCL/PVA nanofibers resulted into a lower inflammation response
leading to a faster proliferation and maturation phases. In agreement with the higher biocompatibility of AgNPs
than SSD, a faster angiogenesis, epithelialization and subsequently, remodeling were observed for the wound
dressings loaded with AgNPs. The group treated with the highest concentration of AgNPs showed the fastest
healing process leading to a final epithelialization with 96% wound closure after 30 days. This study indicated
that AgNPs have higher biocompatibility and regulate wound healing process more efficiently compared to SSD.
PCL/PVA nanofibers conjugated with AgNPs are promising wound dressings for full-thickness wounds.

1. Introduction the healing of chronic wounds.

Chronic wound healing is a long-term treatment which is often
compromised by biofilm contamination. Infection impedes the critical
transition from inflammation to proliferation and delays fibroblast de-
velopment which ensues impaired wound repair (Landén et al., 2016;
Percival et al., 2015). By controlling wound infection and inflammatory
response, a cascade of chemical signaling is initiated to modulate the
proliferative stage and finally, remodeling stage (Murphy and Evans,
2012; Percival et al., 2012; Boateng and Catanzano, 2015). The draw-
backs of using antibiotics via the digestive system has prompted to
develop advanced therapeutic dressings with antimicrobial activity and
apply a localized delivery of antimicrobial agents. Antimicrobial wound
dressings are promising to decrease the risk of infection and accelerate
Silver components are antimicrobial resistant to Gram-negative and
Gram-positive bacteria. For decades, silver sulfadiazine has been used
in the form of cream to control wound infection. Recently, Mohseni
et al. developed highly porous wound dressings loaded with silver
sulfadiazine and evaluated their antimicrobial activity and bio-
compatibility at different concentrations (Mohseni et al., 2016). It was
purported that concentration of silver components should be optimized
to provide a balance between biocompatibility and antimicrobial ac-
tivity of wound dressings. In vivo study is still required to evaluate the
effect of SSD concentration on a real model of wound healing process.
AgNPs are other silver components which have attracted attentions to
develop new generation of antimicrobial agents. Similar to SSD, they
are resistant to a wide range of bacteria and have shown great potential

⁎
Corresponding authors.
E-mail addresses: shamloo@sharif.edu (A. Shamloo), hamideh_moravvej@yahoo.com (H. Moravvej).

https://doi.org/10.1016/j.ijpharm.2019.04.068
Received 1 November 2018; Received in revised form 22 April 2019; Accepted 23 April 2019
Available online 24 April 2019
0378-5173/ © 2019 Elsevier B.V. All rights reserved.
M. Mohseni, et al.
in managing wound infections. They have been loaded into a cotton
fabric to make it effective against different species of organism (Hebeish
et al., 2014). The fabric treated with the concentration of 250 ppm
indicated similar results compared to Dermazin cream. AgNPs were also
loaded into biopolymers to prompt rapid dermal regeneration (Singh
and Singh, 2014). Chitin membranes containing 100 ppm indicated
strong antimicrobial activity against common wound pathogens. Col-
lagen-chitosan hybrid scaffolds loaded with 10 ppm also indicated an-
timicrobial activity along with promoted wound healing by regulating
fibroblast migration and macrophage activation (You et al., 2017).
AgNPs were also loaded into Collagen-alginate biocomposites at dif-
ferent concentrations (0.4 mM, 0.8 mM and 1 mM) (Zhang et al., 2018).
It was shown that by increase of AgNPs concentration, toxicity against
fibroblast cell and antimicrobial activity against Staphylococcus aureus
and Escherichia coli increased. These studies indicated that anti-
microbial activity and biocompatibility are highly dependent to the
concentration of Ag components and contradict each other. From a
regeneration perspective, toxicity of antimicrobial agents has been al-
ways a controversial issue and should be controlled to achieve enough
biocompatibility (Ge et al., 2014; Ning et al., 2015). This requires
comprehensive in vitro toxicity and antimicrobial activity assays as well
as in vivo wound healing modelling to evaluate concentration-depen-
dent features and finally, optimize the concentration of Ag components.
In advanced tissue engineering therapeutic, wound dressings con-
tribute in regeneration process directly and act as temporary substrates
for cells and growing tissue. This adds additional important features for
wound dressings. They require an adequate architecture to provide a
cell-friendly environment for growing tissue (Shamloo et al., 2015).
They need to be highly porous to allow for an efficient diffusion of
nutrients and oxygen leading to a high cell viability (Boateng et al.,
2008; Braghirolli et al., 2014). They should also be enough hydrophilic
to adhere to wound closure and keep it moisturized with wound fluid
(Li et al., 2013; Boateng et al., 2008). Furthermore, they should indicate
a robust mechanical behavior supporting enough handling and flex-
ibility close to native skin tissue. (Braghirolli et al., 2014; Fang et al.,
2008). Electrospinning is as a simple and reproducible fabrication
technique to develop highly porous microstructures (Jun et al., 2018).
Nanofibers can be well tuned to mimic the native microenvironment
and provide a cell-friendly substrate. Electrospinning also allows to
fabricate composite structures, which gives more flexibility to optimize
mechanical and physical properties of the substrate.
In our previous study, a highly porous wound dressing was devel-
oped from polycaprolactone (PCL) and polyvinyl alcohol (PVA) nano-
fibers with a good handling, flexibility and hydrophilicity which pro-
vides requisite physical properties for wound dressings (Mohseni et al.,
2016). PVA is inherently hydrophilic without enough integration in
aqueous media (Yao et al., 2013). Addition of optimized volume frac-
tion of PCL nanofibers as a hydrophobic polymer to PVA nanofibers via
co-electrospinning improves the mechanical stability of wound dres-
sings while keeping the hydrophilic property. In the current study, PCL/
PVA nanofibers were conjugated with a uniform distribution of AgNPs
and SSD at different concentrations. A series of in vitro and in vivo ex-
periments were conducted to compare the potential of these two groups
of silver components in wound healing process. The biocompatibility of
wound dressing was evaluated over 7 days in vitro culture of fibroblast
cells, and their antimicrobial activity was assessed against S. aureus by
the inhabitation zone method. An in vivo model was conducted to
evaluate and compare the potential of developed antimicrobial wound
dressings in a full-thickness wound. This study provides a platform for a
direct comparison between SSD and AgNPs at different concentrations.
351
International Journal of Pharmaceutics 564 (2019) 350–358
2. Materials and methods
2.1. Scaffold fabrication
2.1.1. Material
PCL (Mn = 80KDa), chitosan (medium molecular weight), AgNO3
(99.9% purity) and SSD were purchased from Sigma-Aldrich. PVA
(Mn = 72KDa), chloroform, acetic acid and methanol were purchased
from Merck.
2.1.2. Synthesis of chitosan-Ag nanoparticles
0.15 mg chitosan was dispersed in 5 mL of a 0.1 M AgNO3 solution
and stirred for 6 h at 50 °C to form AgNPs by reducing Ag+ with chit-
osan. 1 h after the progress was initiated, the color of the solution
changed from colorless to yellowish brown showing the formation of
AgNPs. Precipitated Nanoparticles were washed with acetone 5 times
and dried at 100 °C.
2.1.3. Electrospinning
PCL was dissolved in a 1:1 solution of chloroform and methanol at
the concentration of 10 wt% for 3 h. A 10 wt% solution of PVA was
prepared by dissolving PVA in distilled water at 80 °C and stirring for
4 h. AgNPs were dispersed in 10 wt% solution of PVA at the con-
centrations of 0.1 (AgNP1), 0.5 (AgNP5), 1 (AgNP10) wt% of PVA so-
lution and stirred for 24 h at room temperature. As described in our
previous study, SSD was added to the PVA solution at concentrations of
0.1 (SSD1), 0.5 (SSD5), 1 (SSD10) wt% of PVA solution and stirred for
6 h at room temperature to obtain a homogeneous solution (Mohseni
et al., 2016). Eelectrospinning parameters were optimized to obtain
continuous and uniform nanofibers (Fig. 1). PCL nanofibers were de-
veloped with the flow rate of 3 mL/h at the electric potential of 27 kV.
PVA nanofibers incorporated with AgNPs or SSD were fabricated at the
flow rate of 1 mL/h under the electric potential of 21 and 25 kV, re-
spectively. The speed of collector was adjusted at 1000 rpm. Scaffolds
were kept in a desiccator at 25 °C and 20% humidity for 2 days to re-
move any residual solvents.
2.2. Fiber characterization
Nanofibers were examined under scanning electron microscopy
(SEM, XL30 model, Philips) at the voltage of 25 KV to investigate the
morphology of nanofibers. The samples were gold sputter-coated using
a JEOL fine sputter coater for 75 s and 8 mA current. The field emission
electron microscopy (FESEM) was utilized to observe silver nano-
particles. Elemental map analysis was also performed to see the dis-
tribution of nanoparticles within the fibers. In order to measure fiber
diameters and nanoparticle sizes, the images were analyzed with
ImageJ (NIH) software by measuring 10 fields of view. Dynamic light
scattering (DLS, Microtrac, USA) was also performed to measure the
size of nanoparticles. X-ray differentiation (XRD) analysis was con-
ducted to confirm the formation of AgNPs. The XRD patterns of nano-
fibers containing AgNPs were recorded by Cu K’α radiation generated at
40 kV and 30 mA (Spectro, xepos, Ametek).
Tensile test was performed to evaluate the elastic modulus of the
wound dressings and compare it with the skin mechanical properties.
The rectangular samples with the width of 0.5 cm and initial length of
3 cm were put between two grips and stretch by the rate of 5 mm/min
using universal tensile machine (H10KS, Tinius Olsen, Horsham, PA,
USA).
2.3. Antibacterial activity
The antibacterial activity of the scaffolds against S. aureus was
evaluated by the inhabitation zone method. The conditions were con-
ducted according to a previous study on commercialized wound dres-
sings containing silver components to have comparable results (Kohta
M. Mohseni, et al.
Fig. 1. SEM photographs of a) PCL, b) SSD1, c) SSD5, d) SSD10, e) AgNP1, f) AgNP5, g) AgNP10. h) FESEM photograph of AgNPs conjugated with PVA nanofibers.
and Ohyabu, 2015). A bacterial solution with a concentration of
6.8 × 105 colony-forming units per milliliter (CFU/mL) was prepared
by monitoring the optical density of the solution under spectro-
photometer. Scaffolds were cut into disc shapes with the diameter of
1.5 cm and placed on solid agar containing 1 mL of bacterial solution in
90 mm diameter plates. The agar plates were incubated for 24 h at
37 °C. Inhabitation zone was measured to determine the antibacterial
activity.
2.4. Cytotoxicity evaluation
The cytotoxicity of the scaffolds was carried out using MTT assay as
an indirect cytotoxicity test. Human fibroblast cell was cultured in a
75 cm2 culture flask containing complete medium (Dulbecco’s modified
Eagle’s medium, DMEM) supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin. Cells were incubated under a
humidified atmosphere at 37 °C and 5% CO2. The culture media was
changed every 3 days. When the cells reached the confluence of 80%,
they were detached by trypsin-EDTA and counted using Neubauer lam.
Scaffolds were cut into disc shapes with the dimension of 24-well plate
and sterilized under ultraviolet light for 2 h on each side. A culture plate
treated with polyester used as the control group. Fibroblast cells with
the density of 5 × 104 cell were seeded on each scaffold and each
controlled well. MTT [3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenylte-
trazolium] assay was performed to quantify the live cells. 5 mg of MTT
(Sigma) was dissolved in 1 mL of PBS solution and sterilized by fil-
tering. The culture medium of the 24 well plates were replaced with
50 µl of MTT solution and 500 µl of fresh culture medium and incubated
to form formazan crystals by mitochondrial dehydrogenases. After 4 h,
the medium was removed, and 200 µl Dimethyl sulfoxide (DMSO) was
added to each well to dissolve the formazan crystals. The optical density
of the solution was obtained using an Elisa plate reader at 570 nm.
2.5. In vivo experiments
In vivo experiments were conducted to have a more reliable results
and comparison between antimicrobial agents. 70 male Wistar rats
(180–200 g) were prepared and categorized into 7 groups randomly.
The protocol was performed according to the Shahid Beheshti of
352
International Journal of Pharmaceutics 564 (2019) 350–358
Medical Science University Guide for the Care and Use of Laboratory
Animals. All animals were sedated via injection of 50 mg/kg Ketamine
hydrochloride and 5 mg/kg diazepam. Each rat’s dorsal hair was shaved
and the dorsum of each rat was marked for contact burns. Operation
site was disinfected using 10% Povidone-iodine and 70% ethanol so-
lution and imposed into water steam for 20 s. The steam rises from a
custom made aluminum pipe with square cross-section profile
(2 cm × 2 cm). After that, the burned site was debride according to the
marked area, and full thickness wound was developed. The sterile
wound dressing was implanted into wound bed and covered with a
layer of gauze. The bandage was secured in place with tape to preserve
the scaffold from falling. The negative control group was covered just
with gauze without any scaffolds. The positive control was treated with
SSD cream. Using camera and Image J software, the area of wound
closure ( Ai ) at 10, 20, 30 was assessed, and the ratio of closed area to
initial area ( A0 ) of wound closure was calculated according to the fol-
lowing formula (Cukjati et al., 2001):
A0 − Ai
Closed area (%) = × 100
A0
For histological analysis, 5 animals from each group were sacrificed
by chloroform at 10 days at 30 days. The whole part of wound bed was
excised and immersed into 10% formalin. Pathologic analysis was
performed by hematoxylin and eosin staining to study skin regenera-
tion.
3. Results
3.1. Fiber characterization
Utilizing SEM analysis, morphological characterization indicated
that average diameter of PVA fibers incorporated with AgNPs are re-
duced as their concentration increased (Table 1). This observation can
be explained by the fact that the addition of AgNPs increases the
electrical conductivity of the solution, which affects on the required
potential to form a Taylor cone and overcome the surface tension of the
liquid (Gora et al., 2015; Li et al., 2013). On the contrary, addition of
SSD increased the fiber diameter (Table 1), which can be explained by
high molecular weight and relatively low polarity of sulfadiazine. Using
M. Mohseni, et al. International Journal of Pharmaceutics 564 (2019) 350–358

Table 1
Average fiber diameter of PCL, PVA with different concentration of AgNPs and SSD.
Nanofiber PVA AgNP1 AgNP5 AgNP10 SSD1 SSD5 SSD10 PCL

Average fiber diameter (nm) 360 ± 10 250 ± 15 230 ± 15 190 ± 20 450 ± 10 530 ± 20 700 ± 20 370 ± 10

Fig. 2. a) FESEM of Silver nanoparticles. b) DLS analysis of silver nanoparticles showing the average diameter of 16.39 nm.

Fig. 3. Elemental mapping analysis of PVA nanofibers conjugated with AgNPs.

353
M. Mohseni, et al.
Fig. 4. XRD pattern of AgNPs incorporated with PVA nanofibers.
FESEM analysis, average diameter of nanoparticles was measured
10 nm (Fig. 2, a). To acquire a more accurate measurement for the size
of nanoparticles, DLS analysis was conducted. The mean size of AgNPs
at optimum condition was recorded 16.39 nm and the range of nano-
particles was from 13.4 to 24.7 nm (Fig. 2, b). Elemental mapping
analysis also showed homogenous distribution of AgNPs within the PVA
fibers. The presence of AgNPs into nanofibers was also confirmed by
evaluating the crystal structures of scaffolds utilizing XRD analysis
(Fig. 4). Diffraction peaks at 2θ = 33°, 40° and 44.5° are indexed to
(1 1 1) and (2 0 0) planes of crystalline silver (Madhumathi et al., 2010;
Vimala et al., 2010).
Elastic modulus of wound dressings was calculated as the initial
slope of tensile tests (Table 2). PCL/PVA scaffolds indicated
56.07 ± 5.1 MPa. Addition of silver components made the PCL/PVA
scaffolds more flexible. AgNP10 with the elastic modulus of
15.61 ± 0.35 MPa had the lowest elastic modulus.
3.2. Antibacterial test
The antimicrobial activity of scaffolds containing AgNPs and SSD
was assessed by evaluating the inhibition zone against S. aureus (Fig. 5).
In contrast to the control group (PCL/PVA), the samples containing
AgNPs or SSD had a clear inhibition zone confirming their anti-
microbial properties. The smallest and largest inhibition zones were
observed for AgNP1 (1.5 ± 0.2 mm) and SSD10 (4.2 ± 0.3 mm), re-
spectively.
By comparing the samples with similar concentrations of SSD and
AgNPs, it was revealed that these two groups of silver components have
a very close antimicrobial activity. However, this activity is strongly
dependent on the concentration of silver components. As the con-
centration of SSD or AgNPs increased, the inhibition zone increased, but
not with a linear correlation. Increasing the concentration from 1 wt%
to 5 wt% caused a 2-fold extension of inhibition zone while increasing
the concentration from 5 wt% to 10 wt% increases the inhibition zones
slightly, less than 10%. The antimicrobial activity of the wound dres-
sings was also compared with commercialized wound dressings
(Table 3). It was revealed that PCL/PVA nanofibers conjugated with
Table 2
Elastic modulus of scaffolds under uniaxial tensile test.
Scaffold Elastic modulus (MPa)
PCL/PVA 56.07 ± 5.1
SSD1 36.19 ± 1.7
SSD5 27.52 ± 1.1
SSD10 18.14 ± 0.79
AgNP1 29.25 ± 1.2
AgNP5 20.73 ± 0.33
AgNP10 15.61 ± 0.35
354
International Journal of Pharmaceutics 564 (2019) 350–358
SSD and AgNPs provides highly stronger antimicrobial activities.
3.3. Cytotoxicity studies
Indirect cytotoxicity test was performed using MTT assay with
human fibroblast cells at 1, 3 and 7 days to evaluate the biocompat-
ibility of wound dressings (Fig. 6). The control group (PCL/PVA)
showed the highest biocompatibility, whereas, the addition of SSD or
AgNPs decreased cell viability, which indicates the effect of toxicity of
particles against fibroblast cells (Fig. 6). With a direct comparison of
SSD and AgNPs at similar concentrations, it was concluded that AgNPs
has higher biocompatibility than SSD over 7 days of in vitro culture.
Compared to SSD, AgNPs showed more than 40% and 30% cell viability
at the concentration of 5 wt% and 10 wt%, respectively. Interestingly,
the biocompatibility of AgNP10 is even higher than SSD5, which in-
dicating significant differences in biocompatibility of these two groups
of silver components.
3.4. In vivo experiments
The wound dressings with the concentration of 5 wt% and 10 wt%
were chosen for in vivo implantation since 1 wt% showed very low
antimicrobial activity. The scaffolds indicated a good handling along
with high adhesion to the wound bed. This hydrophilicity property
allows having a moisturized wound bed with the wound fluid, which
contains various cytokines and growth factors and provides a suitable
circumstance for the cell proliferation. The wound healing progress was
assessed through wound closure measurement at 10th, 20th and 30th
day (Figs. 7 and 8) and histological analysis at 10th and 30th day
(Fig. 9).
Compared to the control group, the group treated with PCL/PVA
dressings indicated 26%–54% larger area of closed wound closure over
30 days showing the positive effect of hydrophilic wound dressings to
keep the wound bed moisturized with the wound fluid. Furthermore,
the groups treated with antimicrobial wound dressings showed higher
healing rate compared to the group with SSD cream or only PCL/PVA
dressings. Additionally, the increased concentration of both SSD and
AgNPs from 5 wt% to 10 wt% enhanced healing rate significantly. In
the first 10 days, AgNP10 and SSD10 showed 58% and 47% bigger area
of closed wound closure compared to AgNP5 and SSD5, respectively.
However, at a similar concentration of SSD and AgNPs, the groups
treated with AgNPs indicated a larger closed area over 30 days.
In agreement with wound closure measurements, the histological
analysis indicated a faster healing rate for the groups treated with
wound dressing containing AgNPs and SSD. By comparing the control
and PCL/PVA groups with antimicrobial wound dressings at 10th day,
it was observed that control and PCL/PVA groups have significantly
more inflammatory cells, and the presence of silver components lead to
a reduction of the inflammatory response. Additionally, the increase of
antimicrobial agents accelerated angiogenesis as the SSD10 and
AgNP10 indicated second angiogenesis after10 days while other groups
were still at initial stages of angiogenesis.
The absence of antimicrobial agent in control and PCL/PVA groups
caused a delay in wound healing stages leading to the abnormal and
loose dermal matrix with the lack of mature collagen fibers after
30 days. The groups with higher concentration of Ag components
showed mature dermal matrix with more collagen fibers and fibroblast
cells. By comparing the length of the epithelial layer, it was concluded
that AgNP10 has the maximum length of epithelial layer with mature
collagen fibers, while the control group has the minimum epithelial
layer with weak collagen fibers. In agreement with the wound closure
measurement, the groups with AgNPs indicated more epithelialization
at the same concentration of SSD.
M. Mohseni, et al. International Journal of Pharmaceutics 564 (2019) 350–358

Fig. 5. Antimicrobial characterization of wound dressings against Staphylococcus aureus using the inhibition zone measurement technique (n = 5).

4. Discussion has also attracted attention due to exclusive characteristics. However,

In our previous studies, we have developed platforms for enhancing
the wound healing process (Mohseni et al., 2016; Shamloo et al., 2018).
Here, we aimed to develop antimicrobial wound dressings loaded with
SSD or AgNPs and make a direct comparison between these two groups
of antimicrobial agents to evaluate their efficiency in wound healing
process. Silver sulfadiazine is one of the most popular antimicrobial
agents used extensively in wound healing. However, there is a limited
number of studies focusing on wound dressings loaded with SSD. AgNPs
there is still a controversial issue regarding the toxicity and bio-
compatibility of silver nanoparticles. This study provides a platform to
compare antimicrobial activity and toxicity of SSD and AgNPs as well as
their potential in full-thickness wounds.
PCL/PVA/SSD and PCL/PVA/AgNPs wound dressings were fabri-
cated through the co-electrospinning method, a reproducible technique
to develop highly porous scaffolds with micro and nano-structures. In
the first part of the study, MTT assay and an antimicrobial test were
conducted at various concentrations of SSD and AgNPs. It was found

Table 3
Antimicrobial activity of commercialized wound dressings against Staphylococcus aureus using inhibition zone measurement technique (Kohta and Ohyabu, 2015).
Commercialized wound dressings Biohesive Ag Aquacel Ag Algisite Ag Mepilex Ag PolyMem Ag

Inhibition Zone (mm) 0.2 ± 0.1 0.6 ± 0.2 1 ± 0.2 ND ND

355
M. Mohseni, et al.
Fig. 6. In vitro biocompatibility of wound dressings loaded with different concentrations of AgNPs via MTT assay of fibroblast cells after 1, 3, 7 days (n = 5).
that antimicrobial scaffolds are resistant against S. aureus, and their
activity is enhanced by increasing the concentration of SSD or AgNPs. It
was also observed that SSD and AgNPs have very similar antimicrobial
activity at similar concentrations. The biocompatibility of the wound
dressings were also evaluated though in vitro fibroblast cell culture for
7 days. Cellular proliferation was reduced by increasing the con-
centration of SSD and AgNPs. However, at a similar concentration, SSD
showed more toxicity against fibroblast cells compared to AgNPs.
Subsequently, in a similar range of antimicrobial activity, AgNPs are
less toxic, and probably better candidates for antimicrobial wound
dressings. At the second part of the study, the capability of the wound
dressings were examined through in vivo experiments using a chronic
wound healing model. PCL/PVA nanofibers showed a good flexibility
and hydrophilicity allows keeping the wound closure moisturized with
wound fluid, which is enriched with cytokines and growth factors. The
wound dressings loaded with silver components indicated efficient
antimicrobial activity, which caused a noticeable reduction in in-
flammatory response along with accelerating proliferative and re-
modeling phases. Furthermore, the increase of silver components from
Fig. 7. Wound closure measurement at 10, 20 and 30 days showing the rate of re-epithelialization (n = 5).
356
International Journal of Pharmaceutics 564 (2019) 350–358
5 wt% to 10 wt% indicated accelerated angiogenesis and epithelializa-
tion significantly. By comparing the proficiency of SSD and AgNPs at a
similar concentration, it was concluded that AgNPs have faster healing
rate, probably caused due to the higher biocompatibility observed
during the in vitro study. AgNP10 showed the fastest epithelization and
dermal matrix regeneration with mature collagen fiber mimicking the
normal tissue structure. These results are in agreement with a recent
clinical study, where nano-silver and silver sulfadiazine were compared
for treatment of II burn wound (Liu et al., 2017). It was concluded that
nano-silver significantly promotes wound healing procedure and reg-
ulates the infection and epithelization more efficiently compared to
silver sulfadiazine. In another recent study, the negative effects of SSD
on rabbit ear wound model has been reported. SSD impaired re-
epithelization and caused hypertrophic scar formation (Qian et al.,
2017). These studies and our recent comparison between SSD and
AgNPs indicated that AgNPs are more promising antimicrobial agents
and lead to a faster and efficient reepithelization.
M. Mohseni, et al. International Journal of Pharmaceutics 564 (2019) 350–358

Fig. 8. The photographs of wound closures during the healing process after 10, 20 and 30 days.

357
M. Mohseni, et al. International Journal of Pharmaceutics 564 (2019) 350–358

Conflict of interest

None.

References

Boateng, J.S., et al., 2008. Wound healing dressings and drug delivery systems: a review.
J. Pharm. Sci. 97 (8), 2892–2923.
Boateng, J., Catanzano, O., 2015. Advanced therapeutic dressings for effective wound
healing—a review. J. Pharm. Sci. 104 (11), 3653–3680.
Braghirolli, D.I., Steffens, D., Pranke, P., 2014. Electrospinning for regenerative medicine:
a review of the main topics. Drug Discovery Today 19 (6), 743–753.
Cukjati, D., Reberšek, S., Miklavčič, D., 2001. A reliable method of determining wound
healing rate. Med. Biol. Eng. Comput. 39 (2), 263–271.
Fang, J., et al., 2008. Applications of electrospun nanofibers. Chin. Sci. Bull. 53 (15),
2265.
Ge, Y., et al., 2014. Nanomedicine: Principles and Perspectives. Springer.
Gora, Aleksander, et al., 2015. Silver nanoparticle incorporated poly(L-lactide-co-glyco-
lide) nanofibers: evaluation of their biocompatibility and antibacterial properties. J.
Appl. Polym. Sci.
Hebeish, A., et al., 2014. Antimicrobial wound dressing and anti-inflammatory efficacy of
silver nanoparticles. Int. J. Biol. Macromol. 65, 509–515.
Jun, I., et al., 2018. Electrospun fibrous scaffolds for tissue engineering: viewpoints on
architecture and fabrication. Int. J. Mol. Sci. 19 (3), 745.
Kohta, M., Ohyabu, Y., In vitro parallel evaluation of antibacterial activity and cyto-
toxicity of commercially available silver-containing wound dressings, 2015.
Landén, N.X., Li, D., Ståhle, M., 2016. Transition from inflammation to proliferation: a
critical step during wound healing. Cell. Mol. Life Sci. 73 (20), 3861–3885.
Li, G., et al., 2013. Super hydrophilic poly (ethylene terephthalate)(PET)/poly (vinyl
alcohol)(PVA) composite fibrous mats with improved mechanical properties pre-
pared via electrospinning process. Colloids Surf. A: Physicochem. Eng. Aspects 436,
417–424.
Li, Chenwen, et al., 2013. Silver nanoparticle/chitosan oligosaccharide/poly(vinyl al-
cohol) nanofibers as wound dressings: a preclinical study. Int. J. Nanomed.
Liu, Z., et al., 2017. The efficacy of nano-silver and silver sulfadiazine for degree II burn
wound: a meta-analysis. Biomed. Res. 28 (9), 3880–3885.
Madhumathi, K., et al., 2010. Development of novel chitin/nanosilver composite scaffolds
for wound dressing applications. J. Mater. Sci.: Mater. Med.
Mohseni, M., et al., 2016. Antimicrobial wound dressing containing silver sulfadiazine
with high biocompatibility: in vitro study. Artif. Organs 40 (8), 765–773.
Murphy, P.S., Evans, G.R., 2012. Advances in wound healing: a review of current wound
healing products. Plast. Surg. Int. 2012.
Ning, C., et al., 2015. Concentration ranges of antibacterial cations for showing the
highest antibacterial efficacy but the least cytotoxicity against mammalian cells:
implications for a new antibacterial mechanism. Chem. Res. Toxicol. 28 (9),
1815–1822.
Percival, S.L., et al., 2012. A review of the scientific evidence for biofilms in wounds.
Wound Repair Regener. 20 (5), 647–657.
Percival, S.L., McCarty, S.M., Lipsky, B., 2015. Biofilms and wounds: an overview of the
evidence. Adv. Wound Care 4 (7), 373–381.
Qian, L.-W., Fourcaudot, A.B., Leung, K.P., 2017. Silver sulfadiazine retards wound
healing and increases hypertrophic scarring in a rabbit ear excisional wound model.
J. Burn Care Res. 38 (1), e418–e422.
Shamloo, A., et al., 2018. Accelerated full-thickness wound healing via sustained bFGF
delivery based on a PVA/chitosan/gelatin hydrogel incorporating PCL microspheres.
Int. J. Pharm. 537 (1–2), 278–289.
Shamloo, A., Mohammadaliha, N., Mohseni, M.J.J.o.b., 2015. Integrative utilization of
microenvironments, biomaterials and computational techniques for advanced tissue
engineering. J. Biotechnol. 212, 71–89.
Singh, R., Singh, D., 2014. Chitin membranes containing silver nanoparticles for wound
dressing application. Int. Wound J. 11 (3), 264–268.
Vimala, K., et al., 2010. Fabrication of porous chitosan films impregnated with silver
nanoparticles: a facile approach for superior antibacterial application. Colloids Surf.
B: Biointerfaces 248–258.
Yao, R., et al., 2013. A biomimetic physiological model for human adipose tissue by
Fig. 9. The histology analysis after 10 and 30 days of healing process using
adipocytes and endothelial cell cocultures with spatially controlled distribution.
hematoxylin and eosin staining of regenerated tissue. Biomed. Mater. 8 (4), 045005.
You, C., et al., 2017. Silver nanoparticle loaded collagen/chitosan scaffolds promote
wound healing via regulating fibroblast migration and macrophage activation. Sci.
Acknowledgements Rep. 7 (1), 10489.
Zhang, H., et al., 2018. Silver nanoparticles-doped collagen–alginate antimicrobial bio-
The authors acknowledge Sharif University of Technology for sup- composite as potential wound dressing. J. Mater. Sci. 53 (21), 14944–14952.

porting this study.

358