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Chitosan and Silver Sulfadiazine

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Wound Dressing...

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Trends Biomater. Artif. Organs, 30(1), 32-40 (2016) http://www.sbaoi.org/tibao

Original Article

Chitosan and Silver Sulfadiazine Immobilization onto

Silicone Membrane for Wound Dressing Applications

Elham Babaiea,b,*, Hamid Mirzadehb, Hamid Keshvarib, Atefeh Soloukb, Azadehsadat Hashemi Doulabib
a
Department of Bioengineering, University of T oledo, P.O. Box: 43606/ 3390, Toledo, Ohio
b
Department of Biomedical Engineering, Amirkabir University of T echnology (Tehran Polytechnic),
P.O. Box: 15875/4413, T ehran, Iran

Received 12 January 2016; Accepted 7 June 2016; Published online 10 June 2016

Bilayer wound dressing with antibacterial capability was prepared via two-step oxygen plasma treatment using
polydimethylsiloxane (PDMS) and chitosan incorporated with silver sulfadiazine (AgSD) for treating acute burn
wounds. An AgSD-incorporated chitosan membrane was immobilized onto PDMS via acrylic acid (AAc) as a spacer. To
reach the maximum immobilization of chitosan and antibiotic of AgSD, different treatment times were applied to
obtain a maximum grafting density of AAc. The time of 35 s and 180 s were chosen as plasma pretreatment time and
plasma polymerization time, respectively. Physical characterization of the prepared wound dressing via ATR-FTIR,
SEM, contact angle and inductively couple plasma methods confirmed the immobilization of chitosan incorporated
with AgSD. The composition with maximum graft density of spacer was chosen as an optimum formulation for further
studies due to suitable surface properties. Biocompatibility and antibacterial assesses were also performed, demonstrating
this new type of bilayer wound dressing incorporated with AgSD possessing graft density ca. 87 g.cm-2 may be effective
in the treatment of infected wounds by promoting cell responses and decreasing the risk of infection.

Introduction acceleration of re-epithelialization and regeneration of normal

Autograft, allograft, or synthetic skin substitutes are of different
ways for burn wound treatment. Due to the antigenicity or the
limitation of donor sites, polymeric wound dressing could
obviate these limitations. Therefore, to protect a skin defect
from infections and dehydration in the intervening period
between hospitalization and grafting, temporary closure of a
wound with the use of biopolymeric material has become
ordinary recently [1, 2]. Among the wound dressings, bilayer
artificial skin or wound dressing composed of a dense top
layer (skin layer) and a lower porous layer (sublayer) may be
an excellent and promising one [2]. The skin layer such as
polydimethylsiloxane (PDMS) which possess physiological
inertness, low toxicity, low modulus and good mechanical
properties can prevent bacterial penetration and dehydration
of the wound surface [3, 4] while the sublayer such as chitosan
is designed to achieve high adsorption for fluid drainage of the
wound and infiltration by fibroblasts for tissue regeneration.
Chitosan is well known for accelerating the healing of wounds
in humans while stimulated the migration of
polymorphonuclear (PMN) and mononuclear cells due to
possessing special functional groups. This suggests the
skin that minimizes scar formation in wounded areas [5-7]. In
artificial skin approach, covalently immobilization of sublayer
like chitosan onto skin layer like PDMS is the efficient and
reliable method reported earlier [8-10]. In order to immobilize,
synthetic polymers of skin layer (such as PDMS) often
required selective modification to introduce specific functional
groups of spacer like AAc to the surface for the binding of
biomolecules [11-13]. Graft polymerization is an attractive
way in which a desired monomer can be grafted onto the skin
layer before immobilization step. Among the common methods
in graft co-polymerization, the two-step plasma (TSP) graft
co-polymerization is a prominent and efficient technique
developed by our group [3]. In this TSP technique, a polymeric
substrate treated with plasma immersed in monomer solution,
and then undergoes a second step of plasma treatment that
leads to co-polymerization of preadsorbed monomer. This
technique possess benefits like lower graft polymerization time
length, no controlling requirement on pH of monomer solution,
higher grafting amount, and producing more homogeneous
morphology and topology of the grafted surface compared to
the other grafting method including one step plasma or laser
technique.

*
Coresponding author: Dr. Elham Babaie;
Besides of promoting cellular function, fluid drainage and good
E-mail: Elham.babaie@rockets.utoledo.edu mechanical properties required for potentially ideal wound

32
 Chitosan and Silver Sulfadiazine Immobilization onto Silicone Membrane for Wound Dressing Applications
dressing, risk of infection beneath the dressing is another issue
associated with dressing [14, 15]. Infectious organisms
preferentially invade wounds beneath dressing materials, leading
to serious infections. This requires removal of the wound dressing
and excision of cutaneous wounds. Thus, wound dressings or
artificial skins containing antibiotic agents have been developed
to decrease wound infection, and the laborious replacement of
wound dressings that can avoid damage to the newly formed
epithelium caused by replacement [2]. In the present study, we
developed a bilayer wound dressing that consists of an upper
skin layer of PDMS and a porous sublayer of chitosan that
have the ability to control the release of antibiotic agent. Silver
sulfadiazine (AgSD), an effective and widely used antibiotic
agent for burn injuries in humans [16], is used as an antibacterial
drug for the treatment of infected wounds. Acrylic acid (AAc)
employed as a spacer to covalently immobilized chitosan onto
silicone. AAc was simultaneously grafted onto the surface of
PDMS films using two-step oxygen plasma treatment (TSPT).
Then the mixture of chitosan/AgSD was immobilized onto AAc
grafted silicone. The in vitro biocompatibility, antibacterial
activity and physical characterization of bilayer wound dressing
are evaluated. To the best of our knowledge, there is no report
in the literature to discuss about bilayer chitosan/AgSD
immobilized onto AAc grafted silicone and the properties as a
potential film for wound dressing applications.
Materials and methods
The silicone rubber, Silastic MDX4-4210 medical grade
elastomer was purchased from Dow Corning Corp (Midland,
MI). The procedure of preparing silicone film was described
elsewhere [3]. The AAc was bought from Fluka (Buchs,
Switzerland). AAc was redistilled under vacuum condition to
make it free from the inhibitor [3]. N-(3-dimethyl aminopropyl)
N2-ethyl carbodiimide hydrochloride (EDC) was purchased from
Merck and used for activating COOH groups on the AAc grafted
silicone. Chitosan (MW = 400KD and 85% deacetylated) was
obtained from Sigma Aldrich, USA. Silver sulfadiazine was
obtained by Iran Najoo lab. Other reagents were reagent grades
and used without any further purification.
Plasma treatment step
TSPT were carried out onto silicone samples according to our
Figure 1: The Change of graft amount (g.cm-2) during
oxygen plasma treatment time 60 W, 6 10-1 mbar (n= 3,
meanSD)
previous works [3, 17, 18]. Briefly, Nano-RF-PC (Diener
Electronic GmbH, Germany) apparatus was utilized for both
plasma pretreatment and copolymerization of silicone films.
The films were placed on the bottom of the reaction chamber,
which was evacuated to 6 10-1 mbar, and pretreated with 60
W of oxygen plasma up to 35 second. Then, the plasma
pretreated films were immersed in aqueous monomer solutions
with the given ratios of AAc for up to 30 min at room
temperature, and dried at the same temperature. The dried
plasma pretreated silicone films with a preadsorbed layer of
reactive monomer of AAc on their surfaces were placed into the
reaction chamber for plasma graft copolymerization for up to 3
min. The time of treatment was chosen based on our previous
results to obtain maximum graft density (GD) as summarized
in Table 1 [3]. Then, by using Soxhlet extraction in distilled
water for 72 h the residual monomers and homopolymers were
removed.
Determination of GD
The carboxyl group density on the PAAc-g-PDMS surface was
measured by a colorimetric method using toluidine blue O
staining [13]. Grafted samples were immersed in an aqueous
solution of toluidine blue (5 10-4 M) for 6 h at 30 C. Then
samples were rinsed with an aqueous solution of NaOH (5 10-
3
M) in order to remove uncomplexed dye. A standard series was
done with seven different concentrations between 4 10-6 and
5 10-5 M, which allows us to determine the concentration of
decomplexed toluidine blue using the molar extinction coefficient.
Decomplexation of toluidine blue occurs by immersing samples
in an aqueous solution of acetic acid (50 vol.%) for 24 h.
Concentration of decomplexed toluidine blue is measured via
UV-VIS spectrophotometry (Shimudza, 1650PC, Japan) at 633
nm. A different formulation of silicone plasma treated film was
shown in Table 1.
Immobilization of CS/AgSD
PAAc-g-PDMS films were placed in 10 mg.mL-1 of an aqueous
solution of (EDC) at room temperature for 2 h to activate
carboxyl groups in the grafted PAAc chains. The activated PAAc-
g-PDMS films were immersed into CS and AgSD in 0.5% (v/v)
lactic acid solution (Table 2) at 4 C for 24 h to allow the
activated carboxyl groups in the grafted PAAc chains and amino
groups in the CS to form covalent bonds. During immobilization
step, the pH of CS solution was 3.0. The AgSD concentration
in CS solution was 1% (w/w) (in relation to CS mass). Silver
sulfadiazine concentration was chosen based on previous study
that showed a solubility threshold of AgSD in CS solution [19].
All the PDMS-g-PAAc-CS/AgSD samples were rinsed with
distilled water many times and then the samples were stored in
desiccators at 4 C before use.
Inductively couple plasma methods (ICP)
To evaluate the presence of AgSD in wound dressing, silver
concentrations were measured in a immobilization solution
containing only CS and AgSD (Table 2) before and after soaking
PDMS-g-AAc (Max sample refer to Table 1) into the solution
by inductively coupled plasma (ICP-OES, VISTA-PRO-
VARIAN) method. This ICP apparatus has 0.06 ppm silver
assay accuracy. The difference in silver concentration of the
immobilization solution before and after of immobilizing step
was considered as AgSD loading in wound dressing film.
Contact angle and surface tension measurements
The static contact angles of the untreated film, PAAc-g-PDMS
33
 E. Babaie, H. Mirzadeh, H. Keshvari, A. Solouk, A.H. Doulabi

Figure 2: ATR-FTIR of (a) unmodified silicone (control), (b) silicone grafted AAc and immobilized CS/AgSD (Max-g-
CS/AgSD) at 60 W and 0.6 mbar

Figure 3: SEM micrographs of (a) unmodified silicone (control), (b) oxygen plasma treated silicone (3 min, 60 W, 0.6
mbar) (c) PDMS-g-AAc (Max, 60 W, 0.6 mbar) (d) Max-g-CS/AgSD; scale bar 6 m

34
 Chitosan and Silver Sulfadiazine Immobilization onto Silicone Membrane for Wound Dressing Applications

films and also immobilized with CS/AgSD were measured by

the sessile drop method using Kruss G10 goniometer (Kruss
GmbH, Germany) contact angle measurement equipment. A 5
L double distilled water droplet was used for each point and
the contact angle was recorded after 1 min [13]. The average
values of three measurements on different points of each sample
were recorded. Moreover, double distilled water and
diiodomethane attributed to polar and disperse parts of the
sample were used to calculate surface tensions using Owens-
Wendt equation [20].

ATR-FTIR measurement

Attenuated total reflectance Fourier transform infrared

Figure 4: SEM cross-sectional micrograph of Max-g-CS/
AgSD, magnification 200, scale bar 150 m
spectroscopy (ATR-FTIR) was used in order to confirm PAAc
and CS grafting onto silicone plasma treated film in comparison
with untreated film. A KRS5 prism (Bruker Optik GmbH,
Germany) FTIR spectrometer was applied with incident contact
angle 45 and progressive scanning range from 4000 cm-1 to 600

Figure 5: Cell adhesion, onto: (a) untreated silicone (b) Min-g-CS/AgSD (c) Med-g-CS/AgSD (d) Max-g-CS/AgSD
(e) tissue culture plate polystyrene 400 and number of cell adhered vs GD, n=3, meanSD, Pvalue compared to
untreated silicone

35
 E. Babaie, H. Mirzadeh, H. Keshvari, A. Solouk, A.H. Doulabi
cm-1.
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed on gold-
coated samples using a Polaron sputter coater. A Cambridge S-
360 SEM operating typically at an accelerating voltage of 10
kV in secondary electron mode to ensure a suitable image
resolution was employed for morphology measurements of
untreated film, plasma treated film, PAAc-g-PDMS film and
also immobilized samples.
In vitro assay
The immobilized silicone films with different PAAc graft
densities were immersed into sterilized saline for 12 h just before
cell culture. The mouse fibroblast cells (L929) were gently gifted
from National Cell Bank, Pasteur Institute of Iran. The cells
were maintained in RPMI-1640 growth medium (Sigma Chemical
Company, St. Louis, USA), supplemented with 10% heat-
inactivated fetal bovine serums (Gibco, BRL), 100 IU mL-1
streptomycin. A routine subculture was employed to prepare
the cell line [21]. The cells were incubated in a humidified
atmosphere of 5% CO2 at 37 C. After one week incubation, the
monolayer was harvested by trypsinization. The samples were
placed into each well using a multi-well plate with 5 mL of cell
suspension with 4105 cell.mL-1 concentration and seeded in
Figure 6: SEM micrographs showing fibroblast culture for 2 days on the surface (a) untreated silicone (control) and
plasma treated silicone including (b) Min-g-CS/AgSD; (c) Med-g-CS/AgSD; (e) Max-g-CS/AgSD
36
each well, keeping one well as a control without any sample,
and then maintained them in the incubator for 48 h. The samples
were removed from the well, washed with phosphate buffer
saline solution twice, placed on a glass slide and fixed with
ethanol and then stained with 5 % Giemsa (Sigma-Aldrich). All
the samples were air-dried and then cover slips were mounted
on them. The samples were examined by an inverted light
microscope (E200, Nikon, Japan).
Antibacterial activity
The quantitative antimicrobial activities of the unloaded and
AgSD loaded films were determined using a viable cell count
method. The results were presented as surviving bacteria and
population reduction in logarithmic scale on the test pathogenic
bacterium. All film samples (CS and Max-g-CS/AgSD) were cut
into square pieces (1.5x1.5 cm2) and placed in individual sterile
flasks containing the test bacteria, Escherichia coli; E. coli (ATCC
8739) at a concentration of 5.2x106 CFU.mL-1 in the Nutrient
Broth solution (Merck, Germany). The inoculated broth was
adjusted photometrically at 600 nm to a cell density equivalent
to approximately 0.5 McFarland standards (1.5108 CFU/mL).
The suspensions were then diluted in Nutrient Broth solution
to reach the final concentration of about 106 CFU.mL-1. The
tubes were kept in an incubated shaker at 37C and 100 rpm.
During the incubation, the cell viability count of the medium
was measured every hour for 6 h. At the intervals, 1 mL of the
 Chitosan and Silver Sulfadiazine Immobilization onto Silicone Membrane for Wound Dressing Applications

solution was diluted with 9 mL of sterilized distilled water, and
decimal serial dilution was performed and repeated. Then 100
L of the solution was taken out and quickly spread on the
surface of the plate containing nutrient agar (Merck, Germany).
After inoculation, the plates were kept at 37C, and the colonies
were counted after 24 h. The number of colonies on each plate
was counted and reported as CFU.mL-1. An inoculum of cell
suspension in a flask with no film sample was used as a control.
The population reduction of the test organism were calculated
using equation (1) [22]:
Log population reduction = Log cell count of control - Log
survivor count on sample (1)
Statistical Analysis
Analysis of Variance (ANOVA) and linear regression were the
main statistical tools used for data analysis. The unpaired
students t-tests were used for all statistical analyses. Results
are expressed as meanstandard deviation (SD).
Results and Discussion
Determination of graft density (GD)
of untreated silicone and surface modified silicone films were
summarized in Table 3 as water contact angle and surface tension.
The contact angle of silicone surface decreased due to grafting
of PAAc and also immobilizing of CS/AgSD onto surface of
plasma treated silicone. These phenomena could be associated
with introducing hydrophilic groups such as carboxyl and amine
functional groups onto surface of the silicone film. The presence
of these functional groups may possibly improve the
hydrophilicity of biomaterials surface and suggest additional
interactions with water molecules that influence the wettability
of the films. As presented in Table 3, the more increase in
plasma treatment time, the more decrease in contact angle,
demonstrating that these materials have good hydrophilicity
thereby they are suitable for cell supporting [23]. Previous
studies revealed that the fibrobroblast cells show maximum
adhesion to the surface of materials with contact angle in the
range of 60and 80 [24]. Thus, the as-prepared membranes
have suitable surface hydrophilicity for effective contribution
in cellular activity. Changes in the surface tensions of the samples
are also presented in Table 3. More GD of AAc caused the more
amount of CS immobilization which was reported earlier [8].
According to the results, grafting of hydrophilic monomer (AAc)

The AAc grafting onto silicone films was carried out to develop
a surface which is carrying a high density of carboxyl groups.
The overall process of AAc grafting through TSPT involved the
pretreatment of silicone film with oxygen plasma and subsequent
exposure to AAc solution. Produced polar groups (hydro
peroxide radicals) on the surface of chemically inert silicone due
to plasma pretreatment helped it out to physically react with
hydrophilic monomer (i.e. AAc) via hydrogen bonding, and the
produced peroxide groups may act as initiators in
copolymerization step. We chose a different pretreatment time
and the polymerization time of plasma to produce optimum
conditions in order to get the maximum amount of GD by
introducing maximum peroxides on the surface. Peroxides are
known to be the species responsible for initiating the graft
copolymerization when silicone reacts with AAc [13]. In the
next step, the dried plasma pretreated silicone films with a
preadsorbed layer of AAc monomer on their surfaces were placed
into the plasma reaction chamber for plasma graft
copolymerization. During propagation step of the plasma
copolymerization, the preadsorbed AAc react with radicals,
which create as a result of plasma treatment, and graft onto the
surface of the film until the copolymerization reaction
terminates. In fact, before the second step of plasma treatment
co-polymerization would not occur as also reported earlier [3].
The amount of grafted copolymer was sensibly affected by
pretreatment time length and the polymerization time length.
The relationship between the amount of grafted copolymer per
square centimeter and plasma pretreatment duration and
polymerization time is plotted in Fig. 1. As shown in Fig. 1
with increasing plasma treatment time the amount of GD
increased from 28.10510.147 g.cm-2 for the Min sample to
32.2999.163 g.cm-2 for the Med sample, and 86.96910.634
g.cm-2 about the Max sample. Based on results of our previous
study, there is an optimum t in treatment time. Further increment
above optimum point affects unfavorably the amount of GD.
This can be explained by the fact that further increase in time
causes plasma etching rather than co-polymerization [3].
Figure 7: Survival of E. coli (Log10 CFU.mL1) during 6 h
Surface wettability and surface tension exposure to CS and Max-g-CS/AgSD film loaded with
The contact angle test is used to obtain hydrophilic or AgSD, (a). Population reduction of E. coli (Log10 CFU.
hydrophobic surface properties. The changes in hydrophilicity mL1) during 6 h exposure to CS and Max-g-CS/AgSD

37
 E. Babaie, H. Mirzadeh, H. Keshvari, A. Solouk, A.H. Doulabi
Table 1: AAc grafted PDMS via TSPT with variable process time
with fixed power of 60 (W) and AAc concentration 30% (v/v)
and hydrophilic group (CS/AgSD) onto the surface of silicone
films increased the surface tension from 22.670.32 to
40.351.28 (n= 3, p < 0.005). On the other hand, p (polar part
of surface tension) also significantly increased for untreated
silicone and PDMS-g-AAc-g-CS/AgSD with maximum amount
of GD, respectively, indicating an increment in the polar groups
existing on the surface.
ATR-FTIR spectra
The presence of the grafted AAc and CS was confirmed by
comparing the ATR-FTIR spectra of unmodified and modified
silicone film (Fig. 2). A peak at 1715 cm-1 was observed in the
spectrum of the Max-g-Cs/AgSD sample (Table 2) that was
due to the grafted carboxyl groups (C=O) onto the silicone
surface in comparison with unmodified silicone (Fig. 2 (a, b)).
The two characteristic absorption bands of CS appearing at
consisted of two bands (medium intensity) at 1645 cm-1 and
1542 cm-1; corresponds to the grafted amine groups of CS (amide
I and amide II) onto the surface of Max-g-CS/AgSD (Fig. 2 (a,
b)) [8]. However, the intensity of amide band at 1645 cm-1 was
lowered that could not be detected in FTIR spectra likely due
to adsorption of Ag+. In the adsorption process amino groups
become protonated resulted in changing the intensity of amide
band. The presence of CH2OH groups of polysaccharide of
chitosan is attributed to the peak at 1450 cm-1 which is also
another site of Ag+ sorption [25, 26] while the intensity is very
low due to sorption.
SEM images
Surface morphology of control and modified silicone was
evaluated using SEM micrographs of surface of the films as
illustrated in Fig. 3. SEM micrographs of sample indicated that
oxygen plasma pretreatment made the surface of silicone became
cleaner and smoother due to the plasma etching effect since
small molecules and occasional fragments which were attached
onto the surface were removed. Furthermore, some cracks, which
are shown in Fig. 3 (a, b) could be attributed to the silica-like
layer [3]. However, by grafting PAAc onto silicone plasma
treated film, the surface of silicone became rougher in comparison
with control and oxygen plasma treated silicone (Fig. 3 (c)).
Table 2: Silicone grafted with AAc and immobilized with
CS and AgSD mixture
38
Immobilization of CS/AgSD solution onto the surface of PDMS-
g-AAc changes the surface morphology of silicone which is
grafted by PAAc (Fig. 3 (c, d)). The cross-section of the grafted
layer was observed in SEM micrographs (Fig. 4). According to
Fig. 4 the thickness of the grafted layers was directly depend on
grafted amount. This statement suggests that an increase in the
grafted amount lead to increase the thickness of the grafted
layer. As shown in Fig. 4, cross section of the grafted film is
about 5 m thickness, which is comparable with what already
published [3].
ICP method
The presence of AgSD drug which is known as an antibacterial
agent in the wound dressing was confirmed via ICP methods.
Silver sulfadiazine was dissolved in CS and lactic acid solution.
AgSD bonded with CS and produced chelate structure with CS
[25, 26]. Chitosan contains NH2 functional groups which makes
it capable of binding with silver. The Max-g-CS/AgSD membrane
has 2.5 ppm AgSD content at film with 1 cm x 2 cm.
Cell culture and microscopy images
Affinity of cells for adhesion to the surface is an excellent
indication for studying of cytocompatibility of biomaterials.
The effects of the three different PAAc densities grafted onto
silicone surface and immobilized with CS and AgSD were
quantitatively and qualitatively evaluated on cell adhesion, and
are shown in Fig. 5 (a-f). The mean number of cells cultured on
different surfaces was quantified by Image Pro Plus software
(version 6.0.0.260). As shown in Fig. 5 with increasing GD of
PAAc to increasing treatment time onto the surface of silicone
immobilization of CS and silver sulfadiazine, the more
hydrophilic group such as carboxyl and amine group were
introduced onto the surface of wound dressing, and the more
cells were attached as consequently. It is known that
hydrophilicity property strongly correlates with protein
adsorption, and also cell adhesion. It was suggested that
hydrophilic substrates influence adsorption of serum proteins
in the quality, amount, and conformation that favors adhesion
and subsequent growth [27]. Those factors cause more cell
adhesion on hydrophilic surfaces. In our work, according to
Table 3, the Max-g-CS/AgSD with the lowest amount of water
contact angle ( = 63 6.12) resulted in the most number of
adhering cells which was depicted in Fig. 5 (d). This formulation
showed the best behaviour; however, there is a less significant
difference in cell number between control and Min-g-CS/AgSD
and Med-g-CS/AgSD samples (p <0.05) (Fig. 5 (a, b, c)) in
comparison with the Max-g-CS/AgSD (p <0.005). On the other
hand, after 48 h of fibroblast culture, dense cellular monolayer
covered the Max-g-CS/AgSD samples which are similar to the
tissue culture polystyrene plate; although, the morphology of
 Chitosan and Silver Sulfadiazine Immobilization onto Silicone Membrane for Wound Dressing Applications
Table 3: Surface tension and water contact angle of untreated silicone, PDMS-g-AAc and Min-g-AAc-g-CS/AgSD,
Med-g-AAc-g-CS/AgSD, Max-g-AAc-g-CS/AgSD
n = 3, meanSD, *p < 0.005 compared to untreated silicone
cells in contact with the Max-g-CS/AgSD sample is more conical
than those in tissue culture polystyrene plate (Fig. 5 (d, e)).
The Max-g-CS/AgSD showed greater tendency for entering into
a rapid proliferative growth phase, build up critical cellcell
interactions and grow to confluence on the membranes.
For finding better insight regarding the state of adhered cells,
the morphology of fixed cells was investigated with SEM. Fig.
6 shows the typical SEM micrographs of the adhered cells on
the PAAc-g-PDMS immobilized with CS and AgSD compared
with the unmodified silicone. Anchorage-dependent cells such
as fibroblast need to adhere in order to proliferate [28].
As depicted in Fig. 6 (a), the carboxylic acid functional groups
of PDMS-g-PAAC had a negative effect on the adhesion,
spreading of fibroblast cells, while the amino groups of chitosan
immobilized on the surfaces, showed better adhesion, spreading
(Fig. 6 (b,c,d)). Fortunately, the fibbroblast cells were grown
properly in all chitosan immobilized samples. They were flat
and elongated with spindle-shaped morphology on the surface
of the immobilized membranes confirming their excellent ability
to support cell growth and proliferation. While, the cells in
Med-g-CS/AgSD show more cytoplasm extension comparing
with Min-g-CS/AgSD whereas the round cells can be seen onto
the surface of untreated silicone. The Max-g-CS/AgSD sample
presented the best results among treated membranes (Fig. 6
(d)). Surface properties, especially surface energy have great
effect on the type of protein adsorption and also their
orientation. Protein adsorption is a determining step for the
first phase of cell attachment and further control of cell
morphology, as well as their proliferation capacity. In addition,
it has been proved that since polar and disperse components of
surface energy is a key factor for interfacial interaction, an
optimal distribution of polar and disperse components of
surface energy should be reached [28]. As can be seen from the
surface energy results (Table 3), the Max-g-CS/AgSD samples
have more amounts of cell attachment, more elongated fibroblast
cells and large pseudopodia-like structure, and also it can be
assumed increasing silver ion concentration has no adverse effect
on cell response in all modified samples.
Antibacterial activity
Using wound dressing membranes with promising antibacterial
activity boosts their performance of protection of wounded
area from bacteria likley provide faster healing. By close
inspection of antibacterial dressing materials available in the
market, silver is popular as a powerful broad spectrum
antimicrobial agent to control infection of the eyes, burns, acute
and chronic wounds. For instance, silver containing wound
dressings were found to kill gram-negative bacteria gram-positive
bacteria [25]. To monitor the effectiveness of the cumulative
antibiotic agent release from the films as wound dressings,
antimicrobial activity of CS films and CS loaded with AgSD
were quantitatively evaluated. The surviving bacteria and
population reduction of the films are depicted in Fig. 7 (a,b). As
mentioned in the standard method, at least a 1 Log reduction of
bacterial load is required to claim antibacterial property [29].
All films inhibited the growth of this test strain and reduced the
number of E. coli by approximately at least 3 Log CFU.mL1
comparing to the positive control at each interval. The AgSD-
loaded CS film with maximum GD (Max sample) exhibited higher
antibacterial activity than that of CS film against E. coli. The
CS-AgSD could kill most of the bacteria within 6 h, whereas;
the CS film was bacteriostatic on the bacteria concentration of
about 5.2106 CFU.mL-1. By comparison of CS-AgSD films to
the control without films, a decrement of >7 log cycles after 6 h
of cultivation was observed. In the presence of the AgSD-loaded
CS films, high bacterial inoculations of 5.2106 CFU.mL-1 were
decreased by 99.99% after 1 h and this decrement will continue
to 6 h. It is reported that a slow AgSD releasing rate from the
designed wound dressing allows for better trapping of silver in
the wound than can be achieved with a traditional cream. It also
reduces the potentially cytotoxicity caused by silver [2].
Penetration of Ag through cell wall of bacteria, possibly causes
DNA loses its replication ability and denaturation of cellular
proteins that is one mechanism for antibacterial effect of Ag
[25].
Conclusion
Plasma functionalized surfaces of PDMS with different surface
COOH density used successfully for covalent immobilization
of CS and AgSD to obtain a specific cell response and
antibacterial activity by enhancing the hydrophilicity of surfaces.
The positive effect of the increasing plasma treatment time was
confirmed by a successful fibroblast response with respect to
film with a maximum grafting density of AAc that showed high
cell adhesion properties and also the most cytoplasmic extension.
The presence of AgSD significantly improved the antibacterial
activity of the wound dressing compared to CS film. The findings
of this study are expected to be promising antibacterial wound
dressing for burn treatment.
Acknowledgement
The authors gratefully acknowledge the Iran national science
foundation for financial support and also wish to thank to Dr.
Morteza Daliri from National Institute of Genetic Engineering
and Biotechnology for his helps in cell culture tests.
39
 E. Babaie, H. Mirzadeh, H. Keshvari, A. Solouk, A.H. Doulabi
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