Journal of Drug Delivery Science and Technology 60 (2020) 101962

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Journal of Drug Delivery Science and Technology

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

Research paper

A novel wound dressing material: Pullulan grafted copolymer hydrogel via

UV copolymerization and crosslinking
Humeyra Mert *, Bengi Özkahraman *, Hüdanur Damar
Hitit University, Faculty of Engineering, Department of Polymer Engineering, Cevre Yolu Avenue, 19030, Corum, Turkey

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

Keywords: This study presents the preparation and characterization of pullulan grafted copolymer hydrogel as a new wound
Pullulan dressing material. In this regard, pullulan grafted copolymer hydrogel (PGH) was prepared via UV copolymer­
Hydrogel ization and crosslinking reactions of acrylic acid and itaconic acid onto pullulan. Ampicillin sodium salt loaded
Drug delivery
PGH was also prepared in the same manner with the addition of the drug in the reaction medium. In-vitro drug
Wound dressing
release experiments showed that 67% of the loaded drug was released from the PGHs after 7 days. Swelling
Antibiotic release
studies performed at different pH conditions confirmed the high swelling capacities of PGHs (i.e., 700%). Be­
sides, MTT assay showed that L929 cell viability could overpass 80% for PGHs. These results revealed that the
prepared non-cytotoxic PGHs are promising candidates for wound dressing applications.

1. Introduction Recently, the hydrogels have received considerable attention since

The wound recovery, as a dynamic biological process in the human
body, is accomplished through four phases (i.e., hemostasis, inflamma­
tion, proliferation, and remodeling). The completion of the phases in the
proper sequence and time is essential for a successful wound healing
process [1]. Many factors can complicate the wound healing process,
leading to improper or impaired and prolonged wound healing [2].
Therefore, it is very important to treat the wound promptly according to
the wound care recommendations to complete the healing process
without complications such as infections, tissue necrosis and scar
formation.
The wound dressings have been used for a long time to facilitate the
wound recovery process. They are divided into four groups according to
their physical form (i.e., ointment, film, foam and gel), and one can
choose the appropriate wound dressing depending on the type, depth,
location and grade of the wound [3]. An ideal dressing, regardless of its
type, is expected to show certain properties, such as biocompatibility
and non-toxicity. Besides, some other features such as the ability to
remove the excess exudate and to protect the wound from external ef­
fects, and gas permeability contribute significantly to the rapid wound
recovery [4]. On the other hand, transparency, which contributes to
both wound healing process and patient comfort by allowing visual in­
spection of the wound without removing the dressing, is another desired
feature of an ideal wound dressing [5,6].
they fulfill the most of the aforementioned criteria of an ideal wound
dressing [7]. Their lightly crosslinked and highly hydrophilic nature,
which allows them to mimic the soft tissues, facilitates the absorption of
the excess exudate thereby preventing the accumulation of exudate in
the wound bed. Besides, the proper water content of the hydrogels re­
duces the risk of bacterial infection by allowing them to control the
moist environment of the wound bed [8]. Among the various types of
hydrogels, polysaccharide-based hydrogels have attracted great interest
as wound dressing materials since they have superior properties
compared to their synthetic counterparts [9]. The features such as
biocompatibility and affordable prices distinguish the
polysaccharide-based hydrogels as potential dressing aids [10,11].
Pullulan as a microbial polysaccharide is produced by Aureobasidium
pullulans [12]. In addition to the previously mentioned common ad­
vantages of the polysaccharide-based hydrogels, the pullulan-based
hydrogels are colorless, tasteless, odorless, and flexible with excellent
mechanical properties [13]. The relatively poor biological activity of
pullulan, which limits its utilization in biomedicine field, can be over­
come by the graft polymerization of different monomers onto pullulan
[14,15]. The monomers, which are used in these graft polymerization
reactions, are selected by considering the properties of the final product
such as biocompatibility, biodegradability, non-toxicity, etc. It is known
that the incorporation of itaconic acid (IA) monomer into polymers
provides a unique hydrophilic character to the material [16]. Hence the

* Corresponding authors.
E-mail addresses: hmertbalaban@hitit.edu.tr (H. Mert), bengiozkahraman@hitit.edu.tr (B. Özkahraman).

https://doi.org/10.1016/j.jddst.2020.101962
Received 14 May 2020; Received in revised form 29 June 2020; Accepted 21 July 2020
Available online 4 September 2020
1773-2247/© 2020 Elsevier B.V. All rights reserved.
H. Mert et al.
hydrogels, which have improved water absorption capacities, can be
prepared by the use of IA as a co-monomer [17–19]. In addition, the
presence of IA in the network increases the dissolved oxygen values of
hydrogels, thereby making these IA containing hydrogels particularly
advantageous for the wound dressing applications [20]. Moreover, IA
content in the hydrogel increases the porosity of the structure due to the
electrostatic repulsion between the carboxylate groups of IA, thereby
enabling the sustainable drug release [21]. However, the copolymeri­
zation of IA with appropriate monomers is more common due to the
relatively low solubility of IA in water [16–22]. One potential como­
nomer, that is also biodegradable, is acrylic acid (AA). Although there
are several reports on the preparation and utilization of acrylic
acid-itaconic acid copolymers [17,22], pullulan grafted AA-IA co­
polymers have not been reported yet.
The synthetic methodology, which is utilized for the preparation of
hydrogels, is of vital importance since it directly affects the performance
of the wound dressing material. UV polymerization is a powerful tech­
nique to prepare hydrogels, especially due to its mild reaction conditions
[23]. It can be carried out in the presence of a small amount of initiator
and crosslinking agent without the need for a reaction accelerator,
thereby reducing the toxicity of the prepared hydrogels [24]. Therefore,
the hydrogels prepared by using UV polymerization are particularly
advantageous for the biomedical applications, including wound dress­
ings [24–26]. However, there are limited number of studies concerning
the utilization of UV crosslinking and copolymerization technique in the
preparation of pullulan-based hydrogels for the wound dressing appli­
cations [27].
Recently, the hydrogel wound dressings, which have the capability
of controlled antibiotic delivery, have received great attention since
they provide high local concentrations of antibiotic while lowering the
overall serum concentrations [28,29]. The antibiotic loaded wound
dressings release the drug on the wound site at higher amounts, thereby
minimizing the risks of systemic toxicity that might occur in traditional
intravenous methods [28–32]. Comprehensive reviews on the utilization
of the controlled drug delivery for tissue repair and regeneration can be
found in Refs. [31,32].
The current literature knowledge summarized above has prompted
us to design an antibiotic loaded pullulan grafted copolymer hydrogel by
UV copolymerization technique. In this design, it was aimed for the
hydrogel to be composed of biocompatible building blocks and to
exhibit high swelling capacity and transparency. Therefore, in this study
pullulan grafted poly(acrylic acid-co-itaconic acid) (Pu-g-p(AA-co-IA))
composite hydrogels were proposed as wound dressing material for the
first time in the literature. The novel hydrogels were synthesized via UV
crosslinking graft copolymerization of AA and IA onto pullulan, in the
presence of N,N′ -methylenebisacrylamide as the crosslinking agent and
ammonium persulfate as the initiator. Antibiotic loaded pullulan grafted
poly(acrylic acid-co-itaconic acid) (Pu-g-p(AA-co-IA)-D) hydrogels were
prepared in the same manner by using ampicillin sodium salt as a model
drug to investigate the drug carrying ability of the proposed hydrogels.
Thereby prepared PGHs were characterized by FT-IR, SEM, DSC and
TGA analyses. Swelling and in-vitro cytotoxicity studies were performed
to reveal the performance of Pu-g-p(AA-co-IA) as wound dressing ma­
terial. In-vitro drug release experiments revealed that 67% of the loaded
drug was released from the Pu-g-p(AA-co-IA)-D hydrogels after 7 days.
The results revealed that the proposed Pu-g-p(AA-co-IA) hydrogel could
be acceptable as a promising candidate for the wound dressing
applications.
2. Experimental
2.1. Materials
Acrylic acid (AA), ammonium persulfate (APS), ampicillin sodium
salt (Amp) as a model drug, potassium phosphate monobasic and
phosphate buffer tablet were acquired from Sigma-Aldrich. N,N′ -
2
Journal of Drug Delivery Science and Technology 60 (2020) 101962
methylene bisacrylamide (BIS) and itaconic acid (IA) were merchan­
dised from Merck. Sodium phosphate dibasic anhydrous and pullulan
(Pu) were supplied from Carlo Erba and Jkchemical, respectively. Fetal
bovine serum (FBA) and Dulbecco’s modified Eagle’s medium (DMEM)
were received from Caprion. 3-[4, 5-dimethyltetrazolium bromide
(MTT) and Penicillin-Streptomycin were purchased from Serva and
Pen&Strep, respectively. All the reagents were chemically pure grade
and used as obtained. Deionized water was used as solvent.
2.2. Preparation of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D hydrogel
films
Pu-g-p(AA-co-IA)) hydrogels were synthesized via UV crosslinking
graft copolymerization of AA and IA onto pullulan, in the presence of BIS
as crosslinking agent and APS as initiator. To the aqueous solution of
pullulan (2%: w/v); APS (0.043 g, 0.18 mmol), AA (4 mL, 58 mmol), IA
(0.4 g, 3 mmol), BIS (0.01 g, 0.06 mmol) were added, respectively.
Thereby obtained homogenous solution was poured into glass petri
dishes and exposed to UV light for 2.7 h in Spectrolinker XL-1000A UV
Crosslinker equipped with five 8 W/365 nm tubes. After removing from
the petri dishes, the resulting Pu-g-p(AA-co-IA) hydrogels were washed
with distilled water several times to remove the unreacted monomer and
dried in the vacuum oven at 40 ◦ C for 48 h. Ampicillin sodium salt,
which is one of the broad spectrum antibiotics, was used as model drug
due to its high solubility in aqueous solutions [28]. Hence, drug-loaded
Pu-g-p(AA-co-IA) hydrogels (Pu-g-p(AA-co-IA)-D) were prepared in the
same manner as described above by adding Amp (0.1 wt%) directly to
the reaction medium. The same isolation and purification protocol as
Pu-g-p(AA-co-IA) hydrogel were applied for Pu-g-p(AA-co-IA)-D
hydrogels.
2.3. Characterization of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D
hydrogel films
DSC analyses were performed between − 50 and 400 ◦ C at a heating
rate of 10 ◦ C/min under nitrogen flow of 30 mL/min using Mettler –
Toledo DSC1/700 equipment. All data were collected from the second
heating cycle, and thermal transition temperatures were calculated as a
midpoint of the thermogram. TGA analyses were performed between 25
and 600 ◦ C at a heating rate of 10 ◦ C/min under nitrogen flow of 30 mL/
min using Mettler – Toledo DSC1 HT TGA-DTA equipment. The FT-IR
spectra were assessed by a MATTSON 1000 FTIR spectrophotometer.
The Amp release from Pu-g-p(AA-co-IA)-D hydrogel into phosphate
buffered saline (PBS) was monitored by Schimadzu uv-1800 UV/VIS
spectrophotometer. SEM analyses were performed using FEI Quanta 450
FEG.
2.4. Swelling study of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D
hydrogel films
The swelling ratios of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D
hydrogel films were determined gravimetrically under different pH
conditions (i.e., pH 2.1, 5.2 and 7.4) for 24 and 48 h. Completely dry and
pre-weighed PGH was placed in the test medium maintained at 37 ◦ C.
The swollen PGH was taken out from the solution and the excess water
on the hydrogel film surface was wiped with filter paper. All measure­
ments were made in triplicate. The swelling ratio (SR%) was determined
by equation (1) [33]:
WS − WD
SR% = X100 (1)
WD
where WS and WD are the weights of the swollen and dry hydrogels,
respectively.
H. Mert et al.
2.5. Drug release properties of Pu-g-p(AA-co-IA)-D
50 mg of Pu-g-p(AA-co-IA)-D hydrogel film was placed in 25 mL of
PBS at pH 5.2. The resulting dissolution was placed in shaking water
bath (WITEG – WSB Series) at 75 rpm and 37 ◦ C. One-milliliter aliquots
were withdrawn from the solution medium at appropriate time intervals
and replaced with the same amount of fresh PBS to maintain a constant
volume of the release medium. The amount of Amp, which was released
from Pu-g-p(AA-co-IA)-D hydrogel, was determined by using UV–Vis
spectrophotometer at 255 nm. The cumulative release (%) was deter­
mined by equation (2):
3 ∑
Cumulative release% = [Cn + Cn− 1 ]x100
10
where Cn and Cn-1 are the amount of the drug release at specific time n
and n-1, respectively [34].
Fig. 1. Schematic representation of the synthesis of Pu-g-p(AA-co-IA) hydrogel.
Journal of Drug Delivery Science and Technology 60 (2020) 101962
2.6. In vitro cytotoxicity evaluation
MTT assays for the cytotoxicity evaluation of the Pu-g-p(AA-co-IA)
and Pu-g-p(AA-co-IA)-D hydrogels were performed as follows. L929
fibroblast cells were seeded in 96-well plate. The plates were kept in the
CO2 incubator at a concentration DMEM with L-glutamine and 10% FBS
supplemented with 1% Penicillin-Streptomycin at 37 ◦ C in 5% CO2 for
24 h. The culture medium was changed every 3 h in a dark condition.
Following this step, 100 μl isopropanol–HCl was added to each well to
dissolve the formed dark blue formazan crystals. The wells were read by
the ELISA plate reader using a SpectraMax 190 microplate reader
(Molecular Devices, Sunnyvale, CA) at 570 nm. The number of living
(2) and dead cells was counted with a cell counter. The cytotoxicity test was
expressed as the relative cell viability (%) according to the following
equation (3):
[A]sample
Cell viability% = X 100 (3)
[A]control
where [A]sample is the absorbance of the test sample and [A]control is the
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H. Mert et al. Journal of Drug Delivery Science and Technology 60 (2020) 101962

absorbance of the control sample at 570 nm. Each assay was repeated
three times.

3. Results and discussion

3.1. Characterization of Pu-g-p(AA-co-IA) hydrogels

As detailed in the previous section, this study presents the prepara­

tion and characterization of Pu-g-p(AA-co-IA) and Amp loaded Pu-g-p
(AA-co-IA) hydrogels as wound dressing material. For this purpose,
acrylic acid and itaconic acid, which are biocompatible monomers, were
successfully grafted onto the linear polysaccharide pullulan backbone by
UV copolymerization technique in the presence of BIS as the crosslinking
agent and APS as the initiator. Representative reaction scheme for the
preparation of PGHs was shown in Fig. 1.
FT-IR analyses were used to assess the chemical structure of Pu-g-p
(AA-co-IA) and Pu-g-p(AA-co-IA)-D hydrogels. Fig. 2 shows the overlaid
FT-IR spectra of Pu, Amp, Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D.
The main characteristic bands of Pu were detected to be: O–H stretch­
ing (3325 cm− 1), C–H stretching (2928 cm− 1), C–O–C stretching (1642
cm− 1), C–O–H bending (1638 cm− 1), and C–O–C stretching (1146 cm− 1)
[35]. The successful graft copolymerization of AA and IA onto pullulan
was verified by the appearance of a new peak at around 1715 cm− 1
which was assigned to the stretching vibration of the C=O bonds [28].
Also, the stretching vibration band of C–H bonds at about 2925 cm− 1
have increased considerably [27]. The spectra also show a weak band at
2150 cm− 1 due to the presence of –C–N group of the crosslinking agent
BIS [36]. The spectrum of Amp exhibited characteristic peak at 1600
cm− 1, which was assigned to the C–C stretching of aromatics. For Pu-g-p
(AA-co-IA)-D, characteristic peak of the drug appeared at around
1600–1650 cm− 1 with a minor contribution of Amp [37–39].
DSC analyses were performed to investigate the thermal behaviors of
Pu, Pu-g-p(AA-co-IA), Pu-g-p(AA-co-IA)-D and ampicillin sodium salt as
the selected drug (Fig. 3). Two exothermic peaks were observed for Pu-g-
p(AA-co-IA) hydrogel which are at 137 and 220 ◦ C, while pure pullulan
thermogram displayed one broad exothermic peak at 90 ◦ C indicating
the presence of hydrogen bonding [37] and a second exothermic peak at
280 ◦ C associated with the melting of pullulan in the literature [40,41].
This thermal behavior of Pu-g-p(AA-co-IA) hydrogels indicates the suc­
cessful incorporation of pullulan to the polymeric network structure.
The DSC thermogram of the pure drug showed a broad exotherm,
ranging from 60 to 130 ◦ C due to the dehydration and a sharp
exothermic peak at about 228–231 ◦ C related to the melting point [38,
39]. The absence of the sharp exothermic dehydration peak of Amp in
the thermogram of Pu-g-p(AA-co-IA)-D hydrogel evidenced to the ho­
mogenous dispersion of the crystalline drug through the polymer
matrice.
TGA analyses were carried out to evaluate the mass loss profiles of
Pu, Pu-g-p(AA-co-IA), Pu-g-p(AA-co-IA)-D and Amp (Fig. 4). The TGA
thermogram of the drug showed two thermal transition zones due to the
removal of humidity and decomposition in a temperature range of
30–215 ◦ C and 380–600 ◦ C, respectively [42]. The Pu-g-p(AA-co-IA) and
Fig. 2. FTIR spectra of Amp, Pu, and Pu-g-p(AA-co-IA)-D.
Fig. 3. DSC thermograms of Amp, Pu, Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-
IA)-D.
Fig. 4. TGA thermograms of Amp, Pu, Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-
IA)-D.
Pu-g-p(AA-co-IA)-D showed almost the same two-zone thermal stability
behavior. The initial mass losses at 138–200 ◦ C resulting from the first
step degradation of PGHs were calculated to be 28%. The major deg­
radations of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D were observed at
325 to 450 ◦ C and calculated as 40% and 45%, respectively. This result
can be explained with the Amp content which degrades in the same
temperature region [38].
Fig. 5 shows the representative SEM images of the cross-sections of
Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D hydrogel films. The image of
Pu-g-p(AA-co-IA)-D hydrogel film indicates that the crystalline drug
could be homogenously dispersed in the polymeric network. Besides,
Fig. 5d clearly shows the smooth, compact and free surface of Pu-g-p
(AA-co-IA)-D hydrogel film. On the other hand, it was observed that the
prepared hydrogels are transparent enough to enable the visual in­
spection of the wound without removing the hydrogel film (i.e., Fig. 6).
3.2. Swelling study
An ideal hydrogel wound dressing is expected to remove the excess
exudate from the wound bed while preserving the moisture of the skin.
Besides, it is expected to facilitate the penetration of the active in­
gredients into the wound site, to protect the wound against bacterial
contamination and to relieve the pain by its cooling effect [43]. These
characteristics, which contribute to the wound healing and patient’s
comfort in a great extent, are strongly related to the proper water con­
tent of the hydrogel. Hence, the swelling capacity of a hydrogel plays a
crucial role in the performance of the hydrogel in wound dressing ap­
plications. In this regard, the swelling tests were performed for Pu-g-p
(AA-co-IA) and Pu-g-p(AA-co-IA)-D hydrogels. The swelling capacities of
PGHs were investigated at different pH conditions, namely pH 5.2 and
7.4, which simulate the wound exudate and the skin layer, respectively.
Swelling studies were also performed at pH 2.1 to reveal the response of
PGHs containing acrylic acid and itaconic acid to acidic environments.
As shown in Fig. 7, the swelling degrees of both Pu-g-p(AA-co-IA) and
Pu-g-p(AA-co-IA)-D at pH 5.2 and 7.4 were higher compared to those of

4
H. Mert et al.
Fig. 5. SEM images of Pu-g-p(AA-co-IA) (cross-section; magnification, ×1000) (a), Pu-g-p(AA-co-IA)-D (cross-section; magnification, ×1000) (b) Pu-g-p(AA-co-IA)
(cross-section; magnification, ×10000) (c) and Pu-g-p(AA-co-IA) (surface; magnification, ×1000) (d).
Fig. 6. The optical image of Pu-g-p(AA-co-IA).
at pH 2.1. This is due to the presence of carboxylic acid groups in the
hydrogel structure which are not ionized in the acidic medium (pH 2.1).
Once the hydrogels have been exposed to neutral (pH 5.2) or basic (pH
7.4) medium, carboxylic acid groups have turned into carboxylate ions,
thereby increasing the swelling capacities of hydrogels [44,45]. Namely,
Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D hydrogels showed 700 and
850% swelling capacities at pH 5.2, respectively. Since this pH value
corresponds to the pH of the body fluids, such as blood and wound
exudate, it can be said that the proposed PGHs could hold about 7–8
times more body fluid compared to their own weight.
5
Journal of Drug Delivery Science and Technology 60 (2020) 101962
Fig. 7. The swelling degrees of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA)-D.
3.3. In vitro drug release study
In vitro release profile of Amp from Pu-g-p(AA-co-IA)-D hydrogel was
investigated in a conditioned medium that mimics the wound environ­
ment (i.e., pH 5.2 and temperature 37 ◦ C). The cumulative release rate of
Amp was observed as 5.36 mg drug/g PGH after 4 days. Pu-g-p(AA-co-
IA)-D hydrogel showed an Amp burst release of 42% in the first 7 h and
an Amp release of 67% in the following 168 h (i.e., Fig. 8). Indeed, these
results indicate that the most of the drug could be incorporated into the
hydrogel network successfully and the PGHs, which are prepared in this
study, are within the ideal scope of the control release system.
Drug release mechanism of Amp was determined with different
mathematical models: zero-order (4), first-order (5) and Higuchi (6).
The modeling is described by following mathematical expressions:
qt = q0 + k0 t (4)
In(qt ) = In(qeq ) − k1 t (5)
qt = kH t1/2 (6)
where, qt is the mass of the released drug in time (t), q0 is the total drug
mass of the release in the medium (usually q0 = 0), qeq is the equilibrium
H. Mert et al.
Fig. 8. In vitro drug release profile of Pu-g-p(AA-co-IA)-D at 0–167 h (a) and 0–8 h (b).
mass of released drug, k0 is the zero order rate constant and k1 is the first
order rate constant model and kH is the Higuchi kinetic rate constant
model [46].
In vitro release data of Pu-g-p(AA-co-IA)-D was fitted to the above-
mentioned kinetic models. The correlation coefficients (R2) of these
mathematical models, which were calculated by using the different
model parameters given in Table 1, show that the drug release profile of
the Pu-g-p(AA-co-IA)-D hydrogel is amenable with the Higuchi model.
Thus, it was concluded that Pu-g-p(AA-co-IA)-D hydrogel releases the
drug based on the Fick’s law of diffusion [47].
The drug release rate of Pu-g-p(AA-co-IA)-D hydrogel was found to be
quite close to the drug release rates reported in the studies summarized
below. Sultanova et al. investigated the Amp release of polycaprolactone
nanofibers prepared via modified coaxial electrospinning method. They
reported that the sample released the 85% of Amp in the first 4 h period
and regarded this behavior as burst release, and 95% release of Amp was
achieved after 24 h at pH 7.4. Besides, drug release kinetic data of the
nanofibers was closer to zero-order kinetic model due to considerable
burst release effect [37]. In another work, acrylamide grafted pul­
lulan/poly(vinyl alcohol) interpenetrating polymer network hydrogels
were prepared by Soni et al. [48], and pirfenidone release was investi­
gated. The cumulative release of the drug was found to be 45–80% and
65–100% at the end of 2 h and 8 h, respectively. The in vitro pirfenidone
release rate of polymer formulations were observed to be dependent on
the drug loading capacity and the crosslinker concentration. Constantin
et al. synthesized thermo-sensitive pullulan-g-poly(N-iso­
propylacrylamide) with indomethacin-loaded nanoparticles for drug
delivery systems. The drug release from the grafted polymer in acetate
buffer solution was determined to be 60–85% in 6 h at 37 ◦ C. In addition
to this, the drug release kinetic data was found to be best fit to
Korsmeyer-Peppas among the main kinetic models, namely zero-order,
first-order, Higuchi, Korsmeyer-Peppas and Hixson-Crowell [49].
3.4. MTT study
The non-cytotoxicity of the drug delivery system is a prerequisite for
the success in wound dressing applications. Thus, the biocompatibility
of Pu-g-p(AA-co-IA) and Pu-g-p(AA-co-IA) was evaluated via MTT assays
using the mouse fibroblast cell line L929 for 24 h. Based on the MTT
assay represented in Fig. 9, it can be said that the blank and drug loaded
PGHs do not have an adverse effect on the L929 cell viability.
It was observed that, as the polymer concentration increased from
Table 1
The release parameters for Pu-g-p(AA-co-IA)-D.
Zero Order First Order Higuchi
Equation Equation Equation
Samples code k0 R2 k1 R2 kH
Pu-g-p(AA-co-IA)-D 0.4677 0.705 0.4688 0.639 0.9354
Journal of Drug Delivery Science and Technology 60 (2020) 101962
Fig. 9. L929 cell viability (%) at 37 ◦ C for 24 h. Results are presented as means
± SDs (n = 3) (p*< 0.5).
0.25 to 1 mg/mL, the cell viability (cell growth) decreased from 99.7 to
81.6%. The percentages of cell viability of Pu-g-p(AA-co-IA)-D hydrogel
at concentrations 0.25, 0.5 and 1 mg/mL were determined to be 135.4,
155.6 and 137.4%, respectively. These results, which are quite compa­
rable with the data in the literature, indicate that the prepared hydrogels
have good cytocompatibility [50,51]. Although the drug free Pu-g-p
(AA-co-IA) exhibited lower cytotoxicity than the drug loaded Pu-g-p
(AA-co-IA), L929 cell viability was above 80% for both samples [52]. As
a result, PGHs were considered to be non-cytotoxic on the cell viability
of L929 [53].
4. Conclusion
In this study, pullulan grafted copolymer hydrogels were prepared
via easy-to-apply UV copolymerization and crosslinking technique
which do not require temperature control, removal of oxygen, reaction
accelerator or pH-adjustment agents. Antibiotic loaded counterpart of
the aforementioned PGH was also prepared to reveal the controlled
release capability of PGHs. The successful incorporation of pullulan with
the polymeric network structure and the homogenous dispersion of the
crystalline drug through the polymer matrice were confirmed by using
appropriate analysis techniques. Swelling studies performed at 37 ◦ C
and different pH conditions simulating the skin layer and the wound
environment reveal that the prepared PGHs have over 700% swelling
capacities. Besides, MTT assay conducted using the mouse fibroblast cell
line L929 showed that L929 cell viability was above 80% for PGHs. More
importantly, it was observed that the Pu-g-p(AA-co-IA)-D hydrogel re­
leases 67% of the loaded drug after 7 days. Hence, the proposed PGHs
can be regarded as promising candidates for future wound dressing
applications.
R2 Disclosure statement
0.820
No potential conflict of interest was reported by the authors.
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H. Mert et al. Journal of Drug Delivery Science and Technology 60 (2020) 101962

Author statement [19] J.S. Vuković, M.M. Babić, K.M. Antić, J.M. Filipović, S.T. Stojanović, S.J. Najman,
S.L. Tomić, In vitro cytotoxicity assessment of intelligent acrylate based hydrogels
with incorporated copper in wound management, Mater. Chem. Phys. 175 (2016)
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gation, Resources, Formal analysis, Writing - original draft, Writing - [20] J.S. Vuković, M.M. Babić, K.M. Antić, M.G. Miljković, A.A. Perić-Grujić, J.
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