European Polymer Journal 134 (2020) 109853

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European Polymer Journal

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Chitosan/PVA hydrogels incorporated with green synthesized cerium oxide T

nanoparticles for wound healing applications
⁎
Katayoon Kalantaria, ,1, Ebrahim Mostafavia,1, Bahram Saleha, Pooneh Soltantabarb,
⁎
Thomas J. Webstera,
a
Department of Chemical Engineering, Northeastern University, Boston, MA 02115, USA
b
Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA

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

Keywords: In this paper, we report the synthesis and evaluation of a polyvinyl alcohol/chitosan (PVA/chitosan) hydrogel
Chitosan incorporated with green synthesized cerium oxide nanoparticles (CeO2-NPs) using a Zingiber officinale extract as
Wound healing the reducing, capping and stabilizer agent for wound healing applications. The PVA/chitosan/CeO2-NPs hy-
CeO2 nanoparticles drogel was synthesized via the freeze-thaw technique with 0 to 1% (wt) 5 nm CeO2-NPs. Results revealed that
Hydrogel
the hydrogels containing 0.5% of CeO2-NPs showed better antibacterial activities after just 12 hr (with MRSA but
Human dermal fibroblasts
not E. coli) and healthy human dermal fibroblast viabilities up to 5 days (more than 90%) compared to the
Antibacterial
MRSA control group. Therefore, these chitosan/PVA hydrogels incorporated with Ce-NPs could be a potential candidate
as a robust wound dressing agent that impressively may decrease wound infections without resorting to the use
of antibiotics.

1. Introduction
The largest organ and first line of defense for the human body is the
skin, which can be easily damaged by trauma, surgery and burns [1].
The skin provides a protective barrier due to its dense surface and layers
with corneous structure; for a wound, such a perfect barrier is vital [2].
An ideal wound covering should have good mechanical strength to
satisfy clinical requirements (1–2 MPa) for the strength of membranes
and flexibility as well as an ability for wound exudate absorbance, and
furthermore, the dressings should not stick onto the surface of wounds
as that could hamper the repair process. Additionally, they should have
a porous structure, so the exchange of gases (such as oxygen) can easily
take place [3]. While traditional wound dressings are still the best
sellers in the market, using novel wound coverings including foams,
nanofibers, and hydrogels is of high demand to provide anti-infective,
anti-inflammatory, and pro-wound healing properties [4].
Hydrogels are 3D polymeric networks, which have been used suc-
cessfully in numerous biomedical fields [5,6]. They act as a substitute
extracellular matrix and can create a moist environment to promote the
process of wound healing [7,8]. Several physical and chemical methods
have been applied for the fabrication of hydrogels, such as radiation
with gamma and UV rays and freeze-thaw processes [1]. The freeze-
thaw technique does not need any toxic or chemical additives and leads
to the formation of simple hydrogels, which have a strong ability to
adsorb exudates from wounds. The exudate accumulation at the surface
of the wound results in a delayed healing process and then an increase
in the risk of bacterial growth [9]. Different biopolymers have been
used in the synthesis of hydrogel wound dressings. For instance, in the
case of hydrogels, chitosan and PVA are mostly used because of their
biocompatibility, low price and non-toxicity. Chitosan is a natural
polysaccharide, which is usually applied in the field of tissue en-
gineering [10]. However, chitosan shows low mechanical strength and
a simple technique to conquer this problem is blending it with other
polymers such as PVA [11]. PVA has food water-solubility with several
applications in drug release, tissue engineering and biomedicine [12].
Chitosan/PVA hydrogels are suitable candidates for wound dressing
applications. Recently, these nanocomposites have been prepared by
the incorporation of some NPs such as Ag and ZnO to biopolymers, and
the resultant material used as a wound dressing [1]. Using nano-
particles in the wound dressings structure may result in quick healing of
chronic and acute wounds because of their chemical stability, anti-in-
flammatory and antibacterial properties. Moreover, generally nano(bio)
composites possess high mechanical properties, low permeability
against water and vapor, and superior thermal resistance [13].

⁎
Corresponding authors.
E-mail addresses: k.kalantari@northeastern.edu (K. Kalantari), th.webster@neu.edu (T.J. Webster).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.eurpolymj.2020.109853
Received 14 April 2020; Received in revised form 9 June 2020; Accepted 14 June 2020
Available online 20 June 2020
0014-3057/ © 2020 Elsevier Ltd. All rights reserved.
K. Kalantari, et al.
Studies have shown that cerium oxide nanoparticles exhibit pro-
oxidative properties or antioxidant properties depending on their pH
environment. CeO2-NPs can prevent reactive oxygen species (ROS)
accumulation and cellular damage, and reduce inflammation to provide
for more suitable local healing [14]. They have revealed excellent
properties like biocompatibility, antibacterial and autocatalytic [15].
Several methods have been used for the fabrication of CeO2-NPs, such
as sonochemical, microwave-hydrothermal assisted, and co-precipita-
tion. These methods are expensive, complicated, time consuming, and
use hazardous chemicals such as hydrogen peroxide. However, green
and low cost fabrication techniques are required to fabricate nano-
particles in adequate quantities. Plant assisted synthetic methods have
been shown to be eco-friendly and less hazardous [16,17]. As an ex-
ample, Zingiber officinale (Z. officinale) is an ancient plant which has
been used as a flavor in various beverages and food [18]. Zerumbone is
the main compound in the extract which is currently being used and
studied for anti-cancer and anti-viral applications [19].
In this paper, we report for the first time, a green and simple method
for the preparation of CeO2-NPs at room temperature using the Z. of-
ficinale extract as the reducing and stabilizing agent. We then in-
corporated these CeO2-NPs into a chitosan/PVA hydrogel produced by
a simple freeze-thaw technique. The hypothesis of this study is that the
addition of CeO2-NPs to chitosan, a well-established carbohydrate in
medicine, will provide antibacterial properties to antibiotic resistant
bacteria for wound healing applications. Physicochemical properties
along with the biocompatibility, and antimicrobial activities of the
hydrogels were analyzed to demonstrate their ability to serve as im-
proved wound dressing materials in vitro. The overview of this work is
shown in Fig. 1.
2. Materials and methods
2.1. Green synthesis of CeO2-NPs using the Z. Officinale extract
Z. officinale (fresh) was obtained from Star Market (a local store) in
Boston, USA. Briefly, 200 g of the Z. officinale rhizome was washed
carefully, dried, and cut into small parts. Then, they were boiled in
400 mL of distilled water for 20 min and the aqueous extract of Z.
Fig. 1. Schematic illustration of the hydrogel preparation.
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European Polymer Journal 134 (2020) 109853
officinale was filtered. To separate any solid substrates, the resulting
aqueous extract was centrifuged at 5000 rpm for 10 min, then the su-
pernatant was stored at −4 °C. 8.68 g of Ce(OH)3·6H2O (Sigma Aldrich,
USA) was added to 200 mL of the extract and stirred for 2 h. Then, 5 mL
of NaOH (30 mM) was added drop-wise and the mixture was stirred for
2 h. The blackish-brown CeO2-NPs were collected using a centrifuge at
6000 rpm for 6 min. The obtained NP pellet was washed several times
with MilliQ water and finally dried at 60 °C for 8 h and further annealed
in a furnace (Thermo Scientific/Blue M BF51766C, USA) at 500 °C for
2 h. The yellowish nanoparticles were stored at room temperature for
more characterization and experiments.
2.2. Hydrogel preparation
PVA/Chitosan/CeO2-NPs hydrogels were prepared by a freeze-thaw
technique (Fig. 1). Briefly, 4 g of PVA (M.W. 86000, Fisher Scientific)
were added into ultra-pure MilliQ water and heated at 60 °C for 30 min.
The mixture was then stirred for 3 h at 90 °C to completely dissolve the
PVA. Then, 0.5 g of chitosan was dissolved in a solution of 2 v% acetic
acid. The beaker was covered and sealed before blending for 1 h to
achieve complete dissolution. Then, 3 solutions of PVA: Chitosan at a
weight ratio of 4:1 were mixed, dried and degassed in a vacuum drier
for 1 h. Finally, CeO2-NPs with different concentrations (0%, 0.5% and
1%) were suspended in ultra-pure MilliQ water and sonicated for
10 min. The CeO2-NPs nanosuspension was added drop-wise to the
hydrogels and stirred vigorously for 2 h. Then, the solutions were
poured into a sterile 24 well-plate. The plates were sealed and kept for
20 h at −20 °C and then thawed for 8 h at room temperature. This cycle
was repeated six times. In this method, the hydrogen bonding between
chitosan and PVA helped to create a 3D structure during the process of
nucleation and growth in the interpenetrating network of the polymer.
When freezing happened in the polymer chain, the nucleuses formed
inside the polymer solution. When thawing, the chains in polymer
formed near the nucleuses in a systematic manner to have crystalline
structures [1].
K. Kalantari, et al.
2.3. CeO2-NP characterization
TEM (JEM 3010 – JEOL, Japan) and SEM (JEOL JSM-5400, Japan)
were used for characterization of the morphology and size of the CeO2-
NPs. Their crystalline nature was analyzed by an X-ray diffractometer
(Empyrean, PANalytical, Netherlands). The formation of the CeO2-NPs
was observed by UV–Vis spectroscopy (Shimadzu UV 2401pc) in the
200–450 nm region. Dynamic Light Scattering was used for de-
termining the stability of the CeO2-NPs (colloidal) using a Malvern
Nano ZS/HeNe 633 nm laser (Malvern, UK). A Fourier transform in-
frared spectrophotometer (Spotlight-400, Perkin Elmer) was used in the
range from 400 to 4000 cm−1 to analyze the chemical groups.
2.4. Hydrogel characterization
2.4.1. Hydrogel gel fraction
The hydrogels were dried for 24 h in a fume hood followed by
drying at 50 °C until W0 as the constant weight was reached. Then,
hydrogels were soaked in distilled water for 24 h to reach We as an
equilibrium swelling weight. Then, the samples were dried at 50 °C for
48 h to a constant weight (We). The gel fraction was defined as (W0)/
(We) based on the following equation [20]:
Gel content(%) = (We/ W0) × 100 (1)
2.4.2. Hydrogel swelling ratio
The swelling ratio (S) of the hydrogels was found using the con-
ventional gravimetric technique and Eq. (2) [21]:
Ws − Wd
S (%) = × 100
Wd (2)
where Ws and Wd are the hydrogel weight after swelling and in the dry
state, respectively.
2.4.3. Hydrogel water vapor transmission rate (WVTR)
The swollen hydrogels (Ws) were kept in an incubator with 40%
humidity at 37 °C. After regular time intervals, the weight was mea-
sured (Wt). The percentage of water loss was determined using Eq. (3):
Ws − Wt
Water lost(%) = × 100
Ws − Wd (3)
2.4.4. Hydrogel porosity measurements
The porosity of the hydrogels was analyzed by the liquid displace-
ment technique. Three samples were used from each type of hydrogel.
The hydrogels were immersed into ethanol for 48 h until they were
saturated by absorption and afterwards the hydrogels were re-weighed
[22]. The hydrogel porosity was measured by Eq. (4) [23]:
Ww − Wd
Porosity = (%)
Ww − Wl (4)
where Wd is the hydrogel dry weight, Ww the wet hydrogel weight, and
Wl the weight of the hydrogel in ethanol.
2.4.5. Contact angle measurements
To evaluate the wettability of the hydrogels, the contact angle (CA)
between the surface of the hydrogel and a drop of ultrapure water was
measured using a Krϋss, Model DSA 100 apparatus. Briefly, a flat piece
of the hydrogel was fixed on a glassy slide. Then, a drop of 16 μL ul-
trapure water was dropped onto the hydrogel and the image was taken
after 2 s at room temperature. All hydrogel contact angles were mea-
sured in triplicate (n = 3).
2.4.6. In vitro mammalian cell culture
Primary human dermal fibroblast (HDF) cells (Lonza, CC-2509,
AMP, containing ≥ 500,000 cells; population numbers < 10) were
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European Polymer Journal 134 (2020) 109853
cultured in a humidified atmosphere (37 °C and 5% CO2) in Dulbecco’s
Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham,
MA) supplemented with 10% v/v fetal bovine serum (FBS; ATCC® 30-
2020™, American Type Culture Collection, Manassas, VA) and 1% pe-
nicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA). When
cells were 70% confluent, they were trypsinized and used for seeding on
the scaffolds. For this, the hydrogels were cut into squares
(4.5 mm × 4.5 mm). Prior to in vitro cell seeding, hydrogels were
sterilized by soaking in ethanol (70%) for half an hour followed by UV
exposure on each side for 30 min. Finally, each hydrogel was seeded
with 95,000 cells. 2D seeded hydrogels were kept at 5% CO2 and 37 °C
over 5 days for cell viability, adhesion, and proliferation assays as de-
scribed next.
2.4.7. Cell viability
An Invitrogen live/dead assay kit was utilized to find the viability of
the cells seeded on the hydrogels for 1, 3 and 5 days post-seeding using
the provided manufacturer’s instructions. Briefly, after each time
period, cells on the hydrogels were stained with ethidium homodimer-1
(EthD-1, 2 μL/mL in DPBS) for determining dead cells and calcein AM
(0.5 μL/mL in PBS) for determining live cells. Following this, the cells
were incubated (15 min at 37 °C), then the stain was removed, and the
cells were washed with DPBS three times. Finally, the images of the
stained cells were taken by an inverted fluorescence microscope (Zeiss
Axio Observer Z1, Carl Zeiss Microscope, Thornwood, NY) and images
were analyzed using Image J software. The percentage of cell viability
was found by dividing the number of live or dead cells by the total
number of cells.
2.4.8. Cell metabolic activities
The cellular metabolic activity was determined using a PrestoBlue
cell viability kit (Thermo Fisher Scientific) following the manufacturer’s
protocol. In brief, the PrestoBlue solution was added to the wells in-
cluding hydrogels and media at 1, 3 and 5 days post-seeding to con-
stitute 10% of the whole media in the wells. Following this, the cells
were incubated for 45 min at 37 °C and 5% CO2. Fluorescence intensity
of the solutions was recorded using a microplate reader (Bio-Tek Inc.) at
590 nm emission and 530 nm excitation.
2.4.9. Antibacterial properties using CFU assays
Antibacterial activity of the hydrogels against gram-negative E. coli
(ATCC 25922) and gram-positive methicillin-resistant Staphylococcus
aureus (MRSA, ATCC 33591) was investigated according to the colony
forming unit (CFU) method. For this, prior to the experiment, the hy-
drogels were cut into small cylindrical and identical pieces (D = 5 mm,
t = 3 mm) in triplicate and placed in a 48 well plate (separate wells)
and sterilized under UV light for 30 min. After 24 h of incubation of a
single colony of the bacteria strain in tryptic soy broth (TSB, 22,092
Sigma-Aldrich, USA), the optical density (OD) of the resulting bacterial
suspension was adjusted to the absorbance of 0.52 @ 562 nm, which
corresponds to a density of 109 CFU/mL. Next, this bacteria suspension
was diluted to 106 CFU/mL. In the next step, 500 µL of the resultant
suspension was added to each well of 24 well plates containing the
hydrogels and the well plate was incubated at 5% CO2 and 37 °C for
24 h. After incubation, the hydrogels were washed with PBS (3X) to
remove all non-adherent bacteria on the surface of the hydrogels.
Finally, for the CFU assay, the hydrogels were vortexed (3000 rpm,
15 min) with 500 µL of PBS to release all bacteria from the hydrogel
into the solution. Bacterial suspensions were then diluted serially in PBS
over 5 logarithmic dilutions (100x; 1,000x; 10,000x; 100,000x; and
1,000,000x). Then, three 10 μL drops of each dilution were seeded on
agar-TSB plates, which were then incubated for 9 h at 37 °C and 5%
CO2. Finally, the number of bacterial colonies found on each agar plate
was counted, and the dilution factor was used to calculate CFU values.
K. Kalantari, et al.
Fig. 2. Physico-chemical characterization of the CeO2-NPs. (A) UV–vis spectrum, (B) XRD diffraction patterns, (C) zeta potential results and (D, E) TEM images of
CeO2-NPs, showing a particle size with a mean diameter of 5 nm with regular morphology.
2.4.10. Statistical analysis
Each experiment was completed at least three times and data ana-
lyzed using a two-way ANOVA test with GraphPad Prism 6.0 software.
Error bars were obtained as the mean ± standard deviation (SD) and
statistics indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and
****p < 0.0001, n = 3.
3. Results and discussion
3.1. CeO2-NP characterization
The UV spectra of the CeO2-NPs (3 mg dispersed in 10 mL of double
distilled water) can be seen in Fig. 2A. The absorption spectra showed
an intense peak at around 290 nm with the formation of CeO2-NPs
[17,24]. Moreover, the appearance of two peaks at 258 and 320 nm was
related to the oxidation states of Ce3+ and Ce4+ that confirmed CeO2-
NPs formation [16]. Generally, the transfer of charge between the states
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European Polymer Journal 134 (2020) 109853
of (2p) and (4f) of O and Ce, respectively, in O2− and Ce3+ was pre-
viously reported for CeO2-NPs in the UV absorption region [25]. Based
on our previous study, the Z. officinale extract did not show any peaks in
the UV–Vis spectrum [18]. Furthermore, the absorbance of the solution
containing CeO2-NPs at a 24 h interval was monitored and no major
changes in absorbance were found during storage.
Fig. 2B shows the well defined diffraction peaks from the X-ray
diffractograms. No other phase of ceria or other crystalline compounds
were observed in the samples studied [26]. The XRD peaks were located
at a 2θ of 28.60, 35.90 and 48.70 corresponding to the (1 1 1), (2 0 0)
and (2 2 0) planes of the CeO2-NPs, respectively. Other peaks located at
2θ of 55.90, 60.02, 67.80, and 75.80 corresponded to the (3 1 1),
(2 2 2), (4 0 0), and (3 3 1) planes of CeO2-NPs, respectively. The
standard diffraction peaks indicate the face-center cubic phase of CeO2-
NPs [17].
The stability and surface properties of the synthesized nanoparticles
are generally measured by zeta potential. Mainly, the more absolute
K. Kalantari, et al.
Fig. 3. Gel content (A), porosity (B), swelling behavior of the hydrogels (C) and water contact angle measurements (D). Error bars show the means standard error,
n = 3, * p < 0.01.
zeta potential value ( ± ve), the more the repulsion force between the
NPs to aggregate and, thus, the greater stability of the nanoparticles.
Particles with a zeta potential value more than ± 30 mV are typically
considered stable [27]. The zeta potential of the synthesized CeO2-NPs
was measured to be – (50) mV, which provides evidence that the na-
noparticles were stable in the colloid suspensions, Fig. 2C. The size and
shape of the CeO2-NPs were also examined by TEM. The nanoparticles
were of uniform size and appeared to be quasi-spherical in shape but
large aggregates of nanoparticles were observed as in Fig. 2D and E.
The particles were found to be ~5 nm in diameter.
3.2. Hydrogels
3.2.1. Gel content
For the quantitative evaluation of the cross-linking degree, the gel
content of the fabricated hydrogels was evaluated. From Fig. 3A, the gel
content was 80%, 73% and 78% for the 0, 0.5% and 1% hydrogels,
respectively. With increasing the amount of CeO2-NPs to 0.5% weight
in the hydrogel structure, the gel content decreased but with more
amounts of CeO2-NPs (1%), gel content increased as well which showed
an increase in the interaction between the functional group of CeO2-NPs
and the polymer chains. In other research, Khorasani et al. reported that
with an increase in ZnO nanoparticles, gel content increased as well.
They found that the gel content varied between 60% and 87% with an
increase in the amount of ZnO nanoparticles [1].
3.2.2. Porosity
The porous structure of the hydrogels can help to take high volumes
of exudates from the wound surface. It helps nutrient distribution as
well and makes an excellent environment for the adhesion and growth
of cells. Here, adding CeO2-NPs to hydrogels increased the porosity in
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European Polymer Journal 134 (2020) 109853
the structure of the hydrogels, and the porosity was almost the same in
the range of 81–90%, Fig. 3B. The highly porous structure of the hy-
drogels would support the adsorption of wound exudates from the
surface of the wounds to aid in wound healing.
3.2.3. Swelling behavior
Water is the main component of blood (90%) and good swelling
properties of hydrogels allow them to absorb a high volume of water
from the blood to stop bleeding [2]. Here, the fabricated hydrogels had
good swelling behavior. PVA/chitosan hydrogels have numerous hy-
drophilic groups which show good water accessibility. For less dis-
solution of the mentioned polymer networks, physical or chemical
bonds between the chains of the polymer can be created. Hydrogels can
absorb water to become completely swollen, which is common and
possibly desirable to mimic living tissues (such as skin) with the same
physical properties [28,29].
Swelling tests were completed at room temperature to estimate the
hydrogel swelling capacity in water. The increase in the swollen hy-
drogel weight was directly attributed to the swelling period. Thus,
through more incubation time, the weight of the swollen hydrogel in-
creased. Fig. 3C shows the effect of CeO2-NPs on the swelling behavior
of the hydrogels. Obviously, swelling increased sharply by the con-
tinuation of incubation time for up to 5 h and then leveled off. More-
over, the presence of CeO2-NPs in the hydrogels caused their network to
enlarge. A significant variation in the hydrogel swelling capacity was
noted when they were incorporated with CeO2-NPs. The CeO2-NPs
embedded in the gel led to network expansion of the hydrogel. The
significant increase in the hydration degree was related to the surface
charge of the colloidal CeO2-NPs. Furthermore, increasing the CeO2-NP
amount enhanced the swelling ability of the host hydrogel. Due to the
high hydrophilicity of PVA together with the loosened and
K. Kalantari, et al.
macromolecular chains of chitosan, the fabricated PVA/chitosan hy-
drogels showed a good swelling ratio. Adding more CeO2-NPs to the
structure of the hydrogels increased the swelling ratio slightly. The
hydrogel network expanded with an increase in the amount of nano-
particles, which led to the creation of pores and vacancies within the
network of the hydrogel and an increment in water absorption [30].
3.2.4. Contact angles
Generally, wettability analysis provides information about the hy-
drophilicity/hydrophobicity of the surface and molecular mobility at
the solid/water/air interface. A water contact angle on an ideal wound
dressing surface should be < 90° to possess suitable ability to absorb
wound exudates [31]. As shown in Fig. 3D, by adding 0.5 and 1 wt%
CeO2-NPs to the hydrogels, the contact angle decreased. Adding CeO2-
NPs increased hydrophilicity and, thus, it led to an increased tendency
to absorb water and contact angles decreased. Without any chemical
modification, CeO2-NPs are hydrophobic because of its unique elec-
tronic structure that prevents it from forming hydrogen bonds with
interfacial water [32]. However, as confirmed via the FTIR spectra, the
ceria nanoparticles were coated with polysaccharides and there was a
surface layer of hydrophilic polysaccharides, which is the major com-
pound in the Z. officinale extract, rendering the surface of the CeO2-NPs
hydrophilic [33].
3.2.5. FTIR spectrum analysis
In Fig. 4, the FTIR spectra of chitosan, PVA and CeO2-NPs (Fig. 4A)
and hydrogels (control, 0.5%, and 1%) (Fig. 4B) are compared. The
spectrum of chitosan shows some peaks at 1655 and 1578 cm−1 related
to stretching CeO, and bending NeH, respectively. The absorption
peak of chitosan at 1050 cm−1 is related to stretching CeO and a broad
and strong peak between 3300 and 3500 cm−1 implies the stretch-
ing vibration of N-H and O-H [34,35]. The PVA spectrum shows a broad
absorption peak at 3000–3700 cm−1 attributed to OeH groups and
different characteristic peaks at wave numbers of 2900 cm−1 stretching
(CeH), 1416 cm−1 scissoring (CH2 ), and 1100 cm−1 stretching (CeO)
[30]. For CeO2-NPs, the absorption band around 3400 cm−1 is related
to the stretching vibration OeH, and the absorption band at 1500 cm−1
is because of the scissor bending mode of associated water. CeO2-NPs
included absorption peaks at 537, 718, 1060, and 1500 cm−1. The band
at 848 cm−1 corresponds to a metal–oxygen bond [36]. The absorbance
Fig. 4. FTIR spectra of: (A) pure chitosan, PVA, and CeO2-NPs and (B) Hydrogels (0, 0.5% and 1% CeO2-NPs).
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European Polymer Journal 134 (2020) 109853
peaks are attributed to Ce-O bond formation that are in the range of
800–400 cm−1 (IR range) of crystalline CeO2 active phonon modes
[24]. Several small peaks at 1450, 1200, 900 and 800 are obvious
signals of heterocyclic compounds including alkaloids and flavonoids,
the active substances of Z. officinale, which act as capping agents [37].
For the control hydrogel (0%), the broad band from 3500 to
3100 cm−1 revealed the existence of eNH2 and eOH stretching vi-
brations [38]. It can be seen that almost all related peaks of pure
chitosan and PVA were also present in the FTIR spectrum of the fab-
ricated hydrogels. The bands corresponding to the OH and NH2 group
stretching modes became sharper and broader in the IR spectra of the
hydrogels (0.5 and 1%). This result shows the assembly of chitosan with
CeO2-NPs in the NH2/OH group. In addition, the intensity of bands at
1000–1500 cm−1 increased. This variation is probably related to the
complexation process that occurs between Ce and the amine groups
[39]. The absorbance peak at 570 cm−1 is ascribed to the formation of
the Ce-O bond; moreover, the peak intensity in the IR range of
800–400 cm−1 for crystalline CeO2 active phonon modes increased
[24].
3.2.6. SEM analysis
Scanning electron microscopy was used to analysis the morphology
of the hydrogels. Cross-sectional images of the hydrogels are shown in
Fig. 5A–D. It can be seen that all of the hydrogels have a 3-D network
structure. There are interconnected micropores in the structure of the
hydrogels, which were distributed uniformly and allowed the water
vapor or other gasses to penetrate and permeate throughout the hy-
drogels to aid in wound healing.
Fig. 5 confirms the incorporation of CeO2-NPs within the structure
of the hydrogels. Noticeably, the network topologies of the control
sample seem to be appreciably different from the hydrogels including
CeO2-NPs. It may be inferred that CeO2-NPs are assembled or attached
inside the hydrogel networks as well as on the surface. Moreover, the
addition of CeO2-NPs led to an increase in average pore size and an
increase in pore density. Higher micro-porosity is appropriate for ef-
fective CeO2-NP immobilization in the structure of the hydrogel. The
average pore size was 0.562 ± 0.32 μm, 0.104 ± 0.03 μm and
0.077 ± 0.02 μm for the hydrogels with 1%, 0.5% and controls, re-
spectively.
K. Kalantari, et al.
Fig. 5. Cross-section SEM images of hydrogels with 0 (A), 0.5% (B), and 1% CeO2-NPs (C), and respective pore sizes (D).
3.3. Cell viability and proliferation assays
Viability assays are well known as the vital steps in toxicology in-
cluding the response of cells to a toxicant or new material. Furthermore,
these assays provide information on cellular metabolic activities sur-
vival or death [40]. Here, we used high sensitivity luminescence-based
and fluorescent based assays to determine cellular response to CeO2-
NPs. Cell viability was also studied with the Live/Dead viability/cyto-
toxicity kit (Fig. 6A). No evidence was observed about the damage in
cell membranes following their treatment with hydrogels (0.5% and
1%), which clearly indicated viability comparable to that of the control
ones. The steady increase in cell proliferation on the hydrogels can be
attributed to the incorporated CeO2-NPs (or components of it) that
would have released in the cell culture medium. Other studies have
reported that cerium nanoparticles improved cell survival by de-
creasing the intracellular levels of ROS produced because of metabolic
activities, which otherwise cause cellular damage [41,42]. Higher cell
densities were observed on the hydrogel loaded with CeO2-NPs with
fewer cells adherent on the control hydrogel after 3 and 5 days of
culture. The cell viability and proliferation of HDF cells on hydrogels
over a period of 5 days were studied by a presto blue assay (Fig. 6 B). It
was observed that cells are viable and proliferated on both hydrogels
(0.5% and 1%) and demonstrated no significant toxicity. An insignif-
icant reduction in the viability of cells at higher concentrations of CeO2-
NPs (1%) might be because of the overexpression of proinflammatory
factors that might have an effect on the functions of cells [43,44].
Nevertheless, the mentioned effect was marginal since there was no
detectable change in cell density on the hydrogels with 1% CeO2-NPs
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European Polymer Journal 134 (2020) 109853
compared to the nearest lowest concentration used (0.5%). This clearly
showed that CeO2-NPs at an optimized concentration could promote
HDF cell adhesion and proliferation on the hydrogels, necessary first
steps for wound healing [45].
3.4. Metabolic activity of cells
The HDF cell metabolic activity increased consistently throughout
the culture duration, as demonstrated by the commercial PrestoBlue
assay (Fig. 6C). The metabolic activity of the cells, reflecting the rate of
HDF cell proliferation, increased through the use of a higher percentage
of CeO2-NPs in the hydrogel. In addition, the metabolic activity of cells
continuously increased during the 5 days of cell culture. As reported by
other research, the hydrogels demonstrated no significant toxicity even
after 5 days meaning that the prepared hydrogels were well tolerated
by HDF cells [46,47]. These results showed that hydrogels could effi-
ciently support the growth and proliferation of HDF cells without eli-
citing any in vitro cytotoxic responses. As shown in Fig. 6C, there were
no statistically significant decreases in cell viability during the 5 days
after seeding. In contrast, on day 3, the HDF cell metabolic activity on
the hydrogels with 1% CeO2-NPs was significantly higher. These are
impressive results especially considering that no growth factors or cy-
tokines were added to the hydrogels, thus, warranting further in-
vestigation.
3.5. Antibacterial activities
The antimicrobial activities of the hydrogels were examined against
K. Kalantari, et al. European Polymer Journal 134 (2020) 109853

Fig. 6. In vitro HDF cytocompatibility study on the various hydrogels: (A) Representative live/dead images from HDF cells seeded on different hydrogels at days 1 and
5 post-seeding. (B) Cell viability quantity for HDF cells seeded on different hydrogels. (C) Quantification of metabolic activity of HDF cells, relative cell numbers by
the PrestoBlue assay at days 1, 3, and 5 post-seeding on different hydrogels. Error bars show means standard error, asterisks mark significance levels of p < 0.05 (*),
p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****), ns = not significant, n = 3.

Fig. 7. Antimicrobial activity of the hydrogels. Colony forming unit (CFU) tests representing the effect of CeO2-NPs incorporation into hydrogels after 12 h against
gram-negative and positive (E. coli and MRSA, respectively). The results revealed the significant inhibitory effect of the hydrogel with 0.5% CeO2-NPs on the growth
of MRSA on hydrogels. Data are represented as the mean ± SD (p < 0.05 (*), ns = not significant (n = 3)).

E. coli, as gram-negative, and MRSA, as gram-positive, bacteria which
are the most common wound-infecting microorganisms. The inhibitory
activities of CeO2-NPs on microbial growth were investigated using
enumeration of colony forming units (CFU). The results indicated that
the hydrogels with 0.5% CeO2-NPs could effectively inhibit the growth
of MRSA when compared to the 0% and 1% hydrogels. However, for E.
coli, there was no significant difference between the different hydrogels
(Fig. 7). Significant differences were detected in the sensitivity of var-
ious opportunistic microorganisms to the presence of CeO2-NPs. The
presence of CeO2-NP hardly oppressed the viability of E. coli. A certain

8
K. Kalantari, et al.
reduction in the number of microorganisms in the suspension in the
presence of the hydrogels was detected only after 24 h of incubation.
Other factors besides size and aggregation of nanoparticles, bioavail-
ability, surface area, structural distortion, and growth medium used can
affect nanoparticle inhibition/toxicity [48,49]. Aggregation and/or
agglomeration can change the CeO2-NP physical properties and their
biological reactivity as well. The lack of the reliable effect of growth
inhibition of microorganisms in the presence of the hydrogel (1%)
should be noted which may be associated with a high concentration of
particles and their agglomeration that defined the ability of Ce3+ to
interact with phosphates.
However, the noted 85% reduction in MRSA colonization through
the use of 0.5 CeO2-NPs is of high importance especially considering the
aforementioned high HDF cell viability and that no antibiotics were
used. As has been well documented, due to the growing concern of
antibiotic resistant bacteria (like MRSA), approaches to inhibit bacteria
colonization on wound healing materials without resorting to antibiotic
use is greatly desired.
The presence of CeO2 did not change the viability of E. coli most
likely due to the more complex structure of the gram-negative bacteria
membrane: except for the outer layer of murein, a peptidoglycan layer
is located in the periplasm and the outer membrane containing lipo-
polysaccharides [50]. The preservation of the stability of the lipopoly-
saccharide layer is provided by divalent calcium cations. No significant
impact of CeO2-NPs on the viability of E. coli could be due to many
reasons. There is a thick layer of peptidoglycan in the membrane of
gram-positive bacteria including linear chains of polysaccharides with
short peptides which together make a rigid structure, so the CeO2-NP
penetration is difficult. CeO2-NPs with positively charged surfaces
showed significantly more toxicity and bioaccumulation than the ne-
gatively or neutral charged NPs [51]. The interaction between nano-
particles and bacteria not only inhibits the growth of bacteria but also
induces the generation of ROS, which leads to cell death. It has been
established that gram-positive bacteria are more sensitive to ROS than
gram-negative bacteria [52]. Future studies will optimize the interac-
tion between CeO2-NPs and E. coli to inhibit their colonization as well.
4. Conclusions
In current study, CeO2-NPs were fabricated via a green method in
order to decrease the use of toxic compounds in their synthesis by using
the Zingiber officinale extract. Then, PVA/CS hydrogels containing 0.5
and 1% CeO2-NPs were successfully produced for wound dressing ap-
plications. The freeze-thaw technique was used to improve bio-
compatibility and to have a porous structure in the fabricated hydro-
gels. Results showed that adding CeO2-NPs improved the hydrogel
swelling properties and as a result, increased the porosity in the hy-
drogels. Also, the contact angle results indicated a good capability of
the hydrogels for water absorption. Results further showed that all
samples were biocompatible and nontoxic to human dermal fibroblasts
up to 5 days, and were antibacterial to MRSA after just 12 hr, a key
bacteria resistant to methicillin that infects wounds today. Collectively,
all of these results provide significant promise for the use of the present
green CeO2-NPs in hydrogels to improve wound healing, which should
be further studied.
CRediT authorship contribution statement
Katayoon Kalantari: Investigation, Visualization, Data curation,
Writing - original draft. Ebrahim Mostafavi: Investigation,
Methodology, Visualization, Data curation, Writing original draft,
Writing - review & editing. Bahram Saleh: Methodology, Visualization.
Pooneh Soltantabar: Writing - review & editing. Thomas J. Webster:
Supervision, Writing - review & editing.
9
European Polymer Journal 134 (2020) 109853
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The author would like to thank Northeastern University (NEU),
Boston, MA, USA for the financial support of this research.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.eurpolymj.2020.109853.
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