Materials Science & Engineering C 131 (2021) 112538

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Materials Science & Engineering C

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

A balanced charged hydrogel with anti-biofouling and antioxidant

properties for treatment of irradiation-induced skin injury
Jiamin Zhang a, Yingnan Zhu c, Yumin Zhang a, Wenjing Lin a, Jia Ke b, Jianfeng Liu a,
Lei Zhang b, *, Jinjian Liu a, *
a
Key Laboratory of Radiopharmacokinetics for Innovative Drugs, Chinese Academy of Medical Sciences, and Institute of Radiation Medicine, Chinese Academy of
Medical Sciences & Peking Union Medical College, Tianjin 300192, PR China
b
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems
Bioengineering (MOE), Tianjin University, Tianjin 300350, China
c
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China

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

Keywords: Skin injury caused by large doses of ionizing radiation is the common and severe side effect of radiotherapy.
Balanced charged hydrogel However, its therapeutic efficacy is always hindered by early reactive oxygen species generation, repetitive
Alginate inflammatory microenvironment and bacterial infection risk. Herein, we report an anti-biofouling hydrogel with
Anti-biofouling
anti-inflammation and anti-oxidative properties for the treatment of irradiation-induced skin injury. The anti-
Irradiation-induced skin injury
Wound dressing
biofouling hydrogel can be achieved by balancing oppositely charged alginate, hyaluronic acid (HA) and poly­
lysine (PLL) at the optimal ratio, which effectively resist protein and bacterial adhesion, and evades immune
response. Moreover, curcumin and epigallocatechin gallate (EGCG) can be facially encapsulated and substan­
tially released from the hydrogel. Results showed that the resulting AHP-Cur/EGCG hydrogel can significantly
weaken the development of skin injury and accelerate its healing process by alleviating inflammation, scav­
enging ROS and promoting angiogenesis. Therefore, the findings presented in this work provide an effective
strategy for clinical management and treatment of ionizing radiation-induced skin injury.

1. Introduction healing process, resulting in repetitive and uncontrolled inflammatory

With the rapid development of nuclear technology, accidental nu­
clear leakage induced injury still poses a potential risk for human health
by exposing them to high doses of ionizing radiation (IR) [1–3]. Skin, the
largest and most radiosensitive organ with a short renew cycle (~26
days), is more vulnerable to be injured by IR and resulting in acute or
chronic IR-induced skin injury [4–6]. The main symptoms of IR-induced
skin injury include erythema, dry desquamation, dyspigmentation, hair
loss and ulceration, even leading to amputation [7]. It has been reported
that over 90% of patients with radiotherapy would suffer from IR-
induced skin injury, which is always posed the major challenge for the
post-operation management of cancer patients [8–10].
Generally, the healing process of IR-induced skin injury is more re­
fractory and complex than that of normal wounds. Common wound
healing processes can be divided into four overlapping phases, including
hemostatic, inflammation, proliferation and tissue regeneration [11].
While the high dose of irradiation can break this ordered sequence of
responses and ongoing cellular excessive proliferation. This is because
the ray of IR can interact with water (~70% in body tissues) and
generate multiple reactive oxygen species (ROS), which can continu­
ously cause cellular DNA damage and induce cell apoptosis. Moreover,
the overproduction of hydroxyl radicals would lead to an inflammatory
and high oxidative stress microenvironment, which greatly delayed the
damage healing. Furthermore, the open sores and decreased immunity
tend to induce skin infection and even cause deep tissue necrosis
[12–14]. Therefore, early elimination of inflammation, improvement of
antioxidation as well as avoidance of infection are critical factors for the
successful treatment of IR-induced skin injury.
Necrotic skin debridement, traditional dressing with corticosteroids
and/or anti-inflammatory agents are the commonly used treatment for
IR-induced skin injury in clinics [15,16]. Though many advanced ther­
apeutic strategies have been explored to promote IR injury healing, such
as cytokine therapy and stem cells treatment, low-intensity laser therapy
and hyperbaric oxygen therapy, the wound dressing remains the most

* Corresponding authors.
E-mail addresses: lei_zhang@tju.edu.cn (L. Zhang), liujinjian@irm-cams.ac.cn (J. Liu).

https://doi.org/10.1016/j.msec.2021.112538
Received 25 May 2021; Received in revised form 22 October 2021; Accepted 3 November 2021
Available online 5 November 2021
0928-4931/© 2021 Elsevier B.V. All rights reserved.
J. Zhang et al.
commonly used strategy [17–20]. Studies have demonstrated the moist
environment could provide an optimal condition for cell proliferation,
migration and promote the healing process [21]. Therefore, according to
the concept of moist wound healing, various hydrogels, such as
polysaccharide-based hydrogel, polymeric hydrogel, and glycopeptide
hydrogel, etc., have been developed as the wet wound dressing to
accelerate wound healing, due to their high-water content and 3D
network structure comparable to the native tissue matrix [22–26].
However, most of these hydrogel dressings were always susceptible to
undesirable biofouling (the non-specific adhesion and accumulation of
proteins, cells and bacteria), resulting in a secondary injury and infec­
tion risk when frequently replacing hydrogel dressings.
Recently, zwitterionic hydrogels with excellent biocompatibility and
anti-biofouling property have emerged as a new type of nonstick wet
wound dressing, owing to their superhydrophilicity and neutral charge
property [27]. Studies demonstrated that zwitterionic hydrogel could
efficiently promote the healing processes of the burn wound and acute
full-thickness wound by accelerating epithelialization and eliminating
inflammation [28–30]. Moreover, Zhang et al. present a dual detection
zwitterionic hydrogel which can real-time monitor the wound parame­
ters of the chronic diabetic wound and promote its healing [30]. How­
ever, to our knowledge, anti-biofouling hydrogel as wound dressing for
the treatment of the IR-induced skin injury have not been reported yet.
Therefore, the anti-biofouling property and biocompatibility of the
antifouling hydrogel motivated us to explore its effect on IR-induced
skin injury.
Alginate hydrogel is one of the commonly used wound dressings due
to its hydrophilicity, biocompatibility and easy gelation properties
[31,32]. However, the immunogenicity and electrostatic interaction
make it susceptive to stick on impaired tissues, resulting in secondary
injury when replacing the dressing. Therefore, inspired by the macro­
scopically neutral charge of zwitterionic material, we seek to prepare an
Fig. 1. (A)The schematic of the one-step preparation of the antioxidants (Curcumin and EGCG) loaded balanced charged hydrogel and its application as the pro-
healing wound dressing for the treatment of IR induced skin injury in mice.
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Materials Science & Engineering C 131 (2021) 112538
alginate-based neutral charged hydrogel by balancing oppositely
charged polymers. The hydrogel combined the anti-biofouling merit of
zwitterionic hydrogel with the antioxidant for a rapid repair of the IR-
induced skin injury. Negatively charged alginate (Alg) and hyaluronic
acid (HA) were used as the hydrogel matrix and the aqueous soluble
polymer polylysine (PLL) with positive charge was used to modulate
charge to balance. Natural polyphenol derivative curcumin and
(-)-epigallocatechin gallate (EGCG) have been used as antioxidative and
anti-inflammatory agents for reduction of chronic inflammation
[33–37]. Therefore, in this work, curcumin and EGCG were further
encapsulated into the hydrogel via a one-step gelation for the treatment
of IR-induced skin injury. Results showed that the balanced charged
hydrogel (Alg/HA-PLL0.038) could be achieved when the oppositely
charged polymers in optimal ratio of alginate, HA and PLL at 1/
0.25:0.038 (w/w). Animal studies showed that this multifunctional
hydrogel dressing could relieve the development and promote the
repairment of IR-induced skin injury in a mice model by eliminating
inflammation and ROS, as well as accelerating angiogenesis (Fig. 1).
2. Experimental section
2.1. Materials
Alginate sodium, hyaluronic acid (HA) and polylysine (PLL) were
purchased from Sigma-Aldrich (USA). Barium chloride (BaCl2, 99.9%
metals basis) was obtained from Macklin Biochemical Technology Co.,
Ltd. (Shanghai, China). Curcumin (Cur) and (-)-epigallocatechin gallate
(EGCG) were obtained from TCI Inc. (Japan). Fluorescein
isothiocyanate-labelled bovine serum albumin (BSA-FITC), CCK-8 kit,
Masson's trichrome kit, and hematoxylin and eosin kit (H&E) were
purchased from Solarbio Science & Technology (Beijing, China). The
regents for bacteria culture were obtained from Oxoid (UK). The
J. Zhang et al.
Surgidrape Adhesive Membrane was purchased from 3 L Company
(Jiangxi, China). Rabbit anti-CD31 antibody and rabbit anti-CD 68
antibody were brought from Abcam (Cambridge, U.K.) Other chemical
regents were all obtained from Tianjin Chemical Regent Company
(Tianjin, China).
2.2. Preparation of polyelectrolyte hydrogels
Alginate powders were first purified before using in this work [38].
AH-PLL hydrogels were prepared by mixing the negatively charged
polyelectrolytes (alginate and HA) with the positively charged poly­
electrolyte (PLL). Briefly, 0.25 ml HA solution (1%, w/v) was added into
1 ml alginate solutions (1%, w/v) with a ratio of 1:0.25 (w/w). Then, PLL
solutions (1%, w/v) were slowly added into the Alg/HA solutions with
high-speed stirring and then further crosslinked by BaCl2 solutions
(10%, w/v) with the proportion of Ba2+, alginate being 1:2 (w/w). The
Zeta-potential of each hydrogel sample was measured by Zetasizer Nano
(Malvern, U.K.). Finally, five hydrogel formulations were prepared by
tuning the mass ratios between the Alg/HA and PLL, including 1:0.25:0,
1:0.25:0.018, 1:0.25:0.038, 1:0.25:0.06, 1:0.25:0.1. After gelation,
hydrogels were washed with 0.9% NaCl solutions three times for further
experiments.
Curcumin and EGCG loaded hydrogel were fabricated via a one-step
gelation process. Curcumin (1 mg/ml) alone or with EGCG (0.5–1 mg/
ml) powder was added to the Alg/HA-PLL mixture solution (1 ml). After
fully blending, curcumin was well dispersed in the solution, and the
EGCG was completely dissolved in the Alg/HA-PLL mixture solution.
Then, 300 μl BaCl2 solutions were added for the gelation process.
Finally, the curcumin-loaded hydrogel (AHP-Cur), EGCG-loaded
hydrogel (AHP-EGCG) and the curcumin and EGCG loaded hydrogels
(AHP-Cur/EGCG) were obtained.
2.3. Characterization
The water contents of the hydrogel samples were measured by the
mass loss method. Hydrogel samples were weighed and noted as mw.
Then, the samples were dehydrated under vacuum at 60 ◦ C for 3 d and
weighed again, noted as md. All samples were measured in quintupli­
cate. The water content values were calculated by the following
equation:
Water content = (mw − md )/mw × 100% (1)
The compressive modulus tests of the hydrogel samples were
measured by a caliper microcomputer control mechanical tester (WDW-
5, Beijing, China). Hydrogel samples were punched into 8 mm-diameter
disks (2 mm thickness). Disk samples were compressed at a rate of 0.5
mm/min. The compressive modulus was calculated from the initial 10%
strain to avoid any complications in case the top plate had not been
completely touching the samples when compression began.
2.4. Protein adsorption
FITC-labelled BSA was used in this test to evaluate the surface pro­
tein adsorption property of the hydrogel samples. All the hydrogel
samples were punched into 5 mm diameter disks and incubated with
FITC-labelled BSA solutions (0.5 mg/ml) for 30 min away from the light.
Then the samples were washed with 0.9% normal saline solutions 3
times and observed under an inverted fluorescence microscope (Nikon
Eclipse Ti–S, Japan). The relevant fluorescence intensity was normal­
ized to the area of the observed hydrogel using Image J software (n = 5).
2.5. Hemocompatibility tests
To evaluate the hemocompatibility of the hydrogel samples, blood
cell adhesion and hemolysis assays were used in this test. For blood cell
adhesion tests, hydrogel samples (1 cm diameter) were transferred into
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Materials Science & Engineering C 131 (2021) 112538
24-well plates and incubated with whole blood for 30 min at 37 ◦ C.
Then, the hydrogel samples were gently rinsed with 0.9% normal saline
solutions 3 times and observed (n = 5). For hemolysis assay, hydrogel
samples were co-incubated with fresh blood in 5 times 0.9% NaCl so­
lution. Deionized water and 0.9% NaCl solution diluted blood were used
as positive control group and negative control group, respectively. After
being incubated at 37 ◦ C for 30 min, samples were centrifugated at 1000
rpm for 10 min. Then the supernatants were collected, and their ab­
sorbances were measured at 541 nm. The hemolysis ratio (HR) was
calculated by the Eq. (2):
/( )
Hemolysis ratio (HR) = (As − An ) Ap − An × 100% (2)
where As, Ap and An represent for the absorbance of samples, positive
control, and negative control at 541 nm, respectively.
2.6. Bacterial adhesion
Gram-negative bacteria, Escherichia coli (E. Coli) was used to evaluate
the bacterial adhesion on the surface of hydrogels. Before hydrogel
preparation, all the materials were sterilized, and hydrogels were pre­
pared in aseptic conditions. Hydrogel samples (1 cm diameter) were
incubated with bacterial suspension (1 × 104 cell/ml) for 2 h under
orbital stirring. Then the hydrogel samples were transferred into a new
plate and gently rinsed 3 times with 0.9% NaCl solution to remove the
free bacteria. Finally, to remove the adherent bacterial cells from the
sample surface, the samples in fresh solution were then sonicated with a
frequency of 40 Hz for 15 s at room temperature. The last washing so­
lution (100 μl) of each hydrogel was spread on LB agar plates and
incubated at 37 ◦ C for 24 h. Finally, the bacterial colony was counted,
and colony-forming units were calculated (n = 5).
2.7. Curcumin and EGCG releasing behavior
Hydrogel disk samples (8 mm in diameter, 5 mm in thickness) were
immersed in 10 ml PBS solution, mimicking the physiological environ­
ment, and incubated at 37 ◦ C in a shaking bath. 5 ml PBS solution was
collected at each predetermined timepoint and 5 ml fresh PBS solution
was added. The release behaviors of curcumin and EGCG from the AHP-
Cur/EGCG hydrogel and the resultant solution after gelation were
measured by the HPLC.
2.8. Hydroxyl radical scavenging assay
The antioxidative ability of hydrogel samples was measured by
DPPH (2,2- diphenyl-1-picrylhydrazyl hydrate) assay[39,40]. Briefly,
AH-PLL, AHP-Cur, AHP-EGCG and AHP-Cur/EGCG hydrogel samples
(0.5 g) were first immersed and incubated in medium for 48 h. 2 ml
DPPH solution (0.1 mM) in 60% ethanol mixed with 2 ml different
hydrogel sample immersed solutions and incubated at 37 ◦ C for 30 min
away from the light. The absorbance of the DPPH (A0) and samples (As)
were detected at 520 nm. The scavenging rate (%) was calculated ac­
cording to Eq. (3).
Scavenging Rate (%) = (A0 − AS )/A0 × 100% (3)
2.9. Cytotoxicity analysis
For cell and in vivo experiments, alginate and HA solution were pre-
filtered by 0.22 μm sterile membrane and the curcumin and EGCG
powder were sterilized under UV for 30 min. All hydrogels were pre­
pared in a vertical flow clean bench. The cytotoxicity of antioxidants-
loaded hydrogel samples was measured by CCK-8 assay. NIH-3 T3
fibroblast cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum and peni­
cillin/streptomycin (1%, v/v) and seeded onto a 96-well plate at a
J. Zhang et al.
concentration of 5 × 105 cells/ml (100 μl). AH-PLL, AHP-Cur, AHP-
EGCG and AHP-Cur/EGCG hydrogel samples were first immersed and
incubated in medium for 24 h, and then used for cytotoxicity analysis.
The extraction medium was added to a 96-well plate after discarding the
culture medium. After 24 h, CCK-8 reagents were added and the
absorbance of the final purple suspensions was measured with a
microplate reader (Tecan infinite 200 PRO, Switzerland) at 450 nm.
2.10. In vivo implantation and treatment of radiation-induced mice skin
injury
All animal experiments were performed in compliance with the
guidelines of the Administration of Experimental Animals (Tianjin,
revised June 2004) and approved by the Animal Ethics Committee of
IRM-CAMS. Female BALB/c mice (8 weeks, 20 g) were obtained from the
Laboratory Animal Center of the Academy of Military Medical Sciences
(Beijing, China). To evaluate the anti-inflammatory property of the three
AH-PLL hydrogels, hydrogels with different charges were subcutane­
ously implanted into the mice for 1 week and 3 months. Two hydrogel
samples (5 mm diameter) were implanted at each side of the dorsal
region, respectively. At each time point, five implanted samples of each
hydrogel formulation were harvested for further statistical significance
studies.
To set up a radiation-induced skin injury model, the mice were
anesthetized and exposed to their hind limb skin to 40 Gy of X-ray at a 2
Gy/min for 20 min (Biological X-ray irradiator, Rad Source, RS 2000).
After exposure, all mice were observed daily and well housed for 10 d.
When the hair in the irradiation site began to lose, then exposure sites
were covered with as-prepared round shape hydrogels (8 mm in diam­
eter), and then fixed with the Surgidrape Adhesive Membrane. Mice
were divided into four groups: AH-PLL, AHP-Cur, AHP-EGCG, AHP-Cur/
EGCG, and commercial wound dressing (Duoderm®) treatment control.
The hydrogel dressings were changed every two days. On days 0, 2, 4, 6,
8, 10, 12 and 14 post-treatment, wound areas were observed and
photographed.
2.11. Histological analysis
At each predetermined time point, mice were sacrificed through CO2
asphyxiation. For implanted hydrogel samples, the surrounding tissues
and samples were harvested together at 1 week and 3 months after
implantation, and the hydrogel treated impaired skin tissues were
collected at 3 d, 7 d and 14 d after treatment. Subsequently, all samples
were fixed with 4% paraformaldehyde at 4 ◦ C overnight and dehydrated
with 30% sucrose solutions. Then, embedded into an optical cutting
temperature compound at − 20 ◦ C and cut into 6 μm sections using
freezing microtome (Leica, Germany). For histological analysis, H&E
staining and Masson's trichrome staining were used to evaluate the
inflammation and healing processes, and the images were obtained by
upright microscope (Leica, Germany). Macrophage infiltration and
neovessels formation were studied by immunofluorescence stain. The
macrophage expression and endothelial cells in the samples were eval­
uated by incubating the sections with the rabbit anti-CD68 and rabbit
anti-CD31 primary antibodies (dilution 1:100, Abcam). Alexa Fluor 594
goat anti-rabbit and Alexa Fluor 488 goat anti-rabbit (dilution 1:200,
Abcam) were used as secondary antibodies. Finally, the sections were
stained and mounted by the 4,6-diamidino-2-phenylindole
Fluoromount-G (Southern Biotech, U.K.). After 10 min incubation
away from the light, sections were observed under a fluorescent
microscope.
2.12. Enzyme-linked immunosorbent assay (ELISA)
To quantify the amount of IL-10, TNF-α and Arg-1 in the skin tissues
after 7 d and 14 d treatment with different dressing, IL-10 (Abcam, cat:
ab108870), TNF-α (Abcam, cat: ab208348) and Arg-1(Abcam, cat:
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Materials Science & Engineering C 131 (2021) 112538
ab269541) ELISA kits were used. Briefly, we harvest the skin tissue
samples of the injury site after treatment for 7 and 14 d and grind the
tissues. After centrifugation at 5000 rpm for 10 min, the supernatants
were collected. Then the amount of IL-10, TNF-α and Arg-1 were
measured according to direction of the ELISA kit.
2.13. Statistical analysis
Quantitative data was expressed as mean ± standard deviation (SD).
Statistical comparisons were made by two-sample Student's t-test. P
value <0.05 was considered statistically significant.
3. Results and discussion
3.1. Preparation and characterization of the hydrogel samples
Inspired by the anti-biofouling mechanism of zwitterionic materials,
we prepared a new type of antifouling hydrogel by balancing the
oppositely charged polymers alginate, HA and PLL (Fig. S1). It has been
reported that oppositely charged polyelectrolytes could aggregate and
assemble together through electrostatic interactions [41]. However,
oppositely charged polymers with high charge density could also induce
aggregation and form flocculent precipitate [42]. Therefore, in this
work, we incorporate viscous HA solution with low charge density into
alginate solution at the ratio of 1/0.25 (alginate/HA, w/w) to weaken
the strong interaction between alginate and PLL. Five different formu­
lations of Alg/HA-PLL solution (Alg/HA-PLL0, Alg/HA-PLL0.018, Alg/
HA-PLL0.038, Alg/HA-PLL0.06, Alg/HA-PLL0.1) were obtained by mixing
Alg/HA mixture with PLL at different ratios. Finally, the solutions were
further crosslinked with barium chloride (Ba2+) at room temperature.
A high-water content of hydrogel is beneficial to the wound healing
process, which can keep the wet healing environment. Therefore, we
first characterized the water content of the hydrogel samples by the mass
loss method. It could be found that no obvious changes in water content
of these five hydrogel samples (Fig. S2A). Moreover, all of them
exhibited high water content values (> 97%). This result indicated that
the incorporation of PLL did not influence the water content of hydrogel
samples. Afterward, the compressive modulus of hydrogel samples was
also evaluated (Fig. S2B). It was shown that the compressive modulus of
the hydrogel decreased with the increase of PLL from 0.0518 MPa to
0.0225 MPa. This may be because the competitive consumption of
carboxylic acid groups (COO− ) from alginate by positively charged
polymers would weaken the crosslinking behavior between alginate and
Ba2+. The uniform structure of the hydrogel samples was not obviously
influenced by the higher positively charged polymer incorporation,
though the higher contents resulted in lower compressive modulus.
3.2. In vitro anti-biofouling properties of the hydrogel
Biomedical materials with anti-biofouling properties are highly
favorable for many biomedical applications [43]. Biofouling (e.g. bio­
molecules, cells or bacteria) adhesion on biomedical materials will
induce a cascade of biological responses and impede their function and
cause severe side effects. Therefore, to investigate the anti-biofouling
properties of the hydrogel samples, protein adsorption tests, bacteria
adhesion tests and hemocompatibility analysis were performed in this
work. Protein adsorption on the surfaces of materials was the first step
when the implants contact tissues and then induced a cascade of im­
mune responses [44].
Protein adsorption performances on all hydrogel samples were
evaluated by fluorescence protein (BSA-FITC) adsorption tests. It could
be noted that no obvious green fluorescence could be observed on Alg/
HA-PLL0.038 hydrogels (Fig. 2A). Conversely, other hydrogel samples
with positive charge or negative charge showed different degrees of
green fluorescence adsorption on their surfaces. The strongest fluores­
cence was achieved when the hydrogel with more net charge (Fig. 2B).
J. Zhang et al. Materials Science & Engineering C 131 (2021) 112538

Fig. 2. Protein adsorption on the surfaces of five hydrogel formulations. (A) BSA-FITC adsorption on the surfaces of different charged hydrogel samples, Green: BSA-
FITC. (B) The relative fluorescence intensity of the different hydrogel samples (n = 5). (C) The zeta potential of the different hydrogel samples. Scale bar =200 μm.
Data were presented as mean ± SD. Comparisons among multi-groups were analyzed by one-way ANOVA with post-hoc Bonferroni’s test. *P < 0.05, **P < 0.001,
***P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

This may be because biomolecules (e.g. proteins) with charges can be
adsorbed on the charge-bearing surfaces via electrostatic and hydro­
phobic interactions. Among the five formulations, the Alg/HA-PLL0.038
hydrogel exhibited the best anti-protein adsorption property, indicating
this hydrogel may be the neutral charged hydrogel. To further confirm
this assumption, we measured the zeta potential of these hydrogel
samples. As expected, the zeta potential of Alg/HA-PLL0.038 was close to
zero (− 1.3 ± 0.7 mV), consistent with the results of BSA protein
adsorption test (Fig. 2C). This result demonstrated that the balanced
charged hydrogel could be achieved when the oppositely charged
polyelectrolytes at optimal ratio, further confirming the balanced
charged strategy.
Bacterial adhesion on the biomedical material surface is the main
cause of the formation of biofilm, which would induce inflammatory and
even cause secondary infection after contact with tissues. An ideal
wound dressing should be able to resist bacterial adhesion and invasion.
Thus, we further evaluated the anti-biofouling properties of hydrogel
samples by E. Coli and S. aureus adhesion tests. As shown in Fig. 3A and
Fig. S3, negligible E. Coli or S. aureus adhesion could be observed on the
surfaces of Alg/HA-PLL0.038 hydrogel. In contrast, severe bacterial
adhesion was observed on either negatively charged hydrogels or posi­
tively charged hydrogels. Interestingly, bacterial adhesion on the posi­
tively charged hydrogel is less than that on the negatively charged
polymer (Fig. 3B). This may be because the positive charge of hydrogel
could interact with the negatively charged bacterial membrane, leading
to bacterial membrane destruction. These data demonstrated that the
balanced charged hydrogel could prevent protein adsorption as well as
prevent bacterial adhesion on their surfaces, which is highly desirable in
the development of wound dressing materials.
Hemocompatibility is also a critical factor for the development of
biomedical materials. The adsorption of fibrinogen will induce platelet
aggregation and activation, resulting in blood clotting and even causing
thrombus. Therefore, in this work, blood cell adhesion and hemolysis
analysis of different charged hydrogel samples were evaluated. As ex­
pected, no obvious blood cells adhesion or blood clotting formation on
the surfaces of the balanced charged Alg/HA-PLL0.038 hydrogel sample,
thanks to its excellent anti-protein adsorption property (Fig. 4A). While
other hydrogel samples, especially the hydrogels with more positive

Fig. 3. Bacterial adhesion tests on the surfaces of five hydrogel formulations. (A) Digital images of relative E. coli adhesion on the surfaces of different charged
hydrogel samples; (B) the Colony-Forming Units of E. coli adhesion on different hydrogel samples, respectively.

5
J. Zhang et al. Materials Science & Engineering C 131 (2021) 112538

Fig. 4. Hemocompatibility of the five formulations of different charged hydrogel samples. (A) Fresh blood cell adhesion tests on surfaces of different types of
hydrogel samples. (B–C) the hemolysis ratios of these hydrogel samples.

charges, showed a little blood cells adhesion, due to the electrostatic results indicated that all hydrogel samples could not elicit severe he­
interaction between the charged hydrogel and cell membrane. More­ molysis, meanwhile, the balanced charged Alg/HA-PLL0.038 hydrogel
over, the hemolysis assay showed that all the hydrogels could not evoke showed the best hemocompatibility.
serious hemolysis, exhibiting a lower hemolysis rate than the detection
line of biomedical hemolysis rate (<5%) [45]. And the supernatant of
balanced charged hydrogel was the cleanest (Fig. 4B and C). The above

Fig. 5. (A) H&E staining images of different charged Alg/HA-PLL hydrogel samples (Alg/HA-PLL0, Alg/HA-PLL0.038 and Alg/HA-PLL0.1) 1 week after subcutaneous
implantation in mice. Masson's trichrome staining images of the different charged hydrogel samples (B) 1 month and (C) 3 months after subcutaneous implantation in
mice, scale bar = 200 μm. Red arrows indicate the formation of fibrous capsule. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

6
J. Zhang et al.
3.3. In vivo biocompatibility of the hydrogels
In vivo inflammation responses to the implants were the main threat
to the wound healing process. The in vivo biocompatibility of the
different charged Alg/HA-PLL hydrogels (Alg/HA-PLL0, Alg/HA-
PLL0.038, Alg/HA-PLL0.1) were evaluated by subcutaneously implanted
hydrogel samples in BABL/c mice. All of the hydrogel samples passed
the detection of endotoxin test before being used in in vivo experiments
via limulus amebocyte lysate test. Generally, acute inflammation often
occurs at the early 3 weeks after implantation [46]. Therefore, we first
investigated the early immune responses to implants after 1-week and 1-
month implantation. Representative H&E staining and Masson's tri­
chrome staining images revealed that negligible inflammatory cells
accumulated at the interfaces of the balanced charged Alg/HA-PLL0.038
hydrogels, corresponding to the in vitro anti-biofouling properties results
(Fig. 5A and B). In contrast, it could be found that lots of inflammatory
cells and collagen deposited on the interfaces between hydrogel samples
and those unbalanced charged hydrogels, especially the positively
charged hydrogels. These results suggested that the balanced charged
hydrogel could elicit less inflammation than those charge bias hydrogel
samples.
Moreover, we further evaluated the long-term performance of the
hydrogels after implantation by investigating the capsule formation
around the hydrogels after implantation for 3 months. Masson's tri­
chrome staining images showed that all the positively charged hydrogels
were encapsulated by a thick collagenous capsule (Fig. 5C). While the
negatively charged Alg/HA-PLL0 hydrogel was also surrounded by a
fibrous capsule. As expected, there was no obvious capsule formation
around the balanced charged Alg/HA-PLL0.038 hydrogel. The above re­
sults proved that the balanced charged hydrogel with anti-biofouling
and anti-FBR capacities can effectively reduce the risk of infection and
inflammation, which could be an ideal wound dressing candidate.
Fig. 6. (A) The morphology of AH-PLL and AHP-Cur and AHP-Cur/EGCG. (B) Cell viability of hydrogel samples. (C)The releasing behavior of curcumin and EGCG
from the hydrogel. (D-E) Hydroxyl radical scavenging activities of AH-PLL, AHP-Cur, AHP-EGCG and AHP-Cur/EGCG. (D) The digital images of hydrogel samples
incubated with DPPH for 30 min in the dark and the (E) scavenging rate of hydrogel samples. Comparisons between any two groups were analyzed by a two-tailed
Student's t-test. *P < 0.05, **P < 0.001, ***P < 0.0001.
7
Materials Science & Engineering C 131 (2021) 112538
3.4. Curcumin and EGCG releasing behavior
For the treatment of IR-induced skin injury, besides the anti-
biofouling properties, antioxidative activities and anti-inflammation
were also required for an ideal wound dressing. Curcumin is a natu­
rally derived low molecular weight polyphenol, exhibiting multiple
biological functions, such as antioxidative activities, anti-inflammatory
or anti-tumor activities[34,47]. However, curcumin is a hydrophobic
molecular which cannot well dissolve in an aqueous environment, which
may influence its releasing rate from the hydrogel. Recently, numerous
efforts have been made to improve the solubilization of curcumin, such
as the development of different delivery systems [33]. While it has also
been reported that high concentrations of polyphenol material could
suppress cell growth [48]. Therefore, we introduced another effective
and commonly used hydrophilic antioxidant EGCG into balanced
charged Alg/HA-PLL0.038 hydrogel for the sustainable releasing antiox­
idant from the hydrogel matrix, decreasing the dosge of curcumin and
reducing decrease the cell suppression of the polyphenol antioxidants
[49,50].
For the subsequent treatment experiments of IR-induced mice skin
injury, we named Alg/HA-PLL0.038 hydrogel without or with antioxi­
dants as AH-PLL, AHP-Cur, AHP-EGCG and AHP-Cur/EGCG, respec­
tively. As shown in Fig. 6A, it could be found that the transparent AH-
PLL hydrogel became yellow after incorporating curcumin and EGCG,
indicating the successful encapsulation of the antioxidants could not
affect hydrogel morphology (Fig. S4). Moreover, the cytotoxic tests
showed cell viabilities of curcumin and/or low concentration EGCG (0.5
mg/ml) loaded hydrogels were higher than 80%, indicating that these
hydrogels were not cytotoxic (Fig. 6B). Notably, it could be found that
the higher EGCG content, the cytotoxic will be increased. Therefore, we
choose EGCG at a concentration of 0.5 mg/ml for further in vivo tests.
Furthermore, the curcumin and EGCG releasing behaviors were
J. Zhang et al.
measured and shown in Fig. 6C. It could be found that both curcumin
and EGCG could continuously released from the AHP-Cur/EGCG
hydrogel in 48 h, however, the EGCG (95.2 ± 1.8% at 48 h) is faster
than that of the curcumin (46.1 ± 2.9% at 48 h). This is mainly due to
the different hydrophilic and hydrophobic properties of ECGC and
curcumin, and EGCG is more easily released in aqueous environment.
Next, the antioxidant activity of these different hydrogel dressings
was also investigated. Rapid scavenging excessive ROS is important for
the wound healing process. As shown in Fig. 6D-E, the AHP-Cur
hydrogel extract solution exhibited a very slow scavenging rate. This
may be due to the hydrophobic property of the curcumin, which cannot
fast release from the hydrogel. Both the AHP-EGCG hydrogel and AHP-
Cur/EGCG hydrogel showed fast scavenging rates, meanwhile the AHP-
Cur/EGCG hydrogel had the best hydroxyl radical capacity. From the
long-term antioxidant experiments of the AHP-Cur/EGCG, we could be
found that the addition of ECGC could significantly improve the short-
term antioxidant capacity of the AHP-Cur hydrogel. And the AHP-Cur/
EGCG showed the best long-term antioxidant capacity at 24 h, higher
than that of AHP-ECGC (Fig. S5). Therefore, the combination of hy­
drophilic and hydrophobic antioxidants could significantly improve the
antioxidant release from the hydrogel and enhance the antioxidant
property of the AH-PLL hydrogel.
3.5. The therapeutic effects of hydrogel dressing on the radiation-induced
skin injury
To investigate the therapeutic effects, AH-PLL and AHP-Cur, AHP-
EGCG and AHP-Cur/EGCG hydrogel dressings were applied to the IR-
induced local skin impaired region 10 d after 40 Gy irradiation
(Fig. 7A). Then, the dressings were replaced every 2 d and retrieved the
tissues after 3, 7, and 14 d treatment. As shown in Fig. 7B, it could be
noted that all the tissues exhibited typical erythema and depilation
states before treatment. The wound surfaces in both AH-PLL and control
groups became red ulcers and/or transparent exudation with the times
after treatment. In contrast, the wound gradually healed of the mice
treated with AHP-Cur/EGCG. And all the hydrogel groups were better
Fig. 7. In vivo treatment of the IR-induced skin injury. (A) The time schedules of the treatment procedure. (B) The photographs of healing process of the IR-induced
skin lesion region treated with different hydrogel dressings. (C) H&E staining images of the skin tissues after different hydrogel dressings treatment for 3 d, 7 d, 14 d,
Scale bar = 200 μm.
8
Materials Science & Engineering C 131 (2021) 112538
than the control group, indicating the moist hydrogel could slow the
development of the skin injury.
To further evaluate the cell infiltration and regeneration of the
epidermis, H&E staining was performed at 3, 7 and 14 d after hydrogel
treatment. As shown in Fig. 7C, H&E images indicated that the AHP-
Cur/EGCG treatment groups alleviated the skin injury development
and obviously boosted the healing process of the IR-induced lesion.
Additionally, the epidermis and the hair of the mice treated with AHP-
Cur/EGCG were well regenerated at 14 d after treatment, but other
groups were still not healed. In particular, the epidermis and tissues in
skin lesion of control group were incomplete. The IR-induced skin injury
did not show an obvious visible wound at the early stage (14 d after
exposure to IR). Then, the skin began typical erythema is localized to the
radiation field and the skin is noticeably red, edematous, warm, and
tender by the third or fourth week of radiation [7]. Therefore, on the 3
days, the skin was still intact and with the injury development, the skin
began to be damaged.
3.6. The regeneration and vascularization of the damaged skin tissues
Vascularization is an important process for tissue recovery and
regeneration. In this stage, new capillaries developed from the micro­
vasculature and promoted cell proliferation and mitigation, further
establishing cell-cell communications [51]. Moreover, sufficient blood
supply could provide oxygen and nutrients, which are essential for
wound healing process. Therefore, we assay the effect of hydrogel
dressing on neovascularization by immunohistochemical staining CD31
antibody, a capillary marker, 7 and 14 d after treatment. As shown in
Fig. 8A, more new blood vessels could be observed on the AHP-Cur/
EGCG hydrogel treatment group compared with the control group and
AH-PLL group on 7 d after treatment. Meanwhile, microvessels were
increased over time, while the control group still had limited blood
vessels, which were fewer than the other groups at 14 d after treatment.
Chronic inflammation is an unfavorable process for tissue regener­
ation, which would delay the wound healing process. Macrophages are
the main commander of immune response regulation [52]. Therefore,
J. Zhang et al.
Fig. 8. Immunostaining images of the skin injury region against (A) CD31 and (B) CD 68 7 d and 14 d after different hydrogel dressings treatment. (C-E) ELISA
analysis of relative TNF-α, IL-10 and Arg-1 expression in the skin injury region 7 and 14 d after treatment, respectively. DAPI stained in blue, CD31 stained in red and
CD68 stained in green. Scale bar = 100 μm. Data were presented as mean ± SEM. Comparisons between any two groups were analyzed by a two-tailed Student's t-
test. *P < 0.05, **P < 0.001, ***P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
we further evaluated the macrophage infiltration at the impaired region
by immunohistochemical staining CD68, a macrophage maker, at 7 and
14 d after treatment. A large number of macrophages were found at the
tissues in all hydrogel treated mice at 7 d. While there were still lots of
macrophages that could be observed at 14 d in control and AH-PLL
hydrogel treatment groups (Fig. 8B). In comparison, macrophages
were significantly decreased and only a few could be observed at the
tissue treated with AHP-Cur-EGCG hydrogel at 14 d. Furthermore, we
also investigated the pro-inflammatory macrophage (TNF-α) and anti-
inflammatory macrophage (IL-10 and Arginase-1) expression by ELISA
assay (Fig. 8C-E). Notably, the pro-inflammatory factor TNF-α expres­
sion of AHP-Cur, AHP-EGCG and AHP-Cur/EGCG hydrogel treated mice
were lower than that of the control group and AH-PLL hydrogel group,
both on 7 d and 14 d. While the AHP-Cur/EGCG hydrogel treated mice
showed the lowest TNF-α expression, which was similar to that of
normal tissues at 14 d. Moreover, a high anti-inflammatory factor IL-10
and Arginase-1 expression on the AHP-Cur/EGCG hydrogel treated mice
at 7 d indicated that the AHP-Cur/EGCG hydrogel could eliminate
inflammation and began to promote the tissue healing.
4. Conclusion
In summary, we successfully developed a balanced charged hydrogel
wound dressing AHP-Cur/EGCG for the treatment of IR-induced skin
injury. The balanced charged hydrogel as the matrix of the wound
dressing could effectively resist protein adsorption, bacterial adhesion
and showed excellent hemocompatibility. Moreover, they could also
evade immune recognition and long-term capsule formation after 3
months implantation in mice model. Furthermore, the introduction of
antioxidants could impart the wound dressing rapid ROS scavenging
capacity. Most importantly, the AHP-Cur/EGCG hydrogel could signif­
icantly relieve the development of IR-induced skin injury and accelerate
wound healing by eliminating inflammation and improving vasculari­
zation than that of the control group. Therefore, the new hydrogel
9
Materials Science & Engineering C 131 (2021) 112538
dressing materials hold a great potential clinical application for man­
agement of the IR-induced skin injury.
CRediT authorship contribution statement
Jiamin Zhang: Conceptualization, Methodology, Writing-original
draft, Project administration. Yingnan Zhu: Investigation, Validation,
Formal analysis. Yumin Zhang: Investigation, Formal analysis. Wenj­
ing Lin: Investigation, methodology. Jia Ke: Investigation, Formal
analysis. Jianfeng Liu: Supervision Resources, Funding acquisition,
Writing-review & editing. Lei Zhang: Conceptualization, Funding
acquisition, Writing-review & editing. Jinjian Liu: Conceptualization,
Resources, Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
This study was financially supported by the National Natural Science
Foundation of China (Nos. 82001963, 21621004, 22078238,
21961132005), PUMC Youth Fund and the Fundamental Research
Funds for the Central Universities (No. 3332020058), the Non-profit
Central Research Institute Fund of Chinese Academy of Medical Sciences
(2018PT35031) and the National Science Fund for Distinguished Young
Scholars of Tianjin (18JCJQJC47300).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.msec.2021.112538.
J. Zhang et al. Materials Science & Engineering C 131 (2021) 112538

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