LWT - Food Science and Technology 171 (2022) 114147

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LWT
journal homepage: www.elsevier.com/locate/lwt

Fabrication, characterisation, and application of green crosslinked sodium

alginate hydrogel films by natural crab-shell powders to achieve drug
sustained release
Hui Wang a, Jinming Liu a, b, Xu Fan a, Jing Ren a, Qian Liu a, Baohua Kong a, *
a
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China
b
Patent Examination Cooperation Jiangsu Center of the Patent Office, CNIPA, Suzhou, Jiangsu, 215000, China

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

Keywords: In this study, natural crab-shell particles (CSP) and deacetylated crab-shell particles (dCSP) were used as
Sodium alginate hydrogel film crosslinking agents at the present of glucolactone to obtain high strength mono-crosslinked and bi-crosslinked
Green crosslinking sodium alginate (NaAlg) hydrogel films, respectively. The results indicated that the CSP in the system could
Crab-shell particles
occur Ca2+ crosslinking, and the addition of dCSP could help to form both Ca2+ crosslinking and polyelectrolytes
Drug release
interactions with NaAlg. The remaining unreacted CaCO3 could enhance the thermal stability of the films due to
the high thermally stable CaCO3. The surface hydrophobicity, gel properties, mechanical strength and thermal
stabilities of those bi-crosslinked NaAlg hydrogel films were better than those of neat NaAlg film and mono-
crosslinked NaAlg hydrogel films, which overcome the inferior thermo-mechanical properties for traditional
hydrogel films. In addition, the releasing time of those prepared hydrogel films for diclofenac sodium was higher
than 36 h, which illustrated sustained drug releasing activity. The CSP modified NaAlg hydrogel film with 0.5%
GDL content exhibited the highest cumulative release amount. The proposed approach is a promising green
crosslinking technique to develop high performance NaAlg hydrogel films that can realize active ingredients and
drugs sustained release in food packaging and medical wound dressing areas.

1. Introduction in numbers of applications such as wood dressing, tissue engineering

Bio-based hydrogel films have garnered considerable interest
compared with petroleum-based materials owing to the increasing
awareness of environmental aspects over the years. Biodegradable
polymers such as starch, lignin, cellulose, and carbon nanofibers have
been widely used to design hydrogel films by many green principles such
as cycle of freezing and thawing, natural crosslinking, self-assembly, and
plasma polymerization (Llorens-Gámez et al., 2020). Among these
polymers, sodium alginate (NaAlg) as a kind of natural, abundantly
available, cost-effective, nontoxic, and biocompatible polysaccharide
exhibits excellent absorption ability, closure capacity, mechanical
resistance, and moisture retention rate (Dowling et al., 2011).
NaAlg is composed of β-D-mannuronic acid (M) and ɑ-L-guluronic
acid (G), which is extracted from brown algae and has been gradually
and extensively used to form biodegradable hydrogel film. Those three-
dimensional NaAlg hydrogel films can be easily formed by chemical or
physical crosslinking (Comaposada et al., 2015), which are widely used
(Martin et al., 2015), biomedicine (Neto et al., 2016), drug delivery
(Ahmed, 2015), biomacromolecule immobilisation (Volodkin et al.,
2004), active food packaging (Liu et al., 2021) and other fields (Iglesias
et al., 2020). Nevertheless, the lower crosslinking strength restricts its
application. In recent years, several methods have been reported to in­
crease its crosslinking strength, such as introducing interpenetrating
polymer networks (Li, Dou, Feng, & Zhang, 2013; Serrano-Aroca &
Llorens-Gámez, 2017) and reinforcing via other polymer (Khoushabi
et al., 2015). Increasing the crosslinking content is the most straight­
forward method among those modification strategies. The most uni­
versal chemical crosslinking method is combined with bivalent cations
such as Cu2+, Zn2+, and Ca2+ to form stable ‘egg box’ structure during
heating (Li et al., 2022; Zhou et al., 2018), however, those bivalent
cations are usually coming from non-natural substance (Li et al., 2021).
Although physical crosslinking methods, such as that for the formation
of polyelectrolytes using polyanions and polycations, are prospective
solutions to obtain hydrogel films, the polyelectrolytes interaction is

* Corresponding author.
E-mail address: kongbh63@hotmail.com (B. Kong).

https://doi.org/10.1016/j.lwt.2022.114147
Received 7 July 2022; Received in revised form 25 October 2022; Accepted 4 November 2022
Available online 5 November 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
H. Wang et al.
insufficient to achieve hydrogel with the desired mechanical strength.
Multiple crosslinking method is widely used to enhance the crosslinking
degree of NaAlg hydrogel films (Zhang et al., 2022). More over, most
multiple crosslinking methods of NaAlg hydrogel films declared are
induced by two or more compounds. However, the study of multiple
crosslinking method for NaAlg hydrogel films induced by a single nat­
ural source has been reported rarely.
Crab-shell particles (CSP), which are the main waste of crustacean
products, are consist of approximately 80% calcium carbonate (CaCO3)
and 20% chitin. CSP are effective natural reinforcing agents that can
enhance the physical properties of polymer (Ramdani et al., 2014).
CaCO3 can be gradually dissociated under acidic conditions and provide
Ca2+ for NaAlg to form a crosslinked hydrogel network (Tang et al.,
2020). Therefore, CSP were expected to be regarded as natural active
crosslinking agents to supply Ca2+. Furthermore, the –NH2 group of the
chitin in CSP can be exposed by deacetylation and thereby easily be
protonated to generate –NH+ 3 , which was wished to interact with the
–COO- in NaAlg to form a polyelectrolyte in acidic conditions (Chen
et al., 2017). Consequently, the introduction of deacetylated CSP (dCSP)
in NaAlg can lead to the bi-crosslinking of NaAlg, which help to improve
the performance of NaAlg hydrogel films and ensure effective utilization
of agricultural by-products. However, none of the existing studies have
examined about the effect of CSP or dCSP on the crosslinking of NaAlg.
Considering these aspects, in this study, CSP and dCSP were used as
crosslinking agents, and D-(+)-Gluconic acid δ-lactone (GDL) was added
to release Ca2+. This method represents a novel green technique to
obtain bi-crosslinked NaAlg hydrogel films. The structure, gel proper­
ties, mechanical properties, and thermal stability of mono-crosslinked
and bi-crosslinked NaAlg hydrogel films were characterized. In addi­
tion, the drug loading and release abilities were investigated, in which
diclofenac sodium was selected as a model drug. Furthermore, these
hydrogel films can be served as novel environmentally friendly materials
for biomedical applications.
2. Materials and methods
2.1. Materials
NaAlg (pharmaceutical grade, AR: 98%) and GDL (BR = 99%) were
purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai,
China). Diclofenac sodium and sodium hydroxide were supplied by
Aladdin, Inc. (Shanghai, China). Integrated shells were extracted from
crabs (Portunid from the East China Sea). All the reagents were used as
received without further purification.
2.2. Preparation of NaAlg hydrogel films
2.2.1. Treatment of CSP and dCSP
The treatment procedure of CSP and dCSP were based on our pre­
vious study (Liu et al., 2022). Prior to the chemical treatments, the crab
shells were physically crushed into fine particles by using a superfine
grinder for 10 min (GTM-300, Qingxin Powder Machinery Company,
Yixing, Jiangsu province, China). The resulting particles were treated
with NaOH (5% w/w) and constantly stirred for 5 h at 65 ◦ C to remove
proteins. The pigment constituents were removed using a pure ethanol
solution and stirred at room temperature for 6 h, followed by filtrating
until no obvious filtrate coming out. After that, the precipitate was
washed with deionised water until neutral and dried in a vacuum oven at
60 ◦ C for 12 h to obtain milk-white like CSP.
The preparation of dCSP was conducted by the following procedure.
10 g CSP and 25 mL NaOH (33% w/w) solution were added to a 100 mL
three neck flask equipped with a magnetic stirrer, reflux condenser, and
thermometer, and stirred at 100 ◦ C for 3 h. The mixture was poured into
ice water solution after cooling to room temperature, and washed
several times with deionised water until neutral. Pale yellow dCSP was
obtained. A laser size detector (MICROTRAC, USA) was used to measure
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LWT 171 (2022) 114147
the particle size of the resulting particles, and the average particle size of
CSP and dCSP was 9.71 and 7.45 μm, respectively.
2.2.2. Preparation of hydrogel films
First, 6 g GDL was dissolved in 10 mL distilled water at room tem­
perature to obtain 0.6 g/mL GDL solution for later use. Then, 0.6 g
NaAlg powder was added into 30 mL distilled water at 25 ◦ C and stirred
until completely dissolved to obtain a homogeneous solution containing
2% (w/v) NaAlg. Moreover, CSP and dCSP (0.3 g, each) were mixed with
NaAlg solution individually, followed by the addition of 1% (w/v)
glycerol and was vigorously stirred for 2 h. After that, 0.25 mL, 0.35 mL,
and 0.45 mL (0.5% w/v, 0.7% w/v, and 0.9% w/v GDL to distilled water
in film-forming solution) prepared GDL solution was slowly added into
each aqueous dispersion, respectively. Those CSP and dCSP modified
NaAlg films with the absence of GDL were used as control samples,
namely NaAlg/CSP and NaAlg/dCSP blend films, respectively. Finally,
the entire aqueous dispersion was poured into a Petri dish with 90 mm in
diameter and allowed to evaporate at 25 ◦ C for 72 h after venting
through a sonicator to eliminate air bubbles. All the hydrogel films were
peeled off and placed in an incubator (25 ◦ C and 50% relative humidity)
for 48 h to achieve equilibrium. Those samples were labelled as NaAlg/
CSP/GDL0.5, NaAlg/CSP/GDL0.7, NaAlg/CSP/GDL0.9, NaAlg/dCSP/
GDL0.5, NaAlg/dCSP/GDL0.7, and NaAlg/dCSP/GDL0.9, respectively.
2.3. Characterization
2.3.1. Particle size determination
The particle size distributions of the CSP and dCSP were measured by
a laser particle analyzer (MICROTRAC, USA) basing on wet method from
Konert with some modification at room temperature (Konert & Van­
denberghe, 1997).
2.3.2. FTIR analysis
The structure of CSP, dCSP, NaAlg, and the prepared hydrogel films
was analyzed through FTIR spectroscopy using a Thermo Fisher FTIR
spectrometer according to the method by Liu et al. (Liu et al., 2022). The
spectra were recorded at a resolution of 4 cm− 1 over a 400− 4000 cm− 1
wavenumber range with 32 scans.
2.3.3. X-ray diffraction analysis
The X-ray diffraction (XRD) analysis of the CSP, dCSP, and prepared
films was performed using an X’Pert Pro diffractometer (PA Nalytical
B⋅V., The Netherlands) basing on the report by Wang et al. (Wang et al.,
2014). A diffraction range of 5◦ –90◦ (2θ) was selected. Further more, the
CaCO3 content was investigated using XRD basing on the intensity for
the diffraction peak at 29.5o (2θ) according to the literature reported by
Kim & Kang, in which the CaCO3 content in the films without the
addition for GDL was used as control assays (Kim & Kang, 2008).
2.3.4. Surface hydrophilicity analysis
The water contact angles (WCA) of the blend and hydrogel films
were recorded at 25 ◦ C by using a contact angle analyzer (Model DMs-
401, Kyowa Interface Science Co. Ltd., Tokyo) in the terms of the
study by Jia et al. (Jia et al., 2022). A MilliQ water drop with a volume of
approximately 3 μL was added to the surface of the measurements, and
the equilibration time was 3 s.
2.3.5. Scanning electron microscopy
The morphologies characterization of those two types of freeze-dried
hydrogel films after hydration was performed using a scanning electron
microscopy (SEM) (Hitachi, Japan) according to the research reported
by Ramdani et al. at an accelerating voltage of 10 kV (Ramdani et al.,
2014). Before the evaluation, all the samples were thinly coated with
gold. The surface views of the hydrogel films (freeze dried) were taken at
magnification of 100 × .
H. Wang et al.
2.3.6. Gel strength
The gel strength (N) was analyzed using a probe (P/0.5) attached to a
TA. XT Plus texture analyzer (Stable Micro Systems, London, UK). All the
dried hydrogel films were allowed to fully swell in a phosphate buffer
solution (PBS, 0.05 mol/L, pH 7.4) for 24 h at 37 ◦ C before the test and
later compressed using the probe of the texture analyzer at a rate of 1
mm/s. The compression was terminated when the gels ruptured.
2.3.7. Swelling property
The swelling ratio of the dried sample films was determined by
weighing the films before and after immersion in a beaker with 30 mL
Total amount of drug − remaining drug in the solution
Drug loading efficiency (%) =
Total amount of drug
PBS (0.05 mol/L, pH 7.4) basing on the method by Tanuma et al.
(Tanuma et al., 2010). All of the prepared films (10 mm × 20 mm) were
immersed in PBS and allowed to swell at 37 ◦ C for 24 h. Subsequently,
the samples were extracted from the PBS, and the dried films were
weighed. The swelling ratio of the hydrogel films was determined as
Ms − Mi
Swelling ratio(%) = × 100 (1)
Mi
where Mi is the initial dry mass of a hydrogel film, and Ms is the mass of
a fully swollen wet film.
2.3.8. Gel fraction
The gel content of the films was determined using a gravimetric
method (Niu et al., 2019). All the films (10 mm × 20 mm) were
immersed in a beaker with 30 mL PBS (0.05 mol/L, pH 7.4) for 24 h at
37 ◦ C. Subsequently, the samples were extracted from the PBS and dried
at 60 ◦ C until the weight of the films became a constant value. The gel
fraction of the samples was calculated as
Wf
Gel fraction(%) = ×% (2)
Wi
where Wi is the initial mass of the film, and Wf is the final mass of the
dried hydrogel films after immersion.
2.3.9. Mechanical properties
All the films were cut to a rectangular shape (length and width of 50
mm and 10 mm, respectively) to calculate the elastic modulus (EM),
tensile strength (TS), and elongation at break (EB) from the stress–strain
curves obtained using a TA. XT Plus C texture analyzer (Stable Micro­
systems, UK) according to the method by Sun et al. (Sun et al., 2020).
The test speed and initial distance were 1 mm/s and 35 mm,
respectively.
2.3.10. Thermal analysis
The thermal behaviors were analyzed using a differential scanning
calorimeter DSC (TA instruments, USA), in which the melting temper­
ature (Tm) of the films was obtained (Liu et al., 2022). The films (5 mg)
were placed in an aluminium pan and heated from 20 to 170 ◦ C at a rate
of 10 ◦ C min− 1 in a nitrogen atmosphere. The thermal stability were
recorded using a thermogravimetric analyzer (TGA) (NETZSCH, Ger­
many) at a constant heating rate (10 ◦ C/min) from 30 to 600 ◦ C in the
nitrogen atmosphere.
2.3.11. Diclofenac sodium loading and release
The diclofenac sodium loading and release abilities of the hydrogel
films were investigated according to the reported literature (Zactiti &
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LWT 171 (2022) 114147
Kieckbusch, 2009; Afkhami et al., 2016). The drug loading capacity can
describe the drug absorption and the drug storage ability of hydrogel
films in PBS. NaAlg/CSP/GDL and NaAlg/dCSP/GDL hydrogel films
with the same size (10 mm × 20 mm) were placed in a beaker with 15
mL PBS and 7.5 mg diclofenac sodium at 37 ◦ C for 24 h. After the
samples were removed, the concentration of diclofenac sodium in the
solution was analyzed at 307 nm by using an ultraviolet–visible
(UV–Vis) spectrophotometer. The amount of remaining drug in the so­
lution was calculated according to the predetermined calibration curve.
The drug loading efficiency of the hydrogel films was calculated using
the following equation:
× 100 (3)
The drug-loaded hydrogel films were immersed in 15 mL PBS at
37 ◦ C for 5 days. Samples (3 mL) were collected at different time in­
tervals and replaced with an equal amount of fresh PBS (0.05 mol/L, pH
7.4). The fresh PBS solution was used as blank assay. The characteristic
peak at 307 nm was considered to analyze the drug release. The drug
release of the hydrogel films was determined using the following
equation:
amount of released drug
Drug release (%) = × 100 (4)
amount of loaded drug
2.3.12. Cell viability analysis
The cytotoxicity of different hydrogel films was determined by a Cell
Counting Kit-8 (CCK-8) assay according to the method by Mndlovu et al.
(Mndlovu et al., 2019). All the sampels were sterilized by UV irradiation
for 1 h, then soaked in PBS (7.4) at 37 ◦ C for 24 h obtain extract solution.
Fibroblast cells (HF90) (Shanghai Institute of Biochemistry, China) were
seeded in 96-well plate contain Dulbecco’s modified Eagle’s medium
(DMEM), which supplemented with fetal bovine serum and
penicillin-streptomycin. Then the fibroblast cells were incubated in a 5%
CO2 incubator for 24 h. Different filtered solution was added to the
96-well plate and incubated for another 24 h. After that, 200 μL CCK-8
solution was added to each well for 4 h. The optical density (OD) was
measured at 450 nm. The fresh PBS solution and pure diclofenac sodium
solution was used as control and treatment group, respectively. The cell
viability analysis of the hydrogel films was determined using the
following equation:
As − Ab
Cell viability (%) = × 100 (5)
Ac − Ab
where Ab and Ac were the absorbance of black and control, and As is the
absorbance of samples. Further more, a Laser Scanning Confocal Mi­
croscope Model (SP2, Leica Co. Ltd., Wetzlar) was used to obtain the
microscopic morphology of cells.
2.4. Statistical analysis
The quantitative data were represented as mean value ± SD. Three
parallel experiments were conducted for each sample at least. Statistical
analyses were performed using the Statistix software package. More­
over, the significance level was analyzed using the Tukey test. Origin
and SigmaPlot were used to plot the results.
H. Wang et al.
Fig. 1. FTIR spectra of CSP (A) and dCSP (B) modified NaAlg hydrogel films; X-ray diffraction patterns of CSP (C) and dCSP (D) modified NaAlg hydrogel films.
3. Results and discussion
3.1. FTIR analysis
Results of the FTIR analysis of the NaAlg, GDL, and crosslinked
NaAlg hydrogel films are presented in Fig. 1. As shown in this figure, the
hydrogen bonding for the chitin polysaccharides in the CSP was
observed at 3698 cm− 1, However, a new peak was observed at 3642
cm− 1, which was attributed to the hydrogen bonding between the –NH2
and CO2−3 in the dCSP. Those results illustrated the successful prepara­
tion of CSP and dCSP (Liu et al., 2022; Ramdani et al., 2014). More over,
the characteristic peaks of NaAlg occurred at 3262 cm− 1, 2929 cm− 1,
1599 cm− 1, and 1406 cm− 1, corresponding to the O–H stretching vi­
brations, C–H banding, and asymmetric and symmetric stretching vi­
brations of –COO− , respectively (Chen et al., 2021). For
NaAlg/CSP/GDL and NaAlg/dCSP/GDL hydrogel films, the peaks cor­
responding to the O–H stretching vibration at 3262 cm− 1 shifted to a
lower wavenumber region and the C–H banding at 2929 cm− 1 shifted to
higher wavenumber with the increase in the GDL content. The peaks of
–COO− asymmetric stretching vibrations for NaAlg/CSP/GDL and
NaAlg/dCSP/GDL hydrogel films shifted from 1599 cm− 1–1594
cm− 1and 1595 cm− 1, respectively. Those changes indicated the chang­
ing of intermolecular and intramolecular hydrogen bonds. The absorp­
tion peak of GDL located at 1727 cm− 1 disappeared. In addition, the
peaks at 1085 cm− 1 and 1023 cm− 1, corresponding to C–O–C stretching,
shifted to a higher wavenumber region for those two kinds of crosslinked
hydrogel films. This phenomenon could be attributed to the successful
release of Ca2+ by GDL and realization of ionic crosslinking with NaAlg
in the composite hydrogel films (Sun et al., 2020). It is worthy to note
that the peaks corresponding to –COO− symmetric stretching vibrations
shifted to higher wavenumbers (from 1406 cm− 1–1409 cm− 1) for
NaAlg/dCSP/GDL hydrogel films. This phenomenon indicated the
occurrence of physical crosslinking and strong polyelectrolytes
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LWT 171 (2022) 114147
interactions between –COO− and –NH+ 3 in the prepared
NaAlg/dCSP/GDL hydrogel films (Farno et al., 2021; Tang et al., 2020).
The FTIR analysis results demonstrated that CSP and dCSP crosslinked
NaAlg hydrogel films were successfully prepared.
3.2. XRD analysis
XRD patterns of the prepared NaAlg crosslinked hydrogel films are
presented in Fig. 1 (C) and (D). A broad peak was observed at 2θ = 20.6◦
for NaAlg, which was the characteristic peak. The diffraction peak of
CSP/dCSP, corresponding to CaCO3, was located at 2θ = 29.5◦ (Shah
et al., 2018). Additionally, a typical peak of chitosan was noted at 2θ =
20.5◦ for dCSP, which demonstrated the occurrence of the deacetylation
reaction (Smitha et al., 2005). The intensity of CaCO3 decreased but did
not disappear with the increasing content of GDL in the NaAlg/CSP/GDL
and NaAlg/dCSP/GDL hydrogel films. This result indicated that CaCO3
in the composite hydrogel films occurred in two forms (ionic and min­
eral state). However, the characteristic peak of chitosan became smooth
after crosslinking. This phenomenon illustrated that polyelectrolyte
crosslinking did not drastically alter the chemical structure of chitosan
but changed the crystal structures (Mndlovu et al., 2019).
3.3. SEM observation
The morphology of freeze-dried hydrogel films after hydration are
shown in Fig. 2. The neat NaAlg film and non-crosslinked NaAlg/CSP
and NaAlg/dCSP films gradually dissolved in the hydration process for
the reason that the calcium with mineral state could not form salt bridge
without the addition of GDL. It is difficult to form crosslinking network
structure and could not conduct the hydration procedure. Thus, the
microstructure could not be evaluated through SEM (Sun et al., 2020).
As shown in this figure and Table 1, the porosity of the prepared
hydrogel films and the CaCO3 content declined as GDL content
H. Wang et al. LWT 171 (2022) 114147

Fig. 2. Surface morphology of samples subjected to freeze-drying cycling after hydration: (a) NaAlg/CSP/GDL0.5; (b) NaAlg/CSP/GDL0.7; (c) NaAlg/CSP/GDL0.9;
(d) NaAlg/dCSP/GDL0.5; (e) NaAlg/dCSP/GDL0.7; (f) NaAlg/dCSP/GDL0.9.

Table 1
The initial moisture content, CaCO3 content, WCA value, and gel properties different samples.
Sample Initial moisture content (%) CaCO3 content (%) WCA value (O) Gel strength (N) Swelling ratio (%) Gel fraction (%)

NaAlg 27.3 ± 1.6a – 43.85 ± 1.62g – – –

NaAlg/CSP 13.4 ± 0.8cd 14.84a 55.64 ± 1.32f – – –
NaAlg/dCSP 12.6 ± 1.3cd 14.95a 57.62 ± 1.03f – – –
NaAlg/CSP/GDL0.5 18.6 ± 2.7b 14.60 ± 0.33a 65.04 ± 1.08e 10.83 ± 2.83a 5140.70 ± 333.81a 47.14 ± 1.76a
NaAlg/CSP/GDL0.7 15.7 ± 0.8bcd 13.12 ± 0.29b 73.32 ± 1.25c 19.78 ± 2.20a 4413.60 ± 185.18b 52.90 ± 1.96b
NaAlg/CSP/GDL0.9 11.8 ± 0.9d 12.81 ± 0.15c 79.28 ± 1.05c 26.24 ± 3.72b 3037.90 ± 220.32c 54.89 ± 2.30b
NaAlg/dCSP/GDL0.5 15.4 ± 1.8bcd 13.49 ± 0.22b 68.94 ± 0.66d 12.49 ± 1.83a 3509.30 ± 298.48a 53.20 ± 4.69a
NaAlg/dCSP/GDL0.7 16.2 ± 1.5bc 11.67 ± 0.18d 78.97 ± 1.61b 22.74 ± 1.16b 3316.11 ± 575.29ab 53.80 ± 0.53a
NaAlg/dCSP/GDL0.9 13.3 ± 0.9cd 10.57 ± 0.11e 85.21 ± 1.20a 28.80 ± 2.80c 2533.00 ± 104.04b 55.78 ± 0.88a

The mean value ± SD in the same columns denoted with different letters are significantly different (P < 0.05); ’-’: can not be measured.

increasing. This might be attributed to the fact that the increased Ca2+
content in the systerm rised the crosslinking point, which improved the
compactness and the restricted the swelling as the GDL content
increasing (Geng et al., 2021).
3.4. Surface hydrophobicity
The WCA of the crosslinked NaAlg hydrogel films is presented in
Fig. 3 and Table 1. The WCA value of the neat NaAlg film was 43.85◦ ,
which confirmed its hydrophilic property. The WCA value for NaAlg/
CSP and NaAlg/dCSP blend composites was slightly higher than that of
neat NaAlg film, which was owing to the fill of self-hydrophobic CSP or
dCSP decreasing the free volume of the polymer network (Zhang et al.,
2022). As also can be seen in this figure, the WCA value increased with
the increasing of GDL content, which is owing to the higher crosslinking
sites from Ca2+ increasing the compactness of the hydrogel film. In
addition, the WCA value of bi-crosslinked NaAlg/dCSP/GDL was higher
than that of mono-crosslinked NaAlg/CSP/GDL, which illustrated the
higher crosslinking density the higher hydrophobicity further. The WCA
value for NaAlg/dCSP/GDL0.9 hydrogel film had a maximum value
(85.21◦ ) among those prepared films, which was 41.36◦ higher than that
of neat NaAlg film. It can be concluded that the surface hydrophobicity
of NaAlg based hydrogel film can be enhanced significantly by the
crosslinking from CSP and dCSP. Thus, the crosslinking enhanced the
hydrophobicity of NaAlg hydrogel film to some content, which is suit­
able to absorb solutions without causing dissolution.
3.5. Gel properties
The neat NaAlg film and non-crosslinked NaAlg/CSP and NaAlg/
dCSP films gradually dissolved in PBS and could not to be tested. Thus,
the gel properties of those samples formed without GDL could not be
obtained. Table 1 represents the gel strength, swelling ratio, and gel
fraction of these crosslinked hydrogel film. However, the crosslinked
films could maintain the integrity of the gel structure even after swelling
for 24 h. With the addition of GDL, the gel strength and gel fraction of
those two types crosslinked films were enhanced, whereas the swelling
degree decreased, related to the increased crosslinking content. All
crosslinked hydrogel films exhibited excellent swelling ratio between
2533% and 5140%, corresponding to high fluid absorbing capacity,
which was particularly useful for wood dressing material in medical
domain. Notably, this value was considerably larger than that of com­
mercial NaAlg hydrogel films (50%–1800%) (Geng et al., 2021; Giz
et al., 2020; Martínez-Gómez et al., 2017). The NaAlg/dCSP/GDL
hydrogel films exhibited a higher gel strength and gel fraction and
inferior swelling behaviour than those of NaAlg/CSP/GDL hydrogel

5
H. Wang et al.
Fig. 3. Water contact angles of the neat CSP and dCSP modified NaAlg hydrogel films.
films at the same GDL content. This result was attributed to the addi­
tional polyelectrolyte interaction in NaAlg/dCSP/GDL hydrogel films
leading to a higher crosslinking density and more compact hydrogel
network structure compared with NaAlg/CSP/GDL (Chen et al., 2021).
These changes strengthened the polymer structure, limited the chain
expansion, and decreased the free volume of the polymer. Therefore, the
quantity of hydrophilic groups dwindled, and the fluid uptake was
reduced (Sun et al., 2020; Tang et al., 2020). This result was consistent
with the FTIR and SEM analyses above.
3.6. Mechanical properties
Table 2 shows the EM, TS, and EB of the prepared films. The neat
NaAlg film exhibited the lowest EM (14.44 MPa) and TS (11.33 MPa) but
the highest EB (59.12%) values, corresponding to a weak deformation
property and excellent flexibility. Compared with those of the neat
NaAlg film, the EM value of the blend films were higher, however, the
EB and TS values were lower. In particular, the introduction of CSP or
dCSP enhanced the strength and deteriorated the ductility of NaAlg due
to the rigid nature of CSP or dCSP and strong hydrogen bonding in­
teractions between NaAlg and the particles (Ramdani et al., 2014).
Among the crosslinked hydrogel films, the EB value of the NaAlg/
CSP/GDL hydrogel films gradually decreased from 36.22% to 28.11%
with the increasing release of Ca2+. However, the EM and TS values
increased to 42.45 MPa and 12.37 MPa, respectively. This result indi­
cated that crosslinking treatment intensified the strength and stiffness of
the material. The same trend was observed for the NaAlg/dCSP/GDL bi-
6
LWT 171 (2022) 114147
crosslinking hydrogel films. The mechanical properties of the NaAlg/
dCSP/GDL films were superior to those of the NaAlg/CSP/GDL films at
the same particle and GDL loadings. In addition, NaAlg/dCSP/GDL0.9
exhibited the highest TS value (16.4 MPa) owing to the highest incre­
ment in the crosslinking sites, which is approximately 2.25 times than
that of silver-loaded hydroxyethylacryl chitosan/NaAlg hydrogel films
(7.3 MPa) (Chalitangkoon et al., 2020). Notably, artificial skin usually
exhibits TS value in the range of 2.5–16 MPa and EB of approximately
70% in the most flexible zones (Hansen & Jemec, 2002).
3.7. Thermal analysis
The thermal properties of the prepared films were examined using
DSC and TGA, and the results are shown in Fig. 4. As shown in Fig. 4 (A)
and 4 (B), all the samples exhibited only one endothermic behaviour,
which was in terms of the melting temperature (Tm). The Tm value of
NaAlg, NaAlg/CSP, and NaAlg/dCSP was 113.55 ◦ C, 114.26 ◦ C, and
119.59 ◦ C, respectively. This increment in Tm value could be attributed
to a stronger hydrogen bonding interactions between NaAlg and CSP or
dCSP and higher meltbility of the particle itself. The Tm values of the
NaAlg/CSP/GDL and NaAlg/dCSP/GDL crosslinked hydrogel films
increased gradually with the increasing GDL content, and the bi-
crosslinked NaAlg/dCSP/GDL films exhibited a higher melting point
than that of the NaAlg/CSP/GDL films at the same GDL level. This result
implied that the formation of polyelectrolytes between components
enhanced the intermolecular interactions of the polymers, which hin­
dered the chain mobility of NaAlg, resulting in a higher melting point
H. Wang et al. LWT 171 (2022) 114147

Table 2 compared with neat NaAlg film. Further more, the NaAlg/dCSP/GDL
The mechanical and thermal properties of samples. films exhibited a higher Td than that of the NaAlg/CSP/GDL films. This
Sample Elastic Elongation at Tensile Td (◦ C) Yc (%) phenomenon occurred might because the bi-crosslinking interaction of
modulus break (%) strength the network structure as well as higher compactness enhanced by mo­
(MPa) (MPa) lecular hydrogen bond in the films limited the decomposition of poly­
NaAlg 14.44 ± 59.12 ± 11.33 ± 212.34 18.33 saccharide segment (Liu et al., 2016). The char yield (Yc, 600 ◦ C) was the
1.41f 2.39a 1.92b ± 0.56c ± 1.12c remaining weight at 600 ◦ C for those prepared films, which could be
NaAlg/ 19.71 ± 39.49 ± 9.99 ± 216.14 48.28 observed in the TGA curves and exhibited a similar tendency as the Td
CSP 2.37ef 0.98bc 0.15b ± 1.28a ± 3.32a
NaAlg/ 25.96 ± 45.89 ± 10.04 ± 216.31 43.98
values. The Yc value of composite films was higher than that of the neat
dCSP 4.91de 2.39b 0.24b ± 1.00a ± 1.98a NaAlg film. However, the Yc of hydrogel films did not exhibit a clear
NaAlg/ 20.25 ± 36.22 ± 10.26 ± 212.65 31.07 variation trend due to the enhancement of the crosslinking degree and
CSP/ 1.27ef 1.90cde 0.87b ± 0.23c ± 1.46c reduction in the amount of calcium in the mineral form with the
GDL0.5
increasing content of GDL. Even so, the thermal stability and flame
NaAlg/ 39.78 ± 29.08 ± 11.40 ± 212.90 34.02
CSP/ 0.77c 3.99ef 0.42b ± 0.49bc ± retardancy of modified NaAlg hydrogel films were higher than those of
GDL0.7 0.95bc neat NaAlg films.
NaAlg/ 42.45 ± 28.11 ± 2.10f 12.37 ± 212.93 29.00
CSP/ 5.45bc 1.87ab ± 0.25bc ±
GDL0.9 1.27bc 3.8. Drug loading and release
NaAlg/ 33.77 ± 38.62 ± 12.74 ± 213.11 28.05
dCSP/ 2.37cd 3.40bcd 0.85ab ± 1.22bc ±
GDL0.5 0.73bc To evaluate the potential application of the NaAlg/CSP/GDL and
NaAlg/ 52.27 ± 33.06 ± 13.87 ± 215.10 26.54 NaAlg/dCSP/GDL hydrogel films in active compound delivery, the
dCSP/ 7.62ab 0.96cdef 2.00ab ± 0.47ab ± diclofenac sodium loading efficiency (%) and cumulative release
GDL0.7 0.66ab
amount were tested and the relevant data are presented in Fig. 5 (A). The
NaAlg/ 58.85 ± 31.22 ± 16.40 ± 215.51 27.81
dCSP/ 2.70a 2.43def 2.25a ± 1.15a ± 2.15a diclofenac sodium loading efficiency of the NaAlg/CSP/GDL and NaAlg/
GDL0.9 dCSP/GDL hydrogel films were enhanced from 25.33% to 38.03% and
from 28.67% to 43.60%, respectively. However, the NaAlg/dCSP/GDL
The mean value ± SD in the same columns denoted with different letters are
significantly different (P < 0.05).
bi-crosslinked films exhibited a considerably higher loading efficiency
compared to that of NaAlg/CSP/GDLD: films, which was attributed that
the additional polyelectrolyte interactions enhanced the diclofenac so­
(Souza et al., 2020).
dium adsorption ability of NaAlg/dCSP/GDL hydrogel films. Similar
The TGA results of the samples are shown in Fig. 4 (C). The
phenomena were observed for citric acid crosslinked NaCMC-HPMC
decomposing stage of all composite films was delayed compared with
hydrogel films, for which the drug loading efficiency increased from
that of neat NaAlg film. All samples presented two stages in weight loss
approximately 35%–55% (Dharmalingam & Anandalakshmi, 2019).
curves. The first stage occurred at 30–200 ◦ C (about 15% of the weight
The diclofenac sodium release profile of the hydrogel films is illus­
loss) related to the loss of water in films, and the initial moisture of those
trated in Fig. 5 (B). The releasing time for the prepared hydrogel films
films was calculated basing on the weight loss rate during this stage. The
was higher than 36 h, which illustrated all of the hydrogel samples
major weight loss occurred in the second stage at 200–300 ◦ C, owing to
exhibited typical slow-release characterization. However, the release
the decomposition of main chains for NaAlg and the polysaccharide in
patterns of diclofenac sodium were significantly influenced by the GDL
CSP and dCSP (Kaya et al., 2016). The temperature corresponding to the
content. Initially, a large amount of diclofenac sodium was observed in
maximum decomposition rate that is defined as decompositon tem­
PBS solution due to the diffusion of diclofenac sodium from the hydrogel
peratre (Td) of the films in the second stage is summarized in Table 2. In
films surface to the PBS. The cumulative release amount of those pre­
case of NaAlg/CSP and NaAlg/dCSP ordinary blend films, the intro­
pared hydrogel films decreased with the increasing GDL content were
duction of CSP or dCSP caused a slight increasing for the Td value of
higher than those of NaAlg/dCSP/GDL hydrogel films. This result could
NaAlg film. This might be owing to the thermostable CaCO3 and the
be explained by the high crosslinking density and hydrophobic property,
decomposition of polysaccharide in CSP and dCSP (the CaCO3 could
owing to which, the solvent could not easily penetrate the films (Niu
remain stable until 600 ◦ C) (Giz et al., 2020; Ramdani et al., 2014).
et al., 2019). Compared with NaAlg/dCSP/GDL hydrogel films,
Furthermore, the crosslinked hydrogel films exhibited lower degrada­
NaAlg/CSP/GDL hydrogel films exhibited higher drug release amount at
tion temperatures than those of ordinary blend films, owing to the
the same GDL content. This phenomenon could be attributed to the drug
preferential decomposition of GDL and the content of CaCO3 decreasing.
adsorption capacity associated with polyelectrolyte interactions in
For this reason, the Td of crosslinked hydrogel films increased slightly
bi-crosslinking hydrogel films. In summary, NaAlg/CSP/GDL0.5

Fig. 4. DSC behaviour for CSP (A) and dCSP (B) modified hydrogel film, TGA (C) curves of the all composite films.

7
H. Wang et al.
Fig. 5. DS loading (A) and release abilities (B) of the NaAlg/CSP/GDL and NaAlg/dCSP/GDL hydrogel films in PBS at 37 ◦ C (P < 0.05); Cell viability analysis (C) and
microscopic morphology (D) of cells after incubated at 37 ◦ C for 24 h.
possessed the highest releasing rate among those other hydrogel films.
Thus, NaAlg/CSP/GDL0.5 hydrogel film is promising candidates for use
in biomedical applications in terms of costs and applications.
3.9. Cell viability analyze
The cell viability analysis and the microscopic morphology of cells
after incubated at 37 ◦ C for 24 h were presented in Fig. 5 (C) and 5 (D).
As shown in Fig. 5 (C), the average percentage of cell viability of NaAlg/
dCSP/GDL hydrogel films were higher than that of NaAlg/CSP/GDL
hydrogel films with crosslinking density increased. This phenomenon
can be explained by the slow drug release ability of bi-crosslinking
hydrogel films. However, the average percentage of cell viability of all
the films was over 90, indicating nontoxic to cell. The cell numbers and
morphology can be observed from Fig. 5 (D). There was no significant
difference of cell numbers and morphology between control and
different samples. The results suggested that the prepared hydrogel films
slightly affected to cell viability and could be a potential NaAlg based
hydrogel films with sustained drug release in biomedical applications.
4. Conclusion
A series of CSP and dCSP crosslinked NaAlg hydrogel films with high
hydrogel strength, excellent swell ratio, and well slow-release ability
were successfully prepared, in which the dCSP and CSP were acted as
crosslinking initiators for NaAlg. The FTIR and XRD results suggested
the occurrence of Ca2+ crosslinking, polyelectrolytic interaction, and
hydrogel bonding interaction in NaAlg/dCSP/GDL hydrogel films. The
remaining unreacted CaCO3 could enhance the thermal stability of the
films. Consequently, gel strength, gel fraction, mechanical properties
and thermal stabilities of NaAlg/dCSP/GDL bi-crosslinking hydrogel
8
LWT 171 (2022) 114147
films were much better than those of NaAlg/CSP/GDL mono-
crosslinking hydrogel films. Furthermore, all of the crosslinked hydro­
gel films exhibited a sustained drug release ability. It is worthy to note
that NaAlg/CSP/GDL0.5 possessed the highest releasing rate among
those other hydrogel films due to the lower crosslinking site making it
easy for the drug to release. This study provides an alternative green
internal crosslinking method that can promote the development of
modern NaAlg based hydrogel films with sustained drug release.
CRediT authorship contribution statement
Hui Wang: Data curation, Writing – original draft, Visualization.
Jinming Liu: Investigation, Formal analysis. Xu Fan: Software, Re­
sources. Jing Ren: Conceptualization, Methodology, Supervision. Qian
Liu: Writing – review & editing. Baohua Kong: Conceptualization,
Writing – review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This study was supported by the Natural Science Foundation of
Heilongjiang Province (LH 2021C048), the Major Science and
H. Wang et al.
Technology Project of Heilongjiang Province (2021ZX12B05), the
financial support from National Natural Science Foundation of China
(31901660), and the Heilongjiang Postdoctoral Financial Assistance
(LBH-Z17214).
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