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International Journal of Biological Macromolecules 185 (2021) 592–603

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules


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

Multifunctional injectable pluronic-cystamine-alginate-based hydrogel as a


novel cellular delivery system towards tissue regeneration
Le Hang Dang a, b, **, Phuong Doan b, Tran Thi Yen Nhi a, c, Dinh Trung Nguyen a, b,
Bich Tram Nguyen d, Thi Phuong Nguyen e, Ngoc Quyen Tran a, b, *
a
Graduate University of Science and Technology, Vietnam Academy of Science and Technology, HCMC, Viet Nam
b
Institute of Applied Materials Science, Vietnam Academy of Science and Technology, HCMC, Viet Nam
c
NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City, Viet Nam
d
Department of Natural Science, Thu Dau Mot University, Thu Dau Mot City, Viet Nam
e
Faculty of Chemical Technology, HCMC University of Food Industry, Ho Chi Minh City, Viet Nam

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

Keywords: This paper presents a new thermal sensitive hydrogel system based on cystamine-functionalised sodium alginate-
Sodium alginate g-pluronic F127 (ACP). The introduction of cystamine to the alginate backbone not only creates a covalent bond
Thermal sensitive hydrogel with pluronic F127 but also provides intrinsic anti-bacterial activity for the resultant hydrogel. The amount of
Cell delivery
water uptake inside the hydrogel remained ~200% for 6 days and the degradation was completed in 12 days in
physiological media. The ACP copolymer solution could form a hydrogel at body temperature (~37 ◦ C) and
could return to the solution phase if the temperature decreased below 25o ◦ C. Fibroblast encapsulated in situ in
the ACP hydrogel maintained their viability (≥90% based on the live/dead assay) for 7 days, demonstrating the
good biocompatibility of the ACP hydrogel for long-term cell cultivation. In addition, three-dimensional (3D)
culture showed that fibroblast attached to the hydrogels and successfully mimicked the porous structure of the
ACP hydrogel after 5 days of culture. Fibroblast cells could migrate from the cell-ACP clusters and form a
confluent cell layer on the surface of the culture dish. Altogether, the obtained results indicate that the thermal-
responsive ACP hydrogel synthesised in this study may serve as a cellular delivery platform for diverse tissue
engineering applications.

1. Introduction intermolecular forces including hydrophobic-hydrophobic interactions


and hydrogen bonds in response to the body temperature, a factor that is
Thermal-responsive hydrogels currently offer an ideal material for very beneficial for medical applications [1–3].
biomedical applications, especially drug delivery systems or cell de­ Thermal-responsive materials have been designed and have great
livery systems for tissue regeneration and mechano-therapeutic wound potential application in tissue engineering, these materials, include poly
dressings [1–4]. Typically, these materials undergo a physical sol-to-gel (N-isopropyl acrylamide) [11], poly(2-oxazoline)s [12], poly(N,N-
transition phase to adapt to the change in temperature [3,4]. In tissue diethyl acrylamide) [13], poly(N-vinyl caprolactam) [14], (poly­
regeneration, in situ gelling systems, which are applied as aqueous so­ ethylene glycol)-based (co)polymers [15], and poly(ethylene oxide)-
lutions and then changed to gel forms after entering the body, have poly(propylene oxide)-poly(ethylene oxide) [16]. In particular, plur­
attracted tremendous interest [2,3,5]. Given their unique properties, onic, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)
several kinds of therapeutic agents [3,4], drugs [6], or cells [7–10] could (PEO-PPO-PEO) [16], has been investigated extensively due to their
be easily mixed with a polymer solution and subsequently encapsulated well-tolerated and low toxic properties. Among them, pluronic F127
into the hydrogel matrix following the increase of temperature at the (Poloxamer 407) has been approved by Food and Drug Administration
local microenvironment upon injection into the body [2,5,7–9]. In (FDA) as an excipient and its solution can switch to the solid phase when
addition, the formation of these hydrogels is based on the expression of the concentration is up to 18% (wt/wt) at the body temperature [2].

* Corresponding author at: Institute of Applied Materials Science, Vietnam Academy of Science and Technology, HCMC, Viet Nam
** Corresponding author at: Graduate University of Science and Technology, Vietnam Academy of Science and Technology, HCMC, Viet Nam.
E-mail addresses: [email protected] (L.H. Dang), [email protected] (N.Q. Tran).

https://doi.org/10.1016/j.ijbiomac.2021.06.183
Received 18 February 2021; Received in revised form 18 June 2021; Accepted 26 June 2021
Available online 30 June 2021
0141-8130/© 2021 Published by Elsevier B.V.
L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

Nevertheless, this hydrogel does not allow well-controlled drug release added into reaction following the addition of NHS (0.00046 mol, 1
because of its low mechanical strength and rapid degradation [2,4]. equiv.) and stirred for 30 min. Cys (0.00046 mol, 1 equiv.) was added
Furthermore, the bare pluronic F127 hydrogel is known as anti-adhesive and stirring was continued for 5 h at RT. The reaction solution was
material, a factor that limits its potential application in tissue engi­ added drop-wise into absolute alcohol. The precipitation was re-
neering [2,17,18]. Thereby, various pluronic modification methods dissolved in water, and then dialysed against in morpholinoethane­
have been reported to improve its applications [17,18]. An interest in sulfonic acid sodium salt (MES) buffer. The resulting solution was
covalent binding with natural polymers such as chitosan [19] or gelatine lyophilized yielding Na-alg-cys.
[20] has exhibited the merits of programmable mechanic properties of
the pluronic hydrogel. 2.3. Synthesis pathway of Na-alg-cys-F127 (ACP)
The aim of this study was to fabricate a novel thermal-responsive and
multifunctional hydrogel that included the grafted materials (pluronic- The hydroxyl groups of pluronic F127 were activated with p-NPC
cystamine-alginate)-termed ACP to extend the applicability of the con­ (NPC-F127-OH) as previously reported [2,4,20]. Na-alg-cys and NPC-
ventional alginate-based hydrogels. Alginate, a well-known natural F127-OH were dissolved separately in water. NPC-F127-OH was then
polysaccharide with a negative charge, is composed of 1,4-linked-D- added drop-wised into Na-alg-cys. The reaction took place at 4 ◦ C in 5 h.
mannuronic acid (MM-blocks) and 1,4-linked-l-guluronic acid (GG- The obtained ACP was dialysed 2 h in absolute alcohol. Subsequently,
blocks) residues in variable proportions [17,21,22]. Alginate hydrogels the solution was dialysed against deionised water (DI) water (3 days),
have a broad-range of application in tissue engineering, due to their and freeze-dried to obtain the ACP copolymer as a white solid.
excellent biocompatibility [17]. Nevertheless, their low degradation
rate and poor controllability are the primary obstacles in the develop­ 2.4. Characterization technique
ment of alginate hydrogels [23]. In practice, alginate is an anti-adhesive
materials [17,18,20]; thus, the alginate chains should be chemically The chemical structure of the products was verified using Fourier
modified with cell adhesion ligands or with the functional groups to transform infrared spectroscopy (FT-IR, Horiba, Japan) and proton nu­
increase cell permeability [24]. The disulfide bond on cystamine is clear resonance (1H NMR, Bruker NMR JCAMP-5.01). The hydrogel
found in cell-surface proteins, and thus this molecule can easily interact morphology was examined via scanning electron microscopy analysis
with cells [25]. Cystamine is the oxidised form of cysteamine. Cystamine (SEM-JEOL JSM-7401 F) and confocal microscope (Andor). The degree
exerts antimicrobial activity by generating reactive oxygen species of grafting was determined based on the concentration of amino groups
(ROS) [26]. An adhesive hydrogel with intrinsic antibacterial activity using l-alanine as an internal standard. 2,4,6-trinitrobenzene sulfonic
and good biocompatibility could find applications as an injectable tissue acid (TNBS, Thermo Fisher Scientific) was used to determination of free
engineering scaffold. Therefore, in this study, cystamine-functionalised amino groups on the obtained products following the instruction of the
alginate was first fabricated to promote interaction with cells, and manufacture.
then conjugated with pluronic F127 to reduce the degradation rate of
the original alginate hydrogel [27,28]. The designed ACP hydrogel was 2.5. Rheological analysis
examined in terms of chemical composition, thermal responsive
behaviour, and swelling property in physiological buffer. Additionally, The ACP copolymer was dissolved in DI water with mild stirring at
the cytocompatibility of the ACP hydrogel was studied with fibroblast 10–15 ◦ C. The obtained solution was then stabilised at 4 ◦ C for 24 h. The
cells. Moreover, to evaluate the potential application of the ACP critical gelation concentration (CGC) and gelation temperature (GT)
hydrogel in tissue engineering, a cell-laden hydrogel assay and migra­ were first determined by the conventional test tube inversion method.
tion assay using 3D fibroblast cell clusters was conducted. The ACP solution (1 ml) was loaded into a glass vial and heated from
10 ◦ C to 50 ◦ C at 5 ◦ C intervals. These vials were equilibrated at each
2. Experiment and methods determined temperature point for 10 min before determining the
behaviour of the ACP solution. If the ACP solution inside the vials could
2.1. Materials not flow within 60 s, it was recognised as the gel stage, otherwise, it was
regarded as a solution. Then, the critical gelation temperature (Tgel)
Sodium alginate (Na-alg, W201502), pluronic F127 (F127, P2443) was determined by small amplitude oscillatory shear (SAO) using the
were purchased from Sigma-Aldrich (St Louis, MO, USA). 1-(3-Dime­ dynamic oscillation test (Thermo HAAKE 6000, Thermo Fisher Scienti­
thylaminopropyl)-3-ethyl carbodiimide (EDC, ≥97%), N-hydrox­ fic, Waltham, MA, USA). Prior to the temperature sweep tests, the strain-
ysuccinimide (NHS, ≥98%), 3-amino-1-propanol (≥98%), p-nitrophenyl dependent experiment was performed from 0.01% to 15% with an
chloroformate (p-NPC, 97%), cystamine dihydrochloride (cys, ≥97%) angular frequency of 1 Hz at GT value, to determine the linear visco­
came from Acros Organics (New Jersey, USA). The cellulose dialysis elastic range (LVR). The temperature sweep was run under a determined
membranes (MWCO 12–14 kDa cut off) obtaining from Repligen (Breda, strain, with an angular frequency of 1 Hz at a heating rate of 1 ◦ C/min
The Netherlands) were used to purify products. Absolute ethanol, from 20 ◦ C to 50 ◦ C.
tetrahydrofuran (THF), and diethyl ether were supplied by Fisher Sci­
entific (New Jersey, USA). For cell culture, Dulbecco's modified Eagle's 2.6. Water uptake and degradation test
Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin
solution (10,000 U/mL), and Sodium bicarbonate originated from The ACP copolymer (mi) was prepared separately in DMEM, PBS 1×,
Sigma-Aldrich (Singapore). Phosphate-buffered saline (1×, pH 7.4) was and DI water at the CGC determined from Section 2.5. The ACP solution
purchased from Gibco. Unless otherwise specified, all other chemicals was kept at 4 ◦ C to make a homogenous solution before transferring it to
were acquired from RCI Labscan (Bangkok, Thai Lan) and Alfa Aesar the vial. The empty vials were weighed and labeled as W. The vial
(Massachusetts, USA). containing 0.5 ml of the ACP hydrogel (Wi) was weighed before adding
10 ml of DMEM or 1× PBS. At the determined time intervals, the
2.2. Synthesis pathway of Na-alg-cys immersed medium was removed, and then a cotton paper was used to
remove excess water in the vials. This vial was weighed (Wf). The degree
Na-alg (1 g) was completely dissolved in a mixture of dimethyl of water absorption into hydrogel was quantified using formula (1).
sulfoxide (DMSO):water (1:1 v/v) at room temperature (RT) under
Wf − Wi
shaking conditions. HCl (1 N) was used to adjust the pH value of the Water uptake (%) = × 100% (1)
Wi − W
solution to the target pH (5.5). EDC⋅HCl (0.00046 mol, 1 equiv.) was

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L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

For the degradation test, ACP copolymer (mi) was dissolved in 0.5 ml dissolving it in complete DMEM at the designed concentration. The so­
1× PBS or DMEM at the CGC point. At each predetermined time point, lution was kept at 4 ◦ C for 2 days to achieve a homogenous solution
the ACP hydrogel was collected and then lyophilized and weighted (mt). before filtering via a cell strainer (100 μm, Biologix). BJ cells (1 × 105
The remaining mass percentage of ACP hydrogel after immersion in each cells/ml) were directly suspended in the ACP copolymer solution and
media was calculated using formula (2): plated on the 24-well plate before incubating in an icebox for 3 min to
mt allow equal spreading. The plate was incubated in a CO2 incubator for 2
Remaining mass percentage = × 100 (%) (2) h before adding 200 μl warm complete DMEM to cover the hydrogel.
mi
Then, 200 μl warm complete DMEM was carefully added into each well
every 48 h. At the determined time points, 200 μl culture medium
2.7. Anti-bacteria activity testing
containing 10 μl of AO (50 μg/ml) and 5 μl of PI (1 mg/ml) was added
into culture plate and incubated for 1 h. The cells were fixed with 200 μl
Escherichia coli (ATCC8739), Salmonella typhimurium (ATCC 14028),
fixation solution (4% paraformaldehyde and 2.5% glutaraldehyde) at RT
Pseudomonas aeruginosa (ATCC27853), and Staphylococcus aureus (ATCC
for 15–30 min and were washed with 1× PBS three times before
6538) were a gift from the Department of Biochemistry, Faculty of
observation under Andor confocal microscope (using 525 nm and 650
Biology and Biotechnology, University of Science, Vietnam National
nm filters).
University, Ho Chi Minh City. A bacterial suspension of each strain at
106–107 colony-forming units (CFU)/ml was inoculated onto nutrient
2.10.2. Migration assay
agar. 20 μl each sample (0.2 g/ml Na-alg, 0.2 g/ml pluronic F127, 0.2 g/
BJ cells were loaded into the ACP hydrogel as described for 3D
ml cystamine, 0.2 g/ml ACP hydrogel, and water) was added separately
protocol. The 24-well plate was placed on the hot plate (37 ◦ C) for 15
on Bacitracin disks (6 mm, Merck, Singapore). Subsequently, these disks
min before adding drop-wise 10 μL ACP solution containing BJ cells.
were put on the surface of the prepared agar and incubated at 37 ◦ C for
300 μl of complete DMEM medium was added when the ACP drop was
24 h. The antimicrobial activity was evaluated by measuring the zone of
formed on the surface of the well. The plate was then incubated in the
transparent inhibition against the test microorganisms.
conditions described in Section 2.9. After 48 h and 120 h incubation, the
The agar dilution method was used to determine the lowest con­
culture medium was removed and 300 μl of DEMEM containing AO/PI
centration of the test compound that prevented bacterial growth. The
dual was added following the incubation for 15 min at 37 ◦ C. The free
stock of each compound was prepared at 0.2 g/ml. A range of two-fold
dye was washed with 1× PBS before taking the images using z-stacks at
serial dilutions (10–1000 mg/ml) was prepared with 20 ml of melted
10× magnification with an Andor confocal.
agar. These agar plates were allowed to solidify at RT before plating 100
μl of diluted inoculum (106–107 CFU/mL) from each strain suspensions.
2.11. Statistical analysis
The results were read after incubating the agar plates at 37 ◦ C for 24 h.

All data are expressed as the mean ± standard error, calculated from
2.8. Adhesion testing of the ACP hydrogel at least three independent replications. The statistical differences be­
tween the means were determined using one-way or two-way analysis of
Tissue adhesion properties of the ACP hydrogel were determined via variance (ANOVA), followed by Tukey's multiple comparison test. P <
lap-shear strength using Material Testing Machine (5kN, Shimadzu, 0.05 was considered statistically significant.
Japan). First, porcine skin samples were collected from the local market
and cut into rectangular sheets (5 × 2 cm) before fixing. 100 μl ACP 3. Results
solution was placed on the prepared skin piece followed by surface
contact with another skin piece for 10 min at 37 ◦ C under normal 3.1. Characterization of the ACP copolymer
pressure 0.1 mN to enhance adhesion. The prepared system was
attached to each side of the Material Testing Machine. The adhesive ACP was synthesised using a three-step synthesis pathway following
strength was recorded if the contact between two skins was completely the amine modification of Na-alg with cystamine and the p-NPC–deri­
separated. The experiment was carried out at a crosshead speed of 10 vated pluronic F127 (Fig. 1).
mm/min with 0.1mN normal force. Fibrin glue was also measured as a In the first route, the carboxyl groups on the Na-alg backbone were
control adhesive under the same conditions. activated to form ester intermediate products that could react quickly
with primary amines. FT-IR was conducted to evaluate the modified
2.9. Cytotoxicity assay carboxylic group on the Na-alg process (Fig. 2). The FT-IR spectrum of
Na-Alg presented a broad band centre at 3411 cm− 1 corresponding to
Human fibroblast cells, BJ cells (ATCC® CRL-2522™) were used for the presentation of hydroxyl groups (-OH), low-intensity bands at 2923
cytotoxicity evaluation. In this study, 0.1% gelatin (PCS-999-027™) was cm− 1 attributed to the symmetric vibration of aliphatic carbon (CH2),
used as the control. For the procedure, 1 ml 0.1% gelatin solution and and peaks at 1619 cm− 1 and 1412 cm− 1 corresponding to symmetric and
200 μl of ACP solution were plated onto a 35 mm culture dish separately. asymmetric stretching (-COO-), respectively [22,29]. In the range
These ACP dishes were incubated at 4 ◦ C for 4 h before incubating at 1294–815 cm− 1, some vibrations are due to ether functional groups (C-
37 ◦ C overnight. The disks were dried under ultraviolet (UV)-light O-C) in glycosidic linkage [21]. The vibration peaks in 890–815 cm− 1
before plating 2 × 105 BJ cells/ml suspended in complete DMEM (10% belong to the C–C and C–O bond in mannuronic acid [21,22] while the
FBS and 0.1% penicillin-streptomycin). The sulforhodamine B Assay Kit peak at 1294 cm− 1–1037 cm− 1 indicate the pyranoid ring in guluronic
(ab235935, Abcam) was used to determine the cell viability while the acid [21] is characterized at. After modification with cystamine, the
live/dead assay was performed based on the dual staining acridine or­ major peaks of Na-alg (O–H, C– – O) are still presented in FT-IR spectra
ange (AO)/propidium iodide (PI) method. The morphology of BJ cells of Na-alg-cys. However, there was a blue shift for symmetric and
was observed under a confocal microscope (Andor) with dual laser asymmetric carboxyl groups compared with the pure Na-Alg, suggesting
channels. the interaction of amine groups with carboxylic groups [30]. The
spectrum showed an additional peak with strong absorption at 2890
2.10. Cellular proliferation and migration assay cm− 1 assigned to the vibration of primary amines along with a small
signal of SH bonding at 2298–1960 cm− 1. Alongside the shift in the
2.10.1. Cellular proliferation study carboxylate mode, the FT-IR spectrum of Na-alg-cys showed the small
The ACP powder was put directly under UV-light for 2 days before shift of guluronic acid and mannuronic acid. Notably, the anomeric

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L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

Fig. 1. Three-step synthesis route of the ACP copolymer.

unit (H1-G) and mannuronic unit (H1-M) at δ = 5.05 ppm and δ = 4.45
ppm [31,32], respectively. This spectra also showed the chemical shift
corresponding to proton C-5 of the guluronic unit [24] (H5-G, at δ =
4.37 ppm) and mannuronic unit [33,34] (H5-M, δ = 4.20 ppm). In
addition, the signals at δ = 3.1 ppm (Ha) and δ = 3.4 ppm (Hb) arise
from the respective proton of -CH2CH2S- and -NHCH2CH2S-, these data
confirm the success of the amine functional Na-alg. Based on the TNBS
assay, the coupled amine content was 54.70 ± 0.36 mg/g of Na-alg-cys,
yielding 78.04% ± 2.31.
In the second route, NPC activated pluronic F127 was synthesised
following the previous reports [2,4,20], and its chemical characteriza­
tion is presented in Fig. 3. This spectrum showed a chemical shift for the
protons of the aromatic ring of p-NPC at δ = 7.41 ppm (H2) and δ = 8.29
ppm (H1). The proton signals of PPO blocks of pluronic F127 are at δ =
1.15 ppm (H4, methyl group (-CH3)) and δ = 3.41 ppm (H3), methylene
group (>CH2), whereas the proton signal of –CH2 –CH2– units of PEO
blocks is at δ = 3.67 ppm. In addition, the chemical shift at δ = 4.44 ppm
(H5) represents to the proton on the methylene group in the ester bond
with p-NPC, CH2-O-NPC. The peak at δ = 4.22 ppm corresponding to
methylene protons of the CH2–CH2–O-Ami moiety, confirming the suc­
cess of the second step of the synthesis.
The final step was the conjugation of NPC-activated pluronic F127
onto the Na-alg-cys resulting in the development of the ACP copolymer.
Fig. 2. FT-IR spectra of pure cystamine, pure Na-alg, Na-alg-cys, and The successful synthesis of the ACP copolymer was also confirmed by
ACP copolymer. FT-IR (Fig. 2) and 1H NMR spectra (Fig. 3). The ACP copolymer repre­
sented all the typical characteristic peaks as present in the FT-IR spec­
peaks of the fingerprint at 1700 cm− 1 and 1243 cm− 1 represent the trum of Na-alg-cys. The introduction of pluronic F127 on the Na-alg-cys
characteristic absorption bands of N-C=O (amine I) and N–H bending backbone changed the intensity of the stretching of ester groups (C=O)
vibrations, proposing the amidation of the carboxylic group of alginate and hydroxyl groups (OH). The proof of a covalent chemical bond be­
molecules [24]. The success of aminated functional Na-alg was further tween pluronic and Na-alg-cys is the presence of the deformation vi­
confirmed via the 1H NMR spectrum in Fig. 3. Both the characteristic brations of NH bonds and stretching vibrations of CN in the FT-IR
proton peaks of Na-alg and cystamine are still presented in the 1H NMR spectrum of ACP copolymer. 1H NMR was used to further confirm this
spectra. The spectrum displayed the anomeric proton on the guluronic result. As shown in Fig. 3, along with the anomeric proton signals of Na-

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L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

Fig. 3. 1H NMR (in DCl3, 500 MHz) spectrum of the activated pluronic F127 (A) and 1H NMR (in D2O, 500 MHz) characterization of Na-Alg-cys (B) and ACP
copolymer (C).

alg, this spectrum shows the chemical shifts corresponding to the proton 3.2. Thermal sensitivity of Na-Alg-cys-F127 solution
of pluronic F127. The peak at δ = 1.03 ppm represents the methyl group
(-CH3) in the PPO block of F127, whereas the peak at δ = 3.70 ppm The behaviour of the ACP copolymer as a function of temperature
corresponds to (–CH2 –CH2–) units of the PEO blocks of F127. There was was further investigated using oscillation thermo-rheometry. The
a shift in the methylene proton (-CH2) in this spectrum, although the amplitude oscillation sweep was first performed to identify the linear
intensity of this proton was reduced. The proton signal of methylene in viscoelastic region (LVR) at which the elastic structure of the hydrogel
(NHCH2-) is disappeared in this spectrum because the characteristic remains intact [2,35]. Within the LVR, the changes in the storage
signal for methyl groups on the PPO block is at the same position, δ = modulus (G′ ) and loss modulus (G′′ ) are mainly affected by temperature
3.40 ppm. These results revealed the formation of the ACP copolymer. [2]. Based on this result (Supporting material, S1), the evolution of the
Regarding the remaining amine content calculated by the TNBS assay, viscoelastic moduli of the ACP copolymer as a function of temperature
the grafting reaction efficiency of the activated pluronic (NPC-F127-OH) was conducted at a frequency of 1 Hz and a strain amplitude of 1%.
on Na-alg-cys backbone was about 44.47% ± 0.74. Fig. 4 shows the thermal behaviour of the ACP copolymer through a
sol-gel transition following the temperature function. The temperature
sweep was exploited to identify the Tgel [4]. Temperature ramp mea­
surements at a fixed frequency (Fig. 4) showed that both the elastic (G′ )

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L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

Fig. 4. Evolution of the dynamic moduli in the temperature sweep experiment of ACP copolymer with various concentrations (% wt/wt). A) 13%, B) 15%, C) 17%
and D) 20%t.

and loss (G′′ ) moduli increased as a function of temperature starting terms of rheology, the solvent caused small variations in the Tgel
from 20 ◦ C and crossed over each other at a critical gel point (Tgel). The (Fig. 5C). The 20% (wt/wt) ACP dispersed in DI water underwent the
sol-gel transition of the ACP copolymer solution was strongly dependent sol-gel transition at 35.1 ◦ C during heating. However, when physiolog­
on the ACP concentration. When the copolymer concentration increased ical buffer (1× PBS, pH 7.4) or culture medium (DMEM) was used to
from 13% (wt/wt) to 20% (wt/wt), the temperature-induced Tgel was prepare the ACP hydrogel (20%, wt/wt), the gelation occurred at a
reduced. Specifically, at 13% (wt/wt), the crossing point was at 41.2 ◦ C, lower temperature compared with DI water. This difference may be due
while at 20% (wt/wt) it was reduced to 35.1 ◦ C. Moreover, the to the interaction of Na-alg with ions in the medium. When the ACP
maximum value of G′ followed the order of concentrations, a phenom­ copolymer is dissolved in this solvent, the cations diffuse into the ACP
enon that is attributed to the decrease in the flexibility of the ACP networks and form ionic interchain bridges [41]. Therefore, the lower
copolymer at higher concentrations. Increasing the ACP copolymer Tgel induced by the medium can be explained by the synergistic effect
concentration leads to a higher density of hydrophobic interactions and between the pluronic F127 and Na-alg chains. Due to cationic agents in
thus a greater capacity to build the microstructure [36,37]. Further­ the buffer media, the water surrounding the pluronic F127 chain may be
more, in response to the temperature change, pluronic F127 induced the reduced as the temperature increases, and the PPO segments become
folding of the alginate backbone, creating a more hydrophobic zone. more hydrophobic and less polar, providing a platform for promoting
Similarly to the jamming-induced gelation in pure pluronic systems gelation [35,40]. In the range of concentration and all solvent tested, the
[38,39], the thermal gelation in the ACP system could be due to the ACP hydrogel (20%, wt/wt) could be a suitable scaffold for cell encap­
packing of micelles within the alginate folding pocket. Subsequently, it sulation processes.
potentially stores the deformation energy and reduces the isotropic gel
[35,40]. Therefore, higher ACP copolymer concentrations require less 3.3. Characterization of porous thermal-sensitive ACP scaffolds
heat energy to induce its aggregation; consequently, the Tgel is lower for
higher ACP copolymer concentrations. The morphology of the ACP hydrogel was visualised by scanning
Besides the influence of the ACP copolymer concentration on Tgel, electron microscopy (SEM) for a dry hydrogel (Fig. 6A) and confocal
the effect of the aqueous medium used to dissolve ACP copolymer on the microscopy for a wet hydrogel (Fig. 6B). The ACP hydrogel had a good
sol-gel transition temperature was investigated. As shown in Fig. 5A, three-dimensional interconnected microstructure based on both forms
20% (wt/wt), the ACP copolymer dissolved in DI water, PBS buffer or of microscopy. In addition, the ACP hydrogel possessed homogeneously
DMEM media undergoes the sol-gel transition in the response to the distributed pores with a size in the range of 30–80 μm, suggesting that
heating condition. Regarding the inverted tube method, there was a the ACP hydrogel may be suitable for cell delivery in tissue engineering
unremarkable difference in the GT range of the ACP solutions prepared [18,29,30].
in different media (Fig. 5B). The 20% (wt/wt) ACP solution in all tested Alongside the required microstructure of hydrogel, the degree and
media appeared to be in the gel state at 25 ◦ C and complete gel devel­ rate of swelling are one of crucial parameters that affect the release rate
opment (G′ over G′′ in oscillation rheology) was achieved above 30 ◦ C. In of solvents and drugs from polymeric hydrogel networks [42]. Swelling

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L.H. Dang et al. International Journal of Biological Macromolecules 185 (2021) 592–603

Fig. 5. (A) Images of inverted glass vials containing ACP copolymer solutions below 20 ◦ C and above 30 ◦ C. (B) The sol-gel transition temperature of the ACP
hydrogels prepared in different solvents (DI water, DMEM, and 1× PBS) using the inverted tube method. The Tgel was investigated using the temperature sweep test
of 20% wt/wt ACP solution (C).

studies were performed in 1× PBS and DMEM. Because the pH of both negative bacteria (E. coli, S. typhimurium, P. aeruginosa) and gram-
media was 7.4, the remaining carboxylic groups on the Alg backbone positive (S. aureus). The zone of inhibition of ACP (20wt/wt%) of
had ionised, leading to increased repulsion between the carboxylate P. aeruginosa, E. coli, S. typhi, and S. aureus were changed from 0 to 16.8
groups; consequently, the ACP hydrogel began to swell at the initial mm, 0 to 11.4 mm, 0 to 2.1 mm, and 0 to 12.3 mm, respectively
immersed stage [17,43,44]. As shown in Fig. 6C, the ACP hydrogel (Fig. 7A–B). P. aeruginosa is known as the most common bacterium
swelled quickly after immersion in both media. Interestingly, disinte­ isolated from chronic wounds, and ACP at 50 mg/ml can be used to
gration did not occur after reaching the maximum point. From day 2 to prevent their visible growth. Also, 50 mg/ml is the minimum required
6, the ACP hydrogel was highly resistant to dissolution. The amount of ACP concentration to inhibit the growth of S. aureus - the major infec­
water uptake was around 200%, indicating an equilibrium in the tious organism in all type of wounds. ACP exposes its sensitivity against
swelling process. Presumably, the ionisation of Na-alg may interact with E. coli and S. typhi at 35 mg/ml and 75 mg/ml, respectively. To examine
cations in the medium resulting in the equilibrium in osmotic pressure, why ACP copolymer has intrinsic antibacterial properties, Na-alg,
thereby controlling the driving force for the penetration of water mol­ pluronic F127, and cys were treated to these bacteria stains in a
ecules [45,46]. However, from day 7, the water content in the ACP similar condition to ACP. Among them, only cys showed a sensitive
hydrogel started to decrease in both media. This effect was likely pattern of antimicrobial activity as ACP hydrogel. The presentation of
because the density of hydrogen bonding with water molecules within cystamine induces the increase of ROS concentration in culture media
the ACP hydrogel network had been exhausted and the 3D microstruc­ leading to dysregulation of small thiol pools, disrupting the function of
ture had become damaged [44]. Of note, the degradation course of the the cell membrane as well as the metabolism inside bacteria; finally,
ACP hydrogel in both immersion media was identical (Fig. 6D), a finding inhibiting the growth of bacteria [25,26]. The Kirby–Bauer (KB, zone of
that is consistent with the water uptake ability of ACP in both media. inhibition) test was performed to determine whether the ACP hydrogel
The mass of lyophilized ACP copolymer was maintained over all 7 days had antimicrobial activity. The ACP hydrogel showed promising anti­
(all P > 0.1). The degradation of the ACP hydrogel in both media started microbial activity against both gram-negative (E. coli, S. typhimurium,
on day 8. In PBS, about 31% of the initial weight of the ACP hydrogel P. aeruginosa) and gram-positive (S. aureus) bacteria. The zone of inhi­
was lost while in DMEM, 28% of the ACP hydrogel mass was lost. There bition of the ACP hydrogel (20%, wt/wt) for P. aeruginosa, E. coli,
was an unremarkable difference in the degradation rate of the ACP S. typhi, and S. aureus changed from 0 to 16.8 mm, from 0 to 11.4 mm,
hydrogels in DMEM or PBS, about 11% per day. After 12 days of im­ from 0 to 2.1 mm, and from 0 to 12.3 mm, respectively (Fig. 7A, B).
mersion in PBS or DMEM, the ACP hydrogel could not be recovered, P. aeruginosa is known as the most common bacterium isolated from
confirming its complete degradation. chronic wounds, and ACP at 50 mg/ml could be used to prevent their
visible growth. In addition, 50 mg/ml was the minimum ACP concen­
tration required to inhibit the growth of S. aureus, which is the major
3.4. Anti-bacterial activity infectious organism in all type of wounds. The minimum ACP concen­
trations required to inhibit the growth of E. coli and S. typhi were 35 mg/
Kirby-Bauer (KB, zone of inhibition) was performed to identify ml and 75 mg/ml, respectively. To examine why the ACP copolymer has
whether ACP hydrogel has antimicrobial activity. In our study, ACP intrinsic antibacterial properties, the antibacterial activity of Na-alg,
hydrogel showed promising antimicrobial activity against both gram-

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Fig. 6. Morphology of the ACP hydrogel (20% wt/wt) by SEM (A) and by confocal microscopy using AO dye (B). The water uptake capacity of the ACP hydrogel
(20% wt/wt) when immersed in PBS and DMEM (C). The degradation of the ACP hydrogel (20%, wt/wt) over 12 days reported as the remaining mass (D).

Fig. 7. Antibacterial studies with Escherichia coli (ATCC8739), Salmonella typhimurium (ATCC 14028), Pseudomonas aeruginosa (ATCC27853), and Staphylococcus
aureus (ATCC 6538). (A) Heat map of the zone of inhibition of each separate material (pluronic F127, Na-alg, and cystamine) and the ACP hydrogel. (B) Zone of
inhibition of the ACP hydrogel (20%, wt/wt) and cystamine (24.3 mg/ml). (C) MIC value of the ACP hydrogel and cystamine against four bacterial strains.

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(ACP3). Moreover, increasing the feeding Na-alg-cystamine concentra­


tion in the ACP copolymer from 5% (wt/wt) (ACP3) to 12.5% (wt/wt)
(ACP1) significantly increased the adhesive strength of the ACP hydro­
gel to 396%, evidencing the critical role of the amine functional groups
on alginate in tissue adhesion properties.
Cell vitality is an important criterion to determine whether a mate­
rial could be applied in tissue engineering and regeneration [29,47,50].
Human fibroblasts were used to evaluate the toxicity profile of the
fabricated hydrogel. Culture dishes coated with 0.1% gelatine served as
the control. Both the ACP hydrogel and 0.1% gelatine caused minimal
toxicity; in all cases, BJ cell viability was >90% (Fig. 9A, B). The live/
dead staining showed a no toxic effects in the ACP-coated dish. In both
coating cases, the green nucleated cells (live cells) due to the presenta­
tion of AO were more pronounced while very few cells appeared red
(dead) (Fig. 9C). Fibroblasts cultured on the ACP hydrogel proliferated
quickly and similarly to fibroblasts on the gelatine-coated dish (Fig. 9B,
P > 0.1). After 48 h of incubation, BJ cells covered approximately 70%–
Fig. 8. The adhesive strength of the ACP hydrogel (20% wt/wt) with various 80% of the surface of both dishes. In addition, the morphology of the
amount of the grafted pluronic F127 (from 87.5% to 100%) to porcine tissue. fibroblasts was the typical elongated and spindle-like shape for both
The lap-shear adhesion tests at 37 ◦ C were repeated three times. **P < 0.05. coating conditions (Fig. 9C). These results suggest that ACP hydrogel
provides a good surface for promoting cell proliferation [50]: The ACP
pluronic F127, and cystamine against these bacteria was tested. Among hydrogel did not affect the proliferation, viability, or morphology of BJ
them, only cystamine showed antimicrobial activity similar to the ACP cells, showing the excellent biocompatibility of the ACP hydrogel.
hydrogel. Cystamine increases ROS in the culture medium, leading to
dysregulation of small thiol pools, disrupting the function of the cell
membrane as well as the metabolism inside bacteria and, finally, 3.6. 3D cell culture and cell migration
inhibiting the growth of bacteria [25,26].
To further investigate the cytocompatibility of the ACP hydrogel,
fibroblasts were encapsulated inside the ACP hydrogel matrix. After 48 h
3.5. Adhesion and cytotoxicity properties of the hydrogel scaffolds of culturing, the viability of fibroblasts was 96.7% ± 3.5%, estimated by
the number of green cells divided by red cells. For the long-term culture
In tissue engineering, hydrogels with the ability to adhere to the (168 h), >90% of fibroblasts were primarily stained green, which
adjacent tissue can avoid detachment from target tissues in vivo and demonstrates that most of the cells are viable; hence, the cells were
eventually promote potential biointegration [1,2,47,48]. Hence, we grown in good culture conditions [20,29,51]. After 24 h of incubation,
evaluated the adhesive property of ACP with porcine skin. Fig. 8 in­ BJ cells had adhered to the wall of the ACP hydrogel, and after 120 h, the
dicates that the adhesive strength of the ACP hydrogel to porcine tissues cells had proliferated and extended within the ACP scaffolds. The
significantly increased as the amine functionalisation of Na-alginate growth of fibroblasts increased rapidly and created a layered network
increased from 0% to 12.5% (labeled as ACP1, ACP2, and ACP3). The saturating the hydrogel matrix after 168 h (Fig. 10). Furthermore, the
adhesion force for pure pluronic F127 gel (20%, wt/wt) was about 0.70 fibroblasts culturing inside the ACP hydrogel appeared elongated, a
± 0.16 kPa, in good agreement with reports from the literature [49]. The morphology that is rarely seen under 3D culture conditions [2,47]. The
adhesion force increased 233% (1.63 ± 0.17 kPa) when amine- elongation of fibroblasts cultured inside a hydrogel has been reported
functionalised Na-alg cooperated with 5% (wt/wt) pluronic F127 with a hydrogel system containing gelatine [52] or collagen [53] or

Fig. 9. (A) Viability and (B) quantification of cell number as well as (C) the fluorescence images of BJ cells that were cultured on the ACP hydrogel compared to
Gelatin 0.1% at 2rd day. Scale bar is 100 μm.

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Fig. 10. Human fibroblasts encapsulated in the ACP hydrogel after culturing for 48, 120, and 168 h. The cells were subjected to dual AO/PI staining. The scale bar is
100 μm.

Fig. 11. Z-stack fluorescence images of dual AO/PI staining of cells migrating out of an encapsulated hydrogel droplet (20 μl). A squared-dot circle represents the
formation of the ACP cluster, and the blue arrows indicate the outgrowth cell from the ACP cluster. The scale bar is 200 μm. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

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