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Chitosan nanoparticles loaded hydrogels promote

skin wound healing through the modulation of
reactive oxygen species

Tao Wang, Yan Zheng, Yaping Shen, Yijie Shi, Fang Li, Chang Su & Liang Zhao

To cite this article: Tao Wang, Yan Zheng, Yaping Shen, Yijie Shi, Fang Li, Chang Su & Liang
Zhao (2018) Chitosan nanoparticles loaded hydrogels promote skin wound healing through
the modulation of reactive oxygen species, Artificial Cells, Nanomedicine, and Biotechnology,
46:sup1, 138-149, DOI: 10.1080/21691401.2017.1415212

To link to this article: https://doi.org/10.1080/21691401.2017.1415212

Published online: 13 Dec 2017.

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
2018, VOL. 46, NO. S1, S138–S149
https://doi.org/10.1080/21691401.2017.1415212

Chitosan nanoparticles loaded hydrogels promote skin wound healing through

the modulation of reactive oxygen species
Tao Wanga
, Yan Zhenga
, Yaping Shena
, Yijie Shia, Fang Lia, Chang Sub and Liang Zhaoa
a
School of Pharmacy, Jinzhou Medical University, Jinzhou, PR China; bSchool of Veterinary Medicine, Jinzhou Medical University,
Jinzhou, PR China

ABSTRACT ARTICLE HISTORY

This study developed a novel bioactive chitosan nanoparticle loaded calcium alginate hydrogel to regu- Received 29 September 2017
late inflammation and neovascularization for accelerated wound healing in vivo. It was found that chito- Revised 10 November 2017
san nanoparticles loaded calcium alginate hydrogel exhibited remarkable antibacterial activity. Through Accepted 6 December 2017
the modulation of generation of ROS, it promoted the synthesis and secretion of IL-6 in vascular endo-
thelial cell (VEC), suggesting its potential proinflammatory activation. Further, it promoted VEC invasion, KEYWORDS
metastasis and neovascularization to accelerate wound healing. Inflammation; neovasculari-
zation; chitosan;
nanoparticles; calcium
alginate hydrogel

Introduction Compared with the traditional transdermal drug delivery

systems, nanotechnology-driven therapeutic interventions
Natural (acute) wound healing is a coordinated process that
showed the potential aspects on facilitating the wound heal-
includes several largely overlapping phases that involve an
ing process and ultimately repairing the injured tissue
inflammatory response and associated cellular migration, pro-
[17–21]. Especially, it was reported that a large amount of
liferation, matrix deposition and tissue remodelling [1,2].
ROS were produced via internalization of some nanoparticles,
When the skin injury was initially occurred, haemostasis was
indicating the possibility that NPs may exhibit intrinsic prop-
immediately induced by the assembly of a fibrin clot, and ini-
erties beneficial for wound treatment via NPs mediated ROS
tiated the invasion and recruitment of inflammatory and
production [22–25]. Based on the primary hypothesis of our
other cells for achieving debridement and anti-infection effect
study, we prepared a novel bioactive chitosan nanoparticles
[3–5]. Vascular endothelial cells (VECs) played an important
loaded calcium alginate hydrogel (CS NPs loaded CaAlg
role on the formation of capillary vessels and brought an
hydrogel) with a specific multi-target profile on the regulation
important effect in the final healing of wound. The rapid
of anti-bacteria, inflammation and neovascularization.
growth of VECs formed new blood vessels in the granulation
Moreover, its preliminary possibility for accelerated wound
tissue and supplied nutrients to the facilitate the skin regen-
healing in vivo was further investigated (Figure 1).
eration by enhancing proliferation and migration of fibro-
blasts and keratinocytes for re-establishing the normal
cellular and extracellular matrix (ECM) composition of skin Materials and methods
[6–10]. Therefore, designing therapeutic treatment on the
Materials
regulation of inflammation and neovascularization showed a
specific multi-target profile and had been regarded as a pos- The polymer chitosan (MW, 100–150 kDa; degree of deacety-
sible strategy to improve the wound healing effect. lation, 85%) was acquired from Haixin Biological Product Co.,
Reactive oxygen species (ROS) played important roles in Ltd (Ningbo, PR China); alginic acid with molecular weight of
vascularization and it was reported that some substance approximately 160 kDa was purchased from Qingdao
showed suppression of ROS mediated VEGF signalling and Huanghai Biological Pharmaceutical Co., Ltd (Qingdao, PR
had an anti-angiogenic effect on pathologic retinal neovascu- China); tripolyphosphate (TPP) and calcium chloride were pur-
larization [11–15]. In addition, ROS are generated after tissue chased from Sigma-Aldrich (St. Louis, MO); Dulbecco’s modi-
injury through initiating acute inflammation and some results fied Eagle’s medium (DMEM) and foetal bovine serum (FBS)
indicated that ROS induced ERK activation and led to the were purchased from Gibco Technologies (Logan, UT); all
activation of pro-inflammatory cytokine responding to mech- other chemicals purchased were of analytical grade and were
anical injury in the acute phase [16]. obtained from a variety of vendors. Human umbilical vein

CONTACT Liang Zhao liangzhao79@163.com School of Pharmacy, Jinzhou Medical University, Jinzhou, PR China

These authors contributed equally to this work.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S139

Figure 1. The primary hypothesis of this study. Chitosan nanoparticle loaded calcium alginate hydrogel exhibited antibacterial activity and triggered the generation
of ROS, thus enhancing acute inflammatory response which was conducive to debridement and anti-infection effect. In the stage of skin tissue regeneration, it pro-
moted proliferation and migration of VEC, and led to rapid healing.

endothelial cells (HUVECs) were obtained from the Cell Line Instruments, Malvern, UK) was used to determine the size,
Bank of the Chinese Academy of Sciences (Shanghai, China). morphology and zeta potential of CS NPs. Rheological stud-
ies of CS-CaAlg hydrogel and CS NPs loaded CaAlg hydro-
gel were conducted using rotational rheometer (Anton Paar
Preparation and identification of chitosan nanoparticles MCR302, Graz, Austria). Swelling capacity of gels was
loaded calcium alginate hydrogel also determined by calculating the difference between
According to our previous reports [26], chitosan NPs were the initial weight of the gel (W0) and the weight of
prepared by ionic crosslinking method. Certain amount of absorbent gel (WS) as follows: swelling ratio (SR)
sodium alginate was dissolved in distilled water followed by (%) ¼ 100  (WS – W0)/W0.
the respective addition of CS NPs under stirring until NPs
were fully dispersed in solution. When calcium chloride used
Antibacterial tests
as the best Ca2þ sources for the ionic crosslinking of alginate
was added into the solution, homogeneous alginate hydrogel Different amount of CS NPs loaded CaAlg hydrogel and CS-
cross-linked with Ca(2þ) cations was formed and CS nanopar- CaAlg hydrogel was placed into a tube and incubated with
ticles were loaded into the matrix of calcium alginate hydro- 5 mL of bacterial suspension containing 1  106 CFU of
gel. By comparison, chitosan conjugated calcium alginate Escherichia coli and Staphylococcus aureus for continuous
hydrogel (CS-CaAlg hydrogel) was prepared by the addition shaking for 24 h at 37  C. Sterilized cotton swab was used to
of calcium chloride into the mixture of chitosan and sodium dip into bacteria suspension treated with both gels and
alginate. The preparation of chitosan nanoparticles loaded slowly scratched on the LB-agar growth plates for incubation
calcium alginate hydrogel is shown in Figure 2(A). The char- at 37  C. The optical density (OD) was measured at 600 nm
acterization of CS NPs loaded CaAlg hydrogel was investi- for determining the minimum bactericidal concentrations
gated by affinity-1 infrared spectroscopy (Shimadzu, Kyoto, (MBCs) by using a microplate reader (Tecan, infinite M200,
Japan). Scanning electron microscopy (S-4800, Hitachi, Tokyo, M€annedorf, Switzerland). In addition, the OD at 260 nm was
Japan) was used to observe the morphology of CS NPs also evaluated for checking the leakage of DNA and RNA
loaded CaAlg hydrogel. Zetasizer (Nano ZS90; Malvern from bacteria.
S140 T. WANG ET AL.

Figure 2. Schematic representation of CS NPs loaded CaAlg hydrogel preparation (A). SEM images of CS NPs loaded CaAlg hydrogel (B). FT-IR spectra of pure CS
NPs, CaAlg hydrogel, simple mixture of pure CS NPs and CaAlg hydrogel, and CS NPs loaded CaAlg hydrogel (C). DLS analysis of the obtained CS NPs (D).
Rheological flow curves of CS-CaAlg hydrogel and CS loaded NPs (E). In vitro swelling profile of CS NPs loaded CaAlg hydrogel and CS-CaAlg hydrogel in phosphate-
buffered saline (pH 7.4 and pH 8.0 at 37  C) for 24 h. Data are expressed as the mean ± standard deviation (n ¼ 3) (F).

ROS production measurement Tokyo, Japan), and also quantified using microplate reader
(Synery-2, Biotek, Winooski, VT, USA).
According to our previous reports [27], CS NPs loaded CaAlg
hydrogel and CS-CaAlg hydrogel were co-incubated with
HUVECs for 12 h at 37  C with the treatment of 2,7-dichloro- MTT assay
fluorescein diacetate (DCFH-DA, Sigma, St. Louis, MO, USA)
with cells for about 30 min. Generation of ROS was detected One hundred microlitres suspension of HUVECs at the loga-
by observing the intracellular DCF fluorescence using con- rithmic growth phase was added to each well in the 96-well
focal laser scanning microscopy (FluoView FV10i, Olympus, plate and adjusted the cell concentration to 1  105 cells/well
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S141

at 37  C in a humidified atmosphere of 5% CO2, 95% air. One Western blot assay

hundred microlitres of extracts of CS NPs loaded CaAlg
According to the protocol [27], extracts of CS NPs loaded
hydrogel and CS-CaAlg hydrogel were added into each hole
for incubating with cells at 37  C in a humidified atmosphere CaAlg hydrogel and CS-CaAlg hydrogel containing concentra-
of 5% CO2, 95% air. At different time interval, 20% MTT with tion of CS at 10 and 50 lg/mL as well as the combination of
concentration of 5 mg/mL was added to each well and con- NAC and CS NPs loaded CaAlg hydrogel were incubated with
tinued to incubate for 4 h. One hundred and fifty microlitres HUVECs for 48 h followed by washing twice with ice-cold PBS.
of DMSO was added to each well, and the absorbance was Finally, the level of the targeted proteins was photographed
measured at 490 nm. and analysed by UVP gel analysis system.

Angiogenesis and confocal microscopy Experimental wound model and wounding procedure

The ECM gel solution was thawed out at 4  C and 200 lL of Six- to eight-week-old female SD mice were purchased from
mixture of ECM gel solution and serum free medium (v/v, Beijing Vital River Laboratory Animal Technology Company.
1:1) was added into each well of a 24-well plate. After incuba- All procedures were performed by the guidelines of National
tion for 30 min at 37  C, the ECM solution formed a gel. Institutes of Health Guide for the Care and Use of Laboratory
HUVECs (80 lL per well, about 1  105 cells/well) were Animals and were approved by the Animal Care and Use
cultured in gel loaded-bottom 24-well tissue culture plates Committee of Jinzhou Medical University. Using a dermato-
followed by addition of the extracts of CS-CaAlg hydrogel logical pencil, a circle with a diameter of 2 cm was drawn on
and CS NPs loaded CaAlg hydrogel for continuous incubation the back skin, and the skin was cut using a scalpel and scis-
for 8 h. The tube formation of HUVECs was observed and ana- sors. Wound in all animals was treated with PBS, CS NPs
lysed using a confocal microscope (TCS SP8; Leica loaded CaAlg hydrogel and CS-CaAlg hydrogel separately
Microsystems, Wetzlar, Germany) and imaged at 100 twice daily for consecutive days for two weeks. Wound area
magnification. reducing curves were determined by calculating the ratio of
the initial area and the wound area at the different time. The
mice were sacrificed, then the area around the scar or
Scratch wound healing assay residual wound were isolated and the wound specimens
were prepared as paraffin-embedded sections for histopatho-
HUVEC was cultured in DMEM supplemented with 10% FBS logical analysis.
and seeded into 24-well tissue culture plate until they reach
70–80% confluence as a monolayer. A new 200 lL pipette tip
was used to gently and slowly scratch a straight line to the Results and discussion
monolayer across the centre of the well. After scratching,
Physicochemical characterization of hydrogels
the wells were washed twice with medium to remove the
detached cells followed by replenishing the wells with fresh In this study, we employed a Ca(2þ) cations as the crosslinker
medium. Extracts of CS NPs loaded CaAlg hydrogel and CS- to fabricate a hydrogel system by dispersing CS nanoparticles
CaAlg hydrogel containing the different concentration of CS into alginate aqueous solution, and the solution was formed
were cultured in a tissue culture incubator at 37  C. At differ- into a hydrogel using CaCl2. We observed that CS NPs
ent time interval, the cells were washed twice with PBS, and we prepared were monodisperse spheres, and further
the gap distance can be observed and quantitatively eval- analysis revealed that the diameters of NPs were about
uated under the microscope. 208.4 ± 15.7 nm and positive zeta potential about
24.2 ± 3.9 mV (Figure 2(D)). In addition, the polydispersity
index of NPs was above 0.019, indicating the homogenous
Transwell cell migration assay monodispersion and good stability in media. The investiga-
The invasion of HUVEC was detected by transwell method. tion of the hydrogel microstructure was performed by scan-
Extracts of CS NPs loaded CaAlg hydrogel and CS-CaAlg ning electron microscopy (SEM). CS NPs loaded CaAlg
hydrogel containing the different concentration of CS were hydrogel showed tight stack vertically and had a low poros-
placed into the transwell insert precoated with basement ity, whereas it showed a considerable amount of CS nanopar-
membrane matrigel and incubated with HUVEC for 24 h in ticles (diameter around 200 nm) at the surface of hydrogel
the 37  C, 5% CO2 incubator. At different time interval, insert (Figure 2(B)). The possible interaction between CS NPs and
was removed from the chamber and chamber was turned CaAlg hydrogel was investigated via FTIR spectroscopic ana-
upside-down and gently submerged in PBS several times to lysis of pure CS NPs, CaAlg hydrogel, simple mixture of pure
remove unattached cells. Cells were fixed by submerging CS NPs and CaAlg hydrogel, and CS NPs loaded CaAlg hydro-
insert in 4% paraformaldehyde for 20 min and stained with gel. It is observed in Figure 2(C) that compared with the
1% crystal violet for 30 min (or overnight). Cells were spectra of t simple mixture of pure CS NPs and CaAlg hydro-
removed from unmigrated (top) side by gently scrapping gel, the appearance of two intense and sharp peaks at 1049
this side with a wet Q-tip/cotton swab. Invasiveness was and 1024 cm1 in the spectra of CS NPs loaded CaAlg hydro-
determined by counting cells on the lower surface of the gel indicated CS NPs with the functional groups such as –NH
filter. and –OH as hydrogen bonding donors formed hydrogen
S142 T. WANG ET AL.

bond at the surface of CaAlg hydrogel with the functional induced the massive generation of ROS (Figure 4). With the
groups such as carboxylic group as hydrogen bonding increasing amount of CS at the concentration from 20 to
acceptors. Therefore, the carbon–oxygen electron cloud dens- 100 lg/mL, green fluorescence intensity also continued to
ity on the carboxyl group was increased, and stretching vibra- rise gradually and the productions of intracellular ROS were
tion from –C–O was strengthened and appeared at 1049 and increased from 112.3% to 124.5% in HUVECs treated with
1024 cm1. A flow curve shown in Figure 2(E) is presented by CS-CaAlg hydrogel. On the contrast, when HUVECs were
measuring the viscosity of developed gels from 0.01 to treated with CS NPs loaded CaAlg hydrogel with the different
100 s–1 of shear rate. The results showed that CS NPs loaded concentration of CS, green fluorescence intensity was signifi-
CaAlg hydrogel could be easily spread and located in wound cantly enhanced and the productions of intracellular ROS
site. With the initial minutely increase of shear rate, viscosity were increased from 125.3% to 189.8%. Addition of 10 mM
of both gels decreased rapidly. When the shear rate was oxygen free radical scavenger acetylcysteine (NAC) inhibited
increased gradually, the gel viscosity decreased slowly, indi- the production of oxygen free radical, thus the intensity of
cating that the gels showed the shear thinning property and intracellular green fluorescence was weakened and amount
the viscosity of all the prepared gels decreased with increase of ROS was significantly reduced to 110.0% in cells. It indi-
in shear rate. The results in Figure 2(F) showed that both gels cated that CS NPs loaded CaAlg hydrogel enhanced the
depended on the variation of time required to control the uptake of CS NPs and increased ROS production.
absorbing amount of water. The swelling process of CS NPs
loaded CaAlg hydrogel and CS-CaAlg hydrogel showed a
MTT assay and angiogenesis analysis
similar swelling pattern. The SR of both gels was increased
with the enhancement of pH of medium. It further indicated The MTT results shown in Figure 5(B) demonstrated that CS-
that when pH was increased, the anionic carboxyl group was CaAlg hydrogel and CS NPs loaded CaAlg hydrogel at a cer-
highly ionized and tend to charge repulsion and hydration, tain concentration of CS (ranging from 5 to 100 lg/mL) did
thus enhancing hydrogel swelling. not show cytotoxicity and the promotion on the proliferation
of HUVECs induced by CS-CaAlg hydrogel and CS NPs loaded
CaAlg hydrogel was not obvious, and the viability ratios in
Antibacterial activities of CS NPs loaded CaAlg hydrogel
cells treated with CS NPs loaded CaAlg hydrogel at a certain
The results in Figure 3 show that compared to the control concentration of CS at 10 and 100 lg/mL were 121.3% and
and CS-CaAlg hydrogel, CS NPs loaded CaAlg hydrogel 113.4% at 24 h, respectively.
showed the highest anti-bacteria activity. It is demonstrated Angiogenesis played a normal and vital process in wound
in Figure 3(A) that with the increasing addition of CS NPs, it healing process and capillary-like tube formation of HUVEC
could kill two more kinds of bacteria, which may be part of formed new blood vessels from preexisting vessels and facili-
the reason for the remarkable antibacterial activities. It indi- tated the formation of granulation tissue. The results in
cated that positive charged CS NPs released from gel adhered Figure 5(A) show that compared with control and CS-CaAlg
to the negative charged surface of E. coli and S. aureus cells hydrogel, tube formation in the presence of CS NPs loaded
with a high probability. A strong interaction with both CaAlg hydrogel was significantly accelerated and the branch
Gram-positive and Gram-negative bacteria by electro-static numbers of connected cells in randomly selected fields
interaction might be a key reason that explains the effective depended on a dose to significant increase. On the contrary,
antibacterial activity of CS NPs loaded CaAlg hydrogel. The treatment with the combination of CS NPs loaded CaAlg
MBCs quantitatively evaluated the antibacterial activities and hydrogel and NAC as oxygen free radical scavenger inhibited
were measured by determining the OD600. As shown in tube formation of HUVEC on Matrigel. These results sug-
Figure 3(C), the resulting gel exhibited remarkable antibacter- gested that CS NPs loaded CaAlg hydrogel promoted in vitro
ial activities at very low dosages, with an MBC of 100 lg/mL angiogenesis by enhancing tube formation. Although CS NPs
against E. coli and against S. aureus. As shown in Figure 3(D), loaded CaAlg hydrogel did not show the obvious prolifer-
the amount of DNA and RNA released from the bacteria was ation of HUVEC cell, blood vessel recruitment may be mainly
significantly higher in the CS NPs loaded CaAlg hydrogel by cell migration rather than proliferation of HUVECs.
group than the CS-CaAlg hydrogel group; CS NPs loaded Angiogenesis induced by CS NPs loaded CaAlg hydrogel was
CaAlg hydrogel group also exhibited an increased release of primarily regulated by the generation of ROS.
bacterial DNA and RNA with the increasing addition of CS
NPs. The results above revealed that CS NPs loaded CaAlg
Cell migration and invasion assay
hydrogel group killed G– bacteria (E. coli) and G þ bacteria
(S. aureus) by changing the membrane permeability, while As shown in Figure 6, compared with the control group, the
the disruption in the cell wall was the primary mechanism of migration distance of cells treated with the extracting solu-
action on bacteria. tions of CS-CaAlg hydrogel and CS NPs loaded CaAlg hydro-
gel was higher than that of the control group and showed
the dose dependent pattern. Especially, CS NPs loaded CaAlg
ROS determination
hydrogel induced the higher cell migration and migration
The results showed that compared with control group and rates in contrast with cells treated with CS-CaAlg hydrogel. It
CS-CaAlg hydrogel group, CS NPs loaded CaAlg hydrogel indicated that CS NPs were loaded into the matrix of
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S143

Figure 3. Photographs of culture plates of E. coli and S. aureus after exposure to CS NPs loaded CaAlg hydrogel containing different concentration of CS NPs (A).
Photographs of the culture plates of E. coli and S. aureus after exposure to CaAlg hydrogel as well as CS-CaAlg hydrogel and CS NPs loaded CaAlg hydrogel with the
same concentration of CS at 50 lg/mL (B). OD600 nm of bacterial suspension treated CS NPs loaded CaAlg hydrogel with different concentrations of CS NPs. These
data represent three separate experiments and are presented as the mean values ± SD.
p < .05 versus OD600 nm of E. coli treated with control group, #p < .05
versus OD600 nm of S. aureus treated with control group. (C). Relative release of 260 nm absorbing materials from CS-CaAlg hydrogel and CS NPs loaded CaAlg
hydrogel treated E. coli and S. aureus. These data represent three separate experiments and are presented as the mean values ± SD.
p < .05 CS-CaAlg hydrogel
group versus control group, #p < .05 CS NPs loaded CaAlg hydrogel versus control group (D).

hydrogel and further released, finally internalized into the NPs-mediated ROS generation could be associated with the
HUVEC, thus accelerating the migrating rate of cells. With induction of cell migration. The cell invasion assay results
the increasing amount of CS at the concentration of 20 and also confirmed that compared with cells treated with CS-
100 lg/mL, migrated distance also continued to rise grad- CaAlg hydrogel, the number of CS NPs loaded CaAlg hydro-
ually from 8 to 34 lm in cells treated with CS-CaAlg hydro- gel treated cells on the underside of the membrane was
gel. On the contrast, when cells were treated with CS NPs significantly increased, indicating that cells secreted more
loaded CaAlg hydrogel with CS NPs at the concentration of protease to damage the ECM, therefore enhancing the
20 and 100 lg/mL, migrated distance was significantly invading force of cells. In the same way, the addition of
enhanced from 40 to 84 lm. Interestingly, with the addition NAC effectively prohibited cell invasion and migration. It
of NAC as an inhibitor of oxygen free radical, the degrees also proved that CS NPs loaded CaAlg hydrogel promoted
of cell invasion and migration were significantly reduced in cell invasion and metastasis via the massive production
contrast with cells treated with non-NAC. It suggested that of ROS.
S144 T. WANG ET AL.

Figure 4. ROS generation in cells treated with extracts of CS NPs loaded CaAlg hydrogel and CS-CaAlg hydrogel containing low concentration of CS at 20 lg/mL
and high concentration at 100 lg/mL as well as the combination of NAC and CS NPs loaded CaAlg hydrogel in HUVEC cells.
p < .05, versus ROS levels of untreated
cells at 24 h, the scale bar is 100 lm and applies to all figure parts.

Western blot assay secretion of proinflammatory cytokines in HUVEC in the initial

period of wound healing process.
Interleukin 6 (IL-6) acts as both a pro-inflammatory cytokine
To further justify the effect of CS NPs loaded CaAlg hydro-
and an anti-inflammatory myokine [28]. The results in
gel on angiogenesis in wound healing process, we detected
Figure 7 show that compared with CS-CaAlg hydrogel, CS
the expression of VEGF, MMP-2 and bcl-2 as the primary neo-
NPs loaded CaAlg hydrogel showed the higher potential on
vascularization-relevant proteins via western blot analysis. The
promoting the acute synthesis and secretion of IL-6 in HUVEC
results showed that CS NPs loaded CaAlg hydrogel and
within the initial 12 h of wound injury period in a dose-
dependent manner, suggesting that it had potential acute CS-CaAlg hydrogel had no significant difference on bcl-2 as
proinflammatory activation and was conducive to wound tis- anti-apoptotic protein in HUVEC, illustrating that compared
sue aggregation, sterilization. After using ROS scavenger, IL-6 with the control groups, both gels did not induce the cell
inflammasome signal activation was significantly suppressed, apoptosis. In order to further evaluate invasion and metasta-
indicating the activation of IL-6 mediated inflammasome was sis of cells treated with gels, the expressions of proteins of
partially ROS dependent. Within 48 h, the expression level of the matrix metalloproteinase (MMP) family were investigated
IL-6 in HUEVCs treated with CS NPs loaded CaAlg hydrogel by western blot experiment. It demonstrated that CS NPs
was significantly decreased, suggesting that lower expression loaded CaAlg hydrogel induced the highest expression of
of IL-6 attenuated the pain, inhibited the prolonged inflam- mmp-2 in HUVEC in contrast with control and CS-CaAlg
matory response, and improved the pathologic condition hydrogel. The expressions of mmp-2 were significantly
induced by wound injury. CS NPs loaded CaAlg hydrogel acti- increased with the increasing amount of added CS NPs in
vated a ROS-dependent oxidative stress response and gels. Combination of NAC and CS NPs loaded CaAlg hydrogel
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S145

Figure 5. Tube formation in HUVECs treated with extracts of CS NPs loaded CaAlg hydrogel and CS-CaAlg hydrogel containing low concentration of CS at 20 lg/mL
and high concentration at 100 lg/mL as well as the combination of NAC and CS NPs loaded CaAlg hydrogel in HUVECs (A). HUVEs viability cultured with CS NPs
loaded CaAlg hydrogel (B) and CS-CaAlg hydrogel (C) containing different concentrations of CS after 24 h. Data are presented as means ± SD (n ¼ 3).

down-regulated the expression levels of mmp-2, suggesting migration and adhesion of VECs and thus promoting cell
that CS NPs loaded CaAlg hydrogel facilitated the secretion angiogenesis effects.
of mmp-2 as main migration-relevant proteins and acceler-
ated the breakdown of ECM in physiological processes. The
Full-thickness skin wounds and quantification of healing
results also confirmed that there was a possibility that NPs
promoted the invasion and metastasis of cells via NPs medi- CS-CaAlg hydrogel and CS NPs loaded CaAlg hydrogel were
ated ROS production. The inhibition of ROS with NAC signifi- applied to the wounds twice daily, and wound size was quan-
cantly reduced the degree of cell invasion and migration. tified from images taken on post-wounding days 1, 2, 3, 7, 9,
Finally, CS NPs loaded CaAlg hydrogel induced the highest 12 and 14. The results in Figure 8 show that the rate of
expression of VEGF in HUVEC in contrast with control and CS- wound closure in mice treated with CS NPs loaded CaAlg
CaAlg hydrogel. Combination of NAC and CS NPs down-regu- hydrogel was significantly faster than control group and CS-
lated the expression levels of VEGF by reducing ROS. It CaAlg hydrogel treated group. When injury wound were
revealed that increased intracellular ROS induced the activa- treated with CS NPs loaded CaAlg hydrogel, the rate of
tion of VEGF on the cell membrane to initiate the phosphor- wound closure was increased to 85.6% within seven days and
ylation of the redox-sensitive signalling transduction 98.7% within 14 days, respectively. Similarly, after being
molecules such as P38 and ERK, leading to proliferation, treated with PBS and CS-CaAlg hydrogel, the rates of wound
S146 T. WANG ET AL.

Figure 6. CS NPs loaded CaAlg hydrogel promoted migration and invasion of HUEVCs. Image (A) and quantity (C) of migration and image (B) and quantity (D) of
invasion taken from HUEVC cells incubated for 24 h with extracts of CS NPs loaded CaAlg hydrogel and CS-CaAlg hydrogel containing low concentration of CS at
20 lg/mL and high concentration at 100 lg/mL as well as the combination of NAC and CS NPs loaded CaAlg hydrogel (magnification, 100). The above assays were
quantified. Results were expressed as mean ± SD (n ¼ 3).
p < .05, versus untreated cells at 24 h.

Figure 7. Expression levels of IL-6 protein in HUVEC treated with CS NPs loaded CaAlg hydrogel containing low concentration of CS at 20 lg/mL, middle concentra-
tion of CS at 50 lg/mL and high concentration at 100 lg/mL for 12 h (A). Western blot analyses of the expression levels of IL-6, VEGF, MMP-2 and Bcl-2 in HUVEC fol-
lowing treatment with CS NPs loaded CaAlg hydrogel for 48 h (B).
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S147

Figure 8. Photographs of the appearance of the wounds on days 7 and 14 under the treatment of CS-CaAlg hydrogel and CS NPs loaded CaAlg hydrogel (A).
Wound closure curves after wound sites were treated by CS-CaAlg hydrogel and CS NPs loaded CaAlg hydrogel (B). The results were expressed as mean ± SD (n ¼ 5).

closure were lower and valued at 63.6% and 69.9% within were a large amount of inflammatory cells infiltrating in
seven days, and 81.5% and 95.7% within 14 days, respect- wound site, granulation tissue layer structure was still loose
ively. The results further confirmed that CS NPs loaded CaAlg and few fibroblasts were formed and arranged impacted.
hydrogel promoted wound contraction and accelerated heal- Compared with CS-CaAlg hydrogel, CS NPs loaded CaAlg
ing of wounds. hydrogel did not show acute inflammatory reaction and a
Histological observations (Figure 9) revealed that after small amount of neutrophils and some inflammatory cells
treatment from 1 day to 3 days, in the control group, there were observed in wound site. Moreover, CS NPs loaded CaAlg
S148 T. WANG ET AL.

Figure 9. H&E staining of tissue sections treated with CS NPs loaded CaAlg hydrogel on days 1, 3, 7 and 14 (A). Number of inflammatory cells (B), blood vessels (C)
and fibroblasts (D) on days 1, 3, 7 and 14. Data represent means ± SD (n ¼ 3). #p < .05, CS-CaAlg hydrogel group versus the corresponding control group at the
same day.
p < .05, CS NPs loaded CaAlg hydrogel group versus the corresponding control group at the same day.

hydrogel exhibited significant formation of fibroblast, granu- neutrophil infiltration was visible around the border of epi-
lation tissue and blood vessel at the wound site. After treat- thelium. The structure of granulation tissue with more neo-
ment from 7 days to 14 days, in the control group, the vascularization and fibrous cells still existed. In the wound
wound was not completely closed and reepithelialization was site treated with CS-CaAlg hydrogel, new epidermis and a
not completed, the thinner epithelium was formed and more scab were obviously formed. CS-CaAlg hydrogel induced the
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S149

formation of thicker epithelium containing the infiltration of a angiogenesis and inhibiting inflammation. Am J Transl Res.
certain number of inflammatory cells, high granulation tissue 2017;9:2352–2362.
[8] Tadokoro S, Ide S, Tokuyama R, et al. Leptin promotes wound
content and a few regenerative blood vessels. When CS NPs
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Funding
reduced and stabilized gold nanoparticles: kill cancer cells via
This work was supported by the Liao’ning Educational Committee (No. reactive oxygen species. ACS Appl Mater Interfaces. 2016;8:
L2014339) and the Natural Science Foundation of Liaoning Province (No. 22106–22112.
2014022039, No. 2015020692 and No. 201602337). [23] Latvala S, Hedberg J, Di Bucchianico S, et al. Nickel release, ROS
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