Abstract
The emergence of drug‐resistant bacteria due to excessive antibiotic
use has drawn increasing attention to inorganic nanoparticles for their
broad‐spectrum antibacterial properties. Here, a “green” strategy for
the simultaneous in situ synthesis of silver nanoparticles (AgNPs)
during the photocrosslinking process of casein hydrogels is described.
The in situ photoactivated biomineralization of AgNPs provides
noticeable stability and antibacterial activity, with high photothermal
effect during a sequential near‐infrared laser activation. The
milk‐derived casein is screened out due to its great biomineralization
capability and wound healing activity. Casein‐AgNP hydrogel dressing
shows low swelling, good mechanical properties, and nice
biocompatibility. In animal experiments, casein‐AgNP hydrogel
accelerates wound repair and tissue regeneration after bacterial
infection by regulating immune response. Our work shows that sequential
photoactivation served as a promising strategy for antiinfectious wound
treatment, and casein hydrogel stood as a potential candidate for
in situ biomineralization.
Keywords: antibacterial activities, biomineralization, casein, silver
nanoparticles, wound healing
__________________________________________________________________
A milk‐derived casein protein hydrogel wound dressing with sequential
photoactivatable antisepsis is developed. The first photoactivation
enables rapid in situ hydrogel gelation and AgNP biomineralization; the
second photoactivation enhances bactericidal effect by optothermal. The
synergistic antibacterial strategy demonstrates remarkable efficacy in
significantly accelerating the recovery of infected wounds.
graphic file with name SMSC-5-2500026-g004.jpg
1. Introduction
Skin injury commonly occurs in daily life and is susceptible to wound
infections. Infections can cause serious complications and hinder skin
recovery.^[ [32]^1 ^] Coinfections with multiple pathogenic
microorganisms have been observed in most infected wounds, as well as
the emergence of multidrug‐resistant bacteria, which raises the urgent
need for highly efficient antiseptic treatment. Inorganic
nanoparticles, notably silver nanoparticles (AgNPs), can interact with
various microorganisms (such as bacteria) and impact the growth of
mature bacterial biofilms; therefore, they could be used as
broad‐spectrum antimicrobials.^[ [33]^2 , [34]^3 ^]
In addition to being antibacterial, a wound dressing must act as a
partial skin barrier to avoid secondary injury and provide a favorable
microenvironment for wound healing.^[ [35]^4 , [36]^5 , [37]^6 ^] At
present, a variety of wound dressings is available for this purpose.
Among these, injectable hydrogel dressings possess the advantages of
tissue‐like structures, excellent flexibility, shape adjustability,
diverse components, and degradability, making them competitive
candidates for next‐generation wound dressings.^[ [38]^7 , [39]^8 ,
[40]^9 ^] Endowing hydrogel dressing with AgNPs has become an effective
strategy for antibacterial wound dressing.^[ [41]^10 , [42]^11 ,
[43]^12 , [44]^13 ^]
Conventional methods for mixing AgNPs with pregel polymers have several
limitations: i) residuals of potentially toxic agents during the
synthesis of AgNPs and ii) the risk of AgNP aggregation and
oxidation‐induced loss of efficacy during storage.^[ [45]^14 ^] In situ
biomineralization is an efficient and eco‐friendly process that
facilitates the integration of inorganic materials with organic
macromolecules.^[ [46]^15 , [47]^16 ^] In particular, the synthesis of
metal nanostructures using biomineralizing agents, such as proteins and
other hydrophilic polymers, has attracted considerable interest because
of their superior biocompatibility, conformal interfaces, uniform
distribution, and high stability.^[ [48]^17 , [49]^18 ^] Strategies,
including repetitive freeze‐thawing,^[ [50]^19 ^] ultraviolet (UV)
irradiation,^[ [51]^20 ^] and alkaline conditions,^[ [52]^21 ^] have
been integrated to promote biomineralization of different proteins for
AgNP synthesis. However, the in situ synthesis of AgNP/hydrogels still
shows limited biomineralization efficiency. For example, the formation
of collagen‐AgNP hydrogels spared a 0.5 h incubation under light
exposure.^[ [53]^22 ^]
The strong reducibility of plant polyphenols has been exploited to
improve their biomineralization capacity.^[ [54]^23 , [55]^24 , [56]^25
^] Modifying gelatin with catechol groups enhanced its
biomineralization capacity for in situ AgNP formation.^[ [57]^22 ^] The
highly reactive free radicals originating from the homolytic cleavage
of the photosensitive precursor can also reduce silver cations to
AgNPs. Zaier et al. developed a one‐step photo‐induced method for
synthesizing silver@polymer nanoassemblies on a variety of
substrates.^[ [58]^26 ^] Thus, combining polyphenols and a radical
“initiator” accelerates AgNP synthesis in hydrogels.
In addition to small‐molecule excipients, macromolecular backbones also
play an important role in the biomineralization process. Fu et al.
utilized the tyrosine residues in collagen to reduce Ag^+ under
illumination and formed collagen‐AgNP hydrogels in a single step.^[
[59]^27 ^] Thus, we hypothesized that choosing a suitable native
macromolecular backbone could improve biomineralization. Milk‐derived
proteins have long been known to promote bone mineralization.^[ [60]^28
^] As one of the main milk proteins, bovine casein contains a large
proportion of tryptophan, tyrosine, histidine, and proline residues,
which endows it with certain reducibility.^[ [61]^29 , [62]^30 ^]
However, their ability to synthesize AgNPs has not been explored.
Previous studies in our laboratory have shown that milk‐derived casein
hydrogels have a great ability to bind metal ions^[ [63]^31 ^] and
promote wound healing.^[ [64]^32 ^] Thus, we explored the
biomineralization of casein for AgNP synthesis and its effects on the
management of infected wounds.
In this study, we report an efficient in situ sequential
photoactivatable antibacterial hydrogel wound dressing derived from
protein biomineralization. To screen suitable proteins, we selected
several commonly used protein materials^[ [65]^33 , [66]^34 ^] as
candidates and evaluated their photoactivatable biomineralization
properties for AgNP generation (Figure [67] 1a). As three milk‐derived
proteins (casein, whey protein, and α‐lactalbumin) showed significantly
higher AgNP photosynthesis, we ultimately selected casein due to its
promising wound healing function, as we showed previously.^[ [68]^32 ^]
Resveratrol was added to further enhance the mineralization speed for
in situ AgNP generation. Casein was methacrylated to enhance the
mechanical properties of the hydrogel. Additionally, a photosensitive
initiator of free radicals, lithium
phenyl‐2,4,6‐trimethylbenzoylphosphinate (LAP), was used to accelerate
the AgNP formation.^[ [69]^26 ^] The polymerization of methacrylated
casein (casein‐MA) and the formation of AgNPs were accomplished
simultaneously under the first UV irradiation, which was further
amplified by a second near‐infrared (NIR) irradiation (Figure [70]1b).
The sequential photoactivated casein‐AgNP hydrogel (CASMA‐Ag hydrogel)
was used as a dressing to treat infectious lesions in mice. The wound
tissues were collected for whole genome sequencing to identify the main
pathways modulated by the sequentially photoactivated casein‐based
hydrogel. Our results suggest that the sequentially photoactivated
CASMA‐Ag hydrogel has optimal prospects for application as an
antiseptic wound dressing.
Figure 1.
Figure 1
[71]Open in a new tab
a) Schematic diagram of proteins suitable for photoactivated
biomineralization of AgNP. b) Schematic diagram illustrating the
sequential photoactivation of CASMA‐Ag hydrogel and its application in
infectious wound healing.
2. Results and Discussions
2.1. Photoactivated Formation of CASMA‐Ag Hydrogel
Biomineralization plays a significant role in wound dressing. Inorganic
minerals offer structural reinforcement, support essential cellular
processes, and enhance dressing bioactivity owing to their
multifunctional properties.^[ [72]^35 , [73]^36 ^] For infected wounds,
infection control is a paramount factor influencing healing outcomes.
AgNPs are distinguished from other minerals owing to their excellent
broad‐spectrum antibacterial properties.^[ [74]^37 ^] Additionally,
AgNPs with an appropriate particle size range exhibit good photothermal
effects, which can facilitate infection clearance and accelerate
healing through photothermal therapy.^[ [75]^38 ^]
The biomineralization of different proteins was screened using a
photoactivation method. Compared to other native proteins commonly used
in biomaterials, casein, whey, and lactoglobulin, the three main
components of milk showed the highest biomineralization (Figure [76]1).
To prepare a hydrogel dressing with optimized in situ gelation,
biomineralization, and peroxidation, casein was chosen because we have
previously shown its function in wound repair.^[ [77]^32 ^] To further
promote cross‐linking, casein was methacrylated, which did not impair
AgNP formation (Figure S1, Supporting Information). Macroscopic images
of the CASMA‐Ag1 hydrogel demonstrated the transformation from a
colorless and transparent solution into a brown solid hydrogel after
exposure to 405 nm irradiation for 5 min (Figure [78] 2a).
Figure 2.
Figure 2
[79]Open in a new tab
UV‐induced formation of CASMA‐Ag hydrogel. a) Photograph showing the
sol‐gel transition of CASMA‐Ag1 upon 405 nm UV irradiation for 5 min.
b) Rheology curve. c) Gelation time and d) storage modulus of hydrogels
exposed to 405 nm UV irradiation for 300 s (mean ± SD; *P < 0.05,
***P < 0.001, and ****P < 0.0001; n = 3). e) SEM images and f) pore
size distribution of different hydrogels. g) Mass swelling rate of
hydrogels in PBS within 48 h (n = 3). h) 3D printing of 10% w/v
casein‐MA hydrogel.
The gelation process of casein‐MAa under UV curing was monitored by
rheology to assess the kinetics of the photocrosslinking reaction and
the effect of biomineralization on crosslinking behavior
(Figure [80]2b,d). A certain concentration of casein‐MA was required to
initiate gelation (Figure S2, Supporting Information). Increasing
casein‐MA concentration from 10% to 15% reduced the gelation time from
29.73 ± 2.23 s to 18.57 ± 0.48 s and increased the final storage
modulus from 6.19 ± 0.43 to 12.38 ± 0.84 kPa. To meet the flexibility
requirements of wound dressings, we used 10% casein‐MA in the following
experiments unless otherwise specified.
The addition of resveratrol increased the gelation time by 55% and
decreased the modulus to 5.2 kPa. Gelation was further retarded by
increasing [Ag^+]. However, the gelation time was still within 1 min,
and the storage modulus was >4.0 kPa for all conditions. The negative
influence on the gelation of resveratrol and Ag^+ may have occurred
because of their free radical scavenging properties; resveratrol is
well‐known as a free radical quencher,^[ [81]^39 ^] and Ag^+ can react
with radicals generated from LAP to form AgNPs.^[ [82]^26 ^]
The hydrogel microstructures were observed using scanning electron
microscopy (SEM). Figure [83]2e,f shows that all hydrogels had a porous
structure, with the addition of resveratrol and Ag^+ having larger pore
sizes.
Hydrogels tend to swell in various aqueous solutions, such as wound
exudates, which not only reduces their mechanical properties but also
causes adverse compression of the surrounding tissues in vivo.^[
[84]^40 ^] The swelling ratios were measured at different time points
after soaking the samples in phosphate buffered solution (PBS) for 48 h
(Figure [85]2g). All hydrogels (casein‐MA, CASMA‐Ag0.5, CASMA‐Ag1, and
CASMA‐Ag1.5) showed no obvious swelling during the soaking process,
which is consistent with our previous swelling results for casein
hydrogels crosslinked by the Ru/SPS initiator.^[ [86]^32 ^]
Owing to its fast gelation, stable structure, and resistance to
swelling, photocrosslinkable casein‐MAa is suitable for 3D printing to
build a reliable complex architecture. As shown in Figure [87]2h, the
10% casein‐MA hydrogel precursor solution was successfully cross‐linked
layer‐by‐layer into a stereo model, showing good shape fidelity.
2.2. Optimization of In Situ Photoactivated Biomineralization and
Photothermal Effect
The kinetics of biomineralization were studied by tracing the light
exposure time of AgNP formation (Figure S3, Supporting Information).
The peak strength at ≈410 nm increased with light exposure time until
it reached a plateau, indicating complete mineralization of the AgNPs.
The color shift of the hydrogel during light irradiation indicated the
formation of AgNPs. We analyzed the contribution of each component to
biomineralization in a mixture of pregel solutions by depleting certain
constituents. The photoinitiator LAP and polyphenol resveratrol in the
system triggered AgNP synthesis, but the major effect was caused by
casein‐MA (Figure [88] 3a). In addition, we observed the agglomeration
of silver during photoactivation in groups without casein‐MA,
indicating its role as a stabilizer and dispersant for AgNP synthesis.
The amount of resveratrol also affected the rate of AgNP synthesis
(Figure S4, Supporting Information). However, owing to the limited
solubility of resveratrol, highly concentrated resveratrol did not
significantly increase the synthesis rate of AgNPs.
Figure 3.
Figure 3
[89]Open in a new tab
Biomineralization of AgNPs during initial photoactivation and the
photothermal effect activated by subsequent NIR irradiation. a) AgNPs
generation (OD value at 410 nm) in the presence of different components
in the system (mean ± SD; P < 0.05; n = 3). b) TEM images of AgNPs in
different hydrogels. c) PSD of AgNPs in different hydrogels. d) XRD
patterns of AgNPs. e) EDS showing element composition of CASMA‐Ag1. f)
Wide‐scan XPS spectra of hydrogels. g) High‐resolution XPS spectra of
Ag 3d. h) Schematic diagram of the photothermal conversion mechanism in
CASMA‐Ag hydrogel. i) Photothermal images showing temperature
distribution of different hydrogels under NIR irradiation (3 W cm^−2,
10 min); j) Corresponding temperature change curves from (i). k)
Thermal imaging of a mouse treated with CASMA‐Ag1 hydrogel before and
after NIR irradiation (3 W cm^−2, 10 min).
To directly observe the morphology of AgNPs formed during
photoactivated biomineralization, a transmission electron microscope
(TEM) was used. AgNPs were clearly formed by photoactivation, and the
amount and size of the AgNPs positively correlated with the initial
[Ag^+] (Figure [90]3b). The sizes of the AgNPs were 2.5−25 nm for
CASMA‐Ag0.5, 5−35 nm for CASMA‐Ag1, and 2.5−45 nm for CASMA‐Ag1.5
(Figure [91]3c). The particle sizes of the AgNPs in different hydrogels
had a broad distribution range. AgNPs in CASMA‐Ag0.5, CASMA‐Ag1, and
CASMA‐Ag1.5 present a z‐average diameter of 10.7 nm, 14.4 nm, and
19.2 nm, respectively, which was consistent with the TEM results
(Figure S5, Supporting Information). To investigate the impact of
polymer concentration on the nanostructures, we increased the
concentration of casein‐MAa to 15% w/v for the biomineralization
reaction. The morphology of the AgNPs in the 15% casein‐MA system
resembled a cubic shape, indicating that the growth direction of the
AgNPs was restricted in highly concentrated solutions (Figure S6,
Supporting Information).^[ [92]^41 ^]The results further supported that
a casein‐MA concentration of 10% w/v is more suitable for the in situ
mineralization of AgNPs.
The X‐ray diffraction (XRD) patterns of CASMA‐Ag0.5, CASMA‐Ag1, and
CASMA‐Ag1.5 in Figure [93]3d show four distinct diffraction peaks at
38.12°, 44.30°, 64.45°, and 77.41°, which correspond to the (111),
(200), (220), and (311) crystal faces of the AgNPs, respectively. The
results indicated the generation of metallic Ag with cubic
face‐centered symmetry, confirming the successful generation of AgNPs
in the hydrogels.
The elemental composition of the CASMA‐Ag1 hydrogels was investigated
using energy‐dispersive spectroscopy (EDS). The EDS results indicate
the presence of Ag (Figure [94]3e). The uniform distributions of C, O,
N, and Ag in the CASMA‐Ag1 hydrogels are shown in Figure S7, Supporting
Information. The broad X‐ray photoelectron spectroscopy (XPS) results
in Figure [95]3f do not show any differences in CASMA‐Ag1 after
irradiation for 0, 2, and 5 min. However, the fine scan of Ag 3d
(Figure [96]3g) showed variation with irradiation duration. Considering
the standard spectra from National Institute of Standards and
Technology, the binding energy of Ag^+ was higher than that of Ag^0.
The peaks at 373.5 eV and 367.6 eV were assigned to Ag^+, while those
at 372.4 eV and 366.4 eV were assigned to AgNPs. The AgNPs/Ag^+ ratio
was 1.8 after 2 min of irradiation, and the AgNPs/Ag^+ ratio increased
to 25.6 after 5 min of irradiation. The AgNP peaks became stronger, and
the AgNP/Ag^+ ratio increased with longer photoreduction times. Several
studies^[ [97]^3 , [98]^42 , [99]^43 ^] show AgNPs adhere to and
accumulate on bacterial surfaces, disrupt cell membranes, and cause
structural changes. Moreover, when nanoparticles dissolve in water or
enter cells, a certain amount of Ag^+ is released, which also exhibits
antibacterial function.
Subsequently, the photothermal performance of the CASMA‐Ag hydrogel was
studied. The AgNPs exhibited high absorption of NIR light, which could
be effectively converted into heat (Figure [100]3h).^[ [101]^44 ^]
Figure [102]3i,j shows that the temperature of the CASMA‐Ag0 hydrogel
did not increase significantly under NIR irradiation (3 W cm^−2).
However, after the in situ biomineralization and synthesis of AgNPs,
the photothermal response increased significantly. The temperature
difference (ΔTS) of CASMA‐Ag0.5, CASMA‐Ag1, and CASMA‐Ag1.5 hydrogels
before and after 10 min irradiation reached 22.9, 37.6, and 43.1 °C,
respectively, indicating that the introduction of AgNPs provided
satisfactory photothermal properties to the hydrogels. Furthermore, the
CASMA‐Ag1 hydrogel demonstrated exceptional photothermal stability
despite three cycles of on–off NIR radiation (Figure S8, Supporting
Information). The maximum heating temperature did not show an obvious
decrease, and the ΔTs increased to 36.7, 38.4, and 41.8 °C,
respectively. For the CASMA‐Ag1 group, the regional temperature of the
mouse increased from 30.4 to 53.7 °C under NIR for 10 min
(Figure [103]3k). These results suggest that the designed CASMA‐Ag
hydrogel exhibits excellent NIR photothermal performance, which could
aid in the treatment of bacteria‐infected wounds.
The stability of the AgNPs in the CASMA‐Ag1 hydrogel was assessed by
tracing the characteristic peaks and morphology over time using UV–vis
spectroscopy and TEM. After 20 days of aging time at the storage
temperatures of 37 °C, the UV–vis spectrum of the hydrogel and
morphology of AgNPs remained unchanged (Figure S9, Supporting
Information), indicating the stability of the AgNPs within the
hydrogel.
2.3. Biocompatibility of CASMA‐Ag Hydrogels
Biocompatibility is a critical prerequisite for the applications of
materials in biomedicine.^[ [104]^45 ^] Hence, cytocompatibility and
hemocompatibility experiments were performed to evaluate the
biocompatibility of the CASMA‐Ag hydrogels. As shown in Figure [105]
4a,b, casein‐MA, CASMA‐Ag0, CASMA‐Ag0.5, and CASMA‐Ag1 had high
cytocompatibility, as cell viability was similar to that of the normal
culture. However, free Ag^+ at a concentration of 1 mM killed almost
all cells in the system. Notably, the optical density (OD) values of
the CASMA‐Ag hydrogel (CASMA‐Ag0, CASMA‐Ag0.5, and CASMA‐Ag1) groups
were slightly higher than those of the casein‐MA group in the Cell
Counting Kit‐8 (CCK8) experiment (Figure [106]4c). This may have been
caused by the residual resveratrol, which induces nicotinamide adenine
dinucleotide (NADH) oxidation. As the main component of mitochondrial
respiration, NADH can reduce the water soluble tetrazolium salt WST‐8
to a water soluble dye (formazan) through dehydrogenase in the
mitochondria, which increases absorption at 450 nm.^[ [107]^46 ^] Thus,
the cytotoxicity decreased significantly after Ag^+ was mineralized
in situ to form AgNPs, which were successfully embedded in the casein
hydrogel skeleton, further enhancing biosafety. In addition, increasing
the initial [Ag^+] to 1.5 mM for AgNP formation (CASMA‐Ag1.5 group)
significantly lowered cell viability; thus, we used CASMA‐Ag1 for
subsequent wound treatment experiments.
Figure 4.
Figure 4
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Biocompatible and antioxidant activity of CASMA‐Ag hydrogels. a)
Fluorescence microscopy images of live/dead staining of L929 with
different treatments. b) Quantification of live cell ratio (mean ± SD;
****P < 0.0001; n = 5). c) Cell viability assessed by CCK‐8 assay (OD
at 450 nm) in treated cells (mean ± SD; **P < 0.01, ***P < 0.001, and
****P < 0.0001; n = 3). d) Hemolysis ratios of erythrocytes treated
with different hydrogel extracts (mean ± SD; **P < 0.01, and
****P < 0.0001; n = 3). e,f) ABTS + scavenging activity of different
hydrogels (mean ± SD; **P < 0.01, ***P < 0.001, and ****P < 0.0001;
n = 4). g) ROS levels in L929 cells detected using the DCFH‐DA probe
after exposure to H[2]O[2] and hydrogel extracts. h) Quantitative assay
of intracellular ROS depletion based on fluorescence intensity in L929
cells (mean ± SD; ****P < 0.0001; n = 5). i) O[2] generation in L929
cells detected via Ru(dpp)[3]Cl[2] probe following H[2]O[2] and
hydrogels. j) Quantitative assay of intracellular O[2] depletion by
measuring the fluorescence intensity in L929 cells (mean ± SD;
****P < 0.0001; n = 5).
Hemocompatibility is an important parameter for biomaterials;
therefore, we also tested the hemolytic properties of hydrogels The
hemolysis ratios of CASMA‐Ag0, CASMA‐Ag0.5, and CASMA‐Ag1, and
CASMA‐Ag1.5 were 0.11 ± 0.02%, 1.49 ± 0.13%, 2.50 ± 0.23%, and
7.69 ± 0.39%, indicating that hemolysis rates slightly increased with
increased Ag content. Overall, except for CASMA‐Ag1.5, the hydrogels
exhibited satisfactory hemolysis performance (<5%), with negligible
damage to erythrocytes (Figure [109]4d).^[ [110]^47 ^] Based on the
cytotoxicity and hemolytic tests, CASMA‐Ag1 was used for subsequent
experiments.
In vivo biocompatibility was evaluated by subcutaneous implantation in
mice. We observed that the hydrogel matrix underwent gradual
degradation owing to cellular infiltration overtime, with nearly
complete degradation achieved at 28 d (Figure S10, Supporting
Information). Hematoxylin and eosin (H&E) staining showed no
significant pathological alterations in the major internal organs,
including the hearts, livers, spleens, lungs, and kidneys, of mice with
CASMA‐Ag1 implantation, compared to the control group (Figure S11A,
Supporting Information). Blood biochemical analyses indicated that
liver and kidney functions were not affected by CASMA‐Ag1 implantation
(Figure S11B, Supporting Information). In summary, the CASMA‐Ag1
hydrogel exhibits negligible systemic toxicity, confirming its
outstanding biocompatibility.
2.4. Antioxidant Activity of CASMA‐Ag1 Hydrogel
Overproduction of reactive oxygen species (ROS) during the progression
of an infected wound microenvironment seriously impairs wound
healing.^[ [111]^48 ^] Therefore, wound dressings with antioxidant
activity have a positive effect on the healing of inflammatory wounds.
In theory, resveratrol built into the CASMA‐Ag1 hydrogel can scavenge
free ROS and maintain intracellular metabolism.^[ [112]^39 , [113]^49 ,
[114]^50 , [115]^51 , [116]^52 ^] To evaluate the antioxidant activity
of hydrogels, the scavenging efficiency of the 2,2’‐azinobis
(3‐ethylbenzthiazolin‐6‐sulfonic acid) (ABTS+) stable radical cation
was examined.^[ [117]^53 ^] Figure [118]4e,f shows that, compared with
the PBS group, the pure casein‐MA hydrogel had a certain antioxidant
activity, which is consistent with previous work.^[ [119]^54 ^] The
antioxidant activities of CASMA‐Ag0 and CASMA‐Ag1 were significantly
higher; therefore, the addition of resveratrol enhanced the antioxidant
ability. With the addition of Ag^+, the antioxidant activity of the
hydrogels decreased, indicating that the in situ synthesis of AgNPs
dampened the antioxidant activity of the hydrogels. In conclusion, the
CASMA‐Ag1 hydrogel exhibited excellent antioxidant activity.
We further evaluated the ROS scavenging effect of the CASMA‐Ag1
hydrogel by treating L929 fibroblasts with H[2]O[2] to simulate an
oxidative microenvironment.^[ [120]^55 ^] Intracellular ROS levels were
detected using a ROS probe, 2′,7′‐dichlorodihydrofluorescein diacetate
(DCFH‐DA). L929 cells coincubated with the CASMA‐Ag1 hydrogel emitted
significantly lower green fluorescence than the control group,
indicating its profound ROS scavenging ability (Figure [121]4g,h).
Intracellular changes in [O[2]] were measured using the indicator
Ru(dpp)[3]Cl[2]. As depicted in Figure [122]4i−j, the intracellular
fluorescence of cells treated with the CASMA‐Ag1 hydrogel was
significantly lower than that of cells from the control groups
(p < 0.001), indicating that [O[2]] from cells treated with CASMA‐Ag1
hydrogels was significantly higher than that of cells in the control
groups. Research suggests that the effect of resveratrol on
H[2]O[2]‐mediated death signaling could be attributed to (i) its
ability to maintain a higher intracellular O[2] concentration and (ii)
its ability to block H[2]O[2]‐induced cytosolic acidification, thereby
creating an environment that is nonpermissive for caspase activation
and efficient cell death.^[ [123]^56 ^] The addition of resveratrol not
only enhanced the biomineralizing effect of hydrogels but also
increased hydrogel cytocompatibility and improved the microenvironment
of inflammatory wounds.
2.5. Antibacterial Properties of CASMA‐Ag Hydrogels
The spread plate method was used to assess the antimicrobial
performance of the hydrogels against gram‐negative (Escherchia coli)
and gram‐positive (Staphylococcus aureus) bacteria, which are
responsible for most infections. As presented in Figure [124] 5a−d, in
the PBS group, the total number of bacterial colonies was the same with
or without NIR, indicating that the 3 W 808 nm laser did not affect the
growth of E. coli or S. aureus. On casein‐MA and CASMA‐Ag0 hydrogel
treatments with or without NIR irradiation, the total number of
bacterial colonies was similar.
Figure 5.
Figure 5
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Synergistic antimicrobial efficacy of CASMA‐Ag hydrogels following
sequential photoactivation. a,c) Photographs of E. coli and S. aureus
colonies after different treatments with or without NIR irradiation
(3 W cm^−2, 10 min). b,d) Quantification of bacterial CFUs of E. coli
and S. aureus after different treatments with or without NIR
(mean ± SD; **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 4). e,f)
Fluorescence images of live/dead staining of E. coli and S. aureus
after different hydrogel treatment and their corresponding quantitative
analysis. Green represents live bacteria, and red represents dead
bacteria. (mean ± SD; ****P < 0.0001; n = 3) g) Schematic of S. aureus
collection from wound exudates, along with representative images of
colonies cultured for 48 h following different treatments. h)
Quantitative CFU analysis from (g) (mean ± SD; **P < 0.01,
****P < 0.0001; n = 5).
CASMA‐Ag1 hydrogel decreased the number of viable bacteria of S. aureus
and E. coli by 0.5 orders of magnitude compared to AgNP‐free groups
after 24 h incubation. After NIR irradiation (3 W cm^−2, 10 min), the
photothermal CASMA‐Ag1 hydrogel reduced the number of viable S. aureus
and E. coli by 1.98 and 2.04 orders of magnitude, respectively. The
CASMA‐Ag1 injectable hydrogel, thus, had strong antibacterial
properties under the synergistic effect of sequential UV and NIR
photoactivation.
To further explore the antibacterial properties of the CASMA‐Ag1
hydrogel, live/dead bacteria fluorescence staining by SYTO9/propidiun
iodide (PI) was performed. All live and dead bacteria were stained with
SYTO9 with green fluorescence, whereas red fluorescent (PI) staining
revealed membrane‐damaged bacteria. As shown in Figure [126]5e,
control, casein‐MA, and CASMA‐Ag0, with or without NIR irradiation,
were dominated by green fluorescence and barely showed red
fluorescence. In contrast, red fluorescence was observed in the
CASMA‐Ag1 hydrogel‐treated group, which increased steadily with NIR
treatment. When treated with the CASMA‐Ag1 hydrogel, the viability of
the bacterial cells was significantly reduced to < 33% and < 29% for S.
aureus and E. coli, respectively (Figure [127]5f). Furthermore,
CASMA‐Ag1 + NIR treatment resulted in a > 99% reduction in the
viability of both S. aureus and E. coli. The results demonstrated the
bactericidal effect of the sequentially photoactivated CASMA‐Ag1
hydrogel and the microbicidal effect of each photoactivation step.
2.6. In Vivo Wound Healing of the CASMA‐Ag1 Hydrogel in a Mouse
Full‐Thickness Wound Model
In addition to its effective biocompatibility, the CASMA‐Ag1 hydrogel
had significant antioxidative and antibacterial effects; therefore, we
evaluated the wound‐healing performance of the CASMA‐Ag1 hydrogel
in vivo using a full‐thickness wound model in infected mice. After
infection with S. aureus for 24 h, the animals were randomly assigned
to four groups and treated with PBS (control), casein‐MA, CASMA‐Ag1, or
CASMA‐Ag1 + NIR hydrogel (Figure [128] 6a). The wound tissue fluid was
collected on the second day and evaluated using a coating plate
(Figure [129]5g). The number of bacterial colonies in the CASMA‐Ag1
group was significantly lower than that in the PBS and casein‐MA
groups. The 405 nm light irradiation used for photoactivated gelation
and biomineralization also did not affect the growth of S. aureus
(Figure S12, Supporting Information). CASMA‐Ag1 + NIR exhibited the
highest antimicrobial activity in vivo (Figure [130]5h), indicating the
excellent photothermal antibacterial effect of the hydrogel.
Figure 6.
Figure 6
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CASMA‐Ag1 hydrogel promotes healing of the infected skin wounds in
mice. a) Schematic diagram illustrating the treatment schedule for
C57BL/6 mice. b) Representative images of infected wounds at various
time points following different treatments. c) The variation of wound
area over time for each group. (mean ± SD; *P < 0.05, **P < 0.01,
***P < 0.001, and ****P < 0.0001; n = 5). d) H&E staining showing
histological changes during the wound healing process. e) Masson
staining of the wound tissue on day 10, and f) quantitative analysis of
collagen deposition (mean ± SD; ****P < 0.0001; n = 6).
The healing process of infected wounds in mice treated with various
formulations (PBS (control), casein‐MA, CASMA‐Ag1, and CASMA‐Ag1 + NIR)
was documented by photomonitoring (Figure [132]6b,c). Throughout the
course of the entire treatment, the CASMA‐Ag1 and CASMA‐Ag1 + NIR
groups had significantly higher wound closure rates than the other
groups. On day 10 posttreatment, wounds treated with CASMA‐Ag1 + NIR
achieved a 96.7 ± 1.6% coverage with newly regenerated skin, whereas
wounds in the other groups remained overtly visible. The synergistic
effect of the NIR photothermal response and intrinsic antimicrobial
properties of CASMA‐Ag1 augmented its antimicrobial efficacy and
effectively targeted and eradicated pathogens, thus, accelerating the
wound healing process. Furthermore, the casein hydrogels used in this
study showed decent tissue binding and repair, as reported
previously,^[ [133]^32 ^] which also facilitated wound healing compared
to the control group. Scabs were formed and fell off before healing in
all groups. To demonstrate the fate of the CASMA‐Ag1 hydrogel more
clearly, the fluorescently labeled CASMA‐Ag1 hydrogel was gelled
in situ at the wound site, and the state of the hydrogel was recorded
using a fluorescence stereomicroscope at different time points (Figure
S13, Supporting Information). The fluorescently labeled hydrogel fell
off on about the 7th day.
Pathological staining was used to facilitate a more nuanced analysis of
wound healing (Figure [134]6d). H&E staining on day 7 revealed
substantial inflammatory cell infiltration in the PBS and casein‐MA
groups. A reduction in the number of inflammatory cells was observed in
CASMA‐Ag1 and CASMA‐Ag1 + NIR hydrogel groups. On postoperative day 10,
the fundamental structures of the wound epithelium and dermis were
observed in each group. Masson's trichrome staining results revealed
that both the CASMA‐Ag1 and CASMA‐Ag1 + NIR hydrogel groups
demonstrated denser and more reinforced collagen deposition than the
other groups, while the CASMA‐Ag1 + NIR group exhibited the most
remarkable wound regeneration. Specifically, the epidermis showed
thickening and regularity accompanied by the smallest scar area and
notable fibroblast proliferation (Figure [135]6e,f). Our findings
suggest that the antibacterial and antioxidative effects of the
developed dressings have a potentially positive influence on wound
closure, epidermal resurfacing, and collagen deposition, which
collectively offer substantial support for skin wound repair.
2.7. CASMA‐Ag1 Hydrogel Facilitated Tissue Development and Immune Regulation
During Wound Healing
The potential role of a sequentially photoactivated hydrogel
(CASMA‐Ag1 + NIR) in healing infected wounds was investigated by RNA
sequencing of mouse wound tissues on the 7th day following treatment
with the hydrogel. Principal component analysis (PCA) pointed to a
difference in the transcriptomic landscape between the PBS and
CASMA‐Ag1 + NIR groups (Figure [136] 7a). A volcano plot of the PBS
versus CASMA‐Ag1 + NIR group showed 786 upregulated and 787
downregulated genes (Figure [137]7b). Gene ontology (GO) analysis
indicated that the sequentially photoactivated hydrogel substantially
affected the pathways related to organ and tissue development, cell
growth, and immune processes (Figure [138]7c). Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analysis revealed that the
differentially expressed genes were significantly involved in pathways
associated with immune and inflammatory regulation, including T cell
differentiation, S. aureus infection, B cell and T cell receptor
signaling pathways, and leukocyte transendothelial migration
(Figure [139]7d). We also performed gene set enrichment analysis (GSEA)
of tissue development and inflammation‐related processes
(Figure [140]7e). Tissue development‐related gene sets (HIF‐1 signaling
pathway, VEGF signaling pathway, chemokine signaling pathway, and
cytokine–cytokine receptor interaction) were upregulated in the
CASMA‐Ag1 + NIR group. For the infection and immunity sets, the
enrichment scores of antigen processing and presentation, S. aureus
infection, NF‐κB signaling pathway, Th1 and Th2 cell differentiation,
and Th17 cell differentiation were negative, indicating the
antibacterial and antiinflammatory properties of the CASMA‐Ag1
hydrogel. We further performed a heatmap analysis of the gene set
related to tissue development and immune regulation and performed a
gene–gene interaction network analysis of the differentially expressed
genes with the most significantly expressed genes. Genes associated
with tissue development, such as VEGFa, PPARd, FGF2, and CSF1r, were
significantly upregulated in the CASMA‐Ag1 + NIR group
(Figure [141]7f,g). In contrast, many important genes associated with
immunity and inflammation, including proinflammatory cytokines TNF‐a,
CCL27, CCL12, IL17a, CD4, IL2ra, and LCK, were markedly downregulated
in the CASMA‐Ag1 + NIR group relative to the PBS group
(Figure [142]7h,i). LCK and CD4 are key regulatory nodes of the T‐cell
receptor signaling pathway, and downregulation of CD4 and LCK
expression affects T‐cell activation and signaling.^[ [143]^57 ,
[144]^58 ^] ARG1 and IL‐10, known M2 macrophage markers,^[ [145]^59 ^]
were highly expressed in the CASMA‐Ag1 + NIR group (Figure [146]7h,i).
In addition to defending against pathogens, M2 macrophages clear
apoptotic cells, mitigate inflammation, and promote wound healing.^[
[147]^60 ^]
Figure 7.
Figure 7
[148]Open in a new tab
Wound healing mechanisms of the CASMA‐Ag1 + NIR hydrogel. a) PCA of PBS
and CASMA‐Ag1 + NIR‐treated groups. b) Volcano plot showing differently
expressed genes of PBS versus CASMA‐Ag1 + NIR group. The threshold of
log2 (fold change) was 1.5. c) The GO analysis of differently expressed
genes in PBS versus CASMA‐Ag1 + NIR. d) The KEGG analysis of
differently expressed genes in PBS versus CASMA‐Ag1 + NIR. e) GSEA of
differentially expressed signaling pathways involved in tissue
development, macrophage polarization, and T cell differentiation
following CASMA‐Ag1 + NIR hydrogel treatment. f) Heatmap and g)
gene–gene interaction network of significantly differentially expressed
genes associated with tissue development after CASMA‐Ag1 + NIR hydrogel
treatment. h) Heatmap and i) gene–gene interaction network of
significantly differentially expressed genes associated with the immune
system process after CASMA‐Ag1 + NIR hydrogel treatment. j–m) ELISA
quantification of cytokines in wound tissues: (j) TGF‐β, (k) IL‐6, (l)
TNF‐α, and (m) IL‐10 (mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001,
and ****P < 0.0001; n = 4).
RNA sequencing analysis suggested that the CASMA‐Ag1 + NIR hydrogel
impregnated with AgNPs and resveratrol effectively eliminated bacteria
and concurrently diminished oxidative stress, thereby alleviating
inflammatory responses and aiding tissue repair.^[ [149]^61 , [150]^62
^] To further validate the effect of CASMA‐Ag1 on variations in tissue
development and inflammatory factors, we quantified the amount of
relative cytokines in wound tissues by enzyme‐linked immunosorbent
assay (ELISA) assay: Transforming growth factor‐beta (TGF‐β) is a
multifunctional growth factor that exert pleiotropic effects on wound
healing by regulating cell proliferation and migration,
differentiation, extracellular matrix production, and immune
modulation.^[ [151]^63 ^] Tumor Necrosis Factor‐alpha (TNF‐α) and
interleukin 6 (IL‐6) are proinflammatory factors, and interleukin 10
(IL‐10) is an antiinflammatory factor. We observed the upregulation of
TGF‐β and IL‐10, the downregulation of TNF‐α and IL‐6 in the CASMA‐Ag1
and CASMA‐Ag1 + NIR groups in comparison to the PBS and casein‐MA
groups, with the CASMA‐Ag1 + NIR group demonstrating the most
pronounced differences from the control groups (Figure [152]7j−m).
Immunohistochemical staining of IL‐6 and TNF‐α in wound tissue sections
was performed to study the inflammatory response of different groups.
As shown in Figure S14, Supporting Information, a significant amount of
yellow or brown IL‐6/TNF‐α positive areas were present in the PBS and
casein treatment groups, which were markedly reduced in the CASMA‐Ag1
group and the CASMA‐Ag1 + NIR group. The antiinflammatory effects
observed in the CASMA‐Ag1 + NIR group were mainly attributed to the
combined antimicrobial effects of AgNPs and photothermal therapy, as
well as the inherent antioxidant activity of resveratrol. Collectively,
these actions not only alleviate inflammation but also foster the
transition to the later stages of wound healing, thereby enhancing
overall tissue regeneration. This conclusion is supported by previous
studies that highlighted the multifaceted benefits of integrated
therapeutic approaches.^[ [153]^64 ^] Altogether, our results highlight
the significant antibacterial, antiinflammatory, and wound‐healing
effects of our hydrogel dressings in vivo, with the sequentially
photoactivated group showing particularly promising results.
3. Conclusion
In summary, we developed a sequential photoactivatable antibacterial
CASMA‐Ag hydrogel with an efficient in situ one‐step UV‐induced
biomineralization strategy using casein, resveratrol, and a
photosensitive free radical initiator. Cytocompatibility and hemolysis
assays demonstrated the excellent biocompatibility of the CASMA‐Ag
hydrogel, which is important for wound healing and tissue regeneration.
Owing to the favorable photothermal characteristics and intrinsic
bactericidal properties of the AgNPs, the hydrogel effectively
eradicated both gram‐negative and gram‐positive bacteria. The
incorporation of resveratrol not only accelerated in situ synthesis of
AgNPs but also provided antioxidant and antiinflammatory properties to
the hydrogel, resulting in a significant decrease in the level of ROS
in the cells. In mouse models, the CASMA‐Ag hydrogel with sequential
radiation demonstrated the ability to eradicate bacteria in vivo,
reduce inflammation, and promote wound healing. In the future, we may
translate these findings into large animal studies and clinical
applications with the ultimate goal of developing a multifunctional
wound dressing capable of addressing complex healing challenges. In
conclusion, the sequential photoactivatable in situ biomineralization
strategy is poised to play a pivotal role in antiinfective wound
management and has broad translational prospects.
4. Experimental Section
4.1.
4.1.1.
Biomineralization with Diffident Protein
AgNO[3] was added to a 10% protein solution at a final concentration of
1 mM. 100 μL of each solution was transferred to 96‐well plates and
irradiated under 100 mW cm^−2 and 405 nm UV irradiation for 5 min.
Then, the 96‐well plate was scanned at a wavelength of 410 nm using a
Spark multifunctional microplate reader (TECAN, Swiss Confederation) to
observe AgNP formation.
Preparation of CASMA‐Ag Hydrogels
Casein‐MA (0.2 g) was synthesized (as detailed in the supporting
information) and dissolved in 1.93 mL of PBS, LAP solution was added
(2%, 50 μL), and then Res (100 mg/mL, 10 μL), and various
concentrations of AgNO[3] (0, 100, 200, 300 mM, 10 μL) solution. The
solution was irradiated at 100 mW cm^−2 and 405 nm UV to form hydrogels
and AgNPs, respectively. The hydrogels were denoted as CASMA‐Ag0,
CASMA‐Ag0.5, CASMA‐Ag1, and CASMA‐Ag1.5.
AgNP Adsorption Spectrum
Scanned at wavelengths of 300–700 nm using a Spark multifunctional
microplate reader (TECAN, Swiss Confederation).
XRD
Crystallization of AgNPs in the hydrogel was assessed by XRD (D8
ADVANCE, Bruker, Germany) in reflection mode with a range of
2θ =10°–80°.
TEM
Size and morphology of the AgNPs in the hydrogel were determined by TEM
(JEM 2100 F, JEOL, Japan) at a working voltage of 200 kV using a heated
powder (producer, brand, etc.). All hydrogel samples (CASMA‐Ag0.5,
CASMA‐Ag1, and CASMA‐Ag1.5) were hydrolyzed using protease K to release
AgNPs. The precipitate was retained by centrifugation (12 000 g min^−1,
20 min) and washed with ultrapure water three times.
Particle Size Distribution (PSD)
PSD was determined by dynamic light scattering using a Zetasizer ZCEC
instrument (Malvern Instruments Ltd, UK) with temperature control
(25 °C), which was repeated 10 times for each sample.
XPS
Elemental content in the hydrogel was determined by XPS (Thermo
Scientific K‐Alpha, USA) equipped with a 72 W and 12 kV Al Kα ray
source. Wide‐scanning spectra were measured at a passing energy of
150.00 eV and a step size of 1.00 eV. High‐resolution energy spectra
were collected at an energy of 50.00 eV and a step size of 0.1 eV. The
spectra were fitted and analyzed using Thermo Avantage software v5.948.
SEM
Hydrogels were flash‐frozen in liquid nitrogen, dried in a
freeze‐drier, sprayed with platinum, and observed using a field
emission SEM (SU8010, Hitachi, Japan) equipped with an EDS. Elemental
analysis of CASMA‐Ag1 was performed using EDS.
Photothermal Experiments Under 808 nm NIR
Hydrogel samples (200 μL) were placed into a 48‐well plate under an
808 nm NIR laser (3 W cm^−2, 10 min) with a laser module
(TZ808AD8000‐F100, Anford, China). Simultaneously, an infrared imaging
device (Testo 868, testoAG, Germany) was used to monitor the
temperature changes and obtain photothermal images.
Rheological Property
Rheological properties of hydrogel samples were assessed at 37 °C using
a rheometer (MCR302, Anton Paar, Austria) equipped with a Peltier
element for temperature control and a generator. The CASMA‐Ag solutions
were placed between the plates at 37 °C to fill the gap (0.1 mm). Under
20 mW cm^−2, 405 nm UV irradiation, time sweep oscillatory measurements
were performed at 50 Hz and 1% strain. The point at which the storage
modulus (G′) and loss modulus (G′′) intersect was considered the
gelation point, and the point at which the elastic modulus reached a
plateau was considered complete crosslinking.
Swelling Test
The swelling test was performed as in previous research.^[ [154]^32 ^]
The specific process is described in the Supporting Information.
Stability of the AgNPs in Hydrogel
Hydrogels were covered with aluminum foil and incubated at 37 °C. At
time points of 0, 10, and 20 days, representative batches were
withdrawn for UV–vis and TEM measurements.
Biocompatibility of Hydrogels
Cell biocompatibility and biodegradation were assessed as previously
described.^[ [155]^32 ^] The specific process is described in the
Supporting Information.
Hemolysis Evaluation of Hydrogels
Healthy mice were used to obtain “whole” blood samples through venous
blood collection or retro‐orbital orbitally. Blood was collected in
anticoagulant‐containing tubes with sodium citrate, shaken well, and
centrifuged at 3000 rpm for 15 min to obtain blood cells. The extracts
were prepared by CASMA‐Ag hydrogels in PBS at an extraction rate of
200 mg mL^−1 for 12 h at 37 °C. The different extracts, ultrapure water
(ddH20), and PBS solution were mixed with 20 μL of blood cells each.
The mixture was incubated at 37 °C for 4 h, centrifuged at 3000 rpm for
15 min, and photographed. Absorbance of the supernatant was measured at
542 nm using an enzyme marker.
In Vitro Antioxidant Activity
Antioxidant properties of the hydrogels were measured using an ABTS
[2,2′ ‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid)] assay. The
extracts were prepared in PBS at an extraction rate of 200 mg/ml for
12 h at 37 °C. A Total Antioxidant Capacity Assay Kit was used to
assess the antioxidant activity of the extracts using the ABTS kit
(Beyotime, China). The assay was performed after the incubation of ABTS
with an oxidant to produce ABTS+, which has a relatively stable
blue‐green color measured at 734 nm. The color suppression was compared
with that of Trolox. The assay results are expressed as Trolox
equivalents (mM).
Intracellular ROS (H[ 2 ]O [2 ]) depletion
L929 cells were cultured in hydrogel extracts with 200 μM H[2]O[2] for
24 h. Then, cells were washed with PBS, incubated with the ROS
fluorescence probe H2DCFDA (HY‐D0940, MCE, USA; 5 μM) for 30 min, and
refreshed with PBS, and photographed immediately. Semiquantitative
analysis of intracellular ROS was performed by calculating the
fluorescence intensity.
Intracellular O[ 2 ]evaluation
L929 cells were cultured in hydrogel extracts with 200 μM H[2]O[2] for
24 h. An O[2] probe Ru(dpp)[3]Cl[2] (MX4826, MKBio, China) was added
into the medium at a concentration of 5 μM in the last 4 h. The cells
were washed with PBS and immediately photographed using a fluorescence
microscope (Leica DMI8). The fluorescence of Ru(dpp)[3]Cl[2] was
quenched in the presence of oxygen. Thus, a semiquantitative analysis
of intracellular O[2] was performed by calculating the reduction in
fluorescence.
In Vitro Antibacterial Effect
To evaluate the antibacterial effect with or without NIR irradiation,
200 μL of the hydrogels was added in a 48‐well plate and 50 μL
containing a bacterial suspension of E. coli (ATCC 8099) or S. aureus
(ATCC 25 923) diluted with PBS and was incubated with or without NIR
(808 nm, 3 W cm^−2, 10 min). After a 12 h incubation at 37 °C, the
bacteria were resuspended and diluted 10^−4 times in PBS and then
plated on a Luria–Bertani (LB) agar for 24 h. The colony forming units
per milliliter (CFU/mL) were calculated for each group and compared
with the control. The bactericidal effects of the hydrogels were
verified using the LIVE/DEAD BacLightTM Bacterial Viability Kit
(Sigma‐Aldrich, USA). Briefly, bacterial suspensions from the different
hydrogel treatments were stained with propidium iodide (PI) and SYTO 9
fluorescent dye for 15 min in the dark. The bacteria were then washed
three times with physiological saline to remove excess dye. For each
group, live and dead bacterial cells were observed and visualized using
an inverted fluorescence microscope (IX73; Olympus, Japan). The numbers
of live and dead cells were quantified using ImageJ software.
Mouse S. Aureus‐Infected Cutaneous Wound Model
The performance of the CASMA‐Ag1 hydrogel in wound healing was tested
using an S. aureus‐infected cutaneous wound model. All animal
experiments were approved by the Experimental Animal Management
Committee of Dr. Can Biotechnology (Zhejiang) Co., Ltd. (Reference
Number: 2024DRK0029). Briefly, male C57BL/6 mice (6 weeks old,
19‐21.0 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd.
After anesthetization with pentobarbital (30 mg kg^−1 body weight) and
removal of the dorsal hair, 6 mm diameter full‐thickness skin round
wounds were created, and the suspension of S. aureus (10 μL,
10^8 CFU mL^−1) was immediately dropped onto the wounds to establish
the full‐thickness S. aureus‐infected wound model and randomly divided
into four groups: PBS, casein‐MA, CASMA‐Ag1, and CASMA‐Ag1 + NIR. After
24 h, the control group was treated with PBS, and the other groups were
completely covered with the corresponding hydrogels under 100 mW cm^−2
and 405 nm UV irradiation for 5 min. Then, the wounds in the last group
were irradiated with the NIR laser (3 W cm^−2) for 10 min. After 2 d of
treatment, 1 μL of tissue fluids from wounds with different treatments
were collected and diluted with PBS by 10^4 times. 100 μL diluent was
uniformly coated on an LB medium plate, and the medium plate was placed
in a biochemical incubator at 37 °C for inverted culture. After 24 h,
the number of bacterial cells in the culture medium was determined. The
CFU was calculated using the following formula.
[MATH:
CFU=100×gr
owing colonies :MATH]
Photographs were taken to monitor the wound‐healing process of the
mice. The wound areas were measured using the ImageJ software.
Histology
After 3, 7, and 10 days of treatment, the mice were euthanized, and the
wound skin tissue was collected. All collected samples were fixed in a
4% paraformaldehyde solution and embedded in paraffin to prepare tissue
slides with a thickness of 5 μm, which were stained with hematoxylin
and eosin (H&E), Masson trichrome staining, and for histological
observation using a Leica DM3000 microscope (Germany). In addition,
immunohistochemical staining (TNF‐α and IL‐6) of wound tissue on day 7
was performed. Collagen content (collagen%) was quantified by analyzing
the proportion of aniline blue‐stained area (S[Blue]) in the total
tissue area (S[Tissue]) using the following equation: Pixel areas of
S[Blue] and S[Tissue] were segregated from the original images using
the color threshold function of the ImageJ software.
[MATH:
Collagen%=S<
mrow>BlueSTissue×100% :MATH]
Whole Genome RNA Sequencing
The wound tissues on the 7th day were collected and washed with PBS,
and stored at –80 °C before sequencing. After total RNA extraction
using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA), eukaryotic
mRNA was enriched using oligo (dT) beads. Eukaryotic mRNA sequencing
was performed using an Illumina Novaseq6000 (Gene Denovo Biotechnology
Co., Guangzhou, China). The NEBNext Ultra RNA Library Prep Kit for
Illumina (NEB #7530; New England Biolabs, Ipswich, MA, USA) was used to
construct the cDNA library. The following processes were performed:
total RNA extraction, mRNA enrichment, mRNA fragmentation, random
hexamer‐primed cDNA synthesis, size selection, PCR amplification, and
Illumina sequencing.
ELISA of Cytokines
Wound tissue was collected on day 7 and homogenized to retain the
supernatant. The total protein content in the supernatant was
determined using a bicinchoninic acid (BCA) Protein Assay Kit
(Beyotime, China). The inflammation in the wound was assessed using a
commercial ELISA kit (FANKEWEI, China).
Statistical Analysis
All experiments were performed in triplicate. Quantitative values are
shown as the mean ± standard deviation of at least three independent
experiments. The experimental data were assessed for normality using
the Shapiro–Wilk test before analysis. Comparisons between two groups
were made using a two‐tailed unpaired Student's t‐test, while one‐way
ANOVA was applied to analyze differences among three or more groups,
followed by a Tukey post hoc test. All analyses were completed using
GraphPad Prism 9.0 and Origin 2022. Statistical significance is
indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
Qinchao Zhu and Xuhao Zhou: investigation, methodology,
writing—original draft preparation. Zhidan Wang: investigation,
validation. Daxi Ren: supervision, resources, writing—reviewing and
editing, funding acquisition. Tanchen Ren: formal analysis,
supervision, resources, funding acquisition, writing—reviewing and
editing.
Supporting information
Supplementary Material
[156]SMSC-5-2500026-s001.pdf^ (1.8MB, pdf)
Acknowledgements