Bio-heterojunction-engineered recombinant collagen hydrogel orchestrates multimodal sterilization and immunomodulation for MRSA-infected wound healing
Chongyi Li, Zewen Chang, Yuxi Zhang, Shihong Shen, Lin Liu, Dan Zeng, Daidi Fan
TL;DR
A new hydrogel uses light and chemical reactions to kill drug-resistant bacteria and speed up wound healing without antibiotics.
Contribution
A bio-heterojunction-integrated hydrogel enables triple-modal antibacterial therapy with high efficacy and immunomodulation.
Findings
The hydrogel achieves 99.95% MRSA eradication through photothermal, photodynamic, and POD-like mechanisms.
It accelerates wound closure by 98% in vivo via macrophage polarization and angiogenesis.
The Schottky junction enhances ROS production for synergistic bacterial killing.
Abstract
Multidrug-resistant (MDR) bacterial infections, notably methicillin-resistant Staphylococcus aureus (MRSA), necessitate innovative antibiotic-free wound therapies. Here, a bio-heterojunction-integrated recombinant collagen hydrogel (CAP@MXene/CuTCPP) is designed that synergistically combines photothermal therapy (PTT), photodynamic therapy (PDT), and peroxidase-like (POD-like) activity for multimodal antibacterial action. The borate-bonded dynamically crosslinked hydrogel is composed of polyvinyl alcohol (PVA), 3-aminophenylboronic acid (APBA)-modified recombinant collagen (CF-1552), and MXene/CuTCPP bio-heterojunctions (bio-HJs). Under 808 nm near-infrared (NIR) irradiation, the MXene/CuTCPP bio-HJs exhibit a high photothermal conversion efficiency (44.51%), inducing localized hyperthermia to disrupt bacterial membranes. Importantly, the construction of a Schottky junction at the…
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Taxonomy
TopicsNanoplatforms for cancer theranostics · Wound Healing and Treatments · Hydrogels: synthesis, properties, applications
Introduction
1
The escalating crisis of antimicrobial resistance presents one of the most pressing challenges to modern healthcare systems worldwide [1]. Recent epidemiological data paint an alarming picture, with multidrug-resistant (MDR) bacterial infections already claiming over one million lives annually, a figure projected to increase tenfold by 2050 without intervention [2]. MDR bacteria are more resilient and can easily spread to deeper or unknown wound sites [3]. Among these pathogens, methicillin-resistant staphylococcus aureus (MRSA) has emerged as particularly formidable, exhibiting resistance not only to β-lactam antibiotics but often to multiple antimicrobial classes [4]. Many MDR organisms have developed enhanced virulence mechanisms that enable deeper tissue invasion and biofilm formation, creating physical and metabolic barriers that render infections extraordinarily persistent [5]. The limitations of conventional antibiotic therapies have become increasingly apparent in this context [6]. These shortcomings have driven intense investigation into alternative antimicrobial strategies that employ physical mechanisms or harness host immune responses rather than targeting vulnerable biochemical pathways [7,8].
Phototherapeutic approaches, particularly photothermal therapy (PTT) and photodynamic therapy (PDT) [9], have emerged as promising antibiotic alternatives due to their precise spatiotemporal control, mechanisms less susceptible to resistance development, and capacity for repeated application [10]. These modalities utilize light-activated materials to generate localized hyperthermia or reactive oxygen species (ROS) [11,12]. However, each approach has inherent limitations when applied alone. PTT often requires temperatures that risk collateral tissue damage [[13], [14], [15]], while PDT is constrained by oxygen dependency and limited penetration depth [16]. Emerging evidence suggests that combining these modalities yields synergistic effects, enabling reduced individual doses while maintaining potent antimicrobial activity. However, developing effective delivery platforms for such combined phototherapeutics remains challenging [17]. An ideal system must conform to irregular wound geometries, sustain antimicrobial activity between treatments, support tissue regeneration, and withstand mechanical stresses during use.
Dynamic crosslinked hydrogels offer distinct advantages over conventional dressings and represent promising candidates [18,19]. Unlike static networks, their reversible bonds confer self-healing properties and injectability while preserving structural integrity [20]. Furthermore, their stimuli-responsive nature enables tailored release profiles in response to environmental cues such as pH or enzymatic activity [21]. Collagen-based hydrogels are naturally suited for wound management owing to their inherent biocompatibility and ability to recapitulate critical extracellular matrix components [22]. However, traditional animal-derived collagen carries risks of immunogenicity and pathogen transmission, limiting clinical utility [23]. Recombinant human-like collagen variants overcome these limitations while retaining beneficial biological properties. To address the characteristically poor mechanical properties of collagen hydrogels, composite approaches incorporating synthetic polymers have proven valuable [[24], [25], [26], [27]]. Polyvinyl alcohol (PVA) is particularly effective as a reinforcement agent, forming interpenetrating networks through hydrogen bonding and enhancing overall material toughness, and enabling the prepared hydrogel to possess excellent stability [28].
Integrating functional nanomaterials into the hydrogel matrix further expands the therapeutic potential, enabling more precise treatment of wounds [[29], [30], [31], [32]]. Among these, two-dimensional materials have attracted significant attention due to their exceptional surface-to-volume ratios and unique electronic properties [33]. MXenes, a rapidly expanding family of transition metal carbides and nitrides, demonstrate considerable promise for biomedical applications [34,35], including biosensors [36], PTT [37], drug delivery [38], among others. Their surfaces feature abundant hydrophilic functional groups (-OH, -O, -F) [39], facilitating composite formation via hydrogen bonding, electrostatic interaction and π-π stacking [40,41]. The most extensively studied variant, Ti_3_C_2_T_x_ MXene, exhibits outstanding photothermal conversion efficiency [42], excellent electrical conductivity that facilitates charge separation [43], and intrinsic antibacterial activity through physical membrane disruption [[44], [45], [46]]. However, unmodified MXene nanosheets suffer from rapid oxidation under physiological conditions and limited capacity for reactive oxygen species generation [47].
To address these limitations while capitalizing on MXene's strengths, heterojunction engineering was pursued with organic semiconductors [48]. Porphyrin, termed "the pigment of life" and ubiquitous in nature, represents ideal partner materials [49]. Porphyrins and their metal complexes possess extended π-conjugation systems, strong visible-light absorption [50], and ROS generation capability under illumination [51]. The change in Cu ion valence endowed CuTCPP with peroxidase-like ·OH production capability, exhibiting optimal peroxidase activity under the acidic conditions typical of bacterially infected wounds. Owing to their unique electronic and optical properties, porphyrins find applications across diverse scientific disciplines [52], including photosynthesis, PDT agents [53], biological imaging probes [54], chemical sensors [55], conductive organic materials, and luminescent materials [56]. Copper(II)-meso-tetra(4-carboxyphenyl)porphyrin (CuTCPP) was selected for its catalytic metal centers, surface-binding carboxyl groups, and planar molecular structure promoting strong interfacial contact. Metal ion coordination enhances photoexcitation efficiency, absorption characteristics, and biocompatibility [57]. Prior studies have explored composites such as g-C_3_N_4_/CuTCPP [58], TiO_2_/CuTCPP [59], MXene/BiOBr [60]. While similar bio-heterojunctions have shown promise in photocatalytic applications, their integration into antimicrobial hydrogels remains largely unexplored.
Given the aforementioned considerations, an innovative bio-heterojunction-integrated recombinant collagen hydrogel (CAP@MXene/CuTCPP, hereafter referred to as CAP@MX/CT) consisting of MXene/CuTCPP bio-heterojunctions (bio-HJs) within a dynamically crosslinked network was conceived and fabricated. In this approach, the bio-HJ establishes a precise Schottky junction at the interface, creating a built-in electric field that functions as a molecular electron pump to drive directional electron transfer. This mechanism effectively suppresses charge recombination to significantly amplify ROS quantum yields and POD-like activities [61]. Under NIR irradiation, the resultant localized hyperthermia enhances bacterial membrane permeability, facilitating the rapid internalization of generated radicals to achieve near-total MRSA eradication without inducing resistance. Simultaneously, this multimodal system accelerates wound healing by orchestrating an immunomodulatory transition from M1 to M2 phenotypes and promoting robust angiogenesis, thereby resolving the long-standing sterilization-regeneration trade-off in chronic infection management [62]. Both in vitro and in vivo evaluations authenticate this interface-driven paradigm for the advancement of multifunctional bioactive materials (Scheme 1).Scheme 1. Synthesis and performance of CF-1552-APBA-PVA@MXene/CuTCPP (CAP@MXene/CuTCPP) hydrogel. (a) Schematic diagram of the synthesis of MXene/CuTCPP bio-heterojunctions (MXene/CuTCPP bio-HJs) and CAP@MXene/CuTCPP hydrogel, (b) Mechanisms underlying the antibacterial activity and wound healing promotion of the CAP@MXene/CuTCPP hydrogel.Scheme 1
Results and discussion
2
Synthesis and characterization of MXene/CuTCPP bio-HJs
2.1
The microstructure of MXene, CuTCPP, and MXene/CuTCPP bio-HJs was characterized by SEM. Intercalated MXene exhibited a typical accordion structure (Fig. 1a), while CuTCPP showed multilayer sheet morphology formed by Cu^2+^ coordination (Fig. 1b). After modification, MXene/CuTCPP bio-HJs retained structural similarity to MXene (Fig. 1c). TEM analysis of MXene/CuTCPP bio-HJs (Fig. 1d) revealed a lattice spacing of 1.47 nm, corresponding to the (002) crystal plane of MXene, indicating that CuTCPP modification does not alter MXene's crystal structure. Element mapping of MXene/CuTCPP bio-HJs (Fig. 1e–k) showed Ti and C from MXene alongside Cu and N from CuTCPP, confirming successful CuTCPP loading on MXene. FT-IR spectra of TCPP and CuTCPP (Fig. 1m) displayed N-H stretching at 964 cm^−1^, N-Cu stretching at 999 cm^−1^, and N-H stretching at 3315 cm^−1^, indicating successful Cu^2+^ coordination. After etching and intercalation of Ti_3_AlC_2_, the XRD pattern showed a 2θ shift in the original (002) peak, disappearance of the Al layer peak at 39°, and reduced impurity peaks, confirming formation of 2D Ti_3_C_2_T_x_. Peaks for CuTCPP at (110), (002), and (004) planes indicated its crystal structure. Diffraction peak of MXene/CuTCPP bio-HJs (Fig. 1l) originated from both MXene and CuTCPP, with decreased intensity confirming their successful combination.Fig. 1. Synthesis and characterization of CuTCPP, MXene and MXene/CuTCPP bio-HJs. (a) Scanning electron microscopy of MXene, (b) Scanning electron microscopy of CuTCPP, (c) Scanning electron microscopy of MXene/CuTCPP bio-HJs, (d) Transmission electron microscopy of MXene/CuTCPP bio-HJs, (e-k) Elemental mapping of MXene/CuTCPP bio-HJs, (l) The XRD patterns of MXene and MXene/CuTCPP bio-HJs, (m) FT-IR spectra of TCPP and CuTCPP, (n) XPS survey spectrum, (o-q) high-resolution XPS spectra of the C 1s, O 1s, Cu 2p for MXene/CuTCPP bio-HJs.Fig. 1
XPS characterized the molecular structure and valence states of CuTCPP and MXene/CuTCPP bio-HJs (Fig. 1n). Analysis of C, N, O, F, Ti, and Cu elements in MXene/CuTCPP bio-HJs revealed weaker F and Ti intensities compared to pure MXene, alongside a Cu signal, indicating successful CuTCPP immobilization on MXene. The high-resolution C 1s spectrum (Fig. 1o) showed emerging C-N bonds in MXene/CuTCPP bio-HJs, along with O=C=O and C-C bonds. In the O 1s spectrum (Fig. 1p), peaks were assigned to C=O, C-O, and O-H groups. The Cu 2p spectrum (Fig. 1q) exhibited peaks at 952 eV and 932 eV for Cu(I), and at 954 eV and 934 eV for Cu(II), confirming Cu presence in MXene/CuTCPP bio-HJs. Notably, no Ti-bonding peaks appeared in the O 1s spectrum, further verifying successful synthesis of MXene/CuTCPP bio-HJs.
Synthesis and characterization of CAP@MXene/CuTCPP hydrogel
2.2
CF-1552-APBA and PVA were the main hydrogel components. CF-1552-APBA was obtained via amidation of CF-1552 with APBA using NHS and EDC. FT-IR analysis confirmed the modification (Fig. 2a), showing out-of-plane bending vibrations of aromatic C-H bonds at 770 cm^−1^ and characteristic benzene ring absorption at 1550 cm^−1^. Peaks for m-substituted benzene at 793 cm^−1^ and 702 cm^−1^, along with altered -CO-NH- intensity at 1630 cm^−1^, indicated successful APBA grafting onto CF-1552. ^1^H NMR further confirmed synthesis, with characteristic phenylboronic acid peaks at 7.0-8.0 ppm (Fig. 2b). Hydrogel preparation (Fig. 2g) involved mixing 12.5 wt% CF-1552-APBA and 12.5 wt% PVA to form a gel, with subsequent incorporation of MXene/CuTCPP bio-HJs (1 mg/mL) enhancing cross-linking. FT-IR analysis of the CAP hydrogel (Fig. 2c) revealed a phenylborate bond peak at 1543 cm^−1^, confirming bond formation during cross-linking.Fig. 2. Synthesis and characterization of CAP@MXene/CuTCPP. (a) FT-IR spectra of APBA, CF-1552, CF-1552-APBA, (b) ^1^H NMR hydrogen spectra of CF-1552, CF-1552-APBA, (c) FT-IR spectra of CF-1552-APBA, PVA, CAP hydrogel, (d) Swelling rate of CAP, CAP@MX/CT hydrogels, (e) The degradation profiles of the hydrogel in PBS with different pH values (5.0, 7.4, and 8.5) over 14 days, (f) The cumulative release kinetics of Cu^2+^ ions from the hydrogel under different pH values (5.0, 7.4, and 8.5) over 14 days, (g) Schematic diagram of cross-linking of hydrogel, (h) Self-healing properties of hydrogel, (i) Schematic diagram of adhesion of hydrogel, (j) The adhesion mechanism of the CAP@MX/CT hydrogel, (k) The mechanism of the lap shear test for the adhesion of hydrogels to porcine skin, (l) Viscoelasticity (energy storage modulus Gʹ and loss modulus Gʺ) of CAP, CAP@MX/CT hydrogels, (m) Cyclic strain sweep measurement of single crosslinked CAP@MX/CT hydrogel, (n) Adhesion strength of hydrogels. (Data are presented as mean ± standard deviation (SD). n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 2
Hydrogel pore structure is critical for dressing applications, facilitating exudate absorption while maintaining gas permeability. SEM analysis revealed uniform porous architectures in both CAP and CAP@MX/CT hydrogels (Fig. S1). This interconnected network endowed the hydrogels with high fluid uptake capacity. Moreover, MXene/CuTCPP bio-HJ incorporation enhanced swelling capacity by 60% compared to the CAP hydrogel (Fig. 2d). To evaluate the drug delivery capability, the degradation behavior and Cu^2+^ release kinetics were monitored over a 14 day period (Fig. 2e–f). The hydrogel exhibited a smart, pH-responsive degradation profile attributed to the dynamic borate ester cross-linkages. Under acidic conditions (pH 5.0), typical of bacterial infection sites, the hydrogel degraded rapidly, triggering a burst release of Cu^2+^ ions to facilitate immediate sterilization. This mechanism ensures precise, on-demand delivery of the antibacterial agents when they are most needed. Conversely, under neutral conditions (pH 7.4) characteristic of the healing phase, the hydrogel maintained better structural integrity with a sustained release profile. Crucially, the cumulative concentration of Cu^2+^ even under the most accelerated degradation plateaued at approximately 38 μmol/L, which is significantly below the systemic toxicity threshold, thereby ensuring long-term bioactivity while maintaining high biosafety [63].
The hydrogel exhibited macroscopic self-healing capability (Fig. 2h), with the CAP@MX/CT hydrogel self-healing within 20 s to restore its original integrity. As shown in Fig. 2i, the hydrogel adhered to diverse substrates, including rubber, plastic, skin, and glass, demonstrating intrinsic adhesiveness. Structural stability of the CAP@MX/CT hydrogel was assessed using a rheometer. Storage moduli of CAP and CAP@MX/CT hydrogels exceeded corresponding loss moduli, indicating structural stability (Fig. 2l). Rapid recovery of G′ and G″ in continuous cyclic step-strain tests confirmed excellent self-healing properties, consistent with swift reformation of phenylborate bonds after external disruption (Fig. 2m). A lap shear test using porcine skin (Fig. 2n) quantified adhesion strength, revealing that MXene/CuTCPP incorporation doubled the adhesive force.
Photothermal performance evaluation of CAP@MXene/CuTCPP hydrogel
2.3
MXene/CuTCPP bio-HJs exhibit superior photothermal performance due to the synergistic effect of both components [64]. They display stronger UV absorption than MXene or CuTCPP alone, enhancing heat generation under 808 nm laser irradiation (Fig. 3a). As laser power increased from 0.2 to 0.8 W/cm^2^ (Fig. 3b), hydrogel temperature rose from 48.3 °C to 83.3 °C after 10 min. Considering skin tolerance, temperature levels, and phototoxicity minimization, 0.5 W/cm^2^ was selected for subsequent experiments.Fig. 3. Photothermal properties and peroxidase activity evaluation of CAP@MXene/CuTCPP. (a) UV-vis spectrogram of MXene/CuTCPP bio-HJs, (b) Temperature variation of CAP@MX/CT under laser power of 0.2 W/cm^2^, 0.5 W/cm^2^ and 0.8 W/cm^2^, (c) Temperature changes of CAP hydrogels containing MXene or different concentrations of MXene/CuTCPP bio-HJs under near-infrared laser irradiation at 808 nm (0.5 W/cm^2^), (d) Temperature changes of CAP@MX/CT under near-infrared laser on/off cycles for 5 times, (e-f) Relationship between linear time data obtained during cooling period of CAP@MX/CT hydrogel and ln(θ), (g) Infrared images of CAP hydrogel and CAP@MX/CT hydrogel irradiated by 808 nm (0.5 W/cm^2^) near-infrared laser for 10 min, (h) Peroxidase-like activity of CAP and CAP@MX/CT using TMB as a substrate, (i-l) Effect of Temperature, pH, MXene/CuTCPP bio-HJs concentration and H_2_O_2_ concentration on POD activity of MXene/CuTCPP bio-HJs nanosheets, (m) Comparison of peroxidase-like activity among different hydrogel groups (Data are presented as mean ± SD. n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 3
Different concentrations of MXene/CuTCPP bio-HJs were incorporated into the hydrogel (Fig. 3c). Photothermal performance increased with concentration. At the same laser power density, temperature rises were compared for hydrogels doped with equivalent concentrations of MXene and MXene/CuTCPP bio-HJs. Under 10 min irradiation, the temperature of the MXene/CuTCPP bio-HJ-containing hydrogel was significantly higher than that of the MXene-containing hydrogel. As shown in Fig. 3f, CAP@MX/CT exhibited stable photothermal performance over five heating-cooling cycles. Based on linear relationships from cooling curves, the photothermal conversion efficiency was calculated as 44.51% (Fig. 3e and f).
POD activity of CAP@MXene/CuTCPP hydrogel
2.4
The MXene/CuTCPP biological HJs are formed by depositing CuTCPP on the surface of MXene. The coordination of copper ions enhances the biological activity and light absorption performance of TCPP [65]. Meanwhile, under the catalytic action of copper ions, a type of Fenton-like reaction occurs, exhibiting similar enzymatic activity to that of peroxidase [66]. These biological HJs generate ROS in the presence of H_2_O_2_, disrupting the redox balance and structural integrity of bacteria. Meanwhile, we incorporate MXene/CuTCPP into the hydrogel structure to enhance its therapeutic function for wounds, making the CAP@MX/CT hydrogel not only effective in combating multiple drug-resistant bacteria at the wound site but also excellent in promoting wound recovery [67].
The peroxidase-like activity of CAP@MX/CT exhibited distinct environmental responsiveness. As shown in Fig. 3i, the activity peaked at pH 4.0, which coincides with the acidic microenvironment (pH 4-6) typical of MRSA-infected wounds. This ensures site-specific ·OH generation, enhancing bactericidal efficacy while minimizing damage to healthy tissues. Moreover, enzymatic activity reached its maximum around 40 °C (Fig. 3j), confirming its robust performance under physiological fever conditions or during NIR-induced photothermal heating. The catalytic acceleration at elevated temperatures follows the Arrhenius law, where PTT-generated heat lowers the activation energy for the POD-like reaction. The concentration-dependent plateaus observed in Fig. 3k and l reflect typical Michaelis-Menten kinetics, indicating the saturation of active sites on the nanosheets.
The colorimetric absorption spectra of the hydrogel-TMB system are presented in Fig. 3m. Upon the addition of H_2_O_2_, CAP@MX/CT exhibited the highest absorbance at 652 nm, indicating its superior peroxidase-like activity compared to other cohorts. Notably, the CAP@MXene group showed negligible catalytic activity under identical conditions, confirming that the introduction of CuTCPP and the formation of a Schottky junction are pivotal. This heterostructure facilitates efficient interfacial electron transfer, achieving a catalytic efficiency that significantly exceeds the additive effects of single components. Furthermore, the detectable weak peak observed even in the absence of H_2_O_2_ reveals the system's intrinsic oxidase-like activity, ensuring sustained antimicrobial potential within the wound microenvironment.
In vitro antibacterial activity of CAP@MXene/CuTCPP hydrogel
2.5
Under 808 nm near-infrared irradiation, the CAP@MX/CT hydrogel exhibits potent antibacterial activity through a triple-action mechanism that not only effectively combats wound infections but also helps prevent the development of bacterial resistance [68]. This synergistic approach makes it particularly effective during the critical antibacterial phase of wound healing. The photothermal conversion capability achieves 44.51% efficiency, generating localized hyperthermia that physically disrupts bacterial membranes while simultaneously enhancing the material's peroxidase-like catalytic activity. This thermal effect synergizes with photodynamic action to produce ROS, including ^1^O_2_, ·O_2_^−^, with the MXene/CuTCPP interface further amplifying ROS generation through photo-accelerated electron transfer that yields additional ROS radicals. Furthermore, the nanosheet structure of the bio-heterojunctions contributes a physical antibacterial dimension by mechanically penetrating bacterial membranes through a nano-knife effect.
To validate the synergistic advantage of the bio-HJs, supplemental decoupling assays were performed. The results revealed that the MXene/CuTCPP bio-HJs achieved a significantly higher eradication effect (e.g., 0.38% MRSA viability, Fig. S2), compared to the MXene-only (2.65%) or CuTCPP-only (0.75%) groups (Fig. S2). This provides convincing evidence that the bactericidal potency is driven by the specific Schottky junction structure, which optimizes interfacial charge transfer to amplify ROS generation far beyond simple additive effects. Antibacterial efficacy against S. aureus, E. coli and MRSA followed a clear concentration-dependent trend (Fig. S3). Quantitative analysis (Fig. S3) integrated with cytotoxicity results guided the selection of 1 mg/mL as the optimal concentration for subsequent experiments.
The survival rate of bacteria in the CAP@MX/CT hydrogel antibacterial assay is shown in Fig. 4b by the plate counting method according to Fig. 4a. The survival rate of CAP hydrogels without nanoparticles showed no significant change compared to the Control group (Group I). In contrast, the doped with MXene/CuTCPP bio-HJs exhibited enhanced antibacterial ability. The experiment was divided into six groups: Ⅰ. Control, Ⅱ. CAP, Ⅲ. CAP@MXene/CuTCPP, Ⅳ. CAP@MXene/CuTCPP + NIR, Ⅴ. CAP@MXene/CuTCPP + H_2_O_2_, Ⅵ. CAP@MXene/CuTCPP + H_2_O_2_+NIR.Fig. 4. In vitro antibacterial activity of CAP@MXene/CuTCPP hydrogel. (a) Colony photos of E. coli, S. aureus, MRSA under different treatment schemes and corresponding scanning electron microscope images, (b) Determination of growth activity of different hydrogels against E. coli, S. aureus and MRSA by plate counting method (c) Crystal violet stain of biofilm under different treatment methods, (d) Laser confocal images of biofilm under different treatment methods, (e) Fluorescence microscopic images of detection of intracellular ROS in MRSA by DCFH-DA staining across treatment groups. (Data are presented as mean ± SD. n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 4
Groups treated with NIR only (IV) and the H_2_O_2_ only (V) exhibited moderate antibacterial activity, with survival rates decreasing to 24.15% and 19.57% for S. aureus, 36.36% and 24.24% for E. coli, and 24% and 12% for MRSA, respectively. Under combined NIR and H_2_O_2_ stimulation (VI), PTT, PDT, and POD-like activities synergized, as elevated PTT-induced temperatures disrupted bacterial structures while enhancing ROS generation. These ROS penetrated membranes, destroying internal cellular components and proteins and leading to near-complete inactivation (99.98% for S. aureus, 99.5% for E. coli, and 99.95% for MRSA). SEM analysis revealed morphological changes. In the control (I) and CAP (II) groups, S. aureus showed smooth, spherical forms, while E. coli displayed rod shapes. Exposure to the CAP@MX/CT hydrogel (III) induced shrinkage and folding. Combined NIR and H_2_O_2_ treatment (VI) caused further damage via heat and ROS attack, ultimately disrupting bacterial morphology irreversibly under full synergistic action. MRSA, as multidrug-resistant bacterium, followed the same trend in SEM, progressing from intact cells to complete destruction based on the antimicrobial strategy. These results highlight the CAP@MX/CT hydrogel's significant potential for treating wound infections caused by drug-resistant bacteria.
Bacteria often form structured, defensive biofilms that serve as physical barriers against antimicrobial agents. The effect of CAP@MX/CT on MRSA biofilms was evaluated using crystal violet staining (Fig. 4c). The lightest crystal violet color under combined NIR and H_2_O_2_ treatment (Group VI) indicated the strongest anti-biofilm effect, consistent with antibacterial results. To visualize biofilm destruction directly, the 3D structure of MRSA biofilms after live/dead staining was reconstructed using Confocal Laser Scanning Microscopy (CLSM) (Fig. 4d). Similar to the crystal violet findings, CLSM images showed minimal green (live) and extensive red (dead) fluorescence under NIR + H_2_O_2_ conditions (Group VI), confirming profound biofilm disruption.
Regarding the spatiotemporal localization of ROS at the bacterial level, we employed the cell-permeable fluorescent probe DCFH-DA combined with fluorescence microscopic to track the spatial evolution of oxidative stress. As illustrated in Fig. 4e, while single-mode treatments showed only negligible peripheral signals, the synergistic CAP@MX/CT group displayed intense green fluorescence localized precisely within the bacterial cytoplasm of MRSA. These images validate our proposed antibacterial model where PTT-induced hyperthermia acts as a physical gateway by increasing bacterial membrane permeability, allowing the concurrently generated ROS from the PDT and POD pathways to rapidly penetrate and accumulate intracellularly, resulting in optimal antibacterial performance.
Biocompatibility of CAP@MXene/CuTCPP hydrogel
2.6
Good biocompatibility is essential for the clinical application of hydrogel dressings. This property was evaluated using CCK-8 assays and AO/EB staining. As shown in Fig. 5a, AO/EB-stained L929 human fibroblasts maintained a normal spindle-like morphology, exhibiting strong green fluorescence (indicative of live cells) with negligible red fluorescence (from dead cells). The quantitative cell viability results (Fig. 5c) further confirmed that co-culture with either CAP or CAP@MX/CT hydrogels did not lead to significant cytotoxicity, as cell survival rates showed no notable difference from the control group, with all groups maintaining viability above 95%. A cell scratch assay was performed to assess migratory capacity. Representative images (Fig. 5b) demonstrated progressive wound closure, and the quantified migration rate for the CAP@MX/CT group (Fig. 5d) was significantly higher than that of the CAP group and was 26.3% greater than the control, underscoring its potential to enhance wound healing.Fig. 5. Biocompatibility of CAP@MXene/CuTCPP hydrogel. (a) AO/EB fluorescence staining diagram of CAP and CAP@MX/CT hydrogel, (b) Scratch diagram of cell migration of CAP and CAP@MX/CT hydrogel, (c) Cell survival rate of CAP and CAP@MX/CT hydrogel, (d) Cell mobility of CAP and CAP@MX/CT hydrogel, (e) The number of nodes and (f) The angiogenesis status of CAP and CAP@MX/CT hydrogels, (g) Fluorescence expression of CD86 and CD206 in RAW 264.7 cells after co-culture with CAP and CAP@MX/CT hydrogels, (h) Hemolysis experiment of hydrogel, (i) The content of IL-10 anti-inflammatory factor, (j) The content of IL-6 pro-inflammatory factor. (Data are presented as mean ± SD. n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 5
Furthermore, its pro-angiogenic capability was evaluated. The ability of the CAP@MX/CT hydrogel to promote the formation of vascular-like structures was confirmed by an in vitro tube formation assay, as observed under microscopy and quantified through node analysis (Fig. 5e–f). Blood compatibility was assessed via an in vitro hemolysis test (Fig. 5h). Both CAP and CAP@MX/CT hydrogels demonstrated excellent hemocompatibility, exhibiting hemolysis rates below 5% compared to the negative control (deionized water), thereby meeting the ASTM standard for biomedical materials.
The immunomodulatory function was subsequently investigated. Analysis of macrophage polarization markers (Fig. 5g) revealed that in the Lipopolysaccharide (LPS) group, most cells are in the M1 type pro-inflammatory polarization state. In contrast, the CAP@MX/CT group showed a distinct polarization profile, characterized by low expression of the pro-inflammatory M1 marker (CD86) and high expression of the anti-inflammatory M2 marker (CD206). This shift indicates that the hydrogel promotes the transition from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype. Consistent with this finding, cytokine secretion analysis showed that the level of the anti-inflammatory cytokine IL-10 was lowest in the LPS-stimulated group (Fig. 5i). While the CAP hydrogel had no significant effect, the CAP@MX/CT hydrogel slightly elevated IL-10 levels. Conversely, the level of the pro-inflammatory cytokine IL-6 was highest in the LPS group (Fig. 5j). The CAP hydrogel did not markedly alter IL-6 levels, whereas the CAP@MX/CT hydrogel reduced them to a level even slightly below that of normal, unstimulated cells. Together, these results suggest that the CAP@MX/CT hydrogel effectively modulates RAW264.7 macrophage polarization toward an anti-inflammatory phenotype, thereby suppressing the inflammatory response.
In vivo antibacterial assessment and wound regeneration assessment of CAP@MXene/CuTCPP hydrogel
2.7
To evaluate in vivo therapeutic efficacy, a full-thickness MRSA-infected wound model was established in SD rats (Fig. 6a). Following infection on day −1, treatments commenced on day 0, and the wound closure trajectory was monitored (Fig. 6b). Bacterial clearance was quantitatively assessed via colony counting from wound tissue samples on days 3 and 7. Notably, the synergistic treatment group (Group VI) achieved a near-total elimination of pathogens by day 7, showing significantly lower colony numbers than all other cohorts and confirming potent in vivo antimicrobial activity (Fig. 6c). While the control group exhibited persistent abscesses and delayed healing by day 11, Group VI demonstrated the most accelerated closure rate, reaching 98.81% (Fig. 6e). These results, highlighting the clear superiority of the bio-heterojunction system over the commercial Hydrosorb® dressing, provide a robust pharmacological foundation for the potential clinical translation of the CAP@MX/CT hydrogel.Fig. 6. In vivo antibacterial assessment and wound regeneration assessment of CAP@MXene/CuTCPP hydrogel with added clinical standard antibacterial additives. (a) Model diagram of treatment mode of MRSA bacterial infection wound, (b) Representative photos of wound healing process under different treatment methods, (c) Photographs of colonies at the wound on the day 3 and day 7, (d) infrared images of MXene/CuTCPP bio-HJs hydrogel irradiated by 0.5 W/cm^2^ 808 nm laser for 10 min, (e) Comparison chart of wound healing rate under different treatment methods. (Data are presented as mean ± SD. n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 6
To ensure localized safety, the hydrogel was topically applied strictly to the wound bed, and the collimated 808 nm laser beam was restricted to the infected area. Utilizing real-time infrared thermography (Fig. 6d), the bactericidal temperature (>50 °C) was maintained at the site while ensuring a steep thermal gradient that kept adjacent healthy tissues within a safe physiological range (<42 °C). Quantitative assessment confirmed that the 808 nm laser penetrates the hydrogel with minimal attenuation, as the bottom temperature of a 5 cm thick gel reached 56.2 °C under NIR irradiation (Fig. S6). This exceptional penetration depth, which significantly exceeds the actual wound thickness in the rat model (1.5-2.5 mm), guarantees uniform and sufficient photothermal sterilization across the entire infected area without thermal loss.
In vivo histopathological evaluation of CAP@MXene/CuTCPP hydrogel
2.8
Histopathological evaluation of wound healing was performed on days 3 and 7 post-treatment. Hematoxylin and eosin (H&E) staining on day 3 (Fig. 7a) revealed intensive inflammatory infiltration and severe tissue defects in control groups. Notably, the group receiving combined NIR and H_2_O_2_ stimulation exhibited the most pronounced reduction in inflammatory cells. By day 7, all groups showed diminished inflammatory cell presence. The most effective healing occurred in the group receiving combined NIR and H_2_O_2_ treatment, as evidenced by fibroblast migration into the wound bed, continuous re-epithelialization, and notable hair follicle regeneration. Quantitative (Fig. 7b–c) showed that epidermal thickness on day 7 was lower than on day 3. This transition reflects the resolution of inflammatory edema and the maturation of the neo-epidermis, with the all-material treated group achieving the most physiological structure. Masson's trichrome staining (Fig. 7a–d-e) further confirmed that the NIR + H_2_O_2_ group possessed the highest collagen volume fraction and most organized fiber bundles, underscoring the hydrogel's pivotal role in structural restoration.Fig. 7. In vivo histopathological and immunomodulatory evaluation of CAP@MXene/CuTCPP hydrogel. (a) H&E and Masson stain pictures of wounds on day 3 and day 7 under different treatment methods, (b-c) Day 3 and Day 7 epidermal thickness, (d-e) Day 3 and Day 7 collagen volume fraction, (f) Immunofluorescence plots of CD86, CD206, VEGF, and IL-6 on day 7, (g-j) CD86, CD206, VEGF and IL-6 relative intensity. (Data are presented as mean ± SD. n = 3. p values are assessed by one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).Fig. 7
To elucidate the underlying mechanism, immunofluorescence was performed to evaluate macrophage polarization (via the expression of CD86 and CD206) and the levels of the pro-inflammatory cytokine IL-6 and vascular endothelial growth factor (VEGF). The NIR + H_2_O_2_ group demonstrated the strongest VEGF signals and a significant shift from the pro-inflammatory M1 phenotype (lowest CD86 and IL-6) to the reparative M2 phenotype (highest CD206) (Fig. 7f–j). Mechanistically, this immunomodulatory effect is driven by the ROS-scavenging capacity of the MXene component, which alleviates the oxidative stress blockade common in chronic wounds, thereby permitting the phenotypic switch essential for accelerated tissue regeneration.
Meanwhile, the systemic biosafety of the CAP@MX/CT platform was validated. H&E staining of major organs on day 7 (Fig. S4a) and hematological analysis on day 11 (Fig. S4b–e) showed no pathological changes or markers outside normal physiological ranges. These combined results confirm that the hydrogel provides a safe and highly effective active-healer environment for MRSA-infected wounds.
Finally, it is noteworthy that rodents possess a unique panniculus carnosus layer that facilitates wound closure via mechanical contraction, a process distinct from human re-epithelialization. To ensure the translational relevance of our findings, we prioritized comparative efficacy within our experimental design. Since all cohorts (including the clinical control Hydrosorb®) were subjected to identical contraction forces, the systematic improvement in the CAP@MX/CT group is rigorously attributable to the material's intrinsic bioactivity rather than baseline contraction. The observation of robust neo-epithelialization and organized collagen deposition confirms that the bio-heterojunction promotes authentic biological regeneration rather than mere mechanical wound shrinkage.
Mechanism of photothermal and photodynamic effect on MXene/CuTCPP bio-HJs
2.9
MXene/CuTCPP bio-HJs were prepared by a hydrothermal method involving MXene and CuTCPP, with the primary reaction occurring between surface -OH groups on MXene and CuTCPP. Generated ROS types were identified using electron spin resonance (EPR) spectroscopy. As demonstrated in Fig. 8a–c, ·O_2_^−^, ^1^O_2_ and ·OH were detected, with their production increasing over time under NIR laser irradiation. As shown in the data, MXene/CuTCPP bio-HJs exhibits prominent signals corresponding to ·O_2_^−^ and ^1^O_2_ generation. In contrast, the signal for ·OH is comparatively weak. This is attributed to the limited capture of ·OH generated by the MXene/CuTCPP bio-HJs itself. Notably, the primary production of ·OH by this material occurs under external stimulation by hydrogen peroxide. We then monitored the changes in the characteristic peaks of DPBF (398 nm), NBT (560 nm), and TMB (652 nm), and presented the results in Fig. S5a–c,confirmed this point at the same time. Upon MXene-CuTCPP contact, the electron density profile of MXene/CuTCPP bio-HJs (Fig. 8g) reveals a distinct charge density distribution, with MXene and CuTCPP bound via π-π conjugation.Fig. 8. Antibacterial mechanism of MXene/CuTCPP bio-HJs PDT. (a-c) EPR spectra of ·O_2_^−^, ^1^O_2_ and ·OH of MXene, CuTCPP and MXene/CuTCPP bio-HJs, (d-f) DOS of CuTCPP, MXene, and MXene/CuTCPP bio-HJs, (g) Charge densities of MXene/CuTCPP bio-HJs, (h) Transient photocurrent response (I-t curves) of MXene, CuTCPP, and MXene/CuTCPP bio-HJs under 808 nm irradiation (ON/OFF cycles), (i) Electrochemical Impedance Spectroscopy (EIS) Nyquist plots, (j) Tauc plots of CuTCPP and MXene/CuTCPP bio-HJs, (k-l) UPS spectra of MXene, CuTCPP, and MXene/CuTCPP bio-HJs, (m) Energy scheme before and after contact between CuTCPP and MXene, (n) Mechanism for the enhanced yield of ROS upon 808 nm light irradiation.Fig. 8
The potential difference between MXene/CuTCPP bio-HJs and MXene promotes electron transfer from CuTCPP to the MXene surface, achieving an equilibrium state. Density functional theory (DFT) calculations were performed to analyze electronic properties. As shown in Fig. 8d–f, MXene exhibits abundant electronic states crossing the Fermi level in its density of states (DOS), indicating high conductivity and metallic character. CuTCPP shows a typical semiconductor DOS profile. MXene/CuTCPP bio-HJs retain MXene's excellent conductivity. Band structure analysis provides insight into interfacial electron transfer. Crucially, to validate the kinetic efficiency of this charge separation pathway, we further performed transient photocurrent response and Electrochemical Impedance Spectroscopy (EIS) analyses (Fig. 8h–i). As anticipated, the MXene/CuTCPP bio-HJs exhibited a rapid and robust photocurrent response significantly superior to that of individual components, directly indicating the efficient spatial separation of photo-generated carriers. Corroborating this, the EIS Nyquist plots revealed a markedly reduced charge transfer resistance (Rct) for the heterojunction, validating that the constructed interface facilitates rapid electron transport. Collectively, this kinetic evidence, rigorously confirms that the built-in electric field effectively suppresses electron-hole recombination, thereby maximizing the quantum yield of ROS for synergistic bacterial eradication. The band gaps of CuTCPP and MXene/CuTCPP bio-HJs, calculated using the Tauc plot method ((αhv)^2^ = A (hv−E_g_)), are 2.09 eV and 2.00 eV, respectively (Fig. 8j). Ultraviolet photoelectron spectroscopy (UPS) determined the work function (Φ) and valence band of MXene/CuTCPP bio-HJs. Fig. 8k shows secondary electron cut-off energies of 17.14 eV (MXene), 17.96 eV (CuTCPP), and 16.82 eV (MXene/CuTCPP), corresponding to work functions of 4.08 eV, 3.26 eV, and 4.40 eV, respectively. Thus, the Fermi levels relative to vacuum are 4.08 eV, 3.26 eV, and 4.40 eV. Based on the band gap values and the data in Fig. 8l, the valence band (VB) positions of CuTCPP and MXene/CuTCPP bio-HJs are −5.20 eV and −6.27 eV, respectively. The conduction band (CB) positions of CuTCPP and MXene/CuTCPP bio-HJs are calculated as −3.11 eV and −4.27 eV, respectively. Upon contact, electrons migrate from the material with the lower work function to that with the higher work function, as illustrated in the energy level diagram (Fig. 8m).
Energy level changes before and after contact between MXene and CuTCPP are evident. The catalytic mechanism is illustrated in Fig. 8n. Under 808 nm NIR irradiation, localized surface plasmon resonance (LSPR) in MXene/CuTCPP bio-HJs facilitates electron transfer from CuTCPP's CB to MXene, promoting electron-hole separation and ROS generation.
In contrast to conventional physical mixtures or electrostatic assemblies summarized in Table S1, this study engineered a precise Schottky junction at the bio-heterojunction interface. As quantitatively validated by the UPS-derived band alignment in Fig. 8, the resultant built-in electric field functions as a molecular electron pump that suppresses charge recombination to significantly amplify ROS quantum yields and POD-like catalytic activity. Furthermore, integrating this heterostructure into an active recombinant collagen scaffold instead of passive carriers resolves the sterilization-regeneration trade-off, facilitating a seamless transition from bacterial eradication to orchestrated tissue repair.
Conclusion
3
This study presents a bio-heterojunction-engineered CAP@MX/CT hydrogel that unifies photothermal, photodynamic, and peroxidase-like activities into a single therapeutic platform for MRSA-infected wound treatment. The key finding is that the MXene/CuTCPP Schottky bio-HJs significantly enhance catalytic efficiency by facilitating rapid interfacial electron transfer, which allows for potent antibacterial and anti-biofilm effects at reduced treatment dosages. This multimodal approach achieves complete bacterial clearance while preventing resistance development. Beyond disinfection, the hydrogel's inherent biocompatibility and active immunomodulatory capacity promote rapid tissue regeneration by stimulating cell migration, modulating macrophage polarization, and enhancing angiogenesis. These combined attributes establish this bio-heterojunction hydrogel as an effective solution to overcome the limitations of conventional single-mechanism therapies, offering a promising strategy for the clinical management of chronic drug-resistant wound infections.
Materials and methods
4
Materials
4.1
5,10,15,20-tetra(4-carboxyphenyl)porphine (TCPP), polyvinyl alcohol (PVA), and 3-Aminobenzeneboronic acid (3-APBA) were purchased from Shanghai Aladdin Biochemical Technology Co., LTD. CuCl_2_·2H_2_O and N, N-dimethylformamide (DMF) were sourced from Shanghai Macklin Technology Co., LTD. Ti_3_AlC_2_ was obtained from Beike Nanotechnology Co., LTD. CF-1552 was procured from Xi'an Juzi Biological Gene Technology Co., LTD. 3,3,5,5-tetramethylbenzidine (TMB) was supplied by Beijing Solaibao Biological Co., LTD.
Synthesis and characterization of MXene/CuTCPP bio-HJs
4.2
CuTCPP was synthesized as follows. TCPP (100 mg) and CuCl_2_·2H_2_O (65 mg) were dissolved in a mixture of N, N-dimethylformamide (DMF, 20 mL) and ethanol (5 mL) at 80 °C under vigorous stirring for 5 h. After cooling to ambient temperature, CuTCPP was collected by centrifugation and then washed sequentially with ethanol.
MXene/CuTCPP bio-HJs were prepared as follows. Ti_3_AlC_2_ (2 g) was treated with HF solution (40 wt%, 50 mL). The precipitate was collected by centrifugation. intercalated with 5% tetramethylammonium hydroxide (TPAOH, 25 mL) at room temperature for 12 h, washed repeatedly with deionized water and ethanol, and lyophilized to obtain MXene powder (100 mg) and 100 mg CuTCPP were dispersed in a DMF/ethanol mixture (20 mL:5 mL) via ultrasonication. The suspension was stirred vigorously at 80 °C for 6 h and lyophilized to obtain the solid product.
The microscopic morphology of MXene/CuTCPP bio-HJs was analyzed using scanning electron microscopy (SEM, ZEISS Sigma 300). The microstructure and elemental composition were further examined by transmission electron microscopy (TEM, FEI Talos F200X). Surface chemical bonds were characterized via Fourier-transform infrared spectroscopy (FT-IR, Bruker Vertex 70). Crystal structure was determined by X-ray diffraction (XRD, Rigaku SmartLab SE) with a scanning speed of 5°/min over a 5°–90° range. Elemental valence states were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha).
Synthesis and characterization of CAP@MXene/CuTCPP hydrogel
4.3
The modification of CF-1552-APBA was performed as follows: A PBS solution containing 4.86 wt% CF-1552 was first prepared. Next, EDC (7.2 wt%) and NHS (7.2 wt%) were dissolved in PBS and added to the CF-1552 solution, followed by stirring for 1 h. The pH was subsequently adjusted to 4.75. Then, APBA (1.4 g) dissolved in PBS (4.67 wt%, 30 mL) was incorporated into the mixture. The resulting APBA-CF-1552 was dissolved in PBS to achieve a 12.5 wt% concentration. Separately, PVA was dissolved in ultrapure water to obtain a 12.5 wt% solution. Under vortex mixing, the two solutions rapidly formed a gel, with MXene/CuTCPP bio-HJs enhancing hydrogel crosslinking. All reactions were conducted at 37 °C. Chemical bonding states were evaluated by FT-IR (Bruker Vertex 70, Germany) and ^1^H NMR (Bruker AVANCE 400, Germany), while microscopic morphology was characterized using SEM (ZEISS Sigma 300, Germany). For self-healing assessment, CAP@MX/CT hydrogels were molded into circular shapes, stained with Sudan red, bisected evenly, and rejoined at the interface.
Swelling behavior was analyzed by freeze-drying hydrogels for 72 h to determine initial weight (W_0_), then immersing them in 0.01 M PBS (pH 7.4, 37 °C). At designated intervals, hydrogels were removed, surface moisture was blotted with filter paper, and swollen weight (Ws) was recorded until equilibrium was reached. The swelling ratio (E_S_) was calculated using Equation (1).
Degradation/Release Study. The prepared hydrogel sample (3.5 g, initial wet weight W) was accurately weighed using an analytical balance. It was then immersed in pre-warmed (37 °C) PBS buffers at pH 5.0, 7.4, and 8.5, with a sample-to-buffer volume ratio of 1:20. The system was incubated in a constant-temperature shaker at 60 rpm. At predetermined time points (1, 2, 3, 4, 7, 10, and 14 days), samples were removed from the medium. Surface moisture was gently removed with filter paper before the wet weight (W_o_) was immediately measured. Three parallel samples were tested for each time point. The degradation rate was calculated as Equation (2)
The collected degradation medium was used for pH monitoring and degradation-product analysis. The entire experiment was performed under aseptic conditions, and a blank control (buffer only) was included to correct for evaporation Loss.
Mechanical properties were measured using a rheometer (Anton Paar MCR302, Austria) with 10-mm parallel plates at 1 mm gap. Viscoelastic behavior was assessed by monitoring storage modulus (G′) and loss modulus (G″) under 1% strain (0.1–100 rad/s). Self-healing performance was evaluated via strain amplitude alternation (γ = 1%, γ = 1000%) at 10 rad/s.
Photothermal performance evaluation of CAP@MXene/CuTCPP hydrogel
4.4
The ultraviolet-visible (UV-Vis) absorption spectra of MXene/CuTCPP bio-HJs were measured using a UV-2600 spectrophotometer (Shimadzu, Japan). CAP@MX/CT hydrogels were exposed to 808-nm near-infrared light at varying laser power densities (0.2, 0.5, 0.8 W/cm^2^) for 10 min. Hydrogels containing different concentrations of MXene/CuTCPP bio-HJs (0.5, 1, and 2 mg/mL) were irradiated at 0.5 W/cm^2^ for 10 min. The photothermal properties of CAP, CAP@MXene, and CAP@MX/CT hydrogels were evaluated under 0.5 W/cm^2^ laser irradiation for 10 min. Photothermal conversion efficiency was determined through five heating-cooling cycles of the CAP@MX/CT hydrogel.
POD activity of CAP@MXene/CuTCPP hydrogel
4.5
Catalytic oxidation experiments were conducted using 3,3′,5,5′-tetramethylbenzidine (TMB). A solution containing 10 mg/mL TMB and 10 mM H_2_O_2_ was prepared in 0.2 M sodium acetate-acetic acid (NaAc-HAc) buffer (pH 3.6). The effects of key parameters – including pH, H_2_O_2_ concentration, temperature, and MXene/CuTCPP bio-HJs concentration – on the peroxidase-like activity were systematically investigated.
In vitro antibacterial activity of CAP@MXene/CuTCPP hydrogel
4.6
The antibacterial properties of MXene, CuTCPP, MXene/CuTCPP single-component and mixed-component materials were verified by setting up co-culture experiments with Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus with the material groups. Monoclonal colonies were selected from LB plates and inoculated into liquid LB medium. The cultures were incubated at 37 °C until the optical density at 600 nm (OD_600_) reached 0.5 (approximately 12 h).
The bacterial suspension (OD_600_ = 0.5) was diluted to 10^5^ CFU/mL and added to the experimental groups. After mixing, the samples were incubated at 37 °C for 4 - 6 h. 10-fold serial dilutions were performed, and 10 μL aliquots were plated on agar plates. After overnight incubation, the colonies were counted.
The antibacterial performance of CAP@MX/CT was evaluated against S. aureus, E. coli, and MRSA. Monoclonal colonies were selected from LB agar plates and inoculated into liquid LB medium, and cultured until reaching an optical density at 600 nm (OD_600_) of 0.5 (approximately 12 h). Antibacterial assays were divided into six groups:Control (I), CAP (II), CAP@MXene/CuTCPP (III), CAP@MXene/CuTCPP + NIR (IV), CAP@MXene/CuTCPP + H_2_O_2_ (V), CAP@MXene/CuTCPP + H_2_O_2_+NIR (VI).
Bacterial suspensions (OD_600_ = 0.5) were diluted to 10^5^ CFU/mL, added to experimental groups, mixed, and incubated at 37 °C for 4 – 6 h. Serial 10-fold dilutions were performed, and 10 μL aliquots were plated on agar plates. After overnight incubation, colonies were counted. For SEM analysis, bacteria were fixed with 4% paraformaldehyde (1 h), dehydrated in an ethanol gradient (30%, 40%, 50%, 70%, 90%, 95%; 15 min per step), and imaged.
For biofilm assays, MRSA was cultured in LB medium to OD_600_ = 0.5, centrifuged, and resuspended in tryptic soy broth (TSB). Aliquots (2 mL/well) were transferred to 6-well plates for biofilm formation (48 h). Biofilms were washed with PBS, treated with hydrogel extracts (12 h), and washed again to remove planktonic cells. Samples were stained with SYTO 9/PI Live/Dead viability kit and analyzed by confocal laser scanning microscopy (CLSM; Nikon A1-Rsi, Japan). For crystal violet staining, resuspended bacteria were added to 48-well plates (0.5 mL/well) in duplicate, incubated with 10% crystal violet (1 h), washed with PBS, air-dried, and documented.
MRSA cultures in the logarithmic growth phase were centrifuged (5000 rpm, 5 min), washed with PBS (0.1 M, pH 7.4), and resuspended to a concentration of 1 × 10^8^ CFU/mL. The bacterial suspension was divided into six experimental groups identical to those in the aforementioned experiment. Each group was incubated with DCFH-DA probe (final concentration: 20 μM) at 37 °C in the dark for 30 min. After loading, the bacteria were centrifuged (8000 rpm, 5 min) and washed three times with cold PBS to remove extracellular probe. The pellets were resuspended in PBS, treated with the respective agents, and irradiated with an 808 nm laser (where applicable) for 20-30 min. Fluorescence intensity was immediately observed under a fluorescence microscope (excitation: 488 nm, FITC channel) to quantify intracellular ROS levels.
In vivo antibacterial assessment and wound regeneration assessment of CAP@MXene/CuTCPP hydrogel
4.7
Cells were first cultured in a cell incubator for 24 h, after which the original medium was replaced with hydrogel extract. The cells were then cultured for an additional 24-48 h. The OD values of both the experimental and control groups were measured using the CCK-8 colorimetric method. These experimental procedures were repeated for co-culture periods of 24 h and 48 h. Subsequently, the prepared acridine orange/ethidium bromide (AO/EB) staining agent was applied. After incubation for the specified duration, the cells were examined under a fluorescence microscope and imaged, with the fluorescence intensity being recorded. Viable cells appeared green, while non-viable cells displayed red fluorescence. Cell viability was subsequently calculated using Equation (3).
(test: hydrogel group, control: complete medium)
Cell migration was assessed using L929 cells seeded in 6-well plates (1 × 10^5^ cells/well). At 90% confluence, a scratch was created with a sterile pipette tip, detached cells were removed by PBS washing, and hydrogel extracts were introduced. After 12 h incubation, wound widths were measured, with migration rate determined by Equation (4).
( : Area of wound at 0 h, : Area of wound after 12 h)
In hemolysis tests, RBCs from SD rats were isolated (1500 rpm, 10 min), washed, and resuspended to 2%. Test groups (negative control: PBS + RBCs; positive control: DI water + RBCs; test: extract + RBCs) were incubated at 37 °C for 1 h. After centrifugation (3000 rpm, 10 min), supernatant OD was measured at 545 nm, and hemolysis ratio was computed by Equation (5).
Anti-inflammatory activity was evaluated in RAW264.7 macrophages divided into: control (complete medium), inflammation (LPS 1 μg/mL), and treatment (LPS + extract) groups. After 24 h, supernatants were collected for IL-6/IL-10 ELISA, while cells were fixed in 4% paraformaldehyde for CD86/CD206 immunofluorescence.
In the tube formation assay, human umbilical vein endothelial cells (HUVECs) were seeded onto plates pre-coated with Matrigel at a density of 5 × 10^4^ cells per well and cultured in endothelial cell medium. After incubation at 37 °C under 5% CO_2_ for 6 h, tube-like structures were observed under an inverted microscope. Representative images from three independent experiments were captured. The number of nodes per field was quantified using ImageJ software (National Institutes of Health, USA) to evaluate angiogenic activity.
In vivo antibacterial assessment and wound regeneration assessment of CAP@MXene/CuTCPP hydrogel
4.8
Male Sprague-Dawley (SD) rats (Kunming strain) weighing 200-250 g were utilized in this study. All animal procedures were approved by the Animal Ethics Committee of Northwestern University (Approval No. NWU-AWC-20240712R) and conducted in accordance with institutional guidelines. After one week of acclimatization, MRSA-infected wound models were established. Briefly, rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg), and dorsal hair was removed using electric clippers. Full-thickness excisional wounds (8 mm diameter) were created using sterile biopsy punches, followed by intradermal injection of 50 μL MRSA suspension (1.0 × 10^5^ CFU/mL) at the wound site. The experimental group was divided into Control(I), Hydrosorb® (I®), CAP(II), CAP@MXene/CuTCPP(III), CAP@MXene/CuTCPP + NIR(IV), CAP@MXene/CuTCPP + H_2_O_2_(V), CAP@MXene/CuTCPP + H_2_O_2_+NIR(VI) the above six groups.
For photothermal therapy, wounds in Groups IV and VI were irradiated with an 808 nm laser (0.5 W/cm^2^) for 10 min, during which thermal images were acquired using an infrared camera. Wound areas were photographed daily with a digital caliper, and the healing rate was calculated by Equation (6) based on the initial wound area A_0_ and wound area A_n_ on day n.
( the initial wound area, An: the area on day n)
The prepared water gel samples with uniform cross-sections of 1 cm, 2 cm, 3 cm, 4 cm, and 5 cm were placed in a room temperature environment for 30 min to reach equilibrium. A near-infrared laser with a wavelength of 808 nm was used to vertically irradiate the center position of the upper surface of the water gel at a fixed power density. An infrared thermal imager or an optical fiber thermometer was used to monitor and record the temperature changes at the center point of the upper surface of the samples in real time. After continuous laser irradiation for 10 min, the light source was turned off.
In vivo histopathological evaluation of CAP@MXene/CuTCPP hydrogel
4.9
Wound tissues were collected from Sprague-Dawley rats at postoperative days 0, 1, 3, 5, and 11. The specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for histological examination. Tissue sections were stained with hematoxylin and eosin for morphological evaluation and Masson's trichrome for collagen analysis. Bacterial samples were obtained from wounds on days 3 and 7 and cultured on agar plates. On day 7, wound tissues were processed for immunofluorescence staining of CD86, CD206, IL-6 and VEGF. Major organs and blood samples were collected on day 7 for histological examination and hematological analysis.
Photocatalytic mechanism of MXene/CuTCPP bio-HJs
4.10
After treatment of MXene, CuTCPP, and MXene/CuTCPP bio-HJs with near-infrared light (0.5 W/cm^2^, 808 nm), reactive oxygen species (ROS) generation was detected using electron spin resonance (EPR; E500-9.5/12, Bruker, Germany), ·OH and ·O_2_^−^captured with 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Sigma),^1^O_2_ captured with 2,2,6,6-Tetramethylpiperidinooxy (TEMP; Sigma). Subsequently, we used DPBF to monitor the changes in the absorption peaks of three substances: ^1^O_2_, NBT detection of ·O_2_^−^, and TMB detection of ·OH (with 0.5W/cm^2^ 808 nm laser excitation).
First-principles calculations were performed by the VASP and the Projector Augmented Wave (PAW) method [69].The exchange functional groups were treated by the Perdew-Burke-Ernzerhof (PBE) functional and DFT-D correction. Spin-polarized calculations were performed [70]. The cutoff energy of the plane wave basis function is set to 520 eV. To optimize the geometry and lattice size, the Brillouin zone integral is performed using 1 × 4 × 1 Gamma k-point sampling. Self-consistent calculation applies a convergence energy threshold of 10^−5^ eV [71]. The equilibrium geometry and lattice consistency have been optimized, and the maximum force on each atom is within 0.05 eV/Å.
EIS Analysis. Electrochemical impedance spectra (RST5000) were recorded using an electrochemical workstation in a standard three-electrode system. The measurement was performed at the open-circuit potential with an AC amplitude of 5 mV over a frequency range of 0.1 Hz to 100 kHz. All tests were conducted in 1 M Na_2_SO_4_ aqueous solution.
Photocurrent Measurement. The photoelectrochemical response was evaluated via the amperometric i-t method under intermittent illumination. A 300 W xenon lamp equipped with an optical filter (λ > 420 nm) served as the visible-light source. The working electrode was alternately exposed to light and kept in the dark at 30 s intervals under a constant bias of 0 V (vs. Ag/AgCl). The resulting photocurrent density was recorded to assess the photoinduced charge separation efficiency.
The relationship between the energy and position of the electron orbit in MXene/CuTCPP was measured by ultraviolet photoelectron spectroscopy (UPS). Firstly, the sample was subjected to surface cleaning under the action of an ion source to remove substances that would interfere with the test results. Then, the treated sample was spin-coated for sample preparation. In the UPS test chamber, under the irradiation of a UV light source, electrons would escape and be separated and detected in the energy analyzer. The calculated values of band gap (Eg) and work function (Φ) were calculated by Equation (7), (8)
(hv: incident photon energy*, α*: optical absorption coefficient*, A*: proportionality constant)
(hv: incident photon energy, Ecut-off: secondary electron cut-off energy, EF: fermi level energy)
Statistical analysis
4.11
Each data point was obtained from at least three independent experiments. All quantitative data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software, with significance levels defined as follows: ∗P < 0.05 (significant), ∗∗P < 0.01 (highly significant), ∗∗∗P < 0.001 (extremely significant), and ∗∗∗∗P < 0.0001 (most significant).
CRediT authorship contribution statement
Chongyi Li: Writing – original draft, Visualization, Investigation. Zewen Chang: Writing – review & editing, Visualization, Methodology. Yuxi Zhang: Software, Methodology. Shihong Shen: Visualization, Validation. Lin Liu: Software, Methodology. Dan Zeng: Writing – review & editing, Supervision, Project administration, Conceptualization. Daidi Fan: Supervision, Project administration.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethical statement for animal experiments
The animal study protocol was approved by the Animal Care and Use Committee (ACUC) of Northwest University, protocol number NWU-AWC-20240712R. The study adhered to the guidelines set by the committee. The original document is as follows. This article does not contain any studies with human participants performed by any of the authors.
Declaration of interests
The authors declare the following personal relationships which may be considered as potential competing interests: Lin Liu is currently employed by Xi'an Giant Biogene Technology Co., Ltd.
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