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. 2026 Feb 27;11(9):15005–15018. doi: 10.1021/acsomega.5c12013

A Carboxymethyl Chitosan Hydrogel Loaded with Bacteriophage-Derived Bacteriostatic Peptide Exhibits Antibacterial, Hemostatic, and Wound-Healing Properties

Yujiao Huang 1, Ruolan Liu 1, Xiaohua Shi 1, Zichen Yang 1, Yuzhe Huang 1, Zeyuan Lei 1,*, Dongli Fan 1,*
PMCID: PMC12980258  PMID: 41835539

Abstract

Drug-resistant bacterial infections are a formidable challenge in the treatment of skin and soft tissue wounds due to the limited efficacy of currently available antibiotics. Bacteriophage-derived antimicrobial peptides positioned as promising antibacterial agents offer a new solution to this problem. Herein, we fabricated a composite hydrogel system constructed with carboxymethyl chitosan (CMCS) containing Gp68, an antimicrobial peptide from bacteriophage PA3. The obtained CMCS@Gp68 hydrogel can protect the peptide and control the release of Gp68. The results of in vitro antibacterial assays demonstrated that the hydrogel can induce bactericidal effects on Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) through disrupting bacterial activity. The material characterization demonstrated that the mechanical properties of the hydrogel are acceptable, and the hemostatic property is rapid enough. Furthermore, the cytotoxicity assay and histopathological study also indicated that the material is biocompatible. Finally, the therapeutic effect was further studied in a rat model of full-thickness skin wound infection with P. aeruginosa. Treatment with the CMCS@Gp68 hydrogel markedly decreased bacterial burden at the wound site, suppressed inflammatory cytokine production, and promoted re-epithelialization. In summary, our multifunctional composite hydrogel with antimicrobial, hemostatic, and wound-healing properties and excellent biocompatibility is an ideal therapeutic material for infected wound treatment and regenerative medicine.

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1. Introduction

Wound healing is a well-coordinated cellular participation process and advances in orderly four stages: hemostasis, inflammation, proliferation, and remodeling. However, bacterial contamination can impair the change of these four phases and delay the wound healing. Current clinical treatment strategies like antibiotic therapy and debridement are limited by many clinical situations, especially uncontrollable inflammatory response and diffusion of resistant organisms. , The extensive abuse of antibiotics has directly led to the diffusion of multidrug-resistant organisms, such as methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, and P. aeruginosa, which now pose a severe threat to public health. , It is urgent to prepare novel wound therapeutics with strong antimicrobial activity and low resistance to induction.

Bacteriophages can lyse bacteria by self-replication or hydrolytic enzymes encoded by them. A decade before penicillin’s discovery, bacteriophage therapy was already used to treat bacterial infections. Compared with antibiotics, bacteriophages can self-amplify at infection sites, exhibit high target specificity, , and undergo adaptive evolution against emerging resistant strains, which can decrease the bacterial load at the wound site effectively. Moreover, the high specificity of phages can preserve the beneficial commensal bacteria in the community of wound microbiota, thereby preventing secondary infection caused by dysbiosis. Gp68 is derived from a temperate bacteriophage φPaP3 that specifically targets multidrug-resistant Pseudomonas aeruginosa. Unlike most antimicrobial peptides (AMPs) which directly disrupt bacterial membrane structures or biochemical processes, our study found that Gp68 functions as a bacteriophage-encoded transcriptional enhancer. It specifically binds to the promoter regions of two key regulatory genes (minD and mvaT) in the host bacterium (P. aeruginosa PA3), thereby enhancing the expression of the host’s own negative regulatory system to inhibit bacterial division. Although bacteriophage-derived peptides have shown promising antimicrobial activity, they still have some limitations. The peptides are easily proteolytically degraded in the physiological environment, leading to short half-lives. Moreover, bacteriophage-derived peptides cannot be directly applied to achieve therapeutic concentrations at the wound site.

Hydrogels, which are nontoxic polymeric materials composed of three-dimensional network structures formed through physical or chemical cross-linking of hydrophilic polymers, serve as one of the delivery systems developed to address these challenges. , Its excellent tissue compatibility promotes cellular proliferation and accelerates healing, while the low adhesiveness to nascent tissue minimizes trauma during dressing changes. , Beyond the physical properties, hydrogels have been modified to possess various antimicrobial properties by loading different kinds of agents due to their ability to act as a drug reservoir such as antibiotics, AMPs, and metal-based antimicrobials including silver and copper ions. Among various materials suitable for gel fabrication, chitosan is widely used in wound dressings due to its inherent antibacterial activity, excellent biocompatibility, biodegradability, low immunogenicity, and moisture permeability. Nevertheless, the rigid molecular backbone and high cationic charge density of chitosan restrict its solubility to acidic media, thereby constraining its applications in physiological solutions. Carboxymethyl modification is an efficient approach to break through the limitation of chitosan application on acidic media. By introducing carboxymethyl groups into chitosan molecular chains, CMCS exhibits greatly enhanced water solubility and can form stable gel in a neutral environment. Meanwhile, CMCS retains all the antimicrobial activities and biocompatibility of its parent material chitosan. Moreover, carboxyl groups in CMCS can interact with cationic peptides through electrostatic interactions and enhance drug entrapment and slow release. In summary, carboxymethyl modification transforms chitosan into CMCS with enhanced solubility, biocompatibility, and drug delivery, expanding its biomedical applications.

This study introduces a double-bond modification of carboxymethyl chitosan using glycidyl methacrylate, resulting in the synthesis of glycidyl methacrylate (GMA)-modified carboxymethyl chitosan. We propose loading the Gp68 peptide onto this modified chitosan hydrogel to create a novel wound dressing. The aim is to investigate its effect on wound healing infected with P. aeruginosa, with the ultimate goal of providing new therapeutic strategies and a theoretical foundation for the clinical treatment of P. aeruginosa -infected wounds.

2. Materials and Methods

2.1. Materials

The CMCSMA hydrogel (molecular weight: 150–250 kDa, degree of substitution: 30–40 DS%) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) were procured from Engineering for Life (Suzhou, China). Fetal bovine serum (FBS) was sourced from Gibco (USA). Penicillin–streptomycin and 0.25% trypsin were obtained from BioSharp (Beijing, China). Serum-free culture medium, 4,6-diamidino-2-phenylindole (DAPI) stain, phosphate-buffered saline (PBS), and paraformaldehyde were procured from Solarbio (Beijing, China). The CCK-8 assay kit was acquired from Solarbio (Beijing, China). The Live/Dead Bacterial and Cell Staining Kit were obtained from Beyotime (Shanghai, China). Alkene coupling hydrogel fluorescent dyes were procured from Engineering for Life (Suzhou, China). Mouse fibroblast cells (L929) and S. aureus USA300 were kindly provided by the Department of Plastic Surgery, Xinqiao Hospital, Army Medical University. P. aeruginosa PAO1 and E.coli O157 were kindly provided by the Clinical Research Center, Xinqiao Hospital, Army Medical University.

2.2. Synthesis of Gp68

Gp68 is a peptide encoded by bacteriophage PaP3. It consists of 47 amino acids (MKSPYEAAHERAQMINRLKKLTRMIRVHPDPRWIVERQELIRKLSK), with a coding sequence length of 145 bp and a molecular weight of approximately 5 kDa. The Gp68 polypeptide was synthesized and purified by GUOPING PHARMACEUTICAL (Anhui, China). The tertiary structure of Gp68 was predicted using SWISS-MODEL, and the resulting model was visualized using Pmal. Homology analysis was performed using the Basic Local Alignment Search Tool (BLAST) on the NCBI Web site. The promoter regions of the genes were predicted using the BPROM tool available at SoftBerry.

2.3. Preparation of the CMCS@Gp68 Hydrogel

In this study, carboxymethyl chitosan (CMCSMA) was dissolved in a solution containing the photoinitiator LAP to form a precursor solution with a final concentration of 2% w/v. The antimicrobial peptide Gp68 was then dissolved and uniformly blended into the CMCSMA solution at specified mass ratios, corresponding to final concentrations of 40, 60, and 80 μmol/L. The mixtures were cross-linked under UV light (405 nm, 30 s) to form stable hydrogels. Finally, the resulting composite scaffolds were freeze-dried for 24 h and designated as CMCS@Gp68 hydrogels based on their Gp68 content.

2.4. Characterization of the CMCS@Gp68 Hydrogel

The gelation efficiency was confirmed by photopolymerizing the hydrogel precursor solution in transparent glass vials under 405 nm UV light for 30 s. Subsequently, the obtained materials were tested for their structural integrity using the inverted vial test as the standard for successful gelation. The injectability of the hydrogels was evaluated by the standard syringe injection method. First, the precursor solution was extruded through a 0.4 mm diameter needle, and then, the extrusion uniformity and structural integrity of the expelled hydrogel were checked.

The wet hydrogels were then freeze-dried by a freeze-dryer (Telstar, Spain) for subsequent experiments. Hydrophilicity was evaluated by a contact angle meter (SZ-CAMC32, Shanghai, China). The microstructure of the hydrogels was examined via scanning electron microscopy (SEM, ZEISS Sigma 300, Germany). The crystalline structure was characterized by X-ray diffraction (XRD, Rigaku Ultima IV, Japan). Thermal stability was analyzed by a thermogravimetric analyzer (TGA, Netzsch STA449F3, Germany). Chemical composition was analyzed by Fourier transform infrared spectroscopy (FTIR, Thermo Fisher Scientific Nicolet iS20, USA).

In order to evaluate the swelling capacity of the scaffolds, the samples were weighed to determine the initial dry mass (W 0) and then immersed in PBS at 37 °C for 24 h until swelling equilibrium was reached. After that, the samples were taken out of the PBS and the attached liquid on the surface was removed carefully before weighing the wet mass (W w) . The swelling ratio was calculated according to eq :

swellingratio(%)=Ww−W0W0×100% 1

The scaffolds with an initial dry weight (W 0) were immersed in PBS at 37 °C. At predetermined time points over a 30 day period, samples were retrieved and lyophilized, and their dry weights were recorded as W t. The degradation percentage was then calculated by using eq .

degradation(%)=W0−WtW0×100% 2

2.5. Antimicrobial Activity of Gp68 and CMCS@Gp68 Hydrogels

Antimicrobial activity of Gp68 on P. aeruginosa, E. coli, and S. aureus were performed by the broth culture method. First, the logarithmic growth phase of each bacterial strain was cultured in Luria–Bertani (LB) medium at 37 °C. Then, the bacterial suspensions were resuspended in PBS (10 mM, pH5.5) to contain approximately 5 × 106 CFU/mL. Aliquots (100 μL) of each bacterial suspension were incubated with Gp68 (40, 60, and 80 μmol/L) for 6 h at 37 °C. PBS was used as negative control. Finally, the samples were appropriately diluted, spread on agar plates, and cultured overnight. Finally, the colonies were counted and the percentage of survival was calculated.

The same method was applied to evaluate the antibacterial properties of the hydrogel against P. aeruginosa, E. coli, and S. aureus. After treatment, the samples were stained with a live/dead bacterial staining kit (DMAO/PI) for 30 min and observed under a fluorescence microscope (APEXVIEW APX100, Olympus, Japan) to distinguish dead bacteria (red fluorescence, Ex/Em ≈490/515 nm) from live bacteria (green fluorescence, Ex/Em ≈535/617 nm). All experiments were repeated at least three times.

2.6. Cytotoxicity of Peptide-Based Hydrogels

The cytocompatibility of peptide hydrogels was evaluated using mouse fibroblast L929 cells according to a previously established method. The hydrogels were first extracted in DMEM (pH 7.4 or 5.5) containing 1% penicillin–streptomycin at 37 °C for 24 h (0.1 g mL–1). The resulting extracts were supplemented with 10% FBS and sterilized by filtration through a 0.22 μm membrane. For the live/dead staining assay, L929 cells were seeded in 24-well plates at a density of 3 × 104 cells per well and cultured until reaching 60–70% confluency. Following treatment with the extracts for 24 h, the cells were stained using a Calcein-AM/PI Live/Dead staining kit (YOBIBIO, Shanghai, China) to distinguish viable cells from dead cells. Calcein-AM (green fluorescence, Ex/Em ≈490/515 nm) labels live cells with intact esterase activity, while propidium iodide (PI, red fluorescence, Ex/Em ≈535/617 nm) penetrates only membrane-compromised dead cells. The stained samples were then imaged under an inverted fluorescence microscope (IX73, Olympus, Japan) to assess the cell viability and morphological changes. For quantitative analysis, cells were seeded in 96-well plates at 2 × 104 cells per well, incubated for 24 h, and then treated with the extracts for an additional 24 or 48 h. Cell viability was determined using a CCK-8 kit (ABclonal Technology, China) by measuring the absorbance at 450 nm. Cells in normal medium and cell-free medium served as the negative and blank controls, respectively. The cell viability was calculated by using eq .

cellviability(%)=ODsamples−ODblankODcontrol−ODblank×100% 3

Fluorescent hydrogels were prepared by dissolving the fluorescent dye (EFL-DYE-UF-ENE-R) at an excitation wavelength of 550 nm in LAP at a concentration of 5 mg/mL. The hydrogels were molded into cylindrical shapes with a diameter of 1 cm and a height of 5 mm. The mice were anesthetized, and the hair on their backs was shaved. The samples were then implanted into the dorsal region of the mice (n = 3). Imaging was performed using a subcutaneous small animal in vivo imaging system (Bruker) immediately after surgery (day 0) and at weeks 1 and 2 to observe and document the degradation of the hydrogels in the different groups.

2.7. In Vitro Hemolysis and Whole Blood Clotting Assay of the Composite Hydrogels

The hemocompatibility of the hydrogels was evaluated using fresh sheep blood (Thermo Scientific, China) following established methods with slight modifications. Erythrocyte suspension (5% v/v) was prepared by centrifugation at 2000 rpm for 5 min. Scaffold extracts were obtained by incubating 0.2 g of material in 10 mL of PBS for 48 h. For testing, 500 μL of extract was mixed with 500 μL of an erythrocyte suspension in 1.5 mL centrifuge tubes. After 1 h of incubation, samples were centrifuged (2000 rpm, 5 min), and the absorbance of the supernatant was measured at 540 nm. The test sample absorbance (A t) was obtained by centrifuging the 1 mL erythrocyte suspension and measuring its supernatant. Parallel control samples were prepared by adding 1 mL of PBS (for A n) or 1 mL of deionized water (for A p) to separate 1 mL aliquots of the erythrocyte suspension. All values were then applied to eq to compute the hemolysis rate.

hemolysisratio(%)=At−AnAp−An×100% 4

The blood coagulation capacity was assessed as previously reported. In detail, 30 mg of each sample and gauze control were incubated with 100 μL of whole blood for 10 min at 37 °C. Next, 10 μL of 0.2 M CaCl2 was added into the tube to induce clotting, and 10 mL of PBS was added subsequently. The absorbance of the supernatant at 540 nm (A s) was measured by a microplate reader. A negative control (A 0) was prepared using deionized water instead of the sample mixed with 100 μL of whole blood and 10 μL of a 0.2 M CaCl2 solution. Finally, the blood clotting index (BCI) was calculated by eq .

BCI(%)=AsAo×100% 5

2.8. In Vivo Hemostatic Performance

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Army Medical University, and all animal procedures were performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. In order to assess the hemostatic performance of hydrogels in noncompressible wounds, male Sprague–Dawley rats (200–250 g, n = 3 per group) were used, and two hemorrhage models were established, including the tail amputation model and liver incision model, to investigate the hemostatic efficacy of CMCS and CMCS@Gp68 hydrogels in comparison with the untreated group and gauze group as two controls.

Circular-shaped CMCS and CMCS@Gp68 hydrogels with a 1 cm diameter were cut according to the wound area. Under anesthesia with sodium pentobarbital, rats were anesthetized and subsequently subjected to the following procedures: (1) Liver incision model: the abdominal skin was cut to expose the liver, and a standard 1 cm long incision was made on the hepatic surface. After immediate application of hydrogels, the whole procedure was finished; (2) tail amputation model: the distal one-third part of the tail was cut off, and the cut surface of the tail was immediately applied with gentle pressure of the hydrogel sample. The amount of blood loss was quantitatively detected (n = 3 per group) after complete hemostasis was achieved.

In addition, to explore the possible underlying hemostatic mechanism, the platelet adhesion experiment was further performed. Briefly, the whole blood (100 μL) was dropped on the sample surface and further cultured at 37 °C for 1 h. Then, the nonadherent parts of blood were removed by gently rinsing with PBS. After that, the sample was fixed in 4% paraformaldehyde, dehydrated with a graded ethanol series, and finally observed by scanning electron microscopy. The morphology and density of platelet adhesion were further analyzed.

2.9. Wound Healing Assay

The in vivo experiments were performed according to previously reported protocols, with slight modifications. All female Sprague–Dawley rats (body weight 220–250 g) were allowed to acclimate for 7 days and randomly divided into four groups: PBS, Mupirocin (purchased from Sino-American Tianjin Smith Kline & French Laboratories Ltd., China), CMCS, and CMCS@Gp68 (60 μmol/L). A round full-thickness depilation wound with a diameter of 10 mm was made on the dorsal part of each rat. Each wound was inoculated with 10 μL of a Pseudomonas aeruginosa suspension (108 CFU) and then air-dried naturally. Finally, Tegaderm transparent film (3M, USA) was used to cover and fix the wound to avoid disturbance from animals. The experiments were performed 48 h after infection. Then, the wound was applied with 100 μL of the corresponding formulation topically once per day: PBS, Mupirocin (20 mg/g), CMCS, and CMCS@Gp68 (60 μmol/L). The length and corresponding area of the wound were photographed on days 2, 4, 7, and 15 after treatment. The wound healing rate was then analyzed and calculated by ImageJ software. ,

Furthermore, wound tissues were harvested on day 7 for bacterial load assessment. The tissues were homogenized in PBS, and the homogenates were subjected to colony-forming unit (CFU) counting. Additional wound tissue samples were collected on days 7 and 15, fixed in 4% formaldehyde, embedded in paraffin, and processed for histological and immunohistochemical evaluations, including hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and immunohistochemical analysis.

2.10. Statistical Analysis

All experiments were carried out in triplicates. Data are shown as mean ± standard deviation (SD). The sample size (n) for each experiment was indicated in the corresponding figure legends. All data were analyzed using GraphPad Prism (GraphPad Software, Inc., USA). The differences were considered statistically significant when P < 0.05.

3. Results and Discussion

3.1. Synthesis and Antimicrobial Evaluation of Gp68 Peptide

Following established protocols, a multidrug-resistant Pseudomonas aeruginosa strain PA3 was initially isolated from clinical burn specimens, after which its temperate bacteriophage φPaP3 was subsequently purified for further characterization. Through screening of the phage genome, we identified and synthesized in vitro a 47-amino-acid peptide (theoretical molecular weight: ∼5 kDa), which we designated as Gp68. This peptide is predicted to adopt a characteristic helix-turn-helix structural motif (Figure S1). Typically, α-helical AMPs with this architecture can integrate into the phospholipid bilayer via their hydrophobic segments, forming pore-forming channels that cause cytosol leakage and cell death. , Despite this structural prediction, our prior research established that Gp68 exerts antibacterial effects by inhibiting bacterial division and repressing pyocyanin biosynthesis (Figure S2).

Furthermore, in vitro antimicrobial effects of antimicrobial peptide Gp68 were systematically investigated in this study by using P. aeruginosa, E. coli, and S. aureus. The results showed that Gp68 had obvious antibacterial activity in a concentration-dependent manner. After 6 h of contact, more than 99% of the bacteria of these three species were killed at the maximum concentration of 80 μmol/L (P < 0.001). Notably, when the concentrations of Gp68 were 40 and 60 μmol/L, the bacterial elimination rates for the tested strains also exceeded 60% and 80%, respectively (Figure A–F). And the possible reason why Gp68 has antimicrobial activity may lie in the helix-hairpin structure and more importantly the ability to inhibit the division of bacteria. , In addition, live/dead staining experiments also offered direct visual evidence for the above results. For the control group, most of the suspended bacteria of the three species presented green fluorescence, which demonstrated that there was no antimicrobial activity. However, when the live bacteria were treated with 40, 60, and 80 μmol/L Gp68, the red fluorescence intensity obviously increased along with the concentration, and there was great bacteria damage, which further validated its strong antibacterial effect on three types of bacteria (Figure G).

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Synthesis and analysis of the antimicrobial properties of Gp68 peptide. (A) Photographs of surviving P. aeruginosa clones on agar plates after exposure to various concentrations of the Gp68 peptide for 6 h. (B) Antibacterial activity of Gp68 against P. aeruginosa. (C) Photographs of surviving E. coli clones on agar plates after exposure to various concentrations of the Gp68 peptide for 6 h. (D) Antibacterial activity of Gp68 against E. coli. (E) Photographs of surviving S. aureus clones on agar plates after exposure to various concentrations of the Gp68 peptide for 6 h. (F) Antibacterial activity of Gp68 against S. aureus. (G) Live/dead staining of P. aeruginosa, E. coli, and S. aureus after contact with the concentrations of the Gp68 peptide for 6 h. (The statistical analysis was measured by one-way ANOVA. *P < 0.05, ***P < 0.001.)

3.2. Characterization and Performance Evaluation of CMCS@Gp68 Hydrogel

In this study, we fabricated a CMCS hydrogel loaded with the bacteriophage-derived antimicrobial protein Gp68. Both CMCS and CMCS@Gp68 hydrogels exhibited excellent photo-cross-linking behavior. When the photoinitiator LAP was added, the precursor solution underwent an abrupt transition to solid gel in 30 s under 405 nm UV light (Figure A,B). Vial inversion was used to test the gel strength, and no liquid flowed out when the gel was inverted (Figure C), indicating formation of a stable three-dimensional network. CMCS@Gp68 hydrogel (60 μmol/L) had good injectability and was able to be pushed out from the syringe needle (0.4 mm diameter) to print a continuous “SCI” pattern (Figure D), indicating its potential to be injected into uneven or deep wounds. After injection, the hydrogel instantly solidified upon photoinduction and contacted the tissue interface conformally. The covalent network formed via photo-cross-linking endows the material with much stronger mechanical strength and a more stable three-dimensional structure compared to the physical assembled hydrogels assembled by hydrogen bonding.

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Characterization and performance analysis of different hydrogels: (A,B) CMCS and CMCS@Gp68 hydrogels formed by UV irradiation (405 nm, 30 s). (C) Photographs showed the phase transformation of CMCS@Gp68 hydrogels via a vial inversion method. (D) Injectability of CMCS@Gp68 hydrogels through a 26-gauge needle. (E) FTIR spectra of CMCS and 40, 60, and 80 μmol/L CMCS@Gp68 hydrogels. (F) SEM image of hydrogels. (G) XRD of the hydrogels. (H) Swelling ratio of hydrogels. (I) Degradation ratio of hydrogels. The statistical analysis was measured by one-way ANOVA. *P < 0.05, ***P < 0.001, and ns represents no statistical significance.

The wet hydrogels were then freeze-dried by a freeze-dryer for subsequent experiments. Fourier transform infrared (FTIR) spectroscopy was used to characterize the chemical bonds of the scaffolds. The main chemical bonds existing in the untreated sample were C–O–C, –COO–, and C–H. When the peptide was loaded, amide bonds appeared (Figure E). In addition, the CO groups in the amide bonds of peptide interacted with the C–OH groups of modified chitosan in hydrogen bonding, changing the chemical environment of the original C–O–C bonds. Therefore, in the CMCS@Gp68 scaffolds with a Gp68 peptide content of 40, 60, and 80 μmol/L, there appeared a CO absorption peak at about 1700 cm–1. Moreover, with the increase in the peptide content, the shape and position of the C–O–C absorption peak at 1055 cm–1 changed systematically and consistently (Figure E). The SEM results showed that both scaffolds had a uniformly distributed three-dimensional porous structure, and the pore size of CMCS@Gp68 (115.06 μm) was significantly decreased compared with that of CMCS (191.77 μm), which was beneficial for cell adhesion and growth (Figures F and S3). The decrease in pore size of CMCS@Gp68 also matches the enhanced swelling ratio, which might be the structural modification, endowing the material with improved drug loading capacity and enhanced hydrophilicity. Simultaneously, XRD analysis proved that the incorporation of Gp68 did not change the crystalline structure of the hydrogel (Figure G).

The contact angle test results indicated that the contact angles of the two scaffolds did not undergo significant changes and both exhibited hydrophilic properties (Figure S4). And as shown in Figure H, the two scaffolds reached equilibrium swelling within about 24 h, and the ratio was 310% to 368%, which has an advantage in swelling behavior. Meanwhile, the equilibrium swelling ratio increased with the increase of Gp68 peptide concentration. On the one hand, the hydrophilic groups (–OH, –NH2, –COOH) of peptide can increase the whole hydrophilic performance of the network; on the other hand, the molecular chains of peptide may intercalate into the CMCS network, which may decrease the effective cross-linking density of the system.

Moreover, both CMCS and CMCS@Gp68 scaffolds exhibited high thermal stability, which is crucial for their processing and biomedical applications (Figure S5). We investigated the in vitro degradation behavior of CMCS scaffolds with varying Gp68 concentrations in PBS. As shown in the experimental data, clearly when the sample was immersed in PBS, the degradation rate of all scaffolds reached about 40% after only 5 days. This provides strong evidence that partial degradation in the early stage creates an ideal environment for cell proliferation. With the extension of time, the rate of degradation slowed and eventually stabilized at a relatively flat 60–80% after 25 days (Figure I).

3.3. In Vitro Antibacterial Properties of the CMCS@Gp68 Hydrogel

Antimicrobial activity was assessed using P. aeruginosa, E. coli, and S. aureus as model pathogens. CMCS exhibits inherent bactericidal properties through electrostatic interactions between its cationic groups and negatively charged bacterial membranes, resulting in cell inactivation. Plate coating assays showed that Gp68 incorporation into the hydrogel matrix substantially improved antibacterial performance, inhibiting bacterial growth and proliferation. At optimal Gp68 levels, near-complete bacterial suppression (P < 0.05) was observed for all strains (Figure A–D). Live/dead staining further demonstrated a concentration-dependent enhancement of antimicrobial efficacy with increasing Gp68 content (Figure E). The CMCS-Gp68 system likely generates synergistic antibacterial effects by combining two distinct killing mechanisms, thereby providing dual-mode pathogen control.

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In vitro antibacterial properties of the composite hydrogels: (A) Photographs of survival bacteria clones (P. aeruginosa, E. coli, and S. aureus) on agar plates after contact with the different composite scaffolds for 6 h. Statistics of antibacterial rate of different hydrogels against (B) P. aeruginosa, (C) E. coli, and (D) S. aureus. (The statistical analysis was measured by one-way ANOVA. ***P < 0.001, and ns represents no statistical significance.) (E) Live/dead staining of P. aeruginosa and E. coli after contact with the different composite hydrogels for 6 h.

3.4. In Vitro Biocompatibility and Blood Compatibility of the CMCS@Gp68 Hydrogel

The biocompatibility of CMCS hydrogel loaded with different Gp68 concentrations was comprehensively studied for exploring their potential biomedical applications. Live/dead cell staining results demonstrated that cell mortality increased with enhanced Gp68 concentration (Figure A,C). Meanwhile, obvious disruption of the actin cytoskeleton was observed in cells treated with 80 μmol/L Gp68, which were rounded and punctate fluorescence patterns (Figure A,B). All groups exhibited a trend of cellular proliferation over time. CMCS hydrogels loaded with different concentrations of Gp68 all maintained high cell proliferation rates comparable to the CMCS group (P > 0.05), demonstrating their excellent biocompatibility (Figure C). The hydrogel group containing 80 μmol/L Gp68 exhibited a slower cell proliferation rate compared to the other groups, which may be attributed to the excessively high concentration of Gp68 exceeding the safety threshold tolerable by the cells. Based on the comprehensive evaluation of antimicrobial performance and biocompatibility, 60 μmol/L was chosen as the Gp68 loading concentration in the following experiments according to the comprehensive performance.

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In vitro biocompatibility and blood compatibility of the composite hydrogels: (A) Live/dead staining of L929 cells treated with Gp68 hydrogels at different concentrations. (B) Nuclear (blue) and cytoskeleton (green) staining of L929 cells after culture on the different Gp68 hydrogels for 48h. (C) Cytotoxicity of L929 cells cocultured with hydrogels extract medium containing different concentrations of Gp68 for 24 and 48h. (D) Hemolysis ratios of different samples. (E) Quantitative blood clotting index (BCI) results of different composite hydrogels. (F) Hemolysis test images of different samples. (G) Photographs showing gauze and various composite hydrogels after blood contact followed by immersion in PBS for 1 min. (H,I) Representative small animal live imaging images of hydrogel scaffolds degradation. (The statistical analysis was measured by one-way ANOVA. *P < 0.05,** P < 0.01,***P < 0.001, and ns represents no statistical significance.)

Hydrogel evaluation on hemocompatibility demonstrated that all hydrogels exhibited hemolysis rates significantly below the 5% safety limit (Figure D,F), indicating that the erythrocyte membrane was not obviously damaged during the fabrication process and could be applied in the blood contacting field. The Blood Coagulation Index (BCI) can be used as an effective indicator to evaluate the coagulation performance of materials, and the lower BCI value indicates the better clotting performance. The results revealed that the clots of all hydrogel groups exhibited better clotting performance than the gauze control. Especially, the BCI value of 80 μmol/L Gp68 peptide group was the lowest, and the BCI value of the Gp68 group showed significantly enhanced coagulation performance compared with that of CMCS (Figure E,G). Overall, the results indicated that despite the slight cytotoxicity observed at high concentrations of Gp68 (80 μmol/L), the CMCS@Gp68 hydrogels possess excellent blood compatibility and strong hemostatic ability, which makes them suitable for wound care applications.

The in vivo degradation profile of the hydrogels was also followed through EFL-DYE-UF-ENE-R fluorescence labeling. The fluorescence signals of CMCS and CMCS@Gp68 hydrogels retained after subcutaneous implantation on mouse dorsum are shown in Figure H. At 2 h after implantation (day 0), high fluorescence signals were observed at the vicinity of the implant. We observed a gradual decrease in the fluorescence signal with time due to hydrogel degradation. A drop in the fluorescence intensity was more prominent on day 7 and an almost complete background signal was observed on day 14 (completely disappeared, almost total degradation of the material) (Figure H,I). Both types of hydrogels showed similar fluorescence signals, which meant that the Gp68 loading process did not significantly affect the degradation property of the CMCS matrix. Since the relevant therapeutic period for acute wound healing is within 14 days, the time line of the material degradation fits well with the wound healing process.

3.5. In Vivo Hemostatic Properties

The hemostatic properties of the hydrogels were tested in rats under physiological conditions using two classical rat hemorrhage models: liver incision and tail amputation (Figure E). With an increase in the Gp68 concentration, a decrease in the level of blood loss was observed (Figure A–D). The improvement in the hemostatic effect is due not only to the porous structure of the CMCS hydrogel matrix but also to positively charged Gp68, which may attract negatively charged blood cells and promote their aggregation. In addition, Gp68 may speed up the process of fibrin formation by activating coagulation factors. Furthermore, the SEM images showed that the concentration of Gp68 promoted the adhesion of blood cells and platelets in the pores of the three-dimensional network of the hydrogels (Figure F).

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In vivo hemostatic properties: (A) Tail incision and (B) liver amputation models. Blood loss of different hydrogels in the (C) liver incision and (D) tail amputation models. (The statistical analysis was measured by one-way ANOVA. ** P < 0.01, *** P < 0.001.) (E) The schematic illustration of the hemostatic ability of the hydrogels. (F) SEM images of blood cell and platelet adhesion on different hydrogels.

3.6. Promotion of Wound Healing by CMCS@Gp68 Hydrogels

P. aeruginosa is one of the most common pathogenic agents causing skin and soft tissue infections. In order to investigate the wound healing effect of CMCS-based hydrogel dressing, we constructed a full thickness skin wound model infected with P. aeruginosa (1 × 108 CFU). The wound was observed on days 0, 4, 7, 11, and 15, and the tissue was collected on days 0, 4, 7, 11, and 15 (Figure A). Macroscopically, the results showed that the rats in the CMCS@Gp68 hydrogel group had better healing effect on the wound than other groups on day 7 and day 15. The whole process of wound contraction over 15 days was recorded by color tracing (Figure B). Quantitatively, the rate of wound healing in the CMCS@Gp68 group was the highest (Figure C). In addition, on the seventh day, the bacterial load in the wound tissue of the CMCS@Gp68 group was significantly lower than that in the PBS group (P < 0.05, Figure D,E). Rats in the CMCS@Gp68 hydrogel group have better wound healing ability than commercial dressing Mupirocin.

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Promotion of wound healing by CMCS@Gp68 hydrogels: (A) Representative photographs of the infected full-thickness wounds after treatment with PBS, commercial mupirocin, CMCS, and CMCS@Gp68 dressing on days 0, 3, 7, and 14 (n = 3). (B) Schematic diagrams of wound closure progression traced over 15 days. Scale bar: 10 mm. (C) Quantitative analysis of wound closure rate (% of original wound). (D,E) Bacterial count from the wound tissues on day 7. (F) H&E and Masson staining images of wound defects in different groups on day 7 and day 15. (G) Skin thickness statistics. (H) Granulation tissue thickness statistics. (I) Collagen percentage statistics. (The statistical analysis was measured by one-way ANOVA.*P < 0.05, **P < 0.01, ***P < 0.001, and ns represents no statistical significance.)

The wound tissue was harvested for hematoxylin and eosin (H&E) staining and observation. Compared with the normal skin tissue, the wound tissue of P. aeruginosa-infected rats treated with PBS on the seventh day showed thin, loose, and immature granulation tissue with inflammatory cell infiltration and sparse deposition of fibroblasts and blood vessels. The wound untreated on the 15th day formed a moderately thick inflammatory granulation tissue and a large number of newly formed blood vessels, while the CMCS@Gp68 hydrogel treated group showed nearly normal skin tissue on the 15th day. The skin appendages such as hair follicles and sebaceous glands appeared again (Figure F). As appropriate collagen deposition and fibrosis are necessary in the remodeling process of wound tissue, the hydrogel’s ability to regulate these processes was investigated. The newly formed collagen in the wound tissue of all groups was observed by Masson’s trichrome staining. The results showed that on the 15th day, the newly formed collagen content in the CMCS@Gp68 group reached 74.1%, which was slightly higher than that in the normal group (P < 0.05, Figure I).

We next systematically assessed wound inflammation levels via immunohistochemical staining. It has been well-established that during the initial healing phase, inflammation acts as a double-edged sword functioning that removes necrotic tissue and pathogens as well as signaling molecules to recruit and activate subsequent repair cells to induce the proliferation phase. However, if inflammation is excessive or prolonged, it is switched from being constructive to destructive. As shown in Figure A, the IL-10 expression of the CMCS@Gp68 hydrogel group was significantly upregulated compared with that of the control group. As an anti-inflammatory cytokine, IL-10 can inhibit the expression of other pro-inflammatory cytokines and chemokines, suppresses the excessive response of the immune system, and prevents inflammation from running out of control, thereby protecting the tissue from inflammatory damage and maintaining the homeostasis of the immune system. Interestingly, the CMCS@Gp68 hydrogel group also showed downregulated expression of IL-1β and TNF-α (Figure B–D). All of the above results further indicated that the CMCS@Gp68 hydrogel could accelerate wound healing by multiple ways including bacterial clearance, downregulation of pro-inflammatory cytokines and chemokines expression, and upregulation of anti-inflammatory cytokines expression.

7.

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Immunohistochemical staining analysis: (A) Representative images of IL-1β, IL-10, and TNF-α staining. The quantification of IL-1β (B), IL-10 (C), and TNF-α (D) stained tissues on day 7. The statistical analysis was measured by one-way ANOVA. *P < 0.05, **P < 0.01,***P < 0.001, and ns indicates no statistical significance.

4. Conclusions

In summary, this study reports that the composite materials with greatly improved antibacterial properties can be prepared by integrating bacteriophage-derived antimicrobial protein into the hydrogel matrix. The hydrogel with excellent biocompatibility and rapid hemostatic performance can still potently accelerate wound healing by creating a protective microenvironment. By synergizing physical barrier function and precise biology behavior of antimicrobial activity, this bacteriophage-based hydrogel shows great potential in advanced wound management and promising clinical translation in regenerative medicine. The advantage of this system is that it can synergize the physical barrier function and precise biology behavior. This study provides an important theoretical basis and a feasible solution to break bacterial resistance and the clinical translation of new wound dressings.

Supplementary Material

ao5c12013_si_001.pdf (268.9KB, pdf)

Acknowledgments

This research was supported by 2023YQB046 Incubation Program of Young Doctors from Xinqiao Hospital. We also thank FigDraw (www.figdraw.com) for the assistance in creating images, including the Graphical Abstracts, Figure E, and the wound healing schematic in Figure .

The data supporting this study are available within the manuscript.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12013.

  • Diagram of the Gp68 structural simulation; observation results of transmission electron microscopy negative staining of host strain PA3 and host strain PA3 expressing Gp68; morphological and porosity analysis of CMCS and CMCS@Gp68 hydrogels; contact angle of the CMCS hydrogel and CMCS@Gp68 hydrogel (60 μmol/L); and thermogravimetric analysis of CMCS hydrogel and CMCS@Gp68 hydrogel (60 μmol/L) (PDF)

Both authors contributed equally to this work. Yujiao Huang: Data curation, investigation, formal analysis, and writingoriginal draft. Ruolan Liu: Methodology, investigation, and software. Xiaohua Shi: Supervision and writingreview and editing. Zichen Yang: Methodology, investigation, project administration, and writingreview and editing. Yuzhe Huang: Investigation, project administration, and writingreview and editing. Zeyuan Lei: Conceptualization, resources, supervision, writingreview and editing, and funding acquisition. Dongli Fan: Conceptualization, methodology, writingreview and editing, supervision, and funding acquisition.

Ethics approval and consent to participate. This study was approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing Army Medical University, Chongqing, China (AMUWEC2021580). All authors have approved the manuscript and agree for the submission.

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

ao5c12013_si_001.pdf (268.9KB, pdf)

Data Availability Statement

The data supporting this study are available within the manuscript.

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