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. 2025 Aug 28;34:102235. doi: 10.1016/j.mtbio.2025.102235

NIR/pH-responsive arginine-ε-polylysine/black phosphorus nanocomposites for synergistic therapy of bacterial infections

Qian Gao a,1, Ranran Fu a,1, Mengting Li c, Dongbo Guo a, Bingcheng Gan b, Tao Wang b,⁎, Maohua Chen a,⁎⁎
PMCID: PMC12423699  PMID: 40951363

Abstract

The escalating global burden of drug-resistant bacterial infections demands urgent innovation in highly effective, non-invasive antimicrobial strategies. Herein, we developed a multimodal near infrared (NIR) light-responsive bactericidal nanoplatform (BP@EPL-LA) with surface charge adaptability by loading black phosphorus nanosheets with L-Arg-grafted ε-poly(L-lysine) for targeted combinatorial therapy of subcutaneous abscesses. Under physiological conditions, BP@EPL-LA remained a neutral surface charge and demonstrated excellent biosafety both in vitro and in vivo. Upon encountering the acidic infectious environment, BP@EPL-LA rapidly switched to a positive surface charge, promoting deep biofilm penetration and strong electrostatic adhesion to negatively charged bacterial membranes. When irradiated by 660 nm laser, BP@EPL-LA mediated antibacterial photodynamic therapy (aPDT), generating reactive oxygen species (ROS) that oxidized arginine residues to enable controlled nitric oxide (NO) release. BP@EPL-LA effectively dispersed bacterial biofilms and demonstrated broad-spectrum antibacterial activity through the synergistic combination of ROS, NO, and EPL. Moreover, BP@EPL-LA showed remarkable therapeutic outcomes in a mouse model of subcutaneous Methicillin-resistant Staphylococcus aureus abscesses, simultaneously eradicating bacterial infection, reducing inflammation, and promoting tissue healing via enhanced vascularization and collagen deposition. Therefore, BP@EPL-LA overcomes antibiotic resistance barriers and presents a promising therapeutic strategy for effective subcutaneous abscess treatment by integrating NO-potentiated aPDT with EPL's bactericidal action.

Keywords: Nitric oxide gas therapy, Photodynamic therapy, Black phosphorus nanosheets, ε-Polylysine, Synergistic antibacterial effect

Graphical abstract

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Highlights

  • •

    A novel antibiotic-free nanoplatform (BP@EPL-LA) for treating drug-resistant bacterial infections and biofilm-associated abscesses is developed by combining black phosphorus nanosheets with a nitric oxide (NO)-donating polymer L-arginine-grafted epsilon-poly(L-lysine) (EPL-LA).

  • •

    Within this system, EPL-LA not only delivers potent antibacterial activity owing to its high NO donor density but also enhances nanoplatform stability while enabling pH-responsive bacterial targeting via surface charge adaptability.

  • •

    Under near-infrared (NIR) light irradiation, BP@EPL-LA achieves precise NO release, synergistically combining NO-enhanced photodynamic therapy (PDT) with EPL's inherent antimicrobial properties to completely eradicate both drug-resistant bacteria and mature biofilms in vitro and in vivo.

  • •

    BP@EPL-LA significantly accelerates abscess healing by simultaneously suppressing local inflammation and promoting tissue regeneration through enhanced vascularization and collagen deposition at the infection site.

1. Introduction

Bacterial infections-particularly those involving antibiotic-resistant strains-often progress to severe abscesses, a growing global health threat with rapidly escalating incidence rates [1]. Current abscess treatments like surgical drainage and systemic antibiotics carry a high risk of developing resistance and causing recurrent infections [2]. Furthermore, abscesses are often associated with biofilms, which shield bacteria from both antibiotics and the host immune system [3], thereby increasing antibiotic resistance and leading to intractable persistent infections [4]. To address these unmet clinical needs, the development of novel, highly effective therapeutic strategies against abscesses has become an urgent priority.

Antibacterial photodynamic therapy (aPDT) has emerged as a promising non-invasive approach to combat bacterial infections with a minimal risk of developing resistance [5]. It relies on light-activated photosensitizers to generate reactive oxygen species (ROS) in the presence of oxygen, which effectively eliminate planktonic bacteria and biofilms by oxidizing critical cellular components, including lipids, proteins, nucleic acid [5]. As photosensitizers, black phosphorus (BP) nanosheets stand out as two-dimensional single-element materials with exceptional properties for aPDT due to their superior biocompatibility in vivo [6], broad absorption across the visible to near-infrared (NIR) regions, and a high 1O2 quantum yield up to 0.91 [7]. Additionally, BP nanosheets promote skin tissue healing through the activation of endothelial cells [8]. Their unique structures and large specific surface area enable efficient adsorption of small molecules, drugs and polymers, making them as ideal multifunctional nanomaterials for abscess treatment [9,10]. However, BP nanosheets face stability challenges including atmospheric oxidation and solution aggregation that must be addressed for practical applications [11,12]. Moreover, their negatively charged surface can lead to electrostatic repulsion with bacterial cell membranes and biofilms, weakening their interaction and compromising antibacterial performance [10,13,14]. To address these issues, cationic polymers are usually grafted onto BP nanosheets to improve stability and enhance bacterial targeting [14,15]. ε-Polylysine (EPL), a polycationic peptide approved by the FDA, serves as a biocompatible and biodegradable platform for a range of applications in food preservation, drug delivery, and wound dressings [16,17]. EPL exerts broad-spectrum antibacterial effects through electrostatic adhesion to negatively charged bacterial cell membranes, inducing structural damage while avoiding resistance development [18]. Consequently, coating BP nanosheets with EPL can enhance stability, promote biofilm penetration, and amplify antibacterial efficacy through the EPL-aPDT combination. However, cationic surface charge may elevate toxicity to mammalian cells, particularly under light irradiation, due to enhanced cellular uptake [19]. Given that infection sites typically exhibit an acidic microenvironment (5.0–6.5) due to accumulated fermentation metabolites [19,20], developing charge-switchable BP nanocomposites could minimize off-target cytotoxicity while boosting antibacterial efficacy by responding to pH variations.

ROS generated during aPDT possess extremely short diffusion distance (tens to hundreds of nm) and lifespan (3.5 μs), which inevitably limits their antibacterial efficacy [21]. As a result, aPDT alone is often insufficient for complete eradication of microbial infections, necessitating combination therapy to enhance sterilization efficiency and promote tissue repair. Nitric oxide (NO)-based gas therapy has emerged as a promising treatment to combat multidrug resistant bacteria and biofilms through multiple mechanisms—including lipid peroxidation, DNA cleavage, and protein dysfunction—without inducing resistance [22]. NO can reduce EPS and bacterial adhesion by degrading cyclic dimeric guanosine monophosphate (c-di GMP) through phosphodiesterase activation, thereby promoting nanocomposite infiltration into biofilms [23]. Critically, NO potentiates aPDT by reacting with ROS to form highly bactericidal peroxynitrite (ONOO−) and other reactive nitrogen species (RNS) [24], while simultaneously depleting intracellular GSH and helping relieve hypoxia through inhibiting cellular respiration and promoting vasodilation [23,25]. Furthermore, NO accelerates infected tissue healing by stabilizing hypoxia-inducible factor-1α (HIF-1α) protein, modulating inflammatory responses, and stimulating myofibroblast differentiation, collagen deposition, and angiogenesis [26]. Unlike ROS, NO exhibits longer half-life (ca. 5 s) and wider diffusion radius (40–200 μm), enabling broader bactericidal coverage that compensates for ROS limitations [27,28]. The combined oxidative damage from ROS and NO also enhances biofilm and bacterial membrane permeability, increasing bacterial susceptibility to EPL. Therefore, combining NO-enhanced aPDT with EPL can improve abscess treatment outcomes with reduced side effects.

In this study, we designed a pH-responsive, charge switchable nanocomposite (BP@EPL-LA) for biofilm eradication and abscess tissue healing. BP@EPL-LA nanocomposite was synthesized by conjugating L-arginine (LA) to EPL (EPL-LA), followed by its electrostatic adsorption onto BP nanosheets (Scheme 1). Under normal physiological conditions, BP@EPL-LA exhibited a net-neutral surface charge, minimizing nonspecific binding and reducing damage to normal cells. In the acidic microenvironment of infected tissues (pH 5.5), the surface charge switched to a net-positive, promoting deep biofilm penetration and electrostatic interactions with bacterial membranes. Upon NIR activation, BP nanosheets generated ROS through a photodynamic process. Simultaneously, the biocompatible NO precursor EPL-LA released NO in a ROS-triggered manner, enabling spatiotemporally controlled gas therapy. Compared to small-molecule NO donors, macromolecular EPL-LA exhibited superior antibacterial activity, owing to its higher NO payload and enhanced bacterial membrane affinity. The released NO not only improved nanocomposite penetration into biofilms but also synergized with ROS and EPL to achieve potent bacteria and biofilm eradication. Furthermore, BP@EPL-LA accelerated abscess healing by promoting collagen deposition and angiogenesis while demonstrating excellent biosafety in vitro and in vivo. By integrating pH-responsive targeting, NO-enhanced aPDT and EPL-mediated antibacterial action, BP@EPL-LA offers an effective multimodal approach for treating drug-resistant bacteria and biofilm-associated abscesses without inducing drug resistance.

Scheme 1.

Scheme 1

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(A) Schematic illustration of the preparation of BP@EPL-LA nanocomposites. (B) Surface charge-adaptive BP@EPL-LA nanocomplex for effective eradication of multidrug-resistant bacteria and biofilms through NO-enhanced aPDT and EPL's antibacterial action.

2. Experimental section

2.1. Materials

The BP crystals were purchased from HWRK Chemical Co., Ltd. (Beijing, China) and stored in a dark Ar glove box. L-arginine (LA), ε-polylysine (EPL), 1,3-diphenylisobenzofuran (DPBF), 2,2,6,6-tetramethyl-4-piperidone (TEMP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxy succinimide (NHS), 2-morpholine ethanesulfonic acid (MES) buffer, L-ascorbic acid, and crystal violet (CV) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Griess reagent, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcein/PI cell viability/cytotoxicity assay kit were purchased from Beyotime (Shanghai, China).

2.2. Synthesis and characterization of EPL-LA

The EPL-LA polymer was synthesized through a one-step reaction as previously described [29]. Briefly, LA, EDC·HCl, and NHS were dissolved in 100 mL of MES buffer, and the pH was maintained at 6–7 using sodium hydroxide or hydrochloric acid during the 4-h reaction. EPL was then added into the mixture and stirred for 2 days at room temperature. The resulting product was purified by dialysis (MWCO = 1000 Da) followed by lyophilization. The structure of EPL-LA was confirmed by 1H nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). Subsequently, the minimal inhibitory concentration (MIC) was determined by the two-fold serial-dilution method [30]. Specifically, 180 μL of logarithmic-phase bacterial suspension was added to a 96-well round-bottom microplate. After incubation for 2 h, serial dilutions of EPL-LA solution were added to the bacterial suspension, which was then cultured at 37 °C. Each therapeutic agent was prepared in triplicate and all experiments were independently repeated twice. The MIC was defined as the lowest concentration of EPL-LA at which no visible microbial growth was detected.

2.3. Preparation of BP@EPL-LA nanocomposites

BP nanosheets were fabricated via liquid-phase exfoliation as previously described [31]. Briefly, 25 mg of bulk BP was dispersed in 25 mL of N-methylpyrrolidone, which had been purged with nitrogen to remove dissolved oxygen and prevent oxidation during exfoliation. The mixture was then sonicated in an ice bath for 8 h at 25 % amplitude with a 5 s on/5 s off pulse cycle. The low system temperature was maintained by the ice bath. After sonication, the dispersion was centrifuged at 600×g for 20 min to remove unexfoliated bulk BP. The supernatant containing BP nanosheets was carefully collected for further use.

The BP@EPL-LA nanocomposite was prepared through electrostatic bonding. Briefly, BP nanosheets and EPL-LA were dispersed in oxygen-free water under continuous stirring. After 6 h of stirring in the dark, the mixture was centrifuged at 1000×g (4 °C) for 20 min to remove unbound EPL-LA. The resulting product was washed with deionized water and designated as BP@EPL-LA. Similarly, EPL-modified BP nanosheets (BP@EPL) and Rhodamine B -labeled BP@EPL-LA were prepared under identical conditions.

2.4. Characterization of BP@EPL-LA nanocomposites

The morphologies and lattice structures of BP nanosheets and BP@EPL-LA nanocomposites were examined by transmission electron microscopy (TEM, JEM-2100F, Japan). The size distribution and zeta potential were measured using dynamic light scattering (DLS, Nano ZS, Malvern Co., UK). Successful exfoliation of BP nanosheets and EPL-LA conjugation were verified by Raman scattering spectroscopy (XplorRA ONE, France). UV–vis absorption spectra were acquired for BP, BP-LA and BP@EPL-LA using a UV–vis spectrophotometer (UV-2600, Japan) at room temperature. FTIR spectra were collected for LA, EPL, EPL-LA and BP@EPL-LA by FTIR spectrometry (Nicolet iS50, USA).

2.5. Stability and pH-responsive surface charge switchability of BP@EPL-LA nanocomposites

The stability of BP nanosheets significantly influences their phototherapy effect [32]. Therefore, the stability of BP@EPL-LA was assessed in an ambient humid environment. BP nanosheets, BP@EPL, and BP@EPL-LA were dispersed in deionized water and incubated at 25 °C under atmospheric conditions. Absorbance spectra were recorded using UV absorption spectroscopy every 24 h. The colloidal stability of BP@EPL-LA was further evaluated by DLS. BP@EPL and BP@EPL-LA were suspended in phosphate buffer solution (PBS) or cell culture medium containing 10 % fetal bovine serum (FBS). The suspensions were stored at room temperature in sealed, light-protected vials, and their sizes were measured at predetermined time intervals.

The pH-dependent surface charge of BP@EPL-LA was determined by DLS. BP@EPL and BP@EPL-LA were dispersed in a series of phosphate buffers, covering a pH range from 7.4 to 5.0. After 4 h of co-incubation, the zeta-potential of BP@EPL and BP@EPL-LA was measured by DLS.

2.6. In vitro1O2 and NO generation ability of NIR-irradiated BP@EPL-LA nanocomposites

The generation of 1O2 during the photodynamic process was monitored using DPBF as a chemical probe [21]. Briefly, 60 μL of DPBF ethanol solution was added into 600 μL of PBS buffer (pH 5.5) containing either BP@EPL or BP@EPL-LA. After NIR irradiation (660 nm, 0.5 W/cm2) for varying durations, 100 μL of the mixture was transferred to a 96-well plate, and DPBF degradation induced by 1O2 was quantified by measuring absorbance at 410 nm using a microplate reader. Additionally, control experiments were conducted under three conditions: (1) BP@EPL or BP@EPL-LA without NIR irradiation, (2) BP@EPL or BP@EPL-LA with NIR irradiation in the presence of L-ascorbic acid, and (3) EPL, EPL-LA and BP nanosheets with or without NIR irradiation for comparison. To further confirm 1O2 generation from NIR-irradiated BP@EPL and BP@EPL-LA, electron spin resonance (ESR) analysis was performed using TEMP as a trapping agent. Specifically, 10 μL of TEMP in acetonitrile was added into 100 μL of BP@EPL and BP@EPL-LA suspensions (250 μg/mL, pH 5.5), respectively. After 15 min of NIR irradiation, the spectra of spin-trapped 1O2 were detected using an ESR spectrometer.

The generation of NO from BP@EPL-LA under NIR irradiation was quantitatively analyzed using the Griess reagent [1]. Briefly, 200 μL aliquots of BP@EPL or BP@EPL-LA suspensions in PBS (pH 7.4 or 5.5) were mixed with Griess reagent in a 96-well plate. After 20 min of dark incubation at 37 °C, samples were either irradiated with a 660 nm NIR laser (0.5 W/cm2) or kept in the dark as controls. At a predetermined time points, absorbance at 548 nm was recorded by a microplate reader. NO concentrations were calculated by substituting the measured absorbance values into the standard curve of NaNO2.

2.7. In vitro biocompatibility of BP@EPL-LA nanocomposites

The cytotoxicity of BP@EPL-LA was investigated in L929 cells using calcein/PI live/dead staining and an MTT assay [15]. According to the manufacturer's instructions, live cells were stained green by calcein AM, while dead cells were stained red by PI. Briefly, 500 μL of L929 cell suspensions (1.0 × 105 cells/mL) were seeded into a 24-well plate and incubated at 37 °C for 24 h. The medium was then replaced with 500 μL of fresh medium containing varying concentrations of BP@EPL or BP@EPL-LA nanocomposites (0–400 μg/mL). After 24 h of incubation, the cell medium was removed, and 300 μL of the prepared dye was added to each well under dark conditions. After staining for 15 min, the fluorescence images were acquired using a fluorescence microscope. The cell viability was quantitatively assessed by a MTT assay. Briefly, L929 cells were incubated with different concentrations of BP@EPL or BP@EPL-LA at 37 °C for 24 h. The cell medium was then removed, and 100 μL of MTT (0.5 mg/mL) solution was added into each well, followed by a further 4 h of incubation. The supernatants were aspirated, and 100 μL of DMSO was carefully added to each well to dissolve the formed formazan. The plate was gently shaken for 15 min, and the absorbance at 490 nm was measured using a microplate reader.

Moreover, cell proliferation and migration were assessed using a scratch assay [17]. Briefly, L929 cells (5 × 105 cells/well) were seeded in a 12-well plate and cultured at 37 °C until 90 % confluence was reached. A pipette tip was employed to create uniform linear scratches in each well, which were then washed with PBS. BP@EPL or BP@EPL-LA were added to each well for co-incubation. At scheduled time points, the medium was removed, and cells were wished three times with PBS and stained with calcein AM. The cell growth and migration were recorded using an inverted fluorescence microscope. The migration rate was calculated using the following formula: Migration rate (%) = (W0-W1)/W0 × 100 %

where W0 was the width of the gap at 0 h and W1 was the width of the gap at 24 or 48 h.

The in vitro hemolytic activity of BP@EPL-LA was evaluated using fresh blood from female Balb/c mice (6 weeks old) [17]. Briefly, 1 mL of whole blood was diluted with 9 mL of PBS and centrifuged at 3000 rpm for 15 min to isolate red blood cells (RBCs). The RBCs were rinsed ( × 4) and resuspended at 4 % v/v in PBS. Subsequently, 200 μL of RBCs were mixed with 800 μL of BP@EPL-LA nanocomposites suspensions at varying concentrations (50, 100, 200 and 400 μg/mL) and incubated at 37 °C for 4 h in the dark. Deionized water and PBS served as the positive and negative controls, respectively. After centrifugation at 10,000 rpm for 15 min, the absorbance of the supernatant at 542 nm was measured using a UV–vis spectrophotomer. The ratio of hemolysis was calculated according to the following equation:

Hemolysis (%) = (Asample-APBS)/(Awater-APBS) × 100 %

2.8. In vitro antibacterial efficacy of BP@EPL-LA nanocomposites

The antibacterial capacities of nanocomposites were evaluated against Gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Pseudomonas aeruginosa (PA) bacteria following a published protocol [28]. MRSA and PA are strategically selected due to their clinically critical yet fundamentally distinct resistance and biofilm formation mechanisms [33]. The ability of BP@EPL-LA nanocomposites to demonstrate efficacy against both organisms not only validates broad-spectrum potential but also significantly enhances their clinical translatability. Briefly, 500 μL of BP@EPL or BP@EPL-LA nanocomposites solutions at varying concentrations (0–1000 μg/mL) were mixed with 500 μL bacterial suspension (107 CFU/mL, pH 5.5) in a 24-well culture plate. After 30 min of dark incubation, the mixtures were exposed (or not) to 660 nm NIR laser (0.5 W) for 15 min. Subsequently, 100 μL of serially diluted bacterial solution (104 dilution) was dispersed onto agar plates and incubated at 37 °C for 18–24 h. Colony-forming units (CFUs) were quantified, with untreated bacteria serving as the control. All experiments were performed in triplicate.

To further substantiate the bactericidal activity of BP@EPL and BP@EPL-LA, bacterial cells (both MRSA and PA) were harvested by centrifugation (6000 rpm, 3 min) and stained with SYTO 9/PI dyes for 30 min in the dark following a published protocol [34]. The stained bacteria were then washed with sterile PBS twice and visualized with a confocal laser scanning microscope (CLSM). Dead bacterial were stained with red fluorescence (PI), while viable cells were stained with green fluorescence (SYTO 9).

Morphological changes in bacterial cell membranes post-treatment were detected by scanning electron microscope (SEM). The treated bacterial cells were harvested by centrifugation and fixed with 4 % glutaraldehyde overnight at 4 °C. Subsequently, the bacterial cells were dehydrated with a series of ethanol solutions (30–100 %), with 10 min incubation at each concentration. Finally, samples were dried at room temperature and sputter-coated with gold prior to SEM imaging.

Following antibacterial treatment, protein leakage and ATP level changes were analyzed according to established methods [35]. To quantify protein release, bacterial cells were first washed with PBS to remove extracellular proteins, and then treated with various samples. After centrifugation (12000 rpm, 5 min, 4 °C), the supernatant was collected and protein concentration was determined using an enhanced BCA protein assay kit. For ATP measurement, treated bacteria were pelleted by centrifugation following the standard antibacterial protocol. The bacterial pellets were lysed in 200 μL lysis buffer by boiling for 15 min, followed by centrifugation (1000×g, 15 min, 4 °C). ATP levels in the resulting supernatant were then measured using an enhanced ATP detection kit.

The long-term bacteriostatic effect of BP@EPL-LA was also tested by the growth curve method. Specifically, 100 μL of MRSA and PA suspensions (≈105 CFU/mL) were mixed with 100 μL of PBS, BP, BP@EPL, BP@EPL-LA, or NIR-pre-irradiated BP@EPL-LA at pH 5.5. After incubation for 3h and 6 h, the number of bacteria was quantified using the plate-counting method.

2.9. In vitro anti-biofilm efficacy of BP@EPL-LA nanocomposites

The mature biofilms were established using modified published protocols [36]. Briefly, MRSA or PA suspensions were inoculated in 48-well plates and incubated statically at 37 °C for 48 h. After removing planktonic cells and washing with PBS, the developed biofilms was treated with PBS, BP@EPL and BP@EPL-LA (250 μg/mL). Following 4 h of co-culture, the biofilms were irradiation with or without 660 NIR laser for 15 min. Subsequently, the medium was removed and the biofilm was washed with PBS. The remaining biofilm biomass was observed and quantified by CV staining, and bacterial viability within biofilms was visualized using CLSM following SYTO 9/PI live/dead staining [37].

2.10. In vivo abscess-treating efficacy of BP@EPL-LA nanocomposites

All animal procedures were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Hainan University (HNUAUCC-2023-00100). The in vivo antibacterial efficacy of BP@EPL-LA nanocomposites was investigated by a MRSA-infected model. Bacterial infection model was established by subcutaneous injection of MRSA (30 μL, 5 × 108 CFU/mL) into the shaved and disinfected back of 6-week-old female Balb/c mice. One day after infection, 30 MRSA-infected mice were randomly divided into five groups (control, BP@EPL, BP@EPL + NIR, BP@EPL-LA, BP@EPL-LA + NIR groups). Subsequently, 50 μL of PBS, BP@EPL (200 μg/mL), and BP@EPL-LA (200 μg/mL) were administered via subcutaneous injection at the infected abscess site. After 4 h and 24 h of treatment, the mice in the BP@EPL + NIR and BP@EPL-LA + NIR groups were anesthetized and exposed to 660 nm NIR laser (0.5 W/cm2) for 15 min. The abscesses of each mouse were photographed, and body weights were monitored throughout the study. After 12 days post-treatment, mice were euthanized, and the infected tissues were excised and homogenized in sterile PBS for colony counting. For histological evaluation, excised skin tissues were immersed in 10 % neutral formalin for 24 h, paraffin-embedded and sectioned (5 μm thickness). Tissue sections were stained with hematoxylin-eosin (H&E), Masson's trichrome and CD31 respectively. After various treatments, main organs (heart, liver, lung, kidney, and spleen) were collected and fixed in a 4 % formaldehyde solution for H&E staining. Additionally, blood samples were collected for routine blood tests, as well as liver and kidney function assessments.

2.11. Statistical analysis

The experimental data were presented as means ± standard deviation (SD), where each experiment was repeated at least three times. Statistical significance (p < 0.05) was evaluated by using Student's t-test when only two groups were compared. If more than two groups were compared, evaluation of significance was performed using one-way analysis of variance followed by Bonferroni's post hoc test. Statistical significance was set at p < 0.05.

3. Results and discussion

3.1. Synthesis and characterization of polymeric NO donor EPL-LA

The NO-releasing polymer EPL-LA was synthesized in 41 % yield by a nucleophilic reaction between EPL and LA, using EDC/NHS as coupling agents (Fig. S1A) [29]. The chemical structure of EPL-LA was confirmed by 1H NMR and FTIR. In the 1H NMR spectrum (Fig. S1D), characteristic peaks at 3.28–3.43 ppm, 1.43–1.92 ppm, and 2.25–2.55 ppm corresponded to three methylene groups of Arg, confirming the successful grafting of L-Arg onto EPL. The Arg grafting rate (∼14.7 %) was determined by the ratio of two integral signals at 2.25–2.55 ppm (from L-Arg) and 3.55 ppm (from EPL). The FTIR spectrum revealed a band at 1630 cm−1 (Fig. S1E), attributing to the stretching vibrations of the guanido group. The MIC values of EPL and EPL-LA were showed in Table S1. Notably, EPL-LA demonstrated comparable MIC value to EPL, indicating preserved antimicrobial potency against Gram-positive MRSA and Gram-negative PA.

3.2. Preparation and characterization of BP@EPL-LA nanocomposites

The preparation process of BP@EPL-LA nanocomposites was schematically illustrated in Fig. 1A. Bulk BP was first exfoliated into nanosheets using a liquid exfoliation method as previous described [38]. The resulting BP nanosheets were then functionalized with EPL-LA through electrostatic adsorption, which enhanced their circulation stability and endowed them with bacteria-targeting functionality. To optimize the mass ratio of EPL-LA to BP, we systematically evaluated the surface charge characteristics of the nanocomposites. As shown in Fig. 1B, the zeta potential increased from −32.6 to +17.9 mV with increasing EPL-LA content. Surface charge critically influenced nanomedicine stealth properties in vivo, and the neutral nanomedicines (Zeta-potential ≈ ±10 mV) possessed higher biocompatibility and longer circulation time compared to negatively or positively charged nanomedicines [39]. Therefore, BP@EPL-LA with a feed ratio of 10:100 (Zeta-potential = 4.1 mV) was selected for the subsequent experiments. The size distribution and morphology of BP@EPL-LA nanocomposites were investigated by DLS and TEM. The TEM image of BP nanosheets revealed a typical sheet-like morphology with a lateral size ranging from 100 to 200 nm (Fig. 1C), while DLS indicated a hydrodynamic size of 169 nm (Fig. 1F). The photosensitizer BP nanosheets were then surface-functionalized with EPL-LA to form the BP@EPL-LA nanocomposites, which simultaneously conferred pH-responsive bacterial targeting, NO release capability, and inherent EPL-mediated antibacterial activity. BP@EPL-LA exhibited similar morphology with a slight increase in hydrodynamic size to 183 nm. As shown in Fig. 1D, the zeta potential of BP nanosheets increased sharply from −31.6 mV to 1.6 mV upon EPL-LA loading. As shown in Fig. 1C, HR-TEM displayed lattice fringes with d-spacing of 0.26 nm (BP) and 0.24 nm (BP@EPL-LA), matching the (040) crystallographic plane of BP [40]. The structural transformation of BP nanosheet to BP@EPL-LA nanocomposite was also verified by the Raman spectra. As shown in Fig. S2, BP nanosheets displayed three characteristic peaks at 360 cm−1 (Ag1, out-of-plane mode), 437 cm−1 (B2g, in-plane mode) and 465 cm−1 (Ag2, in-plane mode) [40]. In comparison, BP@EPL-LA nanocomposites exhibited similar peaks with a slight red shift, likely attributable to minor thickness changes induced by EPL-LA surface coating. Collectively, these data verified the successful construction of BP@EPL-LA nanocomposites.

Fig. 1.

Fig. 1

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Synthesis and characterization of the BP@EPL-LA nanocomposites for antibacterial applications. (A) Schematic illustration of the synthetic process of BP@EPL-LA. (B) Surface zeta potentials of BP@EPL-LA nanocomposites with varying feed mass ratio of EPL-LA to BP at pH 7.4. (C) TEM (left) and HR-TEM (right) images of BP nanosheets and BP@EPL-LA nanocomposites. (D) Zeta potentials of BP nanosheets, BP@EPL and BP@EPL-LA nanocomposites at pH 7.4. (E) Hydrodynamic diameters of BP nanosheets, BP@EPL and BP@EPL-LA nanocomposites. (F) pH-dependent zeta potential profiles of BP@EPL and BP@EPL-LA nanocomposites. Error bars represent SD (n = 3).

Generally, BP nanosheets are instability when exposed to water or air [41]. Various stabilization strategies, including covalent modification, surface inactivation, and capping layer protection, have been developed to improve their aqueous stability [41,42]. When incubated in an aqueous solution, unmodified BP nanosheets underwent rapid degradation with absorbance decreasing substantially within 48 h, while BP@EPL-LA nanocomposites maintained significantly higher absorbance, demonstrating markedly improved aqueous stability (Fig. S3). Moreover, good dispersion stability represents a critical requirement for both fundamental studies and clinical translation of nanotherapeutics. Prior to biomedical applications, comprehensive evaluation of nanocomposite stability in physiologically relevant media is essential [32,40]. The stability of BP@EPL-LA was assessed in PBS and cell culture media with 10 % FBS (simulating in vitro and in vivo environments) through 24 h of monitoring via DLS. As shown in Fig. S4, the diameters of BP@EPL and BP@EPL-LA nanocomposites remained relatively constant, indicating superior stability in physiological environments.

Furthermore, the pH-induced charge-conversion property of BP@EPL-LA was investigated through zeta potential measurements. As shown in Fig. 1F, when the environmental pH decreased from 7.4 to 5.0, the zeta potential of BP@EPL-LA switched from net-neutral to strongly positive (23.5 mV). This switching behavior could be attributed to the protonation of phosphate on the BP nanosheets and amine groups on the EPL-LA under acidic conditions.

3.3. Evaluation of NIR-triggered ROS and NO release from BP@EPL-LA

As is well known, BP nanosheets can efficiently generate ROS under NIR laser irradiation in aqueous media [7,43]. The photodynamic efficacy of BP@EPL-LA nanocomposites was investigated using DPBF as a 1O2 indicator. BP@EPL-LA nanocomposites led to a gradual decrease in DPBF absorbance at 410 nm with increasing time (Fig. 2A), indicating 1O2 generation. To quantitatively assess 1O2 production, the absorption peaks of DPBF against irradiation time was plot. As shown in Fig. 2B, the absorption intensity of DPBF displayed negligible changes in PBS, BP nanosheets, EPL + NIR and EPL-LA + NIR groups. Approximately 46 % of DPBF was degraded by BP@EPL under 15 min of illumination, which was similar to that of the BP nanosheets + NIR group, indicating that the ROS generation capacity of BP nanosheets was not influenced by EPL loading. In comparison, the percentage of DPBF degradation reduced to 35.6 % for BP@EPL-LA, likely due to ROS consumption in the sequential reaction with LA. Notably, DPBF decomposition was significantly inhibited by adding L-ascorbic acid (a ROS scavenger) to the BP@EPL-LA + NIR group. EPR analysis with TEMP as a radical trapper provided complementary evidence of 1O2 generation. As shown in Fig. 2C, characteristic TEMP-1O2 triplet signals (1:1:1) appeared in both BP@EPL-LA and BP@EPL groups under irradiation, whereas no signals were detected in the absence of irradiation. The triplet signal intensity was higher in the BP@EPL group than in the BP@EPL-LA group, consistent with the DPBF degradation assay results.

Fig. 2.

Fig. 2

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NIR-triggered ROS and NO release from BP@EPL-LA nanocomposites. (A) Time-dependent absorbance spectra of BP@EPL-LA reacting with DPBF under NIR irradiation (660 nm, 500 mW/cm2) in PBS. (B) Kinetic profiles of DPBF oxidation by BP@EPL-LA under varied conditions. (C) ESR spectra of BP nanosheets and BP@EPL-LA nanocomposites under NIR irradiation using TEMP as the 1O2 spin trap. (D) Schematic illustration of the mechanism of NO generation from EPL-LA. (E) NO release from BP@EPL-LA under different conditions. (F) NO release profile from BP@EPL-LA nanocomposites under intermittent NIR irradiation (G) NO release profiles from BP@EPL-LA nanocomposites at various concentrations under NIR irradiation. (H) Zeta potentials of the bacteria before and after incubation with BP@EPL and BP@EPL-LA (1 mg/mL) at pH 7.4 and 5.5. (I) CLSM images showing the interaction of MRSA with BP@EPL-LA nanocomposites (1 mg/mL) at pH 7.4 and 5.5. Error bars represent SD (n = 3).

Recent efforts have focused on developing L-Arg-based nanoplatforms for precise, controlled NO delivery using exogenous triggers such as ROS [44]. Interestingly, NO can remarkably inhibit bacterial growth in skin infections at higher concentrations (>1 μm), while promoting wound healing through myofibroblast proliferation and collagen production during skin reconstruction at lower concentrations (<1 μm) [45,46]. We quantified NO release from the nanocomposites using Griess agents, which detect nitrite (a stable NO oxidation product in solution) through formation of a characteristic red-violet chromophore (λmax ≈ 540 nm) [47]. BP@EPL-LA demonstrated remarkable light-triggered NO generation (Fig. 2D and S6), reaching a cumulative concentration of 5.78 μM after 15 min of NIR irradiation (Fig. S5), which met the antibacterial requirements according to previous reports [46]. Control experiments revealed negligible NO production from either BP@EPL + NIR or BP@EPL-LA groups. When a ROS scavenger L-ascorbic acid was added, BP@EPL-LA displayed negligible NO production under NIR irradiation. These results indicated that NIR irradiation and NO donor were two critical factors to generate ROS and NO by a cascade reaction (Fig. 2E). In addition, BP@EPL-LA exhibited rapid on/off switching behavior in response to intermittent NIR irradiation (Fig. 2F), with immediate NO release initiation upon NIR activation and prompt cessation when irradiation stopped. After three on/off cycles, the cumulative NO release reached 5.98 μM at the 30 min time point, comparable to that of the continuous irradiation group. BP@EPL-LA also showed a concentration-dependent NO production feature (Fig. 2G), enabling fine-tuned NO release through adjustment of nanocomposite concentration, irradiation duration and NIR on/off cycling. These features made BP@EPL-LA as a promising platform for spatially and temporally controlled NO delivery in biomedical applications.

3.4. Bacteria targeting of BP@EPL-LA nanocomposites in vitro

The microenvironment at the infected area is known to be weakly acidic compared to healthy tissues [48]. Bacteria maintain a consistently negative surface charge (−20 to −30 mV) under both acidic and neutral conditions, attributed to the abundance of lipopolysaccharides and teichoic acids on their cell walls (Fig. 2H) [13]. As demonstrated above, BP@EPL-LA acquired a positive potential of 23.4 mV under acidic conditions through protonation of EPL-LA's free amine groups (Fig. 1F). When BP@EPL-LA was introduced to bacteria at pH 7.4, the resulting BP@EPL-LA/bacteria composite retained a negative zeta potential. However, when the pH lowered to 5.5, the composite's zeta potential switched to positive, confirming strong electrostatic interactions between BP@EPL-LA with bacteria (Fig. 2H). To further validate the pH-responsive targeting capability of BP@EPL-LA, its interactions with MRSA at different pH levels were examined by CLSM. As shown in Fig. 2I, only sparse red fluorescence was observed around the bacteria at pH 7.4, whereas abundant BP@EPL-LA were bound to MRSA at pH 5.5. These findings indicated that BP@EPL-LA exhibited pH-dependent surface charge switchability, facilitating bacterial binding at the site of infection and underscoring its potential for precise therapeutic applications.

3.5. In vitro antimicrobial activity of BP@EPL-LA

Encouraged by the acid-responsive targeting and cascade production of the photodynamic effect and ROS-driven NO generation, the antimicrobial properties of BP@EPL-LA nanocomposites were systematically assessed against MRSA and PA under both irradiated and non-irradiated conditions. In the absence of NIR irradiation, BP@EPL-LA exhibited very weak antibacterial activity against MRSA and PA, even at high concentrations up to 500 μg/mL, regardless of the pH conditions (7.4 or 5.5) (Fig. S7). When exposed to laser irradiation, BP@EPL-LA showed a potent dose-dependent bactericidal effect, which was further amplified under acidic conditions. Notably, BP@EPL-LA demonstrated significantly higher bactericidal activity than BP@EPL under irradiation, suggesting a synergistic antibacterial effect of aPDT and NO. Under acidic conditions, the positively charged BP@EPL-LA adhered electrostatically to negatively charged bacteria, shortening the diffusion distance of 1O2 and NO radicals to the bacteria and thus improving bacterial killing efficacy. When the concentration of BP@EPL-LA reached 250 μg/mL at pH 5.5, over 95 % of the MRSA and PA were killed. To further clarify the antibacterial effects of different formulations, 250 μg/mL of BP@EPL-LA was chosen for subsequent sterilization tests.

The antibacterial activities of BP@EPL and BP@EPL-LA against MRSA and PA were evaluated using bacterial colony counting and Live/Dead staining assays [49]. Fig. 3A showed representative images of bacterial colonies and the corresponding quantitative analysis of residual colonies after various treatments. Without NIR irradiation, both BP@EPL and BP@EPL-LA displayed weak bactericidal performance. Additionally, no bactericidal activity was observed for NIR irradiation alone (PBS + NIR). In contrast, the BP@EPL exhibited a dramatic reduction in colonies after 15 min of 660 nm NIR irradiation with a power density of 0.5 W/cm2, with killing efficiencies of 84.3 % (MRSA) and 70.6 % (PA) (Fig. 3B and C), mainly due to the targeted photodynamic effect. The BP@EPL-LA + NIR group demonstrated even greater efficacy, with only a few viable colonies remaining (99.87 % inactivation for MRSA and 96.52 % for PA), underscoring NO's critical role in enhancing aPDT. The Live/Dead staining assay was also performed, in which live bacteria stained green and dead or damaged bacteria fluoresced red. Consistent with colony counting results, negligible bacterial killing was observed in non-irradiated groups, while BP@EPL + NIR induced intense red fluorescence due to aPDT (Fig. 3D). Strikingly, almost all MRSA and PA stained red in the BP@EPL-LA + NIR group, indicating that nearly all bacteria were killed by the synergistic action of aPDT and NO. These findings highlighted the superior antibacterial efficacy of BP@EPL-LA, which resulted from a multifaceted mechanism integrating NO-enhanced aPDT, EPL's inherent antimicrobial activity and pH-dependent bacterial targeting. The synergistic interplay of these factors resulted in highly efficient and selective bacterial elimination at the acidic infection site.

Fig. 3.

Fig. 3

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In vitro antibacterial activity of BP@EPL-LA. (A) Plate Photographs of MRSA and PA treated with PBS, BP@EPL and BP@EPL-LA with or without laser irradiation at pH 5.5. Statistical analysis of antibacterial activity against MRSA (B) and PA (C) following different treatments, assessed via agar plate assay. (n = 5, ∗: p < 0.05 vs other groups) (D) Representative CLSM images of MRSA and PA viability after different treatments, stained with a live/dead viability kit (red: dead cells; green: live cells). (E) SEM images highlighting morphological alterations in MRSA and PA following treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To elucidate the synergistic antibacterial mechanisms of NO-enhanced aPDT, the morphological changes and protein leakage of MRSA and PA were investigated. SEM images revealed the adhesion of BP@EPL and BP@EPL-LA nanocomposites to bacteria (Fig. 3E). After co-cultured with BP@EPL and BP@EPL-LA without irradiation, MRSA and PA maintained their complete spherical or rod-like shapes with smooth surfaces. When exposed to 660 nm NIR irradiation, bacterial treated with BP@EPL exhibited deformed shapes with sunken and wrinkled membrane structures due to the membrane disruption and cytoplasm leakage caused by aPDT. The damage proved even more pronounced in the BP@EPL-LA + NIR group, where complete cellular collapse and structural disintegration were observed. This enhanced destruction suggested that NO potentiated ROS-mediated membrane disruption, leading to accelerated bacterial death.

As ATP and proteins are crucial intracellular components in bacterial cells, their release serves as a reliable indicator of membrane damage [35]. Fig. S10A and B demonstrate significant protein leakage from MRSA and PA following treatment with BP@EPL-LA under NIR irradiation, showing 3.95-fold and 5.33-fold increases in released protein content compared to the PBS control group, respectively, which confirms substantial membrane disruption. Concurrent measurements of intracellular ATP levels (Fig. S8) revealed reductions of up to 75 % in both bacterial strains after BP@EPL-LA treatment with NIR irradiation compared to untreated controls, indicating severe impairment of cellular energy metabolism. These findings collectively demonstrate that while BP@EPL-LA achieves targeted bacterial binding through electrostatic interactions in acidic environments, causing minor membrane perturbations that are insufficient to kill bacteria, its combination with NIR irradiation generates abundant 1O2 and NO in situ. These reactive molecules synergistically induce catastrophic membrane damage (evidenced by extensive protein leakage), disrupt ATP synthesis and maintenance, and ultimately lead to bacterial cell death.

Clinically, even with appropriate antimicrobial treatment, up to 30 % of patients may suffer from chronic or recurrent skin and soft tissue infections [50]. The long-term antibacterial activity of BP@EPL-LA was assessed by a bacteriostasis test. As shown in Fig. S9, BP nanosheets did not displayed obvious bacteriostatic action against MRSA and PA, whereas EPL- and EPL-LA-modified composites significantly suppressed bacterial proliferation. Notably, BP@EPL-LA pre-treated with NIR irradiation exhibited bacterial inhibition comparable to BP@EPL. It was suggested that EPL and its derivatives could not only target bacteria but also inhibit bacterial growth after release from nanocomposites.

3.6. In vitro antibiofilm of BP@EPL-LA

The pathogenic bacteria are prone to form multicellular biofilms, which contribute to drug resistance and complicate infection treatment [3]. Given the promising antibacterial effect of BP@EPL-LA, its NIR-driven biofilm dissipation capacity was evaluated using CV staining assay. Compared to the PBS group, BP@EPL and BP@EPL-LA slightly increased biofilm removal efficacy (Fig. 4A–C). Under NIR irradiation, BP@EPL eliminated 49 % of MRSA biofilms and 53 % of PA biofilms, while BP@EPL-LA removed over 80 % of both MRSA and PA biofilms. These results indicated that NIR-induced 1O2 and NO release from BP@EPL-LA could effectively eliminate the biofilm biomass.

Fig. 4.

Fig. 4

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In vitro antibiofilm activity of BP@EPL-LA. (A) Crystal violet staining of MRSA and PA biofilms after different treatments. (B–C) Quantitative analysis of the residual MRSA and PA biofilm biomass. (n = 5, ∗: p < 0.05 vs other groups) (D) CLSM images of MRSA and PA biofilms stained with calcein-AM/PI following treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The antibiofilm effect of BP@EPL-LA was further assessed using Live/Dead staining, which distinguished live (green fluorescence) and dead (red fluorescence) bacteria. As exhibited in 3D images (Fig. 4D and Fig. S10), BP@EPL or BP@EPL-LA treatment led to weak red fluorescence in both MRSA and PA biofilms, indicating limited membrane damage, attributable to EPL's inherent antibacterial activity. In contrast, BP@EPL + NIR treatment generated intense red fluorescence throughout both biofilms, demonstrating the broad-spectrum bactericidal performance of aPDT against biofilm-embedded bacteria. Most strikingly, BP@EPL-LA + NIR treatment resulted in near-complete red fluorescence in both biofilms, revealing that NO-enhanced aPDT achieved superior biofilm eradication through potent bactericidal action.

3.7. In vivo antibacterial activity of BP@EPL-LA nanocomposites

Encouraged by the in vitro results—which revealed that BP@EPL-LA nanocomposites exhibited pH-responsive properties, favorable biocompatibility, and excellent synergistic antibacterial activity through NO-enhanced aPDT and EPL's antimicrobial action—We further evaluated their therapeutic potential in a mouse model of subcutaneous MRSA abscesses [51]. Fig. 5A outlined the experimental design, from establishing the subcutaneous abscess model to treating and evaluating the therapeutic outcomes. Fig. 5B and C showed representative macroscopic images and quantitative measurements of abscess size changes after different treatments at designated time points. Throughout the treatment, persistent ulcerations and infected abscess were observed in the PBS + NIR, BP@EPL and BP@EPL-LA groups, with abscess area ratios exceeding 30 % by day 12, suggesting incomplete bacterial eradication. In contrast, BP@EPL + NIR group exhibited controlled abscess progression, with the abscess area ratio drastically decreasing to 12.7 % by day 12—a significantly faster recovery compared to both the control and non-NIR groups. However, a scab and swelling were still present in the infected area, indicating that aPDT alone had limited antibacterial efficacy and was insufficient to completely cure MRSA-induced subcutaneous infection. Strikingly, mice treated with BP@EPL-LA + NIR exhibited the most rapid recovery. By day 12, the infected abscess was almost fully healed, with no visible eschar or swollen abscess. This superior healing likely stemmed from the combined action of NO-enhanced aPDT and EPL-mediated antibacterial activity, which not only eradicated bacterial infections but also stimulated keratinocyte proliferation and fibroblast migration.

Fig. 5.

Fig. 5

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Evaluation of BP@EPL-LA therapeutic efficacy against MRSA-induced subcutaneous abscesses in a murine model. (A) Schematic diagram of subcutaneous abscess model development and the treatment workflow. (B) Time-course photographic record of MRSA-infected subcutaneous abscesses in mice treated with different formulations. (C) Quantitative analysis of abscess area over time following treatment. (n = 5, ∗: p < 0.05 vs other groups) (D) Representative MRSA colony formation on agar plates cultured from abscess tissues following treatment. (E) The corresponding colony number of surviving MRSA on the agar plates. (n = 5, ∗: p < 0.05 vs other groups) (F–G) Representative H&E, Masson's trichrome and CD31 immunohistochemical staining images of abscess tissues at day 10 post-treatment. Error bars represent SD (n = 5).

To further assess the superior synergistic antibacterial efficacy of ROS, NO and EPL, infected tissues were harvested after 4 days of treatment, and the number of remaining bacteria was determined by the standard plate counting method. Compared to the PBS + NIR group, bacterial colony counts were reduced to 95.36 % in the BP@EPL group, 84.58 % in the BP@EPL-LA group, 12.35 % in the BP@EPL + NIR group, and 0.07 % in the BP@EPL-LA + NIR group (Fig. 5D and E). These results indicated that BP@EPL-LA nanocomposites could effectively eliminate bacterial at the infection site, owing to the synergistic therapy of ROS, NO and EPL.

The synergistic antibacterial effects of ROS, NO and EPL were further investigated by histological analysis. Tissues surrounding infected sites were collected and examined by H&E staining and Masson's trichrome staining. As displayed in Fig. 5F, PBS + NIR, BP@EPL and BP@EPL-LA groups exhibited severe inflammatory cell infiltration and incomplete epidermal layers. In the BP@EPL + NIR group, inflammatory cell numbers decreased significantly, but the epithelial layer and connective tissue remained defective. In contrast, skin tissues from the BP@EPL-LA + NIR group showed near-normal histoarchitecture, including intact blood vessels and hair follicles resembling healthy skin tissues. Masson's staining revealed the highest collagen fiber (blue) deposition in the BP@EPL-LA + NIR group (Fig. 5G), indicating robust tissue regeneration. Neovascularization, a critical process in abscess healing, was evaluated using CD31-immunohistochemical staining. The BP@EPL-LA + NIR group exhibited significantly elevated CD31 expression compared to the other groups (Fig. 5H), suggesting that the well-healed abscess received an adequate blood supply. Collectively, these observations demonstrated that the BP@EPL-LA nanocomposites eradicated bacterial infections through the synergistic combination of NO-enhanced PDAT and EPL's antibacterial activity while simultaneously promoting tissue repair. This dual action underscored their efficacy and safety for treating subcutaneous abscesses.

3.8. In vitro and in vivo biocompatibility of BP@EPL-LA nanocomposites

While BP@EPL-LA nanocomposites demonstrated excellent bactericidal performance in vitro, good biocompatibility is prerequisite for clinical translation [52]. The cytotoxicity of BP@EPL and BP@EPL-LA nanocomposites was investigated on 3T3 cells (mouse embryo cells) by MTT assays and live/dead staining. Both nanocomposites showed negligible cytotoxicity, with cell viability exceeding 90 % even at 400 μg/mL (Fig. 6A and S10A). Live/dead staining confirmed this finding, with L929 cells maintaining normal spindle-shaped morphology and green fluorescence at all tested concentrations of BP@EPL or BP@EPL-LA. In addition, promoting fibroblast migration is a critical process in tissue repair. After 48 h of co-culture, BP@EPL-LA significantly accelerated fibroblast migration (migration rate = 89 %) compared to the PBS control (Fig. S10B–C)i, indicating a higher potential for expediting abscess healing. This dual functionality—combining antimicrobial efficacy with the promotion of tissue regeneration—made BP@EPL-LA a promising therapeutic candidate for treating infected abscesses.

Fig. 6.

Fig. 6

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Biosafety evaluation of BP@EPL-LA nanocomposites. (A) Cytotoxicity evaluation of BP@EPL and BP@EPL-LA in 3T3 cells. (B) Hemolysis rates of BP@EPL and BP@EPL-LA at different concentrations. (C) Body weight changes in mice throughout the treatment period. (D) H&E staining of major organs (heart, liver, spleen, lung, kidney) in mice after treatment. (E) Blood analysis (WBC, RBC, PLT) in mice after 12-day treatment. (F) Serum liver function indicators (ALT, AST) and (G) kidney function indicators (BUN, CRE) at 12 days post-treatment. Error bars represent SD (n = 3).

For in vivo biomedical applications, BP@EPL-LA must exhibit negligible erythrocyte toxicity. Therefore, their blood compatibility was investigated through a hemolytic test. As displayed in Fig. 6B, the hemolysis rates of BP@EPL and BP@EPL-LA nanocomposites remained below 5 % at all concentrations tested (up to 400 μg/mL), suggesting excellent hemocompatibility. These results indicated that the BP@EPL-LA nanocomposites had outstanding biocompatibility under physiological conditions (pH 7.4), highlighting their significant potential for in vivo applications.

In addition, the biosafety of BP@EPL-LA nanocomposite was systematically evaluated. As shown in Fig. 6C, no significant fluctuations in mouse body weight were observed throughout the treatment, indicating negligible adverse effects. On day 12 post-treatment, mice were executed, and main organs (heart, liver, spleen, lung, and kidney) and blood were collected for histological studies. H&E staining revealed no obvious abnormalities, inflammatory lesions or necrosis among the six treatment groups (Fig. 6D). Blood routine and biochemistry of analyses further confirmed the absence of significant pathological changes (Fig. 6E–G). Collectively, these results confirmed the high biosafety of BP@EPL-LA nanocomposites, supporting their clinical applicability for subcutaneous abscess therapy.

4. Conclusion

In this study, we engineered a multimodal NIR light-responsive bactericidal nanoplatform (BP@EPL-LA) by loading BP nanosheets with polymeric nitric oxide donors (EPL-LA) for combinatorial subcutaneous abscess therapy. BP@EPL-LA demonstrated exceptional physiological stability and pH-triggered charge reversal, enabling deep biofilm penetration and enhanced bacterial adhesion at infected sites. Under NIR irradiation, BP@EPL-LA triggered the release of ROS and NO through a photodynamic process followed by ROS-mediated LA oxidation. The release kinetics could be modulated by varying sample concentration, irradiation duration, and NIR on/off cycling. BP@EPL-LA effectively eradicated MRSA and PA biofilms and killed bacteria within biofilms in vitro through the synergistic action of ROS, NO, and EPL. In vivo treatment of subcutaneous MRSA abscesses with BP@EPL-LA achieved potent bacterial eradication, reduced inflammation, and promoted tissue regeneration via enhanced vascularization and collagen deposition. Additionally, BP@EPL-LA exhibited excellent biocompatibility, with no adverse effects observed in either cellular or animal models. Therefore, BP@EPL-LA represents a promising multimodal therapeutic platform that synergistically combines NO-enhanced aPDT with EPL antimicrobial action for effective subcutaneous abscess treatment, while overcoming the limitations of conventional antibiotic resistance.

CRediT authorship contribution statement

Qian Gao: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Ranran Fu: Formal analysis, Data curation. Mengting Li: Supervision, Methodology, Investigation. Dongbo Guo: Supervision, Methodology, Investigation, Funding acquisition. Bingcheng Gan: Visualization, Resources. Tao Wang: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Maohua Chen: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (32360238, 52203160, 52362006), Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2024-33, SCKJ-JYRC-2024-34), Hainan Provincial Natural Science Foundation (524RC479), Edible Fungus Innovation Team of Sichuan Province (SCCXTD-2025-7) and Agricultural Science and Technology Innovation Program (34-IUA-06 and SZ202403).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.102235.

Contributor Information

Tao Wang, Email: wangtao03@caas.cn.

Maohua Chen, Email: maohuachen@hainanu.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (8.1MB, docx)

Data availability

Data will be made available on request.

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