Chronic rhinosinusitis (CRS) is a persistent inflammatory condition affecting in the sinus mucosa and nasal cavity. It is one of the most common diseases in Otorhinolaryngology-Head and Neck Surgery, which has the characteristics of high incidence, prolonged course, and difficulty in recovering [1]. CRS is often characterized by runny nose, nasal congestion, head and facial pain, reduced sense of smell, and other symptoms, seriously affecting the quality of daily life of patients [[2], [3], [4], [5]]. The pathological mechanism of CRS is complicated, which is generally believed to be related to the disorder of microflora in paranasal sinuses, disorders of the mucociliary transport system, abnormal anatomical structure of paranasal sinuses, allergies, and imbalance of immune signal pathway [[6], [7], [8], [9], [10], [11]]. For a long time, it has been agreed that bacterial infection is one of the most important causes of CRS [12]. With the widespread use of antibiotics, Methicillin-resistant Staphylococcus Aureus (MRSA) has become an important pathogen of infection [[13], [14], [15]]. Conventional antibiotic treatments often failed in patients with MRSA-induced difficult-to-treat rhinosinusitis (DTRS), due to the formation of biofilms and intense inflammatory responses triggered by MRSA, necessitating the exploration of alternative therapies. Although new antibiotics are being developed, they are still analogous to previous drugs and causing damages to systemic organs for a long time. Hence, there is an urgent need to develop new drugs and treatments in the clinic.

Nitric oxide (NO) has been proven to be an excellent broad-spectrum antimicrobial candidate, with the advantages of ability to combat biofilms without promoting drug resistance [16,17]. Some studies have shown that NO could disrupt bacterial biofilms by inducing lipid peroxidation and oxidative stress reaction within bacteria, indicating its potential therapeutic role in DTRS [16,17]. Therefore, the application of exogenous NO to the bacterial infection parts seems to be an effective treatment [18]. However, the clinical application of NO faces challenges due to the instability of NO donors, which complicates their storage, delivery, and controlled release in the body. Controlling the release site, concentration, release rate and continuous release time of NO is the key to develop their therapeutic efficiency. Some reports have designed methods of external stimulation, such as laser irradiation, ultrasound, and radiation, but these approaches remain difficult to control, compounded by the fact that most carriers initially release high levels of NO, which decreases gradually over time, making it difficult to sustain for sterilization [[19], [20], [21], [22], [23], [24], [25], [26], [27]]. We believe that animal body temperature is a relatively controllable factor, which is limited in a certain range and sustainable, thus making it possible to use thermal stimulation to achieve NO release in the treatment of MRSA-induced DTRS. Metal-polyphenol network (MPN) is a supramolecular lattice structure material formed by the coordination of metal ions and natural polyphenols, since polyphenols and metal ions form complexes, which have good drug-carrying performance, and polyphenols also have the function of stabilizing carriers. Based on these and excellent bioadhesion and biocompatibility, it is commonly used in films, microcapsules, and gels [[28], [29], [30], [31], [32], [33], [34]]. In addition, due to the antioxidant and antimicrobial activities of polyphenols, MPN can inhibit microbial growth, scavenge free radicals, and have low cytotoxicity, which has great potential for development in the biomedical field [[35], [36], [37], [38]].

Herein, a new NO material was designed by coating GSNO with MPN, which has the photo-thermal conversion function. The physicochemical properties of GSNO-MPN were characterized, and a NO release assay was evaluated. The in vitro antibacterial and anti-biofilm activities against MRSA were investigated, and GSNO-MPN was applied to a rat model of MRSA-induced difficult-to-treat rhinosinusitis (DTRS) for the first time to evaluate healing activity. Overall, our proposed approach involves utilizing the stable and controllable factor of animal body temperature to induce release of NO for treating MRSA-induced DTRS. This method aims to maintain therapeutic NO levels consistently over time, offering a potential breakthrough in managing this complex condition.

The ferric chloride hexahydrate solution and Tannic Acid (TA) were purchased from Energy Chemical (Shanghai, China). The Nitric Oxide Assay Kit was purchased from Shanghai Beyotime Biotechnology Corporation (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and Trypsin were obtained from Invitrogen Gibco (USA). The human nasal mucosal epithelial cell line was acquired from Guangzhou Baohui Biotechnology Corporation (Guangzhou, China). Methicillin-resistant ∗Staphylococcus aureus∗ (MRSA, ATCC 43300) was provided by East China Jiaotong University. Tryptone Soya Broth (TSB) was sourced from Beijing g-clone Biotechnology Corporation (Beijing, China). The Apoptosis Fluorescence Hoechst 33342/PI Staining Kit was procured from Beijing Solarbio Science & Technology Corporation (Beijing, China). Deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ) was used in all experiments. All animal experiments were conducted in compliance with the ARRIVE guidelines and performed in accordance with the UK Animals (Scientific Procedures) Act 1986, EU Directive 2010/63/EU, the NIH Guide for the Care and Use of Laboratory Animals, or equivalent internationally recognized standards.

The metal-polyphenol network (MPN) was synthesized according to a previously described method. A ferric chloride hexahydrate solution was prepared at a concentration of 0.1665 g/mL; 54 μL of this solution was added to a flask containing 9 mL of deionized water, followed by the addition of an equimolar amount of tannic acid (TA) relative to iron. The mixture was left to react for 15 min, then centrifuged (3000 rpm, 10 min). The supernatant was discarded, and the precipitate was washed three times with physiological saline. After another round of centrifugation, the sediment was collected and dried.

S-Nitroso glutathione (GSNO) was synthesized based on a previously reported method [39]. Briefly, reduced glutathione (GSH) and sodium nitrite were dissolved in HCl solution at 0 °C (final concentrations: 0.625 M GSH, 0.625 M sodium nitrite). The mixture was stirred at 300 rpm for 40 min in an ice bath. Excess cold acetone was added to precipitate GSNO, which was then washed once with 80 % acetone, twice with 100 % acetone, and three times with diethyl ether. The resulting solid was freeze-dried and stored at −20 °C.

One day before each nasal administration, equal masses of MPN, GSNO, and a physical mixture of MPN + GSNO were separately suspended in PBS to final concentrations of 1 mg/mL, 1 mg/mL, and 2 mg/mL, respectively. The suspensions were sonicated for 10 min and stored at 4 °C overnight. Before use, each suspension was vortexed thoroughly.

GSNO-MPN and MPN suspensions were prepared as described above and sonicated for 10 min. After filtration, the supernatant was transferred to a sample dish for particle size analysis using a particle size analyzer. Data were collected and graphed accordingly.

The MPN (1 mg/mL) and GSNO-MPN (2 mg/mL) suspensions were prepared as described. For each sample, a few microliters of the well-dispersed suspension was deposited onto a clean TEM grid and air-dried under ambient, dust-free conditions. Morphological characterization was then performed using Transmission Electron Microscopy (TEM).

MPN and GSNO were mixed at a 1:1 mass ratio and dispersed to form a suspension. The NO concentration in the solution was quantified using a commercial assay kit according to the manufacturer's instructions and converted to mass.

MPN and GSNO were prepared and mixed at different mass ratios under ultrasonic agitation to form suspensions. For comparison, solutions of GSNO alone and a mixture of tannic acid and GSNO were prepared at equivalent concentrations. NO release was measured periodically using the Griess method as per the kit instructions. Absorbance was recorded at 546 nm, and NO concentrations were determined against a standard curve. NO release profiles of the different formulations were plotted and compared at 0 °C, 18 °C, and 37 °C.

MRSA was thawed and inoculated onto a TSB agar plate, followed by incubation at 37 °C for 24 h. A single colony was inoculated into 10 mL of TSB medium in a 50 mL centrifuge tube and cultured at 150 rpm, 37 °C for 12 h. The bacterial suspension was centrifuged at 5000 rpm for 5 min; the supernatant was discarded, and the pellet was washed three times with sterile PBS to remove residual medium. Finally, the bacteria were resuspended in 10 mL of PBS, and the concentration was adjusted to 1 × 108 CFU/mL using McFarland turbidity standards.

All experimental materials were sterilized by UV irradiation. Based on previous findings, GSNO exhibits minimal NO release and decomposition at 260 nm. Although post-synthesis storage did not guarantee sterility, UV irradiation at 260 nm for 30 min was applied to achieve sterilization while minimizing material degradation. Bacterial suspensions were diluted to 1 × 108 CFU/mL and co-cultured with different UV-sterilized materials at 37 °C for 24 h. After serial dilution with sterile PBS, 100 μL of each dilution was spread on TSB plates and incubated at 37 °C for 12 h. The antibacterial effect was evaluated by counting colony-forming units (CFU) [40].

Bacterial morphology after treatment was observed using SEM. Bacteria were co-cultured with different materials for 24 h, harvested by centrifugation, and washed with sterile PBS. The pellets were fixed in 2.5 % glutaraldehyde at 4 °C for 12 h, then dehydrated through a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, and 100 %). Samples were placed on sterile slides, pre-cooled at 4 °C for 5 h, freeze-dried, and sputter-coated with gold before SEM observation [41].

Bacteria were treated with different materials, and the supernatant was collected by filtration through a 0.22 μm membrane. The optical density at 260 nm was measured using a Multiskan microplate reader to assess nucleotide leakage [27].

A 50 μL aliquot of bacterial suspension was added to a 96-well plate, followed by 200 μL of TSB medium, and incubated at 37 °C for 48 h with medium replacement every 12 h. After incubation, the biofilm was washed three times with saline and treated with different materials for 24 h. The remaining biofilm was washed twice and stained with 0.1 % crystal violet for 30 min. Excess dye was removed by washing, and the absorbance at 540 nm was measured to quantify biofilm biomass.

Bacteria were co-cultured with different materials for 24 h, collected by centrifugation, and resuspended in buffer. According to the Calcein-AM/PI staining kit instructions, the bacterial suspension was mixed with the staining solution and incubated at 37 °C for 30 min in the dark. Stained bacteria were observed under a fluorescence microscope, and the overall survival rate was analyzed.

The cytotoxicity of MPN, GSNO, and GSNO-MPN was evaluated using the CCK-8 assay on human nasal epithelial cells (HNEpC) and DC cells. Cells in the logarithmic growth phase were seeded into 96-well plates at 5 × 103 cells per well and cultured for 24 h. The medium was replaced with fresh medium containing the test materials at final concentrations of 0.25, 0.5, 1, 2, 4, and 8 μg/mL (0.5, 1, 2, 4, 8, and 16 μg/mL for GSNO-MPN), with three replicates per concentration. After 24 h of incubation, cells were washed with PBS, and CCK-8 solution was added. Following 2 h of incubation, the absorbance at 450 nm was measured, and the cell growth inhibition rate was calculated.

For apoptosis analysis, HNEpCs were treated with different drug concentrations for 24 h, harvested, and stained using an apoptosis fluorescence staining kit according to the manufacturer's instructions. Apoptosis was observed under a fluorescence microscope [42].

Thirty male Sprague-Dawley rats (200–250 g) were selected. A PVA medical sponge loaded with MRSA (0.1 mL, 1 × 108 CFU/mL) was inserted into the left nasal cavity to establish the DTRS model. To sustain inflammation, 100 μL of MRSA suspension (1 × 108 CFU/mL) was administered into the left nasal cavity twice weekly for three weeks. Symptoms such as nasal discharge and nose rubbing were observed during modeling, and a sinusitis symptom score was recorded (Table 1). A score greater than 5 indicated successful model establishment [42].

Rats were randomly divided into five groups: healthy, control (untreated), MPN, GSNO, and MPN-GSNO. Except for the healthy group, all rats received daily intranasal spray of 100 μL of the respective drug solution for 10 consecutive days. The DTRS and healthy groups received saline nasal spray.

Body weight, diet, and clinical symptoms were monitored daily throughout the treatment period, and sinusitis symptom scores were recorded.

Precision pH test strips (range 6.4–8.0) were cut into 2 mm × 20 mm pieces. Rats were restrained, the nasal vestibule was disinfected, and the test strip was inserted into the nasal cavity in contact with the mucosa. After 30 s, the strip was removed, and the pH value was determined by comparison with a standard color chart. Measurements were performed three times for each nasal cavity, and the average value was recorded.

Rats were restrained, and a murine temperature probe was inserted into the nasal cavity for 10 s per measurement. Three consecutive measurements were taken per session, and the mean value was calculated for each group. The probe was disinfected with alcohol after each use.

The concentrations of TNF-α, IL-5, and IL-6 in serum and nasal lavage fluid were measured using ELISA kits according to the manufacturer's instructions. Results are expressed in pg/mL.

Twenty-four hours after the last treatment, rats were anesthetized, and nasal mucosa, lung, brain, kidney, spleen, liver, and heart tissues were collected. Nasal mucosa was used for histopathological and immunohistochemical analysis; other organs were used for histopathology and in vivo toxicity assessment.

Animals were perfused with 4 % paraformaldehyde, and heads were excised and fixed overnight. After removal of eyes, skin, and muscle, the mandible was detached. Specimens were decalcified in EDTA, and coronal sections (5 μm) were prepared from the posterior plane of the incisors to the anterior plane of the first molar. Sections were dehydrated, embedded in paraffin, and stained with H&E. Inflammatory cell infiltration in the submucosa was graded as follows: 0 (absent), 1 (mild, scattered cells), 2 (moderate), or 3 (severe, diffuse infiltration).

To track the in vivo distribution of GSNO-MPN, the nanoparticles were conjugated with the fluorescent dye IGC. Following intranasal administration, fluorescence signals were monitored at predetermined time points to determine the spatiotemporal distribution and metabolic pathway of GSNO-MPN.

ICR mice (6–8 weeks old) were anesthetized with 2 % isoflurane and administered 40 μL of GSNO-MPN (2 mg/mL) via nasal spray. In vivo imaging was performed immediately, and at 1, 2, 4, 8, 24, 48, and 72 h post-administration using an optical imaging system (excitation: 780 nm, emission: 822 nm, exposure: 10 s). The average radiant efficiency of regions of interest was calculated. After the final time point, major organs were harvested and imaged ex vivo to examine drug distribution.

All data are presented as means ± standard deviation, and statistical significance of the data was evaluated using the One-way ANOVA method in IBM SPSS Statistics V27. P-values <0.05 were considered significant differences (∗p < 0.05).