Sprayable lipid-based lubricated hydrogel coating for alleviating catheter-related mucosal damage and pain

Abstract

Medical catheters are essential for clinical diagnosis and therapy, yet their insertion and retention inevitably cause mucosal damage and pain. Conventional catheters compromise comfort and risk secondary inflammation, underscoring the need for advanced catheter surface coatings. Herein, we developed a sprayable lipid-based lubricated hydrogel coating (Oxy@Lipo/Gel) by integrating oxybuprocaine-loaded liposomes (Oxy@Lipo) with an oxidized hyaluronic acid/carboxymethyl chitosan (OHA/CMCS) hydrogel. The OHA/CMCS hydrogel enables rapid spray-induced gelation, while Oxy@Lipo acts as a carrier for the local anesthetic oxybuprocaine. Oxy@Lipo/Gel not only enhances catheter hydrophilicity, reduces interfacial friction and nonspecific protein adsorption, but also exhibits pH-responsive drug release, self-healing capacity, and appropriate degradability. It is worth noting that Lipo dramatically augments the hydrogel's lubricity by reconstruction of phospholipid lubricating interface upon friction-induced migration and surface exposure of Lipo. Furthermore, Oxy@Lipo/Gel-coated endotracheal tube significantly minimize intubation-induced heart rate fluctuations of rhesus monkey, alleviate airway mucosal injury and pain responses effectively, and preserve post-extubation feeding willingness. Mechanistically, Oxy@Lipo/Gel mitigates mucosal damage and pain by activating the Nrf2 pathway for antioxidant defense, inhibiting the pro-inflammatory MAPK cascade, and facilitating M2 macrophage polarization. Collectively, Oxy@Lipo/Gel emerges as a promising strategy to mitigate catheter-related mucosal damage and pain, holding substantial potential for clinical translation and application.

Graphical abstract

Highlights

• •The sprayable lipid-lubricated hydrogel coating (Oxy@Lipo/Gel) enables rapid in-situ gelation, pH-responsive drug release, favorable biosafety, and appropriate degradability for catheter lubrication.
• •This coating exhibits dynamic self-lubrication behavior, reducing catheter-related mucosal damage and pain in rhesus monkeys.
• •This coating mitigates mucosal damage and pain by activating the Nrf2 pathway, inhibiting the pro-inflammatory MAPK cascade, and facilitating M2 macrophage polarization.

1. Introduction

Medical catheters (e.g., endotracheal tubes, urinary catheters, central venous catheters) are indispensable in perioperative management and critical care [1]. However, during insertion and prolonged retention, inevitable friction arises at the catheter-mucosa interface, causing undesirable tissue injury (manifested as mucosal edema, erosion, and hemorrhage) and distressing symptoms like post-extubation sore throat and urethral irritation [2,3]. These complications not only directly compromise patient comfort but also raise the risk of secondary infections (e.g., catheter-associated bloodstream infections) and extend hospital stays, thereby posing substantial challenges for clinical practice [4,5]. Current clinical lubrication approaches, mainly relying on liquid paraffin and normal saline, are far from ideal. Liquid paraffin achieves moderate friction reduction yet bears prominent limitations: its mineral oil composition prevents mucosal absorption, and accidental aspiration may induce lipid pneumonia [6]. Moreover, poor in vivo degradability leaves residual mineral oil, which triggers persistent mucosal irritation and increase the risk of local inflammation and fibrosis [7]. Conversely, normal saline exhibits favorable biocompatibility but lacks durable lubrication, given that its lubricating potency declines rapidly during catheter manipulation and thus fails to provide sustained protection against friction-induced injury.

To overcome the drawbacks of conventional lubricants, recent research has shifted toward advanced catheter lubrication strategies. For instance, Bai et al. developed a mucosa-mimetic conformal hydrogel coating [8], Li et al. engineered an HA/F127 coating [9], and Liu et al. fabricated a hydrogel coating with water-responsive Janus adhesion and acidity-triggered antibacterial treatment on medical devices [10]. While these coatings demonstrate lubricating capabilities, they encounter notable bottlenecks: complex synthesis processes impede scalable production; excessive additives (e.g., toxic cross-linking agents) raise biosafety concerns; most importantly, few formulations integrate lubrication with therapeutic functions, limiting their clinical translation. Therefore, there is an urgent demand for a next-generation catheter lubricant that integrates superior lubricity, biocompatibility, degradability, and targeted therapeutic effects (e.g., mucosal analgesia).

Hyaluronic acid (HA) and carboxymethyl chitosan (CMCS) stand out as ideal candidates for medical coating, supported by their well-established advantages. As a key extracellular matrix component, HA boasts excellent biocompatibility, water retention, and mucosal adhesion. Oxidized hyaluronic acid (OHA), in particular, introduces aldehyde groups (-CHO) to enable dynamic covalent cross-linking [11]. CMCS, a water-soluble chitosan derivative, preserves chitosan's biocompatibility and antibacterial properties while providing abundant amino groups (-NH₂) for cross-linking reactions [12]. Importantly, OHA and CMCS form a hydrogel via a Schiff base reaction between -CHO and -NH₂—this synthesis avoids toxic catalysts, and the hydrogel's reversible imine bonds endow it with intrinsic self-healing properties [13,14]. Previous studies have demonstrated that the OHA/CMCS hydrogel possesses favorable biocompatibility, self-healing capacity, and biodegradability. Its rapid hydration and gelation enable it to function as an effective physical barrier, with sprayable or spreadable properties favoring clinical translation [15,16]. Building on these merits, it was hypothesized that the OHA/CMCS hydrogel could be engineered as a carrier for catheter lubricants, leveraging its intrinsic biocompatibility and lubricity to overcome the drawbacks of conventional lubricants.

To further optimize the performance of the OHA/CMCS hydrogel, liposomes (phospholipid bilayer vesicles) may offer complementary functionalities. As well-established drug carriers, liposomes are capable of encapsulating both hydrophilic and hydrophobic therapeutic agents, protecting payloads from degradation, and mediating controlled release [17]. More importantly, recent evidence has indicated that liposomes markedly enhance hydrogel lubrication: their phospholipid bilayer mimics natural synovial fluid lubricants, and under the friction forces of catheter insertion and manipulation, liposomes migrate to the coating surface, forming a continuous lubricating film that minimizes direct catheter-mucosa contact [[18], [19], [20]]. Based on this mechanism, we incorporated liposomes into the OHA/CMCS hydrogel to develop a lipid-lubricated hydrogel coating, aiming to synergistically improve lubrication durability and establish a versatile drug-loading platform.

Beyond improving lubrication, mitigating catheter insertion-induced mucosal pain remains another critical clinical priority. To address this, we selected oxybuprocaine (Oxy), a widely used ester-based local anesthetic, as the therapeutic agent [21]. Oxy exhibits excellent mucosal penetration and rapid analgesic effects by blocking sodium channels in nerve ending [22], making it ideal for relieving catheter-associated mucosal pain (e.g., post-extubation sore throat). However, its short in vivo half-life limits sustained analgesic effects. To address this drawback, Oxy was encapsulated into liposomes, which were further embedded within the OHA/CMCS hydrogel. It was hypothesized the porous network of the hydrogel would retard drug diffusion, whereas the liposomal bilayer would further modulate release kinetics to realize prolonged local analgesia for catheter-associated pain.

In this work, we fabricated a sprayable lipid-lubricated hydrogel coating (Oxy@Lipo/Gel) by integrating Oxy-loaded liposomes into an OHA/CMCS hydrogel to dramatically alleviate mucosal injury and pain induced by indwelling medical catheters (Scheme 1). The OHA/CMCS hydrogel presented baseline lubrication and excellent biocompatibility; in addition, it prolonged Oxy release from the coating. Imine bonds were formed via Schiff base reactions, endowing the hydrogel coating with intrinsic self-healing capability and appropriate degradability. Oxy@Lipo not only provided sustained analgesia by releasing Oxy but also improved the hydrogel's lubricity by reconstructing a lipid biofilm on the coating surface under frictional contact with adjacent mucosa. Importantly, we systematically elucidated the lubrication performance and underlying mechanisms of the liposome-reinforced coating for various catheter types, confirming the dynamic self-lubrication behavior of liposomes (i.e., migration toward the coating surface under frictional loading). Moreover, we validated that Oxy@Lipo/Gel-coated endotracheal tubes effectively alleviated mechanical mucosal injury and catheter-related mucosal pain in rhesus monkeys. Additionally, the coating's modulatory effects on oxidative stress, macrophage polarization, and inflammatory signaling pathways were thoroughly characterized. Collectively, this work aims to provide a novel, clinically translatable coating that reduces catheter-associated complications and improves patient outcomes.

Scheme 1.

Fabrication of the sprayable Oxy@Lipo/Gel coating and its application in the rhesus monkeyendotracheal intubation model. (a) Preparation of OHA/CMCS hydrogel, followed by combination with oxybuprocaine (Oxy)-loaded liposomes to fabricate the Oxy@Lipo/Gel coating. (b) Schematic illustration of the self-lubrication mechanism for the lipid-based hydrogel coating: when liposome microreservoirs on their surfaces experience frictional wear, more underlying microreservoirs are exposed, thereby forming lipid boundary layers on the coating surface. (c) In vivo application of the Oxy@Lipo/Gel coating in the rhesus monkey endotracheal intubation model. Created with BioRender.com.

2. Methods

2.1. Materials

Hyaluronic acid (HA, MW: 1500∼2500 kDa), Calcein-AM/propidium iodide (PI) live/dead assay kit, bovine serum albumin (BSA), and fibrinogen were purchased from Macklin (Shanghai, China). Carboxymethyl chitosan (CMCS, MW: 400 kDa, carboxymethyl substitution: 80%), sodium metaperiodate (NaIO₄), ethylene glycol, cholesterol, chloroform, oxybuprocaine, fluorescein isothiocyanate (FITC), and rhodamine B were obtained from Aladdin (Shanghai, China). Dioleoyl phosphatidylcholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-mPEG₂₀₀₀) were supplied by SunLipo NanoTech (Shanghai, China). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime (Shanghai, China). Artificial saliva (pH 6.6) and urine (pH 6.5) were acquired from Yuduo Biotechnology Co., Ltd. (Shanghai, China). ROS assay kit and DAPI were purchased from Servicebio (Wuhan, China). Liquid paraffin, polyvinyl chloride (PVC) endotracheal tubes, laryngeal masks, rubber urinary catheters, silicone urinary catheters, silicone gastric tubes, central venous catheters, shunt tubes, and stainless steel bars were all provided by the Department of Anesthesiology, The Third Affiliated Hospital of Sun Yat-sen University.

2.2. Synthesis of oxidized hyaluronic acid (OHA)

OHA was prepared via periodate oxidation [23]. Briefly, 4 g of HA was dissolved in 400 mL of double-distilled water (ddH₂O) with stirring. Subsequently, 4 g of NaIO₄ (pre-dissolved in 40 mL of ddH₂O) was added dropwise to the HA solution. After reacting at room temperature (RT) for 2 h, 4 mL of ethylene glycol was added dropwise, and the mixture was further stirred at RT for another 2 h. The collected solution was dialyzed against ddH₂O for 3 days (dialysis medium replaced daily). All synthesis steps were conducted under light-shielded conditions. Finally, the dialyzed solution was freeze-dried for 3 days to obtain OHA. ¹H nuclear magnetic resonance (¹H NMR) spectroscopy was used to confirm the structural characteristics of HA and OHA.

2.3. Preparation of OHA/CMCS hydrogel

Aldehydes and primary amines undergo a condensation reaction via nucleophilic addition-elimination. The amine nucleophilically adds to the aldehyde to form a hemiaminal intermediate, which afterwards eliminates a molecule of water to yield an imine, i.e. Schiff base [24]. This mechanism enables OHA's aldehydes and CMCS's amines to undergo Schiff base reactions, driving self-assembly and hydrogel formation. After systematic optimization of OHA/CMCS concentration ratios (details in Table S1), 6 wt% OHA and 2 wt% CMCS were identified as optimal formulations. Spray-mixing these two solutions at a 1:1 (v/v) ratio successfully yielded a sprayable OHA/CMCS hydrogel with rapid gelation kinetics.

2.4. Preparation and characterization of liposomes

Liposomes were prepared via the conventional thin-film hydration method [25]. Briefly, DOPC, DSPE-mPEG₂₀₀₀, and cholesterol (at a molar ratio of 70:5:25) were dissolved in chloroform within a 50 mL round-bottom flask to achieve a total lipid concentration of 1 mg/mL. The organic solvent was evaporated using a rotary evaporator (37 °C water bath) to form a lipid film, which was further dried under vacuum for 2 h. The dried film at the flask bottom was redispersed in ddH₂O and sonicated for more than 15 min to form liposomes, which were stored at 4 °C for further use.

In order to obtain oxybuprocaine (Oxy)-loaded liposomes (Oxy@Lipo), Oxy was firstly dissolved in chloroform to achieve a concentration of 20 mg/mL. Afterwards, lipid components (DOPC/DSPE-mPEG₂₀₀₀/cholesterol, 70:5:25 molar ratio) were mixed, followed by the addition of Oxy solution at a 2:1 (Oxy:lipids) mass ratio. Oxy@Lipo was prepared via the thin-film hydration procedure with excess Oxy removed by dialysis. To prepare DiI-labeled fluorescent liposomes (DiI@Lipo), DiI (in chloroform) was added to the lipid mixture at a molar ratio of 1:200 (DiI to total lipids). DiI@Lipo was obtained using the same protocol, followed by dialysis to removed excess dye.

Dynamic light scattering (DLS) combined with laser Doppler electrophoresis (Litesizer 500, Anton Paar, Austria) was employed to characterize the size distribution of liposomes at pH 7.4, 6.4 and 5.4, alongside their average hydrodynamic diameter and zeta potential during a 7-day storage stability test. Transmission electron microscopy (TEM; Thermo Scientific, Talos F200i S) was applied to characterize their morphological features. To determine the drug loading content (DLC) and drug loading efficiency (DLE) of Oxy, dried Oxy@Lipo was dissolved in methanol, and the absorbance of the solution at 310 nm was obtained from the absorption spectrum (Fig. S1). DLC and DLE were calculated using the following equations [26]:

DLC (%) = (Mass of loaded Oxy / Total mass of Oxy@Lipo) × 100%

DLE (%) = (Mass of loaded Oxy / Initial mass of Oxy) × 100%

2.5. Preparation and characterization of Lipo/Gel coating

To prepare lipid-lubricated hydrogels, 10 mg/mL liposomes (with or without Oxy loading) were added to OHA and CMCS solutions, respectively. Lipid-based hydrogels were fabricated via co-spraying the two precursor solutions at a volume ratio of 1:1 (v/v).

Scanning electron microscopy (SEM; Phenom S-2000, Netherlands) was used to characterize the morphology of OHA/CMCS hydrogel (Gel) and Lipo/Gel. Energy dispersive spectroscopy (EDS) was employed to analyze the elemental mapping of Lipo/Gel. Additionally, the distribution of DiI-labeled Lipo and FITC-labeled Gel in Lipo/Gel was imaged using a fluorescence microscope (Ni-U, Nikon, Japan). The injectability was evaluated via a 21 G syringe needle, and the self-healing property of Lipo/Gel was also characterized. Furthermore, the gelation properties of different solution components were assessed by tube inversion method.

To evaluate the dynamic rheological behaviors of Gel and Lipo/Gel, strain sweep tests were conducted at a constant frequency of 1 Hz over a strain range of 10⁻² to 10³%. Additionally, angular frequency sweep tests were performed at a constant strain of 1% over a frequency range of 10¹ to 10⁵ rad/s. Time-dependent changes in storage modulus (G′) and loss modulus (G″) were immediately recorded after mixing the Lipo-containing OHA and CMCS precursor solutions. The shear-thinning behaviors of Gel and Lipo/Gel were characterized, and continuous step-strain measurements of Lipo/Gel were performed at 1 Hz (duration: 3 min).

2.6. In vitro drug release of Oxy@Lipo/Gel coating

Drug release experiments were conducted over 72 h according to previous reports [27]. Briefly, 1 mL of Oxy-loaded liposome (Oxy@Lipo) or Oxy@Lipo/Gel suspension was transferred into a dialysis bag (molecular weight cutoff (MWCO): 3500 Da, Sigma-Aldrich, USA) and then immersed in a 10 mL vial. Phosphate-buffered saline (PBS, pH 7.4 or 6.4) was slowly added to the vial, followed by incubation in a shaking incubator (37 °C, 40 rpm). At preselected time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h), 200 μL of dialysate was collected and immediately replaced with 200 μL of fresh PBS to maintain constant volume. The concentration of released Oxy was quantified according to the absorbance at 310 nm. Linear calibration curve (R² > 0.99, Fig. S1) wasestablished using standard Oxy solutions and then applied to determine the Oxy concentrations in all test samples.

2.7. In vitro hemolysis test of Lipo/Gel coating

Blood (2 mL) was collected from the abdominal aorta of mice using EDTA as an anticoagulant and diluted with 1 mL of PBS. The mixture was centrifuged at 2500 rpm for 5 min, after which the erythrocytes were collected and a 2% erythrocyte suspension was prepared. One milliliter of the 2% erythrocyte suspension was aliquoted into six sterile test tubes. Tube 1 (negative control) received 1 mL of PBS (2%), tube 2 (positive control) received 1 mL of ddH₂O, and tubes 3–6 received 1 mL of the Lipo/Gel solution at concentrations of 2, 4, 6, and 8 mg/mL, respectively. After incubation at 37 °C for 2 h, the solution clarity and residual erythrocytes at the bottom of each tube were observed. Finally, the tubes were centrifuged at 1500 rpm for 5 min, and the absorbance of the supernatant was measured at 410 nm using a microplate reader (MOX200, BioTek, USA). All samples were analyzed in three independent repetitions. The hemolysis rate was calculated using the following formula [28]:

Hemolysis rate (%) = [(Absorbance of treatment group − Absorbance of negative control group) / (Absorbance of positive control group − Absorbance of negative control group)] × 100%

2.8. In vitro cell biocompatibility of Lipo/Gel coating

Cell viability was evaluated by CCK-8 kit assay. Briefly, L929 cells, HUVEC cells or BEAS-2B cells were seeded into 96-well plates at a density of 1 × 10⁵ cells/well and cultured overnight to allow for cell attachment. Lipo/Gel solutions with different concentrations were then added to the above wells. After 24 h or 48 h of co-incubation, cells were gently rinsed three times with 37 °C pre-warmed PBS (10 mM, pH 7.4) to remove residual Lipo/Gel. Next, 10 μL of CCK-8 reagent, diluted 1:10 in serum-free medium according to the per manufacturer's protocol, was added to each well. The plates were then incubated at 37 °C for 2 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader.

L929 cells, HUVEC cells or BEAS-2B cells were seeded into 24-well plates at a density of 8 × 10⁴ cells/well and allowed to adhere overnight. The cells were then treated with different concentrations of Lipo/Gel solution for 24 h or 48 h (consistent with the CCK-8 assay). After treatment, the cells were washed twice with 37 °C pre-warmed PBS (10 mM, pH 7.4) to remove residual Lipo/Gel. Cells were then stained with a working solution containing 2.0 μM calcein-AM and 4.5 μM propidium iodide (PI), and incubated at 37 °C for 15 min in the dark. Finally, fluorescence images were captured using an inverted fluorescence microscope (Ti2-U, Nikon, Japan).

2.9. In vitro swelling behavior and degradability of Lipo/Gel coating

Lipo/Gel coatings with identical mass were prepared, with three parallel replicates set for each time point. Briefly, all specimens were freeze-dried to a constant weight to record their dry mass (W_d). Subsequently, the dried samples were immersed in pH 6.6 artificial saliva at 37 °C and incubated in a thermostatted shaker oscillating at 30 rpm to simulate physiological in vivo fluid conditions. At predetermined time intervals (0, 1, 3, 6, 12, 20, 24, 28, 32, 36, and 48 h), specimens were collected, excess surface fluid was carefully blotted using filter paper, and their wet mass (W_s) was rapidly measured before immediate re-immersion into the medium. The swelling ratio (SR) was calculated according to the following equation: SR (%) = [(W_s−W_d)/W_d]×100%.

The preliminary degradation behavior of the Lipo/Gel were performed according to the previous method [29]. Briefly, Rhodamine B-labeled Lipo/Gel samples were placed individually into wells of 6-well plates. The samples were then immersed in one of three different media: PBS (pH 7.4), artificial saliva (pH 6.6), or artificial urine (pH 6.5). At predefined time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, and 24 h), the morphological changes of each sample were photographed.

Furthermore, quantitative mass loss measurement was performed to systematically evaluate the degradation profiles of Lipo/Gel coating in various media. Coatings of identical mass were randomly assigned to three groups (n = 3): pH 7.4 PBS, pH 6.6 artificial saliva and pH 6.5 artificial urine. Each specimen was placed into a pre-weighed 15 mL centrifuge tube (empty mass: m₀), freeze-dried and reweighed (m₁) to determine the initial dry mass: m_0,dry = m₁−m₀. After addition of 5 mL of the corresponding medium, samples were incubated at 37 °C under orbital shaking at 30 rpm. At predetermined time intervals (0, 1, 3, 6, 12, and 24 h), specimens were collected and separated via a 100 μm filter; excess surface solvent was gently blotted with filter paper. Following freeze-drying, tubes were weighed again (m₂) to calculate the residual dry mass (m_t,dry = m₂−m₀). The mass loss percentage was calculated via the following formula: Mass loss (%) = [(m_0,dry−m_t,dry)]/m_0,dry×100%. Time-dependent mass-loss curves were subsequently generated.

2.10. In vitro protein adsorption and anti-bacterial adhesion of Lipo/Gel coating

Protein adsorption was assessed on PVC or polyurethane (PU) tubes (7 mm diameter, 5 mm length), either coated or uncoated with Lipo/Gel. Following PBS rinse, samples were placed in a 48-well plate and incubated with BSA or fibrinogen solutions (1 mg/mL in PBS) at 37 °C for 2 h. Samples were then transferred to a fresh 48-well plate, rinsed with PBS, and then treated with 500 μL BCA working reagent. Absorbance was measured at 562 nm using a microplate reader. Protein concentrations were calculated according to the standard curve.

In addition, the potential anti-bacterial adhesion property of the Lipo/Gel coating was evaluated using Escherichia coli (E. coli), a primary pathogen responsible for clinical catheter-associated infections. Coated or bare PVC substrates (0.5 cm × 0.5 cm) were immersed in bacterial suspension (1 × 10⁶ CFU/mL) and statically incubated at 37 °C for 0.5, 1 and 2 h. Post-incubation, specimens were carefully harvested and rinsed three times with sterile PBS, then vortexed in 1 mL sterile PBS for 5 min to dislodge adherent bacteria. The OD₆₀₀ of the resultant bacterial suspension was determined to calculate the relative bacterial adhesion rate (%).

2.11. Evaluation of Lipo/Gel coating adhesion to catheters and hydrophilicity improvement

Various medical catheters (PVC, silicone, latex, and PU) were divided into saline-treated or Lipo/Gel-sprayed groups. After cleaning with deionized water and drying, a 10 μL Rhodamine B-labeled deionized water droplet was gently dispensed onto each sample surface via micropipette. Droplet morphologies were photographed immediately to preliminary evaluate substrate hydrophilicity. Static contact angles were further quantified using a LAUDA Scientific LSA100S-T goniometer (Germany) at 25 ± 1) °C, to precisely determine the improved hydrophilicity of Lipo/Gel-coated various flat substrates (PVC, silicone, latex, and PU). Measurements were performed on all substrates pre- and post-spray-coating.

FITC-labeled Lipo/Gel was uniformly deposited onto diverse medical catheters (PVC endotracheal tubes, laryngeal masks, rubber/silicone urinary catheters, silicone gastric tubes, central venous catheters and shunt tubes) and flat substrates composed of PVC, silicone, latex and PU to obtain coatings approximately 50 μm in thickness. Following drying the coated catheters at 25 °C for 30 min, bright-field and fluorescence microscopic images were acquired to preliminarily assess coating adhesion via fluorescent uniformity.

To simulate clinical intubation procedures, coated PVC catheters (n = 3) underwent cyclic bending into curvatures consistent with physiological airway morphology and ±180° axial torsion, repeated over three cycles, and fluorescence imaging was used to observe the structural integrity of coatings. To characterize coating erosion stability under physiological conditions, the initial fluorescence intensity and uniformity of PVC catheters and flat substrates coated with Lipo/Gel (n = 3) were determined prior to incubation in pH 6.6 artificial saliva. Samples were shaken continuously at 30 rpm and 37 °C for 30 min to mimic in vivo fluid flushing. After residual saliva was rinsed off with PBS, post-treatment fluorescence intensity was tested to calculate the fluorescence retention rate of the coatings.

For interfacial peel strength testing, Lipo/Gel coatings were prepared on PVC, silicone, latex and PU substrates and compressed at 8 kPa and 37 °C for 3 min. A universal mechanical testing machine (MTS C42.503, USA) was adopted to conduct 180° peel tests at a stretching rate of 10 mm/min (n = 3), quantifying the interfacial bonding strength between coatings and different substrates.

2.12. Tribological test

The lubrication properties of saline, Lipo, Gel, Lipo/Gel, liquid paraffin, and soaked-Lipo/Gel (pre-soaked in pH 6.6 artificial saliva for 30 min) were characterized via linear reciprocating sliding tests on a Bruker UMT-3 universal material tester. Meanwhile, the coefficients of friction (CoF) for saline, artificial saliva, Gel and Lipo/Gel were further measured with artificial saliva serving as the base lubricant. Specifically, a ball-on-disk configuration was employed, with a PE sphere (8 mm diameter) sliding against a flat PVC substrate. The applied load and sliding speed were set to 5 N and 5 mm/s, respectively, with a 600 s test duration. Each test was repeated at least three times. The CoF was calculated as the mean value from the steady-state segment of the friction curves. Post-testing, wear tracks on the PVC substrate surface were characterized under a bright-field microscope (Ni-U, Nikon, Japan) to quantify wear width. Additionally, Gel and Lipo/Gel were collected for macroscopic observation and SEM imaging. Furthermore, the distribution of DiI-labeled liposomes on Lipo/Gel surfaces, both pre- and post-friction, was visualized under a fluorescence microscope (Ni-U, Nikon, Japan).

2.13. In vivo systemic toxicity of Oxy@Lipo/Gel coating

To evaluate the acute systemic toxicity of the Oxy@Lipo/Gel, 6-week-old male C57BL/6 mice (18∼22 g) were obtained from Zhuhai BesTest Bio-Tech Co.,Ltd. (Guangdong, China) and housed in ventilated cages under a 12 h light-dark cycle. Six mice were randomly divided into Saline and Oxy@Lipo/Gel groups (n = 3 per group). All animals were anesthetized with 1% isoflurane via inhalation (Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.). Subsequently, 0.5 mL of Oxy@Lipo/Gel (10 mg/mL) was subcutaneously administered into the dorsal region of mice in the experimental group, while the ones in the control group received an equal volume of 0.9% saline. At 24 h post-injection, blood samples were collected via retro-orbital enucleation from the mice under deep anesthesia. Serum levels of creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (Cr) were quantified. The mice were then euthanized via an overdose of isoflurane and transcardially perfused with 20 mL of 0.9% saline. Major organs (heart, liver, spleen, lung, kidney, and brain) were collected and stained with hematoxylin and eosin (H&E), followed by histological observation under an optical microscope (Ni-U, Nikon, Japan). All mice-related procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University (Approval No.: SYSU-IACUC-2025-001035).

2.14. Efficacy of the Oxy@Lipo/Gel coating in the rhesus monkey tracheal intubation model

Rhesus monkeys were obtained by Guangdong Landau Biotechnology Co., Ltd. (Experimental Animal License No.: Guangdong Landau Biotechnology SYXK 2022-0281). The monkeys were housed individually in hot-dip galvanized cages of standard dimensions. The housing environment was maintained at 16∼26 °C (daily variation ≤4 °C) and 40∼70% relative humidity, with 8∼10 air exchanges per hour. Natural light was supplemented by incandescent lamps to maintain a 12L:12D cycle. The animal study protocol was approved by the company's IACUC (Approval No.: LDACU 20250302-01).

To establish an rhesus monkey endotracheal intubation model, nine male rhesus monkeys (6∼8 years old) were randomly assigned to three groups: Saline, Oxy@Gel, and Oxy@Lipo/Gel (n = 3 per group). Preoperatively, monkeys were fasted for 8 h with both food and water restricted, followed by intramuscular anesthesia using 10 mg/kg ketamine hydrochloride (Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.). After sedation, animals were positioned on the operating table for continuous monitoring of electrocardiography (ECG), non-invasive blood pressure and peripheral oxygen saturation (SpO₂). Subsequently, under direct laryngoscopy, endotracheal tubes with an inner diameter of 4.0 mm pre-coated with saline, Oxy@Gel or Oxy@Lipo/Gel corresponding to group assignment were inserted into the trachea. Each tube was fixed to the mandible, and mechanical ventilation was initiated with a ventilator (AM-100, Chengdu Techman, China) under inhalation anesthesia of 2∼3% isoflurane (Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.). Ventilator parameters were fixed at a tidal volume of 6∼8 mL/kg and a respiratory rate of 20∼25 cycles/min.

Following 2 h of mechanical ventilation, animals were extubated once spontaneous breathing, protective reflexes and consciousness returned. Heart rate was continuously monitored throughout the perioperative period. After clearance of laryngeal secretions, a 2.8-mm visual bronchoscope (TIC-SD-I, Zhejiang UE, China) was utilized to examine the glottis and trachea at three predetermined time points: prior to intubation, immediately after extubation, and 3 days post-extubation. On postoperative day 3, sesame seed-sized airway mucosal biopsies were harvested from the airway under direct bronchoscopic visualization. All harvested tissues were preserved for pathological examination of tracheal mucosa and transcriptome sequencing analysis.

The mucosal injury score was applied to quantify the severity of glottic and tracheal lesions in rhesus monkeys after extubation. A five-point grading scale was established with detailed criteria listed below [30]: Grade 1: Intact and smooth mucosa with no hemorrhage or edema; Grade 2: Mild mucosal edema in the absence of ulceration and hemorrhage; Grade 3: Mucosal edema accompanied by marked hyperemia and erythematous swelling; Grade 4: Edematous and hyperemic mucosa with scattered ulceration and focal hemorrhage; Grade 5: Severe mucosal erythema and swelling coupled with widespread ulceration and hemorrhage.

Food intake of rhesus monkeys measured 24 h before intubation was defined as the baseline value. Throughout the experiment, preferred feed was provided at scheduled time points, and feeding behaviors were carefully recorded. A blinded experimental design was employed to improve assessment objectivity and minimize observational bias. Furthermore, all feeding manipulations were performed by a single experienced animal caretaker to avoid stress-related behavioral reactions triggered by unfamiliar operators. To quantify feeding willingness of the monkeys, a validated Feeding Willingness Score system was applied [8]. Each monkey started with a baseline score of 3. One bonus point was awarded for each of the following positive behaviors: active food solicitation, competition for feed at the feeding trough, and immediate ingestion once preferred feed was provided. Conversely, one point was deducted per adverse behavioral item: no response to offered feed, backward hip retreat posture, and food sniffing without subsequent consumption. Final individual scores ranged from 0 to 6, serving as an integrated indicator of feeding willingness.

2.15. Transcriptome sequencing analysis

Tracheal mucosal tissues were collected from rhesus monkeys in saline (Control) and Oxy@Lipo/Gel (Treat) groups (n = 3 per group). After rinsing with pre-cooled PBS, total RNA was extracted using Yeasen kit (Cat. No. 10606ES60) according to the manufacturer's instructions, involving tissue homogenization, RNA isolation via chloroform phase separation, isopropanol precipitation, and washing with 75% ethanol. RNA purity was verified by Nanodrop spectrophotometer (OD₂₆₀/OD₂₈₀: 1.8∼2.0; OD₂₆₀/OD₂₃₀  ≥ 1.5) and integrity (RIN ≥7.0) was assessed using an Agilent 2100 Bioanalyzer with DNA 1000 and RNA 6000 Nano Kits. Qualified samples were then sent to Yunhong Life Technology (Huzhou, China) for library preparation. rRNA was depleted using the Illumina TruSeq Stranded Total RNA Kit. Libraries were quantified by RT-qPCR (concentration >2 nM) and subjected to paired-end 150 bp (PE150) sequencing on BGI T7 platform, achieving a minimum depth of 6 Gb per sample. Raw sequencing data, converted to FASTQ format via CASAVA, were quality-filtered to obtain clean reads by fastp. These reads were then aligned to rhesus monkey reference genome (Mmul_10) using STAR. Gene expression levels were quantified as FPKM by StringTie. Differentially expressed genes (DEGs) were analyzed via edgeR, DESeq2, and DEGseq (adjusted P-value <0.01, fold change < 2/3 or > 3/2) to explore the association between Oxy@Lipo/Gel treatment and macrophage M2 polarization. The raw data were deposited in the NCBI GEO database (accession: GSE301389).

2.16. Reverse transcription quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from frozen rhesus monkey tracheal mucosal tissues using TRIzol reagent (Invitrogen). Briefly, approximately 100 mg tissue was homogenized in 1 mL TRIzol with a TissueLyser II (Qiagen) and ceramic beads (30 Hz, 2 min). After adding chloroform (200 μL), the homogenate was centrifuged (12,000 × g, 4 °C, 15 min). The upper aqueous phase was transferred to a new tube, mixed with 500 μL isopropanol, and incubated at −20 °C for 30 min for RNA precipitation. The sample was then centrifuged again (12,000 × g, 4 °C, 10 min). The obtained RNA pellet was washed with 75% DEPC-treated ethanol, air-dried, and resuspended in RNase-free water. Genomic DNA was eliminated with DNase I (Thermo Fisher, 37 °C, 30 min), followed by purification using RNA Clean & Concentrator-5 Kit (Zymo Research). RNA concentration and purity were assessed via Nanodrop 2000 spectrophotometer (Thermo Fisher). Only qualified samples were used for RT-qPCR analysis. All primer sequences are listed in Table S1. Experiments were performed with 3 independent biological replicates per group.

2.17. Western blot analysis

Total protein was extracted from rhesus monkey tracheal mucosal tissues by homogenizing the samples in ice-cold RIPA lysis buffer (Solarbio) containing 1 mM PMSF (Solarbio) and phosphatase inhibitor cocktail (Roche). After incubation on ice for 45 min, the lysates were centrifuged at 12,000 × g for 15 min at 4 °C to collect the supernatants. Protein concentration was quantified via BCA assay kit (Thermo Fisher). For electrophoresis, 30 μg of protein was mixed with 5× loading buffer and denatured at 95 °C for 10 min. Samples were then separated on 10% SDS-PAGE gels [80 V for 30 min (stacking gel), 120 V for 90 min (separating gel)]. Subsequently, proteins were transferred onto PVDF membrane (Millipore) at 100 V for 90 min. The membranes were blocked with 5% non-fat milk in TBST at RT for 1 h and then incubated with specific primary antibodies overnight at 4 °C. After three 10 min-washes with TBST, the membranes were probed with HRP-conjugated secondary antibodies (1:5000, Invitrogen) for 1 h at RT, followed by another series of three TBST washes. Protein bands were visualized with ECL kit (Millipore, Cat. No. WBULP) and imaged via ChemiDoc system (Bio-Rad). Protein molecular weight markers (ABclonal Technology) were used for calibration, with β-actin or GAPDH served as internal reference. Gray values were analyzed via ImageJ software. Antibody details are provided in Table S2. All experiments were repeated independently at least 3 times.

2.18. Flow cytometry analysis

Single-cell suspensions were prepared from rhesus monkey tracheal mucosal tissues. Briefly, minced tissues were digested in DMEM supplemented with 0.2% collagenase Ⅳ, 0.05% trypsin, 0.1% hyaluronidase at 37 °C for 45 min with gentle shaking. The digest was then filtered through a 70 μm cell strainer (BD Falcon) to obtain single-cell suspensions. Cells were collected by centrifugation at 300 × g for 5 min at 4 °C, and cell pellets were washed twice with PBS under the same centrifugation conditions.

For ROS detection, the prepared cells were incubated with 10 μM DCFH-DA (Beyotime) in the dark at 37 °C with shaking (150 rpm) for 20 min. After incubation, cells were washed three times with PBS (300 × g, 5 min each) to remove excess probe and resuspended at a density of 1 × 10⁶ cells/mL. The ROS fluorescent signal was detected via Beckman CytoFlex S flow cytometer (FITC channel). Unstained cells served as a negative control.

To phenotype the macrophages, single-cell suspensions were blocked with 10% FBS (Gibco) at RT for 30 min. Cells were then incubated with primary antibodies [anti-CD68, anti-CD86, CD206/mannose Receptor (D-1) for 1 h at 4 °C in the dark with gentle agitation. After washing three times with PBS (300 × g, 4 °C, 5 min each), cells were stained with fluorescent secondary antibodies for 30 min at RT in the dark. Following another three washes, cells were resuspended in 2% FBS solution at a density of 1 × 10⁶ cells/mL. Flow cytometry was performed on a CytoFlex S flow cytometer (Beckman Coulter). Data were analyzed via CytoExpert software to determine the expression levels of macrophage markers (CD68, CD86, CD206). All antibody used are detailed in Table S2, and experiments were conducted at least three biological replicates per group.

2.19. Immunofluorescence staining

Paraffin-embedded rhesus monkey tracheal mucosal tissue sections (4 μm) were deparaffinized and rehydrated. After fixation in 4% paraformaldehyde for 30 min at RT and three 5-min washes with PBS, sections were permeabilized with 0.3% Triton X-100 for 15 min and washed again. Non-specific binding was blocked by incubating with 10% goat serum (Solarbio) for 30 min at RT. Primary antibodies (rabbit anti-TNF-α, mouse anti-IL-1β, mouse anti-IL-6) were applied to incubate the sections overnight at 4 °C. Following three washes with PBS, sections were incubated with secondary antibodies (Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 594/647 anti-mouse IgG) for 1 h at RT. After sections were rewashed three times with PBS, nuclei were counterstained with DAPI for 5 min (1 μg/mL, Solarbio) at RT and excess DAPI was removed by PBS washing. Finally, sections were mounted with an anti-fade medium (Beyotime), and imaged using a Zeiss LSM 880 laser scanning confocal microscope. All primary antibodies were pre-verified for cross-reactivity with M. mulatta antigens. For negative controls, the primary antibodies were replaced with PBS. Detailed antibody information is provided in Table S2. For each group, at least 5 fields of view were analyzed.

2.20. Statistical analysis

Statistical results were expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical differences between groups were analyzed by using one-way ANOVA followed by Bonferroni's post hoc test. SPSS 23.0 and Origin 2025 software were used for statistical analyses. A P-value of < 0.05 was considered statistically significant (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

3. Results and discussion

3.1. Synthesis and characterization of OHA/CMCS hydrogel

OHA was synthesized via periodate oxidation of HA. In this reaction, NaIO₄ selectively oxidizes the hydroxyl groups (-OH) on HA to form dialdehyde groups, affording aldehyde-modified HA (OHA). The oxidation degree, which exhibits an inverse correlation with the molecular weight of the resulting product, can be regulated by the mass ratio of HA to NaIO₄ [31,32]. To obtain a suitable oxidation degree for subsequent experiments, the HA/NaIO₄ mass ratio was fixed to 1:1 in this study, and the target OHA was according obtained. The primary structural distinction between pristine HA and oxidized OHA lies in the conversion of vicinal dihydroxyl groups (-OH) into aldehyde functionalities (-CHO) (Fig. 1A), and such structural transformation was further verified via ¹H NMR spectroscopy. Compared with the ¹H NMR spectrum of raw HA, the OHA spectrum presents two prominent variations: (i) a newly emerged characteristic peak assigned to aldehyde protons (green marker, Fig. 1B), which unambiguously verifies the successful generation of -CHO groups; (ii) obvious changes in peak shapes and signal intensities within the range of 2.0∼5.0 ppm, indicative of the modified chemical environment of the polymeric backbone upon oxidative modification (Fig. 1B). On the basis of the acquired ¹H NMR spectra of OHA, the aldehyde substitution degree of OHA was calculated to be 26.24%.

Fig. 1.

Characterizations of the Oxy@Lipo/Gel coating. (A, B) Chemical structures (A) and ¹H nuclear magnetic resonance (¹H NMR) spectra (B) of hyaluronic acid (HA) and oxidized hyaluronic acid (OHA). (C, D) Fourier transform infrared (FTIR) spectra of HA, OHA, CMCS, and OHA/CMCS Gel. (E, F) Size distribution (E) and zeta potential (F) of liposomes (Lipo) and oxybuprocaine-loaded liposomes (Oxy@Lipo). (G) Representative transmission electron microscopy (TEM) micrographs of Oxy@Lipo. (H) Schematic illustrating the fabrication of the Oxy@Lipo/Gel coating via incorporation of Oxy-loaded liposomes into OHA/CMCS hydrogel. Created with BioRender.com. (I) Representative scanning electron microscopy (SEM) micrographs of pure OHA/CMCS Gel and Lipo/Gel. Red arrows indicate liposome microreservoirs. (J) Energy-dispersive X-ray spectroscopy (EDS) analysis of Lipo/Gel. (K) Fluorescence microscopy images of DiI-labeled liposomes (red), FITC-labeled hydrogel (green), and the merged image of Lipo/Gel. (L) Injectability assessment of RhB-stained Lipo/Gel: the composite can be smoothly extruded from a syringe via a 21 G needle. (M) Digital photographs of individual precursors: OHA solution (left) and CMCS solution (right). (N) Self-healing performance of Lipo/Gel labeled with RhB and methylene blue: cut hydrogel pieces spontaneously reconnect and heal after physical contact, verifying its favorable self-repair capacity.

In this work, CMCS was chosen because it is compatible with the designed shear-responsive lubrication mechanism and features superior mucosal adaptability, desirable gelation performance and controllable drug-loading capacity for clinical catheter coatings. Subsequently, a sprayable OHA/CMCS hydrogel was fabricated via mixing aqueous OHA and CMCS solutions. After systematic concentration optimization, the optimal formulation consisting of 6 wt% OHA and 2 wt% CMCS (mixed at a volume ratio of 1:1) was finalized, owing to its ultrafast gelation within 10 s upon spraying (Table S3). Fourier-transform infrared (FTIR) spectroscopy was utilized to characterize the chemical structures of OHA, CMCS, and the formed OHA/CMCS hydrogel (Fig. 1C and D). The FTIR spectrum of OHA displayed a characteristic absorption peak at 1726 cm⁻¹ originating from the stretching vibration of aldehyde moieties, which stemmed from oxidative cleavage of the cis-diol C-C bonds in native HA and verified the successful preparation of oxidized OHA. Following hydrogel formation via Schiff-base crosslinking between OHA and CMCS, the characteristic aldehyde absorption at 1726 cm⁻¹ vanished entirely. Meanwhile, a new absorption peak emerged at 1636 cm⁻¹, which was attributed to the stretching vibration of imine linkages (-N=C-) generated during the Schiff-base reaction. Collectively, these spectroscopic evidence corroborates the successful synthesis of OHA and its covalent crosslinkage with CMCS to yield Schiff-base crosslinked hydrogel.

3.2. Synthesis and characterization of Lipo/Gel coating

Liposomes were fabricated via the conventional thin-film hydration method [25]. Blank liposomes (denoted as Lipo) were prepared from DOPC, DSPE-mPEG₂₀₀₀, and cholesterol at a fixed molar ratio of 70:5:25. Oxybuprocaine-loaded liposomes (denoted as Oxy@Lipo) were further prepared by incorporating oxybuprocaine (Oxy) into the above lipid mixture at a drug-to-lipid mass ratio of 2:1. The DLE and DLC of Oxy@Lipo were determined to be 91.96% and 53.18%, respectively, indicating favorable drug-loading capacity (Table S4). DLS analysis revealed that blank Lipo possessed an average hydrodynamic diameter of 184.69 ± 0.51 nm with a polydispersity index (PDI) of 17.85%. After Oxy encapsulation, the mean particle size of Oxy@Lipo rose to 210.67 ± 5.61 nm (PDI = 19.54%) (Fig. 1E). The corresponding zeta potentials of Lipo and Oxy@Lipo were −7.38 ± 0.52 mV and −6.39 ± 0.41 mV, respectively (Fig. 1F). The distinct particle size increment combined with negligible variation in surface potential indirectly verified the successful encapsulation of Oxy inside liposomal vesicles. Both liposomal formulations displayed satisfactory colloidal stability; no obvious fluctuations in particle size or PDI were observed after seven days of storage in ddH₂O (Fig. S2). Furthermore, the average particle sizes of liposomes remained at 218.7 ± 3.7 nm, 204.8 ± 5.4 nm and 209.0 ± 7.9 nm at pH 7.4, 6.4 and 5.4 (Fig. S3), respectively, confirming robust physical stability across the tested pH gradients. Such pH-insensitive behavior originated from the near-neutral nature of all constituent lipids (DOPC, DSPE-mPEG₂₀₀₀, and cholesterol), which minimizes electrostatic alteration upon ambient pH shift. TEM images showed that Oxy@Lipo presented a typical circular lipid bilayer structure with obvious hollow core features, consistent with the classic morphology of drug-loaded liposomes (Fig. 1G).

Inspired by prior investigations demonstrating the capability of liposomes to improve the lubricating performance of hydrogels [18], liposomes were incorporated into the OHA/CMCS hydrogel for lubrication optimization. As shown in Fig. 1H, composite coatings consisting of oxybuprocaine-loaded liposomes embedded within hydrogel (denoted as Oxy@Lipo/Gel) were successfully fabricated. SEM images revealed that the OHA/CMCS hydrogel possessed a homogeneous porous network; liposomes were evenly distributed throughout the pore cavities and along the hydrogel skeleton without apparent aggregation (Fig. 1I). EDS mapping verified the existence of C, N, O, Cl and P across the Lipo/Gel composite. Specifically, the detected phosphorus (P) originated from phospholipid constituents of liposomal bilayers, further corroborating effective liposome incorporation into the hydrogel network (Fig. 1J). Fluorescence microscopy displayed distinct red fluorescence originating from DiI-tagged liposomes homogeneously dispersed within the green FITC-stained hydrogel framework (Fig. 1K). Such fluorescence distribution was consistent with SEM and EDS findings, confirming the uniform and stable integration of liposomes into the hydrogel coating.

Subsequently, the macroscopic morphology and physical properties of Lipo/Gel coating were systematically characterized. The Lipo/Gel coating exhibited excellent injectability, as it could readily pass through a standard 21G syringe needle (Fig. 1L). Furthermore, benefiting from the rapid in situ gelation of the OHA/CMCS precursors upon spraying, the Lipo/Gel coating also demonstrated robust sprayability, supporting its feasibility for clinical local delivery applications (Fig. 1M). Notably, the Lipo/Gel coating possessed intrinsic self-healing capability. When two separate pink and blue gel blocks were brought into contact, they spontaneously fused into an integrated, crack-free structure within several minutes, indicating that the coating could autonomously restore its structural integrity after mechanical damage (Fig. 1N). This self-healing property is attributed to the dynamic reversibility of the imine linkages (Schiff base), synergistically assisted by non-covalent interactions and polymer chain mobility within the hydrogel network [33]. Medical catheters require short- or long-term contact with the human tissues, and their surface coatings must withstand operational friction, physiological erosion, and minor impact damage while maintaining structural integrity —this prevents safety hazards associated with exposure of the catheter substrate [34]. The inherent self-healing property of Lipo/Gel coatings directly addresses this critical requirement for durability and safety, enabling the material to autonomously repair minor damage and preserve its functional function in situ.

The gelation behaviors of Lipo, OHA, CMCS, blank OHA/CMCS hydrogel (Gel), and blank liposome-incorporated hydrogel (Lipo/Gel) aqueous solutions were first qualitatively characterized at RT using the test tube inversion method [27]. Sol-gel transition was exclusively observed in Gel and Lipo/Gel formulations composed of both OHA and CMCS, whereas individual Lipo, OHA, or CMCS aqueous solutions were incapable of hydrogel formation (Fig. 2A). Subsequent rheological characterizations were performed on Gel and Lipo/Gel samples. In strain and frequency sweep measurements (Fig. 2B and C), the storage modulus (G') and loss modulus (G'') of the two samples displayed nearly identical values under low strain and frequency conditions with marginal discrepancies. Specifically, Lipo/Gel possessed marginally improved G′ stability, weakened frequency dependence, and a slower growth rate of G'', demonstrating that embedded liposomes barely alter the intrinsic rheological features of the host hydrogel yet moderately elevate its structural stability. Time sweep measurements (Fig. 2D) verified fast sol-gel transition for both hydrogel systems, where the G′ value of Lipo/Gel progressively rose and finally reached a plateau following the same trend as pristine Gel, which verified unchanged gelation kinetics upon liposome incorporation. Steady-shear viscosity measurements against shear rate (Fig. 2E) confirmed prominent shear-thinning behavior for both formulations. Remarkably, Lipo/Gel exhibited statistically higher viscosity across the full tested shear-rate range, especially under low-shear environments; such prominent rheological divergence was ascribed to the introduction of liposomes. Benefiting from this viscosity enhancement, Lipo/Gel acquires favorable static structural stability at rest and instant fluidization under high shear loading, endowing it with promising application prospects. Step-strain recovery tests (Fig. 2F) corroborated comparable thixotropic characteristics for both hydrogels: the crosslinked networks were disrupted under large-amplitude strain and rapidly reconstructed upon strain reduction. Collectively, Gel and Lipo/Gel share analogous rheological signatures including rapid gelation, shear-triggered injectability/sprayability, and favorable thixotropic recovery, with statistically meaningful disparities limited to viscosity-related parameters. The negligible deviation in modulus values confirms that liposome incorporation does not impair the core physicochemical performance of the parent hydrogel, and the elevated viscosity of Lipo/Gel originates directly from the incorporated liposomal components.

Fig. 2.

In vitro rheological performance, cytotoxicity and degradability of the Lipo/Gel coating. (A) Gelation assessment of different solution components via the tube inversion test; photographs display OHA, CMCS, Lipo, OHA/CMCS Gel and Lipo/Gel. (B) Strain-sweep rheology measured at a fixed frequency of 1.0 Hz and a strain range of 10⁻²∼10³%. (C) Angular frequency-sweep tests carried out at a constant strain of 1% across an angular frequency range of 10¹∼10⁵ rad/s. (D) Time-dependent changes in storage modulus (G′) and loss modulus (G″) following the mixing of Lipo-containing OHA and CMCS, demonstrating rapid sol-gel transition. (E) Shear-thinning behavior of Gel and Lipo/Gel; the inset shows the injectability of the coating. (F) Continuous step-strain measurements on Lipo/Gel (duration: 3 min) at a fixed frequency of 1.0 Hz. (G) Micrographs of erythrocytes incubated with Lipo/Gel in 2% PBS. PBS and ddH₂O were set as negative and positive controls, respectively. (H) Hemolysis rate of Lipo/Gel in erythrocyte-PBS suspension; n = 3, mean ± SD. (I, J) In vitro cytocompatibility evaluation of L929 cells cultured with Lipo/Gel for varied incubation durations: cell viability quantified via CCK-8 kits (I), and live/dead fluorescence staining with calcein-AM/PI (J); n = 6, mean ± SD; ns, no significant differences. (K) Photographic monitoring the in vitro degradation of Lipo/Gel over 24 h in PBS (pH 7.4), artificial saliva (pH 6.6), and artificial urine (pH 6.5).

In conclusion, the injectable, sprayable and self-healable Lipo/Gel coating serves as a versatile candidate to ameliorate the surface performance of clinical medical catheters. These integrated functional properties address the inherent drawbacks of traditional catheter surfaces via providing surface lubrication, resisting physiological erosion, and preserving structural integrity. Moreover, the intrinsic rapid gelation, shear-responsive processability and rapid thixotropic recovery further facilitate clinical compatibility and in-service performance of modified catheters.

3.3. Biocompatibility and degradability of the Lipo/Gel coating

Biocompatibility constitutes an essential prerequisite for the prospective clinical translation of Lipo/Gel coating. Accordingly, systematic biocompatibility evaluations were performed through a combination of in vitro hemocompatibility testing, in vitro cytotoxicity assays, and in vivo biosafety assessments. First, hemolytic performance was quantified by incubating Lipo/Gel coating specimens with purified red blood cells (RBCs). The measured hemolysis ratios of all experimental groups varied between 0.67% and 1.74% (Fig. 2G and H), all values were substantially lower than the clinically recognized safety threshold of 5% [35], thereby verifying the favorable hemocompatibility of the coating. Subsequently, in vitro cytotoxicity of the lipo/Gel coating was assessed using L929 cells, HUVEC cells and BEAS-2B cells. After 24 h and 48 h incubation with serially diluted Lipo/Gel extracts at concentrations ranging from 5 to 20 mg/mL, quantitative CCK-8 measurement and qualitative live/dead fluorescence staining consistently demonstrated negligible detrimental impacts on the viability of all three cell lines (Fig. 2I and J; Fig. S4). For in vivo biosafety validation, a subcutaneous implantation mouse model was adopted. Briefly, Lipo/Gel samples were surgically implanted into the dorsal subcutaneous tissue of experimental mice. At 24 h post-implantation, histological observation of major visceral organs and routine serum biochemical analysis indicated no obvious abnormal toxic responses relative to the control group (Fig. S5), further confirming the in vivo biosafety of the Lipo/Gel coating.

Apart from favorable biocompatibility, the swelling performance of the Lipo/Gel coating is another crucial physicochemical index for practical application. The swelling profile of Lipo/Gel was characterized in pH 6.6 artificial saliva, which was formulated to simulate the physiological microenvironment of oral and respiratory tracts. The coating underwent rapid initial hydration and swelling, reaching an equilibrium swelling ratio of approximately 3000% driven by the unfolding of crosslinked polymeric networks. Thereafter, the ionic components present in artificial saliva, together with dynamically reversible Schiff base linkages induced rearrangement and partial collapse of polymer chains; consequently, the swelling ratio progressively declined and plateaued within a range of 400% to 470% after 32∼48 h of incubation (Fig. S6). No obvious structural disintegration was observed for the hydrogel even after 48 h of continuous immersion. These results demonstrate that the Schiff base-crosslinked OHA/CMCS matrix possesses a compact and stable 3D network architecture capable of modulating water uptake and swelling kinetics. Such tunable swelling behavior effectively avoids coating rupture and detachment from catheter surfaces, whereas moderate water absorption sustains robust interfacial adhesion between the Lipo/Gel coating and underlying catheter substrate.

The in vitro degradability of the Lipo/Gel coating was explored in three distinct physiological fluids, namely pH 7.4 PBS, pH 6.6 artificial saliva, and pH 6.5 artificial urine. Degradation profiles were quantified by monitoring the residual mass loss of specimens. As shown in Fig. S7, the mass loss of Lipo/Gel surpassed 50% after 6 h of incubation in all three media. At 24 h post-incubation, the corresponding degradation ratios were determined to be 68.88 ± 3.17% in PBS, 74.37 ± 3.33% in artificial saliva, and 62.19 ± 1.74% in artificial urine, respectively. These findings confirm that the Lipo/Gel hydrogel possesses tunable degradability, with its degradation rate closely related to the surrounding environment. Complementary digital photographic evidence further confirmed visible bulk degradation of the Lipo/Gel within 6∼8 h across all tested physiological media (Fig. 2K). These in vitro degradation profiles are well-aligned with the clinical requirements for short-term implanted or body-contacting medical catheters, as they strike a critical balance between two key performance criteria: sustaining the coating's functional efficacy (e.g., lubrication, pain relief) during clinical use and mitigating the risk of complications associated with persistent residual debris in the body.

Notably, the present Lipo/Gel coating is specifically tailored for short-term endotracheal intubation during general anesthesia, rather than for long-term indwelling catheterization. Clinically, routine general anesthesia procedures typically last 2∼6 h, while even the majority of complex surgical interventions are completed within 6∼8 h. Consequently, the 6∼8 h degradation window of the Lipo/Gel coating perfectly overlaps with the entire indwelling period of endotracheal tubes for the vast majority of surgeries. This temporal matching ensures that the coating maintains stable lubricating and analgesic properties throughout the entire surgical procedure, without requiring prolonged structural integrity beyond clinically required duration. Collectively, the Lipo/Gel coating demonstrates favorable biocompatibility and well-tunable, clinical scenario-matched degradability. These core physicochemical and biological characteristics lay a solid foundation for subsequent preclinical and clinical translational research.

The release kinetics of Oxy from Lipo and Lipo/Gel were systematically characterized. A biphasic release profile, which features an initial rapid release followed by a sustained release phase, was observed for both formulations. Specifically, cumulative Oxy release from Lipo reached approximately 50% within 12 h of incubation, whereas drug release from Lipo/Gel proceeded at a markedly slower rate (Fig. 3A). This indicates that the OHA/CMCS hydrogel further extends Oxy release via its network diffusion-retarding effect, laying a foundation for long-acting analgesia.

Fig. 3.

In vitro hydrophilicity, drug release behavior, protein adsorption resistance, and medical device adhesion of the Lipo/Gel coating. (A, B) Cumulative in vitro release profiles of Oxy from Lipo and Lipo/Gel in PBS (pH 7.4) (A), and from Lipo/Gel in PBS buffers with different pH values (B). (C, D) Relative adsorption rates of bovine serum albumin (BSA) (C) and fibrinogen (Fib) (D) on polyvinyl chloride (PVC) or polyurethane (PU) tubes with/without Lipo/Gel coating. (E) Photographs showing the wettability transition of various medical tubes (PVC, silicone, latex, PU) from hydrophobic (left) to hydrophilic (right) after Lipo/Gel spray-coating. (F) Static contact angles of various flat substrates (PVC, silicone, latex, PU) before and after Lipo/Gel spray-coating. (G) Optical images (top row, Control) and corresponding fluorescence images (bottom row, Coating) of medical devices partially spray-coated with FITC-labeled Lipo/Gel (1 mg/mL): i) PVC endotracheal tube; ii) laryngeal mask; iii) rubber urinary catheter; iv) silicone urinary catheter; v) silicone gastric tube; vi) central venous catheter; vii) divergent tube; viii) stainless steel bar. n = 3, mean ± SD; ∗∗∗P < 0.001.

Beyond the inherent sustained-release property of OHA/CMCS hydrogel confirmed above, the fabricated Lipo/Gel also possessed favorable pH-responsive release capability for Oxy. Oxy exhibited distinct pH-responsive release behavior, with markedly accelerated drug release under acidic conditions as verified in Fig. 3B (faster release at pH 6.4 versus pH 7.4). To further mimic the physiological oral environment, we assessed Oxy release at saliva-mimicking pH 6.6, which also yielded enhanced release rates under this weakly acidic condition. To uncover the underlying structural origin of such pH responsiveness, hydrogel microstructure was visualized by SEM. As presented in Fig. S8, hydrogel pore size gradually enlarged and the network became more porous with declining pH. Such morphological change originates from acid-triggered hydrolysis of Schiff base bonds within OHA/CMCS, which loosen the crosslinking network and structurally accounts for the observed pH-dependent release of Oxy [36].

3.4. Hydrophilicity and adhesion of the Lipo/Gel coating to diverse medical devices

Biofilm formation, a primary trigger of catheter-associated infections, is initiated by protein adsorption onto the surface of medical catheters [37]. Accordingly, the antifouling performance of the Lipo/Gel coating was evaluated via in vitro protein adsorption assays. The adsorption rates of both fibrinogen and BSA on Lipo/Gel-coated catheters were significantly lower than those on unmodified catheters (P < 0.001, Fig. 3C and D). Furthermore, as illustrated in Fig. S9, the E. coli adhesion rate for the Lipo/Gel-coated group was markedly lower than that of the control group following 1∼2 h of incubation (P < 0.05). These results demonstrated that PVC catheters modified with the Lipo/Gel coating efficiently suppress early bacteria adhesion; such observations align with the protein adsorption inhibition data confirm the favorable antibacterial potency of the coating. This effective antifouling performance during the early stage of biofilm development renders the Lipo/Gel coating a promising candidate for reducing the incidence of catheter-associated infections and improving the clinical safety of indwelling catheters.

The surface hydrophilicity of catheter samples was assessed via static water contact angle measurements [38]. As visualized in Fig. 3E, spray application of the Lipo/Gel coating substantially improved the spreading performance of water droplets on the curved surface of various commercial medical catheters (PVC, silicone, latex, PU). Quantitative static contact angle tests were further performed on corresponding flat substrates fabricated from identical raw catheter materials (PVC, silicone, latex, PU); statistical analysis confirmed that Lipo/Gel modification markedly reduced substrate contact angles (P < 0.001, Fig. 3F). Collectively, these findings demonstrate that the Lipo/Gel coating efficiently enhances the hydrophilicity of catheter materials, and this improved hydrophilic property is expected to reduce interfacial frictional resistance.

In addition, the adhesion and distribution of the Lipo/Gel coating on various medical catheter surfaces were visualized via FITC labeling. Firstly, the coating adhered uniformly to all tested catheters (including PVC, PU, silicone, and latex) with no obvious detachment (Fig. 3G), confirming its favorable compatibility with common catheter materials. Secondly, to mimic the mechanical stress encounted during clinical intubation, FITC-labeled Lipo/Gel-coated PVC catheters were subjected to sequential bending and torsion tests, and the coating integrity was evaluated by fluorescence imaging. As evidenced in Fig. S10A, no cracking, delamination or peeling was detected upon bending; only negligible localized surface deformation appeared at high-stress regions. These results demonstrate that the coating exhibits favorable mechanical durability against clinically relevant clinical manipulations. Thirdly, to recapitulate the in vivo physiological environment and preclude undesired damage arising from excessive mechanical shear, dynamic perfusion assays using pH 6.6 artificial saliva were implemented to characterize coating stability. Representative images in Fig. S10A demonstrate that the Lipo/Gel coating preserved favorable structural integrity following 30 min of mild saliva perfusion relative to bare unperfused PVC catheter, without apparent shedding or bulk peeling; this observation confirms robust interfacial adhesion under physiologically relevant simulated conditions. For further validation, parallel perfusion trials were conducted on FITC-labeled Lipo/Gel-coated PVC substrates under identical settings (Fig. S10B). The coating maintained full structural integrity with minimal detachment, accompanied by a fluorescence retention rate above 99%, which was statistically indistinguishable from the untreated non-perfused control (Fig. S10C).

Furthermore, quantitative peel tests were implemented to quantify the interfacial bonding strength between the Lipo/Gel coating and substrate specimens fabricated from four raw materials (PVC, silicone, latex, PU). From the results presented in Fig. S11, the average peel strength for coating-substrate assemblies reached 464.6 ± 14.6 J/m², 525.5 ± 24.5 J/m², 478.7 ± 15.6 J/m² and 566.7 ± 14.8 J/m², respectively. Meanwhile, complementary SEM characterizations were performed on Lipo/Gel-coated PVC specimens prior to and post friction abrasion. According to Fig. S12, the coating maintained structural integrity upon friction, featuring only trivial localized abrasion without bulk delamination or crack formation, corroborating favorable coating–substrate interfacial bonding against frictional load. Cross-sectional SEM images revealed a well-integrated coating-PVC boundary devoid of obvious voids or interfacial separation, further validating persistent adhesion over the whole friction cycle. Collectively, these results highlight superior interfacial robustness of the Lipo/Gel coating against various mechanical loads mimicking practical clinical scenarios, which adequately fulfills the functional requirements of indwelling catheters during intubation and mucosal contact.

In summary, the Lipo/Gel coating achieves pH-responsive sustained Oxy release. Through enhancing surface hydrophilicity, suppressing nonspecific protein adsorption, conferring potent antibacterial activity, and securing robust interfacial adhesion, the Lipo/Gel coating equips commercial catheters with superior biofunctionalities, which offers great potential for clinical translation.

3.5. Interfacial lubrication properties of the Lipo/Gel coating

Although liposomes were incorporated into the OHA/CMCS hydrogel to facilitate Oxy loading and sustained release, another core objective was to improve the lubricating properties of the hydrogel coating. To systematically verify the lubrication-enhancing effect of liposomes embedded within the hydrogel coating and elucidate the underlying mechanism, tribological tests were performed on different lubricant samples. A UMT-3 tribometer was employed to conduct linear reciprocating motion tests, with the CoF were recorded in real-time (Fig. 4A). Compared with the saline group, both the Gel and Lipo/Gel coatings yielded a marked reduction in CoF (P < 0.001); importantly, the Lipo/Gel group exhibited an even lower CoF compared with the Gel group (Fig. 4B and C). Meanwhile, optical microscopy was utilized to characterize the wear width of coated substrates, and the diminished wear width observed for the Lipo/Gel group agreed well with the CoF variation trends (Fig. 4D and E). These preliminary findings confirm that liposomes incorporation effectively improves the intrinsic lubricating capability of the hydrogel matrix. To further evaluate the translational potential of Lipo/Gel coatings, free liposome suspension and liquid paraffin (a commercially available clinical lubricant) were set as extra control groups to compare the lubricating performance of diverse formulations. The results revealed that free liposome suspension exhibited superior lubrication than saline yet inferior lubricity relative to the Gel and Lipo/Gel group. Importantly, the Lipo/Gel coating displayed markedly lower CoF values than bare Gel and free liposome suspension alike, further confirming the improved lubrication stemming from liposome incorporation. Furthermore, no statistically significant difference in CoF was observed between Lipo/Gel and liquid paraffin (Fig. S13A), indicating that the composite coating delivers lubricating efficacy comparable to conventional clinical lubricants.

Fig. 4.

Lubrication performance of the Lipo/Gel coating. (A) Schematic of the UMT-3 tribological test. Created with BioRender.com. (B) Coefficient of friction (CoF)-time curve for the Saline, Gel, and Lipo/Gel groups. (C) CoF histograms for three experimental groups. (D) Bright-field micrographs of wear tracks on PVC substrates following testing with saline, Gel, and Lipo/Gel. (E) Quantitative analysis of wear width across three groups. (F) Schematic illustrating the self-lubrication mechanism for lipid-loaded hydrogel: when liposome microreservoirs on the hydrogel surface undergo frictional wear, additional underlying microreservoirs are exposed, thereby forming lipid boundary layers on the surface. Created with BioRender.com. (G) SEM micrographs of Lipo/Gel before and after friction testing. (H) DiI-labeled liposomes on the Lipo/Gel surface before and after friction testing. (I) Quantification of fluorescence intensity (for DiI-labeled liposomes); n = 6, mean ± SD; ∗∗∗P < 0.001.

Given the complex physiological microenvironment of mucosal tissues in vivo, artificial saliva was adopted as a simulated lubricating medium to re-evaluate the environmental adaptability of Lipo/Gel coatings by quantifying the CoF of all test groups. The results showed that artificial saliva inherently exhibits better lubricity over saline; nevertheless, both the Gel and Lipo/Gel coatings achieved markedly lower CoF values than pure artificial saliva and saline when tested in this physiological medium (Fig. S13B). These findings verify that the Lipo/Gel coating maintains excellent lubricating performance under saliva-simulated physiological conditions, highlighting its promising prospects for in vivo applications.

Considering the clinical reality that indwelling catheters are persistently exposed to bodily fluids, we further explored how prolonged soaking in artificial saliva affects the lubricating properties of the Lipo/Gel coating. After 30 min immersion in artificial saliva, the pre-soaked Lipo/Gel coating retained a low average CoF of 0.053, with no statistically significant difference relative to the untreated counterpart (average CoF = 0.048). These results reveal negligible degradation in lubricating performance following prolonged hydration (Fig. S13C). Combined with our earlier static water contact angle data, the coating is capable of generating a robust surface-hydrated layer to lower interfacial frictional resistance after immersion, further indicating the durable lubrication of Lipo/Gel in fluid microenvironments and satisfying the practical requirements for clinical translation.

Collectively, liposome modification substantially enhances the lubricating performance of hydrogels, and the Lipo/Gel coatings exhibit lubrication efficiency equivalent to that of clinical liquid paraffin. The composite coating maintains excellent lubricity under both conventional and artificial saliva-simulated physiological conditions, with no obvious lubrication decline after long-term immersion in body fluids. Benefiting from favorable physicochemical and lubricating properties compatible with in vivo physiological environments, the developed Lipo/Gel coating holds great promise for clinical lubrication applications such as biomedical catheters.

The improved lubrication is presumed to originate from a dynamic self-lubricating effect conferred by embedded liposomes (Lipo). Upon medical catheter insertion and long-term indwelling in vivo, frictional contact against surrounding tissues gradually wears the coating and prompts the outward exposure of embedded Lipo. The phospholipid bilayer of exposed Liposomes subsequently assemble into a continuous lubricating film at the frictional interface, which isolates direct contact between the catheter and luminal mucosa and cuts down interfacial frictional resistance, consequently reducing the CoF (Fig. 4F). This proposed mechanism was further validated by post-friction surface characterizations. SEM observations confirmed markedly increased Lipo exposure across worn coating surfaces (Fig. 4G). Consistently, post-friction fluorescence characterization displayed remarkably elevated liposome-specific fluorescent signals on the coating surface (Fig. 4H and I). Taken together, these results verify friction-triggered migration and surface enrichment of Lipo, solidifying the existence of a phospholipid-based lubricating interfacial layer.

In conclusion, our work presents a multifunctional Lipo/Gel coating featuring three core innovations. First, we establish a mutually reinforcing system in which liposomes improve the lubricity of hydrogel, while the hydrogel matrix extends the sustained-release performance of drug-loaded liposomes. This design overcomes the limitation of “single-function optimization” found in conventional medical catheter coatings and constructs a synergistic system integrating “lubrication and damage reduction alongside sustained drug release”, thus to realize dual benefits combining physical protection and pharmacological treatment. Second, we elucidate a “dynamic self-lubricating” mechanism, wherein friction induces the exposure of liposomes and facilitates the formation of a boundary-lubricating phospholipid film. Third, the Lipo/Gel coating exhibits robust adhesive capacity and stable functional performance on various catheter substrates, greatly enhancing its practical translational applicability. Based on these encouraging in vitro findings, we further evaluate the coating's therapeutic efficacy and biosafety using in vivo animal models.

3.6. Efficacy of the Oxy@Lipo/Gel coating in endotracheal intubation model

Given their high genetic homology to humans, non-human primates serve as valuable animal models for preclinical research [39]. To simulate clinical tracheal intubation under general anesthesia, a rhesus monkey endotracheal intubation model was established (Video S1). Evaluations were performed from three perspectives: (i) monitoring the monkeys’ vital signs (primarily heart rate) throughout the perioperative period (Video S2); (ii) evaluating airway mucosal injury pre- and post-intubation via flexible bronchoscopy (Video S3); and (iii) assessing the lubricating and analgesic properties of the Oxy@Lipo/Gel-coated catheter by observing spontaneous feeding behavior of monkeys following extubation (Fig. 5A).

Fig. 5.

Efficacy of Oxy@Lipo/Gel coating in a rhesus monkey tracheal intubation model. (A) Experimental procedure schematic of rhesus monkeys. Created with BioRender.com. (B) Bronchoscopic images of tracheal intubation using a coated endotracheal tube in anesthetized, supine rhesus monkeys. Mechanical ventilation was provided with continuous physiological signal recording. (C) Heart rate fluctuations in anesthetized rhesus monkeys during tracheal intubation. (D) Bronchoscopic images of the glottis and trachea pre-intubation, immediately post-extubation, and 3 days post-extubation. Blue arrows indicate mucosal swelling and congestion. (E) Semi-quantitative assessment of tracheal injury severity based on bronchoscopic images; n = 3, mean ± SD; ∗∗∗P < 0.001; ns, no significant differences. (F) (Left) A schematic illustrating post-extubation active feeding behavior observation in rhesus monkeys for pharyngeal discomfort evaluation. Created with Doubao Software. (Right) Representative photographs of rhesus monkeys and their feeding willingness post-extubation. (G) Comparison of feeding intention scores among groups; n = 3, mean ± SD; ∗P < 0.05; ∗∗∗P < 0.001; ns, no significant differences. (H) H&E-stained sections of rhesus monkey tracheal mucosa post-extubation and (I) quantitative analysis of histological injury severity; n = 6, mean ± SD; ∗∗∗P < 0.001.

As shown in Fig. 5B and C, the Oxy@Lipo/Gel group exhibited the narrowest range of heart rate fluctuations during intubation, suggesting that the lubricating coating reduced mechanical damage and mucosal stimulation. Direct bronchoscopic examination identified mucosal edema, contusion, and hemorrhage of the glottis and trachea at three predefined time points: before intubation, immediately after extubation, and on the day 3 after extubation (Fig. 5D). Semi-quantitative scoring of mucosal injury further confirmed that endotracheal intubation almost invariably induced mechanical mucosal damage. Nonetheless, such injury was significantly attenuated in both the Oxy@Gel and Oxy@Lipo/Gel groups, with the Oxy@Lipo/Gel group demonstrating the most prominent protective efficacy (Fig. 5E).

The Oxy@Lipo/Gel group also exhibited the narrowest range of heart rate fluctuations during tracheal extubation (Fig. S14). Post-extubation spontaneous feeding behavior was recorded as a functional indicator of pharyngeal discomfort. On the days 1 and 3 post-extubation, monkeys in the control group (catheters lubricated with normal saline) displayed reduced spontaneous feeding due to post-extubation sore throat (Video S4). In sharp contrast, feeding activity of monkeys from the Oxy@Lipo/Gel group remained nearly comparable to their pre-intubation baseline (Video S5). These results confirm that endotracheal intubation elicits postoperative pharyngeal pain and discomfort (Figure S15, Fig. 5F); by contrast, sustained Oxy release affords local mucosal analgesia, and superior lubrication of the coating lessens mechanical trauma, jointly mitigating intubation-triggered mucosal injury and associated discomfort (Fig. 5F and G). H&E staining analysis was conducted on airway mucosal biopsies harvested via bronchoscope forceps. Histological results revealed severe disruption of mucosal cellular architecture and aggravated epithelial damage following intubation in the control group, whereas the Oxy@Lipo/Gel coating markedly attenuated such mucosal lesions (Fig. 5H and I), validating the coating's tissue-level protective capacity.

In summary, comprehensive physiological, behavioral and histological evidence collectively demonstrates that the Oxy@Lipo/Gel coating markedly alleviates intubation-associated mechanical mucosal injury and relieves resultant postoperative sore throat and pharyngeal discomfort.

3.7. Transcriptome sequencing reveals inflammation-related pathways in Oxy@Lipo/Gel coating-alleviated mucosal injury

To explore the potential molecular mechanism underlying the protective effect of Oxy@Lipo/Gel against airway mucosal damage, RNA sequencing (RNA-seq) analysis was performed on tracheal mucosal tissues from rhesus monkeys in two groups: the Control (saline-lubricated catheters) and the Treat group (Oxy@Lipo/Gel-coated catheters).

Transcriptomic analysis revealed distinct gene expression profiles between the groups. Principal component analysis (PCA; Fig. 6A) revealed clear intra-group sample clustering, with significant separation between the two groups along the first principal component (PC1, 35.37% of variance) and the second principal component (PC2, 27.48% of variance). This confirms substantial global differences in gene expression. A Venn diagram showed 5513 and 5103 genes specifically expressed in the Control and Treat groups, respectively, with 7704 genes shared between them (Fig. 6B, Fig. S16), indicating that Oxy@Lipo/Gel induces distinct transcriptional profiles. Heatmap analysis (Fig. 6C) enabled intuitive visualization of group-specific differential gene expression patterns, corroborating the PCA findings.

Fig. 6.

Transcriptomic profiling identifies differentially expressed genes (DEGs) in tracheal mucosa of rhesus monkeys following distinct interventions. (A) Principal component analysis (PCA) of tracheal mucosa specimens from saline control and Oxy@Lipo/Gel treatment groups; n = 3. (B) Venn diagram illustrating the counts and overlapping relationships of DEGs across experimental groups. (C) Heatmap of significantly altered DEGs [fold change (FC) > 2, P < 0.05] among groups. (D) Volcano plot of DEGs: red dots indicate significantly upregulated genes (e.g., MRC1, ARG1), blue dots indicate significantly downregulated genes (e.g., NOS2, CD86), and gray dots indicate non-significant expression changes. (E) Protein-protein interaction (PPI) network of macrophage polarization-related genes via the STRING database. (F) Gene Ontology (GO) enrichment of DEGs; the top 20 enriched GO terms (biological process/BP, cellular component/CC, molecular function/MF) ranked by adjusted P-value are displayed. (G) KEGG pathway enrichment of DEGs; the top 10 pathways ranked by adjusted P-value are plotted, with prominent enrichment observed in MAPK, NF-κB, JAK-STAT, Nrf2 and IL-17 signaling cascades. (H) Bubble plot of expression and enrichment analysis for DEGs in multiple signaling pathways, revealing the multi-pathway interaction network linked to macrophage polarization. (I) Reactome-based gene set enrichment analysis (GSEA) depicting the enrichment distribution of DEGs across biological processes, cellular components and molecular functions.

To clarify the biological functions of differentially expressed genes (DEGs), functional enrichment and pathway analyses were performed. First, a volcano plot (Fig. 6D) quantified global DEGs distribution: among the total 18,320 detected genes, 1260 genes were defined as significantly differentially expressed (P < 0.05 and |log₂FC| > 1), of which 579 were markedly downregulated. Next, protein-protein interaction (PPI) analysis (Fig. 6E) mapped the interaction network of key DEGs, which uncovered prominent interconnections among core candidates and offered critical clues for screening hub genes governing airway mucosal injury. Subsequently, a circular enrichment plot (Fig. 6F) systematically annotated DEGs from three GO categories, namely biological process (BP), cellular component (CC), and molecular function (MF); this plot simultaneously visualized enriched functional terms, the count of mapped genes, and corresponding statistical significance. Finally, a pathway enrichment bubble plot (Fig. 6G) pinpointed prominently enriched signaling cascades, including MAPK, NF-κB, JAK-STAT, Nrf2 and IL-17 pathways. Bubble size and color separately represent gene counts and enrichment significance, implying that these signaling axes potentially mediate the mucosal protective activity of Oxy@Lipo/Gel against intubation-induced airway injury.

To further analyze core signaling pathway activity, the expression of key genes in the MAPK, NF-κB, Nrf2, IL-17 and JAK-STAT pathways was detected (Fig. 6H), with their expression trends and statistical significance (P-values) clearly displayed. Meanwhile, gene set enrichment analysis (GSEA) identified pathways with significant enrichment differences between the Treat and Control groups: ECM-receptor interaction, mucin type O-glycosylation, vascular smooth muscle contraction, and protein digestion and absorption (Fig. 6I). Specifically, ECM-receptor interaction promotes airway mucosal repair via controlling epithelial cell adhesion, migration and proliferation to remodel extracellular matrix and reconstruct impaired epithelial barriers; mucin-type O-glycosylation stabilizes mucus barrier integrity by modifying airway mucin physicochemical features to intercept pathogens and reduce mucosal damage. Regulating vascular smooth muscle contraction relieves mucosal edema and improves local microcirculation for tissue regeneration; upregulated protein digestion and absorption increase nutrient supply to furnish energy and raw materials required for rapid epithelial proliferation and wound repair. These pathways likely mediate mucosal protection and repair.

In conclusion, transcriptome sequencing analysis indicates that Oxy@Lipo/Gel exerts its mucosal protective effect by regulating key inflammatory (e.g. MAPK, Nrf2, IL-17 signaling) and tissue-repair (e.g. ECM-receptor interaction) pathways, thereby remodeling the gene expression profile of tracheal mucosal tissue. These findings provide the molecular-level insights into the airway mucosal protective mechanism of Oxy@Lipo/Gel and offer theoretical references for subsequent in-depth exploration of specific therapeutic targets and the design of medical catheter coatings.

3.8. Oxy@Lipo/Gel coating alleviates mucosal injury by Nrf2/MAPK-dependent regulation of oxidative stress and macrophage polarization

The aforementioned RNA-seq results suggest that inflammatory signaling pathways may play a key role in the airway mucosal protection afforded by Oxy@Lipo/Gel. To further validate this mechanistic hypothesis, a series of in vitro experiments were conducted. First, intracellular ROS levels were assessed using the DCFH-DA probe via flow cytometry. The fluorescence histogram (Fig. 7A) showed an obvious leftward shift (decreased fluorescence intensity) in the Oxy@Lipo/Gel group compared with the control group. Quantitative analysis confirmed a significant decrease in ROS levels following treatment (P < 0.05, Fig. 7B), indicating that Oxy@Lipo/Gel alleviates oxidative stress by reducing ROS production.

Fig. 7.

Multidimensional analysis of macrophage oxidative stress and polarization phenotypes. (A, B) Flow cytometry detection of ROS levels [FL1-A: DCFH-DA fluorescence intensity (reflecting ROS production); FL2-A: positive cell proportion]. Pink (Control) vs. blue (Treat, Oxy@Lipo/Gel) histograms show changes in ROS levels in the Treat group. n = 3, mean ± SD; ∗P < 0.05. (C) qPCR analysis of mRNA expression levels of M1 polarization markers (CD86, Nos2) and M2 polarization markers (Mrc1, Arg1). n = 3, mean ± SD; ∗∗P < 0.01; ∗∗∗P < 0.001. (D, E) Western blot validation of Oxy@Lipo/Gel's role in regulating M2 macrophage polarization: Protein expression of macrophage surface marker CD86, M1 marker iNOS, and M2 markers CD206/ARG1; β-actin served as the internal control. n = 3, mean ± SD; ∗P < 0.05; ∗∗P < 0.01. (F) Schematic diagram of rhesus monkey tracheal tissues and experimental design for flow cytometry. Created with BioRender.com. (G) Flow cytometry scatter plots of tracheal tissue cells labeled with CD68 (macrophage marker). (H, I) Quantitative bar graphs of the CD86⁺ or CD206⁺ cell ratio in the two groups. n = 3, mean ± SD; ∗P < 0.05. (J) Immunofluorescence staining of tracheal mucosa in rhesus monkeys post-extubation; (K) Quantitative analysis of relative fluorescence intensity for TNF-α, IL-6, and IL-1β; n = 6, mean ± SD; ∗∗∗P < 0.001.

Subsequently, the modulatory effect of Oxy@Lipo/Gel on inflammatory responses was explored by detecting the expression of key genes and proteins. qPCR results showed that Oxy@Lipo/Gel treatment significantly downregulated the mRNA levels of pro-inflammatory markers CD86 (P < 0.01) and Nos2 (P < 0.001), whereas the coating treatment upregulated the transcripts of anti-inflammatory markers Mrc1 (P < 0.01) and Arg1 (P < 0.01) compared with the control group (Fig. 7C). Such phenotypic shift was further verified at the protein level by western blotting (Fig. 7D and E), which showed decreased CD86 (P < 0.01) and iNOS (P < 0.05) expression alongside increased CD206 (P < 0.01) and ARG1 (P < 0.01) following treatment. Collectively, these results indicate that Oxy@Lipo/Gel facilitates macrophage polarization from a pro-inflammatory towards an anti-inflammatory phenotype.

To further verify such polarization at the cellular level, primary macrophages were isolated from rhesus monkeys' tracheal tissue and analyzed by flow cytometry (experimental design outlined in Fig. 7F). Flow cytometric analysis revealed a significantly reduced proportion of pro-inflammatory CD86⁺CD68⁺ macrophages (P < 0.001), while the proportion of anti-inflammatory CD206⁺CD68⁺ macrophages rose significantly relative to controls (P < 0.001, Fig. 7G–I). Consistent with the aforementioned qPCR and western blot data, these cellular findings further confirm that Oxy@Lipo/Gel exerts prominent modulatory effects on macrophage polarization. Finally, immunofluorescence staining and quantitative analysis of core pro-inflammatory cytokines revealed substantially reduced relative fluorescence intensities of TNF-α, IL-6, and IL-1β (all P < 0.001) in the treatment group relative to controls (Fig. 7J and K). Collectively, these results confirm that Oxy@Lipo/Gel effectively inhibit the secretion of these inflammatory cytokines and curbs subsequent cascaded amplification of inflammatory responses.

Furthermore, to confirm that the Nrf2 and MAPK signaling pathways predicted via transcriptome sequencing mediate the mucosal protective effects of Oxy@Lipo/Gel, we quantified the expression of core Nrf2-pathway proteins, including nuclear translocation of Nrf2 and its downstream antioxidant effector HO-1. As illustrated in Fig. S17, relative to the control group, Oxy@Lipo/Gel coating treatment markedly facilitated Nrf2 nuclear translocation and upregulated HO-1 levels, verifying robust activation of the Nrf2 antioxidant pathway in good agreement with transcriptomic pathway enrichment findings. We next quantified the phosphorylation ratios of key MAPK components (p-p38/p38, p-JNK/JNK, p-ERK/ERK) to substantiate the transcriptome-implied changes in MAPK signaling. As shown in Fig. S18, coating treatment significantly reduced the phosphorylation of p38, JNK and ERK, thereby blunting aberrant overactivation of the pro-inflammatory MAPK cascade and furnishing direct molecular evidence supporting the coating's anti-inflammatory activity. Collectively, these results highlight the dual antioxidant and anti-inflammatory bioactivities of the Oxy@Lipo/Gel coating.

Collectively, these findings provide compelling evidence that Oxy@Lipo/Gel confers robust airway mucosal protection by regulating Nrf2 and MAPK signaling cascades to initiate a sequential protective cascade: (i) activation of the Nrf2 antioxidant pathway suppresses intracellular ROS production and alleviates oxidative stress; (ii) restrained MAPK overactivation combined with reduced ROS drives macrophage polarization from pro-inflammatory M1 toward anti-inflammatory M2 phenotype; (iii) M2-biased macrophage polarization further attenuates the secretion of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β and blocks inflammatory cascade amplification. These results validate RNA-seq predictions that Nrf2 and MAPK pathways serve as core regulators of airway mucosal defense. Characterization of this upstream-to-downstream molecular cascade strengthens the translational prospect of Oxy@Lipo/Gel coating and highlights the Nrf2/MAPK axis and macrophage polarization as actionable therapeutic targets for designing next-generation anti-inflammatory coatings for medical devices.

4. Conclusion

In summary, we fabricated a multifunctional Oxy@Lipo/Gel coating that combines sustained, localized oxybuprocaine delivery to achieve prolonged mucosal analgesia with liposome-boosted hydrogel lubrication, specifically alleviating catheter-related mucosal injury and procedural pain. The OHA/CMCS hydrogel confers the coating outstanding biocompatibility, degradation kinetics tunable for short-term indwelling catheters, substantially improved hydrophilicity, good resistance to non-specific protein adsorption and antibacterial adhesion, and favorable interfacial lubrication. Furthermore, its imine moieties endow the hydrogel with pH-responsive drug release, self-healing capacity, and long-lasting lubrication. Meanwhile, Oxy@Lipo mitigates procedural pain via released oxybuprocaine and prominently improves hydrogel lubricity by reconstructing an interfacial lipid film under frictional stress. Critically, endotracheal catheters coated with our engineered Oxy@Lipo/Gel effectively curb intubation-triggered hemodynamic fluctuations, reduce airway mucosal edema and hemorrhage, and maintain post-extubation feeding activity in rhesus monkeys by easing pharyngeal irritation. The underlying mechanism relies on modulation of upstream Nrf2 and MAPK signaling cascades, initiating a sequential protective response via three core biological actions: (i) Nrf2 antioxidant pathway activation to curb excessive ROS generation; (ii) suppression of abnormal pro-inflammatory MAPK signaling to facilitate M2 macrophage polarization; (iii) M2-dominant polarization-driven downregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Importantly, this versatile formulation can be readily modified for diverse perioperative medical devices such as urinary and central venous catheters, establishing a material-based strategy against catheter-associated complications including mucosal damage, phlebitis and postoperative pain. Collectively, the Oxy@Lipo/Gel coating holds great promise for clinical translation and widespread use to improve safety and patient comfort during minimally invasive interventions.

Ethics approval and consent to participate

• 1.All mice-related procedures were approved by the Animal Care Committee of Sun Yat-sen University (Approval No.: SYSU-IACUC-2025-001035).
• 2.The rhesus monkey animal study protocol was approved by the Landau Biotechnology SYXK's Institutional Animal Care and Use Committee (IACUC) (Approval No.: LDACU 20250302-01).

CRediT authorship contribution statement

Sufang Chen: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Haixia Wang: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. Jingyi Du: Investigation, Methodology, Software. Jinyan Guo: Data curation, Methodology. Jing Yang: Funding acquisition, Methodology. Ni Ding: Methodology. Zhenjia Lin: Methodology. Yuzhuo Hei: Methodology. Yu Tao: Funding acquisition, Validation, Writing – review & editing. Ziqing Hei: Funding acquisition, Writing – review & editing. Gangjian Luo: Writing – review & editing. Mingqiang Li: Conceptualization, Funding acquisition, Validation, Visualization, Writing – review & editing. Weifeng Yao: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing.

Declaration of competing interest

No potential conflict of interest relevant to this article was reported.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Data will be made available on request.