Tracheal intubation is a crucial clinical intervention for respiratory management [1]. Although it is indispensable for patients who lose autonomous respiration due to anesthesia, serious injury and coma, it brings about various depressing side effects [2]. Airway injury occurs in almost every patient and is the most common cause of tracheal stenosis. Endotracheal tube (ETT) obstruction is a life-threatening event accompanied by hypoxia, hypercapnia, and respiratory acidosis [3]. Ventilator-associated pneumonia (VAP) is an iatrogenic pulmonary infection that results from pathogen colonization on ETT surfaces [4]. Current solutions to alleviate the intubation-related complications mainly focus on improving clinical intubation procedures, such as optimizing intubation skills, using antibiotics [5], and exerting local anesthesia. However, the ameliorative effect is limited since intubation-related complications are highly related to the ETT itself that lacks the requisite biological performance for intubation, such as water lubricity, antifouling and antibacterial properties [6].

The close relevance between the intubation-related complications and the ETT surface has put forward a great demand for surface modification [7–11] of commercial catheters [12–14]. Surface coatings have evoked an intense interest in realizing the surface functionalization of ETTs [15,16]. Alves et al. immobilized ciprofloxacin (CIP) and/or chlorhexidine (CHX) onto poly(vinyl chloride) (PVC) surfaces through polydopamine (PDA)-based functionalization. The coating exhibited sustained CIP/CHX release to prevent the formation of antibiofilm [17]. Naranjo et al. coated ETTs with mucins purified from porcine gastric mucus. It promoted hydration lubrication and provided the ETT surface with strong resistance toward lipid deposition and cell colonization [18]. A photocurable coating using quaternary benzophenone-based amide was reported to inhibit the formation of biofilms in vivo [19]. Nevertheless, these coatings require complicated preparation procedures, which are not convenient or affordable for handling. In some cases, ultraviolet (UV) crosslinking was employed. Owing to the limited penetration depth, the functionalization of the intraductal surface is challenging [20]. Therefore, it is highly anticipated to develop a simple and scalable approach to confer the full surfaces of ETTs with multiple functionalities to meet the clinic indwelling requirements.

Human airway mucus (HAM), which is composed of two interactional mucins (MUC5B and MUC5AC), lines the epithelium of the respiratory tract to perform crucial barrier functions [21]. It maintains airway hydration to facilitate transport, and traps the inhaled particulates and pathogens to protect the respiratory tract from infection. Here, we developed an artificial respiratory mucus (ARM) coating with HAM-like biofunctions to make ETT an airway-friendly interventional device for endotracheal intubation. As shown in Fig. 1a, to mimic the two-component feature of HAM, the ARM coating is achieved by combining carboxymethyl chitosan (CMCS) with methyl cellulose (MC) through strong intramolecular coupling. CMCS is an amphoteric chitosan derivative with excellent hydrophilic, biocompatible, and antimicrobial properties [22,23]. MC is a type of nonionic cellulose ether that has excellent moisture retention, dispersion, thickening and film-forming properties [24–27]. The chemical structure and composition of the ARM coating were studied to verify its feasibility for decorating ETT. The transparency, hydrophilicity, lubricity, cytocompatibility, antifouling and antibacterial properties of the ARM coating were investigated in detail. Effects of the ARM-coated ETT on endotracheal intubation were assessed via a clinically relevant cynomolgus monkey model in vivo. Together, we demonstrated that the ARM coating not only effectively mitigated stress responses and intubation-associated airway injury but also exhibited good antifouling and antibacterial performance to maintain mechanical ventilation. It is believed that the ARM coating has direct implications for surface engineering of ETTs and other biomedical devices.

The surface-rich groups of CMCS and MC (such as carboxylic acid, amino, and hydroxyl groups) facilitate their intermolecular interactions, specifically as hydrogen bonding between the amino and carboxyl of CMCS and the hydroxyl of MC [28]. As a hydrophilic adhesive, MC plays a thickening role in forming a mucus-like viscoelastic complex with CMCS [29]. The rheological results reveal that at a constant content of CMCS, the viscosity of the aqueous solution is obviously increased with increasing MC (Fig. S1a in Supporting information). However, excessively high viscosity disables the fluidity of the mixture solution to compromise the formation of the coating. The total concentration of 10 wt% for CMCS and MC (CMCS: MC = 1:1) is optimized to obtain an appropriate fluidity of the mixed solution (Fig. S1b in Supporting information). To generate the active sites for molecular deposition, oxygen plasma treatment was conducted on the ETTs. The decoration of the ARM coating on ETT was implemented via a simple one-step dip-coating approach (detailed information is described in Supporting information). For comparison, the CMCS and MC coatings were prepared using the same procedures.

Fluorescence microscope observation illustrates that the ARM coating enables a full surface decoration for ETT (Fig. 1b). The cross-sectional fluorescence image shows that the thickness of the coating layer is uniform on both the external and internal surfaces of ETT. At high magnification, as observed by scanning electron microscope (SEM), the ARM coating is tightly bonded with the ETT surface, which is due to the strong hydrogen bonds and electrostatic attraction. Compared to the smooth surface of the pristine ETT, the surface of the coated ETT is slightly fluctuated. The thickness of the CMCS, MC, and ARM coatings is 0.5, 2, and 45 µm, respectively (Fig. 1b and Fig. S2 in Supporting information). The coating thickness of the internal and external surfaces is comparable, confirming the high structural consistency. Fourier transform infrared (FTIR) spectra of both the external and internal surfaces of the coated ETTs show the —N—H peak at 3430 cm⁻¹ and the —C═O peak at 1586 cm⁻¹ of CMCS, as well as the —C—O—C— peak of MC at 1050 cm⁻¹. The presence of the —O—H peak at 3250 cm⁻¹ proves the formation of hydrogen bonds between molecular chains (Fig. 1c). The surface chemical composition of the coatings was further analyzed via X-ray photoelectron spectroscopy (XPS). The —COOH and —C—O—C— groups are observed from the O KLL, O 1s and C 1s spectra, indicating the existence of CMCS and MC in the ARM coating (Fig. S3 in Supporting information) [30]. The highly conformal decoration of the ARM coating on the complicated ETT surfaces is demonstrated in Fig. 1d. The ARM coating fully covers the surface of the ETT, particularly the balloon and tip with intricate geometries.

The intermolecular interaction between CMCS and MC facilitates the formation of the ARM coating on the ETT surface. The ARM coating has the highest adsorption rate, almost twice that of the CMCS coating. Correspondingly, the ARM coating possesses the utmost hydration proficiency in comparison to the pristine, CMCS, and MC coatings (Fig. 2a). The optical transparency of ETT is indispensable for bronchoscope-assisted intubation and observation. As shown in Fig. 2b, the transparency of the ARM coated substrate is comparable to that of the pristine substrate. The visible light transmittance of the ARM coating is 82% in the dry state and further increases in the wet state. The surface hydrophilicity of the coated ETTs is depicted in Fig. 2c. The water contact angle (WCA) of the pristine ETT is 67.4°, whereas it decreases for the coated ETTs. The ARM coating has the lowest WCA (21.8°), indicating its good hydrophilicity. The wetting property relies on the interaction between the coating materials and water molecules, yielding a strongly bound water layer at the interface [31].

To evaluate the lubrication performance of the ARM coating, tribological measurement was carried out in a reciprocating sliding contact mode in water. The coefficient of friction (CoF)-time plots are shown in Fig. 2d and the average CoF was calculated according to the measured frictional force. Compared to the pristine substrate, the CMCS and MC coatings lead to a slight decrease in CoF (Fig. 2e). The ARM coating shows the strongest lubricity with a CoF of only 0.05, which is one-fifth of the pristine substrate. In addition, scratches on the ARM coating were difficult to observe after tribological testing (Fig. 2f), indicating its high abrasive resistance.

Infection caused by bacteria is one of the fatal complications in endotracheal intubation. The antibacterial activity of the coatings was assayed using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as the representative Gram-positive and Gram-negative bacteria, respectively. The evaluation of the MC coating was excluded because of its lack of the intrinsic antibacterial property [32]. As shown in Fig. 3a, no inhibition is observed for the pristine ETT, while the number of colonies of the two bacteria reduces obviously after coculture with the coated ETTs. The plate counting results display that the bacterial mortality of the ARM coating against E. coli and S. aureus reaches 88% and 70%, respectively, indicating the superior killing ability to the CMCS coating (Fig. 3b). It is attributed to the ease of fixing more CMCS in the ARM coating. Live/dead bacterial staining shows that most of the bacteria are stained red (Fig. 3c), which is consistent with the results of plate colony counting. The anti-fouling property was evaluated by the binding of bovine serum albumin (BSA) on the coating. Compared to that of the pristine ETT, the amount of BSA adsorbed by the ARM coating decreases by 64% (Fig. 3d and Fig. S4 in Supporting information). The bacterial adhesion on the ARM coating is obviously reduced (Fig. S5 in Supporting information).

The in vitro cytocompatibility of the coatings was evaluated via the cell counting kit-8 (CCK-8) assay (Fig. 3e). The CMCS and ARM coatings yield the viability of the cells higher than 85% after 24 and 48 h of cultivation, showing comparable viability to that of the pristine sample (P < 0.05). Furthermore, bronchial epithelial BEAS-2B cells were isolated and seeded on the coated ETTs. As shown in the live/dead assay (Fig. 3f), the ARM coating does not alter the morphology of the cells, resulting in a typical spindle shape and very few dead cells. Taken together, the ARM coating with good biocompatibility leads to a significant improvement in the antibacterial and antifouling properties of ETTs.

To assess the effect of the ARM coating on endotracheal intubation, we established a cynomolgus monkey model as illustrated in Fig. 4a. All the animal experimental procedures were approved by Guangzhou Medical University, China (Approved No. 2020–049). To simulate the clinical scenario, the monkeys were anesthetized with sevoflurane, and the ETT was catheterized into the airway through the glottis. The duration of ventilation was maintained for 8 h. Fig. 4b plots the change in the heart rate during intubation as a direct evidence of stress response. The pristine group shows a fast increase in the heart rate, peaking at approximately 145 times/min. An elevated heart rate is an independent risk factor for cardiovascular complications such as heart failure and arrhythmia. In clear contrast, the stable heart rate of the ARM group demonstrates the prominent ability of the ARM coating to mitigate intubation stimulation. Obvious edema, congestion, and bleeding of the glottis and trachea after extubation are observed in the pristine and CMCS groups through fiberoptic bronchoscopy (Fig. 4c and Fig. S6 in Supporting information). The trachea is severely injured according to the airway injury score (Fig. 4d). The remarkable hydration lubricity of the ARM coating effectively decreases friction-induced damage, leading to the lowest airway injury score (P < 0.05). Accordingly, three kinds of typical pro-inflammation cytokines (including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6) show the lowest expression levels in the ARM group, which are 47.1%, 54.8%, and 42.5% of those in the pristine group, respectively (P < 0.05, Fig. 4g and Fig. S7 in Supporting information).

The feeding behavior of the monkeys after intubation was further recorded to evaluate the effect of the ARM coating on physiological events. As depicted in Figs. 4e and f, the pristine and CMCS groups show distinct feeding-suppressant behavior, in which the monkeys even refused to eat. Notably, the monkeys in the ARM group have a strong willing to consume food on Day 1 after surgery, showing a comparable feeding intention score to that of the monkeys before intubation. Bradykinin (BK), Substance P (SP), and prostaglandin E2 (PGE2) are essential mediators of pain transmission and inflammatory response [33]. Compared to the pristine group, the expression of BK, SP, and PGE2 is decrease by 22.5%, 25.7%, and 24.9%, respectively (Fig. 4h). These findings indicate that the ARM coating is effective to alleviate pain symptoms and has no adverse impact on normal eating behavior.

ETT obstruction caused by a foreign body is documented in clinic, but has not been well solved yet. We evaluated the mechanical ventilation performance of the coated ETT (Fig. 5a). In the first 180 min, the airway pressure does not obviously change in the pristine and ARM groups. However, with an intubation time above 180 min, the airway pressure rises more significantly in the pristine group than in the ARM group. A similar phenomenon is recognized from the real-time profile of tidal volume, which remains stable for the ARM group during intubation (Fig. 5b) [34]. The increased airway pressure and decreased tidal volume of the pristine group emphasize that the normal ventilation is gradually disturbed with the prolonged intubation by using the pristine ETT. Considering the rather long intubation time in practical applications, complete ETT obstruction, as a fatal event, is highly likely to occur for pristine ETT. As shown in Fig. 5c, the amount of airway secretions adsorbed the ARM-coated ETT reduces 71.3% compared to those on the pristine ETT. The outstanding antifouling property endows the ARM coating with the ability to hinder the occurrence of ETT obstruction.

It has been reported that mechanical ventilation is implicated in 83% of cases of nosocomial pneumonia cases. Bacterial colonization on the inner surface of ETTs is a prominent predisposing factor for VAP, where Klebsiella pneumoniae poses threats to the respiratory system [35]. Fig. 5d shows that compared to the pristine group, the ARM group enhances the relative sterilization ratio against Klebsiella pneumoniae by nearly five times. The expression of inflammatory factors on the inner surface of ETTs was evaluated. In comparison to the pristine group, the expression of IL-1β, IL-6, and TNF-α in the ARM group is reduced by 71.4%, 82.1% and 67.1% respectively (Fig. 5e). These results suggest that the high potential of the ARM coating to resist foreign pathogen invasion in vivo.

In summary, we described an ARM coating to endow ETT with multifunctional performance. The ARM coating was formed via strong intramolecular coupling between CMCS and MC, showing uniformly conformal decoration on the outer and inner surfaces of ETT by optimizing the viscosity of the coating solution. The ARM coating exhibited remarkable hydrophilic, lubricative, and antifouling capabilities. In vitro experiments confirmed the excellent antibacterial activity and biocompatibility of the ARM coating. A cynomolgus monkey intubation model further demonstrated the beneficial effect of the ARM coating on declining intubation-related tissue injury, inflammation, and obstruction in vivo. Moreover, the ARM coating significantly inhibited Klebsiella pneumoniae growth and also restrained the biofilm formation and expression of inflammatory factors caused by bacterial infection. Overall, the ARM coating is promising for surface modification of commercial ETT towards comfortable endotracheal intubation.

Declaration of competing interest

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

CRediT authorship contribution statement

Jun-Yang Wang: Writing – original draft, Investigation. Yu-Qing Wei: Methodology, Investigation. Qing-Ning Wang: Methodology, Investigation. Zhi-Guo Wang: Validation, Methodology. Rui Hong: Software, Formal analysis, Data curation. Lisha Yi: Software, Methodology. Ping Xu: Visualization, Validation, Data curation. Jia-Zhuang Xu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Zhong-Ming Li: Supervision, Resources, Project administration. Baisong Zhao: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 52203046 and 82171219), Sichuan Science and Technology Program (No. 2023NSFSC1944), West China Nursing Discipline Development Special Fund Project, Sichuan University (No. HXHL21007), the China Postdoctoral Science Foundation (No. 2023M742483), the National Natural Science Foundation of Guangdong (No. 2024A1515012881).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110559.