siRNA- and Steroid-Eluting Endotracheal Tubes Enhance Mucosal Healing by Modulating Immune, Microbial, and Mechanical Responses in Injured Airways

Introduction: : Endotracheal intubation is a commonly performed procedure, yet it is often associated with laryngeal injury which can impair speaking, swallowing, and breathing. If left untreated, these injuries can result in progressive scarring and the development of glottic or subglottic stenosis, conditions that frequently require surgical intervention to re-establish airway patency. Early therapeutic intervention during the acute phase of laryngeal wound healing may offer a critical window to mitigate fibrosis and preserve airway and vocal fold function. In this study, we evaluate a novel dual drug-eluting endotracheal tube (ETT) coated with a composite, mucosal-compatible hydrogel designed for localized delivery of dexamethasone and siRNA targeting smad3. This dual combination therapy aims to suppress pro-fibrotic signaling, particularly TGFβ-1, and promote regenerative healing following intubation-induced airway trauma.

Materials and

Methods: : This injured animal model was approved by the Bridge Preclinical Testing Services Institutional Animal Care and Use Committee (protocol BPTS-21-01). ETTs were first coated with dexamethasone-loaded polycaprolactone (PCL) fibers via electrospinning, then embedded in four-arm polyethylene glycol acrylate matrix (to minimize mucosal epithelium insult) containing smad3 silencing siRNA. Traumatic intubation was simulated in swine under endoscopic visualization and uncoated (regular), dexamethasone coated, or composite coated (dexamethasone + PEG hydrogel with siRNA targeting smad3) were placed in the injured airway for 3, 7, or 14 days. Laryngotracheal tissues were evaluated using indentation testing for biomechanical properties, immunohistochemistry for cellular and molecular markers, cytokine profiling to assess inflammatory responses, and 16S rRNA sequencing to characterize microbial communities. Endotracheal tube (ETT) biofilm formation was visualized and assessed using scanning electron microscopy (SEM) and micro-computed tomography (µCT) models.

Results, Conclusions, and Discussions:: Mechanical, immunological, and microbial responses to ETT placement varied by ETT coating composition and anatomical subregion. Indentation testing revealed differences in tissue stiffness across laryngotracheal subregions, correlating with alterations in extracellular matrix proteins, including collagen IV, laminin, and elastin (Figure 1). Macrophage polarization shifted from a pro-inflammatory M1 phenotype to an anti-inflammatory, tissue-reparative M2 phenotype in animals treated with the composite coated ETT, which included both dexamethasone and siRNA targeting smad3 (Figure 2A and 2B). These findings were supported by cytokine profiling, indicating reduced pro-inflammatory markers and elevated repair-associated cytokines (Figure 2C).

Microbiome analysis via 16S rRNA sequencing demonstrated that microbial community composition and functional pathways varied by ETT type and over time (Figure 3C-E). Notably, dexamethasone coated and composite coated ETTs were associated with shifts in energy metabolism pathways and a decrease in taxa associated with mucosal adhesion. Biofilm formation on ETT surfaces, visualized through SEM and µCT, showed increased bacterial presence on electrospun fiber coatings, likely due to elevated surface roughness (Figure 3A). While these coatings were effective in reducing inflammation, they may also promote epithelial abrasion and microbial colonization.

Mucin expression patterns also changed in response to ETT type (Figure 3B and 3F). Dexamethasone delivery promoted early increases in MUC5B, a mucin associated with mucosal protection, followed by a later shift toward MUC5AC, linked to goblet cell metaplasia and inflammatory responses. These shifts suggest dynamic epithelial remodeling driven by local drug delivery and surface topography.

These findings demonstrate that ETT coating composition plays a critical role in shaping airway healing through modulation of immune responses, microbiome, mucosal barrier integrity, and biomechanical properties. This work provides insight for biomaterial design to prevent fibrosis and improve intubation outcomes.

Acknowledgements and/or References (Optional)::