Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that controls epithelial ion transport. As a result, mucus secretions in epithelial tissues become abnormally thick and sticky. This particularly affects the respiratory tract, where mucociliary clearance is impaired, chronic bacterial colonization and biofilm formation are promoted, and a sustained pro-inflammatory environment is established [1].

Airway inflammation in CF involves elevated production of interleukin-8 (IL-8) by epithelial cells, leading to massive infiltration of neutrophils and macrophages [2]. Neutrophils, the predominant immune cells in the CF lung, contribute to tissue damage through the release of proteolytic enzymes, reactive oxygen species (ROS), pro-inflammatory cytokines, and the formation of neutrophil extracellular traps [3]. Macrophages adopt a hyperinflammatory phenotype, which impairs bacterial clearance and the resolution of inflammation [4].

Management of CF requires combined therapeutic strategies. Although oral CFTR modulators improve lung function and quality of life in patients with responsive mutations [5], such therapies fail to resolve persistent infection and inflammation, especially in advanced disease stages [6]. Tobramycin (TB), a first-line inhaled antibiotic against Pseudomonas aeruginosa, has limited efficacy due to poor biofilm penetration and the inherent tolerance of mucoid strains [7,8]. Anti-inflammatory agents such as ibuprofen have shown benefits [9], but are underused due to adverse effects and the need for therapeutic drug monitoring [10].

New therapeutic approaches that combine antimicrobial and anti-inflammatory activities are increasingly pursued to overcome these limitations. Thymus vulgaris essential oil (EOT), rich in thymol, exhibits antioxidant and anti-inflammatory properties, yet its clinical application is hindered by low aqueous solubility, chemical instability, thermosensitivity, and volatility [11]. Encapsulation of essential oils or their active components into nanoparticles (NP) enhances physicochemical stability [12].

Inhaled NP offer an attractive platform for pulmonary delivery, enabling sustained release, improved local retention, and reduced systemic exposure [13,14]. Moreover, NP allow the co-delivery of antibiotics and anti-inflammatory or antioxidant agents, providing a unified strategy to address the multifactorial nature of CF. However, the structural complexity and adhesive characteristics of mucus—mediated by hydrophobic, electrostatic, and hydrogen-bonding interactions with mucins—often hinder NP diffusion [15]. Therefore, the design of NP capable of efficiently penetrating the viscous mucus of CF airways remains a critical challenge.

Previous work demonstrated that solid lipid nanoparticles (SLN) containing archaeolipids from the archaea Halorubrum tebenquichense exhibit enhanced behavior in mucosal environments, maintaining high levels of cellular uptake in the presence of mucus [16]. Moreover, archaeolipid-containing NP showed superior penetration into P. aeruginosa biofilms compared to their conventional counterparts [17]. Based on these findings, nanostructured archaeolipid carriers (NAL) co-loaded with EOT and TB (NAL-EOT/TB) were developed, resulting in stable, nebulizable formulations intended for pulmonary delivery, showing enhanced anti-biofilm and antioxidant activities compared to the free compounds [18].

Given the complex pathophysiology of CF lung disease, evaluating such formulations requires physiologically relevant in vitro models that mimic P. aeruginosa biofilm infections. While traditional biofilm assays on abiotic substrates allow for high-throughput screening, these assays lack the cellular context and physiological complexity of the host environment [19]. Additionally, the interaction between NP and neutrophils—key players in CF pathogenesis—should be considered to obtain a broader understanding of therapeutic potential [20].

This study presents a comprehensive in vitro evaluation of NAL-EOT/TB as a multifunctional therapeutic strategy aimed at simultaneously addressing bacterial biofilms, airway inflammation, and the challenge of mucus penetration. The formulation was evaluated for its ability to diffuse through artificial mucus (AM), protect airway epithelial cells exposed to biofilms, and modulate neutrophil-mediated immune responses, aiming to explore its therapeutic potential for chronic pulmonary infections in CF.