PEGylation modulates the pulmonary behaviour and antibacterial efficacy of tobramycin liposomes in advanced in vitro lung models

Pulmonary infections caused by P. aeruginosa remain challenging to treat because physiological lung barriers limit antibiotic distribution and local availability at the site of infection. Here, we investigated how PEGylation influences the behaviour and antibacterial performance of tobramycin-loaded liposomes across distinct pulmonary microenvironments. Non-PEGylated (LP-TOB) and PEGylated (LP-PEG-TOB) liposomes were prepared, physicochemically characterised and compared with free tobramycin (TOB) using complementary in vitro assays and advanced alveolar and bronchial air-liquid interface co-culture models. Both liposomal formulations displayed suitable physicochemical properties, high colloidal stability and preserved antibacterial activity against P. aeruginosa. PEGylation significantly altered formulation behaviour by reducing epithelial uptake, increasing mucus-associated localisation and promoting faster tobramycin release. These differences resulted in microenvironment-dependent antibacterial responses following bacterial challenge. LP-PEG-TOB achieved greater control of extracellular bacterial burden than LP-TOB in both pulmonary models, with its distribution within the mucus compartment potentially contributing to its performance in the bronchial model. In contrast, LP-TOB demonstrated superior control of the intracellular bacterial signal in macrophages, consistent with its greater cellular interaction. All formulations were non-cytotoxic and attenuated infection-associated inflammatory responses. High-content imaging further revealed that antibiotic exposure mitigated infection-induced cellular and nuclear morphological alterations in macrophages. Overall, these findings demonstrate that PEGylation is a key determinant of liposome distribution and antibacterial performance across pulmonary microenvironments. This study provides a mechanistic basis for tailoring liposomal surface properties to the intended site of action in inhaled antibiotic therapy.