Assessment of pulmonary delivery efficacy of archaeal tetraether lipids based ICG- and DiR-loaded liposomes for antitumoral photodynamic therapy

Lung cancer remains a leading cause of cancer-related deaths worldwide, highlighting the urgent need for novel therapeutic strategies, whereby pulmonary delivery offers a more promising approach, as it delivers the therapeutic moiety directly to the lungs, reducing systemic toxicity. Among emerging novel treatment strategies, photodynamic therapy (PDT) has emerged as a minimally invasive approach for treating various diseases, including lung cancer. However, the practical application of photosensitizers remains challenging due to their poor stability and limited availability at the targeted site. In this study, archaeal tetraether lipid (TEL)-based liposomes were developed to encapsulate indocyanine green (ICG) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), two near-infrared (NIR) fluorescent photosensitizers, for inhalable, lung-targeted PDT. Liposomes were prepared using the thin-film hydration method with a lipid composition of 1,2-dipalmitoyl-sn‑glycero-3-phosphocholine (DPPC), cholesterol (Chol), and hydrolyzed glycerol dialkyl nonitol tetraether (hGDNT) in a molar ratio of 85:10:5 and DPPC, Chol, and polar lipid fraction E (PLFE) in a molar ratio of 85:10:5. Physicochemical characterization demonstrated that both hGDNT- and PLFE-based liposomes were in the nanometer size range with good monodispersity. Compared to hGDNT-liposomes, PLFE-based formulations were generally smaller, more uniform, and exhibited greater surface charge stability. All formulations exhibited excellent colloidal stability for up to two weeks (14 days), maintaining their physicochemical characteristics both during storage and after aerosolization using a vibrating-mesh nebulizer. A spherical nanoscale morphology was revealed by atomic force microscopy (AFM). Alterations in membrane properties induced by the incorporation of the drug were highlighted by phase imaging, confirming successful encapsulation and structural modulation, whereas transmission electron microscopy (TEM) revealed the unilamellarity of the liposomal formulations. PDT assessment on A549 cells revealed that DiR-loaded hGDNT-lipsomes (DiR-hGDNT-LPs) exhibited greater phototoxicity with a 50 % inhibitory concentration (IC₅₀) of 9.65 µg/mL, compared to 16.45 µg/mL for ICG-loaded-hGDNT-liposomes (ICG-hGDNT-LPs) upon NIR irradiation. Confocal laser scanning microscopy analysis demonstrated that DiR-hGDNT-LPs and ICG-hGDNT-LPs were efficiently taken up by A549 cells, with fluorescence predominantly localized in the cytoplasm, indicating effective intracellular retention. Intracellular reactive oxygen species (ROS) generation was confirmed using the 2',7'-dichlorofluorescin diacetate assay, which exhibited strong light-dependent ROS production upon NIR radiation. These results suggest that TEL liposomes are highly stable, efficient nanocarriers that enhance the therapeutic potential of photosensitizers for non-invasive lung-targeted PDT.