Inhalable nanocapsules for lung delivery of Pirfenidone and Bacterioruberin: Modulating macrophages to target pulmonary fibrosis.

Abstract

In this work, we developed an inhalable polymeric-lipid nanocapsule formulation with mucus-penetrating properties to co-encapsulate Pirfenidone (Pfd) and the carotenoid Bacterioruberin (BR), aiming to enhance anti-inflammatory, antioxidant, and antifibrotic effects by modulating pulmonary macrophage activity. Nanocapsules (pNC-BR-Pfd) were prepared via double emulsion technique, exhibiting a Z average of 171 ± 47 nm and a Z potential of -45 ± 5 mV. Transmission electron microscopy and small angle X-ray scattering analyses revealed predominant particle sizes around 50 nm. Raman spectroscopy confirmed the successful co-encapsulation of Pfd and BR. Pfd release assay showed that 18 ± 9 % of Pfd was encapsulated, displaying a biphasic release pattern: an initial burst attributed to surface-adsorbed drug, followed by sustained release from the lipidic core. pNC-BR-Pfd preserved their physicochemical properties after nebulization using a vibrating mesh nebulizer. Surface PEGylation significantly enhanced penetration through artificial lung mucus, both in a Transwell assay and in mucus-covered A549 and THP-1 cell cultures. In LPS-activated THP-1 macrophages, pNC-BR-Pfd reduced intracellular reactive oxygen species and suppressed IL-6 and TNF-α levels by approximately 3-fold at 10 μg/mL Pfd and 0.64 μg/mL BR. In contrast, free Pfd only partially inhibited IL-6 and had no effect on TNF-α. Conditioned media from LPS-activated THP-1 treated with pNC-BR-Pfd also suppressed MRC-5 fibroblast matrix metalloproteinases (MMPs) and hydroxyproline release, and significantly inhibited cell migration. While free Pfd reduced MMPs and hydroxyproline levels, but did not alter fibroblast migration. Altogether, these results demonstrate that pNC-BR-Pfd is a technically stable and pharmacologically enhanced inhalable formulation with promising anti-inflammatory, antioxidant, antifibrotic, and mucus-penetrating properties.