Iloprost-loaded inhalable Nano into Micro (NiM) formulations for treating hyper-inflammation in a pre-clinical model of cystic fibrosis airway epithelial cells

To address the hyper-inflammation associated with cystic fibrosis (CF) lung disease, we evaluated the efficacy of inhalable Nano into Micro (NiM) formulations carrying a repurposed anti-inflammatory agent, i.e. Iloprost (ILO), a prostacyclin analogue. Nasal epithelial brushings were obtained from 15 individuals with CF bearing at least one F508del mutation. The isolated cells were expanded using the conditional reprogramming culture method and subsequently differentiated into a mucociliary epithelium at air-liquid interface (ALI) cultures capable of producing endogenous mucus. ALI cultures were exposed to lipopolysaccharide (LPS), either alone or in combination with NiM formulations - entrapping pegylated or non-pegylated nanoparticles - carrying ILO (named NiM-PEG-ILO and NiM-ILO, respectively). The treatment with NiM-PEG-ILO significantly reduced mRNA levels of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, and IL-1β compared to LPS stimulation alone. Furthermore, a marked downregulation of miR-145, miR-146a, and miR-17 levels was observed relative to the LPS-only group. Cytofluorimetry analysis carried out by using NiM samples entrapping fluorescent pegylated and unpegylated nanoparticles loaded with ILO showed that both nanoparticles NiM formulations were internalized by cells in a concentration-dependent manner. Our results demonstrate that inhalable NiM-based formulations delivery system loaded with ILO can efficiently attenuate inflammation in a pre-clinical model of human airway epithelium.