(F-233) Delivery of Anti-microRNA-21 by Lung-Targeted Lipid Nanoparticles for Pulmonary Fibrosis Treatment

Introduction:: Pulmonary fibrosis is a prevalent complication among COVID-19 patients. Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with a low survival rate. Currently there are no effective anti-fibrosis therapies except lung transplantation. MicroRNAs (miRs) have been identified as key regulators in the development and progression of pulmonary fibrosis, which are promising therapeutic targets. For instance, miR-21 has been reported to promote pulmonary fibrosis in IPF patients and bleomycin-treated mice by regulating epithelial-mesenchymal transition and mediating fibrogenesis. Blocking miR-21-associated fibrogenic pathways represents a promising therapy for pulmonary fibrosis. However, there are no microRNAs-based therapies that offer lung-targeting capabilities, strong anti-fibrotic effects and high translational potential. To overcome these challenges, a lung targeted, lipid nanoparticles-based formulation of anti-miR-21 has been developed for pulmonary fibrosis.

Materials and Methods:: The anti-miR-21 encapsulated lipid nanoparticles (LP-anti-miR-21) were prepared. The physiochemical properties of LP-anti-miR-21 (size, zeta potential, encapsulation efficiency and morphology) were characterized. We established an in vitro lung fibrosis model using TGF-β-treated normal human lung fibroblast (NHLF) cells. The cellular uptake efficiency of LP-anti-miR-21 was determined by flow cytometry and confocal microscopy. The in vitro therapeutic efficacy was evaluated by quantifying the levels of fibrotic markers via qRT-PCR and western blot. The cytotoxicity was measured by alamarBlue assay. The ex vivo efficacy was determined by measuring the contractile force of NHLF microtissues. For in vivo model, intra-tracheal intubation of bleomycin was used to induce pulmonary fibrosis in C57/BL6 mice. The biodistribution of LP was evaluated by IVIS imaging system. The in vivo therapeutic efficacy was evaluated by measuring fibrotic markers using qRT-PCR, western blot and IHC staining.

Results, Conclusions, and Discussions:: LP-anti-miR-21 had a multilamellar structure with a hydrodynamic diameter of 101 nm and a zeta potential of +29.4 mV. The encapsulation efficiency of anti-miR-21 was 99.5%. LP-anti-miR-21 enhanced cellular uptake of anti-miR-21 by 435-fold and significantly down-regulated key fibrotic markers (miR-21, COL1A1, α-SMA, and FN1) in vitro (Fig 1a). LP-anti-miR-21 reduced the elevated contractile force generation in NHLF microtissues. The biodistribution study confirmed the lung-targeting capability of LP-anti-miR-21 (Fig 1b). The in vivo therapeutic efficacy study revealed significant reductions in the expression of key fibrotic markers following LP-anti-miR-21 treatment (Fig 1c). Histological examination revealed the reduction in ECM accumulation and fibrosis severity. The delivery system exhibited no toxicity, suggesting its safety as a therapeutic modality. Our results demonstrated the potential of LP-anti-miR-21 as a promising therapeutic strategy for pulmonary fibrosis.

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