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Rademacker, S.* ; Müller, J.T.* ; Kromer, A.P.E.* ; Carneiro, S.P.* ; Merkel, O.M.

Nanoparticle-mediated siRNA delivery in human epithelial lung cells cultured at the air-liquid interface.

In: RNA Therapeutics. Berlin [u.a.]: Springer, 2025. 417-437 (Methods Mol. Biol. ; 2965)

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The approval of ONPATTRO® in 2018 resembled a milestone for small interfering RNA (siRNA) therapies, introducing the first siRNA lipid nanoparticle (LNP) into clinical use. This breakthrough has improved research efforts in siRNA-based therapeutics. Similarly, benefits like scalability and adaptability have led to enormous research also in polymeric siRNA delivery systems, leading to polyplexes or micelleplexes after complexation. However, extrahepatic delivery, such as pulmonary administration of these therapies, remains poorly understood and is under active investigation. To enable high-throughput screening while following the 3R principles and minimizing in vivo studies, advanced cell culture systems like the air-liquid interface (ALI) model are being developed. ALI cell culture replicates key physiological features of the lung, including mucus and surfactant production, tight junction formation, and the establishment of a pseudostratified monolayer. These properties make ALI models highly suitable for sophisticated in vitro studies of pulmonary delivery systems. This chapter provides a comprehensive overview of ALI models, including their establishment, cultivation, microscopy-based characterization, and applications in evaluating nanoparticle uptake and gene knockdown efficiency using polymeric nanoparticles and LNPs.

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