From aerosol to alveoli: materials, methods, and models to bridge barriers to respiratory delivery of RNA

Delivery formulations that facilitate mRNA transfection in the respiratory system aim to provide a flexible platform to address or prevent respiratory disease in a patient-accessible, targeted manner. Recent clinical trials in cystic fibrosis, respiratory oncology, and mucosal vaccination have exposed the unique physicochemical and immunological challenges that differentiate respiratory and parenteral mRNA strategies. This review examines the nanomaterials, barriers, and experimental models central to the rational development of aerosolized mRNA medicines. We begin with an overview of the most promising classes of devices and nanomaterial formulations used for respiratory delivery. We then analyze the physical barriers to nasal and lung delivery, spanning delivery-associated physical stresses and anatomical matrices, and present the major chemical and biological strategies developed to overcome them. A parallel analysis addresses the immunological barriers of the respiratory mucosa, including the innate and adaptive cell populations and the sensing pathways that mRNA delivery engages. Immunoengineering approaches to improve the tolerability of repeat dosing are discussed, including nucleoside modification and alternative RNA constructs, degradable and anti-inflammatory material chemistries, and co-administration of standard-of-care pulmonary drugs. Finally, we address methodologies for the investigation of the nanomaterial-mucosal interface using biophysical characterization techniques, and evaluate preclinical models spanning in vitro mucus mimics, air-liquid interface cultures, ex vivo precision-cut lung slices, in vivo rodent and non-rodent species, three-dimensional (3D) nasal and lung cast replicas, and mathematical models of the intranasal airways and lung. We propose an interdisciplinary bioengineering approach that combines the expertise of chemists, immunologists, and pulmonologists to enable clinical translation of respiratory mRNA medicines.