Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19

[Image: see text] Delivering medication to the lungs via nebulization of pharmaceuticals is a noninvasive and efficient therapy route, particularly for respiratory diseases. The recent worldwide severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic urges the development of such therapies as an effective alternative to vaccines. The main difficulties in using inhalation therapy are the development of effective medicine and methods to stabilize the biological molecules and transfer them to the lungs efficiently following nebulization. We have developed a high-affinity angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD-62) that can be used as a medication to inhibit infection with SARS-CoV-2 and its variants. In this study, we established a nebulization protocol for drug delivery by inhalation using two commercial vibrating mesh (VM) nebulizers (Aerogen Solo and PARI eFlow) that generate similar mist size distribution in a size range that allows efficient deposition in the small respiratory airway. In a series of experiments, we show the high activity of RBD-62, interferon-α2 (IFN-α2), and other proteins following nebulization. The addition of gelatin significantly stabilizes the proteins and enhances the fractions of active proteins after nebulization, minimizing the medication dosage. Furthermore, hamster inhalation experiments verified the feasibility of the protocol in pulmonary drug delivery. In short, the gelatin-modified RBD-62 formulation in coordination with VM nebulizer can be used as a therapy to cure SARS-CoV-2.