Development of Lipid-based Nanoformulations of Macrophage Targeting immunotherapies for Oral Inhalation Using a Continuous Manufacturing Approach and Design of Experiments

Lung diseases, including chronic inflammation, infection, and cancer, affect millions of people worldwide and often involve dysregulated immune responses. Macrophages, key immune cells in the lung, can either promote healing or contribute to disease progression, making them promising targets for therapeutic intervention. This research explores the development of inhalable nanomedicine designed to reprogram lung immune cells using messenger RNA (mRNA) delivery. Because mRNA molecules are fragile, they must be encapsulated in protective carriers such as lipid nanoparticles to ensure stability and effective delivery to target cells. The study employed automated, continuous manufacturing methods and a design-of-experiments (DoE) approach to optimize the formulation of mRNA-loaded lipid nanoparticles. Using microfluidic technology—similar to systems used in COVID-19 vaccine production—multiple lipid compositions and formulation variables were systematically evaluated to identify the optimal nanomedicine formulation. Key quality attributes, including particle size, mRNA encapsulation efficiency, and cellular uptake, were measured to assess performance. Statistical modeling and experimental validation identified specific lipid components that improved nanoparticle size, stability, and delivery efficiency to macrophage-like cells in vitro. The optimized formulation demonstrated strong cellular uptake and effective mRNA translation in macrophage cell models, suggesting potential for targeted immune cell reprogramming. These findings establish a scalable strategy for developing inhalable mRNA nanomedicines and lay the groundwork for future in vivo studies to treat lung diseases using non-invasive inhalation-based therapies.