Development of naked pDNA inhalation powder formulation with high transfection efficiency using hyaluronic acid and phenylalanine as excipients

Nucleic acid-based inhalational drugs hold great clinical promise, but development has been limited by poor transfection efficiency among other factors. We previously described a plasmid (p)DNA powder without vector (naked pDNA powder) prepared for inhalation by spray-freeze-drying and containing hyaluronic acid (HA) as an excipient that demonstrated high transfection efficiency in the lungs. In the present study, we describe modified formulations in which HA is partially replaced by hydrophobic amino acids to enhance dispersibility in air and HA molecular weight is optimized for greater transfection efficiency. In vivo experiments in mice revealed that L-phenylalanine (Phe) in combination with HA conferred higher transfection efficiency than other hydrophobic amino acids. Furthermore, both in vivo and in vitro assays demonstrated that 50 kDa HA conferred higher transfection efficiency than other molecular weights and that the naked pDNA powder composed of 73% HA (50 kDa) and 25% Phe yielded the highest transfection efficiency among powders investigated. A higher relative Phe ratio enhanced aerosol performance but yielded lower transfection efficiency. This trade-off highlights the importance of comprehensively evaluating inhalational powders for both intrinsic transfection efficiency and aerosol performance.