Development of Inhalable Plasmid DNA Dry Powders Using Cryomilling of Electrospun Nanofiber Mats for Pulmonary Gene Delivery

This study aimed to develop inhalable dry powder formulations of naked plasmid DNA (pDNA) for pulmonary gene delivery using an electrospinning (ES) technique. Nanofiber mats comprising polyvinyl alcohol (PVA), pDNA encoding firefly luciferase, and either D(-)-mannitol (Man) or lactose monohydrate (Lac) were fabricated and subsequently cryomilled into fine, respirable particles. Agarose gel electrophoresis revealed partial degradation of pDNA during both ES and milling processes, with Lac-based nanofiber mat and powder showing greater pDNA integrity than Man-based formulations. Intratracheal administration of the ES-derived powders in mice led to successful in vivo gene expression, with Man-based powders milled for 0.5 min yielding the highest luciferase activity. Pulmonary imaging using indocyanine green showed that dry powders exhibited extended lung residence compared to aqueous formulations, likely due to improved mucosal adhesion and slower dissolution. Remarkably, the ES-generated pDNA powders demonstrated superior transfection efficiency over both naked pDNA and pDNA-polyethyleneimine complexes, despite some loss in pDNA integrity. These findings highlight the importance of dispersibility and lung retention in achieving effective pulmonary gene transfer. The ES approach represents a promising platform for producing inhalable pDNA powders, offering a non-invasive gene therapy option for respiratory diseases.