Development of aerosolizable mRNA incorporated chitosan nanoparticles for efficient lung delivery

Direct delivery of Messenger RNA (mRNA) via inhalation to the lungs could revolutionize the treatment of various pulmonary diseases. By utilizing the target's cellular machinery to produce therapeutic proteins, this approach bypasses the challenges and side effects associated with traditional systemic administration. This non-invasive approach holds the potential to improve the treatment of genetic and infectious pulmonary diseases. This study aimed at the development of Chitosan-tripolyphosphate (CS-TPP) nanoparticles, known for their biocompatibility and ability to encapsulate nucleic acids, as a means for inhalation delivery of mRNA. The study established an in-house, cost-effective approach for mRNA extraction. The mRNA-incorporated CS-TPP nanoparticles (NPs) were prepared using ionotropic gelation with two formulation approaches based on differing concentrations of chitosan and chitosan to mRNA (w/w) ratios. These NPs were then characterized by their particle size and encapsulation efficiencies. Aerodynamic properties were assessed using a vibrating mesh nebulizer and passing through a Next-generation impactor, revealing optimal aerodynamic diameters between 1-5 µm. Transfection efficiency and cellular uptake were evaluated in lung adenocarcinoma (A549) and epithelial (BEAS-2b) cell lines, with protein expression monitored via confocal imaging. Immunogenicity studies indicated a dose-dependent cytokine release, which could be pivotal for therapeutic outcomes. These findings underscore the potential of CS-TPP mRNA NPs in pulmonary drug delivery systems. The study successfully developed and characterized CS-TPP nanoparticles for inhalable mRNA delivery. These nanoparticles demonstrated successful aerosolization, cellular uptake, and protein expression. Further research is required to optimize nebulization parameters and chitosan-mediated immunogenicity to ultimately enhance safety and therapeutic potential for clinical development.