Intranasal Delivery of mRNA Polyplexes via Rayleigh Breakup Aerosols: An <em>In Vitro</em> Method for Nasal Deposition and Functional Testing

Intranasal delivery of mRNA therapeutics is a promising strategy for vaccination and treating respiratory diseases, offering direct immune activation at the site of pathogen entry. However, conventional aerosolization methods (e.g., ultrasonic or high-pressure nebulizers) deteriorate non-viral mRNA vectors through excessive shear forces, causing mRNAs to lose their structural integrity and biological activities. A Rayleigh breakup nasal atomizer was used to gently aerosolize polyethyleneimine (PEI)-mRNA vectors into uniform droplets. Green Fluorescent Protein (GFP)-encoding mRNA was formulated into cationic polyplexes and characterized pre- and post-aerosolization. The Rayleigh breakup process forms a continuous micro-jet of droplets with minimal shear, thereby preserving the physicochemical properties of the nanoparticles. Consistent particle size, low polydispersity index, and stable zeta potential before and after aerosolization were observed, confirming that the physicochemical properties of mRNA polyplexes were well preserved via Rayleigh breakup for aerosolization. Using an Alberta Idealized Nasal Inlet (AINI) model of the nasal airway, the PEI-mRNA aerosols were delivered. The aerosolized mRNAs were primarily deposited in the turbinate regions. Negligible fractions were found in the nasopharynx or lung-equivalent sections. In addition, the post-aerosolized mRNA polyplexes were successfully delivered to A549 human lung epithelial cells and produced detectable GFP expression. This protocol demonstrates a non-destructive intranasal mRNA delivery method using Rayleigh breakup aerosolization. It effectively maintains the physicochemical properties and biological functions of non-viral mRNA vectors, atomizing the aqueous phase into droplets of appropriate sizes for targeted nasal deposition. This protocol reveals a novel approach for effectively aerosolizing mRNAs and evaluating their regional deposition in the nasal cavity.