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

We worked on and validated a non-destructive and gentle method to deliver nasal mRNA vaccines via Rayleigh breakup aerosolization. Current nebulizers damage fragile mRNA vectors. Our protocol demonstrates a low-shear aerosolization approach that preserves mRNA and integrity.

To begin the device setup, assemble the Rayleigh breakup nasal atomizer device with the Alberta Idealized Nasal Inlet, or AINI model. After placing the required components in the Next Generation Impactor, attach the calibrated flow meter to the Next Generation Impactor. Next, use Parafilm to seal the device properly to the Next Generation Impactor.

Connect the outlet of the impactor to a vacuum pump to draw airflow through the nasal passages. Adjust the pump controls until the flow meter reads 7.5 liters per minute. Check if the airflow of the air pump is delivering 7.5 liters per minute through the AINI and turn off the air pump.

Remove the calibrated flow meter and attach the AINI component in its place. Ensure that all connections, model sections, and tubing to the pump are airtight. Then gently mix the prepared polyethyleneimine RNA suspension to ensure homogeneity while avoiding bubble formation.

Draw the prepared sample into the atomizer reservoir and load it into the atomizer device. After turning on the air pump and attaching the syringe pump to the atomizer, insert the Rayleigh breakup atomizer nozzle into the entrance of the AINI nostril at an angle of 45 degrees. Activate the Rayleigh breakup atomizer to begin aerosolization.

Turn off the air pump promptly after spraying to prevent deposited droplets from being drawn further downstream or drying unevenly. Then carefully disassemble the AINI model into its individual anatomical segments to collect deposited nanoparticles from each region. Next, identify and prepare to collect deposits from six compartments, including the atomizer device, the nasal vestibule section, the turbinate section, the olfactory region, the nasopharynx section, and any downstream capture, such as attached Next Generation Impactor stages or a final filter.

For each compartment, use a measured volume of cell culture medium to wash out the deposited material and recover the samples into a separate container. Repeat the sample collection step for all parts of the device. Then retrieve the liquid from the Next Generation Impactor stages if used by rinsing the stages with DMEM to trap fine particles.

Z-average diameters of polyethyleneimine mRNA polyplexes remained within the expected range of 136 to 160 nanometers before aerosolization and 145 to 162 nanometers after aerosolization, indicating that Rayleigh breakup did not cause significant size changes. The zeta potential and peak 1 mean diameters by intensity remained largely unchanged before and after aerosolization. Numerous blue-stained nuclei were observed in cells from the turbinate region indicating successful DAPI staining of A549 cells.

Green fluorescing cells were observed in A549 wells after exposure to aerosolized messenger RNA, indicating successful transfection. Merged DAPI and green fluorescent protein signal in A549 cells confirmed transfection feasibility and validated cellular uptake of the mRNAs. The green fluorescent protein signal was observed in HEK293T cells exposed to the aerosolized mRNA, confirming that the method produced detectable reporter expression in a second epithelial cell line that served as a positive control.

We showed that Rayleigh breakup preserves mRNA nanoparticle integrity while enabling targeted turbinate deposition and functional gene expression. Our findings advance the field by establishing a fully-validated and non-destructive intranasal mRNA aerosolization framework, enabling a safer and more effective respiratory delivery research.