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

Summary

This protocol presents Rayleigh breakup as a mild process for aerosolizing non-viral mRNA vectors. Regional deposition for aerosolized mRNA vectors is evaluated by the Alberta Idealized Nasal Inlet (AINI) model. Green fluorescence protein (GFP) expression in human lung cells represents maintenance of physicochemical and biological functions of post-aerosolized mRNA vectors.

Abstract

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.

Introduction

Delivering vaccines to the nasal mucosa (the entry portal for many respiratory pathogens) can elicit strong local immunity and block infections at their source. The success of intranasal flu vaccines and recent approval of intranasal COVID-19 vaccines underscore the effectiveness of the nasal route for inducing both mucosal and systemic immune responses¹. Intranasal delivery of mRNA vaccines is therefore attracting great interest as a strategy to prevent respiratory viral infections. A major technical challenge, however, is converting fragile mRNA polyplexes into an inhalable aerosol without compromising their stability.

Protocol

NOTE: Culture HEK293T and A549 cell lines in complete medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2% penicillin-streptomycin prior to PEI-mRNA transfection. Passage both cell lines approximately every 48 h upon reaching confluency. Seed cells at a density of 40,000 cells per cm² and culture for ~48 h. Remove the culture medium and wash cells with phosphate-buffered saline (PBS) immediately before transfection to remove residual serum and standardize transfection conditions. Autoclave all cell culture flasks, 24-well plates, PBS, pipettes, and pipette tips prior to use.

Results

As a methods-focused manuscript, detailed results are not the primary aim; however, representative findings are presented to illustrate the expected outcomes of each part of the protocol. Figures mentioned below show example data (e.g., graphs or images) supporting the method's validity, but no new quantitative analyses beyond demonstration are included.

Droplet size and deposition pattern:
The Rayleigh breakup atomizer preserved the physicochemical properties of the mRNA .......

Discussion

In this protocol, before aerosolizing the formulation, the mRNA is branched with PEI to enhance cellular uptake. This serves as a surrogate for LNP-mRNA delivery during in vitro validation^10,15. PEI-mRNA conserves most functions of LNP formulations, including important biological and functional characteristics¹⁰. The positively charged amine groups in PEI bind negatively charged mRNA through electrostatic interactions, forming nan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL centrifuge tubes	Sigma-Aldrich Company Limited	CLS430829-500EA
50–1000 μL pipette tips	Sigma-Aldrich Company Limited	Z740031-1000EA
A549 Cell Line	American Type Culture Collection	ATCC CRM-CCL-185
Air Flow Meter	N/A	N/A	Generic air flow meter may be used.
Alberta Idealized Nasal Inlet (AINI) model	TSI Incorporated (via Copley Scientific, UK)	0001-01-8540
Autoclave indicator tape	Sigma-Aldrich Company Limited	Z744090
DAPI	Sigma-Aldrich Company Limited	D1306
DMEM (Dulbecco's Modified Eagle Medium)	Gibco, Thermo Fisher Scientific	11966025
Ethyl alcohol, pure	Sigma-Aldrich Company Limited	32221-M	Purchased via Merck
Fetal Bovine Serum (FBS)	Gibco, Thermo Fisher Scientific	A5256701
Formaldehyde	Sigma-Aldrich Company Limited	F8775
HEK293T Cell Line	American Type Culture Collection	ATCC CRL-3216
Heraeus HERAsafe HS 12 Biosafety Cabinet	Heraeus	N/A
Next Generation Impactor	MSP's, a Division of TSI	0170-01-1000	This product is also featured in the Copley Catalog, part number 5201.
Nikon DS-Qi2 Monochrome Microscope Camera	Nikon	N/A
Nunc Cell-Culture Treated Multidishes	Thermo Fisher Scientific	142475
Nunc EasYFlask cell culture treated flasks	Thermo Fisher Scientific	156340
Parafilm	Sigma-Aldrich Company Limited	P7793-1EA
Penicillin-Streptomycin (10,000 U/mL)	Gibco, Thermo Fisher Scientific	15140148
Phosphate-buffered saline (PBS) Tablets	Thermo Fisher Scientific	18912014
Polyethylenimine hydrochloride	Sigma-Aldrich Company Limited	764965-1G	Purchased via SLS
Rayleigh Jet Nasal Atomizer	Resyca BV Enschede	Soft Nasal Spray Adaptor
RNAse free water	Thermo Fisher Scientific	J60610.EQC
Sizing Cuvette	Malvern Panalytical	ZEN0040
The Folded Capillary cell	Malvern Panalytical	DTS1070
Zetasizer Pro	Malvern Panalytical	Zetasizer Pro
Zetasizer Ultra-Pro ZS Xplorer software	Malvern Panalytical	N/A