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.

Rayleigh breakup aerosolization offers a gentle alternative by operating on a different physical principle. In a Rayleigh breakup device, a pressurized liquid stream is forced through a micro-scale orifice to form a continuous micro-jet. The jet naturally breaks into uniformly sized droplets due to the Plateau-Rayleigh instability². Surface tension causes the liquid column to fragment at a characteristic wavelength about twice the nozzle diameter. Unlike ultrasonic nebulization, this process introduces virtually no turbulent shear; droplets are naturally formed from the self-instability of the jet, not from external shattering forces. As a result, nanoparticle formulations experience minimal stress. It has been shown that using Rayleigh breakup can aerosolize lipid nanoparticle (LNP) mRNAs with no significant change in their size, surface charge, or encapsulation efficiency pre- vs. post-spray^3,4,5. In this method, the PEI-mRNA remains intact and functional after aerosolization, in contrast to the deterioration seen with harsher nebulization techniques.

Another advantage of Rayleigh breakup is the control over droplet size. The generated droplets are relatively large (on the order of 20-30 µm). This droplet size is ideal for nasal delivery: particles >10 µm in aerodynamic diameter tend to deposit in the nose and upper airways rather than drifting into the lungs³. By producing droplets mostly above this threshold, the Rayleigh atomizer naturally targets deposition to the nasal cavity. Targeting is further enhanced by using the Alberta Idealized Nasal Inlet (AINI) model to optimize administration techniques. The AINI is a 3D-printed nasal airway replica that accurately predicts in vivo spray deposition patterns⁶. Using this model, it has been found that spraying at an angle of 45° into the nostril largely increases deposition to the turbinates⁷. The administration angle significantly affects the deposition pattern⁷. Deposition in non-target areas like the nasopharynx or lungs is undesirable, as it can cause throat irritation, bad taste, or systemic side effects⁸. In this protocol, these conditions were replicated (a 45° spray angle and controlled inhalation airflow) to maximize turbinate deposition of the mRNA aerosol³. Previous data shows more than half of the delivered mRNA mass can be focused into the turbinate region under optimal conditions, while <10% reaches the nasopharynx or beyond³. This targeted delivery is expected to improve vaccine efficacy by concentrating the dose at the inductive sites of immunity (which are well-vascularized and rich in immune cells and simultaneously minimizing off-target deposition that could lead to waste or adverse effects.

In this methods article, the experimental procedure for aerosolizing mRNA via Rayleigh breakup is described in detail, assessing regional depositions in the AINI model, and evaluating the biological activity of the deposited mRNA in vitro. A reporter mRNA encoding GFP is used to visualize transfection outcomes in human cells. HEK293T cells are chosen to test the functionality of the aerosolized complexes. To enhance cellular uptake of the aerosolized mRNA in vitro, the mRNA is branched with PEI to form PEI-mRNA polyplexes prior to adding them to cells. PEI is a cationic polymer transfection agent that can improve mRNA delivery efficiency, and an N:P (nitrogen: phosphate) molar ratio of 24:1 was used here based on previous optimization for gene delivery. PEI is used only for the cell-culture validation step in this study and is not part of the aerosol delivery to an actual nose (in vivo one would rely on the LNP-mRNA alone, but in cell culture, this helps maximize gene expression for detection).

This protocol highlights a new approach for intranasal mRNA vaccine delivery that addresses the stability issue of aerosolizing mRNA. Rayleigh breakup has only recently been applied to nucleic acid delivery in a few studies^3,4,5,9, and this work provides a detailed methodological guide to implement this technique. By following this step-by-step procedure, researchers can reproduce the gentle aerosolization of mRNA nanocarriers and assess their deposition and transfection characteristics. The Protocol section below is organized into sequential parts: (1) PEI-mRNA formulation; (2) Nanoparticle characterization (size and zeta potential) before and after aerosolization; (3) Operation of the Rayleigh breakup atomizer and AINI nasal deposition procedure. Key considerations for success, such as maintaining RNase-free conditions and proper device alignment, are noted. The Results and Discussion include representative outcomes, advantages of the method over conventional approaches, potential applications (e.g., nasal vaccines for influenza or COVID-19^10,11,12), as well as limitations and troubleshooting tips. This comprehensive methodology aims to enable reproducible intranasal mRNA delivery experiments and to accelerate the development of needle-free mucosal RNA vaccines.

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.

1. PEI-mRNA formulation

1. Forming PEI-mRNA complexes (for in vitro transfection)
   NOTE: This step is only required if PEI is used to boost transfection in cell culture.
   1. Calculate the required PEI amount for the desired N:P molar ratio.
      NOTE: N:P = 24 (meaning 24 moles of positively charged amines per mole of negatively charged phosphates on mRNA) is used; and an amount of 10 µg mRNA would require ~31 µg PEI to form PEI-mRNAs at this N/P ratio.
   2. Prepare a diluted PEI solution (e.g., 1 mg/mL stock in water) and autoclaved.
   3. Dilute the mRNA to an appropriate concentration (e.g., 0.2 µg/µL).
   4. Add the PEI solution to the mRNA solution while gently mixing (do not vortex).
      NOTE: The mixture may be in a small tube; pipette up and down carefully or flick the tube for mixing.
   5. Incubate at room temperature for ~20 min to allow electrostatic binding of PEI to mRNA.
      NOTE: If not using PEI, the naked mRNA can be aerosolized directly. The additional PEI greatly improved gene expression in A549 cells, but it is optional and might not be used for actual in vivo delivery due to toxicity considerations.

2. Nanoparticle characterization (size and zeta potential)

NOTE: Set up the measurement parameters in the software and save as method before using the analyzer to measure the size or zeta potential. The method setup is required to run the instrument, and the parameters are dependent on the measuring environment, the cuvette used, and the sample's properties.

1. Dynamic light scattering (DLS) pre-aerosolization
   1. Using a particle size analyzer, load a diluted sample of the formulation into a disposable sizing cuvette (e.g., 500 µL at 0.2 µg/µL).
   2. Turn on the analyzer and open the software¹³ on the connected computer.
   3. Click on the Measure button at the top of the window.
   4. Click on the blue File icon at the top of the window to select the preset size method.
   5. Rename the protocol to match the sample name.
   6. Press the lid button of the analyzer to open the chamber.
   7. Insert the cuvette into the chamber.
   8. Click the green Run button at the top of the software window. Wait for the measurement to complete¹³.
   9. Click the Analyze button at the top of the software window, and then click the Size button on the result page¹³.
   10. Obtain the average hydrodynamic diameter (Z-average) and the polydispersity index (PDI) from the Statistics Table.
   11. Press the lid button of the analyzer to open the chamber, remove the cuvette, and close the lid.
   12. Dispose of the cuvette and rinse the atomizer with RNAase-free water and with 70% v/v ethanol to disinfect and facilitate drying between measurements of Size.
       NOTE: For the Rayleigh atomizer, ensure no salt deposits or residues remain in the nozzle, as these can alter spray performance.
2. Zeta potential pre-aerosolization
   1. Use the same instrument (or a Zeta potential analyzer), load a diluted sample of the formulation into a folded capillary cell (e.g., 700 µL at 0.2 µg/µL).
   2. Turn on the analyzer and open the software¹³ on the connected computer.
   3. Click on the Measure button at the top of the window.
   4. Click on the blue File icon at the top of the window to select the preset zeta method.
   5. Rename the protocol to match the sample name.
   6. Press the lid button of the analyzer to open the chamber.
   7. Insert the cuvette into the chamber.
   8. Click the green Run button at the top of the software window. Wait for the machine running¹³.
   9. Click the Analyze button at the top of the software window, and then click the Zeta button on the result page¹³.
   10. Obtain the zeta potential (surface charge) by looking at the Statistics Table.
   11. Press the lid button of the analyzer to open the chamber, take out the sample, and close the lid.
   12. Rinse the cuvette and the atomizer with RNAase-free water and with 70% v/v ethanol to disinfect and facilitate drying between measurements of Zeta potential.
       NOTE: For the Rayleigh atomizer, ensure no salt deposits or residues remain in the nozzle, as these can alter spray performance.

3. Rayleigh breakup aerosolization and nasal deposition

1. Atomizer and AINI setup
   1. Assemble the Rayleigh breakup nasal atomizer device and the Alberta Idealized Nasal Inlet (AINI) model.
      NOTE: The AINI model consists of anatomically representative hollow sections corresponding to different regions of the human nasal cavity (e.g., nostril vestibule, turbinate region, olfactory region, nasopharynx).
   2. Ensure all AINI components are clean and dry before use (residues can alter deposition results). Clean by washing with deionized water⁹ and then with ethanol or an organic solvent.
   3. Attach the AINI to a Next Generation Impactor (NGI) that is connected to a vacuum pump at its outlet to draw airflow through the nasal passages.
   4. Set the flow rate to 7.5 L/min - a flow representing a slow, deep nasal inhalation in an adult⁶. Use a calibrated flow meter (or a rotameter) attached to the induction port of NGI. Adjust the pump until the flow meter reads 7.5 L/min.
      NOTE: Use a flow meter to measure the airflow at the required flow rate for testing.
   5. Maintain this airflow for the duration of the spray. Use Parafilm to seal the device properly to the AINI.
2. Positioning the atomizer
   1. Insert the Rayleigh breakup atomizer's nozzle into the entrance of the AINI nostril (vestibule section) at an angle of 45°.
   2. Secure the atomizer so that it does not shift during operation (all connections, model sections, tubing to pump) are airtight.
3. Loading the sample
   1. Gently mix the prepared PEI-mRNA suspension to ensure homogeneity (avoid creating bubbles).
   2. Draw the sample into the atomizer reservoir.
      NOTE: Depending on the device design, this may involve filling a syringe or pipette tip that attaches to the atomizer.
   3. Load the sample into the device.
      NOTE: For example, remove a central cap or chamber in the atomizer, pipette the sample in, then reassemble the device securely. Ensure there are no leaks and that the device is properly primed if required.
4. Generating the aerosol
   1. Turn on the airflow (7.5 L/min through the AINI) of the air pump before or at the same time as starting the spray, per the device's instructions.
   2. Activate the Rayleigh breakup atomizer to begin aerosolization.
      NOTE: For a manual syringe-driven atomizer, steadily depress the syringe plunger to force the liquid through the orifice. Spray the entire sample volume in one continuous go if possible. The plunger should be depressed smoothly to generate a stable operating pressure, because the formulation is aerosolized only after being pressurized to an elevated pressure and exposed to a wall shear rate inside the micro-orifice¹⁴. Rapid plunger movements alter pressure and therefore change both the shear rate and the shear-exposure time¹⁴.
   3. Ensure the flow rate remains at 7.5 L/min throughout. The aerosol droplets should travel through the nasal passage model and be deposited at different regions according to their aerodynamic sizes.
   4. Turn off the air pump promptly to prevent already-deposited droplets from being drawn further down or drying out unevenly.
5. Sample collection from AINI
   1. Carefully disassemble the AINI model into its individual anatomical segments to collect deposited nanoparticles from each region.
      NOTE: Before disassembly, note or mark the orientation to keep track of sections.
   2. Collect deposits from six compartments: (i) the atomizer device, (ii) the nasal vestibule section, (iii) the turbinate section, (iv) the olfactory region, (v) the nasopharynx section, and (vi) any downstream capture (e.g., stages of an attached NGI or a final filter representing lung-penetrating fraction)
   3. For each compartment, use a measured volume of cell culture medium to wash out the deposited material.
      NOTE: For example, to collect the turbinate deposition, detach the turbinate section and rinse its inner surfaces thoroughly with 5 mL of Dulbecco's Modified Eagle Medium (DMEM), using a disposable pipette. Gently wash all surfaces, then pour the rinse into a labelled 15 mL centrifuge tube (use a funnel to aid transfer). During rinsing, the segment was held horizontally or slightly tilted to ensure complete internal surface contact while preventing fluid escape. Repeat similarly for the vestibule, olfactory, nasopharynx, and any other sections, using 5 mL fresh DMEM for each and collecting into separate labelled tubes.
   4. Recover any residual sample from the atomizer device.
      NOTE: For a syringe-type, rinse the syringe and nozzle into a tube.
   5. Retrieve the liquid from the NGI (cascade impactor) stages if the AINI was connected to an NGI to trap any fine particles (e.g., by rinsing the stages with DMEM).
   6. Cap the tube with the collected liquid.
      NOTE: Avoid losing droplets on bench surfaces or via evaporation.
6. Post-collection handling
   1. Immediately after collection, the PEI-mRNA samples were separately placed onto cells for transfection.
      NOTE: Can leave the PEI-mRNA samples in fridge for ~1-2 h (up to 6 h maximum) before using them for transfection. A certain volume (maximum 1 mL) is drawn from each collected PEI-mRNA sample for characterizing the post-aerosolized vectors (i.e., DLS and zeta measurements).
7. Cleaning the apparatus
   1. Clean the atomizer and AINI model thoroughly after each run to prevent cross-contamination of subsequent experiments.
   2. Rinse all AINI sections and the atomizer parts with deionized water to remove media and nanoparticles, then with 70% ethanol to disinfect and facilitate drying.
      NOTE: If the device or model has small orifices, flush them with a syringe.
   3. Dry all parts to air completely (or place in a drying cabinet at low heat) before reassembling. For the Rayleigh atomizer, ensure no salt deposits or residues remain in the nozzle, as these can alter spray performance.

4. Cell imaging

NOTE: After transfection, remove the transfection medium and replace it with fresh complete medium to terminate exposure. Culture cells for 48 h to allow reporter expression and cell recovery. At ~80% confluency, fix cells with 4% formaldehyde for 8 min, then stain nuclei with 4',6-diamidino-2-phenylindole (DAPI; 300 nM) for 5 min followed by 2-times PBS wash prior to imaging.

1. Fluorescence imaging
   1. Use a fluorescence microscope with filter sets for DAPI and GFP.
   2. Begin with the Blue channel (excitation wavelength = 385 nm, emission wavelength = 477.5 nm) to visualize DAPI-stained nuclei.
   3. Switch to the Green channel (excitation wavelength = 488 nm, emission wavelength = 525 nm) to visualize GFP-positive cells.

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