Name: Intranasal Delivery of mRNA Polyplexes via Rayleigh Breakup Aerosols: An In Vitro Method for Nasal Deposition and Functional Testing (Video) | JoVE Journal
Uploaded: 2026-01-20T13:00:00.000+00:00
Duration: 5 min 50 s
Description: Learn a non-destructive method for intranasal mRNA delivery using Rayleigh breakup aerosolization with the AINI model. This protocol preserves mRNA integrity and enables targeted nasal deposition for functional gene expression studies.

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5 minutes

The authors acknowledged the financial support from the King&#39;s Undergraduate Research Fellowship (KURF) and the Student Opportunity Fund provided by King&#39;s College London.

NOTE: Culture HEK293T and A549 cell lines in complete medium consisting of Dulbecco&#39;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 cm&#178; 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
<ol>
	<li>Forming PEI-mRNA complexes (for in vitro transfection) 
	NOTE: This step is only required if PEI is used to boost transfection in cell culture.
	<ol>
		<li>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;&#160;and an amount of 10 &#181;g mRNA would require ~31&#160;&#181;g PEI to form PEI-mRNAs at this N/P ratio.</li>
		<li>Prepare a diluted PEI solution (e.g., 1 mg/mL stock in water) and autoclaved.</li>
		<li>Dilute the mRNA to an appropriate concentration (e.g., 0.2 &#181;g/&#181;L).</li>
		<li>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&#160;or flick the tube for mixing.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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&#39;s properties.
<ol>
	<li>Dynamic light scattering (DLS) pre-aerosolization
	<ol>
		<li>Using a particle size analyzer, load a diluted sample of the formulation into a disposable sizing cuvette (e.g., 500 &#181;L at 0.2 &#181;g/&#181;L).</li>
		<li>Turn on the analyzer and open the software13 on the connected computer.</li>
		<li>Click on the Measure button at the top of the window.</li>
		<li>Click on the blue File icon at the top of the window to select the preset size method.</li>
		<li>Rename the protocol to match the sample name.</li>
		<li>Press the lid button of the analyzer to open the chamber.</li>
		<li>Insert the cuvette into the chamber.</li>
		<li>Click the green Run button at the top of the software window. Wait for the measurement to complete13.</li>
		<li>Click the Analyze button at the top of the software window, and then click the Size button on the result page13.</li>
		<li>Obtain the average hydrodynamic diameter (Z-average) and the polydispersity index (PDI) from the Statistics Table.</li>
		<li>Press the lid button of the analyzer to open the chamber, remove the cuvette, and close the lid.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Zeta potential pre-aerosolization
	<ol>
		<li>Use the same instrument (or a Zeta potential analyzer), load a diluted sample of the formulation into a folded capillary cell (e.g., 700 &#181;L at 0.2 &#181;g/&#181;L).</li>
		<li>Turn on the analyzer and open the software13 on the connected computer.</li>
		<li>Click on the Measure button at the top of the window.</li>
		<li>Click on the blue File icon at the top of the window to select the preset zeta method.</li>
		<li>Rename the protocol to match the sample name.</li>
		<li>Press the lid button of the analyzer to open the chamber.</li>
		<li>Insert the cuvette into the chamber.</li>
		<li>Click the green Run button at the top of the software window. Wait for the machine running13.</li>
		<li>Click the Analyze button at the top of the software window, and then click the Zeta button on the result page13.</li>
		<li>Obtain the zeta potential (surface charge) by looking at the Statistics Table.</li>
		<li>Press the lid button of the analyzer to open the chamber, take out the sample, and close the lid.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Rayleigh breakup aerosolization and nasal deposition
<ol>
	<li>Atomizer and AINI setup
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#160;testing.</li>
		<li>Maintain this airflow for the duration of the spray. Use Parafilm to seal the device properly to the AINI.</li>
	</ol>
	</li>
	<li>Positioning the atomizer
	<ol>
		<li>Insert the Rayleigh breakup atomizer&#39;s nozzle into the entrance of the AINI nostril (vestibule section) at an angle of 45&#176;.</li>
		<li>Secure the atomizer so that it does not shift during operation (all connections, model sections, tubing to pump) are airtight.</li>
	</ol>
	</li>
	<li>Loading the sample
	<ol>
		<li>Gently mix the prepared PEI-mRNA suspension to ensure homogeneity (avoid creating bubbles).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Generating the aerosol
	<ol>
		<li>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&#39;s instructions.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Turn off the air pump promptly to prevent already-deposited droplets from being drawn further down or drying out unevenly.</li>
	</ol>
	</li>
	<li>Sample collection from AINI
	<ol>
		<li>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.</li>
		<li>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)</li>
		<li>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&#39;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.</li>
		<li>Recover any residual sample from the atomizer device. 
		NOTE: For a syringe-type, rinse the syringe and nozzle into a tube.</li>
		<li>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).</li>
		<li>Cap the tube with the collected liquid. 
		NOTE: Avoid losing droplets on bench surfaces or via evaporation.</li>
	</ol>
	</li>
	<li>Post-collection handling
	<ol>
		<li>Immediately after collection, the PEI-mRNA samples were separately placed onto cells for transfection. 
		NOTE: Can leave the PEI-mRNA samples in fridge&#160;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).</li>
	</ol>
	</li>
	<li>Cleaning the apparatus
	<ol>
		<li>Clean the atomizer and AINI model thoroughly after each run to prevent cross-contamination of subsequent experiments.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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&#39;,6-diamidino-2-phenylindole (DAPI; 300 nM) for 5 min followed by 2-times PBS wash prior to imaging.
<ol>
	<li>Fluorescence imaging
	<ol>
		<li>Use a fluorescence microscope with filter sets for DAPI and GFP.</li>
		<li>Begin with the Blue channel (excitation wavelength = 385 nm, emission wavelength = 477.5 nm) to visualize DAPI-stained nuclei.</li>
		<li>Switch to the Green channel (excitation wavelength = 488 nm, emission wavelength = 525 nm) to visualize GFP-positive cells.</li>
	</ol>
	</li>
</ol>

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<li>Geall, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-viral+delivery+of+self-amplifying+RNA+vaccines%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Non-viral delivery of self-amplifying RNA vaccines.</a> Proc Natl Acad Sci U S A. 109 (36), 14604-14609 (2012).</li>
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<li>Method of aerosolizing nanoparticle formulations. China patent. , CN116723892A (2023).</li>
<li>Casper, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyethylenimine+%28PEI%29+in+gene+therapy%3A+current+status+and+clinical+applications%5BTitle%5D)+AND+%22J+Control+Release%22%5BJournal%5D)">Polyethylenimine (PEI) in gene therapy: current status and clinical applications.</a> J Control Release. 362, 667-691 (2023).</li>
<li>Jin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+experimental+approach+to+measure+nebulized+droplet+deposition+pattern+and+deposition+fraction+in+an+idealized+mouth-to-throat+model%5BTitle%5D)+AND+%22Phys+Fluids%22%5BJournal%5D)">A novel experimental approach to measure nebulized droplet deposition pattern and deposition fraction in an idealized mouth-to-throat model.</a> Phys Fluids. 35 (8), (2023).</li>
<li>Darquenne, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aerosol+deposition+in+health+and+disease%5BTitle%5D)+AND+%22J+Aerosol+Med+Pulm+Drug+Deliv%22%5BJournal%5D)">Aerosol deposition in health and disease.</a> J Aerosol Med Pulm Drug Deliv. 25 (3), 140-147 (2012).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Turbinate-homing+IgA-secreting+cells+originate+in+the+nasal+lymphoid+tissues%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues.</a> Nature. 632 (8025), 637-646 (2024).</li>
<li>Debus, H., Baumhof, P., Probst, J., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+messenger+RNA+using+poly%28ethylene+imine%29-poly%28ethylene+glycol%29-copolymer+blends+for+polyplex+formation%3A+biophysical+characterization+and+in+vitro+transfection+properties%5BTitle%5D)+AND+%22J+Control+Release%22%5BJournal%5D)">Delivery of messenger RNA using poly(ethylene imine)-poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties.</a> J Control Release. 148 (3), 334-343 (2010).</li>
<li>Brandtzaeg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+of+nasopharynx-associated+lymphoid+tissue+for+vaccine+responses+in+the+airways%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Potential of nasopharynx-associated lymphoid tissue for vaccine responses in the airways.</a> Am J Respir Crit Care Med. 183 (12), 1595-1604 (2011).</li>
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</ol>

The authors have nothing to disclose.

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 validation10,15. 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...

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-spray3,4,5. 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 &#181;m). This droplet size is ideal for nasal delivery: particles &#62;10 &#181;m 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&#176; 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&#176; 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 &#60;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 studies3,4,5,9, 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-1910,11,12), 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.

<table><tbody><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich Company Limited</td><td>CLS430829-500EA</td><td></td></tr><tr><td>50&amp;ndash;1000 &amp;mu;L pipette tips</td><td>Sigma-Aldrich Company Limited</td><td>Z740031-1000EA</td><td></td></tr><tr><td>A549 Cell Line</td><td>American Type Culture Collection</td><td>ATCC CRM-CCL-185</td><td></td></tr><tr><td>Air Flow Meter</td><td>N/A</td><td>N/A</td><td>Generic air flow meter may be used.</td></tr><tr><td>Alberta Idealized Nasal Inlet (AINI) model</td><td>TSI Incorporated (via Copley Scientific, UK)</td><td>0001-01-8540</td><td></td></tr><tr><td>Autoclave indicator tape</td><td>Sigma-Aldrich Company Limited</td><td>Z744090</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich Company Limited</td><td>D1306</td><td></td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Gibco, Thermo Fisher Scientific</td><td>11966025</td><td></td></tr><tr><td>Ethyl alcohol, pure</td><td>Sigma-Aldrich Company Limited</td><td>32221-M</td><td>Purchased via Merck</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, Thermo Fisher Scientific</td><td>A5256701</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich Company Limited</td><td>F8775</td><td></td></tr><tr><td>HEK293T Cell Line</td><td>American Type Culture Collection</td><td>ATCC CRL-3216</td><td></td></tr><tr><td>Heraeus HERAsafe HS 12 Biosafety Cabinet</td><td>Heraeus</td><td>N/A</td><td></td></tr><tr><td>Next Generation Impactor</td><td>MSP&#039;s, a Division of TSI</td><td>0170-01-1000</td><td>This product is also featured in the Copley Catalog, part number 5201.</td></tr><tr><td>Nikon DS-Qi2 Monochrome Microscope Camera</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Nunc Cell-Culture Treated Multidishes</td><td>Thermo Fisher Scientific</td><td>142475</td><td></td></tr><tr><td>Nunc EasYFlask cell culture treated flasks</td><td>Thermo Fisher Scientific</td><td>156340</td><td></td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich Company Limited</td><td>P7793-1EA</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco, Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) Tablets</td><td>Thermo Fisher Scientific</td><td>18912014</td><td></td></tr><tr><td>Polyethylenimine hydrochloride</td><td>Sigma-Aldrich Company Limited</td><td>764965-1G</td><td>Purchased via SLS</td></tr><tr><td>Rayleigh Jet Nasal Atomizer</td><td>Resyca BV Enschede</td><td>Soft Nasal Spray Adaptor</td><td></td></tr><tr><td>RNAse free water</td><td>Thermo Fisher Scientific</td><td>J60610.EQC</td><td></td></tr><tr><td>Sizing Cuvette</td><td>Malvern Panalytical</td><td>ZEN0040</td><td></td></tr><tr><td>The Folded Capillary cell</td><td>Malvern Panalytical</td><td>DTS1070</td><td></td></tr><tr><td>Zetasizer Pro</td><td>Malvern Panalytical</td><td>Zetasizer Pro</td><td></td></tr><tr><td>Zetasizer Ultra-Pro ZS Xplorer software</td><td>Malvern Panalytical</td><td>N/A</td><td></td></tr></tbody></table>

intranasal delivery of mrna polyplexes via rayleigh breakup aerosols: an in vitro method for nasal deposition and functional testing

Intranasal Delivery of mRNA Polyplexes via Rayleigh Breakup Aerosols: An In Vitro 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.

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 ...

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.

Intranasal delivery of mRNA therapeutics is a promising strategy for vaccination and treating respiratory diseases, offering direct immune activation at ...

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.

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Source: Ullah, I., et.al. A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System. J. Vis. Exp. (2019)This video demonstrates a precise intranasal administration technique for delivering neuron-targeting peptide-siRNA complexes to the mouse brain using a specialized positioning device. Anesthetized mice are secured in a head-down-and-forward position to optimize siRNA inhalation while preventing lung drainage. The targeting peptide...

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Learn a non-destructive method for intranasal mRNA delivery using Rayleigh breakup aerosolization with the AINI model. This protocol preserves mRNA integrity and enables targeted nasal deposition for functional gene expression studies.

Intranasal Delivery of mRNA Polyplexes via Rayleigh Breakup Aerosols: An In Vitro Method for Nasal Deposition and Functional Testing (Video) | JoVE Journal

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

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