Impact of Formulation and Process Parameters on the Stability and Bioactivity of RNA-Loaded Lipid Nanoparticles during Nebulization

Abstract

During the pandemic, lipid nanoparticles (LNPs) became widely established as RNA nanocarriers, and hold the promise of future targeting of a broad variety of previously untreatable diseases. LNPs are mostly administered invasively via intramuscular or intravenous injections. Given the lung’s large surface, high vascularization and low nuclease abundance, inhalation offers a promising alternative for both local and systemic delivery of LNPs. Vibrating mesh nebulizers present a patient-friendly, high-dose delivery platform. However, the nebulization process imposes thermal and mechanical stress on the LNP formulation. This study contributes to a better understanding of how nebulization affects the physicochemical properties and biological activity of LNPs, depending on formulation and process parameters. We investigated the impact of formulation and process variables such as temperature, concentration, buffer type, and RNA modality on LNP properties including particle size distribution, zeta potential, in vitro activity, and RNA integrity. While aggregating, siRNA LNPs protected the encapsulated RNA from degradation, and preserved biological function. In contrast, after the nebulization of mRNA LNPs the cargo was degraded and the biological function diminished. This observation can possibly be attributed both to the higher sensitivity of mRNA toward physical and chemical degradation, and the cargo-dependent morphology of LNPs. While demonstrating that siRNA LNPs preserved their most important characteristics, namely RNA integrity and biological function, our findings emphasize the need for route-specific optimization of LNPs, which need to meet different critical quality criteria when used for inhalation rather than injection.

Introduction

Over the last decades, and especially accelerated by the COVID-19 pandemic, RNA has become a focus in the search for new active pharmaceutical ingredients (APIs). Among the various naturally occurring RNA types, two important pharmaceutical modalities are small interfering RNA (siRNA) and messenger RNA (mRNA), used either to silence or to express target genes transiently. (Xu et al., 2021, Merkel and Kissel, 2011, Kandil et al., 2019)
RNA offers potential treatment or prevention of disease targets previously untreatable combined with a comparably short development time. As RNA molecules are hydrophilic, charged, comparably large, prone to hydrolysis, and substrates for ubiquitous nucleases, stability and delivery challenges must be overcome for clinical applications. (Merkel et al., 2011, Lam et al., 2012)

Delivery vectors such as lipid nanoparticles (LNPs) protect RNA cargo from degradation, facilitate cellular uptake, and promote endosomal escape. (Kandil et al., 2019) LNPs are complex formulations, typically consisting of an ionizable lipid that electrostatically interacts with the RNA cargo, complemented with rigidity regulating cholesterol, stabilizing PEGylated lipids, as well as helper lipids. (Hald Albertsen et al., 2022)

Currently, all RNA LNP formulations approved by health authorities are administered either intravenously, as in case of Onpattro®, or intramuscularly, as performed with the SARS-CoV 2 vaccines Comirnaty® and Spikevax®. (Curreri et al., 2023) Systemically administered LNPs predominantly accumulate in the liver. (Chen et al., 2016) Specific organ targeting following systemic delivery has been reported with specific lipid components as formulated in SORT LNPs (Dilliard et al., 2021, Wei et al., 2023), although significant research is still required to bring this mechanism to the patient and safety concerns are raised about clogging of lung capillaries. (Omo-Lamai et al., 2024)

Due to its large surface, high vascularization, and low occurrence of nucleases, the lung is an excellent site for local and direct RNA delivery. (Kandil et al., 2019, Lam et al., 2012, Carneiro et al., 2023) Additionally, clinical and preclinical RNA medicines show promise in a broad variety of lung diseases, including infectious diseases such as COVID-19, pneumonia, and tuberculosis, as well as non-transmittable diseases, including bronchial asthma, chronic obstructive lung disease, lung fibrosis, or lung cancer. (Hald Albertsen et al., 2022, Omo-Lamai et al., 2024) Local application of powder or liquids is less invasive, requires a lower dose, and leads to fewer side effects, all of which could also improve patient adherence.

In contrast to other lung administration devices, nebulization does not require breath-hand coordination or deep inspiration, thus making it suitable for patient groups struggling with coordination or compromised inspiration. Additionally, nebulization does not require a propellant, and larger doses can be delivered compared to dry powder inhalers and metered dose inhalers. (Newman, 2005, Neary et al., 2024, Elphick et al., 2015) Among the three main types of nebulizers – jet, ultrasonic and vibrating mesh – the latter is the most commonly used one for RNA nebulization. (Neary et al., 2024, van Rijn et al., 2023, Kim et al., 2022, Lokugamage et al., 2021) In vibrating mesh nebulizers, a piezoelectric crystal vibrates the mesh, resulting in the aerosolization of the aqueous drug formulation. This process, however, increases device and reservoir temperature, generates shear stress and overall energy intake, and can lead to instability, RNA cargo release, particle aggregation and decrease in biological function. (van Rijn et al., 2023, Kim et al., 2022, Lokugamage et al., 2021, Zhang et al., 2020, Klein et al., 2021, Hertel et al., 2014)

This work systematically investigates the effect of vibrating mesh nebulization on the physicochemical characteristics and biological activity of established RNA LNP formulations. It focuses on the influence of formulation and process factors including buffer substances, LNP concentration, temperature, nebulization device and RNA type to contribute to a better understanding of how nebulization impacts LNP integrity and function.
With LNPs containing siRNA and mRNA, two of the currently most relevant RNA types are compared. These two types differ regarding size and stability. To understand the influence of the choice of device, two different commercially available vibrating mesh nebulizers were investigated: the PARI eflow rapid and the Aerogen Pro.

While both devices produce aerosols in the desired droplet size range of 1 – 5 µm (Muller et al., 2025), they differ in geometry, residual volume and energy intake. The Aerogen nebulizer is designed to nebulize vertically or slightly angled with the feed solution on top of the vibrating mesh, with almost no residual volume remaining in the reservoir. Due to its geometry, the PARI device retains a larger residual volume – most likely developed to buffer the temperature increase due to energy intake during nebulization (Hertel et al., 2014). Additionally, the mesh is oriented in a 90-degree angle regarding the feed solution. With 35 ±12 J/g, the overall energy intake has been determined to be larger for the PARI device compared to the Aerogen device with 18 ± 6 J/g (van Rijn et al., 2023).

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2. Material and Methods

2.1. LNP preparation

Enhanced green fluorescent protein (EGFP) gene was used as a reporter gene for both siRNA and mRNA LNPs. Commercial GFP siRNA (Table 1) was obtained from Merck (Darmstadt, Germany), and mRNA (sequence in the supplement) was obtained from Ribopro (Oss, The Netherlands).

Three different LNP formulations were prepared based on the Onpattro®-, Comirnaty®-, and Spikevax®-formulations. The different lipids were separately dissolved in absolute ethanol (Thermo Fisher Scientific, Waltham, USA), combined in specific lipid molar ratios, and diluted to 2 mM for siRNA LNPs, and 1 mM for mRNA LNPs (Table 2). RNA was diluted in 25 mM sodium acetate (Merck, Darmstadt, Germany) buffer at pH 4.0. For siRNA LNPs, the solutions were mixed using the Impingemet Jet Mixer NanoScaler (Knauer, Berlin, Germany), or a herringbone mixer (microfluidic ChipShop, Jena, Germany) operated with syringe pumps at a total flow rate of 3 ml/min following a ratio of 3:1 (RNA:lipids solution) to achieve a Nitrogen-to-Phosphate (N/P) ratio of 3. mRNA LNP precursors were formulated via a T-Mixer (Techlab, Braunschweig, Germany) operated with syringe pumps following the flow rate parameters stated above, adjusted to N/P 6. Dialysis was performed in Pur-A-Lyzer Maxi 3500 dialysis kits (Merck, Darmstadt, Germany) against PBS (Thermo Fisher Scientific, Waltham, MA, USA) or 0.9 % NaCl (Merck, Darmstadt, Germany) at 4°C overnight. The resulting LNPs were filtered with a 0.2 µm Supor ® Membrane filter (PALL, New York, NY, USA) and stored at 4°C.

Lipid type	Lipid name	Molar ratio (% mol)
Onpattro®-like (siRNA)
Ionizable lipid	O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29- tetraem-19-yl)- 4-(N,N-dimethylamino)butanoate (D-Lin-MC3-DMA; MedChemExpress, Monmouth Junction, NJ, USA)	50.0
Helper lipid	1,2-distearoyl-sn-glycero-3phosphocholine (DSPC, Avanti Polar Lipids, Alabaster, AL, USA)	10.0
PEG lipid	1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG; Avanti Polar Lipids, Alabaster, AL, USA)	1.5
Sterol	Cholesterol (Merck, Darmstadt, Germany)	38.5
Comirnaty®-like (mRNA)
Ionizable lipid	((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315; Avanti Polar Lipids, Alabaster, USA)	46.3
Helper lipid	DSPC	9.4
PEG lipid	6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0159; Avanti Polar Lipids, Alabaster, USA)	1.6
Sterol	Cholesterol	42.7
Spikevax®-like (mRNA)
Ionizable lipid	1-Octylnonyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102; MedChemExpress, Monmouth Junction, USA)	50.0
Helper lipid	DSPC	10.0
PEG lipid	DMG-PEG	1.5
Sterol	Cholesterol	38.5

Katrin F. Wiebe, Stefan Schneid, Werner Hoheisel, Wolfgang Frieß, Olivia M. Merkel, Impact of Formulation and Process Parameters on the Stability and Bioactivity of RNA-Loaded Lipid Nanoparticles during Nebulization, European Journal of Pharmaceutical Sciences, 2025, 107383, ISSN 0928-0987, https://doi.org/10.1016/j.ejps.2025.107383.