Systematic screening of excipients to stabilize aerosolized lipid nanoparticles for enhanced mRNA delivery

Abstract

Aerosolized lipid nanoparticles (LNPs) delivering mRNA are an attractive strategy for use in local, inhalable therapy to treat patients with lung diseases. However, a major barrier to delivering aerosolized mRNA LNPs is the shear forces encountered during aerosolization. These forces lead to significant morphology changes and subsequent decrease in efficacy of mRNA delivery. To best retain the physicochemical properties of mRNA LNPs during aerosolization, we took a formulation-based strategy to stabilize LNPs. We used a design-of-experiment (DOE) approach to comprehensively screen rationally chosen excipients at multiple concentrations. Excipients were carefully selected based on their use in clinically approved inhaled products or their ability to support lipid membrane properties. These excipients were added to the same mRNA LNP composition after formulation, were subsequently characterized, and used to transfect human lung cells at air–liquid interface. From this systematic screen, we identified that the addition of our lead candidate, poloxamer 188, best stabilizes LNP size throughout aerosolization and enhances mRNA expression after aerosolization. Additional morphological studies of the inclusion of poloxamer 188 in LNPs suggests that the excipient lowers aerosolization induced fusion or aggregation of particles without altering the internal structure. Our results indicate that poloxamer 188 can support aerosolized mRNA LNP delivery by maintaining LNP size and significantly enhancing therapeutic nucleic acid delivery to lung cells.

Introduction

Delivery of nucleic acids is promising for treatment of infections, vaccination, and gene editing of disease-causing
mutations. Nucleic acids, such as messenger RNA (mRNA), are highly tunable and upon delivery into cells, encode instructions for cells to produce their own proteins of interest for therapy, enabling the cell to act as a “living pharmacy”.1

However, when free mRNA is delivered in vivo, it is subject to degradation by nucleases and can trigger the host immunesystem, greatly diminishing therapeutic dosing.2–5 Therefore, it is imperative that the mRNA is encapsulated within a carrier such as a lipid nanoparticle (LNP), polymeric nanoparticle, or viral vector.1 Of particular interest, LNPs have risen to popular ity as carriers for mRNA due to the development of mRNA LNP vaccines against SARS-CoV-2 during the COVID-19 pandemic.6–8 Many other mRNA LNP formulations have been created for treatment of cancer, infectious diseases, and genetic diseases.9–20

Patients with genetic lung diseases such as cystic fibrosis, alpha-1 antitrypsin deficiency, and primary ciliary dyskinesia
are strong candidates for mRNA LNP therapies to correct disease-causing mutations using gene replacement or gene
editing technologies.21,22 Treatments for genetic lung diseases can be directed to the lungs via systemic or local delivery. Systemic delivery often consists of intravenous injection which allows for immediate bioavailability of the therapeutic at the site of action but faces many challenges such as hepatic uptake, binding of plasma proteins to the surface of the LNPwhich limits cellular uptake, and off-target uptake in other organs.23–30 Local delivery of therapies involves directing treatment to the lungs via inhalation. Inhaled delivery remains a promising avenue to deliver LNPs to the lungs as the therapy can bypass liver and kidney clearance and increase the concentration of the therapeutic in the area of interest.30 In addition, biodistribution studies have shown that lipid-based carriers remain in the lungs with no noticeable uptake into circulation, reducing off target effects in all other organs.19,31

Inhaled LNP therapies are transformed from a liquid storage form into aerosol droplets via multiple aerosolization methods including nebulization. However, aerosolization can induce shear stress on the structure of the LNP, significantly reducing mRNA delivery and causing fusion or aggregation of particles.32–37 As a result, it is critical to better maintain the LNP structure during aerosolization to retain mRNA for effective delivery. The structure of most LNPs generally consists of four main components: (1) ionizable lipids, which are neutrally charged at physiological pH but become protonated in acidic environments to facilitate endosomal escape; (2) phospholipids, also referred to as helper lipids, that aids in maintaining the LNP structure and enhancing endosomal escape; (3) polyethylene glycol (PEG)-lipids that contributes to particle size, steric hindrance, and colloidal stability; and (4) cholesterol, which helps maintain membrane integrity and rigidity.38 Multiple aerosolized mRNA-LNP clinical trials for genetic diseases in the lungs have been initiated within the last five years but have been met with limited success or have yet to be evaluated for effectiveness, indicating a need for further development of aerosolized mRNA LNPs.39–43 Current research has moved to improve the efficacy of aerosolized LNPs by screening novel lipid structures, altering buffer solutions, or optimizing lipid components.20,32,33,36,44–46

A few research groups have added an additional component to the base LNP formulation such as a permanently cationic lipid or an inactive substance used to aid delivery, traditionally termed an excipient.35,47,48 The excipients used in previous studies improved mRNA expression after aerosolized delivery compared to LNPs without excipients, but it is unclear if this improvement in delivery was due to the excipient or to the change in buffer system.47,48 There
was an observable size change in the LNPs after aerosolization in both studies, which indicates the LNPs are still susceptible to shear stress even in the presence of excipients. An important observation in these studies is that the excipient-doped LNPs were only tested for transfection after aerosolization. It is important to measure changes in mRNA LNPs both before and after aerosolization to determine if there are undocumented morphological changes, which would reduce the delivered dose and ultimately, adversely affect therapeutic efficacy.

Wewill explore changes in LNP structure during aerosolization using a design of experiments (DOE) approach. This
approach allows us to systematically identify select excipients that can help retain the physicochemical properties of mRNA LNPs before and after nebulization and consequently, improve mRNA delivery. DOE is used to condense a large multiparameter space and systematically evaluate relationships between input variables and responses.32,44,48 Here, we use DOE to draw conclusions between factors and identify the optimal conditions for excipient-doped LNPs. Excipients have been previously shown to provide chemical and physical stability to inhaled products, making them a promising route for stabilizing aerosolized LNPs.49 As part of our study, excipients were added to our previously optimized aerosolized mRNA LNP composition to further improve its ability to deliver mRNA after nebulization.33 By characterizing the effect each excipient had on the mRNA LNP size, morphology, permeation, and mRNA transfection after aerosolization, we were able to identify a lead excipient candidate able to best retain the performance of mRNA LNPs for aerosolized delivery.

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Materials

The ionizable lipid SM-102 (heptadecan-9-yl 8-{(2-hydroxyethyl) [6-oxo-6 (undecyloxy)hexyl]amino}octanoate, BP-25499) was purchased from BroadPharm. The helper lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 850355) was purchased from Avanti Polar Lipids. The polyethylene glycol (PEG) anchored lipid, DMPE-PEG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PM-020CN), was purchased from NOF America Corporation. Cholesterol was purchased from Sigma-Aldrich (C3045).

Excipients l-Arginine HCl ((S)-2-amino-5-guanidinopentanoic acid hydrochloride, A121639) and Leucine (H-Leu-OH, A197119) were purchased from AmBeed. Poloxamer 188 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), 15759) was purchased from Sigma-Aldrich. Poloxamer 407 (Pluronic®-F127, 59000) was purchased from Biotium. Methylparaben, NF (ME163) and propylparaben, NF, BP, EP (PR133) were purchased from Spectrum Chemicals. Polysorbate 20 (Tween 20™ Ultrapure, J20605) was purchased from Thermo Scientific Chemicals. Branched PEG (4-arm PEG Acrylate 20K, 4ARM-ACLT) was purchased from JenKem Technology.

Source: Brittany J. Heiser, Mae M. Lewis, Meysam Mohammadi Zerankeshi, Emily K. Netemeyer, Ashlee M. Hernandez, Alexander E. Marras and Debadyuti Ghosh, Systematic screening of excipients to stabilize aerosolized lipid nanoparticles for enhanced mRNA delivery, RSC Pharmaceutics, Received 28th February 2025, Accepted 18th June 2025, DOI: 10.1039/d5pm00061k, rsc.li/RSCPharma, Cite this: DOI: 10.1039/d5pm00061k