(M1230-03-17) Stabilizing mRNA Lipid Nanoparticles for Aerosolized Delivery Using Excipients

Purpose: Delivery of nucleic acids is promising for the 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. Lipid nanoparticles (LNPs) delivering mRNA have been explored for use in local, inhalable therapy to treat patients with lung diseases. A successful method for inhaled administration of mRNA LNPs utilizes a vibrating mesh nebulizer to aerosolize LNPs. The vibrating mesh nebulizer possesses the lowest temperature change of the therapeutic in solution with the lowest therapeutic loss as compared to jet and ultrasonic nebulizers. However, a major barrier to delivering nebulized mRNA LNPs is the shear forces encountered during aerosolization, which leads to significant morphology changes and subsequent decrease in efficacy of mRNA delivery. This work aims to improve the delivery efficacy of mRNA through the addition of excipients to LNPs post-formulation.

Methods: LNPs were formulated using microfluidic mixing of the organic and aqueous phases. The organic phase contained 10 mg/mL SM-102, DPPC, DMPE-PEG and cholesterol in 100% ethanol at molar ratios of 0.45, 0.20, 0.01, and 0.34, respectively. The aqueous phase consisted of nanoluciferase (NLuc) mRNA diluted in 50 mM sodium citrate buffer at a pH of 3.0. The organic and aqueous phases were combined using a NanoAssemblr Benchtop Microfluidic System at a respective ratio of 1:3 and a flow rate of 9 mL/minute. LNPs were formulated at varying mRNA concentrations ranging from 37.5 ng/µL to 50 ng/µL and dialyzed against 1X PBS overnight. Excipients were then added to the same base mRNA LNP composition, titled B1, using 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. The excipient-added LNPs and LNPs without excipients were aerosolized using the Aerogen Solo vibrating mesh nebulizer. Particle diameter, polydispersity, zeta-potential, encapsulation efficiency, morphology, and transfection of human lung cells at air-liquid interface were then characterized before and after aerosolization of all LNP samples. The criteria for successful stabilization of LNPs include minimal size change due to aerosolization, minimal morphology changes due to aerosolization, and improved cellular expression of mRNA after aerosolization as compared to LNPs without excipients.

Results: NLuc mRNA, a luminescent reporter molecule that is ~150X more sensitive than firefly luciferase, was encapsulated within our B1 formulation. The B1 formulation was split into 23 different tubes and seven different excipients (E2-E8) were added at varying concentrations of low, middle (mid), or high. A previously discovered excipient, E1, was used as a positive control to compare efficacy of our excipients. The luminescence bioactivity from luciferase expression in Calu-3 cells cultured at air-liquid interface was measured after a 24-hour incubation with B1 and excipient-added LNPs (Figure 1). As seen in previous literature, LNPs without excipients had a ~2.3 fold loss of mRNA expression after aerosolization. The best performing excipient-added LNP, high E2, showed a 3.3-fold increase in luminescence intensity as compared to aerosolized B1 without excipients. In addition, the E1 excipient control on our B1 LNP resulted in a 2.8-fold increase in luminescence after aerosolization compared to B1 without excipients after nebulization. However, the high concentration of E2 still resulted in higher delivery of mRNA to cells compared to the E1 excipient control. From this systematic screen, a lead candidate was identified, E2, which best stabilizes LNP size throughout aerosolization, as measured by dynamic light scattering (Figure 2). Results indicate that upon the addition of high E2, middle E7, high E7, middle E8, or high E8 to LNPs, there was no significant change in LNP size throughout aerosolization. Additional morphological studies of the inclusion of E2 in LNPs using cryogenic electron microscopy (cryo-EM) suggests that the excipient lowers aerosolization induced fusion of particles (Figure 3). There is extensive particle fusion visualized in the aerosolized B1 sample without excipients, whereas images taken of the high E2 and low E3 aerosolized LNPs display fewer fused particles. The images from cryo-EM provide strong supporting evidence that E2 can better help stabilize the size and structure of LNPs during aerosolization.

Conclusion: In this study we explored the potential of adding excipients to mRNA LNPs post-formulation to stabilize LNP structure during aerosolization. We utilized a DOE approach to screen twenty-one excipient groups and compared the mRNA expression, size changes, and LNP morphology to controls before and after aerosolization. Our best performing excipient-added LNP, E2, displayed superior mRNA expression in lung cells, no significant size changes, and minimal particle fusions as compared to our non-excipient control LNP. While further studies are needed to understand poloxamer location in LNP formulations, the success of E2 addition to mRNA LNP aerosolized delivery provides a promising future for inhaled LNP therapies to treat pulmonary diseases.


Figure 1: Excipient-added LNP cellular expression of bioactivity before and after aerosolization. Graph displays quantification of luminescence 24 hours after NLuc mRNA transfection of Calu-3 cells at air-liquid interface. B1 and E1 serve as controls for excipient screening. Significance is relative to aerosolized B1 (n = 3; mean ± standard deviation [SD], ***p < 0.001, ****p < 0.0001; two-way ANOVA with alpha=0.05).


Figure 2: Dynamic light scattering measurements of LNP size before and after aerosolization. B1 and E1 serve as controls for excipient screening. Significance is determined between non-aero and aero samples (n = 3; ns=no significance; two-way ANOVA with alpha=0.05).


Figure 3: Cryo-EM images of B1, high E2, and low E3 before and after aerosolization (scale bar=100 nm).