Combinatorial development of nebulized mRNA delivery formulations for the lungs

Abstract

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

Fig. 1 |. Formulation optimization of LNPs for nebulized delivery using DOE.

a, Molar ratios of the ionizable lipid, helper phospholipid and cholesterol were varied along with the identity of the ionizable lipid and phospholipid. b, Molar ratios of the resultant 19 LNP formulations with T1–1 representing the original formulation previously developed in our laboratory around which the formulation design is centred. c, In vitro transfection efficiencies of formulations before (blue) and after (red) nebulization in A549 cells. FFL mRNA (50 ng) was delivered for pre-nebulized formulations, normalized by total mRNA in solution as measured by Ribogreen, and volume-matched doses of post-nebulized formulations. Mean ± s.d., n = 3 wells per group. RLU, relative light unit. d, Encapsulation efficiency of FFL mRNA before (blue) and after (red) nebulization. Mean ± s.d., n = 3 technical replicates. e, Lung luminescence 6 h after nebulized delivery of 0.5 mg FFL mRNA to mice demonstrating that T1–5 significantly increasing luminescent signal compared with the original T1–1 formulation. Representative images of lungs imaged by IVIS are shown. One-way analysis of variance (ANOVA) with post hoc Tukey test, mean± s.d., n = 3 mice for PBS, n = 5 mice for others.

Fig. 2 |. Excipients and buffer modifications for improved LNP stability and in vivo delivery.

a, Schematic of the workflow for evaluating addition of excipients and buffer modifications to improve LNP nebulization. b,c, Bioluminescence in lung 6 h after nebulized administration of 0.5 mg FFL mRNA with T1–5 and PEG excipients showing significant increase over no excipients (b) (n = 3 mice for Ficoll and Dextran, n = 4 mice for all others; final excipient concentration 20% w/v for the disaccharides and 2% w/v for all other excipients), and overnight dialysis of T1–5 into 100 mM NaAc buffer, pH 5.2, improving luminescent signal over dialysis into 0.9% saline or PBS (c) (n = 4 mice for saline, n = 5 mice for all others). Mean ± s.d., one-way ANOVA with post hoc Tukey test. d, DLS measurements of LNP size before and after nebulization demonstrating reduction in size increase following nebulization for T1–5 formulation when bPEG20K is added to PBS and further reduction when PBS is replaced with NaAc buffer in the presence of bPEG20K. Mean ± s.e.m., n = 6–10 repeated measurements.

Fig. 3 |. Synthesis and screening of a biodegradable lipid library.

a, Four-step synthetic sequence, comprising esterification, oxidation and aldol reaction, for making our ionizable lipid library. Tails are made in the aldol reaction (step 3). DIPEA, N,N-diisopropylethylamine; DCM, dichloromethane; STAB, sodium triacetoxyborohydride. b, Individual tails and amine components for combinatorial reaction. c, Schematic of difference between (1) classic submerged culture (undifferentiated cells, no tight junctions, non-physiological environment) and (2) ALI cultures. d, Representative H&E-stained histology images of 3-week-old large (top) and small (bottom) airway ALI cultures. Scale bars, 50 μm. e, Delivery of 1 μg FFL mRNA using T1–5-formulated, microfluidically mixed, non-nebulized C12–200 LNPs, 2–13 days after airlift. n = 3 ALI culture wells per day and airway cell line type, two-way ANOVA with Dunnett’s multiple-comparisons test, mean ± s.e.m. f, Screen of the top 22 A549 hits for delivery to small-airway ALI cultures sorted by sum of log luminescence in small and large airways. n = 4 ALI culture wells per airway cell line type, that is, eight wells total per lipid, 500 ng mRNA per well, mean ± s.e.m. g, Structures of two top hits, IR-117-17 and IR-19-Py.

Fig. 4 |. In vivo testing and in vitro–in vivo comparison of top lipids.

a, Nebulized mRNA delivery to mouse lung after 1 mg dose delivered via nose cone, imaged at peak expression (6 h for LNPs, 24 h for hPBAE, 48 h for NLD1). Mean ± s.d., n = 3 for PBS, NLD1, IR-117-17-ALA, n = 4 for all others, one-way ANOVA with post hoc Tukey test. Left: all nebulized NPs, right: zoomed-in image showing significant NLD1 delivery compared with PBS. b–e, Correlation of in vivo nebulized lung delivery data from a to FFL delivery to ALI cultures (500 ng per well) using handmixed non-nebulized LNPs (b) (no significant correlation, P = 0.37; ALI culture measurements given in Supplementary Fig. 12), FFL delivery to A549 cells (20 ng per well) using handmixed non-nebulized LNPs (c) (no significant correlation, P = 0.85), FFL delivery to ALI cultures (500 ng per well) using microfluidically mixed post-nebulized LNPs (d) (ρ = 0.64, P = 0.0022), and FFL delivery to A549 cells (20 ng per well) using microfluidically mixed post-nebulized LNPs (e) (ρ = 0.658, P = 0.0076). In b–e, correlation P values were calculated using an F-test, mean ± s.e.m., n ≥ 3 ALI cultures wells for each measurement.

Fig. 5 |. Pharmacokinetics and pharmacodynamics of top-performing LNPs.

a, Dose–response study of T1–5 LNPs comprising the two biodegradable ionizable lipids, IR-117-17 and IR-19-Py, compared with the non-biodegradable lipid C12–200. Mean ± s.d., n = 3 mice per dose. b,c, Time course of protein expression in the lung (b) and nose (c) of mice following nebulized delivery of 1 mg mRNA. Mean ± s.d., n = 5 mice per experimental group. d, Representative whole-body images of mice 6 h after nebulized delivery of 1 mg mRNA. e, Repeat-dosing of 1 mg of mRNA every 3 days. Mean ± s.d., n = 5 mice per experimental group. f, Luminescence in the lungs of Scnn1b-Tg mice, a model of the CF phenotype, following nebulized delivery of 1 mg of FFL mRNA with either IR-117-17 LNPs or hPBAE. Mean ± s.d., n = 3 for PBS, n = 8 for all others, one-way ANOVA with post hoc Tukey test.

Fig. 6 |. Evaluating functional mRNA delivery to lung epithelium with IR-117-17 LNPs in the Ai14 mouse model.

a, Schematic of Cre delivery experiment to quantify transfected cells in Ai14 mice. A total of 9 mg of Cre mRNA evenly divided over the course of three doses was nebulized to mice using IR-117-17 LNPs or hPBAE. PBS was nebulized to mice as a control. Created with BioRender.com b, Representative images of large and small airways with or without nebulized IR-117-17 LNP treatment. Left column: co-visualization of transfected cells (tdTomato+) with DAPI. Right column: co-visualization of transfected cells (tdTomato+), club cells (CCSP+) and ciliated cells (acetylated tubulin (AcTub)+, AcTub is membrane-localized, hence colour is not present throughout the cell). Green arrows indicate transfected club cells (tdTomato+ CCSP+), purple arrows indicate transfected ciliated cells (tdTomato+ AcTub+). c, Quantification of transfection of large and small airways by IR-117-17 LNPs and hPBAEs. n = 3 mice; individual points are averages over ≥5 large airways per mouse and ≥25 small airways per mouse; mean ± s.e.m., two-way ANOVA. d, Some epithelial regions in the lung had near 100% transfection.