Inhalable Dry Powders for Lung mRNA Delivery

Abstract

Despite great promise, application of mRNA therapeutics in the lung has proven challenging. Many groups have reported success instilling liquid mRNA formulations in animal models, but direct intratracheal administration of large liquid quantities to the human lung presents significant safety and distribution concerns. To accomplish safe and effective mRNA delivery to the lung, formulations must be prepared for dosing via inhalation. An inhaled mRNA delivery system for the lung must be both robust enough to survive inhalation conditions and potent enough to deliver mRNA upon reaching the lung. In this work dry powder lipid nanoparticle formulations are developed, using spray-freeze-drying, to produce stable, biologically active, inhalable dry powders for mRNA delivery. The final powders have suitable aerosolization properties, with mean mass aerodynamic diameter (MMAD) of 3-4 microns, and fine particle fraction (FPF) ≈40%, allowing for efficient mRNA delivery to the deep lung following inhalation. Importantly, the formulations developed here are suitable for use with different ionizable lipids. Four different ionizable lipid-based formulations are evaluated as powders, and all exhibit in vivo pulmonary mRNA delivery equal to that of instilled liquid formulations. These results lay promising groundwork for the eventual development of an inhalable mRNA dry powder therapeutic.

Keywords: dry powder; lipid nanoparticles; mRNA delivery; pulmonary delivery.

Figure 1

Process Optimization and Cryoprotectant Selection for Dry Powder Formation. a) Schematic of the powder preparation process. Powders are first synthesized via microfluidics with the inclusion of a sugar cryoprotectant/bulking agent. The resultant LNPs are then sprayed into a fine mist which is flash frozen and collected in a liquid nitrogen bath. The flash‐frozen droplets are then lyophilized into a powder which can be dosed in vivo to rats via tracheal cannulation. b) Representative scanning electron microscopy images of particles spray‐freeze‐dried at various total mRNA solution concentrations. At higher concentrations the particles aggregate into larger structures, whereas at lower concentrations the particles form discrete leaf‐like shapes. All images were taken at 1000x magnification with a 10 µm scalebar. c) Comparisons of volumetric sizes of powder particles prepared with each of the sugars as measured by Sympatec HELOS. All are within the expected range to have an appropriate MMAD for lung deposition. d) Activity comparison of powders prepared with each of the sugars using the ionizable lipid A4‐T7. Powders were dissolved into water and dosed to HeLa cells. Cryoprotectant concentrations are reported as % w/v at formulation. e) Solubility comparison of powders prepared using two established lipid formulations and each of the sugars. All graphs are presented as the mean ± 1 standard deviation. P values are presented as determined by one‐way ANOVA with Tukey's post‐hoc correction.

Figure 2

Formulation Optimization of LNPs for Dry Powder Production. a) Table of sizes and encapsulation efficiencies for various non‐powdered standard LNP formulations. Formulation ratios are expressed as the molar ratio of ionizable lipid: helper lipid: cholesterol: pegylated lipid. b) Encapsulation efficiency of mRNA in powders prepared with either DOTAP or their published zwitterionic helper lipid formulation. c) Luciferase transfection activity in HeLa cells of powders prepared with either DOTAP or their published zwitterionic helper lipid formulation. d) Comparison of cKK‐E12 LNP sizes before and after SFD and resuspension in water when formulated with varying amounts of DMPE‐PEG2k. e) Luciferase transfection activity in HeLa cells of cKK‐E12 powders prepared with varying amounts of DMPE‐PEG2k at formulation. All graphs are presented as the mean ± 1 standard deviation. P values are presented as determined by one‐way ANOVA with Tukey's post‐hoc correction for groups with multiple comparisons, and Student's T test for groups without multiple comparisons.

Figure 3

Aerosolization of mRNA LNP Powders. a) Next Generation Impactor (NGI) Mean Mass Aerodynamic Diameter (MMAD) and Fine Particle Fraction (FPF) measurements of powders prepared with each of the commercial lipids. Graphs show the percentage of powder deposited on each stage of the impactor (Ind. = Induction, Pre. = Pre separation, numerals indicate the plate stages in order). MMAD is calculated by a trapezoidal approximation of the flowrate‐corrected stage cutoff values for the impactor (see Table S2 (Supporting Information) for the exact values used) and reported ± 1 SD. The FPF is reported as the fraction of dosed powder with MMAD below 5 µm. b) Demonstration of powder insufflation system for in vivo use. Powders form a light aerosol when ejected from a syringe needle.

Figure 4

In‐vivo Dosing of mRNA Dry Powders. a) Demonstration of fluorescent tracer use to confirm successful dosing in two rat lungs with cKK‐E12 powder. Top: Firefly luciferase luminescent signal (proportional to mRNA transfection activity). Bottom: Indocyanine Green (IcG) fluorescent signal (proportional to true administered powder quantity). b) Sample comparison of in vivo activity of powder and liquid instillation of C12‐200 LNPs. The greater luminescent signal in the liquid sample is roughly proportional to the greater observed dose received. (c) Normalized activity comparisons of powders and liquids dosed to rats, calculated as normalized response = ROI (region of interest) luminescent activity/calculated true mRNA dose. All graphs are presented as the mean ± 1 standard deviation. No significant difference was found between potency of liquid and powder dosing for any of the four LNP formulations tested by Student's T Test. All luminescent (radiance) values are reported in units of photons s‐1 cm‐2 sr. All fluorescent (radiant efficiency) values are reported in units of photons s‐1 sr‐1 µW‐1.