Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium

Abstract

Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g^-1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.

Keywords: biomaterials; gene delivery; inhalation; messenger RNA; topology.

Figure 1.

A) hDD90–118 and hC32–118 hyperbranched PBAEs were synthesized via addition of a tri-functional amine, N-methyl 1,3 diaminopropane. B) PBAE polymers were complexed with Cy-5 tagged mRNA encoding for GFP to confirm particle uptake (yellow) and translation to GFP (overlaid image on the right, green) in A549 lung epithelial cells (nuclei, blue), hDD90–118 polyplexes shown. C) Comparison of in vitro transfection efficiency in A549 cells using hyperbranched and linear polymers delivering 0.003 mg mL⁻¹ of luciferase mRNA (n = 3, +SD). D) Polyplex stability at varying mRNA concentration; at pH 7.4, linear PBAEs formulated with mRNA at 0.5 mg mL⁻¹ (red dashed line) become unstable and aggregate into large particles whereas hyperbranched analogues (black dashed line) remain stable nanoparticles below 200 nm. Both linear and branched PBAEs at 0.003 mg mL⁻¹ mRNA remain stable (red and black solid lines, respectively). E) Particle stability at varying pH (DD90, left; C32, right); zeta potential of hyperbranched PBAE polyplexes display higher isoelectric points (black closed circles) compared to linear (red closed squares). A reduction in surface zeta potential occurs at increasing pH which correlates with growth in particle size at pHs above 7.5 for linear PBAEs (red open squares) compared to hyperbranced polymers which remain stable below 200 nm up to pH 8.5 (black open circles).

Figure 2.. Inhalation of mRNA polyplexes.

A) A vibrating mesh nebulizer connected to a whole-body chamber was used to deliver IVT-mRNA encoding for firefly luciferase to mice. The nebulizer generates micrometer sized droplets optimal for lung deposition, containing nanoparticles for intracellular delivery. B) Quantification of luciferase protein/total protein in the lung 24 h after nebulized delivery of 1.0 mg mRNA (p < 0.001, ±S.D, n = 5 – 6). C) Bioluminescence 24 h after inhalation of polyplexes, hDD90–118 vectors produced significantly higher radiance localized to the lung, compared to hC32–118 and bPEI (p < 0.001, +S.D, n = 4). Statistical analysis using one-way ANOVA with post-hoc Tukey test. D) Electron microscopy of hDD90–118 particles before (left) and after (right) nebulization, particles had an average size of 137 nm (±21) and 146 nm (±40), respectively (±S.D, n = 13 – 15, additional images in Figure S6, Supporting Information). E) Particles have narrow size distribution with polydispersity indices of 0.10 before (black) and 0.11 after (red, dashed) nebulization.

Figure 3.

Distribution of protein expression in the lung and cell sub-type transfected with hDD90–118 polyplexes. A) Dissection of mouse lung 24 h after nebulization of hDD90–118-luciferase mRNA polyplexes, bioluminescence is observed throughout all 5 lobes of the lung. B) Uniform radiance is quantified within each lobe (n = 3, ± SD). C) Quantitative assessment of lung cell subtype transfected by hDD90–118 polyplexes was determined using a cre-loxP mouse model designed to express tdTomato only in cells that translate cre-recombinase mRNA. D) Lungs expressing tdTomato fluorescence were analyzed by flow cytometry using markers for endothelial (CD31), epithelial (EpCAM) and immune (CD45) cells (unpaired T-test, **p < 0.01,***p < 0.005, n = 3, + SD).

Figure 4.

Pharmacokinetics of firefly luciferase mRNA translation in mouse lung and toxicity. A) Quantification of luciferase protein at 6, 24, and 48 h post nebulization of hDD90–118 and bPEI polyplexes (n = 3, ± SD). B) Repeat dosing of 1.0 mg of mRNA every 3 d (green arrow) (n = 3–5, ± SD). C) Luciferase protein expression 24 h after inhalation of hDD90–118 polyplexes formulated with 0.25, 0.50, or 1.0 mg of mRNA (n = 3–5, ± SD). D) Weight change in mouse 24 h after nebulization (green arrows). Mice treated repeatedly with bPEI underwent a reduction in weight gain after the third dose (p < 0.05, n = 4–10, ±SD, one-way ANOVA with Tukeys test). E) Serum levels of liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after three doses of mRNA polyplexes or polymer only (p < 0.05, n = 4, ±SD, one-way ANOVA with Tukeys test). F) Histology of lungs after three inhaled doses (day 8) of bPEI or hDD90–118 polyplexes. Alveolar architecture is maintained in both samples, lungs exposed to bPEI display some occurrence of red blood cells but is not considered abnormal (black arrows). Normal alveolar macrophages are present in the lungs of both samples (white arrows). Bronchiolar architecture is maintained in both samples. H&E staining, 20· magnification.