Polymer-Lipid Nanoparticles for Systemic Delivery of mRNA to the Lungs

Abstract

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles.

Keywords: PBAEs; drug delivery; mRNA; nanoparticles; serum stability.

Figure 1

PBAE polymer synthesis and nanoparticle formation. (a) Monomers from top-performing materials identified previously were chosen. Briefly, step-growth polymerization was performed via Michael addition of diacrylates and amines, with dodecyl amine added in the case of terpolymers to form a random copolymer. Chains were subsequently reacted with an end-capping amine. For nomenclature, “DD90-C12-122” refers to a terpolymer synthesized from the DD diacrylate reacted with amine 90 along with the C12 alkylamine, and then end-capped with amine 122 (see Representative Synthesis section in the Supplementary Information). Nanoparticles were then formulated by incorporation of PEG-lipid via hydrophobic interactions. (b) Cartoon depiction of PEGylated, mRNA-loaded terpolymer nanoparticles. (c) Representative cryoTEM image of DD90-C12-122 formulated with PEG-lipid. (d) Representative sizing data from dynamic light scattering of DD90-C12-122 formulated with PEG-lipid.

Figure 2

PBAE serum stability and in vitro efficacy. (a) PEGylated PBAE nanoparticles were stable for approximately 2 hours in PBS at 37°C, as indicated by turbidity measurements. These particles were previously dialyzed in PBS for 2 hours at 4°C, a process which results in non-PEGylated particles aggregating (mean ± SD, n = 3). (b) PEGylated DD90-C12-122 and C32-C12-118, as well as PEGylated C32-118 remained stable following addition of 10% v/v fetal bovine serum, while others aggregated (mean ± SD, n = 3). (c) PBAE terpolymer nanoparticles formulated with luciferase-encoding mRNA promote luciferase protein expression in HeLa cells (50 ng/well mRNA dose), as quantified by cellular luminescence. Luminescence was normalized to live cell signal using a fluorescent live/dead cell assay. In this case, transfection is compared to a commercial polymeric transfection reagent jetPEI. (d) Relative viability of cells treated with PEGylated DD90-C12-122 and jetPEI (N/P=57) **indicates p<0.01, *** indicates p < 0.001 (mean ± SD, n = 4).

Figure 3

In vivo efficacy of PBAE nanoparticles. DD90-C12-122. DD90-C12-122 (formulated with and without 7 mol% PEG-lipid), PEGylated C32-C12-118, and in vivo jetPEI nanoparticles were formulated with luciferase-coding mRNA and delivered via tail vein injection to mice at a dose of 0.5 mg mRNA/kg mouse. (a) Representative luminescent image of excised organs following PEGylated DD90-C12-122 delivery demonstrate protein expression mainly in the lungs. (b) At a dose of 0.5 mg mRNA kg⁻¹ mouse injected intravenously, PEGylated DD90-C12-122, as well as PEGylated C32-C12-118, yielded significantly greater luminescence than a non-PEGylated counterpart and had protein expression comparable to that of jetPEI in the lungs; * indicates p < 0.05, ** indicates p < 0.01. (c) Luminescent radiance is dependent on the dose of PEGylated DD90-C12-122 nanoparticles. (d) Imaging of mouse organs following injection of PEGylated DD90-C12-122 nanoparticles loaded with fluorescent mRNA revealed that nanoparticle biodistribution is similar across major organs (representative fluorescent image shown).