Exploration of biodistribution of mRNA-delivering lipid nanoparticles in the neonatal mouse inner ear

Hearing loss affects millions worldwide, yet hearing aids and cochlear implants have major limitations. mRNA-based gene therapy could address the underlying molecular causes, but lipid nanoparticle (LNP) delivery in the inner ear remains underexplored and its biodistribution has not been systematically characterized. Here we establish and scale up an mRNA-encapsulated LNP formulation using a design-of-experiments (DoE) approach built on predefined critical quality attributes, and map the route-dependent distribution of reporter mRNA expression in the neonatal mouse cochlea. In HEI-OC1 auditory cells, mRNA-loaded LNPs produced a higher proportion of reporter-expressing cells and a more uniform distribution of expression than the cationic reagent Lipofectamine at a matched mRNA dose, while total reporter protein was comparable and metabolic viability was not reduced. We then compared cochlear distribution following round window membrane (RWM) delivery versus scala media administration. RWM delivery produced widespread reporter expression in perilymph-exposed compartments-the spiral ligament, the scala vestibuli and scala tympani regions, the stria vascularis and the spiral ganglion region-with no detectable expression in hair cells. Scala media injection produced a more restricted pattern, largely confined to the cuticular plate of inner and outer hair cells and part of the outer sulcus, with no expression in the spiral ganglion region or auditory nerve, indicating that this route does not distribute beyond the endolymphatic compartment. These results define the cochlear compartments accessible to LNP-mediated mRNA delivery and the dependence of that access on administration route, and provide the delivery map required for the design of LNP-based mRNA strategies in the inner ear.