Polymersomes are nanosized vesicles formed from amphiphilic block copolymers, and have been identified as potential drug delivery vehicles to the inner ear. The aim of this study was to provide targeting to specific cells within the inner ear by functionalizing the polymersome surface with Tet1 peptide sequence. Tet1 peptide specifically binds to the trisialoganglioside clostridial toxin receptor on neurons and was expected to target the polymersomes toward the cochlear nerve. The Tet1 functionalized PEG-b-PCL polymersomes were administered using routine drug delivery routes: transtympanic injection and cochleostomy. Delivery via cochleostomy of Tet1 functionalized polymersomes resulted in cochlear nerve targeting; in contrast this was not seen after transtympanic injection.

Direct drug delivery to the cochlea is associated with the risk of irreversible damage to the ear. In this study, liposome and polymersome nanoparticles (NPs), both formed from amphiphilic molecules (lipids in liposomes and block copolymers in polymersomes), were tested as potential tools for drug delivery to the cochlea via application onto the round window membrane in adult mice (strain C3H). One day after round window membrane application, both types of NPs labeled with fluorescent markers were identified in the spiral ganglion in all cochlear turns without producing any distinct morphological or functional damage to the inner ear. NPs were detected, although to a lesser extent, in the organ of Corti and the lateral wall. The potential of liposome and polymersome NPs as therapeutic delivery systems into the cochlea via the round window membrane was evaluated using disulfiram, a neurotoxic agent, as a model payload. Disulfiram-loaded NP delivery resulted in a significant decrease in the number of spiral ganglion cells starting 2 days postapplication, with associated pronounced hearing loss reaching 20-35 dB 2 weeks postapplication as assessed through auditory brainstem responses. No changes in hair cell morphology and function (as assessed by recording otoacoustic emissions) were detected after disulfiram-loaded NP application. No effects were observed in controls where solution of free disulfiram was similarly administered. The results demonstrate that liposome and polymersome NPs are capable of carrying a payload into the inner ear that elicits a biological effect, with consequences measurable by a functional readout.

Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

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By incorporating ferrocene into the hydrophobic membrane of PEG-b-PCL polymersome nanoparticles it is possible to selectively visualize their core using Transmission Electron Microscopy (TEM). Two different sizes of ferrocene-loaded polymersomes with mean hydrodynamic diameters of approximately 40 and 90 nm were prepared. Image analysis of TEM pictures of these polymersomes found that the mean diameter of the core was 4-5 times smaller than the mean hydrodynamic diameter. The values obtained also allow the surface diameter and internal volume of the core to be calculated.