Alexander H. Johnston

Targeted delivery of Tet1 peptide functionalized polymersomes to the rat cochlear nerve.

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.

Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear

Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A(665) and A(666)) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A(665) and A(666) shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.

Activation of TrkB receptors by NGFβ mimetic peptide conjugated polymersome nanoparticles

Activation of tyrosine kinase receptor B (TrkB), a neurotrophin receptor, has been shown to increase neuronal cell survival and promote regeneration. Stimulation of the TrkB receptor by neurotrophic growth factors has been identified as a possible therapeutic target for the treatment of neurodegenerative disorders. However, growth factor delivery is problematic because of a short half-life in vivo. We have conjugated hNgf-EE, a short peptide mimetic of NGFβ to the surface of polymersome nanoparticles and shown that they are capable of activating the TrkB receptor in vitro in the SHSY-G7 cell line. We propose that polymersomes could act as a scaffold for the delivery of TrkB activating moieties and that the polymersome size and polyethylene glycol surface have been shown to increase in vivo retention time. These multifunctional nanoparticles have potential for the treatment of neurodegenerative disorders by TrkB activation. From the ClinicaL Editor: Tyrosine kinase receptor B activation has been shown to promote regeneration and survival of neurons. However, growth factor delivery to stimulate these receptors remains problematic. The authors demonstrate that a peptide mimetic of NGFβ conjugated to the surface of polymersome nanoparticles is capable of activating the TrkB receptors. These nanoparticles may offer a novel treatment strategy for a variety of neurodegenerative disorders.

Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO4 to quench autofluorescence in the rat inner ear

Fluorescent tags and fluorophore-conjugated molecular probes have been extensively employed in histological studies to demonstrate nanoparticle distribution in inner ear cell populations. However, autofluorescence that exists in the rodent cochleae disturbs visualization of the fluorescent tags and fluorophore labeling. In the present work, we aimed to improve the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO(4) to quench autofluorescence in the rat inner ear. The in vivo study was performed on eight- to nine-month-old rats using confocal laser scanning microscopy, and the in vitro study was carried out with DiI-tagged poly(ethylene glycol) and poly(capro-lactone) polymersomes and different fluorescent-labeling agents using a spectrofluorometer. The nanoparticles were intratympanically administered using either an osmotic pump or transtympanic injection. Abundant autofluorescence was detected in spiral ganglion cells (SGCs), stria marginal cells, spiral ligament fibrocytes (SL) and the subcuticular cytoplasm of inner hair cells (IHCs). Sparsely distributed faint autofluorescence was also visualized in outer hair cells (OHCs). The autofluorescence was eliminated by treatment with 1 mM CuSO(4) (in 0.01 M ammonium acetate buffer) for 70-90 min, while the fluorescent tag in the nanoparticle was absolutely preserved and the labeling fluorescence signals of the molecular probes were mostly retained.

Comparison of the distribution pattern of PEG-b-PCL polymersomes delivered into the rat inner ear via different methods

Abstract Conclusion: Cochleostomy is the most efficient approach in delivering PEG-b-PCL polymersomes (PMs) to the inner ear. PMs can be delivered to the vestibule by transtympanic injection or cochleostomy. Objective: To evaluate the efficiency of delivering PEG-b-PCL PMs into the inner ear using different approaches. Methods: The PEG-b-PCL PMs were administered either by sustained topical round window membrane (RWM) delivery using gelatin sponge pledgets in combination with an osmotic pump, transtympanic injection, or cochleostomy. The distribution of the PMs in the inner ear was observed by confocal microscopy using either whole mount specimens or cryosections. Results: Cochleostomy resulted in distribution of the PMs in the spiral ligament (SL), mesothelial cells beneath the organ of Corti, supporting cells in the organ of Corti, and spiral ganglion cells (SGCs). Transtympanic injection induced uptake of the PMs in the SL and mesothelial cells beneath the organ of Corti. Topical administration showed distribution of the PMs only in the SL. In the vestibulum, transtympanic injection and cochleostomy induced more distribution of the PMs than did topical RWM delivery (p < 0.05, Kruskal-Wallis test).

Quantification of intracellular payload release from polymersome nanoparticles

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.

Drug delivery by nanoparticles — facing the obstacles

There are numerous concepts of nanoparticle mediated drug delivery. The major advantage will be the option of targeted drug delivery to specific target cells thus avoiding high systemic loads of potentially toxic chemicals. Any kind of drug delivery by nanoparticles relies on delivery of the drug into the cell. In most cases that means drug delivery into the cytoplasm, and in some instances delivery of the drug to extracellular domains of transmembrane signalling molecules. Whenever viable cells are confronted with nanoparticles these are ingested by endocytosis rather then passage through the cell plasma membrane. Once inside endosomal vesicles the nanoparticles or at least their drug payload requires release into the cytoplasm in order to exert it’s biological effect. In order to monitor whether a drug delivered by nanoparticles is biologically active a toxic model drug, disulfiram, was chosen as a payload with micelle and liposome nanoparticles. L929 mouse fibroblasts were incubated with these disulfiram loaded naoparticles and cell viability was determined by quantification of celluar reductase activity. Applied nanoparticles are toxic to the cells. However, with respect to the disulfiram payload a 100-fold higher disulfiram concentration is required in comparison to free disulfiram for a biological effect. Hence, the toxic effect is most likely not due to the disulfiram delivered by the nanoparticles but rather to the amount of free disulfiram that is present in the nanoparticle preparation. Therefore it is advised to carefully characterize the nanoparticle suspension for the amount of free payload molecules.