Weikai Zhang

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.

Differential passage of gadolinium through the mouse inner ear barriers evaluated with 4.7T MRI.

Magnetic resonance imaging (MRI), supplemented by contrast agents, is a powerful tool that can be used to visualise the structures of the inner ear in vivo and assess some aspects of physiology, such as the permeability of agents through membranes. The mouse is an excellent animal species for investigating human diseases, including hearing loss but detailed MRI studies with contrast have not been reported. In this work, we aimed to demonstrate the limits of MR imaging resolution of the fine inner ear structures in the mouse and to explore the permeability of the intracochlear barriers to gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) administered by intravenous injection (IV) or intratympanic (IT) routes. Twenty-three female FVB mice were imaged with a 4.7-T MR scanner using both 2D and high resolution 3D sequences. Inner ear region of interest (ROI) signal intensities and perilymph volumes were evaluated. Finer structures were studied using 3D acquisition and reconstruction techniques and comparisons were made to similarly oriented histological sections that were examined by light microscopy. Gd-DOTA enhancement occurred in the perilymphatic compartment and highlighted the contiguous inner ear structures, but enhancement did not appear within the endolymph. The dynamic uptake of Gd-DOTA in the perilymphatic compartments reached an initial plateau 80min after IV administration and continued to slightly increase to a maximum level by 100min. The perilymph volume demonstrated by Gd-DOTA uptake was statistically significantly larger in the IV group (1.72mm(3)) than in the IT group (1.28mm(3)) (p<0.05).

Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells.

Background: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs) for gene delivery to cochlear targets. Methods: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats’ round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration. Results: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine™. Conclusion: These results suggest that HPNPs are potentially an ideal carrier for gene delivery into the cochlea.

Inner ear biocompatibility of lipid nanocapsules after round window membrane application.

Nanoparticle-mediated drug delivery represents the future in terms of treating inner ear diseases. Lipid core nanocapsules (LNCs), 50 nm in size, were shown to pass though the round window membrane (RWM) and reached the spiral ganglion cells and nerve fibers, among other cell types in the inner ear. The present study aimed to evaluate the toxicity of the LNCs in vitro and in vivo, utilizing intact round window membrane delivery in rats. The primary cochlear cells and mouse fibroblast cells treated with LNCs displayed dosage dependant toxicity. In vivo study showed that administration of LNCs did not cause hearing loss, nanoparticle application-related cell death, or morphological changes in the inner ear, at up to 28 days of observation. The cochlear neural elements, such as synaptophysin, ribbon synapses, and S-100, were not affected by the administration of LNCs. However, expression of neurofilament-200 decreased in SGCs and in cochlear nerve in osseous spiral lamina canal after LNC delivery, a phenomenon that requires further investigation. LNCs are potential vectors for the delivery of drugs to the inner ear.

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.

MRI manifestation of novel superparamagnetic iron oxide nanoparticles in the rat inner ear.

AIM Superparamagnetic iron oxide nanoparticles hierarchically coated with oleic acid and Pluronic F127 copolymers (POA@SPION) have shown exceptional T2 contrast enhancement. The aim of the present work was to investigate the MRI manifestation of POA@SPION in the inner ear. MATERIALS & METHODS A total of 26 male Wister rats were selected for testing POA@SPION administered through intracochlear, intratympanic and intravenous routes. MRI was performed with a 4.7 T MR scanner. RESULTS & CONCLUSION POA@SPION can be introduced into the perilymph space, after which it becomes widely distributed and can demonstrate the integrity of the perilymph-endolymph barrier. Positive highlighting of the endolymph compartment against the darkened perilymph was visualized for the first time. POA@SPION passed through the middle-inner ear barriers in only small amounts, but stayed in the perilymph for 3 days. They did not traverse the blood-perilymph barrier or blood-endolymph barrier. The inner ear distribution of POA@SPION was confirmed by histology. POA@SPION is a promising T2 negative contrast agent.

Nanoparticle-based delivery for the treatment of inner ear disorders.

Purpose of reviewThe delivery of targetable synthetic vectors that can carry a variety of drugs, proteins, and nucleic acids, such as DNA and small interfering RNA (siRNA), to mammalian cells is important as a potential therapeutic system that avoids the problems that are associated with viruses. Recent findingsThe so-called multifunctional nanocarriers that are equipped with several functions, such as targetability, shelter from the immune system, and opsonization, and are capable of delivering payload across the nuclear envelope, have been synthesized. To improve transfection efficiency, a group of novel peptides have been attached to the surface of the carrier that will enhance endosomal escape and promote nuclear entry. The targeting of tropomyocin receptor kinase B (TrkB) with ligands enhances uptake in spiral ganglion cell culture. Treatment cargos have included growth factors such as the Math-1 gene, short hairpin RNA, and steroids. The problems with current synthetic nanocarriers are poorer selectivity, internalization, and transfection rate compared with viral vectors. SummaryWithin a few years, when the synthetic vectors have been optimized, the first human drugs/proteins/gene product-based therapies will become available in a phase I study.

Novel endosomolytic peptides for enhancing gene delivery in nanoparticles.

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.

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).