Guinea Pig Round Window Membrane Research Articles

Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies.

Efficient and minimally invasive drug delivery to the inner ear is a significant challenge. The round window membrane (RWM), being one of the few entry points to the inner ear, has become a vital focus of investigation. However, due to the complexities of isolating the RWM, our understanding of its pharmacokinetics remains limited. The RWM comprises three distinct layers: the outer epithelium, the middle connective tissue layer, and the inner epithelial layer, each potentially possessing unique delivery properties. Current models for investigating transport across the RWM utilize in vivo animal models or ex vivo RWM models which rely on cell cultures or membrane fragments. Guinea pigs serve as a validated preclinical model for the investigation of drug pharmacokinetics within the inner ear and are an important animal model for the translational development of delivery vehicles to the cochlea. In this study, we describe an approach for explantation of a guinea pig RWM with surrounding cochlear bone for benchtop drug delivery experiments. This method allows for preservation of native RWM architecture and may provide a more realistic representation of barriers to transport than current benchtop models.

A Benchtop Round Window Model for Studying Magnetic Nanoparticle Transport to the Inner Ear.

The round window membrane (RWM) presents a significant barrier to the local application of therapeutics to the inner ear. We demonstrate a benchtop preclinical RWM model and evaluate superparamagnetic iron oxide nanoparticles (SPIONs) as vehicles for magnetically assisted drug delivery. Guinea pig RWM explants were inset into a 3D-printed dual chamber benchtop device. Custom-synthesized 7-nm iron core nanoparticles were modified with different polyethylene glycol chains to yield two sizes of SPIONs (NP-PEG600 and NP-PEG3000) and applied to the benchtop model with and without a magnetic field. Histologic analysis of the RWM was performed using transmission electron microscopy (TEM) and confocal microscopy. Over a 4-h period, 19.5 ± 1.9% of NP-PEG3000 and 14.6 ± 1.9% of NP-PEG600 were transported across the guinea pig RWM. The overall transport increased by 1.45× to 28.4 ± 5.8% and 21.0 ± 2.0%, respectively, when a magnetic field was applied. Paraformaldehyde fixation of the RWM decreased transport significantly (NP-PEG3000: 7.6 ± 1.5%; NP-PEG600: 7.0 ± 1.6%). Confocal and electron microscopy analysis demonstrated nanoparticle localization throughout all cellular layers and layer-specific transport characteristics within RWM. The guinea pig RWM explant benchtop model allows for targeted and practical investigations of transmembrane transport in the development of nanoparticle drug delivery vehicles. The presence of a magnetic field increases SPION delivery by 45%-50% in a nanoparticle size- and cellular layer-dependent manner. NA Laryngoscope, 134:3355-3362, 2024.

Microneedles Facilitate Small-Volume Intracochlear Delivery Without Physiologic Injury in Guinea Pigs.

Microneedle-mediated intracochlear injection through the round window membrane (RWM) will facilitate intracochlear delivery, not affect hearing, and allow for full reconstitution of the RWM within 48 hours. We have developed polymeric microneedles that allow for in vivo perforation of the guinea pig RWM and aspiration of perilymph for diagnostic analysis, with full reconstitution of the RWM within 48 to 72 hours. In this study, we investigate the ability of microneedles to deliver precise volumes of therapeutics into the cochlea and assess the subsequent consequences on hearing. Volumes of 1.0, 2.5, or 5.0 μL of artificial perilymph were injected into the cochlea at a rate of 1 μL/min. Compound action potential (CAP) and distortion product otoacoustic emission were performed to assess for hearing loss (HL), and confocal microscopy was used to evaluate the RWM for residual scarring or inflammation. To evaluate the distribution of agents within the cochlea after microneedle-mediated injection, 1.0 μL of FM 1-43 FX was injected into the cochlea, followed by whole mount cochlear dissection and confocal microscopy. Direct intracochlear injection of 1.0 μL of artificial perilymph in vivo , corresponding to about 20% of the scala tympani volume, was safe and did not result in HL. However, injection of 2.5 or 5.0 μL of artificial perilymph into the cochlea produced statistically significant high-frequency HL persisting 48 hours postperforation. Assessment of RWMs 48 hours after perforation revealed no inflammatory changes or residual scarring. FM 1-43 FX injection resulted in distribution of the agent predominantly in the basal and middle turns. Microneedle-mediated intracochlear delivery of small volumes relative to the volume of the scala tympani is feasible, safe, and does not cause HL in guinea pigs; however, injection of large volumes induces high-frequency HL. Injection of small volumes of a fluorescent agent across the RWM resulted in significant distribution within the basal turn, less distribution in the middle turn, and almost none in the apical turn. Microneedle-mediated intracochlear injection, along with our previously developed intracochlear aspiration, opens the pathway for precision inner ear medicine.

Pyrolyzed Ultrasharp Glassy Carbon Microneedles.

Polymeric microneedles fabricated via two-photon polymerization (2PP) lithography enable safe medical access to the inner ear. Herein, the material class for 2PP-lithography-based microneedles is expanded by pyrolyzing 2PP-fabricated polymeric microneedles, resulting in glassy carbon microneedles. During pyrolysis the microneedles shrink up to 81% while maintaining their complex shape when the exposed surface-area-to-volume ratio (SVR) is 0.025 < SVR < 0.04, for the temperature history protocol used herein. The derived glassy carbon is confirmed with energy-dispersive X-ray spectroscopy and Raman spectroscopy. The pyrolyzed glassy carbon has Young's modulus 9.0 GPa. As a brittle material, the strength is stochastic. Using the two-parameter Weibull distribution, the glassy carbon has Weibull modulus of 3.1 and characteristic strength of 710 MPa. The viscoelastic response has characteristic time scale of about 10000 s. In vitro experiments demonstrate that the glassy carbon microneedles introduce controlled perforations across the guinea pig round window membrane (RWM) from the middle ear space into the inner ear, without damaging the microneedle. The resultant controlled perforation of RWM is known to enhance diffusion of therapeutics across the RWM in a predictable fashion. Hence, the glassy carbon microneedles can be deployed for mediating inner ear delivery.

Pyrolyzed Ultrasharp Glassy Carbon Microneedles

Polymeric microneedles fabricated via two‐photon polymerization (2PP) lithography enable safe medical access to the inner ear. Herein, the material class for 2PP‐lithography‐based microneedles is expanded by pyrolyzing 2PP‐fabricated polymeric microneedles, resulting in glassy carbon microneedles. During pyrolysis the microneedles shrink up to 81% while maintaining their complex shape when the exposed surface‐area‐to‐volume ratio (SVR) is , for the temperature history protocol used herein. The derived glassy carbon is confirmed with energy‐dispersive X‐ray spectroscopy and Raman spectroscopy. The pyrolyzed glassy carbon has Young's modulus 9.0 GPa. As a brittle material, the strength is stochastic. Using the two‐parameter Weibull distribution, the glassy carbon has Weibull modulus of 3.1 and characteristic strength of 710 MPa. The viscoelastic response has characteristic time scale of about 10000 s. In vitro experiments demonstrate that the glassy carbon microneedles introduce controlled perforations across the guinea pig round window membrane (RWM) from the middle ear space into the inner ear, without damaging the microneedle. The resultant controlled perforation of RWM is known to enhance diffusion of therapeutics across the RWM in a predictable fashion. Hence, the glassy carbon microneedles can be deployed for mediating inner ear delivery.

Inner Ear Diagnostics and Drug Delivery via Microneedles.

Objectives: Precision medicine for inner ear disorders has seen significant advances in recent years. However, unreliable access to the inner ear has impeded diagnostics and therapeutic delivery. The purpose of this review is to describe the development, production, and utility of novel microneedles for intracochlear access. Methods: We summarize the current work on microneedles developed using two-photon polymerization (2PP) lithography for perforation of the round window membrane (RWM). We contextualize our findings with the existing literature in intracochlear diagnostics and delivery. Results: Two-photon polymerization lithography produces microneedles capable of perforating human and guinea pig RWMs without structural or functional damage. Solid microneedles may be used to perforate guinea pig RWMs in vivo with full reconstitution of the membrane in 48–72 h, and hollow microneedles may be used to aspirate perilymph or inject therapeutics into the inner ear. Microneedles produced with two-photon templated electrodeposition (2PTE) have greater strength and biocompatibility and may be used to perforate human RWMs. Conclusions: Microneedles produced with 2PP lithography and 2PTE can safely and reliably perforate the RWM for intracochlear access. This technology is groundbreaking and enabling in the field of inner ear precision medicine.

Epigallocatechin gallate-loaded tetrahedral DNA nanostructures as a novel inner ear drug delivery system.

The study of drug delivery systems to the inner ear is a crucial but challenging field. The sensory organ (in the inner ear) is protected by the petrous bone labyrinth and the membranous labyrinth, both of which need to be overcome during the drug delivery process. The requirements for such a delivery system include small size, appropriate flexibility and biodegradability. DNA nanostructures, biomaterials that can arrange multiple functional components with nanometer precision, exhibit characteristics that are compatible with the requirements for inner ear drug delivery. Herein, we report the development of a novel inner ear drug delivery system based on epigallocatechin gallate (EGCG)-loaded tetrahedral DNA nanostructures (TDNs, EGCG@TDNs). The TDNs self-assembled via base-pairing of four single-stranded DNA constructs and EGCG was loaded into the TDNs through non-covalent interactions. Cy5-labeled TDNs (Cy5-TDNs) were significantly internalized by the House Ear Institute-Organ of Corti 1 cell line, and this endocytosis was energy-, clathrin-, and micropinocytosis-dependent. Cy5-TDNs penetrated the round window membrane (RWM) rapidly in vivo. Local application of EGCG@TDNs onto the RWM of guinea pigs in a single dose continuously released EGCG over 4 hours. Drug concentrations in the perilymph were significantly elevated compared with the administration of free EGCG at the same dose. EGCG@TDNs were found to have favorable biocompatibility and strongly affected the RSL3-induced down-regulation of GPX4 and the generation of reactive oxygen species, on the basis of 2',7'-dichlorodihydrofluorescein diacetate staining. JC-1 staining suggested that EGCG@TDNs successfully reversed the decrease in mitochondrial membrane potential induced by RSL-3 in vitro and rescued cells from apoptosis, as demonstrated by the analysis of Annexin V-FITC/PI staining. Further functional studies showed that a locally administered single-dose of EGCG@TDNs effectively preserved spiral ganglion cells in C57/BL6 mice after noise-induced hearing loss. Hearing loss at 5.6 and 8 kHz frequencies was significantly attenuated when compared with the control EGCG formulation. Histological analyses indicated that the administration of TDNs and EGCG@TDNs did not induce local inflammatory responses. These favorable histological and functional effects resulting from the delivery of EGCG by TDNs through a local intratympanic injection suggest potential for therapeutic benefit in clinical applications.

Membrane curvature and connective fiber alignment in guinea pig round window membrane

The round window membrane (RWM) covers an opening between the perilymph fluid-filled inner ear space and the air-filled middle ear space. As the only non-osseous barrier between these two spaces, the RWM is an ideal candidate for aspiration of perilymph for diagnostics purposes and delivery of medication for treatment of inner ear disorders. Routine access across the RWM requires the development of new surgical tools whose design can only be optimized with a thorough understanding of the RWM’s structure and properties. The RWM possesses a layer of collagen and elastic fibers so characterization of the distribution and orientation of these fibers is essential. Confocal and two-photon microscopy were conducted on intact RWMs in a guinea pig model to characterize the distribution of collagen and elastic fibers. The fibers were imaged via second-harmonic-generation, autofluorescence, and Rhodamine B staining. Quantitative analyses of both fiber orientation and geometrical properties of the RWM uncovered a significant correlation between mean fiber orientations and directions of zero curvature in some portions of the RWM, with an even more significant correlation between the mean fiber orientations and linear distance along the RWM in a direction approximately parallel to the cochlear axis. The measured mean fiber directions and dispersions can be incorporated into a generalized structure tensor for use in the development of continuum anisotropic mechanical constitutive models that in turn will enable optimization of surgical tools to access the cochlea. Statement of significanceThe Round Window Membrane (RWM) is the only non-osseous barrier separating the middle and inner ear spaces, and thus is an ideal portal for medical access to the cochlea. An understanding of RWM structure and mechanical response is necessary to optimize the design of surgical tools for this purpose. The RWM geometry and the connective fiber orientation and dispersion are measured via confocal and 2-photon microscopy. A region of the RWM geometry is characterized as a hyperbolic paraboloid and another region as a tapered parabolic cylinder. Predominant fiber directions correlate well with directions of zero curvature in the hyperbolic paraboloid region. Overall fiber directions correlate well with position along a line approximately parallel to the central axis of the cochlea’s spiral.

Novel biodegradable Round Window Disks for inner ear delivery of dexamethasone

Drug delivery to the inner ear is an important and very challenging field. The cochlea is protected by several barriers that need to be overcome in the drug delivery process. Local drug delivery can avoid undesirable side effects arising from systemic drug delivery. We developed a biodegradable dexamethasone-loaded Round Window (RW) Disk based on poly(D,L-lactic-co-glycolic acid) (PLGA) for local drug therapy to the inner ear by RW membrane administration by a film-casting method. The optimal drying time was characterized by thermogravimetric analysis and differential scanning calorimetry. In addition, the mass and polymer degradation over time of drug release was measured in vitro showing a total mass loss of 70% after 3 weeks. Dexamethasone release was determined by a RW model setup using a polyethylene terephthalate membrane. We achieved a controlled release over 52 days. Ex vivo implantation of a RW Disk onto a guinea pig RW membrane indicated well-fitting properties of the drug delivery device leading to a close surface contact with the membrane and the successful proof of concept. The developed RW Disks could be new and promising drug delivery device to achieve effective local drug delivery to the inner ear for an extended time.

Novel 3D-printed hollow microneedles facilitate safe, reliable, and informative sampling of perilymph from guinea pigs

BackgroundInner ear diagnostics is limited by the inability to atraumatically obtain samples of inner ear fluid. The round window membrane (RWM) is an attractive portal for accessing perilymph samples as it has been shown to heal within one week after the introduction of microperforations. A 1 µL volume of perilymph is adequate for proteome analysis, yet the total volume of perilymph within the scala tympani of the guinea pig is limited to less than 5 µL. This study investigates the safety and reliability of a novel hollow microneedle device to aspirate perilymph samples adequate for proteomic analysis. MethodsThe guinea pig RWM was accessed via a postauricular surgical approach. 3D-printed hollow microneedles with an outer diameter of 100 µm and an inner diameter of 35 µm were used to perforate the RWM and aspirate 1 µL of perilymph. Two perilymph samples were analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics as part of a preliminary study. Hearing was assessed before and after aspiration using compound action potential (CAP) and distortion product otoacoustic emissions (DPOAE). RWMs were harvested 72 h after aspiration and evaluated for healing using confocal microscopy. ResultsThere was no permanent damage to hearing at 72 h after perforation as assessed by CAP (n = 7) and DPOAE (n = 8), and all perforations healed completely within 72 h (n = 8). In the two samples of perilymph analyzed, 620 proteins were detected, including the inner ear protein cochlin, widely recognized as a perilymph marker. ConclusionHollow microneedles can facilitate aspiration of perilymph across the RWM at a quality and volume adequate for proteomic analysis without causing permanent anatomic or physiologic dysfunction. Microneedles can mediate safe and effective intracochlear sampling and show great promise for inner ear diagnostics.

Drug delivery device for the inner ear: ultra-sharp fully metallic microneedles.

Drug delivery into the inner ear is a significant challenge due to its inaccessibility as a fluid-filled cavity within the temporal bone of the skull. The round window membrane (RWM) is the only delivery portal from the middle ear to the inner ear that does not require perforation of bone. Recent advances in microneedle fabrication enable the RWM to be perforated safely with polymeric microneedles as a means to enhance the rate of drug delivery from the middle ear to the inner ear. However, the polymeric material is not biocompatible and also lacks the strength of other materials. Herein we describe the design and development of gold-coated metallic microneedles suitable for RWM perforation. When developing microneedle technology for drug delivery, we considered three important general attributes: (1) high strength and ductility material, (2) high accuracy and precision of fabrication, and (3) broad design freedom. We developed a hybrid additive manufacturing method using two-photon lithography and electrochemical deposition to fabricate ultra-sharp gold-coated copper microneedles with these attributes. We refer to the microneedle fabrication methodology as two-photon templated electrodeposition (2PTE). We demonstrate the use of these microneedles by inducing a perforation with a minimal degree of trauma in a guinea pig RWM while the microneedle itself remains undamaged. Thus, this microneedle has the potential literally of opening the RWM for enhanced drug delivery into the inner ear. Finally, the 2PTE methodology can be applied to many different classes of microneedles for other drug delivery purposes as well the fabrication of small scale structures and devices for non-medical applications. Graphical Abstract Fully metallic ultra-sharp microneedle mounted at end of a 24-gauge stainless steel blunt syringe needle tip: (left) Size of microneedle shown relative to date stamp on U.S. one-cent coin; (right) Perforation through guinea pig round window membrane introduced with microneedle.

Anatomical and Functional Consequences of Microneedle Perforation of Round Window Membrane.

Microneedles can create microperforations in the round window membrane (RWM) without causing anatomic or physiologic damage. Reliable delivery of agents into the inner ear for therapeutic and diagnostic purposes remains a challenge. Our novel approach employs microneedles to facilitate intracochlear access via the RWM. This study investigates the anatomical and functional consequences of microneedle perforations in guinea pig RWMs in vivo. Single three-dimensional-printed, 100 μm diameter microneedles were used to perforate the guinea pig RWM via the postauricular sulcus. Hearing was assessed both before and after microneedle perforation using compound action potential and distortion product otoacoustic emissions. Confocal microscopy was used ex vivo to examine harvested RWMs, measuring the size, shape, and location of perforations and documenting healing at 0 hours (n = 7), 24 hours (n = 6), 48 hours (n = 6), and 1 week (n = 6). Microneedles create precise and accurate perforations measuring 93.1 ± 29.0 μm by 34.5 ± 16.8 μm and produce a high-frequency threshold shift that disappears after 24 hours. Examination of perforations over time demonstrates healing progression over 24 to 48 hours and complete perforation closure by 1 week. Microneedles can create a temporary microperforation in the RWM without causing significant anatomic or physiologic dysfunction. Microneedles have the potential to mediate safe and effective intracochlear access for diagnosis and treatment of inner ear disease.

3D-Printed Microneedles Create Precise Perforations in Human Round Window Membrane in Situ.

Three-dimensional (3D)-printed microneedles can create precise holes on the scale of micrometers in the human round window membrane (HRWM). An intact round window membrane is a barrier to delivery of therapeutic and diagnostic agents into the inner ear. Microperforation of the guinea pig round window membrane has been shown to overcome this barrier by enhancing diffusion 35-fold. In humans, the challenge is to design a microneedle that can precisely perforate the thicker HRWM without damage. Based on the thickness and mechanical properties of the HRWM, two microneedle designs were 3D-printed to perforate the HRWM from fresh frozen temporal bones in situ (n = 18 total perforations), simultaneously measuring force and displacement. Perforations were analyzed using confocal microscopy; microneedles were examined for deformity using scanning electron microscopy. HRWM thickness was determined to be 60.1 ± 14.6 (SD) μm. Microneedles separated the collagen fibers and created slit-shaped perforations with the major axis equal to the microneedle shaft diameter. Microneedles needed to be displaced only minimally after making initial contact with the RWM to create a complete perforation, thus avoiding damage to intracochlear structures. The microneedles were durable and intact after use. 3D-printed microneedles can create precise perforations in the HRWM without damaging intracochlear structures. As such, they have many potential applications ranging from aspiration of cochlear fluids using a lumenized needle for diagnosis and creating portals for therapeutic delivery into the inner ear.

In-vitro perforation of the round window membrane via direct 3-D printed microneedles.

The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to reliably and precisely deliver therapeutics into the cochlea to treat a plethora of auditory and vestibular disorders. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. A solution to this problem involves the introduction of one or more microscopic perforations through the RWM to enhance the rate and reliability of diffusive transport. This paper reports the use of direct 3D printing via Two-Photon Polymerization (2PP) lithography to fabricate ultra-sharp polymer microneedles specifically designed to perforate the RWM. The microneedle has tip radius of 500 nm and shank radius of 50 μ m, and perforates the guinea pig RWM with a mean force of 1.19 mN. The resulting perforations performed in vitro are lens-shaped with major axis equal to the microneedle shank diameter and minor axis about 25% of the major axis, with mean area 1670 μ m2. The major axis is aligned with the direction of the connective fibers within the RWM. The fibers were separated along their axes without ripping or tearing of the RWM suggesting the main failure mechanism to be fiber-to-fiber decohesion. The small perforation area along with fiber-to-fiber decohesion are promising indicators that the perforations would heal readily following in vivo experiments. These results establish a foundation for the use of Two-Photon Polymerization lithography as a means to fabricate microneedles to perforate the RWM and other similar membranes.

A dual wedge microneedle for sampling of perilymph solution via round window membrane.

Precision medicine for inner-ear disease is hampered by the absence of a methodology to sample inner-ear fluid atraumatically. The round window membrane (RWM) is an attractive portal for accessing cochlear fluids as it heals spontaneously. In this study, we report on the development of a microneedle for perilymph sampling that minimizes the size of RWM perforation, facilitates quick aspiration, and provides precise volume control. Here, considering the mechanical anisotropy of the RWM and hydrodynamics through a microneedle, a 31G stainless steel pipe was machined into wedge-shaped design via electrical discharge machining. The sharpness of the needle was evaluated via a surface profilometer. Guinea pig RWM was penetrated in vitro, and 1 μL of perilymph was sampled and analyzed via UV-vis spectroscopy. The prototype wedge shaped needle was successfully fabricated with the tip curvature of 4.5 μm and the surface roughness of 3.66 μm in root mean square. The needle created oval perforation with minor and major diameter of 143 and 344 μm (n = 6). The sampling duration and standard deviation of aspirated volume were 3 s and 6.8 % respectively. The protein concentration was 1.74 mg/mL. The prototype needle facilitated precise perforation of RWMs and rapid aspiration of cochlear fluid with precise volume control. The needle design is promising and requires testing in human cadaveric temporal bone and further optimization to become clinically viable.

A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration.

This study aimed to investigate the sustained drug release properties and hearing protection effect of polyethylene glycol-coated polylactic acid (PEG-PLA) stealth nanoparticles loaded with dexamethasone (DEX). DEX was fabricated into PEG-PLA nanoparticles using an emulsion and evaporation technique, as previously reported. The DEX-loaded PEG-PLA nanoparticles (DEX-NPs) had a hydrodynamic diameter of 130±4.78 nm, and a zeta potential of −26.13±3.28 mV. The in vitro release of DEX from DEX-NPs lasted 24 days in phosphate buffered saline (pH 7.4), 5 days in artificial perilymph (pH 7.4), and 1 day in rat plasma. Coumarin 6-labeled NPs placed onto the round window membrane (RWM) of guinea pigs penetrated RWM quickly and accumulated to the organs of Corti, stria vascularis, and spiral ganglion cells after 1 hour of administration. The DEX-NPs locally applied onto the RWM of guinea pigs by a single-dose administration continuously released DEX in 48 hours, which was significantly longer than the free DEX that was cleared out within 12 hours after administration at the same dose. Further functional studies showed that locally administrated single-dose DEX-NPs effectively preserved outer hair cells in guinea pigs after cisplatin insult and thus significantly attenuated hearing loss at 4 kHz and 8 kHz frequencies when compared to the control of free DEX formulation. Histological analyses indicated that the administration of DEX-NPs did not induce local inflammatory responses. Therefore, prolonged delivery of DEX by PEG-PLA nanoparticles through local RWM diffusion (administration) significantly protected the hair cells and auditory function in guinea pigs from cisplatin toxicity, as determined at both histological and functional levels, suggesting the potential therapeutic benefits in clinical applications.

Microperforations significantly enhance diffusion across round window membrane.

Introduction of microperforations in round window membrane (RWM) will allow reliable and predictable intracochlear delivery of pharmaceutical, molecular, or cellular therapeutic agents. Reliable delivery of medications into the inner ear remains a formidable challenge. The RWM is an attractive target for intracochlear delivery. However, simple diffusion across intact RWM is limited by what material can be delivered, size of material to be delivered, difficulty with precise dosing, timing, and precision of delivery over time. Further, absence of reliable methods for measuring diffusion across RWM in vitro is a significant experimental impediment. A novel model for measuring diffusion across guinea pig RWM, with and without microperforation, was developed and tested: cochleae, sparing the RWM, were embedded in 3D-printed acrylic holders using hybrid dental composite and light cured to adapt the round window niche to 3 ml Franz diffusion cells. Perforations were created with 12.5-μm-diameter needles and examined with light microscopy. Diffusion of 1 mM Rhodamine B across RWM in static diffusion cells was measured via fluorescence microscopy. The diffusion cell apparatus provided reliable and replicable measurements of diffusion across RWM. The permeability of Rhodamine B across intact RWM was 5.1 × 10(9-) m/s. Manual application of microperforation with a 12.5-μm-diameter tip produced an elliptical tear removing 0.22 ± 0.07% of the membrane and was associated with a 35× enhancement in diffusion (P < 0.05). Diffusion cells can be applied to the study of RWM permeability in vitro. Microperforation in RWM is an effective means of increasing diffusion across the RWM.

Novel flower-shaped albumin particles as controlled-release carriers for drugs to penetrate the round-window membrane.

Controlled-release carriers for local drug delivery have attracted increasing attention for inner-ear treatment recently. In this paper, flower-shaped bovine serum albumin (FBSA) particles were prepared by a modified desolvation method followed by glutaraldehyde or heat denaturation. The size of the FBSA particles varied from 10 μm to 100 μm, and most were 50–80 μm. Heat-denatured FBSA particles have good cytocompatibility with a prolonged survival time for L929 cells. The FBSA particles were utilized as carriers to investigate the release behaviors of the model drug – rhodamine B. Rhodamine B showed a sustained-release effect and penetrated the round-window membrane of guinea pigs. We also confirmed the attachment of FBSA particles onto the round-window membrane by microscopy. The FBSA particles, with good biocompatibility, drug-loading capacity, adhesive capability, and biodegradability, may have potential applications in the field of local drug delivery for inner-ear disease treatment.

Hyaluronic Acid Enhances Gene Delivery into the Cochlea

Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity

We previously demonstrated that an artificial protein, TAT-FNK, has antiapoptotic effects against cochlear hair cell (HC) damage caused by ototoxic agents when applied systemically. To examine the feasibility of topical protein therapy for inner ear disorders, we investigated whether gelatin sponge soaked with TAT-FNK and placed on the guinea pig round window membrane (RWM) could deliver the protein to the cochlea and attenuate aminoglycoside (AG)-induced cochlear damage in vivo. First, we found that the immunoreactivity of TAT-myc-FNK was distributed throughout the cochlea. The immunoreactivity was observed from 1-24 h after application. When Tat-FNK was applied 1 h before ototoxic insult (a combination of kanamycin sulfate and ethacrynic acid), auditory brainstem response threshold shifts and the extent of HC death were significantly attenuated. When cochlear organotypic cultures prepared from P5 rats were treated with kanamycin, TAT-FNK significantly reduced the extent of caspase-9 activation and HC death. These findings indicate that TAT-FNK topically applied on the RWM can enter the cochlea by diffusion and effectively prevent AG-induced apoptosis of cochlear HCs by suppressing the mitochondrial caspase-9 pathway.