Hyaluronic acid-ibuprofen conjugation: a novel ototherapeutic approach protecting inner ear cells from inflammation-mediated damage.

There is a substantial need of effective drugs for the treatment of hearing loss, which affects nearly 500 million individuals globally. Hearing loss can be the result of intense or prolonged noise exposure, ototoxic drugs, infections, and trauma, which trigger inflammatory signaling cascades that lead to irreversible damage to cochlear structures. To address this, we developed and characterized a series of covalent conjugates of anti-inflammatory drugs to hyaluronic acid (HA), for potential use as topical ototherapeutics. These conjugates were tested in in vitro assays designed to mirror physiological processes typically observed with acoustic trauma. Intense noise exposure leads to macrophage recruitment to the cochlea and subsequent inflammatory damage to sensory cells. We therefore first tested our conjugates' ability to reduce the release of inflammatory cytokines in macrophages. This anti-inflammatory effect on macrophages also translated to increased cochlear cell viability. In our initial screening, one conjugate, ibuprofen-HA, demonstrated significantly higher anti-inflammatory potential than its counterparts. Subsequent cytokine release profiling of ibuprofen-HA further confirmed its ability to reduce a wider range of inflammatory markers, to a greater extent than its equivalent unconjugated drug. The conjugate's potential as a topical therapeutic was then assessed in previously developed tympanic and round window membrane tissue permeation models. As expected, our data indicate that the conjugate has limited tympanic membrane model permeability; however, it readily permeated the round window membrane model and to a greater extent than the unconjugated drug. Interestingly, our data also revealed that ibuprofen-HA was well tolerated in cellular and tissue cytocompatibility assays, whereas the unconjugated drug displayed significant cytotoxicity at equivalent concentrations. Moreover, our data highlighted the importance of chemical conjugation of ibuprofen to HA; the conjugate had improved anti-inflammatory effects, significantly reduced cytotoxicity, and is more suitable for therapeutic formulation. Overall, this work suggests that ibuprofen-HA could be a promising safe and effective topical ototherapeutic for inflammation-mediated cochlear damage.

In vitro characterization of novel hyaluronan-antioxidant conjugates as potential topical therapeutics against hearing loss.

Noise-induced hearing loss affects roughly 430 million people worldwide. Current treatment options often require invasive medical procedures, and to date, there are no FDA-approved drug therapies. While the causes can be diverse, noise induced hearing loss is unequivocally associated with oxidative stress and inflammation, and subsequent damage to the inner ear structures. Several studies have shown that various antioxidants such as glutathione, cysteine, and methionine can be used to mitigate oxidative damage from reactive oxygen species; however, these studies relied on invasive or systemic drug delivery methods. This study focused on the development and characterization of a novel series of antioxidant compounds that would be suitable for non or minimally invasive topical inner ear delivery and could mitigate reactive oxygen species associated cellular damage. Specifically, a series of covalent conjugates were synthesized by using hyaluronan as a drug carrier, and methionine, cysteine or glutathione as antioxidant drugs. The conjugates were tested for their ability to readily permeate though in vitro round window membrane and tympanic membrane permeation models, as well as their in vitro internalization into cochlear cells. Our data revealed interdependence between the molecular weight of the hyaluronan carrier, and the tissue and cellular membrane permeation capacity. Subsequent screening of the adequately sized conjugates in in vitro acellular assays revealed the strongest antioxidant activity for the cysteine and glutathione conjugates. These oxidative stress protective effects were further confirmed in cellular in vitro assays. Collectively, the data herein showcase the potential value of these conjugates as therapeutics against oxidative-stress-mediated cellular damage specific to noise-induced hearing loss.

Colorimetric Properties of Bombyx mori Silk Fibroin.

Recent reports highlighted several novel applications for the Bombyx mori silk fibroin (SF), as edible coatings for the preservation of food freshness, smart labels, or packaging materials. This study complements these reports and additionally describes the colorimetric sensing properties of the natural protein that could be explored to enhance the practical value of such applications. Our data show that in response to pH changes, reconstituted SF is able to undergo visible color changes that correlate with the intensity of the stimuli, regardless of its physical format or physical cross-linking state. The intensity of the developed color was proportional to the extent of the protein's hydrolytic degradation. We also found that these pH-driven color changes were reversible and interchangeable, with colorless samples at neutral pH, purple in acidic environments, and yellow under basic conditions. Our mechanistic studies identified tryptophan as being responsible for these colorimetric responses, which could be further intensified by the presence of ionized tyrosine functionalities. In addition, we determined that SF's sensing properties also applied to ultraviolet light exposure. Finally, we showed that the innate sensing capabilities of activated SF can be enhanced via the covalent incorporation of additional tryptophan into the protein. Overall, our results further support the utility of SF for sensing applications.

Manufacturability of a Tetraethyl Orthosilicate-Based Hydrogel for Use as a Single Application Otitis Externa Therapeutic.

Otitis externa, also known as outer ear infection, is a frequent affliction in both humans and animals. The most prevalent treatment for otitis externa is ear drops, but it is difficult to adhere properly to this treatment, causing poor patient compliance and the potential for complications. As a result, we have developed a tetraethyl orthosilicate-based hydrogel for use as single application treatment for otitis externa to increase ease of use and improve patient outcomes. Herein, we investigated the manufacturability of the hydrogel, focusing on several key aspects: formulation repeatability and reproducibility, material source and tolerances, release of a variety of model drugs, and impact of application-specific physiological factors, specifically local pH and enzymatic activity on drug release. Overall, our results indicate that these hydrogels are well suited for production and scalability, as they have a robust manufacturing process, have a wide tolerance for pH level, release a variety of model drugs, and are not impacted by outer ear canal-specific physiological factors.

An Evaluation of the Drug Permeability Properties of Human Cadaveric In Situ Tympanic and Round Window Membranes.

It is estimated that hearing loss currently affects more than 1.5 billion people, or approximately 20% of the global population; however, presently, there are no Food and Drug Administration-approved therapeutics or prophylactics for this condition. While continued research on the development of otoprotective drugs to target this clear unmet need is an obvious path, there are numerous challenges to translating promising therapeutic candidates into human clinical testing. The screening of promising drug candidates relies exclusively on preclinical models. Current models do not permit the rapid high-throughput screening of promising drug candidates, and their relevance to clinical scenarios is often ambiguous. With the current study, we seek to understand the drug permeability properties of the cadaveric tympanic and round window membranes with the goal of generating knowledge that could inform the design and/or evaluation of in vitro organotypic models. The development of such models could enable the early high-throughput screening of topical therapeutic candidates and should address some of the limitations of currently used animal models.

Development and Characterization of an In Vitro Round Window Membrane Model for Drug Permeability Evaluations.

Hearing loss and balance disorders are highly common disorders, and the development of effective oto-therapeutics remains an area of intense research. Drug development and screening in the hearing research field heavily rely on the use of preclinical models with often ambiguous translational relevance. This often leads to failed advancement in the market of effective therapeutics. In this context, especially for inner ear-specific pathologies, the availability of an in vitro, physiologically relevant, round window membrane (RWM) model could enable rapid, high-throughput screening of potential topical drugs for inner ear and cochlear dysfunctions and could help accelerate the advancement to clinic and market of more viable drug candidates. In this study, we report the development and evaluation of an in vitro model that mimics the native RWM tissue morphology and microenvironment as shown via immunostaining and histological analyses. The developed three-dimensional (3D) in vitro model was additionally assessed for barrier integrity by transepithelial electrical resistance, and the permeability of lipophilic and hydrophilic drugs was determined. Our collective findings suggest that this in vitro model could serve as a tool for rapid development and screening of topically deliverable oto-therapeutics.

An In Vitro Model for Characterization of Drug Permeability across the Tympanic Membrane.

Otic disorders, such as otitis media and hearing loss, affect a substantial portion of the global population. Despite this, oto-therapeutics, in particular those intended to treat hearing loss, have seen limited development and innovation. A significant factor to this is likely a result of the inherent costs and complexities of drug discovery and development. With in vitro 3D tissue models seeing increased utility for the rapid, high-throughput screening of drug candidates, it stands to reason that the field of otology could greatly benefit from such innovations. In this study, we propose and describe an in vitro 3D model, designed using a physiologically based approach, which we suggest can be used to estimate drug permeability across human tympanic membranes (TM). We characterize the permeability properties of several template drugs in this model under various growth and storage conditions. The availability of such cost-effective, rapid, high-throughput screening tools should allow for increased innovation and the discovery of novel drug candidates over the currently used animal models. In the context of this TM permeation model, it may promote the development of topical drugs and formulations that can non-invasively traverse the TM and provide tissue-targeted drug delivery as an alternative to systemic treatment, an objective which has seen limited study until present.

Development and Characterization of a Topically Deliverable Prophylactic Against Oxidative Damage in Cochlear Cells.

Hearing loss affects roughly 466 million people worldwide. While the causes of hearing loss are diverse, mechanistically, inflammation and oxidative stress have been identified as major players in hearing loss regardless of pathogenesis. Treatment options remain extremely limited and there is currently no FDA approved drug therapy. Studies indicate that antioxidants such as d-Methionine have shown some protective effects; however, these studies involved systemic or invasive localized delivery methods and highlighted the need for the development of minimally invasive localized therapeutic approaches. Described herein is the development of an antioxidant-conjugated system that shows prophylactic potential against oxidative damage and appears suitable for topical delivery. Specifically, our covalent conjugate of hyaluronan with d-Methionine shows cytocompatibility and protection from oxidative stress in two mouse cochlear cell lines (HEI-OC1 and SV-k1). Mechanistically, the data indicate that the protective effects of the conjugate are due to the hyaluronan-mediated cellular internalization of the antioxidant. Most notably, the conjugate can efficiently permeate through an in vitro round window membrane model without the loss of the attached antioxidant, for subsequent delivery of the therapeutic cargo to the hearing sensory cells. Collectively these findings show that the novel conjugate could be a potential topical preventive agent against hearing loss.