Arrigali EM, Veit JGS, Birru B, Van Tine J, Sandau KC, Barrett-Catton E, Tonnerre Z and Serban MA (2024) In vitro characterization of novel hyaluronan-antioxidant conjugates as potential topical therapeutics against hearing loss. Front. Pharmacol. 15:1355279.

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.

Birru B, Veit JGS, Arrigali EM, Van Tine J, Barrett-Catton E, Tonnerre Z, Diaz P and Serban MA (2024) Hyaluronic acid-ibuprofen conjugation: a novel ototherapeutic approach protecting inner ear cells from inflammation-mediated damage. Front. Pharmacol. 15:1355283.

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.

Sydor MJ, Serban MA. The Application of Fluorescence Anisotropy for Viscosity Measurements of Small Volume Biological Analytes. J. Exp. Theor. Anal. 2023, 1 (2), 86–96.

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 µL and 100 µL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze–thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements’ accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems.

Sandau KC, Arrigali EM, Serban BA, Serban MA. Colorimetric Properties of Bombyx mori Silk Fibroin. ACS Biomater. Sci. Eng. 2023, acsbiomaterials.3c00794.

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.

Veit JGS, Weidow M, Serban MA. A versatile, bioengineered skin reconstruction device designed for use in austere environments. Frontiers in Bioengineering and Biotechnology. 2023; 11, 1-14. https://doi.org/10.3389/fbioe.2023.1208322

Austere environments in which access to medical facilities, medical personnel, or even water and electricity is limited or unavailable pose unique challenges for medical device product design. Currently existing skin substitutes are severely inadequate for the treatment of severe burns, chronic wounds, battlefield injuries, or work-related injuries in resource-limited settings. For such settings, an ideal device should be biocompatible, bioresorbable, promote tissue healing, not require trained medical personnel for deployment and use, and should enable topical drug delivery. As proof of concept for such a device, silk fibroin and an antioxidant hyaluronic acid derivative were chosen as primary constituents. The final formulation was selected to optimize tensile strength while retaining mechanical compliance and protection from reactive oxygen species (ROS). The ultimate tensile strength of the device was 438.0 KPa. Viability of dermal fibroblasts challenged with ROS-generating menadione decreased to 49.7% of control, which was rescued by pre-treatment with the hyaluronic acid derivative to 85.0% of control. The final device formulation was also tested in a standardized, validated, in vitro skin irritation test which revealed no tissue damage or statistical difference from control. Improved topical drug delivery was achieved via an integrated silk fibroin microneedle array and selective device processing to generate crosslinked/through pores. The final device including these features showed a 223% increase in small molecule epidermal permeation relative to the control. Scaffold porosity and microneedle integrity before and after application were confirmed by electron microscopy. Next, the device was designed to be self-adherent to enable deployment without the need of traditional fixation methods. Device tissue adhesive strength (12.0 MPa) was evaluated and shown to be comparable to a commercial adhesive surgical drape (12.9 MPa) and superior to an over-the-counter liquid bandage (4.1 MPa). Finally, the device’s wound healing potential was assessed in an in vitro full-thickness skin wound model which showed promising device integration into the tissue and cellular migration into and above the device. Overall, these results suggest that this prototype, specifically designed for use in austere environments, is mechanically robust, is cytocompatible, protects from ROS damage, is self-adherent without traditional fixation methods, and promotes tissue repair.

Barrett-Catton E, Arrigali EM, Serban BA, Sandau KC, Serban MA. Manufacturability of a Tetraethyl Orthosilicate-Based Hydrogel for Use as a Single Application Otitis Externa Therapeutic. Pharmaceutics. 2022; 14(10):2020. https://doi.org/10.3390/pharmaceutics14102020

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.

Veit JGS, Birru B, Singh R, Arrigali EM, Serban MA. An In Vitro Model for Characterization of Drug Permeability across the Tympanic Membrane. Pharmaceuticals. 2022; 15(9), 1114. https://doi.org/10.3390/ph15091114

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.

Singh R, Birru B, Veit JGS, Arrigali EM, Serban MA. Development and Characterization of an In Vitro Round Window Membrane Model for Drug Permeability Evaluations. Pharmaceuticals. 2022; 15(9), 1105. https://doi.org/10.3390/ph15091105

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.

Veit JGS, Birru B, Wang Y, Singh R, Arrigali EM, Park R, Miller B, Firpo MA, Park AH, Serban MA. An Evaluation of the Drug Permeability Properties of Human Cadaveric In Situ Tympanic and Round Window Membranes. Pharmaceuticals 2022, 15(9), 1037. https://doi.org/10.3390/ph15091037

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.

Arrigali EM, Serban MA. Development and Characterization of a Topically Deliverable Prophylactic Against Oxidative Damage in Cochlear Cells. Front. Pharmacol. 2022, 13(6), 1-14. https://doi.org/10.3389/fphar.2022.907516

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.

Wang Z, Serban BA, Serban MA. Recombinant Silk Fibroin Crystalline Regions as Biomaterial Alternatives to the Full-Length Protein. ACS Biomater. Sci. Eng. 2020, doi: 10.1021/acsbiomaterials.0c01103

Silk fibroin is a natural polymer with a unique repetitive structure that translates to extraordinary properties in terms of processability and mechanical properties. The Bombyx mori silk has a molecular weight of ∼415 kDa and consists of a light chain and a heavy chain. Its heavy chain is organized into 12 crystalline domains. Each of these crystalline domains contains subdomains of ∼70 amino acid containing blocks. It is well understood that the heavy chain of the protein is responsible for its processing versatility and excellent mechanical properties; however, the need for the high number of monomeric repeating units is unclear, and the individual properties of crystalline regions compared to those of the full-length protein are not understood. The work described herein assessed the possibility of using recombinant crystalline regions as alternative biomaterials for applications such as tissue adhesives. Our results indicate that while the two tested substructures do not fully recapitulate the native silk fibroin’s properties, they appear to be a suitable alternative for the production of silk-based medical adhesives.

Serban BA, Barrett-Catton E, Serban MA. Tetraethyl Orthosilicate-Based Hydrogels for Drug Delivery—Effects of Their Nanoparticulate Structure on Release Properties. Gels. 2020; 6(4):38, doi: 10.3390/gels6040038

Tetraethyl orthosilicate (TEOS)-based hydrogels, with shear stress response and drug releasing properties, can be formulated simply by TEOS hydrolysis followed by volume corrections with aqueous solvents and pH adjustments. Such basic thixotropic hydrogels (thixogels) form via the colloidal aggregation of nanoparticulate silica. Herein, we investigated the effects of the nanoparticulate building blocks on the drug release properties of these materials. Our data indicate that the age of the hydrolyzed TEOS used for the formulation impacts the nanoparticulate structure and stiffness of thixogels. Moreover, the mechanism of formation or the disturbance of the nanoparticulate network significantly affects the release profiles of the incorporated drug. Collectively, our results underline the versatility of these basic, TEOS-only hydrogels for drug delivery applications.

Serban BA, Shi K, Alverson JB, Hoody J, Priestley ND, Park AH, Serban MA. A Single Application Cold-Chain Independent Drug Delivery System for Outer Ear Infections. ACS Biomater. Sci. Eng. 2020, 6(10), 5969-5978, doi: 10.1021/acsbiomaterials.0c01223

Outer ear infections (OEs) affect millions of people annually with significant associated healthcare costs. Incorrect administration or noncompliance with the treatment regimen can lead to infection persistence, recurrence, antibiotic resistance, and in severe cases, aggravation to malignant otitis externa. Such issues are particularly pertinent for military personnel, patients in nursing homes, the geriatric population, patients with head or hand tremors, and those with limited or no access to proper healthcare. With the intent of using traditional material science principles to deconvolute material design while increasing relevance and efficacy, we developed a single application, cold-chain independent thixotropic drug delivery system. This can be easily applied into the ear as a liquid and then gels to deliver effective concentrations of antibiotics against bacterial strains commonly associated with OEs. The system maintains thixotropic properties over several stress/no stress cycles, shows negligible swelling and temperature dependence, and does not impact the minimum inhibitory concentration or bactericidal effects of relevant antibiotics. Moreover, thixogels are biocompatible and are well tolerated in the ear. This drug delivery system can readily translate into a user-friendly product, could improve compliance via a single application by the diagnosing healthcare provider, and is expected to effectively treat OEs and minimize the development of antibiotic resistance, infection recurrence, or exacerbation.

Serban MA, Skardal A. Hyaluronan chemistries for three-dimensional matrix applications. Matrix Biol. 2019, May;78-79:337-345. doi: 10.1016/j.matbio.2018.02.010. Epub 2018 Feb 10. PMID: 29438729.

Hyaluronan is a ubiquitous constituent of mammalian extracellular matrices and, because of its excellent intrinsic biocompatibility and chemical modification versatility, has been widely employed in a multitude of biomedical applications. In this article, we will survey the approaches used to tailor hyaluronan to specific needs of tissue engineering, regenerative and reconstructive medicine and overall biomedical research. We will also describe recent examples of applications in these broader areas, such as 3D cell culture, bioprinting, organoid biofabrication, and precision medicine that are facilitated by the use of hyaluronan as a biomaterial.

Johnston ER, Miyagi Y, Chuah JA, Numata K, Serban MA. Interplay between Silk Fibroin’s Structure and Its Adhesive Properties. ACS Biomater. Sci. Eng. 2018, 4(8), 2815-2824, doi: 10.1021/acsbiomaterials.8b00544

Bombyx mori-derived silk fibroin (SF) is a well-characterized protein employed in numerous biomedical applications. Structurally, SF consists of a heavy chain (HC) and a light chain (LC), connected via a single disulfide bond. The HC sequence is organized into 12 crystalline domains interspersed with amorphous regions that can transition between random coil/alpha helix and beta-sheet configurations, giving silk its hallmark properties. SF has been reported to have adhesive properties and shows promise for development of medical adhesives; however, the mechanism of these interactions and the interplay between SF's structure and adhesion is not understood. In this context, the effects of physical parameters (i.e., concentration, temperature, pH, ionic strength) and protein structural changes on adhesion were investigated in this study. Our results suggest that amino acid side chains that have functionalities capable of coordinate (dative) bond or hydrogen bond formation (such as those of serine and tyrosine), might be important determinants in SF's adhesion to a given substrate. Additionally, the data suggest that fibroin amino acids involved in beta-sheet formation are also important in the protein's adhesion to substrates.

Serban BA, Stipe KT, Alverson JB, Johnston ER, Priestley ND, and Serban MA. A Controlled Antibiotic Release System for the Development of Single-Application Otitis Externa Therapeutics. Gels 2017, 3(2), 19 - Featured article in Special issue 'Hydrogels for Drug Delivery', doi: 10.3390/gels3020019

Ear infections are a commonly-occurring problem that can affect people of all ages. Treatment of these pathologies usually includes the administration of topical or systemic antibiotics, depending on the location of the infection. In this context, we sought to address the feasibility of a single-application slow-releasing therapeutic formulation of an antibiotic for the treatment of otitis externa. Thixotropic hydrogels, which are gels under static conditions but liquefy when shaken, were tested for their ability to act as drug controlled release systems and inhibit Pseudomonas aeruginosa and Staphylococcus aureus, the predominant bacterial strains associated with outer ear infections. Our overall proof of concept, including in vitro evaluations reflective of therapeutic ease of administration, formulation stability, cytocompatibility assessment, antibacterial efficacy, and formulation lifespan, indicate that these thixotropic materials have strong potential for development as otic treatment products.

Johnston ER, Smith TN, and Serban MA. Nanoparticulate Poly(glucaramide)-Based Hydrogels for Controlled Release Applications. Gels 2017, 3(2), 17; doi:10.3390/gels3020017

In 2004, D-Glucaric acid (GA) was identified as one of the top value-added chemicals from renewable feedstocks. For this study, a patented synthetic method was used to obtain gel forming polymers through the polycondensation of GA and several aliphatic diamines. The first time characterization and a potential practical application of such hydrogels is reported herein. Our findings indicate that the physical properties and gelling abilities of these materials correlate with the chemical structure of the precursor diamines used for polymerization. The hydrogels appear to have nanoparticulate nature, form via aggregation, are thermoresponsive, and appear suitable as controlled release systems for small molecules. Overall, this study further highlights the versatility of GA as a building block for the synthesis of sustainable materials, with potential for a wide array of applications.

Serban MA. Translational biomaterials-the journey from the bench to the market-think 'product'. Curr Opin Biotechnol. 2016, 40:31-34. doi: 10.1016/j.copbio.2016.02.009

Biomaterial research has been gaining popularity over the past decades and, in parallel, the number of biomaterial-based therapies has been increasing. There is still however, a disconnection between the number of developed biomaterials and those that make it to the market. In this context, a review of the biomaterial development trends in both industry and academia, specifically of biomaterials that were successfully translated from bench side to the clinic and the market, is warranted.