Tinnitus—defined as the perception of sound in the absence of overt acoustic stimulation—is an enigmatic condition that challenges the treatment acumen and management resources of hearing professionals worldwide. The condition represents a theraputic conundrum for clinicians because there is no universal treatment and no known cure.
Establishing a cure or an effective treatment would be analogous to finding the Holy Grail. If discovered, a cure would have immediate impact and desirable consequences, such as permitting a sustained venture into a peaceful and quiet life, allowing the existence—or resumption—of a productive career, and most important, would contribute to improving an overall quality of life.
Of the many different treatment options available for tinnitus suppression (cognitive/behavioral and/or retraining therapies; acoustic, magnetic, and electrical neuromodulatory strategies; pharmacological interventions), we will focus on a pharmacological approach coupled with a unique drug-delivery platform. We followed this path because many pharmaceutical options available in the marketplace have a strong theoretical basis to be successful, with the main limitation being the lack of specificity in reaching targets within the brain at high enough concentrations to be efficacious. At the same time, there must be minimal or no adverse side effects on normal tissue or on cognition, and limited or no impact on other psychological, social, emotional, or biological processes that would negatively affect activities of daily living.
Approach and Collaborations
Herein, we provide the background and theorectical basis for a novel drug-delivery platform that combines nanotechnology, molecular biology, molecular imaging, and pharma as a way to help solve treatment dilemmas associated with tinnitus abatement.
The nanoscience collaboration is channeled through the State University of New York (SUNY) Polytechnic Institute Colleges of Nanoscale Science and Engineering (CNSE) in Albany. Dr. James Castracane, professor and head of the Nanobioscience Constellation, and colleagues Drs. Magnus Bergkvist and Stephanie Curley are the lead scientists and collaborators in this area. The SUNY Polytechnic Institute CNSE is the largest academic institution in the world dedicated to nanotechnology education, fundamental nanoscience research, nanoengineering, nanobioscience, and nanoeconomics and, therefore, is a logical partner in this endeavor. The state-of-the-art facilities, intellectual resources, and the ability to fabricate nanoparticle constructs facilitated the experimental protocols used herein (FIGURE 1).
Dr. Avril-Genene Holt of the Wayne State University Department of Anatomy and Cell Biology and health-research specialist at the John D. Dingell VA Medical Center in Detroit, Michigan, spearheaded the cellular and molecular biology component. (Figure 2). Post-doctoral fellow Dr. Aaron Apawu and other staff members of the Holt Lab contributed to this work. Drs. Cacace and Holt headed the neuroimaging and auditory neuroscience components.
Application of Nanotheranostics
The approach we describe falls under the rubric of nanotheranostics, which represents the combination of diagnostic detection with the goal of targeted drug delivery for therapeutic intent (Funkhouser, 2002; Caldorera-Moore et al, 2001). In theory, this approach enables identification and localization of pathologic site(s) of dysregulation subserving the tinnitus perception and then releases a payload of drugs at the target of interest to abate or silence this condition.
As the prefix “nano” in the term nanotheranostics implies, nanoparticles (NPs) will be used as one of several essential elements in this paradigm. Nanoparticles can take many different forms. In this context, NPs represent very small biochemical objects, typically between 1 and 100 nanometers (nm’s; 10–12 meters) in size, where each NP is surrounded by an external layer that consists of ions, inorganic or organic molecules that enable each NP to behave as a whole unit with respect to its transport and properties in the medium where it is released.
Theranostic approaches in general, and nanotheranostic platforms in particular, take advantage of incorporating several distinct capabilities into one system. Specifically, NPs are fabricated to be multifunctional carriers that are both necessary and sufficient for a drug-delivery platform to succeed. Success is contingent upon the NPs being: (1) biocompatible with the system under consideration (human body and brain); (2) the need to have colloidal stability in aqueous and biological fluids; and (3) the requisite to have minimal or no toxicity to normal tissue.
The driving force underlying this methodology centers on the fact that the exterior surface of these NPs can be functionalized. This means that NPs can be decorated (conjugated) with ligands1 for receptor-targeting in brain tissue. They also can be encapsulated with a contrast agent like manganese or gadolinium, such that when NPs enter the brain, those regions generating or subserving the dysregulated physiology (neuronal hyperactivity/synchrony) can be localized using magnetic resonance imaging (MRI). In addition, their hollow inner core can be loaded with a pharmacological agent so that a payload of drugs can be delivered to a region of interest (ROI) subserving the tinnitus-related neuronal hyperactivity. Thus, linking nanotechnology, molecular biology, molecular imaging, and pharma is an attractive strategy needed to treat multifaceted disorders. This approach is particularly well suited for treating tinnitus, since abatement of this condition is not likely to be a one-size-fits- all scenario.
Put in perspective, the functionalization of NPs is key to meeting the needs of a particular disease state or medical condition. Indeed, it is analogous to the magic bullet concept suggested by Nobel laureate Paul Erlich more than a century ago as a treatment modality for cancer (for a review, see Strebhardt and Ullrich, 2008). At the heart of Erlich’s approach is the notion that specific chemotherapeutic drugs could target cancerous tissue, modify their biological activity to suppress the disease but, at the same time, remain harmless to healthy nonpathologic tissues in the body. We now extend the logic of the magic bullet concept to the treatment of tinnitus using a systems neuroscience approach.
Each segment allocated to this approach is described below, based on accounts, technical details, and experimental data provided by Cacace et al (2018) and Apawu et al (2018), albeit in a more abbreviated manner.
The Nanoparticle (NP) Carrier
Emerging as a premier drug-delivery vehicle, the well-studied MS2 bacteriophage (Valegard et al, 1990; Kovacs et al, 2007; Toropova et al, 2008) is an icosahedral virus (a capsid) that is ~28 nm’s in diameter. A capsid is the protein shell of a virus classified according to its shape and structure. The icosahedral shape approximates a sphere comprising 20 equilateral triangular facades. A unique feature of NP capsids and a central theme of this approach relates to the fact that they can be engineered to be emptied, virus particles having a central hollow core that can carry drugs. From a technical/manufacturing standpoint, the MS2 bacteriophage is straightforward to produce and purify. Moreover, this NP construct remains structurally stable over a broad range of pH values (4.5 to 12.0) and temperatures up to 68 degrees Centigrade (Stonehouse and Stockley, 1993).
Functionalization of NPs
Targeting the Blood-Brain Barrier (BBB)
To be effective, one of the fundamental elements of this approach is the need to get drugs or other large molecules into the central nervous system (CNS) and brain from the general circulation. The BBB, however, represents a formidable road block in this endeavor. In fact, it operates as a dual-edged sword. On the one hand, the BBB prevents neurotoxins and other foreign molecules from entering the CNS, but on the other hand, this protective barrier limits/hinders relatively few diagnostic and drug treatment options necessary to alleviate the burden of those suffering from brain disorders (Partridge, 2005). And there’s the rub.
Briefly, the BBB is composed of ependymal and endothelial cells that line the ventricles and vasculature of the brain. These cells form tight junctions that represent a protective mechanism, analogous to a molecular sieve that prevents the transport of foreign neurotoxic molecules from entering the CNS by passing through the intercellular space between cells. In fact, the BBB prevents approximately 100 percent of large (>400 Da)2 and 98 percent of small molecules (<400 Da) from entering the brain (Partridge, 2005).
One promising strategy to circumvent the BBB takes advantage of endothelial cell receptors at the blood-brain interface (Hersh et al, 2016). Specifically, contemporary research has focused on receptor-mediated transcytosis (RMT), the process by which cells bind
and internalize ligands from the surrounding environment, transport the ligands through the cytosol and exocytose the ligands on the opposite side of the BBB. While receptors for transferrin, insulin, and lipoprotein have all been used as targets, because each element is highly expressed on the endothelial cells of the BBB (Jones and Shusta, 2007), recent work has concentrated on targeting the low-density lipoprotein receptor-related protein 1 (LRP1) (Hertz and Bock, 2002; Demeula et al, 2008a; b; Xin et al, 2011; Bell et al, 2007).
The LRP1 receptor is noteworthy because it has more than 30 distinct ligands (Lillis et al, 2005). One such ligand is the synthetic peptide, angiopep-2 (AP2) (Demeule et al, 2008a; b). Angiopep-2 has a higher transcytosis efficiency than drugs and endogenous ligands, i.e., seven-fold for aprotinin (Jones and Shusta, 2007; Demeule et al, 2008a; b) and 70-fold for transferrin (Demeule et al, 2008a, b). Indeed, the use of AP2 to direct synthetic NP carriers transporting drugs or imaging agents across the BBB has been highly successful (Xin et al, 2011; Huang et al, 2011; Xin et al, 2012a; Xin et al, 2012b; Ren et al, 2012). These AP2-conjugated NPs have been shown to be capable of transport through in vitro BBB models, documented to work in vivo in the rat brain, and have been successful in targeting glioma cells in brain tissue (Demeule et al, 2008b; Ren et al, 2012). Thus, ligands that target endothelial cells are used to decorate the exterior surface of the NP and provide one unique strategy by which NPs can cross the BBB.
Gene Expression Approaches Associated with NP Functionalization and Tinnitus Localization
Another important element specific to our approach is based on gene-expression studies and their distinctive contribution to the dual-targeting delivery system. First described more than a decade ago (Holt, 2006), this approach is based on uncovering specific “over-expressed” genes that might serve as biomarker proxies for tinnitus-related neuronal hyperactivity. Initiated by work on noise-induced tinnitus, Holt and colleagues (2016) have shown that the N-methyl-D-aspartate receptor subunit 2D (NMDAR-2D) is over expressed in nuclei of the dorsal cochlear nucleus and inferior colliculus in rats with behavioral evidence of tinnitus. Therefore, this ionotropic, glutamate-receptor ion-channel protein (NMDA-2D) represents a desirable target for therapeutic intervention (Holt et al, 2016). While the utility of this discovery is very important, we do not wish to imply that other targets or mechanisms are not valuable, only that this is a good starting point to capitalize our current mitigation efforts. For example, in salicylate-induced tinnitus, experimental studies have shown that the N-methyl-D-aspartate receptor subunit 2B (NMDAR-2B) and other biomarkers are over-expressed (Hwang et al, 2011; 2013; Hu et al, 2014; 2016) and they, too, could represent signatures of spontaneous neural hyperactivity and be positioned as NP-ligand decorations. Thus, different tinnitus-induction methods (noise- or drug-based) imply different receptor-targeting mechanisms for a personalized approach to tinnitus abatement.
In order to put gene-expression studies in context, consider the notion that genes are not just passive strands of deoxyribonucleic acid (DNA), but represent tiny chemical manufacturing plants that are controlled by a dynamic set of chemical messengers that travel within and between cells to regulate function or, when things go awry, produce dysfunction/dysregulation, as in the development of tinnitus. Thus, as this work implies, cellular-signaling processes at the molecular level are both helpful and informative as a way to guide treatment.
Nanoparticle Encapsulation with Contrast Agents
By encapsulating NPs with known contrast agents such as gadolinium or manganese, abnormal brain physiology, pathoanatomy, and associated neural pathway dysfunction can be identified and tracked with magnetic resonance imaging (MRI), providing important localizing information for researchers to advance their models and theories and drive intervention strategies forward. The idea for using manganese is based on mounting evidence that this essential metal is an activity-dependent paramagnetic contrast agent. It has already been used in experimental paradigms to study tinnitus (Brozoski et al, 2007; Holt et al, 2010) and has contributed to our understanding of many other types of auditory and non-auditory phenomena (for a review, see Cacace et al, 2014).
Loading the NP with Pharmaceuticals
In the unique NP platform we developed, the hollow inner core of the capsid NP can be loaded with a pharmaceutical agent that can be released in an ROI with the intent of attenuating or neutralizing a prominent pathophysiological substrate subserving the tinnitus percept. With these features in mind, a novel drug-delivery system is born!
An illustration of this entire functionalization, encapsulation, and drug-loading process is shown in FIGURE 3.
Tinnitus, whether induced by noise or a drug such as salicylate, can result in pathologic neuronal hyperactivity across multiple brain regions, ultimately resulting in an over-expression of proteins. These over-produced proteins can be used as tools to guide delivery for NP targets, either to specific brain areas or neurons.
Once identified, specific ligands for an over-produced protein can be conjugated to the exterior surface of the NP.
Many of the regions resulting in altered spontaneous neuronal hyperactivity following the onset of tinnitus are located within the brain, which allows only restricted access via the blood-brain barrier (BBB).
To allow passage into the brain, the exterior surface of the NPs can be decorated with a ligand that would bind to receptors in the blood vessel wall and allow transcytosis across the endothelium into the brain.
Because the NP is encapsulated with an MRI contrast agent and the residual hollow cargo area was filled with a pharmacological agent, the NP could be tracked within the brain and potentially deliver a cargo of drugs to specific brain regions in quantities that could alleviate the symptoms of tinnitus.
There is always a concern that introducing a foreign substance into the body may produce adverse consequences and potentially accumulate in unintended body areas and organ systems. Therefore, the need to address the topic of toxicity and/or reactive changes such as an immune response cannot be ignored.
In the in vivo rat model described by Apawu et al (2018), after venous injection of NPs, toxicity was examined in brain tissue and in major organ systems. Using hematoxylin and eosin stained sections of liver, heart, spleen, and kidney tissue, systemically administered doses of fluorescent-labeled MS2-AP2 NPs (1:10 and 1:40), no deleterious effects on body organs were found. After two weeks, histological assessment of organs other than the brain did not show any NP accumulation. Furthermore, administration of the fluorescent-labeled MS2-AP2 NPs via the tail vein did not alter behavior, appearance, or weight of the experimental animals, even after 12 days, regardless of dose.
In summary, by applying nanotechnology and molecular biologic formulations, we show that it is possible to integrate diagnostic and therapeutic functions under the umbrella of nanotheranostics. Our initial work has shown that MS2 capsids are excellent drug-delivery vehicles capable of self-assembly, forming physically stable structures, and being easily modified with functional moieties. Based upon their small size and the ability to transcytose the BBB, this approach is extremely attractive and particularly well suited for targeting tinnitus-related neuronal hyperactivity.
An Essential Step in Scientific Evolutions
This nanotheranostic approach is a fundamental paradigm shift from currently available methods. It serves as a new component that can be added to the treatment armament of clinicians and complements other factors included within the “Roadmap to a Cure,” a strategic initiative developed by the American Tinnitus Association to inform researchers and other stakeholders about important areas needing further experimental attention in tinnitus research. The concept of nanotheranostics extends important features found within the roadmap with the ultimate vision of discovering a cure for tinnitus.
Integrating diagnostic and therapeutic functions under the rubric of nanotheranostics represents a powerful and attractive paradigm that is well suited for treating tinnitus. What distinguishes this approach from other techniques is the fact that treatment can be individualized—an approach thought by some to be the future standard of care in medicine. A personalized approach would enable clinicians to tailor specific treatments to the biomarkers expressed, either from a single individual and/or from a group of individuals with similar histories (Jotterand and Alexander, 2011).
It is important to emphasize that the unique training and credentialing aspects of assessing hearing function in humans and in providing extensive rehabilitative guidance is the domain of clinical audiology. It has been argued that the audiologist is the likely translational interface between the basic-science laboratory and the clinic, as this individual supervises and directs applications of new technologies, techniques, and methods that ultimately will lead to successful treatment regimens of all types (Cacace, 2016).
This work was supported by a grant from NIH NIDCD R21 DC013895-02 to MB. We are also grateful to Dr. Sumit Dhar for his encouragement and guidance throughout the editorial process.
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