Engineering Biocoatings To Prolong Drug Release from Supraparticles.

Supraparticles (SPs) assembled from smaller colloidal nanoparticles can serve as depots of therapeutic compounds and are of interest for long-term, sustained drug release in biomedical applications. However, a key challenge to achieving temporal control of drug release from SPs is the occurrence of an initial rapid release of the loaded drug (i.e., "burst" release) that limits sustained release and potentially causes burst release-associated drug toxicity. Herein, a biocoating strategy is presented for silica-SPs (Si-SPs) to reduce the extent of burst release of the loaded model protein lysozyme. Specifically, Si-SPs were coated with a fibrin film, formed by enzymatic conversion of fibrinogen into fibrin. The fibrin-coated Si-SPs, FSi-SPs, which could be loaded with 7.9 ± 0.9 µg of lysozyme per SP, released >60% of cargo protein over a considerably longer period of time of >20 days when compared with the uncoated Si-SPs that released the same amount of the cargo protein, however, within the first 3 days. Neurotrophins that support the survival and differentiation of neurons could also be loaded at ∼7.3 µg per SP, with fibrin coating also delaying neurotrophin release (only 10% of cargo released over 21 days compared with 60% from Si-SPs). In addition, the effects of incorporating a hydrogel-based system for surgical delivery and the opportunity to control drug release kinetics were investigated-an alginate-based hydrogel scaffold was used to encapsulate FSi-SPs. The introduction of the hydrogel further extended the initial release of the encapsulated lysozyme to ∼40 days (for the same amount of cargo released). The results demonstrate the increasing versatility of the SP drug delivery platform, combining large loading capacity with sustained drug release, that is tailorable using different modes of controlled delivery approaches.

Fraxicon for Optical Applications with Aperture ∼1 mm: Characterisation Study.

Emerging applications of optical technologies are driving the development of miniaturised light sources, which in turn require the fabrication of matching micro-optical elements with sub-1 mm cross-sections and high optical quality. This is particularly challenging for spatially constrained biomedical applications where reduced dimensionality is required, such as endoscopy, optogenetics, or optical implants. Planarisation of a lens by the Fresnel lens approach was adapted for a conical lens (axicon) and was made by direct femtosecond 780 nm/100 fs laser writing in the SZ2080™ polymer with a photo-initiator. Optical characterisation of the positive and negative fraxicons is presented. Numerical modelling of fraxicon optical performance under illumination by incoherent and spatially extended light sources is compared with the ideal case of plane-wave illumination. Considering the potential for rapid replication in soft polymers and resists, this approach holds great promise for the most demanding technological applications.

Developing the supraparticle technology for round window-mediated drug administration into the cochlea.

The semi-permeable round window membrane (RWM) is the gateway to the cochlea. Although the RWM is considered a minimally invasive and clinically accepted route for localised drug delivery to the cochlea, overcoming this barrier is challenging, hindering development of effective therapies for hearing loss. Neurotrophin 3 (NT3) is an emerging treatment option for hearing loss, but its therapeutic effect relies on sustained delivery across the RWM into the cochlea. Silica supraparticles (SPs) are drug delivery carriers capable of providing long-term NT3 delivery, when injected directly into the guinea pig cochlea. However, for clinical translation, a RWM delivery approach is desirable. Here, we aimed to test approaches to improve the longevity and biodistribution of NT3 inside the cochlea after RWM implantation of SPs in guinea pigs and cats. Three approaches were tested (i) coating the SPs to slow drug release (ii) improving the retention of SPs on the RWM using a clinically approved gel formulation and (iii) permeabilising the RWM with hyaluronic acid. A radioactive tracer (iodine 125: 125I) tagged to NT3 (125I NT3) was loaded into the SPs to characterise drug pharmacokinetics in vitro and in vivo. The neurotrophin-loaded SPs were coated using a chitosan and alginate layer-by-layer coating strategy, named as '(Chi/Alg)SPs', to promote long term drug release. The guinea pigs were implanted with 5× 125I NT3 loaded (Chi/Alg) SPs on the RWM, while cats were implanted with 30× (Chi/Alg) SPs. A cohort of animals were also implanted with SPs (controls). We found that the NT3 loaded (Chi/Alg)SPs exhibited a more linear release profile compared to NT3 loaded SPs alone. The 125I NT3 loaded (Chi/Alg)SPs in fibrin sealant had efficient drug loading (~5 µg of NT3 loaded per SP that weights ~50 µg) and elution capacities (~49% over one month) in vitro. Compared to the SPs in fibrin sealant, the (Chi/Alg)SPs in fibrin sealant had a significantly slower 125I NT3 drug release profile over the first 7 days in vitro (~12% for (Chi/Alg) SPs in fibrin sealant vs ~43% for SPs in fibrin sealant). One-month post-implantation of (Chi/Alg) SPs, gamma count measurements revealed an average of 0.3 µg NT3 remained in the guinea pig cochlea, while for the cat, 1.3 µg remained. Histological analysis of cochlear tissue revealed presence of a 125I NT3 signal localised in the basilar membrane of the lower basal turn in some cochleae after 4 weeks in guinea pigs and 8 weeks in cats. Comparatively, and in contrast to the in vitro release data, implantation of the SPs presented better NT3 retention and distribution inside the cochlea in both the guinea pigs and cats. No significant difference in drug entry was observed upon acute treatment of the RWM with hyaluronic acid. Collectively, our findings indicate that SPs and (Chi/Alg)SPs can facilitate drug transfer across the RWM, with detectable levels inside the cat cochlea even after 8 weeks with the intracochlear approach. This is the first study to examine neurotrophin pharmacokinetics in the cochlea for such an extended period of times in these two animal species. Whilst promising, we note that outcomes between animals were variable, and opposing results were found between in vitro and in vivo release studies. These findings have important clinical ramifications, emphasising the need to understand the physical properties and mechanics of this complex barrier in parallel with the development of therapies for hearing loss.

Auditory nerve responses to combined optogenetic and electrical stimulation in chronically deaf mice.

Objective. Optogenetic stimulation of the auditory nerve offers the ability to overcome the limitations of cochlear implants through spatially precise stimulation, but cannot achieve the temporal precision nor temporal fidelity required for good hearing outcomes. Auditory midbrain recordings have indicated a combined (hybrid) stimulation approach may permit improvements in the temporal precision without sacrificing spatial precision by facilitating electrical activation thresholds. However, previous research has been conducted in undeafened or acutely deafened animal models, and the impact of chronic deafness remains unclear. Our study aims to compare the temporal precision of auditory nerve responses to optogenetic, electrical, and combined stimulation in acutely and chronically deafened animals.Methods. We directly compare the temporal fidelity (measured as percentage of elicited responses) and precision (i.e. stability of response size and timing) of electrical, optogenetic, and hybrid stimulation (varying sub-threshold or supra-threshold optogenetic power levels combined with electrical stimuli) through compound action potential and single-unit recordings of the auditory nerve in transgenic mice expressing the opsin ChR2-H134R in auditory neurons. Recordings were conducted immediately or 2-3 weeks following aminoglycoside deafening when there was evidence of auditory nerve degeneration.Main results. Results showed that responses to electrical stimulation had significantly greater temporal precision than optogenetic stimulation (p< 0.001 for measures of response size and timing). This temporal precision could be maintained with hybrid stimulation, but only when the optogenetic stimulation power used was below or near activation threshold and worsened with increasing optical power. Chronically deafened mice showed poorer facilitation of electrical activation thresholds with concurrent optogenetic stimulation than acutely deafened mice. Additionally, responses in chronically deafened mice showed poorer temporal fidelity, but improved temporal precision to optogenetic and hybrid stimulation compared to acutely deafened mice.Significance. These findings show that the improvement to temporal fidelity and temporal precision provided by a hybrid stimulation paradigm can also be achieved in chronically deafened animals, albeit at higher levels of concurrent optogenetic stimulation levels.

Timing is Everything: Stochastic Optogenetic Stimulation Reduces Adaptation in Retinal Ganglion Cells.

Optogenetics gives us unprecedented power to investigate brain connectivity. The ability to activate neural circuits with single cell resolution and its ease of application has provided a wealth of knowledge in brain function. More recently, optogenetics has shown tremendous utility in prosthetics applications, including vision restoration for patients with retinitis pigmentosa. One of the disadvantages of optogenetics, however, is its poor temporal bandwidth, i.e. the cell's inability to fire at a rate that matches the optical stimulation rate at high frequencies (>30 Hz). This research proposes a new strategy to overcome the temporal limits of optogenetic stimulation. Using whole-cell current clamp recordings in mouse retinal ganglion cells expressing channelrhodopsin-2 (H134R variant), we observed that randomizing inter-pulse intervals can significantly increase a retinal ganglion cell's temporal response to high frequency stimulation.Clinical Relevance- A significant disadvantage of optogenetic stimulation is its poor temporal dynamics which prohibit its widespread use in retinal prosthetics. We have shown that randomizing the interval between stimulation pulses reduces adaptation in retinal ganglion cells. This stimulation strategy may contribute to new levels of functional restoration in therapeutics which incorporate optogenetics.

Spread of activation and interaction between channels with multi-channel optogenetic stimulation in the mouse cochlea.

For individuals with severe to profound hearing loss resulting from irreversibly damaged hair cells, cochlear implants can be used to restore hearing by delivering electrical stimulation directly to the spiral ganglion neurons. However, current spread lowers the spatial resolution of neural activation. Since light can be easily confined, optogenetics is a technique that has the potential to improve the precision of neural activation, whereby visible light is used to stimulate neurons that are modified with light-sensitive opsins. This study compares the spread of neural activity across the inferior colliculus of the auditory midbrain during electrical and optical stimulation in the cochlea of acutely deafened mice with opsin-modified spiral ganglion neurons (H134R variant of the channelrhodopsin-2). Monopolar electrical stimulation was delivered via each of four 0.2 mm wide platinum electrode rings at 0.6 mm centre-to-centre spacing, whereas 453 nm wavelength light was delivered via each of five 0.22 × 0.27 mm micro-light emitting diodes (LEDs) at 0.52 mm centre-to-centre spacing. Channel interactions were also quantified by threshold changes during simultaneous stimulation by pairs of electrodes or micro-LEDs at different distances between the electrodes (0.6, 1.2 and 1.8 mm) or micro-LEDs (0.52, 1.04, 1.56 and 2.08 mm). The spread of activation resulting from single channel optical stimulation was approximately half that of monopolar electrical stimulation as measured at two levels of discrimination above threshold (p<0.001), whereas there was no significant difference between optical stimulation in opsin-modified deafened mice and pure tone acoustic stimulation in normal-hearing mice. During simultaneous micro-LED stimulation, there were minimal channel interactions for all micro-LED spacings tested. For neighbouring micro-LEDs/electrodes, the relative influence on threshold was 13-fold less for optical stimulation compared electrical stimulation (p<0.05). The outcomes of this study show that the higher spatial precision of optogenetic stimulation results in reduced channel interaction compared to electrical stimulation, which could increase the number of independent channels in a cochlear implant. Increased spatial resolution and the ability to activate more than one channel simultaneously could lead to better speech perception in cochlear implant recipients.

Combined optogenetic and electrical stimulation of the sciatic nerve for selective control of sensory fibers.

Introduction: Electrical stimulation offers a drug-free alternative for the treatment of many neurological conditions, such as chronic pain. However, it is not easy to selectively activate afferent or efferent fibers of mixed nerves, nor their functional subtypes. Optogenetics overcomes these issues by controlling activity selectively in genetically modified fibers, however the reliability of responses to light are poor compared to electrical stimulation and the high intensities of light required present considerable translational challenges. In this study we employed a combined protocol of optical and electrical stimulation to the sciatic nerve in an optogenetic mouse model to allow for better selectivity, efficiency, and safety to overcome fundamental limitations of electrical-only and optical-only stimulation. Methods: The sciatic nerve was surgically exposed in anesthetized mice (n = 12) expressing the ChR2-H134R opsin via the parvalbumin promoter. A custom-made peripheral nerve cuff electrode and a 452 nm laser-coupled optical fiber were used to elicit neural activity utilizing optical-only, electrical-only, or combined stimulation. Activation thresholds for the individual and combined responses were measured. Results: Optically evoked responses had a conduction velocity of 34.3 m/s, consistent with ChR2-H134R expression in proprioceptive and low-threshold mechanoreceptor (Aα/Aß) fibers which was also confirmed via immunohistochemical methods. Combined stimulation, utilizing a 1 ms near-threshold light pulse followed by an electrical pulse 0.5 ms later, approximately halved the electrical threshold for activation (p = 0.006, n = 5) and resulted in a 5.5 dB increase in the Aα/Aß hybrid response amplitude compared to the electrical-only response at equivalent electrical levels (p = 0.003, n = 6). As a result, there was a 3.25 dB increase in the therapeutic stimulation window between the Aα/Aß fiber and myogenic thresholds (p = 0.008, n = 4). Discussion: The results demonstrate that light can be used to prime the optogenetically modified neural population to reside near threshold, thereby selectively reducing the electrical threshold for neural activation in these fibers. This reduces the amount of light needed for activation for increased safety and reduces potential off-target effects by only stimulating the fibers of interest. Since Aα/Aß fibers are potential targets for neuromodulation in chronic pain conditions, these findings could be used to develop effective strategies to selectively manipulate pain transmission pathways in the periphery.

Pharmacokinetics and biodistribution of supraparticle-delivered neurotrophin 3 in the guinea pig cochlea.

Hearing loss is the most prevalent sensory disorder affecting nearly half a billion people worldwide. Aside from devices to assist hearing, such as hearing aids and cochlear implants, a drug treatment for hearing loss has yet to be developed. The neurotrophin family of growth factors has long been established as a potential therapy, however delivery of these factors into the inner ear at therapeutic levels over a sustained period of time has remained a challenge restricting clinical translation. We previously demonstrated that direct delivery of exogenous neurotrophin-3 (NT3) in the guinea pig cochleae via a bolus injection was rapidly cleared from the inner ear, with almost complete elimination 3 days post-treatment. Here, we explored the potential of suprapaticles (SPs) for NT3 delivery to the inner ear to achieve sustained delivery over time. SPs are porous spheroid structures comprised of smaller colloidal silica nanoparticles that provide a platform for long-term controlled release of therapeutics. This study aimed to assess the pharmacokinetics and biodistribution of SP-delivered NT3. We used a radioactive tracer (iodine 125: 125I) to label the NT3 to determine the loading, retention and distribution of NT3 delivered via SPs. Gamma measurements taken from 125I NT3 loaded SPs revealed high drug loading (an average of 5.3 µg of NT3 loaded per SP weighing 50 µg) and elution capacities in vitro (67% cumulative release over one month). Whole cochlear gamma measurements from SP-implanted cochleae harvested at various time points revealed detection of 125I NT3 in the guinea pig cochlea after one month, with 3.6 and 10% of the loaded drug remaining in the intracochlear and round window-implanted cochleae respectively. Autoradiography analysis of cochlear micro-sections revealed widespread 125I NT3 distribution after intracochlear SP delivery, but more restricted distribution with the round window delivery approach. Collectively, drug delivery into the inner ear using SPs support sustained, long-term availability and release of neurotrophins in the inner ear.

Effects of localized neurotrophin gene expression on spiral ganglion neuron resprouting in the deafened cochlea.

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

Viral-mediated transduction of auditory neurons with opsins for optical and hybrid activation.

Optical stimulation is a paradigm-shifting approach to modulating neural activity that has the potential to overcome the issue of current spread that occurs with electrical stimulation by providing focused stimuli. But optical stimulation either requires high power infrared light or genetic modification of neurons to make them responsive to lower power visible light. This work examines optical activation of auditory neurons following optogenetic modification via AAV injection in two species (mouse and guinea pig). An Anc80 viral vector was used to express the channelrhodopsin variant ChR2-H134R fused to a fluorescent reporter gene under the control of the human synapsin-1 promoter. The AAV was administered directly to the cochlea (n = 33) or posterior semi-circular canal of C57BL/6 mice (n = 4) or to guinea pig cochleae (n = 6). Light (488 nm), electrical stimuli or the combination of these (hybrid stimulation) was delivered to the cochlea via a laser-coupled optical fibre and co-located platinum wire. Activation thresholds, spread of activation and stimulus interactions were obtained from multi-unit recordings from the central nucleus of the inferior colliculus of injected mice, as well as ChR2-H134R transgenic mice (n = 4). Expression of ChR2-H134R was examined by histology. In the mouse, transduction of auditory neurons by the Anc80 viral vector was most successful when injected at a neonatal age with up to 89% of neurons transduced. Auditory neuron transductions were not successful in guinea pigs. Inferior colliculus responses to optical stimuli were detected in a cochleotopic manner in all mice with ChR2-H134R expression. There was a significant correlation between lower activation thresholds in mice and higher proportions of transduced neurons. There was no difference in spread of activation between optical stimulation and electrical stimulation provided by the light/electrical delivery system used here (optical fibre with bonded 25 µm platinum/iridium wire). Hybrid stimulation, comprised of sub-threshold optical stimulation to 'prime' or raise the excitability of the neurons, lowered the threshold for electrical activation in most cases, but the impact on excitation width was more variable compared to transgenic mice. This study demonstrates the impact of opsin expression levels and expression pattern on optical and hybrid stimulation when considering optical or hybrid stimulation techniques for neuromodulation.

A radiolabeled drug tracing method to study neurotrophin-3 retention and distribution in the cochlea after nano-based local delivery.

Hearing loss is the most common sensory deficit worldwide with no approved therapeutics for treatment. Local neurotrophin delivery into the cochlea has shown great potential in protecting and repairing the sensory cells important for hearing. However, delivery of these factors into the inner ear at therapeutic levels over a sustained period of time has remained a challenge restricting clinical translation. We have developed a method to test the pharmacokinetics of neurotrophin released from porous silica particles called 'supraparticles' that can provide sustained release of neurotrophins to the inner ear.•This report describes a radiolabeling method to examine neurotrophin retention and distribution in the cochlea. The neurotrophin was labeled with a radioactive tracer (iodine 125: 125I) and delivered into the cochlea via the supraparticle system.•Gamma counts reveal drug levels and clearance in the intact cochlea, as well as accumulation in off-target organs (safety test). Autoradiography analyses using film and emulsion permit quantification and visualization of drug distribution at the cellular level. The method has a detection limit of 0.8 pg of radiolabeled neurotrophin-3 in cochlear sections exposed to film.•The tracer 125I with a half-life of 59.4 days can be used to label other drugs/substances with a tyrosine residue and therefore be broadly applicable for long-term pharmacokinetic studies in other systems.

Hybrid optogenetic and electrical stimulation for greater spatial resolution and temporal fidelity of cochlear activation.

OBJECTIVE: Compared to electrical stimulation, optogenetic stimulation has the potential to improve the spatial precision of neural activation in neuroprostheses, but it requires intense light and has relatively poor temporal kinetics. We tested the effect of hybrid stimulation, which is the combination of subthreshold optical and electrical stimuli, on spectral and temporal fidelity in the cochlea by recording multiunit activity in the inferior colliculus of channelrhodopsin (H134R variant) transgenic mice. APPROACH: Pulsed light or biphasic electrical pulses were delivered to cochlear spiral ganglion neurons of acutely deafened mice, either as individual stimuli or as hybrid stimuli for which the timing of the electrical pulse had a varied delay relative to the start of the optical pulse. Response thresholds, spread of activation and entrainment data were obtained from multi-unit recordings from the auditory midbrain. MAIN RESULTS: Facilitation occurred when subthreshold electrical stimuli were applied at the end of, or up to 3.75 ms after subthreshold optical pulses. The spread of activation resulting from hybrid stimulation was significantly narrower than electrical-only and optical-only stimulation (p < 0.01), measured at equivalent suprathreshold levels of loudness that are relevant to cochlear implant users. Furthermore, temporal fidelity, measured as maximum following rates to 300 ms pulse trains bursts up to 240 Hz, was 2.4-fold greater than optical-only stimulation (p < 0.05). SIGNIFICANCE: By significantly improving spectral resolution of electrical- and optical-only stimulation and the temporal fidelity of optical-only stimulation, hybrid stimulation has the potential to increase the number of perceptually independent stimulating channels in a cochlear implant.

Combined optogenetic and electrical stimulation of auditory neurons increases effective stimulation frequency-an in vitro study.

OBJECTIVE: The performance of neuroprostheses, including cochlear and retinal implants, is currently constrained by the spatial resolution of electrical stimulation. Optogenetics has improved the spatial control of neurons in vivo but lacks the fast-temporal dynamics required for auditory and retinal signalling. The objective of this study is to demonstrate that combining optical and electrical stimulation in vitro could address some of the limitations associated with each of the stimulus modes when used independently. APPROACH: The response of murine auditory neurons expressing ChR2-H134 to combined optical and electrical stimulation was characterised using whole cell patch clamp electrophysiology. MAIN RESULTS: Optogenetic costimulation produces a three-fold increase in peak firing rate compared to optical stimulation alone and allows spikes to be evoked by combined subthreshold optical and electrical inputs. Subthreshold optical depolarisation also facilitated spiking in auditory neurons for periods of up to 30 ms without evidence of wide-scale Na+ inactivation. SIGNIFICANCE: These findings may contribute to the development of spatially and temporally selective optogenetic-based neuroprosthetics and complement recent developments in 'fast opsins'.

Targeted therapy of the inner ear.

BACKGROUND: There is experimental evidence that targeted delivery of steroids to the inner ear can protect hearing during cochlear implant surgery. The best protection appears to be achieved through pre-treatment of the cochlea, but the time period required for treatment is long compared with the duration of surgery, and needs further optimization. The stability of hearing thresholds is determined over a 3-month period after hearing preservation cochlear implantation. METHODS: Adult guinea pigs were implanted with a miniature cochlear implant electrode, and pure tone auditory brainstem response (ABR) thresholds were estimated in response to pure tones of 2-32 kHz immediately after surgery and at 1 week, 1 month and 3 months. Spiral ganglion cell (SGC) densities were estimated from mid-modular histological sections of the cochlea. Thirty minutes prior to implantation, a polymeric sponge (Seprapack, Genzyme) was loaded with either a 2% solution of dexamethasone phosphate or normal saline (control) and placed onto the round window. RESULTS: Implantation was associated with an immediate elevation in thresholds across frequencies, with a full recovery below 2 kHz over the next week and a partial recovery of thresholds at higher frequencies. These thresholds remained unchanged for the next 3 months. There was an immediate and sustained reduction in the elevation of thresholds at 32 kHz in dexamethasone-treated animals. SGC densities were greater in steroid-treated animals than controls in the basal turn of the cochlea (at the region of implantation) 3 months after implantation. CONCLUSION: It is concluded that ABR thresholds remain stable for 3 months after cochlear implantation in the guinea pig, and that local application of steroids to the inner ear prior to implantation is an effective method of preserving SGC populations when there is residual hearing at the time of implantation.

New molecular therapies for the treatment of hearing loss.

An estimated 466 million people suffer from hearing loss worldwide. Sensorineural hearing loss is characterized by degeneration of key structures of the sensory pathway in the cochlea such as the sensory hair cells, the primary auditory neurons and their synaptic connection to the hair cells - the ribbon synapse. Various strategies to protect or regenerate these sensory cells and structures are the subject of intensive research. Yet despite recent advances in our understandings of the capacity of the cochlea for repair and regeneration there are currently no pharmacological or biological interventions for hearing loss. Current research focusses on localized cochlear drug, gene and cell-based therapies. One of the more promising drug-based therapies is based on neurotrophic factors for the repair of the ribbon synapse after noise exposure, as well as preventing loss of primary auditory neurons and regrowth of the auditory neuron fibers after severe hearing loss. Drug therapy delivery technologies are being employed to address the specific needs of neurotrophin and other therapies for hearing loss that include the need for high doses, long-term delivery, localised or cell-specific targeting and techniques for their safe and efficacious delivery to the cochlea. Novel biomaterials are enabling high payloads of drugs to be administered to the cochlea with subsequent slow-release properties that are proving to be beneficial for treating hearing loss. In parallel, new gene therapy technologies are addressing the need for cell specificity and high efficacy for the treatment of both genetic and acquired hearing loss with promising reports of hearing recovery. Some biomaterials and cell therapies are being used in conjunction with the cochlear implant ensuring therapeutic benefit to the primary neurons during electrical stimulation. This review will introduce the auditory system, hearing loss and the potential for repair and regeneration in the cochlea. Drug delivery to the cochlea will then be reviewed, with a focus on new biomaterials, gene therapy technologies, cell therapy and the use of the cochlear implant as a vehicle for drug delivery. With the current pre-clinical research effort into therapies for hearing loss, including clinical trials for gene therapy, the future for the treatment for hearing loss is looking bright.

Pharmacokinetics and tissue distribution of neurotrophin 3 after intracochlear delivery.

Neurotrophin therapy has potential to reverse some forms of hearing loss. However, cochlear pharmacokinetic studies are challenging due to small fluid volumes. Here a radioactive tracer was used to determine neurotrophin-3 retention, distribution and clearance after intracochlear administration. 125I-neurotrophin-3 was injected into guinea pig cochleae using a sealed injection technique comparing dosing volumes, rates and concentrations up to 750 µg/mL. Retention was measured by whole-cochlear gamma counts at five time points while distribution and clearance were assessed by autoradiography. Smaller injection volumes and higher concentrations correlated with higher retention of neurotrophin-3. Distribution of neurotrophin-3 was widespread throughout the cochlear tissue, decreasing in concentration from base to apex. Tissue distribution was non-uniform, with greatest density in cells lining the scala tympani and lower density in neural target tissue. The time constant for clearance of neurotrophin-3 from cochlear tissues was 38 h but neurotrophin-3 remained detectable for at least 2 weeks. Neurotrophin-3 was evident in the semi-circular canals with minor spread to the contralateral cochlea. This study is the first comprehensive evaluation of the disposition profile for a protein therapy in the cochlea. The findings and methods in this study will provide valuable guidance for the development of protein therapies for hearing loss.

Novel drug delivery systems for inner ear protection and regeneration after hearing loss.

BACKGROUND: A cochlear implant, the only current treatment for restoring auditory perception after severe or profound sensorineural hearing loss (SNHL), works by electrically stimulating spiral ganglion neurons (SGNs). However, gradual degeneration of SGNs associated with SNHL can compromise the efficacy of the device. OBJECTIVE: To review novel drug delivery systems for preserving and/or regenerating sensory cells in the cochlea after SNHL. METHODS: The effectiveness of traditional cochlear drug delivery systems is compared to newer techniques such as cell, polymer and gene transfer technologies. Special requirements for local drug delivery to the cochlea are discussed, such as protecting residual hearing and site-specific drug delivery for cell preservation and regeneration. RESULTS/CONCLUSIONS: Drug delivery systems with the potential for immediate clinical translation, as well as those that will contribute to the future of hearing preservation or cochlear cellular regeneration, are identified.

Effects of round window dexamethasone on residual hearing in a Guinea pig model of cochlear implantation.

UNLABELLED: To study electric acoustic stimulation, we have developed a model of guinea pig cochlear implantation via a cochleostomy. Thirty minutes prior to implantation, a hyaluronic acid/carboxymethylcellulose bead, loaded with either dexamethasone or normal saline, was placed upon the round window membrane. Animals that did not receive beads acted as controls. Pure-tone auditory brainstem response thresholds were estimated before and after electrode insertion, and 1 and 4 weeks later. Selected cochlear histology was performed. RESULTS: Dexamethasone could be detected in the cochlea for 24 h after cochlear implantation. Thresholds were elevated across frequencies in all animals immediately after surgery. These thresholds recovered completely at and below 2 kHz, and partially at higher frequencies by 1 week after implantation. At 32 kHz, but not the lower frequencies, the presence of dexamethasone had a significant protective effect upon hearing, which increased in magnitude over time. The protection was greatest in difficult implantations where an intractable resistance to electrode insertion was met. There was a persistent foreign body reaction at the site of implantation of saline-treated implanted ears but not in the dexamethasone-treated implanted ears. CONCLUSION: Short-term preoperative delivery of dexamethasone through the round window can protect residual hearing during cochlear implantation, especially during technically difficult surgery.

Gel-Mediated Electrospray Assembly of Silica Supraparticles for Sustained Drug Delivery.

Supraparticles (SPs) composed of smaller colloidal particles provide a platform for the long-term, controlled release of therapeutics in biomedical applications. However, current synthesis methods used to achieve high drug loading and those involving biocompatible materials are often tedious and low throughput, thereby limiting the translation of SPs to diverse applications. Herein, we present a simple, effective, and automatable alginate-mediated electrospray technique for the assembly of robust spherical silica SPs (Si-SPs) for long-term (>4 months) drug delivery. The Si-SPs are composed of either porous or nonporous primary Si particles within a decomposable alginate matrix. The size and shape of the Si-SPs can be tailored by controlling the concentrations of alginate and silica primary particles used and key electrospraying parameters, such as flow rate, voltage, and collector distance. Furthermore, the performance (including drug loading kinetics, loading capacity, loading efficiency, and drug release) of the Si-SPs can be tuned by changing the porosity of the primary particles and through the retention or removal (via calcination) of the alginate matrix. The structure and morphology of the Si-SPs were characterized by electron microscopy, dynamic light scattering, N2 adsorption-desorption analysis, and X-ray photoelectron spectroscopy. The cytotoxicity and degradability of the Si-SPs were also examined. Drug loading kinetics and loading capacity for six different types of Si-SPs, using a model protein drug (fluorescently labeled lysozyme), demonstrate that Si-SPs prepared from primary silica particles with large pores can load significant amounts of lysozyme (∼10 µg per SP) and exhibit sustained, long-term release of more than 150 days. Our experiments show that Si-SPs can be produced through a gel-mediated electrospray technique that is robust and automatable (important for clinical translation and commercialization) and that they present a promising platform for long-term drug delivery.

The effect of polypyrrole with incorporated neurotrophin-3 on the promotion of neurite outgrowth from auditory neurons.

This research aims to improve the nerve-electrode interface of the cochlear implant using polymer technology to encourage neuron survival, elongation and adhesion to the electrodes. Polypyrrole (Ppy) doped with p-toluene sulphonate (pTS) is an electroactive polymer into which neurotrophin-3 (NT3) can be incorporated. Ppy/pTS+/-NT3 was synthesised over gold electrodes and used as a surface for auditory neuron explant culture. Neurite outgrowth from explants grown on Ppy/pTS was equivalent to tissue culture plastic but improved with the incorporation of NT3 (Ppy/pTS/NT3). Electrical stimulation of Ppy/pTS/NT3 with a biphasic current pulse, as used in cochlear implants, significantly improved neurite outgrowth from explants. Using (125)I-NT3, it was shown that low levels of NT3 passively diffused from Ppy/pTS/NT3 during normal incubation and that electrical stimulation enhanced the release of biologically active NT3 in quantities adequate for neuron survival. Furthermore, Ppy/pTS/NT3 and its constituents were not toxic to auditory neurons and the Ppy/pTS/NT3 coating on gold electrodes did not alter impedance. If applied to the cochlear implant, Ppy/pTS/NT3 will provide a biocompatible, low-impedance substrate for storage and release of NT3 to help protect auditory neurons from degradation after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.