Fine control of drug delivery for cochlear implant ...

Hearing, Balance and Communication

ISSN: 2169-5717 (Print) 2169-5725 (Online) Journal homepage: http://www.tandfonline.com/loi/ihbc20

Fine control of drug delivery for cochlear implant applications Alexandra Homsy, Edith Laux, Julien Brossard, Harry J. Whitlow, Marta Roccio, Stefan Hahnewald, Pascal Senn, Pavel Mistrík, Roland Hessler, Teresa Melchionna, Claudia Frick, Hubert Löwenheim, Marcus Müller, Ute Wank, Karl-Heinz Wiesmüller & Herbert Keppner To cite this article: Alexandra Homsy, Edith Laux, Julien Brossard, Harry J. Whitlow, Marta Roccio, Stefan Hahnewald, Pascal Senn, Pavel Mistrík, Roland Hessler, Teresa Melchionna, Claudia Frick, Hubert Löwenheim, Marcus Müller, Ute Wank, Karl-Heinz Wiesmüller & Herbert Keppner (2015) Fine control of drug delivery for cochlear implant applications, Hearing, Balance and Communication, 13:4, 153-159, DOI: 10.3109/21695717.2015.1048082 To link to this article: http://dx.doi.org/10.3109/21695717.2015.1048082

Published online: 29 May 2015.

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Hearing, Balance and Communication, 2015; 13: 153–159

ORIGINAL ARTICLE

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Fine control of drug delivery for cochlear implant applications Alexandra Homsy1, Edith Laux1, Julien Brossard1, Harry J. Whitlow1, Marta Roccio2, Stefan Hahnewald2, Pascal Senn2,3, Pavel MistrÍk4, Roland Hessler4, Teresa Melchionna4, Claudia Frick5, Hubert LÖwenheim5,6, Marcus MÜller5,6, Ute Wank7, Karl-Heinz WiesMÜller7 & Herbert Keppner1, 1Haute

Ecole Arc Ingénierie, HES-SO/ University of Applied Sciences Western Switzerland, La Chaux-de-Fonds, Switzerland, Ear Research Laboratory, University Departments of Clinical Research and Otorhinolaryngology, Head & Neck Surgery, Inselspital, University of Bern, 3University Department of Otorhinolaryngology, Head & Neck Surgery, HUG, Geneva, Switzerland 4MED-EL, Fürstenweg, Innsbruck, Austria, 5Tübingen Hearing Research Centre, Department of Otolaryngology, Head and Neck Surgery, Eberhard Karls University Tübingen, Tübingen, Germany, 6Department of Otolaryngology Head and Neck Surgery, Carl von Ossietzky University Oldenburg, Oldenburg, Germany, and 7EMC microcollections GmbH Sindelfinger, Tuebingen, Germany 2Inner

Abstract Cochlear implants are neuroprostheses that are inserted into the inner ear to directly electrically stimulate the auditory nerve, thus replacing lost cochlear receptors, the hair cells. The reduction of the gap between electrodes and nerve cells will contribute to technological solutions simultaneously increasing the frequency resolution, the sound quality and the amplification of the signal. Recent findings indicate that neurotrophins (NTs) such as brain derived neurotrophic factor (BDNF) stimulate the neurite outgrowth of auditory nerve cells by activating Trk receptors on the cellular surface (1–3). Furthermore, small-size TrkB receptor agonists such as di-hydroxyflavone (DHF) are now available, which activate the TrkB receptor with similar efficiency as BDNF, but are much more stable (4). Experimentally, such molecules are currently used to attract nerve cells towards, for example, the electrodes of cochlear implants. This paper analyses the scenarios of low dose aspects of controlled release of small-size Trk receptor agonists from the coated CI electrode array into the inner ear. The control must first ensure a sufficient dose for the onset of neurite growth. Secondly, a gradient in concentration needs to be maintained to allow directive growth of neurites through the perilymph-filled gap towards the electrodes of the implant. We used fluorescein as a test molecule for its molecular size similarity to DHF and investigated two different transport mechanisms of drug dispensing, which both have the potential to fulfil controlled low-throughput drug-deliverable requirements. The first is based on the release of aqueous fluorescein into water through well-defined 60-mm size holes arrays in a membrane by pure osmosis. The release was both simulated using the software COMSOL and observed experimentally. In the second approach, solid fluorescein crystals were encapsulated in a thin layer of parylene (PPX), hence creating random nanometer-sized pinholes. In this approach, the release occurred due to subsequent water diffusion through the pinholes, dissolution of the fluorescein and then release by out-diffusion. Surprisingly, the release rate of solid fluorescein through the nanoscopic scale holes was found to be in the same order of magnitude as for liquid fluorescein release through microscopic holes.

Key words: cochlear implants, growth factors, diffusion constants, drug delivery systems

Introduction Controlled release studies of specific TrkB receptor agonists such as DHF at low but controlled throughput have to respect strict boundary conditions in the proximity of the dispenser. These boundary

conditions are features such as miscibility of the drug in the surrounding medium liquid, pressure difference across a membrane, and the critical distance to the target. Figure 1a sketches the elementary effects that will occur in a membrane based dispenser

Correspondence: Pavel Mistrik, MED-EL, Fürstenweg, Innsbruck, Austria E-mail:[email protected] (Accepted 1 May 2015) ISSN 2169-5717 print/ISSN 2169-5725 online © 2015 Informa Healthcare DOI: 10.3109/21695717.2015.1048082


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(osmosis based diffusion) and that have to be controlled; Figure 1b indicates the mechanisms that dominate a diffusion-controlled dispensing system when a dry compound is used (solid base drug delivery). In order to induce neuronal sprouting of spiral ganglion neurons towards the implant, inserted within the perilymph filled space of scala tympani, as a first step the threshold of the onset of neuronal outgrowth must be overcome. Our experiments with explant cultures of postnatal mice cochlear spiral ganglion showed increased neurite outgrowth at 100 nM and 1 mM. A value of 300 nM was reported to promote SGN survival in such a culture system by others (4). In the cochlea, neuronal outgrowth may be achieved by gradual increase of the concentration of an active compound in the perilymph-filled gap. As soon as the neurites start growing, a gradient of compound concentration must be established in order to guide the growth of neurites towards the target (electrodes of the implant in the cochlea). Furthermore, the quantity of active compound in the dispenser must be sufficient to maintain the gradient long enough to establish the neuron-electrode junction. The dispensing source for the drug is considered to be the integral area of all openings in the membrane between the well and the considered volume (Figure 1a). This can be either a large number of small

Figure 2. Experimental set-up for the determination of compound flow rate and concentration distribution in case of diffusion through micromiter-sized pores. a ) Sketch of the experimental flow-cell. b) Micrograph of a femto-second laser micromachined hole array in a polymer sheet (polyolephin acrylate) of 50 mm thickness.

pores or a few large pores. Diffusion is considered to be isotropic; hence compound molecules proceed laterally and longitudinally as they cross the gap. Looking at osmosis based diffusion for drug delivery one can distinguish three cases: •• Case 1. The release of active compound (d [active compound]/ dt) is too high: the limited volume of the gap will be filled immediately; passing over the threshold concentration for neurite-growth will be guaranteed; however, the gradient needed for directional growth across the gap will not be maintained.

Figure 1. Schematic representation of controlled release of therapeutics from a dispenser incorporated at the CI electrode array. (a) Sketch for the drug-delivery scenario from a liquidsource well (under the porous membrane) into the upper volume; adjacent neuron cells are located at the other side of the gap. The mechanism is called osmosis based diffusion. (b) Solid phase drug delivery whereby the dry compound in encapsulated by parylene (PPX); hereby the drug release is due to first in-diffusion of H2O; dissolving of compound in H2O; out-diffusion of compound  H2O and diffusion of compound in the gap.

Figure 3. Fluorescein as a test molecule used for the experiments instead of small-molecule TrkB receptor agonists. (a) Chemical formula of fluorescein, molecular weight 332,32 g·M-1 (b) Dihydroxyflavone (DHF), TrkB receptor agonists, with a molecular weight of 238.24 g·M-1.

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Figure 4. Effect of pin-hole density control as a function of the thickness of the PPX layer. There is a relationship expected between the surface topology (size of compound crystals), the PPX thickness, and GF release rate.

•• Case 2. The release of active compound is too small; the diffusion into the gap medium is faster than the active compound supply. In this case the threshold for onset of growth will not be achieved and the gradient does not develop. •• Case 3. The threshold concentration can be achieved and the gradient can be maintained for three days, a typical time-span of an in vitro experiment with spiral ganglion explants. In the case of solid phase drug delivery systems, solid drugs, encapsulated under a polymer commonly used for packaging (thin PPX polymer film (5)), are considered. Such arrangement allows liquid penetration and dissolution of the solid drug (Figure 1b).

The solid drug being now dissolved leaves its reservoir via the same path opened by the liquid when it crossed the encapsulating film. In this case it must be assumed that the porosity is much smaller and should give rise to a much reduced delivery rate compared to the first liquid drug delivery scenario where the pores have diameters in the range of tens of mm.

Experimental considerations Osmosis based drug delivery First, the release of aqueous compound from a reservoir by pure osmosis through micrometer-sized

Figure 5. COMSOL simulation of osmosis based diffusion. (a) Initial state (time 0) of the COMSOL simulation of the liquid concentration in a flow-cell (Modules: ‘CFD’ and ‘transport of diluted species’) that mimics the scenario as sketched in Figure 2a. Red: [c]  3 Mol/m3, blue: [c]  0 M/m3. As test molecule fluorescein, solvent H2O was used. (b) COMSOL simulation of the liquid concentration in the flowcell. The pressure difference of 3 Pa between source and outlet was maintained constant. The diffusion profiles of a small-size compound through the pores of the membrane are indicated.


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pores was analysed. In principle, such a reservoir could be placed into the CI electrode array, and the compound would diffuse into the gap between the array and neural tissue filled by perilymph. Initial values of relevant parameters, such as diffusion constant that comes close to the scenario of Case 3, were estimated using COMSOL computation environment (with ‘CFD’ and ‘transport of diluted species’ modules). Hereby a two-dimensional flow cell arrangement, as shown later in Section 3, was ‘virtually’ set up for simulations. For experimental studies a flow-cell as sketched in Figure 2a was developed. A membrane made from polyolephin acrylate devided the flow-cell into two compartments. These were connected by a lasermachined array of holes in the membrane, as shown by the micrograph in Figure 2b. The diameter of these holes was typically 65 mm. All experiments were carried out using fluorescein as a test molecule. Fluorescein was chosen for its similar molecular weight to DHF (332,32 g·M-1 and 238.24 g·M-1, respectively) (Figure 3). For the monitoring, a fluorescence microscope (SVM 340 with Epi-blue fluorescent camera module from Labsmith, Inc. USA) was used.

by Zen 2011 software (Zeiss, Germany). Post evaluation of the images for measuring the grey scale intensity of the fluorescence images was carried out using Image J 2.0.0-rc-9 software (10). Results and discussion Osmosis based diffusion Results from the COMSOL simulation are given in Figure 5. The initial state, when the source is loaded

Solid phase drug delivery In the second scenario, a release through nanometersized pinholes was investigated. To achieve this, fluorescein was spread in a solid form as tiny crystals over a glass substrate. The crystals of the dry fluorescein were coated using Parylene C (Di-chloro-Para-xylylene) or PPX-C (6,7). PPX coating is highly conformal (8). For example, if a piece of sugar is coated with a PPX layer thicker than 5 mm, the dissolution of the sugar in water will be impossible. However, if the thickness of the coating is kept below 5 mm, problematic sites such as sharp edges or caverns of crystals will not allow perfect PPX overgrowth and hence the water will be able to creep in and dissolve the sugar. This coating technique was applied here, and the thickness of the layers was kept below 5 mm. The scenario of different overgrowth properties of PPX is sketched in Figure 4; hereby it is expressed that the release-relevant pin-hole density depends on the PPX thickness. The fluorescein was dried and coated by a 2-mm thick layer of PPX at the bottom of individual wells in a standard multi-well plate. In a typical fluorescence measurement, each well was immersed in a water bath. Images were acquired using a fluorescence microscope (Axiovert Z1) equipped with a motorized stage and incubation chamber (37°C) using a 10x objective. Fluo Lamp (HXP 120C) images were acquired with a CCD camera (Axiocam MRm, Zeiss, Germany). The microscope is controlled

Figure 6. Experimental estimate of osmosis based diffusion using the flow-cell. (a) Fluorescein emission (grey scale intensity) integrated over a (50 x 50)-mm area as a function of the distance from the membrane. Note, for larger distances the gradients (linear fit of the decay highlighted in pink) are constant. (b) Fluorescein emission intensities of (50  50)-mm areas as a function of the time of observation. The values for different distances become constant.


on the inlet and outlet side; whereas the drain has 0 concentration of fluorescein (blue), is shown in Figure 5a. On the other hand, Figure 5b shows the diffusion results in the absence of any fluid removal/ recycling mechanism at the drain side of the membrane. On the source side the fluorescein concentration is 3 M/m3 (red), with a low flow (3 Pa pressure difference) that guarantees the conservation of the concentration at the source side of the membrane, avoiding local depletion close to the holes. The COMSOL simulation shows that a diffusion coefficient of 109 m2/s governs the molecular transport between both liquids. The osmosis diffusion experiments that are simulated in Figure 5a and 5b were carried out using the flow-cell depicted in the arrangement of Figure 2a. Here, fluorescein in water was used as replacement of small-size TrkB receptor agonists. The results are shown in Figure 6a,b.


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Figure 6 shows fluorescence measurements as a function of the distance from the membrane (Figure 6a) and time (Figure 6b). The fluorescence signal intensity (grey scale intensity) captured by the photo detector in the fluorescence microscope varied in the range between 0 (0 concentration of fluorescein) and 100% (for 25·10-3 M/l at the source side). By analysing curves in Figure 6a after 120 min diffusion time, the grey scale intensity decays from 11% (2.75· 10-3 M/l) at 50 mm to 0% at 150 mm from the source. The concentration gradient of fluorescein is hence 27.5 M/(l·m). The diffusion constant is calculated from the 2800-min curve where at 200 mm distance from the source 10% (2.5 · 10-3 M/l) concentration is measured. The diffusion constant is hence 7.4·10-6 M/(l·s·m). These data are within the scope of envisaged diffusion; after about two days the gradient is always constant at 27.5 M/l·m; however, the required minimum

Figure 7. Time-dependent fluorescence emission from solid fluorescein encapsulated with Parylene. (a) & (b) Fluorescence micrographs taken at the same sample location in time 0 and 18 h later, respectively. The fluorescence intensity and the signal distribution are almost unchanged. It is assumed that no fluorescein was released from the sample and the fluorescence is due to residual water in the dried phase. (c) Initial micrograph taken at another location on the same sample; (d) The same place but 18 h later. Note, a significant increase of the fluorescence signal occurred in specific locations, they indicate an out-diffusion of fluorescein through certain selected spots (pin-holes) on the packaging. Peak 1 is indicated by the solid arrow (result presented in Figure 8a and Figure 8b), peak 2 is indicated by the dashed arrow (result presented in Figure 8b only).


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concentration for the onset of neurite outgrowth is not known and depends on the efficiency of the TrkB receptor agonists. Looking at Figure 6a for the 2800min curve, a reduced concentration of fluorescein is seen for short distances from the membrane. This could be explained by a depletion effect at the source side in proximity to the membrane. The simulation does not show the effect because the depletion is avoided by a flow of fluorescein in the 3 Pa–0 Pa pressure gradient. The delivery rate of the osmosis based drug delivery system fits within the described goal of scope as it should be for release of small-size TrkB receptor agonists from cochlear implants. It may be assumed that if the transport across the membrane is totally due to osmosis, the compound transport is rather diffusion-controlled as it is dependent on the diameter of the pores. This can be inferred from Figure 6a where the signal of the 2800-min curve close to the membrane is reduced due to source problems; however, the gradient of the already released fluorescein is about the same as can be observed at any time earlier. It may be estimated that the compound front, i.e. detectable from the background fluorescence signal, needs six days to cross a 1-mm distance, typical for a gap between the electrode array and neural tissue in the cochlea. When considering such a drug-delivery mechanism, there is one important consideration to be taken into account regarding clinical applicability: the drawback of such osmosis based systems arises from a high risk of contamination via the intrusion of bacteria through the large pores, thus increasing the undesired likelihood of infection. Solid phase drug delivery The mechanism of compound release from a solid phase into the gap is sketched in Figure 1b together with the set-up in Section 2.2. The results of the experimental evaluation are represented in Figures 7 and 8. The time-course of fluorescein release observed during the first 18 h (Figure 7c,d) was analysed in detail by extracting the fluorescence intensity from two selected peaks (indicated by solid and dashed arrows in Figure 7c,d) and is represented in Figure 8. The evaluation using the fluorescence signals depicted in Figure 8 was based on the assumption that the diffusion is isotropic and the detectable signal front can be evaluated as shown in Figure 9. In this solid phase drug delivery, the pores in the PPX layer are extremely small and not visible using conventional surface characterization methods. In this case it is estimated that the compound front (slopes in Figure 8b) needs 11 days to cross a 1-mm distance.

Figure 8. Time resolved plots of the vertical and lateral develop ment of highlighted peaks from Figures 7c,d. (a) The upper left peak (source) in the Parylene-encapsulated dry fluorescein well. (b) Evaluation from the lateral development of two peaks (upper left red and upper right blue) from Figure 7c,d.

Conclusion Different scenarios of low release-rate delivery systems for small-size agonists for TrkB receptor have been set up and compared using fluorescein as test molecule. The principles are very different. COMSOL simulation allowed for the definition of starting parameters for the experimental set-ups. Our first scenario was an osmosis based drug-delivery system through large pores ( 50 mm), which were machined into the membrane separating the source and the drain. It was found that a concentration gradient could be maintained during two days, the detectable threshold of compound at the other side of the gap



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Figure 9. Generation of fluorescence signal from the test molecule diffusing from a pin-hole into solvent. The intensity depends on 1) the absorption coefficient of the exciting light; (2) the absorption coefficient of the re-emitted light; and (3) the concentration and the total quantity of the molecules. Diffusion is assumed to be isotropic.

between implant and wall of the cochlea being reached after six days. The drawback of such osmosis based systems arises from a high risk of contamination via the intrusion of bacteria through the large pores. The second scenario demonstrated drug delivery by using the solid fluorescein encapsulated by a thin 2-mm Parylene packaging layer. At first, a strongly reduced release rate was expected due to a small pore diameter and to that the compound had first to be dissolved by the water and then to diffuse back through the pin-holes. Surprisingly, the release rate was only two times lower than in the osmosis system. The gradient could not be determined by the solid state fluorescein systems due to the much higher concentration at release sites on the dispenser surface. The authors assume that the dissolution of the fluorescein-solid state material creates an enhanced pressure in the vicinity of the pin-hole releasing the compound at high concentration. This effect seems to compensate strongly the difference in pore size. Looking at bacteria contamination risk, this solution appears to be preferable due to much smaller pore size used here.

Acknowledgement This project was funded by the EC under the project NANOCI (FP7) - [NMP.2011 – 281056]

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Glueckert R, Bitsche M, Miller JM, Zhu Y, Prieskorn DM, Altschuler RA, Schrott-Fischer A. Deafferentation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor. J Comp Neurol. 2008;507:1602–21. 2. Shibata SB, Cortez SR, Beyer LA, Wiler JA, Di Polo A, Pfingst BE, Raphael Y. Transgenic BDNF induces nerve fibre regrowth into the auditory epithelium in deaf cochleae. Exper Neurol. 2010;223:464–72. 3. Ramekers D, Versnel H, Grolman W, Klis SF. Neurotrophins and their role in the cochlea. Hear Res. 2012:288:19–33. 4. Yu Q, Chang Q, Liu X, Wang Y, Li H, Gong S, ET AL. Protection of Spiral Ganglion Neurons from Degeneration Using SmallMolecule TrkB Receptor Agonists. JNeurosci. 2013;33: 13042–52. 5. Fortin JB, Lu TM. Chemical vapour deposition Polymerization, the growth and properties of Parylene thin films. Kluwer Academic Publishers, ISBN 1 4020 76886. 6. Charmet J, Banakh O, Laux E, Graf B, Dias F, Dunand A, et al. Solid on liquid deposition. Thin Solid Films. 2010;518:5061–5. 7. Gorham WF. J Polym Sci Part A. Polym Chem. 1966; 4:3027–9. 8. Grattan DW. Parylene at the Canadian Conservation Institute. Can Chem News. 1989;41:25–6. 9. Open source image processing software http://developer. imagej.net/ (webpage accessed on 27th February 2015).