*3.4. Impedance Measurements*

As the function of cochlear or other active implants depends on precise electrical stimulation, and it is to be expected that any coatings of the electrode array will also be deposited on the stimulating contacts, the possible influence of the coating on electrical impedances was investigated. For this purpose, a setup was developed that allowed impedance measurements under reproducible and controlled conditions. The size of the stimulating contacts was chosen to be comparable to the clinically used Cl electrodes. As polymers, and especially the used PLLA, swell slightly by uptake of water after immersion in aqueous solutions [38], samples were just placed in physiological saline and impedances monitored for 24 h. Impedances were stable at very high values, indicating that the PLLA coating acts as insulator independently of the thickness of the coating. Parameters for electrical stimulation were adapted from Peter et al. [39]. The stimulating current was chosen to be safe for spiral ganglion neurons, at least in their in vitro setting. We can only speculate why impedances were reduced on some contacts with the electrical stimulation and on others, no change was detected. Furthermore, cracks in the coating after electrical stimulation were only found at contacts with reduced impedances and always at the transition from the contacts to the surrounding silicone. One possible reason could be the handling of the samples. Silicone is flexible whereas the Pt contacts are rigid. When samples were unintentionally bent during handling, tension within the polymer layer on the surface would be increased especially where the silicone meets the platinum. This could have introduced first cracks that facilitated water uptake and subsequently current flow. In addition, the influence of local voltage peaks at the edges of the Pt contacts cannot be excluded.

Interestingly, impedances of contacts with coatings containing 10% or 20% DCF were different by an order of magnitude right from the first measurement a few seconds after immersion in saline, but stable for at least 24 h. As shown, when there is 20% DCF in the polymer, more than 50% of it is released within 24 h. However, it is very unlikely that this amount of substance is released within a few seconds and the release remains halted for the rest of the first 24 h. Therefore, there must be different explanations for having lower impedances with 20% DCF. It might be that the high amount of the slightly hygroscopic DCF, which is incorporated in PLLA, leads to an increased and/or faster water uptake. This can result in altered spacing between the polymer chains of PLLA, favoring the formation of enlarged pores. Moreover, the increased concentration of the diclofenac sodium salt also increases the ion concentration within the coating, which may lead to increased conductivity and hence lower impedance.

As PLLA acts as insulator, strategies must be developed to avoid coating of contacts or to remove the coating from the stimulating contacts. For the current investigation, masking was additionally added on the contacts before coating. Removal of the masking after coating reliably reduced impedances at contacts as measured at 1 kHz. For a direct comparison with impedances of CI electrodes as measured with the clinical systems using rectangular pulses for the measurements, this approach was additionally taken for measurements of contacts where the coating was removed. Measured values were slightly increased compared to uncoated electrode contacts in the current setting and comparable to known impedance values of animal CI electrodes right before implantation [7] or commercial electrodes shortly after implantation [14].

## **4. Materials and Methods**

## *4.1. Ethical Statement*

The experiments with primary cells were conducted in accordance with the German "Law on Protecting Animals" (§4) and the European Directive 2010/63/EU for protection of animals used for experimental purpose, and registered (no. 2016/118) with the local authorities (Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Oldenburg, Germany).
