Bioelectronic Direct Current Stimulation at the Transition Between Reversible and Irreversible Charge Transfer

Abstract Many biological processes rely on endogenous electric fields (EFs), including tissue regeneration, cell development, wound healing, and cancer metastasis. Mimicking these biological EFs by applying external direct current stimulation (DCS) is therefore the key to many new therapeutic strategies. During DCS, the charge transfer from electrode to tissue relies on a combination of reversible and irreversible electrochemical processes, which may generate toxic or bio‐altering substances, including metal ions and reactive oxygen species (ROS). Poly(3,4‐ethylenedioxythiophene) (PEDOT) based electrodes are emerging as suitable candidates for DCS to improve biocompatibility compared to metals. This work addresses whether PEDOT electrodes can be tailored to favor reversible biocompatible charge transfer. To this end, different PEDOT formulations and their respective back electrodes are studied using cyclic voltammetry, chronopotentiometry, and direct measurements of H2O2 and O2. This combination of electrochemical methods sheds light on the time dynamics of reversible and irreversible charge transfer and the relationship between capacitance and ROS generation. The results presented here show that although all electrode materials investigated generate ROS, the onset of ROS can be delayed by increasing the electrode's capacitance via PEDOT coating, which has implications for future bioelectronic devices that allow longer reversibly driven pulse durations during DCS.

The stimulation protocol consisted of 1 h no stimulation, 1 h stimulation followed by 1 h no stimulation (anode left, cathode right).Pictures were taken every minute, the four pictures in the figure refer to 1, 1.5, 2 and 3 h.The color changes in PANI are only small for 10 µAcm -2 .By increasing the current density by a factor of 100, color changes are easily observed meaning pH changes significantly.The anodic peak is not clearly expressed in Figure S4e which presents the reason why for the calculation of the effective surface area of LIG hPEDOT the scan rate range was reduced to CVs seen in Figure S3e.

Pulsed direct current stimulation
Certain biological applications require consecutive DCS.For instance, pulse widths of 15 min (oscillating field stimulation) were found to guide and support axonal regeneration after spinal cord injury [2].DC is a term typically used to describe a steady, continuous current (i.e., not a time-limited pulse).In the practical context of bioelectronic stimulation, impulses are nevertheless always confined to a certain duration or stimulation time.Thus, in this work the term DCS describes a time-limited current, albeit of a much longer pulse duration (minutes to hours) compared to what is typical in neurostimulation (a few hundred µs).To determine if PEDOT coating delays the onset of H2O2 generation during consecutive DCS we repeated the amperometric measurements of H2O2 while simultaneously applying four 15 min pulses (10 µA/cm 2 ) with 15 min inter-pulse period in the same two-electrode setup as shown in Figure 4a.
The measured H2O2 concentrations and the corresponding voltage recordings are summarized in Figure S11.While the ePEDOT and hPEDOT coatings delay the onset of H2O2 generation during the first pulse, the generation of H2O2 occurs earlier during the three consecutive pulses.Furthermore, during the last three pulses, slightly higher concentrations of H2O2 are generated by all electrode materials except Pt compared to the first pulse.By analyzing the corresponding voltage recordings shown in Figures S11b and S11d, these observations can be explained.The electrodes are polarized during the first pulse and are not able to recover to a non-polarized state during the 15 min inter-pulse period, as evidenced by the increasing voltage values at the start of each consecutive pulse.Therefore, the electrodes reach the voltage required for H2O2 generation earlier during the consecutive pulses than during the first pulse.Impedance spectroscopy EIS was performed using an Autolab potentiostat (PGSTAT 204, Metrohm Autolab B.V., Filderstadt, Germany) in 3 electrode setup with stainless steel (~ 20 cm 2 ) as counter and Ag/AgCl electrode (Ag/AgCl, BASI, USA) as reference.As electrolyte 0.01 M phosphatebuffered saline (PBS, Sigma Aldrich, USA) was used.All electrodes had an area of 0.2 cm 2 .Before EIS the electrode underwent 5 CV cycles (-600 to 900 mV, 100 mV/s).EIS was performed with a 100 mV sine amplitude with 5 points per decade between 0.1 Hz and 100 kHz.

Figure S1 .
Figure S1.PANI coated acrylic sheets were used to identify relative pH changes during DCS.The stimulation protocol consisted of 1 h no stimulation, 1 h stimulation followed by 1 h no stimulation (anode left, cathode right).Pictures were taken every minute, the four pictures in the figure refer to 1, 1.5, 2 and 3 h.The color changes in PANI are only small for 10 µAcm -2 .By increasing the current density by a factor of 100, color changes are easily observed meaning pH changes significantly.

Figure S2 .
Figure S2.Pictures of tilted SIROF and LIG electrodes.PEDOT coating is visible for both base electrodes as the dark coating above the electrode surface.

Figure S8 .
Figure S8.CVs with capacitive part of the current from Dunn method for a) Pt, b) LIG, c) SIROF, d) SIROF ePEDOT and e) LIG hPEDOT all at 1 mVs -1 .Only points with an R-value > 0.95 were considered.

Figure
Figure S9.a) Capacitance from charge of CV. b) Capacitance from charge of CV for b > 0.8 according to Lindquist method.

Figure S11 .
Figure S11.a,c) H2O2 concentration at the cathode during four monophasic pulses of 15 min with 15 min between pulses.c,d) Voltage excursion during the pulsing in 2-electrode setup.

Figure S12 .
Figure S12.a) Impedance of the investigated electrode materials.b) Phase of the investigated electrode materials

Figure S14 .
Figure S14.Measured concentration and recorded voltage for LIG.

Figure S15 .
Figure S15.Measured concentration and recorded voltage for LIG hPEDOT.

Figure S16 .
Figure S16.Measured concentration and recorded voltage for SIROF.

Table S2 .
Linear regression parameters for Trasatti between 1 and 10 mVs -1 to calculate C o q .

Table S3 .
Linear regression parameters for Trasatti between 1 and 10 mVs -1 to calculate