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All-polymeric transient neural probe for prolonged in-vivo electrophysiological recordings

All-polymeric transient neural probe for prolonged in-vivo electrophysiological recordings Biomaterials 274 (2021) 120889 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials All-polymeric transient neural probe for prolonged in-vivo electrophysiological recordings a, 1, 2 a, 1, 3 a a Laura Ferlauto , Paola Vagni , Adele Fanelli , Elodie Genevieve Zollinger , b b a, * Katia Monsorno , Rosa Chiara Paolicelli , Diego Ghezzi Medtronic Chair in Neuroengineering, Center for Neuroprosthetics and Institute of Bioengineering, School of Engineering, Ecole polytechnique f´ ed´ erale de Lausanne, Switzerland Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Switzerland ABSTRACT Transient bioelectronics has grown fast, opening possibilities never thought before. In medicine, transient implantable devices are interesting because they could eliminate the risks related to surgical retrieval and reduce the chronic foreign body reaction. Despite recent progress in this area, the potential of transient bio- electronics is still limited by their short functional lifetime owed to the fast dissolution rate of degradable metals, which is typically a few days or weeks. Here we report that a switch from degradable metals to an entirely polymer-based approach allows for a slower degradation process and a longer lifetime of the transient probe, thus opening new possibilities for transient medical devices. As a proof-of-concept, we fabricated all-polymeric transient neural probes that can monitor brain activity in mice for a few months, rather than a few days or weeks. Also, we extensively evaluated the foreign body reaction around the implant during the probe degradation. This kind of devices might pave the way for several applications in neuroprosthetics. 1. Introduction magnesium, zinc, molybdenum, or iron, dissolve within minutes, hours, or maximum days, once in contact with body fluids [11,18]. This short Building devices able to disappear in the surrounding environment functional window still limits the list of potential medical applications after a programmed lifetime, leaving minimal and harmless traces after for transient devices. their disposal, is a fascinating idea for the development of green elec- To extend the lifetime of transient medical devices, and consequently tronics to preserve the environment by reducing waste production and increase their possible applications, such as for example mid-term recycling processes [1–5]. The same concept is also appealing for monitoring of the brain activity, our strategy is to switch from degrad- medical devices [6–8], such as localised drug release systems [9], power able metals to entirely polymer-based devices. The results show the units [10], external sensors [11] and implantable recording systems fabrication and the in-vitro characterization of all-polymeric transient [12], to eliminate the risks related to surgical retrieval [13] and reduce neural probes (TNPs) allowing prolonged electrophysiological in-vivo the chronic foreign body reaction [14]. recordings up to three months post-implantation. Histological studies In medical applications, the durability of transient devices is highly revealed minimal foreign body reaction both in the short- and long-term. dependent on the degradation time and process of the materials used for the substrate and encapsulation layers and, most importantly, of the 2. Materials and methods functional components exposed to the body. While several biodegrad- able natural or synthetic polymers, such as cellulose [1], silk [15] and 2.1. Fabrication of transient all-polymeric neural probe poly(lactic-co-glycolic acid) [16,17], are often used as substrate and encapsulation materials, so far the choice for the functional elements has Two 4-inch silicon wafers (525-μm thick) were etched (AMS 200, always landed on transient inorganic materials. These materials, such as Alcatel) to obtain the positive mould for the substrate layer and the * Corresponding author. E-mail address: [email protected] (D. Ghezzi). These authors contributed equally to this work. Present address: Department of Physics and Interdepartmental Centre of Industrial Research in Advanced Mechanical Engineering Applications and Materials Technology, University of Bologna, Italy. Present address: Department of Developmental Genetics, Skirball Institute of Biomolecular Medicine, New York University, USA. https://doi.org/10.1016/j.biomaterials.2021.120889 Received 12 November 2020; Received in revised form 26 April 2021; Accepted 6 May 2021 Available online 10 May 2021 0142-9612/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). L. Ferlauto et al. Biomaterials 274 (2021) 120889 negative mould for the encapsulation layer (both structures are 100-μm Scientific), 2 mM L-Glutamine (25030081, Thermo Fisher Scientific) thick). Polycaprolactone (PCL, MW 50,000; 25,090, Polyscience) in and 2.50 μg ml Amphotericin B (15290026, Thermo Fisher Scientific). powder was dissolved in chloroform (C2432, Sigma-Aldrich) at a con- The extraction was performed for 24 h at 37 C and 5% CO . L929 cells centration of 0.25 g ml and was left stirring and heating at 60 C (88102702, Sigma-Aldrich) were plated in a 96-well plate at a sub- overnight over a hotplate. The day after, 6 ml of PCL solution were spin- confluent density of 7000 cells per well in 100 μl of the same medium. coated on both silicon moulds (positive: 150 rpm for 30 s; negative: 200 L929 cells were incubated for 24 h at 37 C and 5% CO . After incuba- rpm for 30 s) pre-treated with chlorotrimethylsilane (92,361, Sigma- tion, the medium was removed from the cells and replaced with the Aldrich) to prevent permanent attachment of the PCL layers. The wa- extract (100 μl per well). After another incubation of 24 h, 50 μl per well fers were then left 30 min in the oven at 75 C to let the chloroform of XTT reagent (Cell proliferation kit 11465015001, Sigma-Aldrich) evaporate. After a cool-down period of at least 2 h, the PCL layer from were added and incubated for 4 h at 37 C and 5% CO . An aliquot of the positive wafer was peeled off, and the design (electrodes, traces and 100 μl was then transferred from each well into the corresponding wells pads) was filled with an aqueous solution of PEDOT:PSS:EG, which is of a new plate, and the optical density was measured at 450 nm by using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS; a plate reader (FlexStation3, MolecularDevices). Clean medium alone M122 PH1000, Ossila) mixed with 20 wt% ethylene glycol (EG; was used as a negative control, whereas medium supplemented with 324,558, Sigma-Aldrich), and cured overnight in an oven at 37 C. The 15% of dimethyl sulfoxide (D2650-5X5ML, Sigma-Aldrich) was used as a PCL layer from the negative wafer, instead, was peeled off after laser- positive control. cutting the openings for the electrodes and pads (Optec MM200-USP). Eventually, the two layers were manually aligned and fused together 2.6. Accelerated degradation over a hotplate at 60 C for a few seconds. A silver-based epoxy (H27D Kit Part A, Epo-Tek) was then applied on the pads and connectors were All-polymeric TNPs were prepared as described above, with the 4- positioned and immobilised with silicone (DC 734 RTV clear, Dow electrode design. After fabrication, samples were weighted to set the Corning). Three types of all-polymeric TNPs were fabricated with a initial weight value before starting degradation. Degradation of each different number of electrodes: 4 electrodes (500-μm in diameter; sample occurred in 10-ml PBS at 37 C and was accelerated by pH in- connector 143-56-801, Distrelec), 1 electrode (500-μm in diameter; no crease: 100 μl of NaOH (2 M NaOH Standard solution, 71,474-1L, Fluka) connector) and 17 electrodes (700-μm in diameter; connector were added to the solution to reach pH 12. At each time point, samples 61001821821, Wurth Elektronik). Samples with fluorescent PEDOT: were first gently dried with a tissue and then let dry completely under PSS:EG were prepared as previously described [19]. Briefly, poly(vinyl vacuum at room temperature for 4 h. Once dry, samples were weighted. alcohol) (PVA, MW 130,000; 563,900, Sigma-Aldrich) was dissolved in Normalised weight for each sample was computed as the ratio between deionization water 5 wt%. A composite solution (25 wt%) of PVA and the weight at each time point and the initial value. PEDOT:PSS:EG was prepared, deposited on the PCL, and baked (50 C, 10 min). (3-Glycidyloxypropyl)trimethoxylane (440,167, 2.7. Animal handling Sigma-Aldrich) was then deposited by vapour deposition at 90 C for 45 min. Afterwards, a solution of fluorescein isothiocyanate (FITC) labelled All experiments were conducted according to the animal authoriza- poly-L-lysine (P3543, Sigma-Aldrich) in phosphate buffered saline (PBS; tions GE13416 approved by the D´ epartement de l’emploi, des affaires 50 μg ml ) was drop-casted on the sample and allowed to react for 2 h ´ ´ ´ ´ sociales et de la sante (DEAS), Direction generale de la sante of the at room temperature. The sample was then rinsed with PBS (0.1 M), Republique et Canton de Geneve in Switzerland and VD3420 approved sodium chloride (0.1 M), and deionised water. ´ ´ by Service de la consommation et des Affaires veterinaires (SCAV) of the Canton de Vaud in Switzerland. All the experiments were carried out 2.2. Scanning electron microscopy during the day cycle. For the entire duration of the experiment, the health condition was evaluated three times a week, and the bodyweight Images were taken with a Schottky field emission scanning electrons was controlled once a week. Experiments were performed in adult (>1- microscope (SU5000, Hitachi) and image post-processing was per- month-old) mice. Male and female C57BL/6J mice (Charles River) were formed with ImageJ. kept in a 12 h day/night cycle with access to food and water ad libitum. White light (300 ± 50 lux) was present from 7 a.m. to 7 p.m. and red 2.3. Electrochemistry light (650–720 nm, 80–100 lux) from 7 p.m. to 7 a.m. Homozygous tm2.1(cre/ERT2)Litt B6.129P2(Cg)-Cx3cr1 /WganJ mice (Stock No: 021,160, tm14 Electrochemical characterization was performed with a three- The Jackson Laboratory) and homozygous B6.Cg-Gt(ROSA)26Sor (CAG-tdTomato)Hze electrode potentiostat (Compact Stat, Ivium) at room temperature. /J mice (Stock No: 007914, The Jackson Laboratory) Each all-polymeric TNP was immersed in PBS (pH 7.4) together with a were kept in a 12 h day/night cycle with access to food and water ad silver/silver chloride reference wire and a platinum counter wire. libitum. White light (300 ± 50 lux) was present from 7 a.m. to 7 p.m. and tm2.1 Impedance spectroscopy (IS) was measured between 1 Hz and 1 MHz no light from 7 p.m. to 7 a.m. Homozygous B6.129P2(Cg)-Cx3cr1 (cre/ERT2)Litt tm14 using an AC voltage of 50 mV. /WganJ mice and homozygous B6.Cg-Gt(ROSA)26Sor (CAG-tdTomato)Hze /J mice were crossed to obtain heterozygous mice bearing a tamoxifen-inducible expression of the tandem dimer Tomato 2.4. Resistance measures fluorescent in microglia in the brain. Mice were injected with tamoxifen Line resistance was measured using a data acquisition and logging dissolved in corn oil (75 mg kg ) to induce the expression at postnatal digital multimeter system (daq6510, Keithley). day 35. Surgery was performed between 2 weeks and 1 month after. 2.5. Cytotoxicity test 2.8. Surgical implantation A test on extract was performed on two all-polymeric TNPs (4-elec- Mice were anesthetised with isoflurane inhalation (induction 1 1 trodes design) sterilised by UV exposure, with a ratio of the product to 0.8–1.5 l min , 4–5%; maintenance 0.8–1.5 l min , 1–2%). Analgesia 2 1 extraction vehicle of 3 cm ml . The extraction vehicle was Eagle’s was performed by subcutaneous injection of buprenorphine (0.1 mg 1 1 minimum essential medium (11090081, Thermo Fisher Scientific) sup- kg ), and a local subcutaneous injection of lidocaine (6 mg kg ) and plemented with 10% foetal bovine serum (10270106, Thermo Fisher bupivacaine (2.5 mg kg ) with a 1:1 ratio. The depth of anaesthesia was Scientific), 1% penicillin-streptomycin (15070063, Thermo Fisher assessed with the pedal reflex, and artificial tears were used to prevent 2 L. Ferlauto et al. Biomaterials 274 (2021) 120889 the eyes from drying. The temperature was maintained at 37 C with a were processed and analysed using MATLAB (MathWorks). After each heating pad during both surgical procedures and recording sessions. The session, the mice were left to recover on a heating pad and later returned skin of the head was shaved and cleaned with betadine. Mice were then to their cage. placed on a stereotaxic frame, and the skin was opened to expose the skull. A squared craniotomy of approximately (4 × 4 mm) was opened 2.11. Euthanasia over the visual cortex (identified by stereotaxic coordinates), and the dura mater was removed. UV-sterilised all-polymeric TNPs were inser- Animals were euthanised with an injection of pentobarbital (150 mg ted in the cortex using a micromanipulator (SM-15R, Narishige). The kg ) under a chemical hood. The chest cavity was opened to expose the probes were always implanted with the PEDOT:PSS:EG layer exposed to beating heart, and a needle was inserted in the left ventricle, while the the caudal part of the brain to clearly discriminate the two sides during right atrium was cut to allow complete bleeding. The animal was image analysis. A reference screw electrode was implanted in the rostral immediately perfused with PBS followed by a fixative solution of 4% side of the cranium. The craniotomy was closed using dental cement, paraformaldehyde (PFA) in PBS. At the end of the procedure, the head of and an extra layer of dental cement was applied to secure the probe. The the animal was removed, and the brain collected and placed in 4% PFA mice were left to recover on a heating pad and later returned to their overnight for post-fixation. cage. 2.12. Histological analysis 2.9. Acute induction of seizure and monitoring of epileptic activity Brain samples were cryoprotected in sucrose 30% and frozen in Immediately after surgery, mice were removed from the stereotaxic optimal cutting temperature compound. 20-μm thick horizontal sections apparatus while still under general anaesthesia, a needle electrode was of the brain were obtained using a cryostat (Histocom, Zug, Switzerland) placed subcutaneously in the dorsal area near the tail as ground, and the and placed on microscope slides. The sections were washed in PBS, four channels of all-polymeric TNPs and the reference electrode were permeabilised with PBS + Triton 0.1% (Sigma-Aldrich), left for 1 h at connected to the amplifier (BM623, Biomedica Mangoni). The signals room temperature in blocking buffer (Triton 0.1% + 5% normal goat were recorded using the WinAver software (Biomedica Mangoni). The serum), and incubated overnight at 4 C with primary antibodies for the recording was started (bandpass filtered 0.1–2000 Hz and sampling glial fibrillary acidic protein (GFAP; 1:1000; Z0334, Dako), the cluster of frequency 8192 Hz) and, after a baseline period, 20 μl of pentylenetet- differentiation 68 protein (CD68; 1:400; MCA1957, Biorad) and neural 1 1 razol (PTZ; 50 mg ml , 45 mg kg ) were delivered via intraperitoneal nuclei (NeuN; 1:500; ABN90P, Millipore). The day after, the sections injection. were incubated for 2 h at room temperature with secondary antibodies (1:500; Alexa Fluor 647 and 488, Abcam), counterstained with DAPI (1:300; Sigma-Aldrich) and mounted for imaging with Fluoromount 2.10. Chronic recordings solution (Sigma-Aldrich). Representative images were acquired with a confocal microscope (LSM-880, Zeiss). For image segmentation and A first acute recording session was carried out immediately after the implantation to check the functionality of the TNPs at the starting point quantification, images were acquired using a slide scanner microscope of the experiment. Chronic recordings were then performed 1- and 2- (VS120, Olympus; 20× objective, pixel size 0.34 μm) and analysed in weeks post-implantation and then every 2 weeks until 12 weeks. The Python, using the scikit-image package (https://scikit-image.org/). mice were dark-adapted for 2 h before each chronic recording session. Mice were anaesthetised with a mixture of ketamine (87.5 mg kg ) and 2.13. Whole-brain imaging xylazine (12.5 mg kg ). The depth of anaesthesia was assessed with the pedal reflex, and artificial tears were used to prevent the eyes from Brain clarification was performed according to a previously ◦ ◦ drying. The temperature was maintained at 37 C with a heating pad described procedure [20]. Briefly, after overnight post-fixation at 4 C in during both surgical procedures and recording sessions. Mice were PFA 4%, the brain was immersed in hydrogel solution (Acrylamide 40% placed on a stereotaxic apparatus in front of a Ganzfeld flash stimulator + VA-044 initiator powder in PBS) at 4 C for three days. The hydrogel (BM6007IL, Biomedica Mangoni) and a needle electrode was placed polymerization was induced by keeping the sample at 37 C for 3 h. subcutaneously in the dorsal area near the tail as ground. The four Then, the brain was passively clarified in 4% sodium dodecyl sulfate channels of all-polymeric TNPs and the reference electrode were con- clearing solution (pH 8.5) for four weeks under gentle agitation at 37 C. nected to the amplifier (BM623, Biomedica Mangoni). The signals were The whole clarified brain was transferred to Histodenz solution at pH 7.5 recorded using the WinAver software (Biomedica Mangoni). First, a (Sigma-Aldrich). Brains were immersed in a refractive index matching baseline recording was obtained for 2.5 min (bandpass filter 0.1–2000 solution (RIMS) containing Histodenz for at least 24 h before being Hz and sampling frequency 8192 Hz). The recording noise was extracted imaged. Brains were glued to a holder and immersed in a 10 × 20 × 45 from the baseline recording using a moving average window of 10 mm quartz cuvette filled with RIMS. The cuvette was then placed in a points; the mean ± s.d. was calculated for each 500 ms epoch, then the chamber filled with oil with (n = 1.45; Cargille). A custom-made mean s.d. of all the epochs was averaged to obtain the noise of the whole light-sheet microscope optimised for labelled clarified tissue was used recording. Channels with a noise level higher than the average plus to image the implant within the mouse brain (COLM, clarity optimised twice the s.d. of the noise at each time point were considered as non- light-sheet microscope [21]). The sample was illuminated (488 and 554 working channels and excluded from further analysis. Local field po- nm) by two digitally scanned light-sheets coming from opposite di- tentials (LFPs) were then acquired for 2.5 min (bandpass filter 0.1–100 rections, and the emitted fluorescence was collected by high numerical Hz and sampling frequency 819.2 Hz). After the recordings, a high pass aperture objectives (Olympus XLPLN10XSVMP, N.A 0.6) filtered filter at 0.5 Hz was applied, and the periodogram was calculated using (Brightline HC 525/50, Semrock) and imaged on a digital CMOS camera the Welch method (window of 4 s) on the whole recording. The area (Orca-Flash 4.0 LT, Hamamatsu) at a frequency ranging between 5 and below the curve between 0.5 and 4 Hz frequencies (corresponding to the 10 frames per second. A self-adaptive positioning of the light sheets delta band) was approximated using the composite Simpson’s rule. The across Z-stacks acquisition ensured an optimal image quality over up to power of the delta band was then divided by the total power (area below 1 cm of tissue. To acquire images of whole samples at lower resolution the periodogram) to obtain the relative power of the delta band. Last, another custom-made light-sheet microscope was used (mesoSPIM, visually evoked potentials were recorded (bandpass filter 0.1–200 Hz mesoscale selective plane illumination microscopy [22]). The micro- and sampling frequency 2048 Hz) upon the presentation of 10 consec- scope consists of a dual-sided excitation path using a fibre-coupled utive flashes (4 ms, 10 cd s m ) delivered at 1 Hz of repetition rate. Data multiline laser combiner (405, 488, 561 and 647 nm; Toptica MLE) 3 L. Ferlauto et al. Biomaterials 274 (2021) 120889 and a detection path comprising a 42 Olympus MVX-10 zoom macro- acquisition was made using custom software written in Python. Z-stacks scope with a 1× objective (Olympus MVPLAPO), a filter wheel (Ludl were acquired at 3 μm spacing with a zoom set at 2x resulting in an 96A350), and a CMOS camera (Orca Flash 4.0 V3, Hamamatsu). The in-plane pixel size of 7.8 μm (2048 × 2048 pixels). The excitation excitation paths also contain galvo scanners for light-sheet generation wavelength was set at 561 nm with an emission 530/40 nm bandpass and reduction of shadow artefacts due to absorption of the light-sheet. In filter (BrightLine HC, AHF). addition, the beam waist is scanned using electrically tuneable lenses (EL-16-40-5D-TC-L, Optotune) synchronised with the rolling shutter of 2.14. Statistical analysis and graphical representation the CMOS camera. This axially scanned light-sheet mode leads to a uniform axial resolution across the field-of-view of 5 μm. Image Statistical analysis and graphical representation were performed Fig. 1. All-polymeric transient neural probes. (a) Picture of a fully assembled TNP with four PEDOT:PSS:EG electrodes. (b) Sketch of the TNP highlighting the three layers: PCL encapsulation, PEDOT:PSS:EG electrodes and PCL substrate. (c) Scanning electron microscopy image of the four electrodes encapsulated in PCL (image ◦ ◦ ◦ tilt 45 ). (d) Magnification of one electrode (image tilt 45 ). (e) Scanning electron microscopy image of the cross-section of one encapsulated trace (image tilt 10 ). (f) Plots of the impedance magnitude (black) and impedance phase angle (grey) of 4 electrodes from a representative TNP. (g) Quantification of the impedance magnitude (left) and impedance phase angle (right) at 1 kHz for all the 4 electrodes from 3 TNPs (respectively in red, blue, and green). (h) Picture of traces with decreasing width: 200 μm, 120 μm, 60 μm, 40 μm and 20 μm. Traces are 6.3-mm long and pads are 2 × 2 mm . (i) Quantification of the trace resistance as a function of the trace width. Kruskal-Wallis: p < 0.0001. Dunn’s multiple comparisons test: 200 μm vs. 120 μm p > 0.9999; 200 μm vs. 60 μm p = 0.0202; 200 μm vs. 40 μm p = 0.0001; 200 μm vs. 20 μm p > 0.0004; 120 μm vs. 60 μm p = 0.5545; 120 μm vs. 40 μm p = 0.0158; 120 μm vs. 20 μm p = 0.0118; 60 μm vs. 40 μm p > 0.9999; 60 μm vs. 20 μm p = 0.6738; 40 μm vs. 20 μm p > 0.9999. (j) Picture of electrodes not encapsulated with decreasing electrode diameter and trace width: 500/200 μm, 300/120 μm, 150/60 μm, 100/40 μm and 50/20 μm (electrode diameter/trace width). (k) Quantification of the impedance magnitude at 1 kHz as a function of the electrode diameter. Kruskal-Wallis: p = 0.6462. (l) Quantification of impedance phase angle at 1 kHz as a function of the electrode diameter. Kruskal-Wallis: p = 0.0002. Dunn’s multiple comparisons test: 500 μm vs. 300 μm p = 0.1578; 500 μm vs. 150 μm p = 0.0009; 300 μm vs. 150 μm p = 0.2790. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4 L. Ferlauto et al. Biomaterials 274 (2021) 120889 with Prism 8 (Graph Pad). The normality test (D’Agostino & Pearson diameter and 20-μm trace width) resulted not connected (Fig. 1j, black omnibus normality test) was performed in each dataset to justify the use arrow), as expected due to the narrow trace. Electrodes with 100-μm of a parametric or non-parametric test. The box plots always extend from dimeter (40-μm trace width) resulted not working because the electrode the 25th to 75th percentiles. The line in the middle of the box is plotted openings in the PCL encapsulation layer closed during the bonding to the at the median. The + is the mean. The whiskers go down to the smallest PCL substrate. Hence, the minimum dimensions reached for the elec- value and up to the largest. In each figure p-values were represented as: trode diameter was 150 μm (60-μm trace width). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Pure PEDOT:PSS electrodes have been previously described for in- vitro applications to record cardiac and neural signals and in-vivo neuromodulation. A flexible all-polymer microelectrode array reported 3. Results impedance magnitude value (1.5 MΩ) at 1 kHz for 120-μm electrodes substantially higher than the ones reported here [35]. The higher 3.1. Probe fabrication and characterization impedance magnitude was caused by the deposition method of PEDOT: PSS in a polymeric channel, which led to surface coating only and not to We fabricated TNPs based on PCL as substrate and encapsulation layers, and PEDOT:PSS doped with EG as a conductive element, by using the complete filling of the channel, thus reducing the electrode surface area. A second device exploring inkjet-printed PEDOT:PSS reported an soft lithographic techniques (Fig. 1a and b and Figs. 1). PCL is a com- mercial biodegradable polyester widely used in biomedical applications impedance magnitude value (19.5 kΩ) at 1 kHz for an electrode area corresponding approximately to the area of a round electrode with a [23–26]. It is easy to process and has a longer degradation time (several diameter of 300 μm comparable to the TNP [36]. In a third report, months or years [23]) compared to most biodegradable polymers. PEDOT:PSS/glycerol electrodes showed an average impedance magni- PEDOT:PSS dispersed in water is commercially available, has excellent tude at 1 kHz of 6.3 kΩ for an electrode area of 0.5 mm (corresponding biocompatibility, has the ability to support cells viability [27,28], and to the area of a round electrode with a diameter of 800 μm) which is can be integrated into softer systems like elastomers [29,30] and similar to the TNP [37]. hydrogels [31,32]. EG doping of PEDOT:PSS is known to improve its conductivity [33,34]. The micro-structured PCL-based substrate Last, cytotoxicity tests performed in-vitro showed a cell viability of 96.9 ± 7.2% (mean ± s.d., 2 probes, 6 replicas per probe) and opened (thickness 60–70 μm) and encapsulation (thickness 60–70 μm) layers have been obtained by replica moulding. The hollow regions of the the way for the in-vivo validation of the devices (Fig. 2). substrate layer have been manually filled with PEDOT:PSS:EG to create electrodes, traces and contact pads (Figs. 1). Electrodes are 500-μm in 3.2. Functional validation in-vivo diameter (Fig. 1c and d) and traces are 200-μm wide (Fig. 1e). At the electrode level, the PEDOT:PSS:EG layer is approximately 3-μm thick, To assess the capability of all-polymeric TNPs in recording neural while at the trace level, it is approximately 1-μm thick (Figs. 2). After activity, we performed in-vivo acute brain recordings in mice upon in- curing of the PEDOT:PSS:EG, the PCL encapsulation layer has been duction of epileptic seizures. All-polymeric TNPs were implanted in the manually flip-bonded to the substrate layer via a low-temperature pro- visual cortex area of anaesthetised mice (Fig. 3a), and the neural activity cess (total probe thickness ranging from 120 to 140 μm). This procedure was recorded both before and after intraperitoneal injection of the ensured a complete filling of the trenches for the conductive traces convulsant PTZ, which is routinely used to test anticonvulsants in ani- (Fig. 1e). IS showed remarkable performances of all-polymeric TNPs mals [38–40]. The transition between resting state and epileptic activity based on PEDOT:PSS:EG, characterised by low impedance magnitude after PTZ injection was clearly detected by the TNPs with an excellent (Fig. 1f) and low impedance phase angle (Fig. 1g) over a wide range of signal to noise ratio (Fig. 3b), thus demonstrating the potential of these frequency (from 10 Hz to 100 kHz), which makes them appropriate for all-polymeric TNPs for monitoring epileptic activity. the recording of both low- and high-frequency neuronal signals, such as Next, we performed chronic in-vivo recordings of baseline activity, LFPs and neural spiking activities respectively. The comparison of LFPs at rest and visually evoked potentials (VEPs) to assess the longevity multiple probes showed very low intra-probe variability. On the other of the device. All-polymeric TNPs were implanted in the visual cortex hand, the inter-probe variability might be explained by the manual area of mice, and their functionality was evaluated through periodic deposition process of PEDOT:PSS:EG resulting in a variable thickness of recordings up to 3 months post-implantation (Fig. 4). The broad-band the conductive layer. To verify that the excellent electrochemical char- noise of recordings was computed from baseline recordings (2.5 min, acteristics of the TNPs were not related to shortcuts between electrodes bandpass filter 0.1–2000 Hz and sampling frequency 8192 Hz). The caused by the porosity of the PCL scaffold, we took advantage of an noise plot (Fig. 4a) shows the evolution of the noise level for each all-polymeric TNP with a modified design embedding 17 electrodes working electrode over time (filled circles). Once an electrode was found (Figs. 3). In this specific device, four traces were interrupted, due to a not properly functional (empty circles), it was excluded from further gap in the PEDOT:PSS:EG layer (Figs. 3a-c). As expected, only the cor- analysis. The applied criteria of exclusion were the following: i) responding electrodes were rightfully not functional (Figs. 3d and e), appearance of periodic artefacts not linked to any biological function thus confirming that shortcuts between electrodes are not present. Be- (red arrow in Fig. 4a); ii) noise level higher than the average plus twice sides electrochemistry, we also measured the averaged line resistance of the standard deviation of the noise at each time point; iii) lack of VEPs. the traces, which was 319.5 ± 75.01 Ω (±s.d., n = 8 traces; Fig. 1h and i) The number of excluded electrodes progressively increased over time for 200-μm wide and 6.3-mm long traces. (Fig. 4b), with 3 out of 16 electrodes still properly functional after three Then, we investigated the minimum dimensions for electrodes and months. LFPs (2.5 min, bandpass filter 0.1–100 Hz and sampling fre- traces that could be reached using these materials and fabrication quency 819.2 Hz) are not strongly affected by broad-band noise because methods. First, we fabricated narrow traces and measured the average of the low pass filter at 100 Hz. On the other hand, LFPs are charac- line resistance (n = 8 traces for each dimension; Fig. 1h and i). As ex- terised by a strong delta rhythm (0.5–4 Hz) due to the animal anaes- pected, the line resistance significantly increased with the decrease of thesia (Fig. 4c). Early after implantation, electrodes showed a narrow the width. It must be noted that for 20-μm wide traces, only 3 out of 8 power spectral density (PSD) in the range of delta rhythm (black traces traces appeared to be connected. Therefore, the resolution limit for in Fig. 4c and black lines in Fig. 4d). Over time, because of the electrode traces is 40 μm. Next, we fabricated TNPs with smaller electrodes and deterioration, the normalised PSD shows activity at higher frequency in traces (Fig. 1j) and tested IS (n = 7 electrodes for each dimension). As electrodes that were previously considered not properly functional expected, the impedance magnitude (Fig. 1k) and the impedance phase because of high broad-band noise (grey line in Fig. 4d). Conversely, angle (Fig. 1l) increased with the reduction of the electrode diameter. electrodes still considered functional showed a narrow PSD (red line in Among the dimensions tested, the smallest electrode (50-μm electrode Fig. 4d). As a consequence, the relative power of the delta band 5 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 2. Cytotoxicity in-vitro. (a) Representative optical images for each condition tested: positive control (left), negative control (middle) and TNP (right). (b) Quantification of the mean (±s.d.) cells viability in the three conditions tested. Positive control 0 ± 1.8% (9 replicas); negative control 100 ± 6.8% (9 replicas); all- polymeric TNPs 96.9 ± 7.2% (2 probes, 6 replicas per probe). One-way ANOVA: F = 853.4 and p < 0.0001. Tukey’s multiple comparisons test: positive control vs. negative control p < 0.0001; positive control vs. neural probes p < 0.0001; negative control vs. neural probes p = 0.4852. Fig. 3. Acute recordings with all-polymeric transient neural probes. (a) Sketch of the TNP implanted in a mouse brain for in-vivo recordings. (b) Detection of epileptic activity after injection of PTZ (black arrow) from the four electrodes of a all-polymeric TNP. The red and blue boxes show an enlargement of the recorded activity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) decreases with the deterioration of the electrodes (Fig. 4e). For each factors could contribute to the deterioration of PEDOT:PSS in-vivo, electrode, early after implantation, a low noise correlates with a high related to both materials properties and interaction with the biological relative power of the delta band. However, at the exclusion point, there environment. A commonly reported problem is the adhesion of PEDOT: is a reduction of the relative delta power which is globally proportional PSS to the substrate [45]. The weak adhesion between PEDOT:PSS and to the increase of noise (linear regression: slope = 1.369, R square = polyesters [46], like PCL, plays a crucial role since PEDOT:PSS might be 0.527). Overall, all-polymeric TNPs retain good in-vivo recording ca- subjected to water-induced swelling and delamination during the pabilities for months after implantation, even if they progressively lose degradation of the PCL scaffold. PEDOT has also been reported to un- functionality and only a few electrodes are still properly functional after dergo degradation by hydrolysis when exposed to salt aqueous solutions three months. [47] or by high concentration of hydrogen peroxide, which is a physi- At this stage, the cause of electrode deterioration is not easy to ological oxidant [48,49]. Macrophages cultured with PEDOT:PSS determine. So far, only a few studies have investigated the chronic generated a significant amount of hydrogen peroxide, that is a signifi - performances of PEDOT:PSS in-vivo [41–44], and the results indicated a cant factor involved in cellular phagocytosis [50]. PEDOT:PSS could reduction in performance after a few months of implantation. Many possibly be degraded by the combination of hydrolysis and presence of 6 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 4. Long-term functioning in-vivo. (a) Noise evaluation as a function of time during in-vivo chronic recordings at fixed time points. Different colours correspond to different mice; filled circles correspond to the electrodes considered functional and thus included in the calculation of the average (±s.d.) noise values (black circles and dashed line), while empty circles correspond to the electrodes considered not correctly working and thus excluded from the calculation of the average noise. The two insets show single baseline recordings (left) and VEPs (right) from all the four electrodes of the TNP implanted in mouse 3 at week 1 and week 6 post- implantation; the red dashed line indicates the light stimulus for VEPs (10 cd s m ). The traces in grey are from electrodes considered not functional. The red arrows show periodic artefacts not linked to any biological function. (b) Plot of the percentage of working electrodes as a function of time during in-vivo chronic exper- iments. (c) Representative example of LFPs recorded from all the electrodes of the TNP implanted in mouse 3 at week 1 and week 6 post-implantation. The traces in grey are from electrodes considered not functional. (d) Representative examples of normalised power spectral densities obtained from the recordings from two electrodes (numbers 2 and 3) of the probe implanted in mouse 3 at week 1 (black traces) and week 6 (black grey and red) after implantation. The trace in grey is from the electrode considered not functional, while the trace in red is from the electrode considered still functional. (e) Correlation between the relative power of the delta band and the noise of the electrodes immediately after implantation (16 electrodes, black circles) or at the time point of exclusion (13 electrodes, empty circles). The black line is the linear regression, while the grey lines the 95% confidence interval. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) hydrogen peroxide following device implantation [45]. segmentation (Figs. 4). Images were rotated to align the probe hori- zontally (Figs. 4a) and converted to binary using a local threshold al- gorithm: all pixels which intensity was above the threshold were 3.3. Evaluation of the foreign body reaction assigned the value of 1, while to the rest of the pixels were assigned the value of 0 (Figs. 4b). A group of adjacent pixels with value 1 was defined To investigate the acute phase of the foreign body reaction induced as a blob, and the area of each blob was calculated, along with the co- by TNPs, we performed a histological paired comparison between all- ordinates of its centroid. The image was divided into 10 × 10 quadrants polymeric TNPs and polyimide (PI) implants, surgically placed in the (Figs. 4c), for each of which the total area of blobs (with their centroid two hemispheres of the brain of the same mice (Fig. 5a). The PI implants included in that quadrant) was computed. Then, the total area occupied were chosen as a reference for non-degradable flexible implants. Also, by blobs in the zone adjacent to the probe (Figs. 4d) was calculated as the implants have been manufactured with similar dimensions to be the cumulative area of all the quadrants included in a rectangle defined more comparable (TNP: ~130-μm thick and 1.3-mm wide; PI: 125-μm by the extremities of the probe (manually specified for each of the im- thick and 0.7-mm wide). Evaluations have been made at 1- and 2- ages), minus the area of the probe itself. The percentage of the area months post-implantation, 6 mice for the first time point and 5 mice occupied by blobs was computed as the area occupied by blobs over the for the second. proximal area. The average percentage of the proximal area occupied by For each mouse, horizontal brain slices were sampled at different the blobs for each sample was used as a parameter to compare the cortical depths (Fig. 5a from 1 to 4) and analysed after image 7 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 5. Foreign body reaction induced by all-polymeric transient neural probes. (a) Sketch of the TNP and PI probe implanted in both visual cortices of a mouse. For each probe, four slices were taken at four depths into the cortical layers. (b,c) Example of GFAP (in red) and CD68 (in cyan) expression in horizontal slices (four depths into the cortical layers indicated by the numbers) obtained from the hemisphere implanted with the TNP (b) and with the PI probe (c) 1-month post- implantation. (d,e) Quantification of the area occupied by GFAP signal (d) and CD68 signal (e) at 1-month and 2-month post-implantation for TNPs and PI probes. Results from all the images at each depth were averaged together: 3 images per depth. (f,g) Quantification of the normalised area occupied by the CD68 signal above (PEDOT:PSS:EG side) and below (PCL-only side) TNPs at the level of the electrode at 1-month (f) and 2-month (g) post-implantation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) experimental groups. Three images for each depth were averaged. The Next, we quantified the spatial extent of the GFAP and CD68 signals staining for the GFAP (Fig. 5b and c) and the corresponding quantifi - around the TNP at each cortical depth. Each image (one per depth) was cation analysis (Fig. 5d) performed on segmented images showed that rotated to align the probe vertically (Fig. 6a). Two regions of interest, the area occupied by the GFAP signal in the zone adjacent to the probe one on the left and one on the right of the probe, were manually defined exhibits a decreasing trend over time for all-polymeric TNPs (p = as rectangles perpendicular to the probe, spanning from the edge of the 0.0178, two-tailed unpaired t-test) and a stable trend for PI probes (p = probe to the border of the image (Fig. 6b). For each of them, the average 0.6924, two-tailed unpaired t-test). Also, the staining for CD68 (Fig. 5b, intensity along the horizontal axis was computed, starting from the edge c), a marker of phagocytic structures in microglia [51], and the corre- of the probe to the border of the image. The average vector of intensities sponding quantification analysis (Fig. 5e) showed that the area occupied was normalised by dividing each point by the maximum value among by the CD68 signal exhibits a slightly increasing trend over time for the two regions of interest and a normalised intensity plot was generated all-polymeric TNPs (p = 0.2897, two-tailed unpaired t-test), while it (Fig. 6c). For each cortical depth, normalised intensity plots from all remains stable for PI probes (p = 0.5905, two-tailed unpaired t-test). mice were averaged (6 mice at 1-month post-implantation and 5 mice at Histological samples of brains implanted with TNPs showed a clus- 2-months post-implantation) to determine the spatial profile. The spatial tering of the CD68 signal at the level of the electrode (slice 4 in Fig. 5b profiles of both GFAP (Fig. 6d and e) and CD68 (Fig. 6f and g) resulted and c), the only point where the PEDOT:PSS:EG is exposed to the tissue. uniform along the vertical axis of the probe (cortical depths) at both 1- Following this observation, we performed a second analysis where the and 2-months post-implantation (Fig. 6d,f and Fig. 6e,g respectively). In segmented images were divided into two parts: the side of the probe order to quantify the extent of the GFAP and CD68 signals, we measured with the PEDOT:PSS:EG exposed and the side of the probe with only the the distance from the probe at which the average profile among the 4 PCL exposed (Figs. 5). For each side, the cumulative area occupied by depths (black lines in Fig. 6d–g) is reduced by 50% (red dots in blobs was calculated along the horizontal axis and normalised by the Fig. 6d–g). The quantification (Fig. 6h) revealed that the GFAP signal is global maximum. The probes were always implanted with the PEDOT: wider 1-month post-implantation (116.82 and 206.16 μm respectively PSS:EG layer exposed to the caudal part of the brain to discriminate the for the left and the right side of the probe) and it is reduced 2-months two sides during image analysis. The analysis confirmed that the CD68 post-implantation (96.55 and 95.18 μm respectively for the left and signal is highly clustered on the side of the PEDOT:PSS:EG electrodes at the right side of the probe), although the reduction is not significant (p both time points (Fig. 5f and g), suggesting an interaction between = 0.3333, two-tailed Mann-Whitney test). This result is in agreement microglia and PEDOT:PSS:EG. with the previous analysis (Fig. 5d). The CD68 signal is restricted to a 8 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 6. Spatial extent of the foreign body reaction. (a) Example of CD68 expression in a horizontal slice obtained from the hemisphere implanted with the TNP at 1- month post-implantation. (b) Definition of the two regions of interest on the left and on the right of the TNP. (c) Average vectors of normalised intensities (top) and normalised intensity plots (bottom). (d,e) Normalised intensity plots for GFAP signal 1-month (d) and 2-months (e) post-implantation. The grey lines correspond to the four cortical depths and the black line is the average. The red dot corresponds to a 50% decrease in the intensity. (f,g) Normalised intensity plots for CD68 signal 1-month (f) and 2-months (g) post-implantation. The grey lines correspond to the four cortical depths and the black line is the average. The red dot corresponds to a 50% decrease in the intensity. (h) Quantification of the distance from the TNP at which the intensity plot shows a 50% decrease. (i) Example of NeuN expression in a horizontal slice obtained from the hemisphere implanted with the TNP at 1-month post-implantation. (j) Quantification of the area occupied by NeuN signal at 1- month and 2-months post-implantation of the TNPs compared to a control (Ctrl) area located far from the TNPs. Each data point corresponds to one cortical depth, average of 3 images per depth. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) small area around the probe (1-month: 85.56 and 62.88 μm; 2-months: evidence suggests that the presence of PEDOT:PSS:EG detached from the 75.60 and 78.00 μm; respectively for the left and the right side of the PCL substrate stimulates phagocytic activity of microglial cells, and that probe), and it does not change from 1- to 2-months post-implantation microglia are participating in the clearance of PEDOT:PSS:EG from the (Fig. 6h; p > 0.9999, two-tailed Mann-Whitney test), in agreement implantation site. Horizontal brain sections at 1-month with the previous analysis (Fig. 5e). post-implantation also revealed microglial cells (in red) localizing in We also quantified the area occupied by NeuN signal (a marker for the region where PEDOT:PSS:EG (in green) is delaminated or directly in neuronal cells) around the TNP at the four cortical depths (Fig. 6i) contact with the brain (Fig. 7d). These cells are CD68 positive (in white), compared to a control area located far from the TNP. The quantification the presence of intracellular phagocytic structures. Based on the results analysis (average of three sections per cortical depth), performed on one discussed above, we hypothesise that implanted TNPs lead to the for- mouse per time point (1- and 2-months post-implantation), showed that mation of a less tight glial scar compared to PI probes. As a consequence, the area occupied by the NeuN signal is not statistically different be- microglia have the space needed to access the probe and phagocyte the tween the two areas (Fig. 6i; 1-month: p = 0.3750, Wilcoxon matched- delaminated PEDOT:PSS:EG flakes. pairs signed rank test; 2-months: p = 0.6250, Wilcoxon matched-pairs signed rank test). 3.4. Probe’s degradation The clustering of the CD68 signal we observed at the level of the PEDOT:PSS:EG electrode (Fig. 5f and g) encouraged us to further One of the most exciting features of transient medical devices is their investigate the interaction between phagocytic microglial cells and natural ability to disappear in the body, which in turns reduces the long- PEDOT:PSS:EG. To this end, we fabricated TNPs with a PCL substrate term foreign body reaction. PCL bulk degradation occurs slowly with a coated with PEDOT:PSS:EG labelled with the fluorescent marker FITC time scale spanning several months or years [23], thus making an [19] and implanted them into the cortex of mice expressing tdTomato in in-vivo assessment in animals extremely challenging. In order to inves- microglia (Fig. 7a). In this experiment, all-polymeric TNPs were not tigate the degradation process of all-polymeric TNPs, we performed encapsulated to enhance interaction between microglial cells and in-vitro accelerated degradation tests, by leaving 3 TNPs immersed in PEDOT:PSS:EG. High-resolution imaging on clarified brains 1-month PBS at 37 C and pH 12 and monitoring their weight over time. Scanning (Fig. 7b) and 2-months (Fig. 7c) post-implantation showed colocaliza- electron microscope images showed the appearance of micro- and tion of PEDOT:PSS:EG (in green) and microglial cells (in red). This macro-cracks over time due to hydrolysis (Fig. 8a), and all-polymeric 9 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 7. | Integration with microglia. a, 3D mesoSPIM image of a clarified brain sample with fluorescent microglia (red) implanted with a TNP with FITC-labelled PEDOT:PSS at 1-month post-implantation. b,c, COLM images of the probe surface, showing fluorescent PEDOT:PSS (in green) and microglia (in red) colocaliza- tion 1-month (b) and 2-months (c) post-implantation. Microglia engulf PEDOT:PSS:EG particles which are detaching from the probe (white arrows). d, Horizontal brain section of mouse brain with fluorescent microglia (in red) implanted with a fluorescent TNP (without encapsulation) at 1-month post-implantation. e,k, Various magnifications of the section in d (corresponding letters) showing phagocytic microglia engulfing PEDOT:PSS:EG (in green). CD68 (in white) is a specific marker for microglial phagocytic structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) TNPs undergo full degradation in about one year of time at pH 12 electrode and neurons, eventually impairing the recording capabilities (Fig. 8b). A comparison between the days to reach a weight loss of 5% at of the device [54,55], and acts as a barrier for growth factors, ions, and pH 12 and at pH 7.4 revealed that the test has an acceleration factor of signalling molecules, thus promoting neural apoptosis and impeding about 2.5. Slow degradation is an appealing feature for TNPs, since it axon regeneration [56,57]. In the case of TNPs, the presence of a limited might allow for better tissue remodelling and reduced chronic trauma at glial scar allows tissue remodelling around the probe, and, as a conse- the implantation site [26,52,53]. To provide evidence of slow degra- quence, neurons (labelled against NeuN in green) are free to relocate dation in-vivo of all-polymeric TNPs, we implanted them in the cortex of within the device, which serves as a support scaffold during the degra- mice and subsequently performed immunofluorescence assays on hori- dation (Fig. 8d). This evidence of the probe slow degradation in-vivo is a zontal brain slices at a fixed time point. Upon long-term implantation (9 promising sign of reduced chronic trauma. months), the all-polymeric TNPs showed a minor chronic glial scar (Fig. 8c), evaluated with staining for astrocytes against GFAP. The for- 4. Discussion mation of a glial scar should be minimised or avoided for several rea- sons: it increases the distance between the recording surface of the In biomedical engineering and medicine, transient devices are a 10 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 8. Degradation of all-polymeric transient neural probes. (a) Scanning electron microscopy images at several magnifications of one all-polymeric TNP obtained ◦ ◦ 191 days after soaking in PBS at 37 C and pH 12. (b) Plot of the normalised probe weight versus time of 3 TNPs during a degradation test in PBS at 37 C and pH 12. The red line is the average of the 3 TNPs. The grey dashed line is the time point (day 42) corresponding to a 5% weight loss. (c) Horizontal brain section from a mouse implanted with a TNP in the cortex. 9-months post-implantation staining against GFAP and NeuN. (d) The same image in (c) with a bright-field overlay to highlight the area occupied by the TNP. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) relatively new concept which has the potential to widen the range of during the recovery period and, possibly, into the effects of the following possible applications of implantable medical devices. We explored this pharmaceutical treatment. Also, the absence of metals in the implanted new concept with using fully polymer-based devices to exploit their portion of the device would provide magnetic resonance imaging unique characteristics and try to overcome some of the current limita- compatibility [29,60,61], allowing the pairing of the electrophysiolog- tions. So far, transient medical devices showed a short functional life- ical monitoring with structural and functional images, therefore time because of the fast degradation of degradable metals in contact obtaining a clearer picture of the brain activity during the recovery with body fluids [11,18]. period. In this work, we showed the fabrication and characterization of a Even though only a few electrodes (approximately 20%) were still polymer-based implantable device conceived to disappear after being operational three months post-implantation, we believe that there is still functional for a few months rather than a few days. A longer device room for fabrication strategies that can improve the device lifetime, such lifetime will allow, for instance, the development of prosthetic devices as enhancing the adhesion between PEDOT:PSS and PCL by using ad- for mid-term monitoring of the brain activity, either before or after ditives or by oxygen plasma treatment. Also, even if a performance surgery in chronic epileptic patients. Nowadays, clinicians make de- comparison with other inorganic and not-transient devices is out of the cisions on localised surgical treatments based on intracranial electro- scope of this study (due to the transient nature of the presented device), encephalography recordings that are typically lasting for only 1 or 2 the electrochemical characterization of all-polymeric TNPs showed a weeks in favour of patient compliance. On the other hand, recent pub- stable performance within a wide range of frequencies, thus allowing us lications in this field revealed that intracranial electroencephalography to perform acute and chronic in-vivo recordings in mice with excellent recordings do not stabilize until several weeks after implantation, due to signal to noise ratio. the invasiveness of the procedure and the inflammatory response of the Recently, not-transient implantable neural probes reached extreme tissue [58,59]. Therefore, an extended chronic monitoring period, miniaturisation to reduce chronic foreign body reaction [62,63]. Mini- together with the possibility to mitigate the inflammatory reaction, aturisation for the TNP is limited by materials properties and fabrication would enable a more accurate representation of the spatiotemporal methods. However, a detailed study of the foreign body reaction variability of the seizures, translating into a better diagnosis for the revealed that all-polymeric TNPs were responsible for a minimal glial patient. Furthermore, a transient device with extended recording scar around the implant. As a consequence, neurons were able to colo- capability would allow for continuous monitoring after the surgical nize the slowly degrading PCL layers, hence promoting tissue remodel- procedure, thus providing essential insights into the brain activity ling: such behaviour is the opposite to what is usually observed with 11 L. Ferlauto et al. Biomaterials 274 (2021) 120889 [3] C. Dagdeviren, S.-W. Hwang, Y. Su, S. Kim, H. Cheng, O. Gur, R. Haney, F. permanent implants of similar sizes, where neurons are depleted from G. Omenetto, Y. Huang, J.A. 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Kim, J. Khalifeh, D. V. Harburg, K. Bean, M. Paskett, J. Kim, Z.S. Zohny, S.M. Lee, R. Zhang, K. Luo, B. Ji, A. Banks, H.M. Lee, Y. Huang, W.Z. Ray, J.A. Rogers, Wireless bioresorbable The authors declare that they have no known competing financial electronic system enables sustained nonpharmacological neuroregenerative interests or personal relationships that could have appeared to influence therapy, Nat. Med. 24 (2018) 1830–1836, https://doi.org/10.1038/s41591-018- the work reported in this paper. 0196-2. [8] C.M. Boutry, L. Beker, Y. Kaizawa, C. Vassos, H. Tran, A.C. Hinckley, R. Pfattner, S. Niu, J. Li, J. Claverie, Z. Wang, J. Chang, P.M. Fox, Z. Bao, Biodegradable and Acknowledgement flexible arterial-pulse sensor for the wireless monitoring of blood flow, Nat. Biomed. Eng. 3 (2019) 47–57, https://doi.org/10.1038/s41551-018-0336-5. ´ ´ This work has been supported by Ecole polytechnique federale de [9] H. Tao, S.-W. Hwang, B. Marelli, B. An, J.E. Moreau, M. Yang, M.A. Brenckle, S. Kim, D.L. Kaplan, J.A. Rogers, F.G. Omenetto, Silk-based resorbable electronic Lausanne, Medtronic PLC and European Commission (Grant agreement devices for remotely controlled therapy and in vivo infection abatement, Proc. 701632). The authors would like to thank Dr. Laura Batti and Audrey Natl. Acad. Sci. Unit. States Am. 111 (2014) 17385–17389, https://doi.org/ Tissot for the support with the brain clarification process and imaging 10.1073/pnas.1407743111. [10] L. Lu, Z. Yang, K. Meacham, C. Cvetkovic, E.A. Corbin, A. Vazquez-Guardado, and Vivien Gaillet for the drawings. M. Xue, L. Yin, J. Boroumand, G. Pakeltis, T. Sang, K.J. Yu, D. Chanda, R. Bashir, R. W. Gereau, X. Sheng, J.A. Rogers, Biodegradable monocrystalline silicon Appendix A. Supplementary data photovoltaic microcells as power supplies for transient biomedical implants, Adv. Energy Mater. 8 (2018) 1703035, https://doi.org/10.1002/aenm.201703035. [11] S.-W. Hwang, C.H. Lee, H. Cheng, J.-W. 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All-polymeric transient neural probe for prolonged in-vivo electrophysiological recordings

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Biomaterials 274 (2021) 120889 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials All-polymeric transient neural probe for prolonged in-vivo electrophysiological recordings a, 1, 2 a, 1, 3 a a Laura Ferlauto , Paola Vagni , Adele Fanelli , Elodie Genevieve Zollinger , b b a, * Katia Monsorno , Rosa Chiara Paolicelli , Diego Ghezzi Medtronic Chair in Neuroengineering, Center for Neuroprosthetics and Institute of Bioengineering, School of Engineering, Ecole polytechnique f´ ed´ erale de Lausanne, Switzerland Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Switzerland ABSTRACT Transient bioelectronics has grown fast, opening possibilities never thought before. In medicine, transient implantable devices are interesting because they could eliminate the risks related to surgical retrieval and reduce the chronic foreign body reaction. Despite recent progress in this area, the potential of transient bio- electronics is still limited by their short functional lifetime owed to the fast dissolution rate of degradable metals, which is typically a few days or weeks. Here we report that a switch from degradable metals to an entirely polymer-based approach allows for a slower degradation process and a longer lifetime of the transient probe, thus opening new possibilities for transient medical devices. As a proof-of-concept, we fabricated all-polymeric transient neural probes that can monitor brain activity in mice for a few months, rather than a few days or weeks. Also, we extensively evaluated the foreign body reaction around the implant during the probe degradation. This kind of devices might pave the way for several applications in neuroprosthetics. 1. Introduction magnesium, zinc, molybdenum, or iron, dissolve within minutes, hours, or maximum days, once in contact with body fluids [11,18]. This short Building devices able to disappear in the surrounding environment functional window still limits the list of potential medical applications after a programmed lifetime, leaving minimal and harmless traces after for transient devices. their disposal, is a fascinating idea for the development of green elec- To extend the lifetime of transient medical devices, and consequently tronics to preserve the environment by reducing waste production and increase their possible applications, such as for example mid-term recycling processes [1–5]. The same concept is also appealing for monitoring of the brain activity, our strategy is to switch from degrad- medical devices [6–8], such as localised drug release systems [9], power able metals to entirely polymer-based devices. The results show the units [10], external sensors [11] and implantable recording systems fabrication and the in-vitro characterization of all-polymeric transient [12], to eliminate the risks related to surgical retrieval [13] and reduce neural probes (TNPs) allowing prolonged electrophysiological in-vivo the chronic foreign body reaction [14]. recordings up to three months post-implantation. Histological studies In medical applications, the durability of transient devices is highly revealed minimal foreign body reaction both in the short- and long-term. dependent on the degradation time and process of the materials used for the substrate and encapsulation layers and, most importantly, of the 2. Materials and methods functional components exposed to the body. While several biodegrad- able natural or synthetic polymers, such as cellulose [1], silk [15] and 2.1. Fabrication of transient all-polymeric neural probe poly(lactic-co-glycolic acid) [16,17], are often used as substrate and encapsulation materials, so far the choice for the functional elements has Two 4-inch silicon wafers (525-μm thick) were etched (AMS 200, always landed on transient inorganic materials. These materials, such as Alcatel) to obtain the positive mould for the substrate layer and the * Corresponding author. E-mail address: [email protected] (D. Ghezzi). These authors contributed equally to this work. Present address: Department of Physics and Interdepartmental Centre of Industrial Research in Advanced Mechanical Engineering Applications and Materials Technology, University of Bologna, Italy. Present address: Department of Developmental Genetics, Skirball Institute of Biomolecular Medicine, New York University, USA. https://doi.org/10.1016/j.biomaterials.2021.120889 Received 12 November 2020; Received in revised form 26 April 2021; Accepted 6 May 2021 Available online 10 May 2021 0142-9612/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). L. Ferlauto et al. Biomaterials 274 (2021) 120889 negative mould for the encapsulation layer (both structures are 100-μm Scientific), 2 mM L-Glutamine (25030081, Thermo Fisher Scientific) thick). Polycaprolactone (PCL, MW 50,000; 25,090, Polyscience) in and 2.50 μg ml Amphotericin B (15290026, Thermo Fisher Scientific). powder was dissolved in chloroform (C2432, Sigma-Aldrich) at a con- The extraction was performed for 24 h at 37 C and 5% CO . L929 cells centration of 0.25 g ml and was left stirring and heating at 60 C (88102702, Sigma-Aldrich) were plated in a 96-well plate at a sub- overnight over a hotplate. The day after, 6 ml of PCL solution were spin- confluent density of 7000 cells per well in 100 μl of the same medium. coated on both silicon moulds (positive: 150 rpm for 30 s; negative: 200 L929 cells were incubated for 24 h at 37 C and 5% CO . After incuba- rpm for 30 s) pre-treated with chlorotrimethylsilane (92,361, Sigma- tion, the medium was removed from the cells and replaced with the Aldrich) to prevent permanent attachment of the PCL layers. The wa- extract (100 μl per well). After another incubation of 24 h, 50 μl per well fers were then left 30 min in the oven at 75 C to let the chloroform of XTT reagent (Cell proliferation kit 11465015001, Sigma-Aldrich) evaporate. After a cool-down period of at least 2 h, the PCL layer from were added and incubated for 4 h at 37 C and 5% CO . An aliquot of the positive wafer was peeled off, and the design (electrodes, traces and 100 μl was then transferred from each well into the corresponding wells pads) was filled with an aqueous solution of PEDOT:PSS:EG, which is of a new plate, and the optical density was measured at 450 nm by using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS; a plate reader (FlexStation3, MolecularDevices). Clean medium alone M122 PH1000, Ossila) mixed with 20 wt% ethylene glycol (EG; was used as a negative control, whereas medium supplemented with 324,558, Sigma-Aldrich), and cured overnight in an oven at 37 C. The 15% of dimethyl sulfoxide (D2650-5X5ML, Sigma-Aldrich) was used as a PCL layer from the negative wafer, instead, was peeled off after laser- positive control. cutting the openings for the electrodes and pads (Optec MM200-USP). Eventually, the two layers were manually aligned and fused together 2.6. Accelerated degradation over a hotplate at 60 C for a few seconds. A silver-based epoxy (H27D Kit Part A, Epo-Tek) was then applied on the pads and connectors were All-polymeric TNPs were prepared as described above, with the 4- positioned and immobilised with silicone (DC 734 RTV clear, Dow electrode design. After fabrication, samples were weighted to set the Corning). Three types of all-polymeric TNPs were fabricated with a initial weight value before starting degradation. Degradation of each different number of electrodes: 4 electrodes (500-μm in diameter; sample occurred in 10-ml PBS at 37 C and was accelerated by pH in- connector 143-56-801, Distrelec), 1 electrode (500-μm in diameter; no crease: 100 μl of NaOH (2 M NaOH Standard solution, 71,474-1L, Fluka) connector) and 17 electrodes (700-μm in diameter; connector were added to the solution to reach pH 12. At each time point, samples 61001821821, Wurth Elektronik). Samples with fluorescent PEDOT: were first gently dried with a tissue and then let dry completely under PSS:EG were prepared as previously described [19]. Briefly, poly(vinyl vacuum at room temperature for 4 h. Once dry, samples were weighted. alcohol) (PVA, MW 130,000; 563,900, Sigma-Aldrich) was dissolved in Normalised weight for each sample was computed as the ratio between deionization water 5 wt%. A composite solution (25 wt%) of PVA and the weight at each time point and the initial value. PEDOT:PSS:EG was prepared, deposited on the PCL, and baked (50 C, 10 min). (3-Glycidyloxypropyl)trimethoxylane (440,167, 2.7. Animal handling Sigma-Aldrich) was then deposited by vapour deposition at 90 C for 45 min. Afterwards, a solution of fluorescein isothiocyanate (FITC) labelled All experiments were conducted according to the animal authoriza- poly-L-lysine (P3543, Sigma-Aldrich) in phosphate buffered saline (PBS; tions GE13416 approved by the D´ epartement de l’emploi, des affaires 50 μg ml ) was drop-casted on the sample and allowed to react for 2 h ´ ´ ´ ´ sociales et de la sante (DEAS), Direction generale de la sante of the at room temperature. The sample was then rinsed with PBS (0.1 M), Republique et Canton de Geneve in Switzerland and VD3420 approved sodium chloride (0.1 M), and deionised water. ´ ´ by Service de la consommation et des Affaires veterinaires (SCAV) of the Canton de Vaud in Switzerland. All the experiments were carried out 2.2. Scanning electron microscopy during the day cycle. For the entire duration of the experiment, the health condition was evaluated three times a week, and the bodyweight Images were taken with a Schottky field emission scanning electrons was controlled once a week. Experiments were performed in adult (>1- microscope (SU5000, Hitachi) and image post-processing was per- month-old) mice. Male and female C57BL/6J mice (Charles River) were formed with ImageJ. kept in a 12 h day/night cycle with access to food and water ad libitum. White light (300 ± 50 lux) was present from 7 a.m. to 7 p.m. and red 2.3. Electrochemistry light (650–720 nm, 80–100 lux) from 7 p.m. to 7 a.m. Homozygous tm2.1(cre/ERT2)Litt B6.129P2(Cg)-Cx3cr1 /WganJ mice (Stock No: 021,160, tm14 Electrochemical characterization was performed with a three- The Jackson Laboratory) and homozygous B6.Cg-Gt(ROSA)26Sor (CAG-tdTomato)Hze electrode potentiostat (Compact Stat, Ivium) at room temperature. /J mice (Stock No: 007914, The Jackson Laboratory) Each all-polymeric TNP was immersed in PBS (pH 7.4) together with a were kept in a 12 h day/night cycle with access to food and water ad silver/silver chloride reference wire and a platinum counter wire. libitum. White light (300 ± 50 lux) was present from 7 a.m. to 7 p.m. and tm2.1 Impedance spectroscopy (IS) was measured between 1 Hz and 1 MHz no light from 7 p.m. to 7 a.m. Homozygous B6.129P2(Cg)-Cx3cr1 (cre/ERT2)Litt tm14 using an AC voltage of 50 mV. /WganJ mice and homozygous B6.Cg-Gt(ROSA)26Sor (CAG-tdTomato)Hze /J mice were crossed to obtain heterozygous mice bearing a tamoxifen-inducible expression of the tandem dimer Tomato 2.4. Resistance measures fluorescent in microglia in the brain. Mice were injected with tamoxifen Line resistance was measured using a data acquisition and logging dissolved in corn oil (75 mg kg ) to induce the expression at postnatal digital multimeter system (daq6510, Keithley). day 35. Surgery was performed between 2 weeks and 1 month after. 2.5. Cytotoxicity test 2.8. Surgical implantation A test on extract was performed on two all-polymeric TNPs (4-elec- Mice were anesthetised with isoflurane inhalation (induction 1 1 trodes design) sterilised by UV exposure, with a ratio of the product to 0.8–1.5 l min , 4–5%; maintenance 0.8–1.5 l min , 1–2%). Analgesia 2 1 extraction vehicle of 3 cm ml . The extraction vehicle was Eagle’s was performed by subcutaneous injection of buprenorphine (0.1 mg 1 1 minimum essential medium (11090081, Thermo Fisher Scientific) sup- kg ), and a local subcutaneous injection of lidocaine (6 mg kg ) and plemented with 10% foetal bovine serum (10270106, Thermo Fisher bupivacaine (2.5 mg kg ) with a 1:1 ratio. The depth of anaesthesia was Scientific), 1% penicillin-streptomycin (15070063, Thermo Fisher assessed with the pedal reflex, and artificial tears were used to prevent 2 L. Ferlauto et al. Biomaterials 274 (2021) 120889 the eyes from drying. The temperature was maintained at 37 C with a were processed and analysed using MATLAB (MathWorks). After each heating pad during both surgical procedures and recording sessions. The session, the mice were left to recover on a heating pad and later returned skin of the head was shaved and cleaned with betadine. Mice were then to their cage. placed on a stereotaxic frame, and the skin was opened to expose the skull. A squared craniotomy of approximately (4 × 4 mm) was opened 2.11. Euthanasia over the visual cortex (identified by stereotaxic coordinates), and the dura mater was removed. UV-sterilised all-polymeric TNPs were inser- Animals were euthanised with an injection of pentobarbital (150 mg ted in the cortex using a micromanipulator (SM-15R, Narishige). The kg ) under a chemical hood. The chest cavity was opened to expose the probes were always implanted with the PEDOT:PSS:EG layer exposed to beating heart, and a needle was inserted in the left ventricle, while the the caudal part of the brain to clearly discriminate the two sides during right atrium was cut to allow complete bleeding. The animal was image analysis. A reference screw electrode was implanted in the rostral immediately perfused with PBS followed by a fixative solution of 4% side of the cranium. The craniotomy was closed using dental cement, paraformaldehyde (PFA) in PBS. At the end of the procedure, the head of and an extra layer of dental cement was applied to secure the probe. The the animal was removed, and the brain collected and placed in 4% PFA mice were left to recover on a heating pad and later returned to their overnight for post-fixation. cage. 2.12. Histological analysis 2.9. Acute induction of seizure and monitoring of epileptic activity Brain samples were cryoprotected in sucrose 30% and frozen in Immediately after surgery, mice were removed from the stereotaxic optimal cutting temperature compound. 20-μm thick horizontal sections apparatus while still under general anaesthesia, a needle electrode was of the brain were obtained using a cryostat (Histocom, Zug, Switzerland) placed subcutaneously in the dorsal area near the tail as ground, and the and placed on microscope slides. The sections were washed in PBS, four channels of all-polymeric TNPs and the reference electrode were permeabilised with PBS + Triton 0.1% (Sigma-Aldrich), left for 1 h at connected to the amplifier (BM623, Biomedica Mangoni). The signals room temperature in blocking buffer (Triton 0.1% + 5% normal goat were recorded using the WinAver software (Biomedica Mangoni). The serum), and incubated overnight at 4 C with primary antibodies for the recording was started (bandpass filtered 0.1–2000 Hz and sampling glial fibrillary acidic protein (GFAP; 1:1000; Z0334, Dako), the cluster of frequency 8192 Hz) and, after a baseline period, 20 μl of pentylenetet- differentiation 68 protein (CD68; 1:400; MCA1957, Biorad) and neural 1 1 razol (PTZ; 50 mg ml , 45 mg kg ) were delivered via intraperitoneal nuclei (NeuN; 1:500; ABN90P, Millipore). The day after, the sections injection. were incubated for 2 h at room temperature with secondary antibodies (1:500; Alexa Fluor 647 and 488, Abcam), counterstained with DAPI (1:300; Sigma-Aldrich) and mounted for imaging with Fluoromount 2.10. Chronic recordings solution (Sigma-Aldrich). Representative images were acquired with a confocal microscope (LSM-880, Zeiss). For image segmentation and A first acute recording session was carried out immediately after the implantation to check the functionality of the TNPs at the starting point quantification, images were acquired using a slide scanner microscope of the experiment. Chronic recordings were then performed 1- and 2- (VS120, Olympus; 20× objective, pixel size 0.34 μm) and analysed in weeks post-implantation and then every 2 weeks until 12 weeks. The Python, using the scikit-image package (https://scikit-image.org/). mice were dark-adapted for 2 h before each chronic recording session. Mice were anaesthetised with a mixture of ketamine (87.5 mg kg ) and 2.13. Whole-brain imaging xylazine (12.5 mg kg ). The depth of anaesthesia was assessed with the pedal reflex, and artificial tears were used to prevent the eyes from Brain clarification was performed according to a previously ◦ ◦ drying. The temperature was maintained at 37 C with a heating pad described procedure [20]. Briefly, after overnight post-fixation at 4 C in during both surgical procedures and recording sessions. Mice were PFA 4%, the brain was immersed in hydrogel solution (Acrylamide 40% placed on a stereotaxic apparatus in front of a Ganzfeld flash stimulator + VA-044 initiator powder in PBS) at 4 C for three days. The hydrogel (BM6007IL, Biomedica Mangoni) and a needle electrode was placed polymerization was induced by keeping the sample at 37 C for 3 h. subcutaneously in the dorsal area near the tail as ground. The four Then, the brain was passively clarified in 4% sodium dodecyl sulfate channels of all-polymeric TNPs and the reference electrode were con- clearing solution (pH 8.5) for four weeks under gentle agitation at 37 C. nected to the amplifier (BM623, Biomedica Mangoni). The signals were The whole clarified brain was transferred to Histodenz solution at pH 7.5 recorded using the WinAver software (Biomedica Mangoni). First, a (Sigma-Aldrich). Brains were immersed in a refractive index matching baseline recording was obtained for 2.5 min (bandpass filter 0.1–2000 solution (RIMS) containing Histodenz for at least 24 h before being Hz and sampling frequency 8192 Hz). The recording noise was extracted imaged. Brains were glued to a holder and immersed in a 10 × 20 × 45 from the baseline recording using a moving average window of 10 mm quartz cuvette filled with RIMS. The cuvette was then placed in a points; the mean ± s.d. was calculated for each 500 ms epoch, then the chamber filled with oil with (n = 1.45; Cargille). A custom-made mean s.d. of all the epochs was averaged to obtain the noise of the whole light-sheet microscope optimised for labelled clarified tissue was used recording. Channels with a noise level higher than the average plus to image the implant within the mouse brain (COLM, clarity optimised twice the s.d. of the noise at each time point were considered as non- light-sheet microscope [21]). The sample was illuminated (488 and 554 working channels and excluded from further analysis. Local field po- nm) by two digitally scanned light-sheets coming from opposite di- tentials (LFPs) were then acquired for 2.5 min (bandpass filter 0.1–100 rections, and the emitted fluorescence was collected by high numerical Hz and sampling frequency 819.2 Hz). After the recordings, a high pass aperture objectives (Olympus XLPLN10XSVMP, N.A 0.6) filtered filter at 0.5 Hz was applied, and the periodogram was calculated using (Brightline HC 525/50, Semrock) and imaged on a digital CMOS camera the Welch method (window of 4 s) on the whole recording. The area (Orca-Flash 4.0 LT, Hamamatsu) at a frequency ranging between 5 and below the curve between 0.5 and 4 Hz frequencies (corresponding to the 10 frames per second. A self-adaptive positioning of the light sheets delta band) was approximated using the composite Simpson’s rule. The across Z-stacks acquisition ensured an optimal image quality over up to power of the delta band was then divided by the total power (area below 1 cm of tissue. To acquire images of whole samples at lower resolution the periodogram) to obtain the relative power of the delta band. Last, another custom-made light-sheet microscope was used (mesoSPIM, visually evoked potentials were recorded (bandpass filter 0.1–200 Hz mesoscale selective plane illumination microscopy [22]). The micro- and sampling frequency 2048 Hz) upon the presentation of 10 consec- scope consists of a dual-sided excitation path using a fibre-coupled utive flashes (4 ms, 10 cd s m ) delivered at 1 Hz of repetition rate. Data multiline laser combiner (405, 488, 561 and 647 nm; Toptica MLE) 3 L. Ferlauto et al. Biomaterials 274 (2021) 120889 and a detection path comprising a 42 Olympus MVX-10 zoom macro- acquisition was made using custom software written in Python. Z-stacks scope with a 1× objective (Olympus MVPLAPO), a filter wheel (Ludl were acquired at 3 μm spacing with a zoom set at 2x resulting in an 96A350), and a CMOS camera (Orca Flash 4.0 V3, Hamamatsu). The in-plane pixel size of 7.8 μm (2048 × 2048 pixels). The excitation excitation paths also contain galvo scanners for light-sheet generation wavelength was set at 561 nm with an emission 530/40 nm bandpass and reduction of shadow artefacts due to absorption of the light-sheet. In filter (BrightLine HC, AHF). addition, the beam waist is scanned using electrically tuneable lenses (EL-16-40-5D-TC-L, Optotune) synchronised with the rolling shutter of 2.14. Statistical analysis and graphical representation the CMOS camera. This axially scanned light-sheet mode leads to a uniform axial resolution across the field-of-view of 5 μm. Image Statistical analysis and graphical representation were performed Fig. 1. All-polymeric transient neural probes. (a) Picture of a fully assembled TNP with four PEDOT:PSS:EG electrodes. (b) Sketch of the TNP highlighting the three layers: PCL encapsulation, PEDOT:PSS:EG electrodes and PCL substrate. (c) Scanning electron microscopy image of the four electrodes encapsulated in PCL (image ◦ ◦ ◦ tilt 45 ). (d) Magnification of one electrode (image tilt 45 ). (e) Scanning electron microscopy image of the cross-section of one encapsulated trace (image tilt 10 ). (f) Plots of the impedance magnitude (black) and impedance phase angle (grey) of 4 electrodes from a representative TNP. (g) Quantification of the impedance magnitude (left) and impedance phase angle (right) at 1 kHz for all the 4 electrodes from 3 TNPs (respectively in red, blue, and green). (h) Picture of traces with decreasing width: 200 μm, 120 μm, 60 μm, 40 μm and 20 μm. Traces are 6.3-mm long and pads are 2 × 2 mm . (i) Quantification of the trace resistance as a function of the trace width. Kruskal-Wallis: p < 0.0001. Dunn’s multiple comparisons test: 200 μm vs. 120 μm p > 0.9999; 200 μm vs. 60 μm p = 0.0202; 200 μm vs. 40 μm p = 0.0001; 200 μm vs. 20 μm p > 0.0004; 120 μm vs. 60 μm p = 0.5545; 120 μm vs. 40 μm p = 0.0158; 120 μm vs. 20 μm p = 0.0118; 60 μm vs. 40 μm p > 0.9999; 60 μm vs. 20 μm p = 0.6738; 40 μm vs. 20 μm p > 0.9999. (j) Picture of electrodes not encapsulated with decreasing electrode diameter and trace width: 500/200 μm, 300/120 μm, 150/60 μm, 100/40 μm and 50/20 μm (electrode diameter/trace width). (k) Quantification of the impedance magnitude at 1 kHz as a function of the electrode diameter. Kruskal-Wallis: p = 0.6462. (l) Quantification of impedance phase angle at 1 kHz as a function of the electrode diameter. Kruskal-Wallis: p = 0.0002. Dunn’s multiple comparisons test: 500 μm vs. 300 μm p = 0.1578; 500 μm vs. 150 μm p = 0.0009; 300 μm vs. 150 μm p = 0.2790. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4 L. Ferlauto et al. Biomaterials 274 (2021) 120889 with Prism 8 (Graph Pad). The normality test (D’Agostino & Pearson diameter and 20-μm trace width) resulted not connected (Fig. 1j, black omnibus normality test) was performed in each dataset to justify the use arrow), as expected due to the narrow trace. Electrodes with 100-μm of a parametric or non-parametric test. The box plots always extend from dimeter (40-μm trace width) resulted not working because the electrode the 25th to 75th percentiles. The line in the middle of the box is plotted openings in the PCL encapsulation layer closed during the bonding to the at the median. The + is the mean. The whiskers go down to the smallest PCL substrate. Hence, the minimum dimensions reached for the elec- value and up to the largest. In each figure p-values were represented as: trode diameter was 150 μm (60-μm trace width). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Pure PEDOT:PSS electrodes have been previously described for in- vitro applications to record cardiac and neural signals and in-vivo neuromodulation. A flexible all-polymer microelectrode array reported 3. Results impedance magnitude value (1.5 MΩ) at 1 kHz for 120-μm electrodes substantially higher than the ones reported here [35]. The higher 3.1. Probe fabrication and characterization impedance magnitude was caused by the deposition method of PEDOT: PSS in a polymeric channel, which led to surface coating only and not to We fabricated TNPs based on PCL as substrate and encapsulation layers, and PEDOT:PSS doped with EG as a conductive element, by using the complete filling of the channel, thus reducing the electrode surface area. A second device exploring inkjet-printed PEDOT:PSS reported an soft lithographic techniques (Fig. 1a and b and Figs. 1). PCL is a com- mercial biodegradable polyester widely used in biomedical applications impedance magnitude value (19.5 kΩ) at 1 kHz for an electrode area corresponding approximately to the area of a round electrode with a [23–26]. It is easy to process and has a longer degradation time (several diameter of 300 μm comparable to the TNP [36]. In a third report, months or years [23]) compared to most biodegradable polymers. PEDOT:PSS/glycerol electrodes showed an average impedance magni- PEDOT:PSS dispersed in water is commercially available, has excellent tude at 1 kHz of 6.3 kΩ for an electrode area of 0.5 mm (corresponding biocompatibility, has the ability to support cells viability [27,28], and to the area of a round electrode with a diameter of 800 μm) which is can be integrated into softer systems like elastomers [29,30] and similar to the TNP [37]. hydrogels [31,32]. EG doping of PEDOT:PSS is known to improve its conductivity [33,34]. The micro-structured PCL-based substrate Last, cytotoxicity tests performed in-vitro showed a cell viability of 96.9 ± 7.2% (mean ± s.d., 2 probes, 6 replicas per probe) and opened (thickness 60–70 μm) and encapsulation (thickness 60–70 μm) layers have been obtained by replica moulding. The hollow regions of the the way for the in-vivo validation of the devices (Fig. 2). substrate layer have been manually filled with PEDOT:PSS:EG to create electrodes, traces and contact pads (Figs. 1). Electrodes are 500-μm in 3.2. Functional validation in-vivo diameter (Fig. 1c and d) and traces are 200-μm wide (Fig. 1e). At the electrode level, the PEDOT:PSS:EG layer is approximately 3-μm thick, To assess the capability of all-polymeric TNPs in recording neural while at the trace level, it is approximately 1-μm thick (Figs. 2). After activity, we performed in-vivo acute brain recordings in mice upon in- curing of the PEDOT:PSS:EG, the PCL encapsulation layer has been duction of epileptic seizures. All-polymeric TNPs were implanted in the manually flip-bonded to the substrate layer via a low-temperature pro- visual cortex area of anaesthetised mice (Fig. 3a), and the neural activity cess (total probe thickness ranging from 120 to 140 μm). This procedure was recorded both before and after intraperitoneal injection of the ensured a complete filling of the trenches for the conductive traces convulsant PTZ, which is routinely used to test anticonvulsants in ani- (Fig. 1e). IS showed remarkable performances of all-polymeric TNPs mals [38–40]. The transition between resting state and epileptic activity based on PEDOT:PSS:EG, characterised by low impedance magnitude after PTZ injection was clearly detected by the TNPs with an excellent (Fig. 1f) and low impedance phase angle (Fig. 1g) over a wide range of signal to noise ratio (Fig. 3b), thus demonstrating the potential of these frequency (from 10 Hz to 100 kHz), which makes them appropriate for all-polymeric TNPs for monitoring epileptic activity. the recording of both low- and high-frequency neuronal signals, such as Next, we performed chronic in-vivo recordings of baseline activity, LFPs and neural spiking activities respectively. The comparison of LFPs at rest and visually evoked potentials (VEPs) to assess the longevity multiple probes showed very low intra-probe variability. On the other of the device. All-polymeric TNPs were implanted in the visual cortex hand, the inter-probe variability might be explained by the manual area of mice, and their functionality was evaluated through periodic deposition process of PEDOT:PSS:EG resulting in a variable thickness of recordings up to 3 months post-implantation (Fig. 4). The broad-band the conductive layer. To verify that the excellent electrochemical char- noise of recordings was computed from baseline recordings (2.5 min, acteristics of the TNPs were not related to shortcuts between electrodes bandpass filter 0.1–2000 Hz and sampling frequency 8192 Hz). The caused by the porosity of the PCL scaffold, we took advantage of an noise plot (Fig. 4a) shows the evolution of the noise level for each all-polymeric TNP with a modified design embedding 17 electrodes working electrode over time (filled circles). Once an electrode was found (Figs. 3). In this specific device, four traces were interrupted, due to a not properly functional (empty circles), it was excluded from further gap in the PEDOT:PSS:EG layer (Figs. 3a-c). As expected, only the cor- analysis. The applied criteria of exclusion were the following: i) responding electrodes were rightfully not functional (Figs. 3d and e), appearance of periodic artefacts not linked to any biological function thus confirming that shortcuts between electrodes are not present. Be- (red arrow in Fig. 4a); ii) noise level higher than the average plus twice sides electrochemistry, we also measured the averaged line resistance of the standard deviation of the noise at each time point; iii) lack of VEPs. the traces, which was 319.5 ± 75.01 Ω (±s.d., n = 8 traces; Fig. 1h and i) The number of excluded electrodes progressively increased over time for 200-μm wide and 6.3-mm long traces. (Fig. 4b), with 3 out of 16 electrodes still properly functional after three Then, we investigated the minimum dimensions for electrodes and months. LFPs (2.5 min, bandpass filter 0.1–100 Hz and sampling fre- traces that could be reached using these materials and fabrication quency 819.2 Hz) are not strongly affected by broad-band noise because methods. First, we fabricated narrow traces and measured the average of the low pass filter at 100 Hz. On the other hand, LFPs are charac- line resistance (n = 8 traces for each dimension; Fig. 1h and i). As ex- terised by a strong delta rhythm (0.5–4 Hz) due to the animal anaes- pected, the line resistance significantly increased with the decrease of thesia (Fig. 4c). Early after implantation, electrodes showed a narrow the width. It must be noted that for 20-μm wide traces, only 3 out of 8 power spectral density (PSD) in the range of delta rhythm (black traces traces appeared to be connected. Therefore, the resolution limit for in Fig. 4c and black lines in Fig. 4d). Over time, because of the electrode traces is 40 μm. Next, we fabricated TNPs with smaller electrodes and deterioration, the normalised PSD shows activity at higher frequency in traces (Fig. 1j) and tested IS (n = 7 electrodes for each dimension). As electrodes that were previously considered not properly functional expected, the impedance magnitude (Fig. 1k) and the impedance phase because of high broad-band noise (grey line in Fig. 4d). Conversely, angle (Fig. 1l) increased with the reduction of the electrode diameter. electrodes still considered functional showed a narrow PSD (red line in Among the dimensions tested, the smallest electrode (50-μm electrode Fig. 4d). As a consequence, the relative power of the delta band 5 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 2. Cytotoxicity in-vitro. (a) Representative optical images for each condition tested: positive control (left), negative control (middle) and TNP (right). (b) Quantification of the mean (±s.d.) cells viability in the three conditions tested. Positive control 0 ± 1.8% (9 replicas); negative control 100 ± 6.8% (9 replicas); all- polymeric TNPs 96.9 ± 7.2% (2 probes, 6 replicas per probe). One-way ANOVA: F = 853.4 and p < 0.0001. Tukey’s multiple comparisons test: positive control vs. negative control p < 0.0001; positive control vs. neural probes p < 0.0001; negative control vs. neural probes p = 0.4852. Fig. 3. Acute recordings with all-polymeric transient neural probes. (a) Sketch of the TNP implanted in a mouse brain for in-vivo recordings. (b) Detection of epileptic activity after injection of PTZ (black arrow) from the four electrodes of a all-polymeric TNP. The red and blue boxes show an enlargement of the recorded activity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) decreases with the deterioration of the electrodes (Fig. 4e). For each factors could contribute to the deterioration of PEDOT:PSS in-vivo, electrode, early after implantation, a low noise correlates with a high related to both materials properties and interaction with the biological relative power of the delta band. However, at the exclusion point, there environment. A commonly reported problem is the adhesion of PEDOT: is a reduction of the relative delta power which is globally proportional PSS to the substrate [45]. The weak adhesion between PEDOT:PSS and to the increase of noise (linear regression: slope = 1.369, R square = polyesters [46], like PCL, plays a crucial role since PEDOT:PSS might be 0.527). Overall, all-polymeric TNPs retain good in-vivo recording ca- subjected to water-induced swelling and delamination during the pabilities for months after implantation, even if they progressively lose degradation of the PCL scaffold. PEDOT has also been reported to un- functionality and only a few electrodes are still properly functional after dergo degradation by hydrolysis when exposed to salt aqueous solutions three months. [47] or by high concentration of hydrogen peroxide, which is a physi- At this stage, the cause of electrode deterioration is not easy to ological oxidant [48,49]. Macrophages cultured with PEDOT:PSS determine. So far, only a few studies have investigated the chronic generated a significant amount of hydrogen peroxide, that is a signifi - performances of PEDOT:PSS in-vivo [41–44], and the results indicated a cant factor involved in cellular phagocytosis [50]. PEDOT:PSS could reduction in performance after a few months of implantation. Many possibly be degraded by the combination of hydrolysis and presence of 6 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 4. Long-term functioning in-vivo. (a) Noise evaluation as a function of time during in-vivo chronic recordings at fixed time points. Different colours correspond to different mice; filled circles correspond to the electrodes considered functional and thus included in the calculation of the average (±s.d.) noise values (black circles and dashed line), while empty circles correspond to the electrodes considered not correctly working and thus excluded from the calculation of the average noise. The two insets show single baseline recordings (left) and VEPs (right) from all the four electrodes of the TNP implanted in mouse 3 at week 1 and week 6 post- implantation; the red dashed line indicates the light stimulus for VEPs (10 cd s m ). The traces in grey are from electrodes considered not functional. The red arrows show periodic artefacts not linked to any biological function. (b) Plot of the percentage of working electrodes as a function of time during in-vivo chronic exper- iments. (c) Representative example of LFPs recorded from all the electrodes of the TNP implanted in mouse 3 at week 1 and week 6 post-implantation. The traces in grey are from electrodes considered not functional. (d) Representative examples of normalised power spectral densities obtained from the recordings from two electrodes (numbers 2 and 3) of the probe implanted in mouse 3 at week 1 (black traces) and week 6 (black grey and red) after implantation. The trace in grey is from the electrode considered not functional, while the trace in red is from the electrode considered still functional. (e) Correlation between the relative power of the delta band and the noise of the electrodes immediately after implantation (16 electrodes, black circles) or at the time point of exclusion (13 electrodes, empty circles). The black line is the linear regression, while the grey lines the 95% confidence interval. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) hydrogen peroxide following device implantation [45]. segmentation (Figs. 4). Images were rotated to align the probe hori- zontally (Figs. 4a) and converted to binary using a local threshold al- gorithm: all pixels which intensity was above the threshold were 3.3. Evaluation of the foreign body reaction assigned the value of 1, while to the rest of the pixels were assigned the value of 0 (Figs. 4b). A group of adjacent pixels with value 1 was defined To investigate the acute phase of the foreign body reaction induced as a blob, and the area of each blob was calculated, along with the co- by TNPs, we performed a histological paired comparison between all- ordinates of its centroid. The image was divided into 10 × 10 quadrants polymeric TNPs and polyimide (PI) implants, surgically placed in the (Figs. 4c), for each of which the total area of blobs (with their centroid two hemispheres of the brain of the same mice (Fig. 5a). The PI implants included in that quadrant) was computed. Then, the total area occupied were chosen as a reference for non-degradable flexible implants. Also, by blobs in the zone adjacent to the probe (Figs. 4d) was calculated as the implants have been manufactured with similar dimensions to be the cumulative area of all the quadrants included in a rectangle defined more comparable (TNP: ~130-μm thick and 1.3-mm wide; PI: 125-μm by the extremities of the probe (manually specified for each of the im- thick and 0.7-mm wide). Evaluations have been made at 1- and 2- ages), minus the area of the probe itself. The percentage of the area months post-implantation, 6 mice for the first time point and 5 mice occupied by blobs was computed as the area occupied by blobs over the for the second. proximal area. The average percentage of the proximal area occupied by For each mouse, horizontal brain slices were sampled at different the blobs for each sample was used as a parameter to compare the cortical depths (Fig. 5a from 1 to 4) and analysed after image 7 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 5. Foreign body reaction induced by all-polymeric transient neural probes. (a) Sketch of the TNP and PI probe implanted in both visual cortices of a mouse. For each probe, four slices were taken at four depths into the cortical layers. (b,c) Example of GFAP (in red) and CD68 (in cyan) expression in horizontal slices (four depths into the cortical layers indicated by the numbers) obtained from the hemisphere implanted with the TNP (b) and with the PI probe (c) 1-month post- implantation. (d,e) Quantification of the area occupied by GFAP signal (d) and CD68 signal (e) at 1-month and 2-month post-implantation for TNPs and PI probes. Results from all the images at each depth were averaged together: 3 images per depth. (f,g) Quantification of the normalised area occupied by the CD68 signal above (PEDOT:PSS:EG side) and below (PCL-only side) TNPs at the level of the electrode at 1-month (f) and 2-month (g) post-implantation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) experimental groups. Three images for each depth were averaged. The Next, we quantified the spatial extent of the GFAP and CD68 signals staining for the GFAP (Fig. 5b and c) and the corresponding quantifi - around the TNP at each cortical depth. Each image (one per depth) was cation analysis (Fig. 5d) performed on segmented images showed that rotated to align the probe vertically (Fig. 6a). Two regions of interest, the area occupied by the GFAP signal in the zone adjacent to the probe one on the left and one on the right of the probe, were manually defined exhibits a decreasing trend over time for all-polymeric TNPs (p = as rectangles perpendicular to the probe, spanning from the edge of the 0.0178, two-tailed unpaired t-test) and a stable trend for PI probes (p = probe to the border of the image (Fig. 6b). For each of them, the average 0.6924, two-tailed unpaired t-test). Also, the staining for CD68 (Fig. 5b, intensity along the horizontal axis was computed, starting from the edge c), a marker of phagocytic structures in microglia [51], and the corre- of the probe to the border of the image. The average vector of intensities sponding quantification analysis (Fig. 5e) showed that the area occupied was normalised by dividing each point by the maximum value among by the CD68 signal exhibits a slightly increasing trend over time for the two regions of interest and a normalised intensity plot was generated all-polymeric TNPs (p = 0.2897, two-tailed unpaired t-test), while it (Fig. 6c). For each cortical depth, normalised intensity plots from all remains stable for PI probes (p = 0.5905, two-tailed unpaired t-test). mice were averaged (6 mice at 1-month post-implantation and 5 mice at Histological samples of brains implanted with TNPs showed a clus- 2-months post-implantation) to determine the spatial profile. The spatial tering of the CD68 signal at the level of the electrode (slice 4 in Fig. 5b profiles of both GFAP (Fig. 6d and e) and CD68 (Fig. 6f and g) resulted and c), the only point where the PEDOT:PSS:EG is exposed to the tissue. uniform along the vertical axis of the probe (cortical depths) at both 1- Following this observation, we performed a second analysis where the and 2-months post-implantation (Fig. 6d,f and Fig. 6e,g respectively). In segmented images were divided into two parts: the side of the probe order to quantify the extent of the GFAP and CD68 signals, we measured with the PEDOT:PSS:EG exposed and the side of the probe with only the the distance from the probe at which the average profile among the 4 PCL exposed (Figs. 5). For each side, the cumulative area occupied by depths (black lines in Fig. 6d–g) is reduced by 50% (red dots in blobs was calculated along the horizontal axis and normalised by the Fig. 6d–g). The quantification (Fig. 6h) revealed that the GFAP signal is global maximum. The probes were always implanted with the PEDOT: wider 1-month post-implantation (116.82 and 206.16 μm respectively PSS:EG layer exposed to the caudal part of the brain to discriminate the for the left and the right side of the probe) and it is reduced 2-months two sides during image analysis. The analysis confirmed that the CD68 post-implantation (96.55 and 95.18 μm respectively for the left and signal is highly clustered on the side of the PEDOT:PSS:EG electrodes at the right side of the probe), although the reduction is not significant (p both time points (Fig. 5f and g), suggesting an interaction between = 0.3333, two-tailed Mann-Whitney test). This result is in agreement microglia and PEDOT:PSS:EG. with the previous analysis (Fig. 5d). The CD68 signal is restricted to a 8 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 6. Spatial extent of the foreign body reaction. (a) Example of CD68 expression in a horizontal slice obtained from the hemisphere implanted with the TNP at 1- month post-implantation. (b) Definition of the two regions of interest on the left and on the right of the TNP. (c) Average vectors of normalised intensities (top) and normalised intensity plots (bottom). (d,e) Normalised intensity plots for GFAP signal 1-month (d) and 2-months (e) post-implantation. The grey lines correspond to the four cortical depths and the black line is the average. The red dot corresponds to a 50% decrease in the intensity. (f,g) Normalised intensity plots for CD68 signal 1-month (f) and 2-months (g) post-implantation. The grey lines correspond to the four cortical depths and the black line is the average. The red dot corresponds to a 50% decrease in the intensity. (h) Quantification of the distance from the TNP at which the intensity plot shows a 50% decrease. (i) Example of NeuN expression in a horizontal slice obtained from the hemisphere implanted with the TNP at 1-month post-implantation. (j) Quantification of the area occupied by NeuN signal at 1- month and 2-months post-implantation of the TNPs compared to a control (Ctrl) area located far from the TNPs. Each data point corresponds to one cortical depth, average of 3 images per depth. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) small area around the probe (1-month: 85.56 and 62.88 μm; 2-months: evidence suggests that the presence of PEDOT:PSS:EG detached from the 75.60 and 78.00 μm; respectively for the left and the right side of the PCL substrate stimulates phagocytic activity of microglial cells, and that probe), and it does not change from 1- to 2-months post-implantation microglia are participating in the clearance of PEDOT:PSS:EG from the (Fig. 6h; p > 0.9999, two-tailed Mann-Whitney test), in agreement implantation site. Horizontal brain sections at 1-month with the previous analysis (Fig. 5e). post-implantation also revealed microglial cells (in red) localizing in We also quantified the area occupied by NeuN signal (a marker for the region where PEDOT:PSS:EG (in green) is delaminated or directly in neuronal cells) around the TNP at the four cortical depths (Fig. 6i) contact with the brain (Fig. 7d). These cells are CD68 positive (in white), compared to a control area located far from the TNP. The quantification the presence of intracellular phagocytic structures. Based on the results analysis (average of three sections per cortical depth), performed on one discussed above, we hypothesise that implanted TNPs lead to the for- mouse per time point (1- and 2-months post-implantation), showed that mation of a less tight glial scar compared to PI probes. As a consequence, the area occupied by the NeuN signal is not statistically different be- microglia have the space needed to access the probe and phagocyte the tween the two areas (Fig. 6i; 1-month: p = 0.3750, Wilcoxon matched- delaminated PEDOT:PSS:EG flakes. pairs signed rank test; 2-months: p = 0.6250, Wilcoxon matched-pairs signed rank test). 3.4. Probe’s degradation The clustering of the CD68 signal we observed at the level of the PEDOT:PSS:EG electrode (Fig. 5f and g) encouraged us to further One of the most exciting features of transient medical devices is their investigate the interaction between phagocytic microglial cells and natural ability to disappear in the body, which in turns reduces the long- PEDOT:PSS:EG. To this end, we fabricated TNPs with a PCL substrate term foreign body reaction. PCL bulk degradation occurs slowly with a coated with PEDOT:PSS:EG labelled with the fluorescent marker FITC time scale spanning several months or years [23], thus making an [19] and implanted them into the cortex of mice expressing tdTomato in in-vivo assessment in animals extremely challenging. In order to inves- microglia (Fig. 7a). In this experiment, all-polymeric TNPs were not tigate the degradation process of all-polymeric TNPs, we performed encapsulated to enhance interaction between microglial cells and in-vitro accelerated degradation tests, by leaving 3 TNPs immersed in PEDOT:PSS:EG. High-resolution imaging on clarified brains 1-month PBS at 37 C and pH 12 and monitoring their weight over time. Scanning (Fig. 7b) and 2-months (Fig. 7c) post-implantation showed colocaliza- electron microscope images showed the appearance of micro- and tion of PEDOT:PSS:EG (in green) and microglial cells (in red). This macro-cracks over time due to hydrolysis (Fig. 8a), and all-polymeric 9 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 7. | Integration with microglia. a, 3D mesoSPIM image of a clarified brain sample with fluorescent microglia (red) implanted with a TNP with FITC-labelled PEDOT:PSS at 1-month post-implantation. b,c, COLM images of the probe surface, showing fluorescent PEDOT:PSS (in green) and microglia (in red) colocaliza- tion 1-month (b) and 2-months (c) post-implantation. Microglia engulf PEDOT:PSS:EG particles which are detaching from the probe (white arrows). d, Horizontal brain section of mouse brain with fluorescent microglia (in red) implanted with a fluorescent TNP (without encapsulation) at 1-month post-implantation. e,k, Various magnifications of the section in d (corresponding letters) showing phagocytic microglia engulfing PEDOT:PSS:EG (in green). CD68 (in white) is a specific marker for microglial phagocytic structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) TNPs undergo full degradation in about one year of time at pH 12 electrode and neurons, eventually impairing the recording capabilities (Fig. 8b). A comparison between the days to reach a weight loss of 5% at of the device [54,55], and acts as a barrier for growth factors, ions, and pH 12 and at pH 7.4 revealed that the test has an acceleration factor of signalling molecules, thus promoting neural apoptosis and impeding about 2.5. Slow degradation is an appealing feature for TNPs, since it axon regeneration [56,57]. In the case of TNPs, the presence of a limited might allow for better tissue remodelling and reduced chronic trauma at glial scar allows tissue remodelling around the probe, and, as a conse- the implantation site [26,52,53]. To provide evidence of slow degra- quence, neurons (labelled against NeuN in green) are free to relocate dation in-vivo of all-polymeric TNPs, we implanted them in the cortex of within the device, which serves as a support scaffold during the degra- mice and subsequently performed immunofluorescence assays on hori- dation (Fig. 8d). This evidence of the probe slow degradation in-vivo is a zontal brain slices at a fixed time point. Upon long-term implantation (9 promising sign of reduced chronic trauma. months), the all-polymeric TNPs showed a minor chronic glial scar (Fig. 8c), evaluated with staining for astrocytes against GFAP. The for- 4. Discussion mation of a glial scar should be minimised or avoided for several rea- sons: it increases the distance between the recording surface of the In biomedical engineering and medicine, transient devices are a 10 L. Ferlauto et al. Biomaterials 274 (2021) 120889 Fig. 8. Degradation of all-polymeric transient neural probes. (a) Scanning electron microscopy images at several magnifications of one all-polymeric TNP obtained ◦ ◦ 191 days after soaking in PBS at 37 C and pH 12. (b) Plot of the normalised probe weight versus time of 3 TNPs during a degradation test in PBS at 37 C and pH 12. The red line is the average of the 3 TNPs. The grey dashed line is the time point (day 42) corresponding to a 5% weight loss. (c) Horizontal brain section from a mouse implanted with a TNP in the cortex. 9-months post-implantation staining against GFAP and NeuN. (d) The same image in (c) with a bright-field overlay to highlight the area occupied by the TNP. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) relatively new concept which has the potential to widen the range of during the recovery period and, possibly, into the effects of the following possible applications of implantable medical devices. We explored this pharmaceutical treatment. Also, the absence of metals in the implanted new concept with using fully polymer-based devices to exploit their portion of the device would provide magnetic resonance imaging unique characteristics and try to overcome some of the current limita- compatibility [29,60,61], allowing the pairing of the electrophysiolog- tions. So far, transient medical devices showed a short functional life- ical monitoring with structural and functional images, therefore time because of the fast degradation of degradable metals in contact obtaining a clearer picture of the brain activity during the recovery with body fluids [11,18]. period. In this work, we showed the fabrication and characterization of a Even though only a few electrodes (approximately 20%) were still polymer-based implantable device conceived to disappear after being operational three months post-implantation, we believe that there is still functional for a few months rather than a few days. A longer device room for fabrication strategies that can improve the device lifetime, such lifetime will allow, for instance, the development of prosthetic devices as enhancing the adhesion between PEDOT:PSS and PCL by using ad- for mid-term monitoring of the brain activity, either before or after ditives or by oxygen plasma treatment. Also, even if a performance surgery in chronic epileptic patients. Nowadays, clinicians make de- comparison with other inorganic and not-transient devices is out of the cisions on localised surgical treatments based on intracranial electro- scope of this study (due to the transient nature of the presented device), encephalography recordings that are typically lasting for only 1 or 2 the electrochemical characterization of all-polymeric TNPs showed a weeks in favour of patient compliance. On the other hand, recent pub- stable performance within a wide range of frequencies, thus allowing us lications in this field revealed that intracranial electroencephalography to perform acute and chronic in-vivo recordings in mice with excellent recordings do not stabilize until several weeks after implantation, due to signal to noise ratio. the invasiveness of the procedure and the inflammatory response of the Recently, not-transient implantable neural probes reached extreme tissue [58,59]. Therefore, an extended chronic monitoring period, miniaturisation to reduce chronic foreign body reaction [62,63]. Mini- together with the possibility to mitigate the inflammatory reaction, aturisation for the TNP is limited by materials properties and fabrication would enable a more accurate representation of the spatiotemporal methods. However, a detailed study of the foreign body reaction variability of the seizures, translating into a better diagnosis for the revealed that all-polymeric TNPs were responsible for a minimal glial patient. Furthermore, a transient device with extended recording scar around the implant. As a consequence, neurons were able to colo- capability would allow for continuous monitoring after the surgical nize the slowly degrading PCL layers, hence promoting tissue remodel- procedure, thus providing essential insights into the brain activity ling: such behaviour is the opposite to what is usually observed with 11 L. Ferlauto et al. Biomaterials 274 (2021) 120889 [3] C. Dagdeviren, S.-W. Hwang, Y. Su, S. Kim, H. Cheng, O. Gur, R. Haney, F. permanent implants of similar sizes, where neurons are depleted from G. Omenetto, Y. Huang, J.A. 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