**4. Hydrogel improves biocompatibility and mechanical properties**

#### **4.1. Improved biocompatibility**

*Hydrogels* such as Alginate are naturally occurring polysaccharides that are typically obtained from brown algae seaweed (**Figure 16**). These water-soluble biomaterials have many versatile properties such as gelling and film-forming. Hydrogels are biocompatible and are commonly used in biomedical or tissue engineering applications. Hydrogels are formed by cross-linking hydrophilic organic components and can respond to specific environmental changes [37]. This makes hydrogels the ideal polymer matrix for controlling the delivery of drugs and bioactive components into a complex biologic system.

**4.2. Improved mechanical properties**

**Figure 3**).

Hydrogels have also been proposed to improve the mechanical properties of the implantable microelectrodes. First, hydrogel coatings can be dehydrated to minimize the initial penetrating profile [41]. Second, the degree of reswelling can be controlled by using different cross-linking molecules to better anchor the position of the probe in the tissue after the implantation [42]. Third, the shear moduli of a hydrogel can be tightly regulated by controlling the cross-linking densities to reduce the stiffness mismatch between the hard silicon-based probes (~100 GPa) and the soft tissue (~10 kPa). Fourth, the uniformity of the hydrogel coating can be controlled by changing the concentration of the solution. Lastly, the hydrogel thickness can be controlled by quickly drying the coated surfaces in air and then overlaying additional layers on top.

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Despite the many advantages of hydrogels, the spatial distribution of neurons (as it relates to recording quality) has been a barrier to the practical implementation of hydrogel coatings on microelectrodes. For instance, neural recordings in the auditory cortex showed a significant loss in functionality (as determined by the number of clearly detectable units and the average signal-to-noise ratios [43]). In fact, in-vivo experiments showed that only 30% of the electrode sites (with 80 μm thick hydrogel coatings) could record detectable signals from surrounding neurons and that the average signal-to-noise ratio dramatically decreased (even for 5 μm thick coatings). The loss in neural recording quality is hypothesized to be caused by the reswelling properties of the dehydrated hydrogel coatings (which can affect the spatial distribution of the target neurons around the implanted electrode). In other words, the low signal detection is hypothesized to be caused by the post-implantation water absorption which pushes the target neurons away from the electrode surfaces as they rehydrate (and beyond the *cylindrical radius* distances in [12] that are required for effective electrode recording, as illustrated in

To solve the hydrogel swelling problem, [44] proposed to modify the electrode design to place the recording sites in closer proximity to the hydrogel's surface (**Figure 17**). The deposition of conducting polymer PEDOT has also been shown to restore some of the lost functionality of electrode sites with thicker hydrogel coatings [43]. Alternatively, fibrin can be selected (rather than Alginate) to be reabsorbed in the surrounding tissues 7 days after the implantation [45].

**Figure 17.** Electrode design with recording sites in closer proximity to the hydrogel's surface. Reproduced from [44].

Alginate hydrogels have been successfully applied as a coating on the Michigan probes. One study [38] showed that biodegradable neurotrophin-eluting hydrogels can be applied on the microelectrode arrays to attract neurites to the surface of the electrodes (for improved neuronelectrode proximity). Hydrogels can also be loaded with nanoparticles of anti-inflammatory agent dexamethasone (DEX) for drug delivery [39]. In-vitro drug release kinetics has revealed that 90% of the DEX can be successfully released from entrapped nanoparticles over 2 weeks. In addition, the impedance of the nanoparticle-loaded hydrogel coatings on microfabricated neural probes were equivalent to the unmodified/uncoated probes controls (indicating that the loaded-nanoparticles do not hinder electrical transport). Most importantly, the chronically implanted electrodes loaded with DEX were shown to maintain impedances in-vivo (unlike the control electrodes that showed increases after 2 weeks of implantation). This improvement in chronic neural recordings indicates the DEX-modified neural probes reduced the amount of glial inflammation via local administration of therapeutic agents. Hydrogels can also be used as a scaffold to encapsulate drug-incorporated biodegradable nanofibers for a more sustained and slower release (to reduce the burst effect [40]).

**Figure 16.** Optical microscopy image of an Alginate hydrogel coating on a Michigan probe. Reproduced from [41].

#### **4.2. Improved mechanical properties**

**4. Hydrogel improves biocompatibility and mechanical properties**

*Hydrogels* such as Alginate are naturally occurring polysaccharides that are typically obtained from brown algae seaweed (**Figure 16**). These water-soluble biomaterials have many versatile properties such as gelling and film-forming. Hydrogels are biocompatible and are commonly used in biomedical or tissue engineering applications. Hydrogels are formed by cross-linking hydrophilic organic components and can respond to specific environmental changes [37]. This makes hydrogels the ideal polymer matrix for controlling the delivery of drugs and bioactive

Alginate hydrogels have been successfully applied as a coating on the Michigan probes. One study [38] showed that biodegradable neurotrophin-eluting hydrogels can be applied on the microelectrode arrays to attract neurites to the surface of the electrodes (for improved neuronelectrode proximity). Hydrogels can also be loaded with nanoparticles of anti-inflammatory agent dexamethasone (DEX) for drug delivery [39]. In-vitro drug release kinetics has revealed that 90% of the DEX can be successfully released from entrapped nanoparticles over 2 weeks. In addition, the impedance of the nanoparticle-loaded hydrogel coatings on microfabricated neural probes were equivalent to the unmodified/uncoated probes controls (indicating that the loaded-nanoparticles do not hinder electrical transport). Most importantly, the chronically implanted electrodes loaded with DEX were shown to maintain impedances in-vivo (unlike the control electrodes that showed increases after 2 weeks of implantation). This improvement in chronic neural recordings indicates the DEX-modified neural probes reduced the amount of glial inflammation via local administration of therapeutic agents. Hydrogels can also be used as a scaffold to encapsulate drug-incorporated biodegradable nanofibers for a more sustained

**Figure 16.** Optical microscopy image of an Alginate hydrogel coating on a Michigan probe. Reproduced from [41].

**4.1. Improved biocompatibility**

186 An Excursus into Hearing Loss

components into a complex biologic system.

and slower release (to reduce the burst effect [40]).

Hydrogels have also been proposed to improve the mechanical properties of the implantable microelectrodes. First, hydrogel coatings can be dehydrated to minimize the initial penetrating profile [41]. Second, the degree of reswelling can be controlled by using different cross-linking molecules to better anchor the position of the probe in the tissue after the implantation [42]. Third, the shear moduli of a hydrogel can be tightly regulated by controlling the cross-linking densities to reduce the stiffness mismatch between the hard silicon-based probes (~100 GPa) and the soft tissue (~10 kPa). Fourth, the uniformity of the hydrogel coating can be controlled by changing the concentration of the solution. Lastly, the hydrogel thickness can be controlled by quickly drying the coated surfaces in air and then overlaying additional layers on top.

Despite the many advantages of hydrogels, the spatial distribution of neurons (as it relates to recording quality) has been a barrier to the practical implementation of hydrogel coatings on microelectrodes. For instance, neural recordings in the auditory cortex showed a significant loss in functionality (as determined by the number of clearly detectable units and the average signal-to-noise ratios [43]). In fact, in-vivo experiments showed that only 30% of the electrode sites (with 80 μm thick hydrogel coatings) could record detectable signals from surrounding neurons and that the average signal-to-noise ratio dramatically decreased (even for 5 μm thick coatings). The loss in neural recording quality is hypothesized to be caused by the reswelling properties of the dehydrated hydrogel coatings (which can affect the spatial distribution of the target neurons around the implanted electrode). In other words, the low signal detection is hypothesized to be caused by the post-implantation water absorption which pushes the target neurons away from the electrode surfaces as they rehydrate (and beyond the *cylindrical radius* distances in [12] that are required for effective electrode recording, as illustrated in **Figure 3**).

To solve the hydrogel swelling problem, [44] proposed to modify the electrode design to place the recording sites in closer proximity to the hydrogel's surface (**Figure 17**). The deposition of conducting polymer PEDOT has also been shown to restore some of the lost functionality of electrode sites with thicker hydrogel coatings [43]. Alternatively, fibrin can be selected (rather than Alginate) to be reabsorbed in the surrounding tissues 7 days after the implantation [45].

**Figure 17.** Electrode design with recording sites in closer proximity to the hydrogel's surface. Reproduced from [44].

#### **4.3. The "Dipping Method"**

**4.4. The "Dropping Method"**

Techniques for coating hydrogels onto microelectrodes include spray coating, brush coating, Dipping Methods [39–45], and micropipetting [38]. Unfortunately, none of these methods can produce uniform coatings in a reproducible manner [46]. Therefore, novel strategies such as the Molding Method (which involves the photopolymerization of hydrogels in a polyethylene tube) have been proposed to provide hydrogel coatings that are more reproducible and have a means to control coating uniformity and thickness (e.g. by varying the polyethylene tubing).

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**Figure 19** proposes a novel procedure called the "Dropping Method" to address the reported limitations of the Dipping Method. First, the microelectrode is permanently attached in one location to eliminate lateral movements (to improve reproducibility [40]). Second, a stage that contains solutions is automatically revolved to eliminate the requirement of approximating movements (to improve the average thickness [44]). Third, the stage is dropped downward to allow gravity to align the droplets and to evenly pull down the edges (to improve the radial thickness [45]). Fourth, the speed of dropping is automated by a digital micromanipulator (to improve the uniformity of the alginate coating around the shank of the probe [40]). Finally, the optimal contact angle is automated on a Dropmeter stage via programmable software (instead of relying on subjective interpretations of a human observer via optical microscopy as in [43]).

**Figure 19.** Dropping Method for hydrogel coating of neural electrodes with a fully-automated procedure.

**Figure 18.** Dipping Method for hydrogel coating of neural electrodes with optical microscopy. Reproduced from [43].

While previous solutions to the hydrogel swelling problem have mostly proposed physical changes to the actual microelectrode or the hydrogel [39–45], most attempts still rely on the "*Dipping Method*" for depositing the hydrogel coatings onto the microelectrodes (as proposed in [41]). **Figure 18** shows the Dipping Method involves the repetitive dipping of electrodes (e.g. via an inverted fine-tooth syringe pump [44]) into an alginate solution followed by immersion in a CaCl2 solution until a desired coating thickness is achieved. The thickness of the hydrogel coating is roughly controlled by monitoring the sample as a function of the number of dips with an optical microscope [43]. For instance, 1 dip is sufficient for a thin coating, and roughly 20 dips are needed to obtain a thick diameter coating. The hydrogel-coated electrodes are then dried in a laminar hood [43] or by exchanging ethanol and drying in an air stream [40].

In practice, the Dipping Method has several problems. First, lateral movements of the neural electrode effect the reproducibility of the alginate coating around the shank of the probe [40]. Second, it is impossible to control the average thickness by just approximating the number of dipping cycles (as verified by ellipsometry data in Figure 4B of [44]). Third, it is impossible to control the maximum radial thickness by just altering the number of dips (as demonstrated in Figure 2C of [45]). Fourth, the speed of dipping effects the uniformity of the alginate coating around the shank of the probe [40]. These limitations of the Dipping Method are confounding and counter-productive when considering that the entire rationale for using hydrogel coatings was to control the mechanical properties of the electrodes in [41]. Furthermore, the Dipping Method requires a human observer to subjectively monitor the entire dipping process with an optical microscope [43]. Therefore, this proposed Dipping Method essentially eliminates one of the primary advantage of the Michigan probes: exploiting semiconductor manufacturing techniques to allow batch fabrication for the high reproducibility of geometrical/electrical characteristics (as highlighted in Section 3.1.2).

#### **4.4. The "Dropping Method"**

**4.3. The "Dipping Method"**

188 An Excursus into Hearing Loss

followed by immersion in a CaCl2

air stream [40].

Section 3.1.2).

While previous solutions to the hydrogel swelling problem have mostly proposed physical changes to the actual microelectrode or the hydrogel [39–45], most attempts still rely on the "*Dipping Method*" for depositing the hydrogel coatings onto the microelectrodes (as proposed in [41]). **Figure 18** shows the Dipping Method involves the repetitive dipping of electrodes (e.g. via an inverted fine-tooth syringe pump [44]) into an alginate solution

**Figure 18.** Dipping Method for hydrogel coating of neural electrodes with optical microscopy. Reproduced from [43].

thickness of the hydrogel coating is roughly controlled by monitoring the sample as a function of the number of dips with an optical microscope [43]. For instance, 1 dip is sufficient for a thin coating, and roughly 20 dips are needed to obtain a thick diameter coating. The hydrogel-coated electrodes are then dried in a laminar hood [43] or by exchanging ethanol and drying in an

In practice, the Dipping Method has several problems. First, lateral movements of the neural electrode effect the reproducibility of the alginate coating around the shank of the probe [40]. Second, it is impossible to control the average thickness by just approximating the number of dipping cycles (as verified by ellipsometry data in Figure 4B of [44]). Third, it is impossible to control the maximum radial thickness by just altering the number of dips (as demonstrated in Figure 2C of [45]). Fourth, the speed of dipping effects the uniformity of the alginate coating around the shank of the probe [40]. These limitations of the Dipping Method are confounding and counter-productive when considering that the entire rationale for using hydrogel coatings was to control the mechanical properties of the electrodes in [41]. Furthermore, the Dipping Method requires a human observer to subjectively monitor the entire dipping process with an optical microscope [43]. Therefore, this proposed Dipping Method essentially eliminates one of the primary advantage of the Michigan probes: exploiting semiconductor manufacturing techniques to allow batch fabrication for the high reproducibility of geometrical/electrical characteristics (as highlighted in

solution until a desired coating thickness is achieved. The

Techniques for coating hydrogels onto microelectrodes include spray coating, brush coating, Dipping Methods [39–45], and micropipetting [38]. Unfortunately, none of these methods can produce uniform coatings in a reproducible manner [46]. Therefore, novel strategies such as the Molding Method (which involves the photopolymerization of hydrogels in a polyethylene tube) have been proposed to provide hydrogel coatings that are more reproducible and have a means to control coating uniformity and thickness (e.g. by varying the polyethylene tubing).

**Figure 19.** Dropping Method for hydrogel coating of neural electrodes with a fully-automated procedure.

**Figure 19** proposes a novel procedure called the "Dropping Method" to address the reported limitations of the Dipping Method. First, the microelectrode is permanently attached in one location to eliminate lateral movements (to improve reproducibility [40]). Second, a stage that contains solutions is automatically revolved to eliminate the requirement of approximating movements (to improve the average thickness [44]). Third, the stage is dropped downward to allow gravity to align the droplets and to evenly pull down the edges (to improve the radial thickness [45]). Fourth, the speed of dropping is automated by a digital micromanipulator (to improve the uniformity of the alginate coating around the shank of the probe [40]). Finally, the optimal contact angle is automated on a Dropmeter stage via programmable software (instead of relying on subjective interpretations of a human observer via optical microscopy as in [43]).

FEM and histological analyses have shown that elevated local strains correspond to increases in gliosis (with a three-fold increase in gliosis at the probe tips compared to other areas of the explanted arrays [19]). In addition, FEM pressure profiles predict that the strain at the tip can be reduced by utilizing more flexible or softer substrates, reducing the opening angle of the probe face, and by promoting tissue integration to reduce excessive adhesion. Therefore, the Dropping Method is recommended for coating hydrogel at the tips of the microelectrodes to optimize the mechanical effects for improved chronic recording stability. Applying hydrogel coatings on the probe tips should stabilize the mechanical interface and lower the interfacial tension with the surrounding biological environment. Constraining hydrogel coating depths to just the tips will also avoid the electrode coverage issue [12]. Finally, the initial penetrating profile should be reduced since only the dehydrated probe tips will reswell in thickness upon water absorption (unlike previous studies that coated the hydrogel layers along the entire length of the neural electrode shanks in efforts to reduce the sustained injury responses).

**Glossary**

information.

**Author details**

Payton Lin1,2\*, Yu Tsao2

Nanomedicine, Taiwan

Hong Kong, Hong Kong SAR

and Li-Wei Kuo3

1 Centre for Biosystems, Neuroscience, and Nanotechnology (CBNN), City University of

2 Academia Sinica, Center for Information Technology Innovation, Taipei, Taiwan

3 National Health Research Institute, Institute of Biomedical Engineering and

\*Address all correspondence to: paytonlin20@gmail.com

*Auditory cortex*: region of the temporal lobe that is responsible for processing auditory

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*Biocompatibility*: ability of a biomaterial to perform with an appropriate host response in the body. *Brain machine interface*: technology that allows direct communication pathways between the brain. *Chronic neural recordings*: long-term neural recordings by implantable (e.g. intracortical) electrodes. *Critical surface area model*: theory of minimizing electrode surfaces to reduce tissue encapsulations. *Cylindrical radius*: neuronal ensembles must lie within ~140 μm of the recording electrode [12]. *Dipping method*: hydrogel coating as a function of the number of dips (via optical microscopy). *Dropping Method*: hydrogel coating automated on Dropmeter stage (via programmable software). *Electrode micromotion*: induced strain from relative movements between the probe and the tissue. *Finite element model*: numerical analysis that can approximate the behavior of mechanical systems. *Glial encapsulation*: formation of a fibrotic encapsulation layer or a glial scar surrounding an implant. *Hydrogel coating*: biomaterials with many versatile properties such as gelling and film-forming. *Inflammatory reactions*: complex biological responses of tissues that protect from harmful stimuli. *Insertion killzone*: region around the shaft where the local neuron density is lower than expected. *Utah electrode array*: 3D arrays consisting of conductive needles (designed at University of Utah). *Mechanical property difference*: theory of minimizing micromotions to reduce tissue encapsulation. *Michigan probe*: planar shanks from semiconductor platforms (designed at University of Michigan). *Microelectrode*: electrical conductor used for recording neural representations in the brain.
