Polysaccharide-based Bioactive Coating for Intracortical Recording Microelectrodes

Introduction:: Invasive neural implants allow for bidirectional communication with the nervous tissue and have demonstrated the ability to record neural activity, stimulate neurons, and sense neuro-active species with high spatial selectivity and resolution. However, upon their implantation, they are exposed to a foreign body response (FBR) which can disrupt the seamless integration of the device with the native tissue and lead to deterioration in device functionality for chronic implantation. Modifying the device surface to incorporate bioactive coatings has been a promising approach to camouflage the device to promote improved integration while maintaining device performance. Existing protein-based coatings are vulnerable to denaturation during processing and are often not active players in reducing the FBR, despite having other beneficial properties. Chondroitin Sulfate (CS) is a naturally occurring polysaccharide in the brain, is commercially available and can offer a more stable, hydrophilic alternative with specific bioactivity for a novel neural electrode interface application. Negatively charged sulfate groups are involved in neurite-growth promoting properties, as shown by in vitro studies [1]. Immobilized CS was also shown to bind key MCP-1 chemokine through its charged sulfate groups [2]. CS can be utilized to sequester MCP-1 and dampen the downstream inflammatory cascade. Due to the abundance of hydroxyl groups in the CS, the hydrophilicity imparted to the surface can help prevent non-specific protein adsorption, the first step in the FBR. This study explores the use of CS as a bioactive coating on silicon-based devices, to dampen the FBR and improve the device-tissue integration for chronic recording applications.


Materials and Methods:: Silicon substrates were functionalized with CS via divinyl sulfone linker chemistry [3]. E18 primary neurons were plated on CS-coated glass coverslips and stained for neurite outgrowth (β-tubulin III marker) which was subsequently quantified using an ImageJ plugin. Similarly, primary microglia were plated on coated substrates and stained (Iba-1) after 3 days in vitro. Iba-1 positive cells were then counted to quantify attachment and growth of microglia. Additionally, microglia were activated via lipopolysaccharide (LPS) to mimic activated microglia phenotype in vitro. Production of nitrite species by LPS-activated microglia was quantified through commercially available Greiss assay. Uncoated and CS-coated non-functional silicon electrodes were bilaterally implanted (3 mm posterior to bregma, 4 mm lateral to midline) in C57BL/6 male mice (n = 5) for 1-week. To perform endpoint histology, the animals were transcardially perfused and brain tissue was cut into 25-micron slices spanning a depth of 100-1900 microns. Intensity of inflammatory markers, such as microglia (Iba-1) and astrocytes (GFAP), was quantified as a function of distance from the implant site.


Results, Conclusions, and Discussions:: CS-coating promotes neurite attachment and growth: Quantified neurite attachment and outgrowth were significantly higher in CS-coated conditions (430% ±25 Welch’s T-test ****p< 0.0001 for 3 independent cultures) as compared to the uncoated control (100%±7). This increase in neurite outgrowth is likely via the side sulfate groups in CS binding to the extracellular matrix receptor integrin αVβ3 on neurons [1].

CS-coating reduces microglia attachment and attenuates activation: Primary microglia had smaller morphology with drastically reduced cellular extensions when grown on CS samples as compared to controls, which promoted normal growth with branching. Moreover, there were significantly lower Iba-1 positive cells on CS-coated substrates as compared to the control (One-way ANOVA Kruskal-Wallis test ****p< 0.0001, 3 independent cell cultures). Preliminary studies show that after LPS stimulation, there is a decrease in supernatant nitrite production in CS-coated conditions, in contrast to the control conditions where nitrite concentration increases. This observation is likely due to the reduction in microglial attachment and growth on the coated surface, suggesting an inhibitory effect of CS-coating on microglia.

Reduced presence of microglia around coated implants in vivo: Background normalized intensity of Iba-1 positive cells was significantly lower around CS-coated implants as compared to uncoated control for up to 70 microns from the implant site (Two-way ANOVA ****p< 0.0001). Although there was no significant difference in GFAP expression between the groups within 80 microns of the implant, there was reduced GFAP expression in the coated group between 90- and 150-micron distance of the implant. These observations indicate an anti-inflammatory effect of the coating around the implant site for acute implantation.

Conclusions

In vitro and in vivo acute studies demonstrate the use of chondroitin sulfate as a bioactive coating for promoting more seamless integration at the neural tissue device interface by reducing local inflammation. Future studies will investigate whether this reduction in inflammation will translate to improved electrophysiological recordings in the nucleus accumbens of mice. This study utilizes the untapped potential of polysaccharide-based coatings which can improve device performance by aiding the seamless integration of neural electrodes with the native tissue


Acknowledgements (Optional): :

References (Optional): : [1] Shida M, Mikami T, Tamura JI, Kitagawa H. Chondroitin sulfate-D promotes neurite outgrowth by acting as an extracellular ligand for neuronal integrin αVβ3. Biochim Biophys Acta Gen Subj. 2019 Sep;1863(9):1319-1331.

[2] Hirose, J., Kawashima, H., Yoshie, O., Tashiro, K., & Miyasaka, M. (2001). Versican interacts with chemokines and modulates cellular responses. The Journal of biological chemistry, 276(7), 5228–5234.

[3] Cheng, F., Shang, J., & Ratner, D. M. (2011). A versatile method for functionalizing surfaces with bioactive glycans. Bioconjugate chemistry, 22(1), 50–57.