Immunomodulatory Gene Therapy to Mitigate Neuroinflammation Following Intracortical Microelectrode Implantation

Introduction: Intracortical microelectrodes implanted in the brain are extensively used both in the clinic with brain-machine interface (BMI) systems and in the lab for neuroscience research due to their unparalleled ability to record action potentials at single neuron resolution. To achieve the precise, accurate, single-neuron level tracking and recording required for the function of BMIs an array of microelectrodes must be implanted directly into the brain. Brain implants show promising effects in the short-term; however, longevity of these devices are heavily dependent on the sustained performance of microelectrodes. Recording quality greatly diminishes due to the Foreign Body Reaction (FBR) and inflammation around implants. This response involves blood-brain barrier (BBB) rupture, infiltration of immune cells and serum proteins, and resident microglia response, leading to device damage and neuronal death. A key mediator of this strong inflammatory response is CD14, which is a co-receptor for Toll-like receptors (TLRs) 2 and 4 present on macrophages, monocytes, and microglia. Activation of this pathway is due to the presence of damage-associated molecular patterns (DAMPs) generated by the destruction of tissue during the implantation of these microelectrodes.
Traditional neutralization of this inflammatory response involves the administration of small molecule therapies to physically inhibit expressed CD14 receptors. We utilize immunomodulatory lipid nanoparticle (immuno-LNP) mediated gene therapy to silence Cd14 expression and suppress the neuroinflammatory response to intracortical microelectrodes. The LNPs are specifically designed to deliver Cd14 targeting siRNA to microglia and infiltrated macrophages, provide protection of its cargo from degradation, have diminished immunogenicity, and have low systemic toxicity.

Materials and

Methods: Lipid nanoparticles loaded with CD14 siRNA were synthesized using SM-102:DSPC:Cholesterol:DSPE-PEG:DMG-PEG:DiR at a molar ratio of 50:10.5:37.7:0.1:1.4:0.3. To introduce representative brain injuries, C57BL/6J mice underwent craniotomies and had microelectrodes implanted into their brains. The mice were given systemic treatments 24 hours before surgery and immediately after with LNPs at a dose of 1 mg of siRNA to 1 kg of body weight. 24 hours after surgery the brains, blood, spleens, and livers were collected from the mice for analysis through flow cytometry. The presence of macrophages, microglia, monocytes, and dendritic cells (among others), nanoparticle uptake within these populations, and CD14 expression were measured throughout the collected tissues to determine the efficacy of the treatments in response to the induced brain injury. Statistical analysis was performed by one-/two-way analysis of variance (ANOVA) and Student’s t-test.

Results, Conclusions, and Discussions: Flow cytometry analysis showed that treatment with the immuno-LNPs demonstrated a significant ability to access important immune populations within the brain and in systemic circulation. Specifically, the activated microglia and infiltrating macrophages in the brain showed 40% of their population to be nanoparticle-positive. Additionally, 35% of the circulating macrophages found in the blood were nanoparticle-positive. The infiltrating macrophages and activated microglia showed significant decreases in the percentage of CD14+ cells in the brain as well as in healthy microglia. Interestingly, neutrophil-like populations showed an increase in CD14 expression in treated groups which points to the possible benefit of a secondary target to be co-administered in future studies. Beyond local brain effects, CD45+ and CD11b+ immune cell populations in the spleen and liver showed significant uptake of the immuno-LNPs demonstrating a robust systemic reach of the treatment.
Our results suggest that silencing of the Cd14 genes can lead to inhibition of the neuroinflammatory response, resulting in significant long-term improvements in intracortical microelectrode performance. Overall this supports the premise that inhibition of central innate immune drivers can impact the early-stage recognition phase of the immune response and prevent deterioration of microelectrode performance by drastically attenuating local innate immune activation at the electrode-tissue interface.

Acknowledgements (Optional): This work was supported by grants from the National Cancer Institute R01CA253627 (E.K.), R01CA278633 (E.K. ), P30CA043703 (E.K.), and the Angie Fowler AYA Cancer Research Fund (E.K.). T.J.M. was supported by the NSF graduate research fellowships program. A.B. was supported by a fellowship from the NIH Interdisciplinary Biomedical Imaging Training Program (T32EB007509).