Introduction 

Temporal lobe epilepsy (TLE) is the most common acquired seizure disorder, often caused by brain injury. Anti-seizure drugs are ineffective in 33% of patients, who are classed as drug-refractory[1]. Furthermore, these systemic treatments fail to tackle the underlying disease pathophysiology. The purinergic receptor P2X7R, highly expressed on inflammatory microglia within injured brain regions, drives chronic inflammation in TLE by inducing the release of neurotoxic cytokines. Systemic P2X7R inhibition produces disease-modifying effects in murine TLE models, seen with pharmacological antagonists[1] as well as silencing RNA[2] (siRNA) which is considered a more targeted gene therapy approach. However, as P2X7R is widely expressed throughout the body, focal delivery is crucial to developing P2X7R-targeting therapies. Therefore, the key aim of this study was to develop a brain-compatible injectable hydrogel carrying siRNA targeting microglial P2X7R (siP2X7R) for focal delivery to the brain for the treatment of TLE. 

Materials and methods 

Fabrication of a photo-crosslinkable biomimetic hydrogel. Methacrylated hyaluronic acid (MeHA) was produced by reacting hyaluronic acid with methacrylic anhydride. Gel biophysical properties and degradation profile were characterised. Non-viral delivery of siRNA targeting microglial P2X7R. siP2X7R was complexed with GAG-binding Enhanced Transduction (GET) peptide[3] to produce peptide-siRNA nanoparticles. These were delivered to reactive microglia at an optimised concentration. P2X7R knockdown and cell phenotype were validated by PCR, and cytokine release quantified by ELISA. Tuning siP2X7R release from MeHA hydrogels. Encapsulated and naked siRNA was loaded into gels at various concentrations and release was quantified over time. Released nanoparticles were delivered to microglia and P2X7R knockdown was validated. 

Results 

MeHA crosslinks when exposed to blue light in the presence of a photo-initiator. In situ photo-polymerisation was confirmed using a soft tissue model.  Hydrogels had a stiffness compatible with neural cells[4], a mesh size suitable for nanoparticle retention, and a rapid degradation profile. P2X7R knockdown of 65% was achieved following delivery of 50 nM siP2X7R nanoparticles to reactive microglia stimulated with LPS and BzATP. P2X7R knockdown ameliorated inflammatory cell phenotype and signalling, as demonstrated by reduced Iba1 mRNA expression and IL-1β cytokine release respectively. GET encapsulation improved the release profile of siRNA from MeHA hydrogels by preventing the initial burst release and prolonging release over time. Released nanoparticles successfully transfected reactive microglia and functional P2X7R knockdown was achieved. 

Discussion 

Injectable hydrogels can act as tuneable drug delivery vehicles for CNS applications that overcome several limitations associated with systemic drugs including difficulty crossing the blood-brain barrier[4]. Here we tuned the physicochemical properties of a hyaluronic acid-based hydrogel to mimic the native brain tissue and to optimise it for use as an siRNA delivery platform. The results of our study indicate that siRNA transfection promotes knockdown of microglial P2X7R which thus reduces the production of neurotoxic cytokines in vitro, and future work will involve confirming the disease-modifying effects of this system in vivo using a well-established murine injury model of TLE. In summary, this gene-activated hydrogel demonstrates potential to act as a novel disease-modifying treatment strategy for post-traumatic TLE patients, with future applications within the wider field of neurotrauma.