Endogenous neural stem cell stimulation and motor recovery after

local delivery of cyclosporine and erythropoietin with a hyaluronan-based hydrogel

Anup Tuladhar1, Jaclyn Obermeyer1,3, Samantha Payne1,3, Sohrab Zand5, Cindi M Morshead1,2,6, Molly S Shoichet1,3,4,6

1: Institute of Biomaterials and Biomedical Engineering, 2: Department of Surgery, 3: Department of Chemical Engineering and Applied Chemistry, 4: Department of Chemistry, 5:Department of Human Biology 6:Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada

Introduction: Neural stem and progenitor cells (NSPCs) found in the adult brain hold promise for neural repair after brain injuries such as stroke. It has been been shown that drugs can be used to stimulate endogenous NSPCs to promote self-repair of the injured brain. Two such drugs are the small molecule immunosuppressant cyclosporine (CsA) and the growth hormone erythropoietin (EPO). In animal models of stroke injury these compounds have been individually found to increase NSPC survival, improve tissue repair and promote functional recovery (CsA); and to improve neurogenesis and angiogenesis (EPO). However, clinical use of these drugs for neurorepair requires overcoming the blood-brain barrier, a specialized structure in the neurovasculature that hinders drug accumulation in the brain after systemic drug delivery.

Local drug delivery can circumvent the blood-brain barrier, avoiding complications due to systemic side effects and dose-limiting toxicities. Unfortunately, the current clinical method causes further tissue damage due to cannula insertion into brain tissue and has an increased risk of infection. Our lab has developed a bioactive hydrogel for local drug delivery. The gel, HAMC, is a physical blend of hyaluronan and methylcellulose. HAMC can be implanted on the brain’s surface without damaging tissue. By mixing drug- loaded polymeric particles into the gel we can locally deliver drugs for several weeks. Herein we investigate local co-delivery of CsA and EPO to stimulate motor recovery and tissue regeneration in stroke-injured rats.

Methods: HAMC hydrogel was mixed with CsA-loaded and EPO-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles and injected onto the ispilateral brain surface of rats with an endothelin-1 stroke. We measured CsA and EPO diffusion over time in dorsoventral brain sections using mass spectrometry and ELISA. Next, we evaluated behavioral recovery in rats treated with drug-loaded HAMC starting at 4 days after stroke. The combination of CsA and EPO was compared to CsA only or EPO only, and the appropriate controls, using 3 assays for sensorimotor function: the Montoya staircase task, forepaw asymmetry task and tapered beam traversing task.

Results: Local delivery with HAMC successfully provided sustained CsA and EPO release to the brain for 28 days post-implant. Drug accumulation in the subcortical NSPC niche was found to be greater than or equal to concentrations found in the brain after systemic administration (CsA) or endogenously (EPO). In the recovery of skilled forepaw use, measured with the Montoya staircase task, only animals treated with the combination with CsA and EPO showed significant recovery compared to untreated controls at day 10 and day 20 post- injury (p<0.05, two-way ANOVA with Dunnett’s post-hoc test), whereas animals treated with either drug alone or with the vehicle (HAMC with blank PLGA microparticles) did not. Data analysis of the remaining sensorimotor assays is on-going.

Conclusions: HAMC hydrogel enabled sustained local delivery of CsA and EPO to the rat brain that resulted in motor recovery after stroke, demonstrating for the first time a synergistic effect of CsA and EPO on motor recovery. On-going work is evaluating endogenous NSPC stimulation and tissue regeneration after CsA and EPO delivery with HAMC.

Acknowledgements: The authors are grateful to the Canadian Institute for Health Research, Ontario Graduate Scholarship, Natural Sciences and Engineering Research Council, Training Program in Regenerative Medicine, Heart and Stroke Foundation and Canadian Partnership for Stroke Recovery for funding.