International Journal of Pharmaceutics 455 (2013) 148–158 Contents lists available at ScienceDirect International Journal of Pharmaceutics journa l h o me pag e: www.elsevier.com/locate/ijpharm Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury Eduardo Ansorena a,1 , Pauline De Berdt a , Bernard Ucakar a , Teresa Simón-Yarza b , Damien Jacobs a , Olivier Schakman c , Aleksandar Jankovski c,d , Ronald Deumens c , Maria J. Blanco-Prieto b , Véronique Préat a , Anne des Rieux a,∗ a Université Catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug delivery Unit, 1200 Brussels, Belgium b University of Navarra, School of Pharmacy, Pharmacy and Pharmaceutical Technology Department, Pamplona, Spain c Université Catholique de Louvain, Institute of Neurosciences (IONS), 1200 Brussels, Belgium d Université Catholique de Louvain, Cliniques Universitaires Mont-Godinne, Service de Neurochirurgie, 5530 Mont-Godinne, Belgium a r t i c l e i n f o Article history: Received 9 May 2013 Received in revised form 15 July 2013 Accepted 17 July 2013 Available online 31 July 2013 Keywords: GDNF Microspheres Spinal cord injury Injectable hydrogel Alginate a b s t r a c t We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere- loaded hydrogels. Intense GFAP (astrocytes), low III tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Neuroprotection and regeneration facilitated by administration of exogenous neurotrophic growth factors has been considered as a potential treatment for spinal cord injury (SCI) (Wang et al., 2008). Brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin 3 and 4/5 (NT3 and NT- 4/5), and fibroblast growth factor (FGF) have already been tested for spinal cord regeneration (Awad et al., 2013). GDNF belongs to the transforming growth factor- superfamily and promotes survival and neurite outgrowth of dopaminergic, motor, peripheral sensory, and sympathetic neurons (Chou et al., 2005). In addition, the upre- gulation of GFR1 and c-Ret gene expression, two GDNF receptors, following SCI may further increase the responsiveness of injured neurons and axons to GDNF (Widenfalk et al., 2001). Iannotti et al. demonstrated that long-term GDNF intrathecal infusion elicited ∗ Corresponding author at: Université Catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug delivery Unit, Avenue Mounier, 73 bte B1.73.12, 1200 Brussels, Belgium. Tel.: +32 2 764 7320; fax: +32 2 764 7398. E-mail address: anne.desrieux@uclouvain.be (A.d. Rieux). 1 Present address: University of Navarra, School of Science, Department of Bio- chemistry and Genetics, Pamplona, Spain. neuroprotective effects on contused adult rat spinal cords, resulting in a substantial degree of tissue and axonal sparing (Iannotti et al., 2004). GDNF mRNA expression is highly upregulated 8 h post-SCI and downregulated from 12 h to 12 weeks post-SCI (Gerin et al., 2011). Therefore, treatments that promote GDNF-mediated sur- vival should be administered by 12 h post-SCI and should last up to 12 weeks (Hill et al., 2008). These data suggest that GDNF treat- ments during the chronic phases, when endogenous GDNF is no longer provided, might be an effective strategy for promoting cell survival (Gerin et al., 2011). Several strategies have been used to deliver GDNF to the central nervous system, e.g., delivery by an infusion pump (Behrstock et al., 2006), gene therapy (Chou et al., 2005), and genetic engineering of cells so that they release GDNF (Lu et al., 2003). However, these techniques present several disadvantages, such as pump refilling necessity, lack of control of the duration of the transgene expres- sion and the viral spread outside the target area, reduced rate of cellular survival within the implanted graft, and immune rejection of the grafted cells by the host tissue (Garbayo et al., 2009). An alternative approach is the encapsulation of GDNF in biodegradable and biocompatible polylactide-co-glycolide (PLGA) microspheres that allow controlled, sustained, and localized delivery in addition to protect the GDNF from enzymatic degradation (Garbayo et al., 2009; Wang et al., 2008). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.07.045