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BDNF-Mimetic Peptide Amphiphiles for Neural Regeneration Alexandra Edelbrock1, Zaida Alvarez-Pinto2 & Samuel Stupp1,2,3 1 Department of Biomedical Engineering, Northwestern University, Evanston, IL 2 Simpson-Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 3 Department of Chemistry and Department of Materials Science and Engineering, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA Statement of Purpose: Neurotrophins are proteins of great clinical interest due to their ability to modulate development, survival, and function of neuronal cells. Brain-derived neurotrophic factor (BDNF), which binds specifically to the tropomyosin-related kinase B (TrkB) receptor, has been shown to promote neuronal survival, differentiation, maturation, re-myelination, and synaptic plasticity [1]. Unfortunately, BDNF protein based therapies have had little success in clinical settings, due to the short half-life (<5 min) of native BDNF protein in serum [2]. Nanofiber scaffolds displaying BDNF-mimetic moieties could provide a support structure and simultaneously promote cell survival at the site of injury over a longer period. The Stupp laboratory has developed a novel class of biomaterials known as peptide amphiphiles (PAs), which self-assemble into nano-fibrous gels upon exposure to physiological divalent ions [4]. PAs are an ideal scaffold for neural regeneration because of their ability to form aligned gels in vivo that mimic the extra cellular matrix structure of the central nervous system (CNS). Here, we present a PA containing a cyclic peptide mimetic (CPM) BDNF sequence, which forms a fibrous scaffold and activates Trk-B in vitro. Methods: All PAs were synthesized by fluorenylmethoxycarbonyl (Fmoc) chemistry using solidphase peptide synthesis. CPM PA (10 mol%) was coassembled with 90mol% of a non-bioactive filler PA. PA solutions were annealed to reach a thermodynamically stable state prior to use. After annealing, 1 wt% PAs were gelled with a solution containing 25 mM CaCl2. Gelled PA materials were imaged using scanning electron microscopy (SEM) to assess their fiber alignment and ability to form fibrous scaffolds. For in vitro studies, primary cortical neurons were dissected from mice at embryonic day 16 and cultured on poly-D-lysine (PDL) coated wells for 14 days before PA treatments were added. Solutions of CPM PA and the cyclic mimetic peptide unattached to PA were diluted in serum-free media to obtain 0.5 µM final concentrations of epitope. The cells were incubated with PA for 1, 2, 4, 6 and 24 hours and the protein was collected to assess TrkB phosphorylation using Western blot. Confocal microscopy was used to analyze changes in cell morphology. Cell survival was assessed using flow-cytometry. Results: SEM showed that when CPM PAs are coassembled with the filler PA, annealed, and gelled with calcium, they form nanofiber networks (Figure 1A). In some cases, these networks become twisted and bundled in the presence of salts. Treatments were dissolved in media and incubated with cells for 1, 3 and 7 days. Cell survival assays showed greater than 90% survival for all conditions at all time points. Confocal microscopy showed that cells took on a normal neuronal morphology (Figure 1B). Western blot (Figure 1C & 1D) showed rapid activation of TrkB with BDNF and no activation with starvation media at time zero. CPM showed delayed activation of the TrkB receptor, but a similar amount of phosphorylation compared to BDNF at 4 and 6 hours. There is a cross-over point where the activation of TrkB was higher at 6 hours with the CPM PA material than with the native BDNF. Activation of TrkB eventually decreases to zero after 24 hours in both conditions. Figure 1: A) SEM image of CPM PA after gelation. Inset: schematic molecular-graphics representation of CPM nanofiber. B) Confocal images of primary cortical neurons exposed to treatments for 24 hours. DAPI = nuclear stain, MAP2 = maturation marker, SMI312 = neurofilament marker. C) Western blot showing amount of TrkB phosphorylation overtime in comparison to native BDNF protein. D) Graphical representation of 1C phosphorylation over time, normalized to total TrkB. Conclusions: The functionalization of PAs with BDNF mimetic cyclic peptides does not inhibit their selfassembly into nanofibers. When added to primary neurons in vitro, this material activates the TrkB receptor after 4 hours of exposure. We hypothesize that this delay is due to the PA material floating in media after addition, and it does not contact the cells right away. Future investigations will identify the optimal concentration and exposure time of the CPM material. Based on these results, we conclude that BDNF-mimetic promotes the survival and neuronal outgrowth after 24 hours in vitro through the activation of the Trk-B signaling pathway. References: [1] Lu B. Nat Rev Neurosci. 2013; 6: 401-416. [2] Bradley WG. Neurology. 1999; 7: 1427-1433. [3] Silva GA. Science. 2004; 5662: 1352-1355.