Sustained Release of Growth Factors from Hyaluronic Acid Hydrogels and Polyethylene-Glycol Microparticles for Axonal Growth

Abstract

The central nervous system, unlike other tissues, lacks the ability to spontaneously regenerate. During spinal cord injury, the disruption of axonal pathways is one of the main causes of functional loss. Neurotrophin-3 (NT-3) and insulin-like growth factor-1 (IGF-1) are two growth factors that have been shown to improve axonal growth and functional recovery in several studies, but there is a lack of effective delivery methods capable of maintaining their biological activity. In this work, we explored the feasibility of using injectable hyaluronic acid (HA) hydrogels and polyethylene glycol-diacrylate (PEG-DA) microparticles as platforms for sustained delivery of bioactive NT-3 and IGF-1, respectively. HA hydrogels were successfully synthesized using a polyethylene-glycol (PEG) dimaleimide crosslinker and characterized chemically and mechanically. The stiffness of the hydrogels was tuned to match that of central nervous tissue. Similarly, PEG-DA MPs were fabricated with acrylic acid as a comonomer and characterized. In the case of HA and NT-3, the drug release was measured, and the delivery mechanism was found to be controlled by electrostatic interactions between the negatively charged HA and the positively charged NT-3. IGF-1 showed a similar interaction when loaded onto and released from the PEG-DA MPs, displaying higher binding when the concentration of acrylic acid present was increased. The presence of affinity binding between the negatively charged polymeric systems and cationic proteins enabled control over the loading and release of the growth factors. The biological activity of the released NT-3 and IGF-1 was confirmed using a dorsal root ganglion assay. The concentration of both growth factors in the media was correlated to the axonal growth of primary sensory neurons, as well as that of stem cell-derived motoneurons and interneurons to more closely match the type of cells that are present in the spinal cord. A synergistic effect on neurite outgrowth was found for the combination of NT-3 and IGF-1, confirmed via phosphorylation of ERK1/2 in the shared downstream signaling pathway of both growth factors. The hydrogel provided an attractive platform for tissue sparing and neural fiber sprouting when neurons were cultured within the scaffolds and were shown to extend neurite processes. The MPs showed high cytocompatibility, suggesting their potential to be translated from in vitro to in vivo. This dual delivery system presents a promising alternative to current treatments for SCI but further work is needed to confirm their effect on in vivo axonal growth and functional recovery.