Modular Molecular-Level Design of Poly(Pro-Estrogen) Scaffolds Enables Controlled Astrocyte Response

Despite extensive efforts aimed at finding a cure, spinal cord injuries (SCI) remain notoriously incurable, resulting in a five-fold mortality risk, and disproportionately impacting vulnerable populations such as veterans and the elderly. Thus, there exists an urgent need for treatments that can offer potential for functional recovery. 17β-estradiol (estrogen, E2) has demonstrated robust neuroprotective properties in countering oxidative stress-induced neurotoxicity in SCI lesions, as well as strong neurotrophic properties to promote axonal growth in numerous studies. However, oral or injected E2 is a suboptimal drug, as systemic administration fails to achieve a therapeutic dose at the injury site, in addition to being contraindicated in male patients. Polymerized pro-drug scaffolds can mitigate these issues; hence, we sought to develop poly(pro-E2) scaffolds with tunable material properties that are capable of providing sustained, targeted delivery of E2 at the site of injury. Further, astrocytes are the most abundant cell type in the central nervous system and play vital roles in regulating axonal growth and remyelination, as well as modulating immune responses and supporting the blood-cerebrospinal fluid barrier – which are key functions essential for promoting spinal cord regeneration after injury. Hence, herein we also sought to study the effect of novel poly(pro-E2) scaffolds on astrocyte behavior, as well as to gauge the biomaterial properties that would lead to optimal astrocyte functionality.<br/><br/>We synthesized pro-E2 as carbonate and ester derivatives and copolymerized monomers with hydrophilic oligoethylene glycol dithiol (EG) and hydrophobic hexylene dithiol (Hex) linkers of equal length to generate poly(pro-E2) polyesters (PE) and polycarbonates (PC) of high M_w (40-60kDa). Faster hydrolysis of polyesters and differential water affinities of linkers resulted in a library of poly(pro-E2) scaffolds with tunable hydrophobicity, ranging from least (PE-EG) to most (PC-Hex) hydrophobic. We quantified thermal characteristics and observed T_g in the physiological range in polymers with Hex-linkers. We analyzed mass-loss and soluble drug elution from scaffolds incubated in PBS at 37C over 6-weeks to verify that polyesters degraded more readily than polycarbonates. Turbidity and profilometry measurements of films indicated bulk erosion. We also observed that incorporation of Hex-linkers led to markedly stiffer and harder scaffolds compared to EG-linkers, demonstrating the ability of linkers to tune mechanical properties.<br/><br/>Next, we performed cell viability studies of mixed primary cortical cells (consisting of neurons and astroglia) cultured on poly(pro-E2) scaffolds for 7-days. None of the polymers displayed significant cytotoxicity compared to control. We studied adhesion of primary cortical astrocytes on scaffolds since robust astrocyte adhesion is vital for their long-term viability and functionality for promoting spinal regeneration. We observed that only scaffolds with Hex-linkers supported sustained astrocyte adhesion. To explain this behavior, we investigated further. SEM data for scaffolds submerged for 7 days showed marked surface roughness with micro- and nano-scale topography for scaffolds with Hex-linkers due to separation of hydrophilic and hydrophobic phases, whereas those with EG-linkers appeared smooth – indicating that astrocytes preferentially adhere to rougher surfaces. Finally, while all polymers exhibited hysteresis during mechanical testing, scaffolds with Hex-linkers demonstrated greater dissipation suggesting increased viscoelastic creep – indicating that astrocytes preferentially adhere to viscoelastic surfaces.<br/><br/>In conclusion, we developed novel poly(pro-E2) scaffolds with tunable material characteristics that can offer sustained, targeted delivery of E2 to spinal lesions and demonstrated that increasing surface roughness and material viscoelasticity can make them conducive to robust astrocyte functionality.