Poster O3 - Modular Molecular-Level Design of Poly (Pro-Estrogen) Scaffolds Enables Controlled Astrocyte Response

Introduction: Despite extensive research efforts aimed at finding a cure, spinal cord injuries 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 the potential of functional recovery. Even slight neurological recovery can have outsized impacts on patients’ quality of life. 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, including in prior work from our laboratory. However, oral or injected E2 is a sub-optimal 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. While previous studies have focused on the effect of E2 delivery on neurons, herein we 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.

Materials and

Methods: Difunctional pro-drugs of E2 were prepared as carbonate and ester derivatives with pendant allyl groups as polymer building blocks. Poly(pro-E2) polymers were synthesized using UV photoinitiated thiol-ene radical addition “click” chemistry with tight stoichiometric control. Monomer and polymer chemical structures were verified with proton nuclear magnetic resonance spectroscopy. Molecular weight (Mw) dispersion and mass loss was characterized using Gel Permeation Chromatography. Hydrophobicity was characterized using goniometry. Thermal characteristics, including glass transition temperature (Tg) were quantified using Differential Scanning Calorimetry. Soluble drug elution was analyzed using fluorescence spectroscopy. Mechanical properties were determined using nanoindentation. Polymer surfaces were imaged using scanning electron microscopy. Primary cortical cells were harvested from P2 Sprague Dawley rat pups. Cell viability and adhesion assays were performed using immunofluorescence with propidium iodide staining and nuclear (Hoechst/DAPI) staining.

Results, Conclusions, and Discussions: 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) [Figure 1] of high Mw (40-60kDa) [Figure 2(i)]. 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 Tg in the physiological range in polymers with Hex-linkers [Figure 2(i)]. 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 [Figures 2(ii)-(iii)]. Turbidity and profilometry measurements of films indicated bulk erosion [Figure 3 (i)-(ii)]. 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 [Figure 3(iii)].

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 [Figure 4(i)]. We studied adhesion of primary cortical astrocytes on scaffolds since robust astrocyte adhesion is vital for their long-term viability and functionality, and consequently for promoting spinal regeneration. We observed that only scaffolds with Hex-linkers supported sustained astrocyte adhesion [Figure 4(ii)]. 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 [Figure 4(i)] – 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 [Figure 4(ii)] – indicating that astrocytes preferentially adhere to viscoelastic surfaces.

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 adhesion.

Acknowledgements (Optional): Devan Puhl, Samuel Ellman, Alex Hsu, Alexis Ziemba, Penelope Philips, Bailey Balouch, Deniz Rende