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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: J Tissue Eng Regen Med. 2020 Nov 21;15(1):14–23. doi: 10.1002/term.3153

Uric acid released from poly(ε-caprolactone) fibers as a treatment platform for spinal cord injury

Nisha K Singh 1,2,#, Salman Khaliq 1,3,#, Mann Patel 1,#, N’Dea Wheeler 4, Sudeepti Vedula 4, Joseph W Freeman 4, Bonnie L Firestein 1
PMCID: PMC7864535  NIHMSID: NIHMS1660886  PMID: 33175472

Abstract

Spinal cord injury (SCI) is characterized by a primary mechanical phase of injury, resulting in physical tissue damage, and a secondary pathological phase, characterized by biochemical processes contributing to inflammation, neuronal death, and axonal demyelination. Glutamate-induced excitotoxicity (GIE), in which excess glutamate is released into synapses and overstimulates glutamate receptors, is a major event in secondary SCI. GIE leads to mitochondrial damage and dysfunction, release of reactive oxygen species (ROS), DNA damage, and cell death. There is no clinical treatment that targets GIE after SCI, and there is a need for therapeutic targets for secondary damage in patients. Uric acid (UA) acts as an antioxidant and scavenges free radicals, upregulates glutamate transporters on astrocytes, and preserves neuronal viability in in vitro and in vivo SCI models, making it a promising therapeutic candidate. However, development of a drug release platform that delivers UA locally to the injured region in a controlled manner is crucial, as high systemic UA concentrations can be detrimental. Here, we used the electrospinning technique to synthesize UA-containing poly(ɛ-caprolactone) fiber mats that are biodegradable, biocompatible, and have a tunable degradation rate. We optimized delivery of UA as a burst within 20 min from uncoated fibers and sustained release over 2 h with poly(ethylene glycol) diacrylate coating. We found that both of these fibers protected neurons and decreased ROS generation from GIE in organotypic spinal cord slice culture. Thus, fiber mats represent a promising therapeutic for UA release to treat patients who have suffered a SCI.

Keywords: glutamate-induced excitotoxicity, neuroprotection, poly(ε-caprolactone) fibers, reactive oxygen species, spinal cord organotypicculture, uric acid

1 |. INTRODUCTION

Spinal cord injury (SCI) is a debilitating traumatic insult to the central nervous system that affects 250,000 to 500,000 worldwide each year and shortens life expectancy (Fehlings, Singh, Tetreault, Kalsi-Ryan, & Nouri, 2014). SCI patients often suffer from pathologies, such as neuropathic pain and compromised motor, sensory, and autonomic function (Fehlings et al., 2014). However, SCI is particularly challenging to treat because it consists of two phases of injury. During the primary phase of injury, mechanical force on the spinal cord physically damages cells and tissue directly. However, this insult is followed by a secondary phase of injury during which a cascade of biochemical events ensues and exacerbates the effects of primary injury. This secondary phase begins just hours after the initial injury and can persist for days to months.

Glutamate, an excitatory neurotransmitter in the central nervous system, contributes significantly to the secondary damage that occurs after SCI (Faden, Demediuk, Panter, & Vink, 1989; Yanase, Sakou, & Fukuda, 1995). Injured neurons release excess glutamate, over-stimulating N-methyl-D-aspartate glutamate receptors and causing ionic imbalances, leading to an increase in intracellular concentrations of Na+ and Ca2+. This results in cell swelling and mitochondrial dysfunction (D. Choi, 1987; D. W. Choi, 1992). Mitochondrial damage and subsequent decreased Adenosine triphosphate production lead to increased production of reactive oxygen species (ROS; Prentice, Modi, & Wu, 2015). In addition, formation of nitric oxide results from high intracellular concentrations of Ca2+. Nitric oxide reacts with superoxide to form toxic peroxynitrite, which then damages mitochondria and eventually leads to cell death (Brenman et al., 1996; Chen et al., 2011). Currently, there is a lack of safe and effective treatments available to curtail damage resulting from glutamate-induced excitotoxicity (GIE) after primary SCI (Y. H.-Kim, Ha, & Kim, 2017). Thus, there is a need for SCI therapeutics that adequately curb secondary damage and improve quality of life.

Uric acid (UA), the most active antioxidant in the plasma (Baillie et al., 2007; Maxwell et al., 1997), is a promising therapeutic candidate for SCI patients. UA confers neuroprotection against GIE as a peroxynitrite scavenger (Scott, Cuzzocrea, Genovese, Koprowski, & Hooper, 2005; Yu, Bruce-Keller, Goodman, & Mattson, 1998) and by upregulating glutamate transporters on astroglia (Du, Chen, Tseng, Eisenberg, & Firestein, 2007). Previous studies have demonstrated the protective role of UA after central nervous system injuries, including traumatic brain injury (Hatefi, Dastjerdi, Ghiasi, & Rahmani, 2016), stroke (Yu et al., 1998), and SCI (Du et al., 2007; Scott et al., 2005). However, while physiological levels of UA are important for protection against oxidative stress, UA imbalances can lead to disease. Kidney disease, hypertension, and gout have been linked to hyperuricemia (Fang, Li, Luo, Wang, & Yang, 2013). Thus, while UA may be an attractive treatment for SCI, delivery and release methods must be carefully considered to avoid detrimental systemic effects. Delivery systems that facilitate local controlled release of UA to the wounded area are ideal; however, there are challenges in developing such a system. Solubility of UA is limited to heated basic solvents, and thus, it is difficult to integrate UA into biocompatible fibers. Consequently, sustained UA release via fibers or nanofibers has not yet been achieved. Therefore, designing a fiber mat for local delivery of UA that circumvents these challenges will provide new opportunities to treat GIE during the secondary phase of SCI.

The use of fiber mats for drug delivery confers many benefits. Polymer fibers, especially, those on the nanoscale, have a very large surface area to volume ratio, can accommodate a wide range of pore sizes and volumes, and have biologically appropriate stiffness and tensile strength, and thus, they are ideal for use in a wide variety of applications (Xue, Wu, Dai, & Xia, 2019). Furthermore, electrospinning, the fiber synthesis method that we employ here, allows for various fiber assemblies and scalability and is a method of choice (Huang, Zhang, Kotaki, & Ramakrishna, 2003), is cost-effective, enables fiber dimension tunability, can produce long continuous fibers (Hekmati, Rashidi, Ghazisaeidi, & Drean, 2013), and constructs fibers from both natural and synthetic polymers (Pakravan, Heuzey, & Ajji, 2011). Electrospun fibers are widely used as drug delivery carriers for antibiotics (Bölgen, Vargel, Korkusuz, Menceloğlu, & Pişkin, 2007), anticancer drugs (Xie & Wang, 2006), and proteins (T. G. Kim, Lee, & Park, 2007). Furthermore, materials, such as poly (ethylene glycol) diacrylate (PEGDA), can be used to coat the fiber mats for additional control over drug release profiles (Hamid & Lim, 2016; Lin & Anseth, 2009). Functionalization with PEGDA slows drug release, improves the mechanical strength of the mats, and inhibits protein adsorption (Hamid & Lim, 2016; Zhao & Harris, 1998).

Using the electrospinning setup shown in Figure 1 and subsequent PEGDA coating, we successfully produced fiber mats containing UA that can be harnessed for biological applications. We synthesized fiber mats for both burst and sustained release of UA and tested our drug delivery systems using a spinal cord organotypic slice culture model platform. Our results demonstrate that UA released from both types of fiber mats effectively reduces levels of cell death and oxidative stress after GIE. Thus, we have developed a new system for localized controlled delivery of UA that may be a viable option for treatment of SCI.

FIGURE 1.

FIGURE 1

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Electrospinning setup for UA fiber synthesis. There are five components to the electrospinning setup: a DC power supply, polymer solution, electrically charged spinneret, a syringe pump, and a grounded collector; UA, uric acid

2 |. MATERIALS AND METHODS

2.1 |. Preparation of UA-containing poly(ɛ-caprolactone) fibers

Electrospinning was performed as we described previously (McKeon-Fischer, Browe, Olabisi, & Freeman, 2015). A 20% (w/v) solution of poly(ɛ-caprolactone) (PCL) (70,000–90,000 Da) in dichloromethane was prepared, and 0.1% and 0.2% UA solutions were added for UA loading. Final solutions were loaded into a 5 ml syringe with a 20 gauge blunt stainless-steel needle and placed into a syringe pump set at an extrusion rate of 5 ml/h. A distance of 18 cm was set between the needle tip and a rotating mandrel (3000 ± 200 rpm) with a 5 cm diameter, which was used to collect the fibers. A 2 kV negatively charged plate was placed behind the rotating mandrel to aid in attracting the positively charged solutions. The positive voltage ranged from 13–17 kV and was adjusted to produce the most stable Taylor cone during the electrospinning process to yield the most consistent fiber morphology in the scaffold.

2.2 |. Coating of PEGDA on UA-PCL fibers

PEGDA (MW 8000 Da) was dissolved in phosphate-buffered saline (PBS) to create a 20% (w/v) solution with vortexing. Photo-initiator solution (300 mg/ml 2,2-dimethoxy-2-phenylacetophenone in 1-vinyl-2-pyrrolidinone) was added to the solution (5% v/v) immediately before UV irradiation as described in (Browe et al., 2017). The fiber mats were placed into a beaker of the solution and then placed under a vacuum for 1 min to remove any air bubbles and ensure efficient covering of all fibers. After 1 min, the mats were removed from the solution, hung vertically to remove excess solution, and then crosslinked via UV irradiation at a wavelength of 365 nm at 30 s intervals using a 3UVTM Lamp.

2.3 |. Release profile of UA

UA-PCL and UA-PCL-PEGDA fiber mats were cut into 2.54 × 2.54 cm cross-sections and were incubated in 10 ml 1X endotoxin-free and calcium- and magnesium-free PBS for 2 h at 37°C. The pH of the PBS was measured at 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 90, and 120 min throughout the duration of the experiment. The pH of the PBS decreases with increasing concentration of UA as it is released from the fiber mats. The Henderson-Hasselbach equation (pH = pKa + log[{A-}/{HA}] where pKa of UA is 5.5) was then applied to these data to determine the concentration of UA released into the PBS at each experimental timepoint.

2.4 |. Assessment of fiber mat swelling

The percent swelling ratio (SR) of the fiber mats was estimated as described previously (Ko et al., 2019). Squares of each fiber mat type (1.9 × 2.2 cm) were weighed (wd, weight of dry fiber mat). The average dry weight of each mat was as follows: 4.9 mg (0.1% UA-PCL), 28.6 mg (0.1% UA-PCL-PEGDA), 5.9 mg (0.2% UA-PCL), and 25.9 mg (0.2% UA-PCL-PEGDA). Each mat was then incubated in 1 ml of 1X PBS for 15 min at 37°C on a rocker. Afterward, each mat was removed from solution and weighed again (ws, weight of swollen fiber mat). Incubation was then resumed, and the mats were reweighed after 2 h (ws). The following equation was used to calculate the percent SR after both 15 min and 2 h: % SR = (ws - wd)/wd × 100. This procedure was performed for up to three cross-sections of each mat type.

2.5 |. Scanning electron microscopy and measurement of fiber diameter and angle

For scanning electron microscopy (SEM) imaging, uncoated (no conductive metal) cross-sections of fiber mats were mounted with conductive acrylic emulsion (Electron Microscopy Sciences) and scanned at 1 kV to minimize beam damage using the in-lens detector on a LEO 1525 FEG-SEM (Carl Zeiss). Images were acquired with an inverted look-up table. Fiber diameter and angle relative to the vertical were quantified in the images of each scaffold type using National Institutes of Health (NIH) ImageJ software as described previously (McKeon-Fischer et al., 2015). Images were inverted to more clearly visualize fiber morphology. For diameter quantification, four to seven of each fiber type were measured. For angle distributions, 25–50 of each fiber type were measured.

2.6 |. Organotypic spinal cord slice culture

All procedures and animal use have been approved by the Rutgers University Institutional Animal Care and Use Committee. Sprague Dawley rat embryos at 16 days of gestation (E16) were extracted via Caesarian section and placed into Gey’s balanced salt solution supplemented with 300 nM kynurenic acid and 0.6% glucose (v/v). Spinal cords were isolated from embryos, and meninges and dorsal root ganglia were removed. Spinal cords were then sliced transversely at a thickness of 350 μm using a McIlwain tissue chopper. Slices from the cervical, lumbar, and thoracic regions were cultured in 12 well plates coated in 1 mg/ml poly-D-lysine and 0.1 mg/ml laminin. Cultures were maintained in serum-free medium (Neurobasal A medium supplemented with 1X B-27, 0.5 mM GlutaMAX, and 25 μg/ml gentamycin) for 7 days in vitro (DIV). Medium (100 μl) was added every other day.

2.7 |. GIE and UA fiber treatment

On DIV7, cultures were treated with 3 mM glutamate dissolved in Locke’s buffer (LB; 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.2) or with LB alone (control) for 1 h. Fiber mats of approximately 250 μm or vehicle (LB) were subsequently placed onto the surface of the slice cultures and remained on the slices for 15 min to achieve an effective dose of 200 μM UA. We determined the timing for an effective dose of 200 μM UA by measuring the pH of a 200 μM UA solution (pH = 6.997). We then used linear regression for each fiber mat release profile to calculate the pH of the surrounding solution at a given time point. The average pH value of all fiber mat types (0.1% and 0.2% UA-PCL and UA-PCL-PEGDA mats) was closest to the pH of a 200 μM UA solution after 15 min of release. After removal of the mats, the cultures were incubated in serum-free medium overnight at 37°C and 5% CO2.

2.8 |. CellROX, 2’,7’-dichlorodihydrofluorescein diacetate, and propidium iodide treatment and fluorescent imaging

For detection of ROS, cultures were incubated with 5 μM CellROX™ Green Reagent or 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) for 1 h. For assessment of cell death, cultures were incubated with 3 μM propidium iodide (PI) for 30 min. Phase contrast and fluorescence micrographs of individual slices were taken using the EVOS FL Microscope (ThermoFisher Scientific) under a 4X objective.

2.9 |. Image processing

Images of organotypic slices that were fluorescently labeled with PI and CellROX™ Green Reagent/H2DCFDA were analyzed using NIH ImageJ software. Using phase contrast images, the tracing tool was used to define the entire slice as the region of interest (ROI) for analysis. The ROI outline was then overlaid on respective fluorescent channel images, and mean gray value (pixel intensity) was subsequently measured to quantify fluorescent signal (Krassioukov et al., 2002). For clarity, individual slices were overlaid on a plain black background for analysis purposes. Slices with clear morphological features, such as the central canal, that could be used to define dorsal and ventral tissue regions were used for additional region-specific analyses of cell death and oxidative stress. For this set of analyses, the dorsoventral axis of each slice was defined at the midpoint between the anterior median fissure on the ventral end and the posterior median sulcus at the dorsal end, and mean gray value was quantified for both dorsal and ventral ROIs. All analyses were performed with the experimenter blinded to the conditions. Statistical analyses of the data were performed using GraphPad Prism six software.

3 |. RESULTS

3.1 |. Sustained release versus burst release of UA from UA-PCL fibers

To test the protective potential of UA treatment for neuroprotection from GIE, we synthesized two types of UA-containing fibers: UA-PCL mats that release UA as a burst and UA-PCL-PEGDA mats that facilitate a sustained release of UA over time. UA was incorporated into PCL and electrospun into fibers; a subset of these mats was coated with PEGDA (Figure 1). SEM images of the fiber mats are shown in Figure 2a. The average diameters of the 0.1% UA-PCL and 0.1% UA-PCL-PEGDA fibers were determined to be 2.97 μm ± 0.78 and 5.61 μm ± 1.0 μm, respectively (Figure 2c), and there is some variation in angle distribution between the two fiber types (Figure 2b). We also assessed mat swelling and found that UA-PCL fiber mats have a higher percent SR than UA-PCL-PEGDA fiber mats (Figure 2d).

FIGURE 2.

FIGURE 2

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Fiber mat properties. (a) Scanning electron microscopy (SEM) images of uncoated cross-sections of UA-PCL and UA-PCL-PEGDA fibers. Scale bars = 20 μm (top) and 2 μm (bottom) (b) Angle distributions of UA-PCL and UA-PCL-PEGDA fibers. (c) Average diameter of UA-PCL and UA-PCL-PEGDA fibers. (d) Percent swelling ratios of UA-PCL and UA-PCL-PEGDA fiber mats after 15-min and 2-h incubations in PBS. Up to three of each mat type were tested; PBS, phosphate-buffered saline; PEGDA, poly(ethylene glycol) diacrylate; PCL, poly(ɛ-caprolactone); UA, uric acid

We then measured decreases in pH of the solution into which UA was released from the fibers to obtain preliminary UA release profiles. We found that UA-PCL fibers released UA into the PBS solution within 30 min (burst), whereas UA-PCL fibers coated with PEGDA gradually released UA over 2 h (sustained) (Figure 3). These release profiles coincide with the observed relative percent SRs, as increased swelling of fiber mats has previously been demonstrated to correlate with higher levels of drug release (Ko et al., 2019).

FIGURE 3.

FIGURE 3

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UA-PCL and UA-PCL-PEGDA fiber release profiles. (a) UA-PCL-PEGDA fibers release UA over 2 h (sustained release) while UA-PCL fibers release UA over 30 min (burst release), as measured by a change in the pH of solution with respect to time. A 2.54 × 2.54 cm fiber placed into phosphate-buffered saline was assayed for the release profile. (b) Concentrations of UA released at different timepoints over 2 h as determined by the Henderson-Hasselbach equation. Inset: after 15 min, the concentration of UA released from the mats ranges from ~300 μM to 1 mM across mat types; PBS, phosphate-buffered saline; PEGDA, poly(ethylene glycol) diacrylate; PCL, poly(ɛ-caprolactone); UA, uric acid

Using the release profiles, we calculated the concentration of UA in the solution over time and determined that at the 15 min time point, the concentration of UA released from UA-PCL-PEGDA fibers was approximately 300 μM (Figure 3b). Preliminary experiments demonstrated that 200 μM soluble UA is effective in scavenging ROS in cultures subjected to GIE (data not shown). Furthermore, previous work by our group and others demonstrated that similar concentrations of UA (100 and 300 μM, respectively) are effective in preserving neuronal viability in dissociated mixed spinal cord cultures after GIE (Du et al., 2007; Scott et al., 2005). Additionally, the concentration range for UA in normal human serum is approximately 200 to 350 μM (Scott et al., 2005). Thus, in later experiments, we applied the fiber mats to spinal cord cultures for 15 min to achieve an effective dose of 300 μM UA. At the 2 h timepoint, the highest concentration of UA that was released among all fiber types was 8 mM. Thus, the concentration of released UA into did not reach the saturation point of 13 mM (Iwata, Nishio, Yokoyama, Matsumoto, & Takeuchi, 1989). This is preferable for our application as excess UA can crystalize and have detrimental effects on tissue.

3.2 |. Levels of cell death and ROS after treatment with UA-releasing fibers

To determine how UA release from the two types of fibers affects cell death and ROS levels after GIE, we implemented an organotypic spinal cord slice model platform. For assessment of cell death, slices were stained with 3 μM PI after injury with 3 mM glutamate and subsequent fiber treatment. We found that injured slices treated with UA-PCL and UA-PCL-PEGDA fibers demonstrated lower levels of cell death compared to those not treated with fibers, contributing to higher overall tissue viability (Figure 4a, b). However, both fibers that facilitate burst or sustained release of UA protected the slices from cell death to the same extent (Figure 4a, b). Furthermore, PCL and PCL-PEGDA fibers alone did not promote cell death (Figure 4a, b). In fact, we observed that uninjured slices treated with PCL or PCL-PEGDA fibers had lower levels of cell death relative to control slices (Figure 4a, b). Thus, the fibers themselves may increase the health and robustness of spinal cord organotypic slices in culture.

FIGURE 4.

FIGURE 4

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Effects of UA fiber mat treatment on cell death and ROS levels after glutamate-induced excitotoxicity. (a) Representative images of organotypic spinal cord slice cultures that were injured with 3 mM glutamate for 1 h, treated with 0.1% UA in PCL (UA-PL) or PCL-PEGDA (UA-PCL-PG) fibers for 15 min, allowed to recover overnight, and subsequently screened for cell death and ROS levels using PI and CellROX™ Green Reagent/H2DCFDA, respectively. Mean fluorescence intensity was quantified for each slice within the defined ROI (white). Slice borders are marked in white as traced from phase contrast images. Scale bar = 500 μm (b) Quantification of mean PI fluorescence intensity of cultures shown in (A) compared to control (c) Quantification of mean CellROX™ Green Reagent/H2DCFDA fluorescence intensity of cultures shown in (A) compared to control. Data were normalized to control (A.U. = arbitrary units), outliers were removed using ROUTS test, and data were subsequently analyzed using one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control, n = 3 independent experiments (4–20 slices per condition per experiment). Error bars = SEM; H2DCFDA, 2’,7’-dichlorodihydrofluorescein diacetate; PBS, phosphate-buffered saline; PEGDA, poly(ethylene glycol) diacrylate; PCL, poly(ɛ-caprolactone); PI, propidium iodide; ROS, reactive oxygen species; SEM, scanning electron microscopy; UA, uric acid

To evaluate how treatment with UA-PCL and UA-PCL-PEGDA fibers affects ROS after GIE, slices were incubated with 5 μM of CellROX™ Green Reagent or H2DCFDA, two ROS-detecting reagents. Injured slices treated with UA-PCL and UA-PCL-PEGDA fibers had lower levels of ROS than those without treatment (Figure 4a, c). In addition, PCL and PCL-PEGDA fibers alone did not affect ROS production (Figure 4a, c). Taken together, our data suggest that the UA released from the fibers facilitate ROS scavenging after GIE and both burst and sustained release of UA from the fibers are equally effective.

3.3 |. Dorsal and ventral region-specific effects of UA release from fibers on cell death and ROS

In the spinal cord, the dorsal horn contains sensory neurons and the ventral horn contains motor neurons (Andrews, Kong, Novitch, & Butler, 2019). To determine if there are differences in the observed effects of UA release from fibers in dorsal versus ventral spinal cord tissue, we analyzed cell death and ROS in these regions in our GIE experiments. The dorsoventral axis of each PI and CellROX™ Green Reagent/H2DCFDA-stained slice was defined, and fluorescent signal in each region was subsequently quantified (Figure 5a). Only slices subjected to GIE and treated with PCL or PCL-PEGDA fibers showed differences in dorsal and ventral cell death: for both treatment groups, there was significantly higher cell death in the dorsal regions of the slices (Figure 5b). However, treatment with UA-releasing fibers decreased cell death to baseline levels, regardless of sensitivity to GIE (Figure 5b). Comparisons of cell death in the dorsal regions of slices in all treatment groups to those in the control group revealed results similar to those observed in total cell death analyses: injured slices treated with UA-PCL and UA-PCL-PEGDA fibers had lower levels of cell death compared to injured slices treated with vehicle or fiber mats lacking UA (Figures 4b and 5b). We observed the same results for cell death in the ventral region (Figure 5b). Upon examination of oxidative stress, we found no significant differences in ROS levels between dorsal and ventral regions of slices within the same treatment group (Figure 5c), and both types of fiber mats reduced ROS levels to baseline levels in dorsal and ventral regions. Our results suggest that UA release from fibers provides protection from and decreases production of GIE-induced cell death and ROS, respectively, in both dorsal sensory and ventral motor spinal cord neurons in slices.

FIGURE 5.

FIGURE 5

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Region-specific effects of UA fiber mat treatment on cell death and ROS levels after GIE. (a) Subset of representative images of dorsal and ventral ROIs (white; traced from phase contrast images) in organotypic spinal cord slice cultures subjected to GIE, UA fiber treatment, PI treatment, and CellROX™ Green Reagent/H2DCFDA, as described previously. Scale bar = 500 μm (b) Quantification of PI mean fluorescence intensity in dorsal and ventral regions of cultures compared to control. (c) Quantification of CellROX™ Green Reagent/H2DCFDA mean fluorescence intensity in dorsal and ventral regions of cultures compared to control. Data were normalized to control (A.U. = arbitrary units). Outliers were removed using ROUTS test, and data were subsequently analyzed using one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test to compare dorsal versus ventral cell death and ROS levels within each experimental group (*), dorsal cell death and ROS levels to control (#), and ventral cell death and ROS levels to control (+), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (p-values follow number of characters for # and +), n = 3 independent experiments (4–20 slices per condition per experiment). Error bars = SEM; GIE, glutamate-induced excitotoxicity; H2DCFDA, 2’,7’-dichlorodihydrofluorescein diacetate; PBS, phosphate-buffered saline; PEGDA, poly (ethylene glycol) diacrylate; PCL, poly(ɛ-caprolactone); PI, propidium iodide; ROI, region of interest; ROS, reactive oxygen species; SEM, scanning electron microscopy; UA, uric acid

4 |. DISCUSSION

This study explored the potential of PCL fibers as a drug delivery platform for UA to treat SCI by assessing cell death and ROS in organotypic spinal cord slices after GIE and treatment with UA-releasing fibers. We tested the effects of our burst and sustained release UA fiber mats on spinal cord organotypic slice cultures to determine whether either treatment results in a better outcome after injury with glutamate. We found that, regardless of release rate, treatment with UA-releasing fiber mats reduced tissue damage from GIE, as evidenced by lower cell death and ROS production. These data are consistent with our previous finding demonstrating that soluble UA reduces damage to neurons caused by GIE (Du et al., 2007). Upon further analysis, we found that UA-PCL and UA-PCL-PEGDA fibers conferred protection to both dorsal and ventral regions of slices and mitigated increased toxicity observed in the dorsal region. Spinal cord slices injured with glutamate and treated with control PCL and PCL-PEGDA fibers had higher levels of cell death in their dorsal regions, suggesting higher sensitivity of sensory neurons to GIE. In fact, dorsal horn neurons are among the most vulnerable to excite-toxic death (Mazzone & Nistri, 2011). Furthermore, hyperactivity and death of dorsal horn sensory neurons is associated with neuropathic pain after SCI (Gwak, Hulsebosch, & Leem, 2017). Thus, it is possible that distinct mechanisms of excitotoxic death affect dorsal and ventral neurons in the spinal cord (Mazzone & Nistri, 2011). However, we demonstrate that treatment with UA-releasing fibers protects both dorsal and ventral spinal cord tissue against GIE.

We estimated that the releasable UA in the fiber mats is approximately 1.3 g, yielding an 8 mM solution after maximal release (Figure 3b). We did not perform an in-depth analysis of the loading efficiency as we are using these fiber mats to study whether burst release of UA versus sustained release of UA promotes greater cell survival. Furthermore, although we did not determine the elastic modulus, we predict our scaffold to have similar properties to those previously reported by our group for 25% PCL-1/10 poly(3,4-ethyl-enedioxythiophene) scaffold (McKeon-Fischer et al., 2015). The data from our study will be used to inform future biomaterials for patients with SCI.

As UA is a well-known antioxidant, it is likely that the protection conferred by UA-PCL and UA-PCL-PEGDA fibers against GIE is attributable, at least in part, to the peroxynitrite scavenging activity of UA. Several research groups have demonstrated neuroprotective effects of UA against damage from excitotoxic insult (Du et al., 2007; Scott et al., 2005; Yu et al., 1998); however, since the rate of UA release from the fiber mats did not affect the extent of protection, additional mechanisms may also be at play. Previously, our laboratory reported that UA upregulates the glutamate transporter EAAT-1 on astrocytes to confer neuroprotection from secondary injury (Du et al., 2007). These transporters facilitate uptake of glutamate by astrocytes from the injury microenvironment, thereby decreasing hyperstimulation of neurons. Thus, fiber-mediated delivery of UA likely protects against GIE via multiple mechanisms.

We decided to use fiber mats to deliver UA to injured neurons due to their versatility, tunability, and conduciveness to biological applications. The ideal incorporation of a drug into a polymer includes nontoxicity, nonimmunogenicity, enhanced permeation and retention rate, high loading capacity, and targeted and controlled delivery (Duncan, 2006). The synthetic polymer, PCL, meets many of these parameters and is a commonly used biomaterial for tissue engineering, wound dressing, and targeted drug delivery systems (Charman, Takakura, Hashida, & Sezaki, 1992; Kopeček, 2003). Electrospun PCL fibers have been extensively studied and used for various biomedical applications because of their slow biodegradation, high biocompatibility, and thermal stability (Charman et al., 1992; Liechty, Kryscio, Slaughter, & Peppas, 2010). Hence, we chose PCL fibers for the development of a novel UA delivery platform.

Our results also support the fact that electrospinning allows for more precise control over the release of UA from PCL fiber mats, as release is controlled by the degradation rate of PCL. Thus, UA release can be optimized by adjusting the concentration and molecular weight of PCL. Coating the PCL with PEGDA reduces hydrolysis of PCL since PEGDA is nondegradable. The release kinetics can be further tailored by changing the molecular weight of the PEGDA being used for coating (Hamid & Lim, 2016), as higher molecular weight yields larger scaffold pore size. Furthermore, PEGDA is hydrophilic and prevents protein adsorption, making it nonimmunogenic and biocompatible (Alcantar, Aydil, & Israelachvili, 2000; Padmavathi & Chatterji, 1996). Our study supports the idea that PEGDA slows the release of UA from UA-PCL fiber mats without detrimental effects on the viability of organotypic spinal cord slices.

5 |. CONCLUSION

In sum, we have developed a novel method for localized UA delivery from a biomaterial and demonstrated that UA-PCL fibers successfully protect spinal cord tissue from excitotoxic injury in an in vitro injury model (Figure 6). Our findings have implications for SCI treatment and further support the viability of UA as an effective therapeutic agent for this application.

FIGURE 6.

FIGURE 6

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Model of the effects of UA fibers on secondary SCI. After the primary mechanical impact occurs during SCI and damages spinal cord tissue, GIE and secondary signaling cascades further exacerbate cell death and ROS release both proximal to and distal to the lesion site. UA released from PCL fibers reduces cell death and ROS levels in spinal cord tissue subjected to GIE and represents a promising therapeutic platform for the treatment of SCI; GIE, glutamate-induced excitotoxicity; PBS, phosphate-buffered saline; PEGDA, poly(ethylene glycol) diacrylate; PCL, poly(ɛ-caprolactone); ROS, reactive oxygen species; SCI, Spinal cord injury; UA, uric acid

ACKNOWLEDGEMENTS

This work is funded in part by the New Jersey Commission on Spinal Cord Research grant #CSCR14IRG005 and CSCR20IRG011 (to B.L.F.). Nisha K. Singh is supported by the National Institutes of Health T32 GM008339-28 from the NIGMS and the New Jersey Commission on Spinal Cord Research Predoctoral Fellowship #CSCR20FEL004. Mann Patel is supported by a fellowship from the Aresty Research Center at Rutgers University. All scanning electron microscopy imaging was performed using equipment provided by the Rutgers New Jersey CryoEM/CryoET Core Facility.

Footnotes

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

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