Poly(pro-curcumin) Materials Exhibit Dual Release Rates and Prolonged Antioxidant Activity as Thin Films and Self-Assembled Particles.

Curcumin is a natural polyphenol that exhibits remarkable antioxidant and anti-inflammatory activities; however, its clinical application is limited in part by its physiological instability. Here, we report the synthesis of curcumin-derived polyesters that release curcumin upon hydrolytic degradation to improve curcumin stability and solubility in physiological conditions. Curcumin was incorporated in the polymer backbone by a one-pot condensation polymerization in the presence of sebacoyl chloride and polyethylene glycol (PEG, Mn = 1 kDa). The thermal and mechanical properties, surface wettability, self-assembly behavior, and drug-release kinetics all depend sensitively on the mole percentage of curcumin incorporated in these statistical copolymers. Curcumin release was triggered by the hydrolysis of phenolic esters on the polymer backbone, which was confirmed using a PEGylated curcumin model compound, which represented a putative repeating unit within the polymer. The release rate of curcumin was controlled by the hydrophilicity of the polymers. Burst release (2 days) and extended release (>8 weeks) can be achieved from the same polymer depending on curcumin content in the copolymer. The materials can quench free radicals for at least 8 weeks and protect primary neurons from oxidative stress in vitro. Further, these copolymer materials could be processed into both thin films and self-assembled particles, depending on the solvent-based casting conditions. Finally, we envision that these materials may have potential for neural tissue engineering application, where antioxidant release can mitigate oxidative stress and the inflammatory response following neural injury.

Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naïve and stimulated cultures.

Astrocytes are responsible for a wide variety of essential functions throughout the central nervous system. The protein markers glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), glutamine synthetase (GS), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and the transcription factor SOX9 are routinely used to label astrocytes in primary rodent cultures. However, GLAST, GLT-1, GS, and SOX9 are also produced by microglia and oligodendrocytes and GFAP, GLAST, GLT-1, and GS production levels are affected by astrocyte phenotypic changes associated with reactive astrogliosis. No group has performed a comprehensive immunocytochemical evaluation to quantify the percentage of cells labeled by these markers in vitro, nor compared changes in staining between cortex- and spinal cord-derived cells in naïve and stimulated cultures. Here, we quantified the percentage of cells positively stained for these six markers in astrocyte, microglia, and oligodendrocyte cultures isolated from neonatal rat cortices and spinal cords. Additionally, we incubated the astrocytes with transforming growth factor (TGF)-ß1 or TGF-ß3 to determine if the labeling of these markers is altered by these stimuli. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Furthermore, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, and ALDH1L1 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST and GS in TGF-ß1-treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models.

Biomaterial Approaches to Modulate Reactive Astroglial Response.

Over several decades, biomaterial scientists have developed materials to spur axonal regeneration and limit secondary injury and tested these materials within preclinical animal models. Rarely, though, are astrocytes examined comprehensively when biomaterials are placed into the injury site. Astrocytes support neuronal function in the central nervous system. Following an injury, astrocytes undergo reactive gliosis and create a glial scar. The astrocytic glial scar forms a dense barrier which restricts the extension of regenerating axons through the injury site. However, there are several beneficial effects of the glial scar, including helping to reform the blood-brain barrier, limiting the extent of secondary injury, and supporting the health of regenerating axons near the injury site. This review provides a brief introduction to the role of astrocytes in the spinal cord, discusses astrocyte phenotypic changes that occur following injury, and highlights studies that explored astrocyte changes in response to biomaterials tested within in vitro or in vivo environments. Overall, we suggest that in order to improve biomaterial designs for spinal cord injury applications, investigators should more thoroughly consider the astrocyte response to such designs.

Removal of Retained Electrospinning Solvent Prolongs Drug Release from Electrospun PLLA Fibers.

A major challenge in developing drug-releasing electrospun nanofibers is obtaining long-term drug release over many weeks with no burst release of drug. Here, we present new methods capable of prolonging the diffusive release of small molecule drugs from electrospun poly-L-lactic acid (PLLA) nanofibers. The methods focus on removal of retained electrospinning solvent through fiber heating, maintaining fibers in a laboratory setting, or a combination of these methods. These post-fabrication methods altered the release characteristics of a model small molecule drug, 6-aminonicotinamide (6AN), from PLLA fibers. Specifically, untreated fibers released 6AN over 9 days, and fibers that underwent a combined treatment of maintenance in a laboratory setting and heating released 6AN over 44 days. The unique and simple method presented here prolongs diffusive release of a small molecule drug from electrospun fibers and has potential to assist in lengthening small molecule drug release from a variety of polymeric nanomaterials.

Nanoparticle Technologies in the Spinal Cord.

Nanoparticles are increasingly being studied within experimental models of spinal cord injury (SCI). They are used to image cells and tissue, move cells to specific regions of the spinal cord, and deliver therapeutic agents locally. The focus of this article is to provide a brief overview of the different types of nanoparticles being studied for spinal cord applications and present data showing the capability of nanoparticles to deliver the chondroitinase ABC (chABC) enzyme locally following acute SCI in rats. Nanoparticles releasing chABC helped promote axonal regeneration following injury, and the nanoparticles also protected the enzyme from rapid degradation. In summary, nanoparticles are viable materials for diagnostic or therapeutic applications within experimental models of SCI and have potential for future clinical use.

Electrospun Fibers for Drug Delivery after Spinal Cord Injury and the Effects of Drug Incorporation on Fiber Properties.

There is currently no cure for individuals with spinal cord injury (SCI). While many promising approaches are being tested in clinical trials, the complexity of SCI limits several of these approaches from aiding complete functional recovery. Several different categories of biomaterials are investigated for their ability to guide axonal regeneration, to deliver proteins or small molecules locally, or to improve the viability of transplanted stem cells. The purpose of this study is to provide a brief overview of SCI, present the different categories of biomaterial scaffolds that direct and guide axonal regeneration, and then focus specifically on electrospun fiber guidance scaffolds. Much like other polymer guidance approaches, electrospun fibers can retain and deliver therapeutic drugs. The experimental section presents new data showing the incorporation of two therapeutic drugs into electrospun poly-L-lactic acid fibers. Two different concentrations of either riluzole or neurotrophin-3 were loaded into the electrospun fibers to examine the effect of drug concentration on the physical characteristics of the fibers (fiber alignment and fiber diameter). Overall, the drugs were successfully incorporated into the fibers and the release was related to the loading concentration. The fiber diameter decreased with the inclusion of the drug, and the decreased diameter was correlated with a decrease in fiber alignment. Subsequently, the study includes considerations for successful incorporation of a therapeutic drug without changing the physical properties of the fibers.

Prefrontal cortical GABAergic dysfunction contributes to age-related working memory impairment.

Working memory functions supported by the prefrontal cortex decline in normal aging. Disruption of corticolimbic GABAergic inhibitory circuits can impair working memory in young subjects; however, relatively little is known regarding how aging impacts prefrontal cortical GABAergic signaling and whether such changes contribute to cognitive deficits. The current study used a rat model to evaluate the effects of aging on expression of prefrontal GABAergic synaptic proteins in relation to working memory decline, and to test whether pharmacological manipulations of prefrontal GABAergic signaling can improve working memory abilities in aged subjects. Results indicate that in aged medial prefrontal cortex (mPFC), expression of the vesicular GABA transporter VGAT was unchanged; however, there was a significant increase in expression of the GABA synthesizing enzyme GAD67, and a significant decrease in the primary neuronal GABA transporter GAT-1 and in both subunits of the GABA(B) receptor (GABA(B)R). Expression of VGAT, GAD67, and GAT-1 was not associated with working memory ability. In contrast, among aged rats, GABA(B)R expression was significantly and negatively associated with working memory performance, such that lower GABA(B)R expression predicted better working memory. Subsequent experiments showed that systemic administration of a GABA(B)R antagonist, CGP55845, dose-dependently enhanced working memory in aged rats. This enhancing effect of systemic CGP55845 was reproduced by direct intra-mPFC administration. Together, these data suggest that age-related dysregulation of GABAergic signaling in prefrontal cortex may play a causal role in impaired working memory and that targeting GABA(B)Rs may provide therapeutic benefit for age-related impairments in executive functions.

Reduced astrocyte viability at physiological temperatures from magnetically activated iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) can generate heat when subjected to an alternating magnetic field (AMF). In the European Union, SPIONs actuated by AMF are used in hyperthermia treatment of glioblastoma multiforme, an aggressive form of brain cancer. Current data from clinical trials suggest that this therapy improves patient life expectancy, but their effect on healthy brain cells is virtually unknown. Thus, a viability study involving SPIONs subjected to an AMF was carried out on healthy cortical rat astrocytes, the most abundant cell type in the mammalian brain. The cells were cultured with aminosilane- or starch-coated SPIONs with or without application of an AMF. Significant cell death (p < 0.05) was observed only when SPIONs were added to astrocyte cultures and subjected to an AMF. Unexpectedly, the decrease in astrocyte viability was observed at physiological temperatures (34-40 °C) with AMF. A further decrease in astrocyte viability was found only when bulk temperatures exceeded 45 °C. To discern differences in the astrocyte structure when astrocytes were cultured with particles with or without AMF, scanning electron microscopy (SEM) was performed. SEM images revealed a change in the structure of the astrocyte cell membrane only when astrocytes were cultured with SPIONs and actuated with an AMF. This study is the first to report that astrocyte death occurs at physiological temperatures in the presence of magnetic particles and AMF, suggesting that other mechanisms are responsible for inducing astrocyte death in addition to heat.

Effect of magnetic nanoparticle heating on cortical neuron viability.

PURPOSE: Superparamagnetic iron oxide nanoparticles are currently approved for use as an adjunctive treatment to glioblastoma multiforme radiotherapy. Radio frequency stimulation of the nanoparticles generates localised hyperthermia, which sensitises the tumour to the effects of radiotherapy. Clinical trials reported thus far are promising, with an increase in patient survival rate; however, what are left unaddressed are the implications of this technology on the surrounding healthy tissue. METHODS AND MATERIALS: Aminosilane-coated iron oxide nanoparticles suspended in culture medium were applied to chick embryonic cortical neuron cultures. Cultures were heated to 37 °C or 45 °C by an induction coil system for 2 h. The latter regime emulates the therapeutic conditions of the adjunctive therapy. Cellular viability and neurite retraction was quantified 24 h after exposure to the hyperthermic events. RESULTS: The hyperthermic load inflicted little damage to the neuron cultures, as determined by calcein-AM, propidium iodide, and alamarBlue® assays. Fluorescence imaging was used to assess the extent of neurite retraction which was found to be negligible. CONCLUSIONS: Retention of chick, embryonic cortical neuron viability was confirmed under the thermal conditions produced by radiofrequency stimulation of iron oxide nanoparticles. While these results are not directly applicable to clinical applications of hyperthermia, the thermotolerance of chick embryonic cortical neurons is promising and calls for further studies employing human cultures of neurons and glial cells.

One-Pot Assembly of Drug-Eluting Silk Coatings with Applications for Nerve Regeneration.

Clinical use of polymeric scaffolds for tissue engineering often suffers from their inability to promote strong cellular interactions. Functionalization with biomolecules may improve outcomes; however, current functionalization approaches using covalent chemistry or physical adsorption can lead to loss of biomolecule bioactivity. Here, we demonstrate a novel bottom-up approach for enhancing the bioactivity of poly(l-lactic acid) electrospun scaffolds though interfacial coassembly of protein payloads with silk fibroin into nanothin coatings. In our approach, protein payloads are first added into an aqueous solution with Bombyx mori-derived silk fibroin. Phosphate anions are then added to trigger coassembly of the payload and silk fibroin, as well as noncovalent formation of a payload-silk fibroin coating at poly(l-lactic) acid fiber surfaces. Importantly, the coassembly process results in homogeneous distribution of protein payloads, with the loading quantity depending on payload concentration in solution and coating time. This coassembly process yields greater loading capacity than physical adsorption methods, and the payloads can be released over time in physiologically relevant conditions. We also demonstrate that the coating coassembly process can incorporate nerve growth factor and that coassembled coatings lead to significantly more neurite extension than loading via adsorption in a rat dorsal root ganglia explant culture model.

Delivery of TGFß3 from Magnetically Responsive Coaxial Fibers Reduces Spinal Cord Astrocyte Reactivity In Vitro.

A spinal cord injury (SCI) compresses the spinal cord, killing neurons and glia at the injury site and resulting in prolonged inflammation and scarring that prevents regeneration. Astrocytes, the main glia in the spinal cord, become reactive following SCI and contribute to adverse outcomes. The anti-inflammatory cytokine transforming growth factor beta 3 (TGFß3) has been shown to mitigate astrocyte reactivity; however, the effects of prolonged TGFß3 exposure on reactive astrocyte phenotype have not yet been explored. This study investigates whether magnetic core-shell electrospun fibers can be used to alter the release rate of TGFß3 using externally applied magnetic fields, with the eventual application of tailored drug delivery based on SCI severity. Magnetic core-shell fibers are fabricated by incorporating superparamagnetic iron oxide nanoparticles (SPIONs) into the shell and TGFß3 into the core solution for coaxial electrospinning. Magnetic field stimulation increased the release rate of TGFß3 from the fibers by 25% over 7 days and released TGFß3 reduced gene expression of key astrocyte reactivity markers by at least twofold. This is the first study to magnetically deliver bioactive proteins from magnetic fibers and to assess the effect of sustained release of TGFß3 on reactive astrocyte phenotype.

Curcumin Release from Biomaterials for Enhanced Tissue Regeneration Following Injury or Disease.

Curcumin, a bioactive phenol derived from turmeric, is an antioxidant, anti-inflammatory, and antibacterial molecule. Although curcumin exhibits beneficial effects in its innate form, it is highly hydrophobic, which leads to poor water solubility and, consequently, low bioavailability. The lack of bioavailability limits curcumin's effectiveness as a treatment and restricts its use in clinical applications. Furthermore, to achieve beneficial, clinically relevant results, high doses of curcumin are required for systemic administration. Many researchers have utilized biomaterial carriers, including electrospun fibers, nanoparticles, hydrogels, and composite scaffolds, to overcome curcumin's principle therapeutic limitation of low bioavailability. By using biomaterials to deliver curcumin directly to injury sites, researchers have harnessed the beneficial natural properties of curcumin while providing scaffolding to support tissue regeneration. This review will provide an in-depth overview of the literature that utilizes biomaterial delivery of curcumin for tissue regeneration in injury and disease models.

Nanomaterial payload delivery to central nervous system glia for neural protection and repair.

Central nervous system (CNS) glia, including astrocytes, microglia, and oligodendrocytes, play prominent roles in traumatic injury and degenerative disorders. Due to their importance, active pharmaceutical ingredients (APIs) are being developed to modulate CNS glia in order to improve outcomes in traumatic injury and disease. While many of these APIs show promise in vitro, the majority of APIs that are systemically delivered show little penetration through the blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) and into the CNS, rendering them ineffective. Novel nanomaterials are being developed to deliver APIs into the CNS to modulate glial responses and improve outcomes in injury and disease. Nanomaterials are attractive options as therapies for central nervous system protection and repair in degenerative disorders and traumatic injury due to their intrinsic capabilities in API delivery. Nanomaterials can improve API accumulation in the CNS by increasing permeation through the BBB of systemically delivered APIs, extending the timeline of API release, and interacting biophysically with CNS cell populations due to their mechanical properties and nanoscale architectures. In this review, we present the recent advances in the fields of both locally implanted nanomaterials and systemically administered nanoparticles developed for the delivery of APIs to the CNS that modulate glial activity as a strategy to improve outcomes in traumatic injury and disease. We identify current research gaps and discuss potential developments in the field that will continue to translate the use of glia-targeting nanomaterials to the clinic.

Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications.

Aligned electrospun fibers provide topographical cues and local therapeutic delivery to facilitate robust peripheral nerve regeneration. mRNA delivery enables transient expression of desired proteins that promote axonal regeneration. However, no prior work delivers mRNA from electrospun fibers for peripheral nerve regeneration applications. Here, we developed the first aligned electrospun fibers to deliver pseudouridine-modified (Ψ) neurotrophin-3 (NT-3) mRNA (ΨNT-3mRNA) to primary Schwann cells and assessed NT-3 secretion and bioactivity. We first electrospun aligned poly(L-lactic acid) (PLLA) fibers and coated them with the anionic substrates dextran sulfate sodium salt (DSS) or poly(3,4-dihydroxy-L-phenylalanine) (pDOPA). Cationic lipoplexes containing ΨNT-3mRNA complexed to JetMESSENGER® were then immobilized to the fibers, resulting in detectable ΨNT-3mRNA release for 28 days from all fiber groups investigated (PLLA+mRNA, 0.5DSS4h+mRNA, and 2pDOPA4h+mRNA). The 2pDOPA4h+mRNA group significantly increased Schwann cell secretion of NT-3 for 21 days compared to control PLLA fibers (p < 0.001-0.05) and, on average, increased Schwann cell secretion of NT-3 by ≥ 2-fold compared to bolus mRNA delivery from the 1µgBolus+mRNA and 3µgBolus+mRNA groups. The 2pDOPA4h+mRNA fibers supported Schwann cell secretion of NT-3 at levels that significantly increased dorsal root ganglia (DRG) neurite extension by 44% (p < 0.0001) and neurite area by 64% (p < 0.001) compared to control PLLA fibers. The data show that the 2pDOPA4h+mRNA fibers enhance the ability of Schwann cells to promote neurite growth from DRG, demonstrating this platform's potential capability to improve peripheral nerve regeneration. STATEMENT OF SIGNIFICANCE: Aligned electrospun fibers enhance axonal regeneration by providing structural support and guidance cues, but further therapeutic stimulation is necessary to improve functional outcomes. mRNA delivery enables the transient expression of therapeutic proteins, yet achieving local, sustained delivery remains challenging. Previous work shows that genetic material delivery from electrospun fibers improves regeneration; however, mRNA delivery has not been explored. Here, we examine mRNA delivery from aligned electrospun fibers to enhance neurite outgrowth. We show that immobilization of NT-3mRNA/JetMESSENGER® lipoplexes to aligned electrospun fibers functionalized with pDOPA enables local, sustained NT-3mRNA delivery to Schwann cells, increasing Schwann cell secretion of NT-3 and enhancing DRG neurite outgrowth. This study displays the potential benefits of electrospun fiber-mediated mRNA delivery platforms for neural tissue engineering.

Development of a Slow-Degrading Polymerized Curcumin Coating for Intracortical Microelectrodes.

Intracortical microelectrodes are used with brain-computer interfaces to restore lost limb function following nervous system injury. While promising, recording ability of intracortical microelectrodes diminishes over time due, in part, to neuroinflammation. As curcumin has demonstrated neuroprotection through anti-inflammatory activity, we fabricated a 300 nm-thick intracortical microelectrode coating consisting of a polyurethane copolymer of curcumin and polyethylene glycol (PEG), denoted as poly(curcumin-PEG1000 carbamate) (PCPC). The uniform PCPC coating reduced silicon wafer hardness by two orders of magnitude and readily absorbed water within minutes, demonstrating that the coating is soft and hydrophilic in nature. Using an in vitro release model, curcumin eluted from the PCPC coating into the supernatant over 1 week; the majority of the coating was intact after an 8-week incubation in buffer, demonstrating potential for longer term curcumin release and softness. Assessing the efficacy of PCPC within a rat intracortical microelectrode model in vivo, there were no significant differences in tissue inflammation, scarring, neuron viability, and myelin damage between the uncoated and PCPC-coated probes. As the first study to implant nonfunctional probes with a polymerized curcumin coating, we have demonstrated the biocompatibility of a PCPC coating and presented a starting point in the design of poly(pro-curcumin) polymers as coating materials for intracortical electrodes.

Altering iron oxide nanoparticle surface properties induce cortical neuron cytotoxicity.

Superparamagnetic iron oxide nanoparticles, with diameters in the range of a few tens of nanometers, display the ability to cross the blood-brain barrier and are envisioned as diagnostic and therapeutic tools in neuro-medicine. However, despite the numerous applications being explored, insufficient information is available on their potential toxic effect on neurons. While iron oxide has been shown to pose a decreased risk of toxicity, surface functionalization, often employed for targeted delivery, can significantly alter the biological response. This aspect is addressed in the present study, which investigates the response of primary cortical neurons to iron oxide nanoparticles with coatings frequently used in biomedical applications: aminosilane, dextran, and polydimethylamine. Prior to administering the particles to neuronal cultures, each particle type was thoroughly characterized to assess the (1) size of individual nanoparticles, (2) concentration of the particles in solution, and (3) agglomeration size and morphology. Culture results show that polydimethylamine functionalized nanoparticles induce cell death at all concentrations tested by swift and complete removal of the plasma membrane. Aminosilane coated particles affected metabolic activity only at higher concentrations while leaving the membrane intact, and dextran-coated nanoparticles partially altered viability at higher concentrations. These findings suggest that nanoparticle characterization and primary cell-based cytotoxicity evaluation should be completed prior to applying nanomaterials to the nervous system.

Altered spatial learning and delay discounting in a rat model of human third trimester binge ethanol exposure.

Ethanol exposure during perinatal development can cause cognitive abnormalities including difficulties in learning, attention, and memory, as well as heightened impulsivity. The purpose of this study was to assess performance in spatial learning and impulsive choice tasks in rats subjected to an intragastric intubation model of binge ethanol exposure during human third trimester-equivalent brain development. Male and female Sprague-Dawley rat pups were intubated with ethanol (5.25 g/kg/day) on postnatal days 4-9. At adolescence (between postnatal days 35-38), these rats and sham intubated within-litter controls were trained in both spatial and cued versions of the Morris water maze. A subset of the male rats was subsequently tested on a delay-discounting task to assess impulsive choice. Ethanol-exposed rats were spatially impaired relative to controls, but performed comparably to controls on the cued version of the water maze. Ethanol-exposed rats also showed greater preference for large delayed rewards on the delay discounting task, but no evidence for altered reward sensitivity or perseverative behavior. These data demonstrate that early postnatal intermittent binge-like ethanol exposure has prolonged, detrimental, but selective effects on cognition, suggesting that even relatively brief ethanol exposure late in human pregnancy can be deleterious for cognitive function.

Designing electrospun fiber platforms for efficient delivery of genetic material and genome editing tools.

Electrospun fibers are versatile biomaterial platforms with great potential to support regeneration. Electrospun fiber characteristics such as fiber diameter, degree of alignment, rate of degradation, and surface chemistry enable the creation of unique, tunable scaffolds for various drug or gene delivery applications. The delivery of genetic material and genome editing tools via viral and non-viral vectors are approaches to control cellular protein production. However, immunogenicity, off-target effects, and low delivery efficiencies slow the progression of gene delivery strategies to clinical settings. The delivery of genetic material from electrospun fibers overcomes such limitations by allowing for localized, tunable delivery of genetic material. However, the process of electrospinning is harsh, and care must be taken to retain genetic material bioactivity. This review presents an up-to-date summary of strategies to incorporate genetic material onto or within electrospun fiber platforms to improve delivery efficiency and enhance the regenerative potential of electrospun fibers for various tissue engineering applications.