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.


Introduction
Glia, which constitute roughly half the cells in the central nervous system (CNS), have essential yet distinct roles in supporting neuronal homeostasis and signal transduction (Allen and Lyons, 2018).In particular, astrocytes, microglia, and oligodendrocytes are necessary for regulating synaptic function, contributing to metabolic support, creating myelin sheaths for signal transduction, and in the CNS immune response (Somjen, 1988;Tomassy et al., 2016;von Bartheld et al., 2016).Glia also are critical players in disease and after traumatic injury, as microglia are the primary source of pro-inflammatory cytokines, astrocytes are regulators of synaptic homeostasis and glial scar formation after injury, and demyelination or changes in myelin thickness by oligodendrocytes alters signal conduction speed (Colonna and Butovsky, 2017;Liddelow and Barres, 2017;Wang S. S. et al., 2018).Due to the fact that neuronal regeneration is highly restricted following CNS injury and disease, glia have begun to emerge as important targets in the development of active pharmaceutical ingredients (APIs) in order to improve clinical outcomes.However, since many APIs do not readily cross the bloodbrain barrier (BBB) to impart their action on glia, nanomaterials have been engineered to carry APIs to the site of action, extend the API release timeframe, and also to impact cellular behavior based on their architectural features (Zhang et al., 2016;Dai et al., 2021).
Nanomaterials, including nanoparticles and nanostructured scaffolds, offer many advantages in the delivery of APIs to CNS glia.Nanoparticles (NPs), composed of polymers, liposomes, inorganic materials, and extracellular vesicles, are used to deliver APIs to glia because they can be administered systemically, engineered to cross the BBB, carry and protect sensitive APIs, and be targeted to cells or regions of interest using antibodies, targeting peptides, and even nucleic acids (Patel et al., 2012;Mann et al., 2016;Zuidema et al., 2016;Furtado et al., 2018;Zhou et al., 2018;Ciciriello et al., 2022;Waggoner et al., 2022).The advantage of NP technologies is that they can be administered systemically; however, in some cases, such as TBI, BBB permeability can decrease over time, which can reduce the accumulation of the API in the CNS at longer time points after injury (Werner and Engelhard, 2007;Mann et al., 2016).In CNS disorders, BBB breakdown often occurs prior to neurodegeneration and persists as the disease progresses (Sweeney et al., 2018a,b).While this can be advantageous for API delivery to the CNS, there are also complex mechanisms, including disrupted BBB transporter expression, inflammation, immune products, and impaired solute transport, which can limit API accumulation in these regions (Sweeney et al., 2018b).NP design needs to consider this when attempting to traverse the injured or diseased BBB to deliver APIs to regions of interest.Nanostructured scaffolds have other advantages, even though they generally must be surgically implanted or injected into the site of interest.The nanoscale topographical features of nanomaterial scaffolds can influence cellular function, migration, and growth; APIs can be delivered from either the surface of the scaffold or incorporated into the scaffold to extend release, and, since the scaffolds are implanted directly at the site, APIs are released locally to glia (Tsui et al., 2019;Puhl et al., 2020Puhl et al., , 2022)).Here, we present the current stateof-the-art in API delivery to CNS glia using nanomaterials, point out the existing gaps in the research, and discuss the potential future developments and advances of this field that will drive nanomaterial delivery of APIs to CNS glia towards the clinic.

Nanoparticle API delivery to astrocytes
Astrocytes are CNS glia that perform core homeostatic functions and whose radiating processes can contact upwards of 1 million synapses in humans (Hasel and Liddelow, 2021).They are integral parts of the BBB where they uptake metabolites such as glucose to fuel active neurons, modulate neurotransmitter concentration in synapses, phagocytose synapses, form part of the glymphatic system, and aid in the homeostatic control of neuronal redox stress (Sofroniew and Vinters, 2010;Hasel and Liddelow, 2021).In the event of injury or pathology, including stab wound injuries, experimental autoimmune encephalomyelitis (EAE), middle cerebral occlusion (MCAO), hypertrophic ciliary neurotrophic factor induction, cortical lesion, spinal cord injury (SCI), Alzheimer's disease, Parkinson's disease, prion disease, Huntington's disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), astrocytes respond via a process termed reactive astrogliosis (Anderson et al., 2014;Pekny et al., 2016;Escartin et al., 2019).This process can be protective, but persistent reactive astrogliosis can become maladaptive, making it a target for APIs (Pekny et al., 2016).Therefore, APIs that act on astrocytes have been developed that target metabolic pathways, transporters and receptors, cell-cell interactions, and even as glia-to-neuron conversion therapies (Lee et al., 2022).Their role in disease and trauma, as well as recent advances in nanomaterial API delivery, make astrocytes a critical cell to target in order to improve clinical outcomes in CNS disease and injury.

Nanomaterial API delivery to astrocytes
Nanomaterial strategies targeting astrocytes, including electrospun fibers, composite hydrogels, and hybrid materials, address two occurrences in the astrocytic response to injury: (1) to reduce glial scar formation, or (2) to mitigate an established glial scar (Jarrin et al., 2021).Often, these materials contain anti-inflammatory API payloads or exogenous stem cells which assist in promoting a neuroprotective phenotype, by diminishing the inhibitory chemical barrier and promoting restoration of the blood-spinal cord barrier.Polymer nanofibers are fabricated by electrospinning, a process that allows for the creation of biodegradable and biocompatible scaffolds that can be used in neural tissue engineering (Schaub et al., 2016;Cheng et al., 2021).Due to their high surface area and structure, electrospun     -(5-2-[2-(5-amino-[1,3,4] thiadiazol-2-yl)-ethylsulfanyl]-   nanofibers mimic the native extracellular matrix of neural tissue and are hence suited to promote neural regeneration (Tian et al., 2015).Nanofibers are attractive as drug depots for astrocytes because their intrinsic material properties can alter astrocyte activation or direct astrocyte growth (Zuidema et al., 2014(Zuidema et al., , 2018)).Improved astrocyte activation outcomes have been demonstrated by employing nanofiber scaffolds alone, incorporating stem cells with nanofibers, and even with conductive nanofibers (Zhao et al., 2018;Shu et al., 2019;Yan et al., 2020;Dai et al., 2023;Xu et al., 2023).As a means to further promote neural repair and mitigation of secondary injury, nanofibers can be loaded with APIs that act on astrocytes due to their porous nature (Zhang et al., 2021).Growth factors and small molecule drugs released from nanofibers have shown the ability to reduce astrocyte activation (Zhang et al., 2018;Bighinati et al., 2020;Sun et al., 2020), decreasing GFAP expression and improving outcomes (Table 2).Nanocomposite hydrogel constructs are used frequently in neural tissue engineering to promote cell adhesion and proliferation, incorporate guidance cues, and provide electrical conductivity in the tissue-supporting scaffold (Madhusudanan et al., 2020).These properties make them especially attractive as injectable materials to deliver APIs to astrocytes.Conductive hydrogels with nanoparticles (Yang et al., 2022) or nanosheets (Chen et al., 2022) following stimulated spinal cord injury demonstrated decreases in GFAP-labeled astrocytes, as well as decreases in chondroitin sulfate proteoglycans and increased neuronal markers (Table 2).Studies using nanofiber hydrogels (NFH) have shown different outcomes, with NFH alone demonstrating no induction of astrocytes (Gonzalez et al., 2022), while increased amounts of astrocytes at the injury site were seen using an NFH construct combined with BMSCs (Li et al., 2020;Haggerty et al., 2022).Nanoparticle hydrogel composites have also shown varying results (Serafin et al., 2022).Hydrogel nanohybrids releasing NGF, diacerein, or chondroitinase ABC have reduced astrocyte activity (Raspa et al., 2021;Gao et al., 2022;Xing et al., 2023), demonstrating the potential of these API-releasing nanomaterials to improve astrocyte outcomes.

Nanoparticle API delivery to microglia
Microglia constitute 5%-10% of total brain cells and are the only true CNS parenchymal macrophages (Aguzzi et al., 2013).Upon CNS injury or disease, microglia adopt an "amoeboid" morphology and are responsible for phagocytosis and elimination of microbes, dead cells, and protein aggregates, and the secretion of soluble factors, including chemoattractants, cytokines, and neurotrophic factors (Colonna and Butovsky, 2017;Li and Barres, 2018).These polarized cells were traditionally categorized as having either toxic (M1) or protective (M2) states; however, accumulating evidence suggests microglial polarization is complex and multidimensional (Ransohoff, 2016a).In fact, single cell sequencing suggests that depending upon their anatomical compartment and pathological environment, microglia display an entire spectrum of functional states, ranging from highly inflammatory and phagocytic to anti-inflammatory and neuroprotective (Sankowski et al., 2022) Persistent pro-inflammatory microglial activation is a component of almost all neurodegenerative diseases (Ransohoff, 2016b).Because of this, many APIs have been developed to target microglia in order to improve outcomes in CNS disorders or after injury.This has prompted researchers to employ nanomaterials as an engineering approach to amplify further the impact of APIs designed for microglia.

Nanomaterial API delivery to microglia
Nanomaterial strategies targeting microglia are focused on nanofibrous scaffolds and hybrid nanostructured materials, often with an immunomodulatory payload to polarize microglia towards an antiinflammatory phenotype in order to promote neuronal protection and repair (Table 2).Microglia have diverse, complex reactions to nanomaterials.3D biodegradable hybrid inorganic nanoscaffolds modulated microglia in vivo to reduce scar formation during stem cell transplantation therapy for SCI (Yang et al., 2018).PCL nanofiber scaffolds coated with self-assembled colloidal graphene implanted in the striatum or subventricular zone of adult rats promoted reduced microglial infiltration (Watson et al., 2017).On the other hand, when primary microglia were cultured on poly(trimethylene carbonate-co-1-caprolactone) nanofibrous scaffolds, there was a reduction in phagocytic capacity, which indicates an inflammatory phenotype (Pires et al., 2015).Microglia were studied with engineered selfassembling (RADA) 4 -IKVAV peptide nanoscaffolds, and in vitro remained viable, phagocytosed the matrix, and remained ramified with high levels of TNF-ɑ and IL-1b and NO expression.When injected intracerebrally, however, the nanoscaffold did not lead to microglial migration, proliferation, or microglia-induced scarring (Koss et al., 2016).The inflammatory behavior of BV2 microglia was significantly reduced when interfaced with graphene nanomaterials compared to conventional polystyrene tissue culture substrates, and 3D graphene foams elicited a significantly milder neuroinflammatory response compared to a 2D graphene film (Fabbri et al., 2021).Nanostructured self-healing hyaluronan and chitosan hydrogel scaffolds injected into the rat brain striatum had negligible microglial activation or neuroinflammation (Liu et al., 2020).The ability of nanomaterials to alter microglial response led researchers to include APIs during development to add a further level of control.Glial cell-derived neurotrophic factor (GNDF)-loaded polydopamine (PDA) nanoparticle-based anisotropic gelatin scaffolds efficiently deliver PDA nanoparticles to scavenge reactive oxygen species and promote the M2 anti-inflammatory polarization in the murine BV2 microglial cell line (Ma et al., 2023).Poly(lactic acid) nanofiber scaffolds with incorporated rat NGF in hyaluronate hydrosol were engrafted with immunoregulatory IL-4 plasmid-loaded aldehyde cationic liposomes and implanted into rats with acute SCI, resulting in a downregulated acute microglial inflammatory response and reduced glial scar formation (Xi et al., 2020).PCL/PSA hybrid nanofiber scaffolds encapsulating methylprednisolone (MP) implanted after transection SCI inhibited microglial inflammatory activation as evidenced by reduced secretion of TNF-ɑ and IL-6 (Zhang et al., 2018).As more advanced nanomaterials are developed, the ability to deliver APIs that modulate the microglial response has therapeutic potential in many CNS disorders.

Nanoparticle API delivery to oligodendrocytes
Oligodendrocytes generate myelin to increase the speed of propagation of axon potentials and provide metabolic support to neurons in the CNS (Simons and Nave, 2015).Unfortunately, oligodendrocytes are vulnerable to reactive oxygen species, hydrogen peroxide, and excitotoxicity from glutamate, and as such, are detrimentally impacted in a range of CNS disorders (Matute et al., 1997;Juurlink et al., 1998;Kuhn et al., 2019;Kenigsbuch et al., 2022;Pandey et al., 2022).The most common causes of oligodendrocyte death in the CNS are trauma, ischemia, or autoimmune attacks, such as multiple sclerosis.However, white matter pathology is also characteristic of other CNS diseases, including Alzheimer's (Love, 2006;Fancy et al., 2011;Assinck et al., 2017;McAleese et al., 2017).Remyelination is a natural regenerative process that has been shown to prevent neurodegeneration and restore function (Duncan et al., 2009(Duncan et al., , 2018)).Therefore, APIs have been studied in order to promote oligodendrocyte remyelination in CNS trauma and disease.Nanomaterial design for API delivery to oligodendrocytes is being studied to capitalize on the synergy between the advantageous properties of the API and those of the material (Russell and Lampe, 2017; Murphy and Lampe, 2018).
Nanoparticles, including polymer, lipid, extracellular vesicles, and inorganic nanoparticles, have been designed to deliver APIs to oligodendrocytes in models of TBI, EAE, focal CNS demyelination, cuprizone-induced demyelination, SCI, and ischemia (Table 1) (Rittchen et al., 2015;Kumar et al., 2018;Lin et al., 2019;Zhang et al., 2019;Osorio-Querejeta et al., 2020;Robinson et al., 2020;Pu et al., 2021).API payloads range from small molecule drugs to proteins, peptides, and miRNA (Fressinaud et al., 2020;Farhangi et al., 2023;Moura et al., 2023).The goal of most NP API therapies directed towards oligodendrocytes is to reduce myelin loss and induce remyelination after injury or disease in order to improve functional outcomes (Table 1).Importantly, NP API delivery to oligodendrocytes has been shown to rejuvenate myelin and improve outcomes in in vivo models of CNS injury and demyelinating disorders (Table 1).While NP API delivery to oligodendrocytes is the least studied of the three most prominent glia in the CNS, the functional benefits demonstrate the potential for developing these NP therapies to improve myelin outcomes in many different CNS pathologies in order to push these treatments towards the clinic.

Nanomaterial API delivery to oligodendrocytes
Nanomaterial strategies that target oligodendrocytes have focused on engineered nanofibrous materials due to their ability to provide an axon-like substrate to promote oligodendrocyte differentiation and myelination.Two fundamental studies pioneered this approach by demonstrating that rat oligodendrocyte progenitor cells (OPCs) cultured on electrospun nanofibers of diameter 500-800 nm proliferated and differentiated into oligodendrocytes and ensheathed the fibers, resembling myelination (Lee et al., 2012).The same group also reported similarly compacted myelination on polystyrene electrospun nanofibers cultured with rodent oligodendrocytes (Lee et al., 2013).Further studies have shown that nanofibers can guide oligodendrocyte orientation that more closely resembles in vivo morphologies, preferentially drive neural stem cells to oligodendrocytes, induce compact myelination, and protect oligodendrocytes following traumatic CNS injury (Tysseling-Mattiace et al., 2008;Cao et al., 2009;Li et al., 2014;Shah et al., 2014;Wang et al., 2015;Ehrlich et al., 2017;Hyysalo et al., 2017;Tupone et al., 2021;Zhang et al., 2022) (Table 2).The ability to mimic the in vivo environment and alter oligodendrocyte response has led to the design of nanomaterials that release APIs to act more specifically on these glia.
Further, hybrid PCL-gelatin nanofiber scaffolds, combined with polyaniline graphene nanocomposites, were incorporated in gelatin to lend conductive properties similar to axons.Chitosan nanoparticles loaded with T3 were incorporated into PCL for sustained release, and    Boroojeni et al., 2020).Primary oligodendrocytes isolated from B16 mice were able to myelinate aligned PCL nanofibers that released PDGF-AA, FGF2, BMP2, and BMP4 (Enz et al., 2019).PCL nanofibers loaded with miR-219, miR-338-3p, and miR-338-5p enhanced the differentiation of primary rat oligodendrocyte progenitor cells and their maturation into RIP+ oligodendrocytes (Diao et al., 2015).Moreover, when hybrid PCL-PSA (polysialic acid) nanofiber scaffolds encapsulating glucocorticoid methylprednisolone were implanted into a transected rat SCI, the methylprednisolone delivered by the hybrid scaffold led to increased survival of oligodendrocytes and enhanced axonal myelination (Zhang et al., 2018).NT-3 is another API that has been used to act on oligodendrocytes, and fibrin nanofibrous scaffolds releasing NT-3 increase oligodendrocyte differentiation of neural progenitor cells (Willerth et al., 2008), while PCLEEP(PCL-coethyl ethylene phosphate)-collagen hybrid nanofibrous scaffolds releasing NT-3 showed extensive oligodendrocyte remyelination with MAG+ structures when implanted into a hemi-cervical incision induced rat spinal cord injury (Nguyen et al., 2017).Future oligodendrocyte-targeting nanomaterial design will seek to devise API-releasing strategies that specifically improve re-myelination after injury and improve myelin integrity in nervous system disorders.

Discussion
Nanomaterials designed to deliver APIs to CNS glia are beginning to emerge as viable therapies to improve outcomes in CNS disorders or after CNS injury.While many API-releasing nanomaterials are still being designed to focus their action on neurons (Kwon et al., 2016;Bruggeman et al., 2018;Zuidema et al., 2020), there is growing evidence that glia should not be overlooked as targets to improve outcomes in CNS injury and disorders (Tables 1, 2).However, in order for nanomaterial-mediated API delivery to glia to become a standard clinical intervention, further advances in engineering such materials are necessary.
Nanoparticle-mediated API delivery holds promise as a systemically administered approach to treat neurological disorders and CNS injuries where direct implantation into the site of action would be detrimental.For such treatments to become commonplace, one of the main areas of improvement is in traversing the BBB and delivering APIs directly to the relevant site of action.This will require a greater understanding of the mechanisms of nanoparticle permeation into the brain, including the importance of NP composition, size, charge, and shape, engineering the adsorbed biomolecular corona to not obstruct NP targeting, targeting the proper cell type once the NPs enter the brain, design of bettertargeting moieties on the external surface of nanoparticles through such processes as in vivo phage display screening, exact API release timelines that induce desired outcomes, and, importantly, a more complete understanding of how to modulate glia to produce desired clinical outcomes (Salvati et al., 2013;Mann et al., 2016;Furtado et al., 2018;Waggoner et al., 2023;Wu et al., 2023).More personalized therapies can be envisioned, where each individual may respond to NPs differently.This may require a battery of different NP constructs to first be administered systematically, and once it is known which NP accumulates to the desired location, potential by using an imaging modality such as magnetic resonance imaging, then that NP construct can be incorporated with the desired API and delivered to the individual.Still, much research is needed to make NP-delivered APIs that act on glia a standard therapy to treat CNS disorders and injuries.
Nanomaterial-mediated API delivery has shown promise in areas where surgical intervention or injection into the site of action to act on glia can be used.These combinatorial nanomaterial-based therapies can simultaneously provide biophysical and biochemical cues to glial cells, eliciting their bioactive responses to facilitate robust neuronal repair and protection in the CNS.For these therapies to be used in the clinic, advances in API release paradigms must be realized, nanomaterial modulation of glia needs to be better understood, surgical implantation techniques optimized, degradation of the implanted material engineered based on the application, and the immune response accounted for not to impart adverse clinical outcomes (Nunes et al., 2012;Huang et al., 2017;Dai et al., 2021).We also envision the potential for nanomaterial therapies to be tailored to each patient to maximize therapeutic efficacy and minimize off-target adverse effects -by varying API release rates, compositions and coatings in nanomaterials design, and even the timeline of the surgical intervention.As advances in NPs, nanomaterials, and API design for targeting glia continue to be realized, there are many avenues for such therapies to improve clinical outcomes in CNS disorders and after CNS injury.

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TABLE 1
Studies that use nanoparticle delivered APIs to target central nervous system glia.

TABLE 1 (
Continued) Studies that use nanomaterials and nanomaterial delivered APIs to target central nervous system glia.