Exosomes as promising bioactive materials in the treatment of spinal cord injury

Abstract

Patients with spinal cord injury (SCI) have permanent devastating motor and sensory disabilities. Secondary SCI is known for its complex progression and presents with sophisticated aberrant inflammation, vascular changes, and secondary cellular dysfunction, which aggravate the primary damage. Since their initial discovery, the potent neuroprotective effects and powerful delivery abilities of exosomes (Exos) have been reported in different research fields, including SCI. In this study, we summarize therapeutic advances related to the application of Exos in preclinical animal studies. Subsequently, we discuss the mechanisms of action of Exos derived from diverse cell types, including neurogenesis, angiogenesis, blood–spinal cord barrier preservation, anti-apoptosis, and anti-inflammatory potential. We also evaluate the relationship between the Exo delivery cargo and signaling pathways. Finally, we discuss the challenges and advantages of using Exos to offer innovative insights regarding the development of efficient clinical strategies for SCI.

Introduction

Spinal cord injury (SCI) is one of the most serious neurological disorders, with a global incidence of 1.2–5.8 and 0.2–13.0 cases per 100 000 population in developed and developing countries, respectively [1,2,3,4,5,6]. Approximately 90% of SCIs are caused by traumatic events, such as traffic accidents, falls, or acts of violence [6]. SCI results in enduring impairments, including paralysis, sensory loss, and long-term complications, including muscle atrophy, joint deformities, infections, atelectasis, pneumonia, venous thromboembolism, dysphagia, chronic pain, pressure ulcer, and psychological distress such as depression [7, 8], thereby accounting for a substantial proportion of the worldwide injury burden of lost productivity and high healthcare costs [5]. Currently, no effective treatment is available to mitigate long-term functional impairments attributed to SCI. Available therapies, such as anti-inflammatory medications, have limited efficacy since they are rapidly eliminated by cerebrospinal fluid, and their bioactivity is diminished [9, 10]. This is partly due to a limited understanding of the intricate pathophysiological processes that occur after SCI and a lack of safe and efficient instruments to regulate the already known therapeutic targets [11].

Pathophysiology of SCI

In large, SCI can be classified into primary and secondary phases [12]. Primary SCI results from physical forces exerted during an initial traumatic event. These forces induce dislocation of the vertebral column, as well as destruction of the vasculature, and compromise the integrity of the blood–spinal cord barrier (BSCB). Following the primary injury, a series of secondary injury events occur, leading to an increase in the volume of spinal cord damage and irritating neurological deficits. During this secondary injury cascade, cell dysfunction and death in the spinal cord occur due to cell permeabilization, pro-apoptotic signaling, and ischemia injury resulting from the breakdown of the microvascular supply. These events occur within minutes of the injury. Additionally, inflammatory cells, such as macrophages, microglia, T cells, and neutrophils, migrate to the area of injury due to the disruption of the BSCB [13]. Secondary neurodegenerative alterations, such as the degeneration of axons and changes in the gray matter caused by the injury or compression, extend both toward the head (rostrally) and tail (caudally) from the location of the initial damage. Moreover, the overstimulation of excitatory amino acid receptors leads to excitotoxicity, which causes neuron and glial cell death through both necrotic and apoptotic processes [14,15,16,17]. Given the above, SCI functional recovery depends on growing neuroplasticity to enhance sprouting and regeneration of spared and injured axons, angiogenesis, preserving the integrity of the BSCB, limiting apoptosis, and decreasing inflammation to boost the potency of residual nerve connections and facilitate the development of new connections between neurons [18].

Characterization and role of exosomes (Exos)

Exos are among the most popular research foci as potential therapeutic agents for overcoming the convoluted pathophysiology process of SCI. Exos are generated by the inward budding of endosomal compartments that later fuse with the plasma membrane, a heterogeneous group of membrane-surrounded particles released by all cell types with a diameter of 30–150 nm [19,20,21]. The isolation methods of Exos progressed as time and technique advanced, from conventional ultracentrifugation, size-based filtration, size-exclusion chromatography, polymer precipitation, and immunoaffinity methods to modern microfluidic-based isolation techniques [22]. Exos has been developed as a new strategy for the non-invasive diagnosis and monitoring of diseases, used as disease markers. Furthermore, Exos can be used as potential non-cell therapeutics. By exchanging functional contents between cells, Exos play fundamental roles in maintaining homeostasis and combatting stress [23]. Additionally, Exos can be engineered to deliver various therapeutic cargos, including short-interfering RNAs (siRNAs), messenger RNAs (mRNAs), microRNAs (miRNAs), antisense oligonucleotides, chemotherapeutic agents, immune modulators, and other bioactive molecules, directly to the desired target [24, 25].

Effects of exos on SCI

In SCI, Exos support many healing mechanisms that have neuroprotective effects by stimulating the regeneration of vessels and nerves and promoting white matter remodeling in the insulted central nerve system (CNS) [26,27,28]. Exos exhibit numerous other advantages, such as good encapsulation and ready penetration of the blood–brain barrier to access the CNS. Furthermore, Exos are important elements of the cellular secretome and are promising options for cell-free therapies because of their potential therapeutic bioactivity, natural compatibility with the body, and ability to target specific cells. This approach helps address concerns regarding the immune response and uncontrolled proliferation or differentiation of cellular transplants. Exos combine the advantages of cell and nanotechnology in drug delivery [29,30,31,32]. Although Exos have advantages in treating SCI, several challenges exist as follows: the potency of Exos large-scale production remains challenging; the off-target effects of Exos remain; systemic clearance limits reach and efficacy of Exos; negatively charged cell membranes and repulsion of Exos induce inefficient uptake; and lysosomal degradation [33, 34].

Current Exo research in SCI treatment mostly focuses on those derived from various stem cell sources as the regenerative abilities of the source cells [35,36,37]. To provide new researchers on Exo with a succinct, up-to-date foundation on SCI, we also included Exos that originate from microglia, macrophages, regulatory T cells (Tregs), Schwann cells (SCs), bone marrow mesenchymal stem cells (BMDMs), plasma, and other substances, which have a beneficial effect on nerve reconstruction by facilitating axonal regeneration and eliminating damaged debris, as well as transporting proteins that actively inhibit inflammation [38,39,40,41,42,43,44,45,46]. Overall, Exos originating from various cells, including mesenchymal stem cells (MSCs), have broad application prospects for SCI [31, 40,41,42, 46].

In this review, we discuss how Exos protect neurons, including the underlying processes and other relevant aspects of Exo treatment for SCI. Currently, the promising treatments for SCI are mostly related to Exos derived from MSCs [47, 48]. We focus on the specific mechanisms exerted by different Exos isolated from various progenitor cells rather than stem cells in the complex pathophysiology of SCI, including human epidural adipose cells, neural stem cells (NSCs), Schwann cells, macrophages, and plasma. We also discuss the prospects and challenges of non-stem cell-derived Exos (Fig. 1). Furthermore, the Exos have been classified into five major functions according to their characteristics and cargos, serving as therapeutic strategies for axonal disruption, vascular injury, BSCB disconnection, cell death, and inflammation, as primarily revealed through substantial preclinical animal research. Additionally, we discuss new strategies and information, including origin, effect, cargo, and the current deep molecular mechanisms of Exo-based treatment and the potential for improving treatment efficiency and preventing long-term disease progression to facilitate the transition to clinical trials. Moreover, we compare the Exos and animal model heterogenous in different studies. This review will help facilitate research on using Exos for the efficient management of SCI in the future.

Fig. 1

Origin and cargo of Exos and the underlying signaling mechanisms in SCI treatment, including neurogenesis, angiogenesis, BSCB preservation, anti-apoptosis, and anti-inflammation. (By Figdraw). BSCB blood–spinal cord barrier; SCI spinal cord injury

Mechanisms of exo-mediated treatment for SCI

Therapeutic strategies tailored to the pathophysiology of SCI are promising for clinical translation. Nevertheless, SCI is a complex condition that involves multiple aspects. To obtain a better therapeutic outcome, it is necessary to address the simultaneous and subsequent pathogenic processes that occur throughout the evolution of the secondary damage (Fig. 2) [13, 16]. Therefore, there is an urgent need for a multitarget therapeutic approach to counteract secondary injury progression. Exos have shown potential for protection due to their wide-ranging effectiveness and have been extensively studied in various preclinical models of SCI.

Recent research indicates that Exos, together with other payloads, such as RNAs, proteins, and medicine, have a strong protective effect. This effect can modify the functioning of recipient cells in the spinal cord system. Furthermore, the bioactivity and biological composition of Exos are contingent on the phenotype of the parent cell from which they are derived and might exhibit variability in response to stimuli and the local microenvironment. Here, we summarized published studies on SCI treatment with Exos by promoting axonal regeneration and angiogenesis while limiting BSCB disruption, apoptosis, and inflammation in the progression of complicated pathology changes in secondary injury post-SCI.

Fig. 2

Mechanism Underlying SCI Changes. Primary and secondary neurodegenerative processes following SCI. The injury leads to the degeneration of the sensory and motor pathways by either anterograde or retrograde axonal degeneration and accompanying demyelination; distant neurons undergo trans-synaptic degeneration. Reproduced with permission [13]. 2019, Lancet Neurology. SCI spinal cord injury; MRI magnetic resonance imaging

Promoting neurogenesis

Adult neurogenesis is essential for maintaining CNS homeostasis and reacting to neurogenic insults. Nevertheless, the adult mammalian spinal cord does not possess the inherent ability for neurogenesis [49]. Nerve regeneration and neurotrophicity are crucial elements in SCI repair and are significant areas of research in SCI treatment [50]. Notably, Exos exhibit considerable potential in SCI neurogenesis therapy [51]. Evidence shows that purified Exos isolated from MSCs and BMDMs, among others, can engage with recipient cells when injected intravenously. This treatment significantly improves bladder dysfunction and motor movement in animal models after SCI by activating NSC differentiation and increasing axonal outgrowth (Fig. 3A) [25, 39, 52, 53].

Growth-associated protein 43 (GAP-43) is a synaptic protein whose expression is significantly elevated during the differentiation of NSCs into cortical neurons. Enhanced production of the neuron-specific proteins microtubule-associated protein 2 (MAP-2) and beta-tubulin III (Tuj1) is vital for the process of NSC differentiation [54,55,56]. Li et al. [57] demonstrated that Exos loaded with overexpressed neuronal growth factor (Exo-oe-NGF) obtained from bone marrow-derived stem cells (BMSCs) enhance the process of NSC differentiation into neuronal cells. Additionally, these Exos promote the neuronal axonal regeneration following SCI that spans a spinal cord cross-section measuring 2 mm in length. This leads to improved locomotor functional recovery, as displayed by a substantial increase in the Basso, Beattie, and Bresnahan (BBB) score compared with SCI mice. This improvement is supported by the upregulated expression of GAP-43, Tuj1, and MAP-2. Jia et al. [58] reported that the levels of Sonic Hedgehog and glioma-associated oncogene homolog 1 increase substantially after injecting BMSC-Exos, along with an increase in GAP-43 expression and the promotion of functional recovery. Moreover, injection with miR-29b Exos and the miR-29b groups significantly promoted SCI (spinal cord contusive injury) characteristics, including increased BBB scores and the numbers of NF200 and GAP-43-positive neurons [59]. Furthermore, Cheng et al. [60] discovered that methacrylated gelatin (GelMA)-Exos stimulated neurogenesis, reduced glial scarring in injury sites, improved the differentiation of Tuj-1-positive neurons, and enhanced axonal outgrowth. Consequently, GelMA-Exos facilitated locomotor functional recovery after SCI (spinal cord contusive injury).

The gene regulatory networks that trigger NSC differentiation at an early stage have been elucidated in part through research on the mechanisms underlying neuronal differentiation and axon regeneration (Fig. 3B) [61]. Based on this, Exos originated from miR-26a-modified MSCs were used to enhance neurogenesis and restrict glial scar formation by activating the phosphatase and tensin homolog deleted on chromosome ten (PTEN)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling cascade (Fig. 3C) [62]. As the extracellular signal-related kinase (ERK)/cAMP response element-binding (CREB) and wingless/integrated (Wnt)/β-catenin pathway also participates in the regulation of neurogenesis (Fig. 4A) [63,64,65], HpMSC-derived Exos could enhance the proliferation of NSCs by activating the MEK/ERK/CREB signaling pathway and increasing the levels of phosphorylation in MAPK/ERK kinase (MEK), ERK, and CREB [63]. Moreover, neuronal differentiation triggered by the novel paclitaxel-delivered MExos–collagen scaffold via the Wnt/β-catenin signaling pathway could effectively instruct NSCs to differentiate into neurons, thereby promoting neuronal regeneration and minimizing scar formation (Fig. 4B) [65, 66].

Additionally, exosomal miRNAs perform an essential function in neuron protection in the initial stages of SCI and promote functional recovery. MiR-133b-modified Exos inhibit ras homolog family member A expression and activate ERK1/2, signal transducer and activator of transcription 3 (STAT3), and CREB, thereby reducing the lesion area, preserving neuronal tissues, and stimulating nerve fiber regeneration after a SCI caused by an aneurysm clip [67]. MiR-151-3p, which targets phospho-protein 53 (p53), is abundant in microglia-derived Exos; this miRNA suppresses the p53/cyclin-dependent kinase inhibitor 1 A (p21)/cyclin-dependent kinase 1 (CDK1) signaling pathway, reduces neuronal apoptosis, and promotes axonal regrowth [38]. The expression levels of miR-199a-3p/145-5p are relatively high in Exos. MiR-mediated knockdown of Cblb, which is specifically targeted by miR-199a-3p, and Cbl, which is specifically targeted by miR-145-5p, subsequently activates the NGF/tropomyosin receptor kinase A (TrkA) downstream pathways AKT and ERK (Fig. 4C) [68]. MiR-431-3p delivered by Exos from a subtype of BMSCs (CD271⁺CD56⁺ BMSC) significantly caused an exacerbation in the length of axon extension and an increase in the number of branches in the axons of the dorsal root ganglion by targeting Repulsive Guidance Molecule Family Member A [69]. EGFR⁺NSC, a subpopulation of endogenous NSC-enriched exosomal miR-34a-5p, can facilitate axonal regeneration at the injured site by directly binding to HDAC6 and inhibiting expression [37]. Moreover, Exos secreted by oxygen- and glucose-deprived astrocytes increased Exo-associated miR-92b-3p to exert a neuroprotective effect [70].

Exos promote neurogenesis through PTEN/phosphatidylinositide 3-kinases (PI3K)/AKT/mTOR, Wnt/β-catenin, MEK/ERK/STAT3/CREB, and NGF/TrkA signaling cascades [71,72,73]. This creates a favorable environment for neurite outgrowth, accelerates NSC differentiation, promotes neuronal survival and axon regeneration, and attenuates glial scar formation (Table 1).

Table 1 Neurogenesis effect following exos treatment in SCI Animal models

Fig. 3

Characteristics of Exos and the Underlying Signaling Mechanisms for Neurogenesis. A) The specific characteristics of Exos that can deliver various types of DNA/RNA/siRNA, protein, and drugs. Reproduced with permission [25]. Copyright 2015, Journal of Controlled Release. B) After axotomy, injured adult CNS neurons present with low regenerative capacity; diverse molecular mechanisms promote axons to become the high regenerative type. Modified from 2018, Annual Review of Cell and Developmental Biology [61]. (By Figdraw.) C) MSC-derived Exos promote axonal regeneration via the phosphatase and tensin homolog (PTEN)/AKT/mammalian target of rapamycin (mTOR) pathway following SCI. Reproduced with permission [62]. Copyright 2021, Stem Cell Research & Therapy. SCI spinal cord injury; CNS central nervous system; MSC mesenchymal stem cell

Fig. 4

Neurogenesis Following SCI. A) Exos extracted from human placental MSCs enhance the proliferation of endogenous neural progenitor cells (NPCs) and promote their differentiation into mature neurons through the activation of MEK/ERK/CREB phosphorylation. Reproduced with permission [63]. Copyright 2021, Stem Cell Research & Therapy. B) Exos extracted from human umbilical cord-derived MSCs (hUC-MSCs) loaded on a multifunctional collagen scaffold (LBMP) promote endogenous NSC migration and differentiation. Reproduced with permission [66]. Copyright 2021, Advanced Healthcare Materials. C) Exos extracted from hUC-MSCs promote neurite outgrowth. Exos secrete high levels of miRNA-199a-3p/145-5p, which specifically target Cblb and Cbl mRNAs to prevent TrkA degradation. Sustained activation of phosphorylated ERK (p-ERK) and p-AKT results in the sustainable expression of NEU-N, neurofilament H (NF-H), and β-tubulin-III. Reproduced with permission [68]. Copyright 2021, Stem Cell Research & Therapy. SCI spinal cord injury; MSC mesenchymal stem cell; NSC neural stem cell

Promoting angiogenesis

To accelerate regeneration following SCI, neurogenesis must be coupled with angiogenesis. In addition to the regeneration of nerve cells, the assistance of the surrounding microenvironment, including blood vessels and the extracellular matrix, is necessary for restoring neural function. Specifically, rejuvenated blood vessel formation plays a significant role in tissue repair [83]. The vasculature can serve as a supportive structure and guide axonal sprouting after injury, thereby promoting axonal guidance [84]. Moreover, vascularization following SCI facilitates nourishment for restoring and sustaining neuronal network stability, which in turn is favorable for functional recovery after SCI [85]. Notably, the administration of MSC-Exos significantly promotes angiogenesis [86].

After SCI, the injured spinal column becomes hypoxic [87]. The preservation of endothelial cells ensures limited secondary injury to the blood vessels following SCI, enabling the provision of vital oxygen and nutrients required for the repair of microenvironment and nerve circuits. Vascular endothelial cells, which are an essential part of the blood vessel wall, increase the absorption of Exos produced by hypoxia-treated MSCs. Mu et al. [31] found that hypo-Exo-treated rats had improved locomotor functional recovery. The hypoxia-inducible factor 1-alpha (HIF-1a) content was significantly increased in hypoxia-stimulated Exos (hypo-Exos), leading to the upregulation of vascular endothelial growth factor (VEGF) in the Exo treatment system, indicating the immense potential of prominent angiogenesis in SCI (a 4.0 ± 0.5-mm spinal cord cross-section and fragments were removed) and repair (Fig. 5A). Regarding VEGF, NSC-Exos are highly expressed VEGF-A and can facilitate the angiogenic capabilities of spinal cord microvascular endothelial cells (SCMECs); promote SCMEC migration, tube formation, and proliferation; mediate pro-angiogenic effects; and promote tissue healing [88]. Exos from M2 macrophages enhance angiogenesis and functional recovery following SCI; this is partially attributed to the activation of the HIF-1/VEGF signaling pathway [89]. Exos that originate from miR-126-modified MSCs enhance the process of microvascular regeneration and human umbilical vein endothelial cell (HUVEC) migration by suppressing Sprouty-related EVH1 domain-containing protein 1 and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) expression [80]. These proteins function as inhibitors of the VEGF pathway [90].

Moreover, several studies have shown that Exos promote blood vessel formation independent of VEGF. For example, macrophage membrane-fused Exo-mimetic nanovesicles (MF-NVs) target the ischemic endothelium and promote angiogenesis (Fig. 5B) [91]. SCs-Exos promote angiogenesis by delivering integrin-β1 through the effect of control VE-cadherin localization and blood vessel stability [92]. The administration of human placenta-derived MSC-Exos (hpMSC-Exos) enhances the process of tube formation and HUVEC migration; moreover, BMS scores improved significantly in the hPMSCs-Exos group [93]. Treatment with Exo-educated macrophages (EEMs), defined as M2-like macrophages generated using Exos isolated from BMSCs, significantly improved the angiogenic activity of HUVECs and facilitated the development of axonal growth in cortical neurons (Fig. 5C) [94].

The JNK/c-Jun, Wnt/β-catenin, and PTEN/mTOR signaling pathways participate in the regulation of angiogenesis. Specifically, unlike those prepared from untreated hMSCs, iron oxide nanoparticle-incorporated Exo-mimetic nanovesicles (NV-IONPs) actuate the JNK and c-Jun signaling pathway, and accumulated NV-IONPs enhance blood vessel formation [95]. In turn, the M2-Exo-derived ubiquitin isopeptidase OTULIN can activate the Wnt/β-catenin signaling pathway by upregulating the expression of β-catenin, which in turn triggers the upregulation of angiogenesis-related genes in SCMECs. These genes are known to be modulated by the Wnt/β-catenin signaling pathway (Fig. 5D) [85]. Subsequently, the use of MSC-Exos containing siRNA (ExoPTEN) reduces PTEN expression and promotes axonal growth and neovascularization [81]. Ultimately, cerebrospinal fluid-derived extracellular vesicles from pigs with SCI promote angiogenesis by activating the PI3K/AKT signaling pathway [96].

In angiogenesis, Exos interact with the PTEN/PI3K/AKT/mTOR, Wnt/β-catenin, and JNK/c-Jun pathways to promote SCMEC migration, tube formation, and proliferation. Particularly, JNK is tightly linked to the release of growth factors; similarly, c-Jun participates in the VEGF receptor 2 signaling axis (Table 2), [97, 98].

Table 2 Angiogenesis effect following exos treatment in SCI Animal models

Fig. 5

Angiogenesis of Exos. A) Hypoxia-stimulated MSC-derived Exos encapsulated in hydrogel promote microvascular and nerve regeneration at the spinal lesion through the upregulation of VEGF. Modified from 2022, Biomaterials Science [31]. (By Figdraw). B) MF-NVs (macrophage membrane-fused Exo-mimetic nanovesicles) target the SCI lesion by binding to ischemic endothelium to exert multiple protective effects. Reproduced with permission [91]. Copyright 2020, International Journal of Molecular Sciences. C) BMSC-derived Exos cultured with macrophages to obtain EEMs. EEMs loaded on hydrogel promote axonal regeneration and angiogenesis in the injured spinal cord. Thus, EEMs accelerate microvasculature regeneration of the spinal cord. Reproduced with permission [94]. Copyright 2021, Frontiers in Cellular Neuroscience. D) Hydrogel-loaded M2-Exos facilitate microvascular regeneration and diminish the lesion area by activating the Wnt/β-catenin pathway following SCI. Reproduced with permission [85]. Copyright 2021, Acta Biomaterialia. SCI spinal cord injury; MSC mesenchymal stem cell; BMSC bone marrow mesenchymal stem cells; EEM Exo-educated macrophage; VEGF vascular endothelial growth factor

Preserving the integrity of the BSCB

The BSCB is crucial in preserving the stability of the microenvironment in the spinal cord; accordingly, BSCB disruption is detrimental to locomotor function recovery. Consistent with this, preserving the integrity of the BSCB can enhance spinal cord tissue repair and lead to movement improvement following SCI [99, 100].

BMSC-Exos help preserve the integrity of BSCB and enhance the process of motor recovery after SCI, partly by regulating tissue inhibitors of the matrix metalloproteinase 2 (TIMP2)/matrix metalloproteinase (MMP) signaling pathway. TIMP2 in BMSC-Exos alleviates BSCB damage by suppressing the MMP pathway, thereby protecting the expression of cell junction proteins (e.g., claudin-5, occludin, zonula occludens-1 (ZO-1), and β-catenin) [100, 101]. Moreover, extracellular vesicles that originate from MSCs can boost the levels of transforming growth factor-beta (TGF-β), TGF-β receptors, and tight junction proteins and decrease the permeability of the BSCB. These vesicles achieved an average BBB score of 7.96 ± 0.83, which was greater than that of the Vehicle group (4.74 ± 0.67) [102]. NSC-Exos containing FTY720, an immunomodulatory agent, can preserve the integrity of the endothelial barrier of SCMECs within a hypoxic environment via the PTEN/PI3K/AKT pathway [103]. Additionally, pericytes are crucial constituents of the neurovascular structure and present with various regulatory effects in preserving the BSCB integrity [104]. BMSC-Exos strengthen the BSCB integrity by preventing aberrant pericyte migration and improving pericyte coverage at the barrier. This is achieved by downregulating the nuclear factor-kappa B (NF-κB) pathway [78]. Moreover, BMSC-Exos can protect BSCB and alleviate edema by suppressing pericyte pyroptosis through the inhibition of the Nod1 inflammasome. This improves the coverage of the pericyte and results in better functional recovery following injury [74]. High expression of MiR-210-5p in pericyte-derived Exos can inhibit the Janus kinase (JAK1)/STAT3 signaling pathway. This inhibition helps to regulate lipid peroxidation levels, improve mitochondrial function, and regulate endothelial barrier function [105].

To preserve the BSCB, Exos regulate the PTEN/PI3K/AKT/mTOR, JAK/STAT3, and TIMP2/MMP signaling pathways to limit the reduction of cell junction proteins, upregulate TGF-β and its receptor, inhibit pericyte migration, improve the rate of pericyte coverage, and inhibit pericyte pyroptosis (Table 3) [74, 100].

Table 3 BSCB protection effect following exo treatment in SCI animal models

Inhibiting apoptosis

SCI is characterized by axonal disruption and neuronal apoptosis and results in profound motor and sensory impairments [106]. Increasing evidence indicates that axonal growth and neuronal apoptosis are important areas of focus during SCI treatment. MiRNAs, which play critical roles in regulating cellular activities, are the predominant nucleic acids found in Exos and have been shown to positively influence the outcome of SCI.

Following SCI, treatment with MSC-miR-338-5p increases cyclic AMP (cAMP) accumulation and cAMP-mediated repressor activator protein 1 (Rap1) activation. The eventual PI3K/AKT pathway activation reduces cell apoptosis and enhances the survival of neurons [107]. BMSC-miR-181c inhibits PTEN and NF-κB signaling, ultimately decreasing the inflammation process and cell apoptosis in the spinal cord tissue and improving SCI (spinal cord contusive injury) [108]. MSC-miR-21/miR-19b depletes PTEN mRNA/protein, significantly promotes axon growth, prevents neuronal apoptosis following nerve injury, and promotes functional recovery; it has also been shown to raise BBB scores in rats with SCI [109]. MSC-miR-21 enhances the locomotor recovery of rats with contusive SCI by inhibiting cell death through the miR-21/PTEN/programmed cell death 4 signaling pathway and activates the JAK/STAT signaling pathways [110, 111]. Exos that originate from hypoxia-conditioned adipose tissue-derived stromal cells (ADSCs) (Hypo-Exos) that are enriched in miR-499a-5p significantly reduce neuronal apoptosis by regulating the c-jun N-terminal kinase 3 (JNK3)/c-jun apoptotic signaling pathway by targeting JNK3 [112]. Conversely, low miR-429 expression in SCI (spinal cord contusive injury) plasma Exos promotes neuronal apoptosis by facilitating PTEN expression and affecting PI3K/AKT signaling [46], whereas human neuroepithelial stem cell-miR-29b downregulates PTEN/caspase-3 expression and subsequently suppresses neuronal apoptosis [113]. BMSC-miR-455-5p directly targets neurite outgrowth inhibitor A (Nogo-A), a myelin-associated axonal growth inhibitory protein, to promote autophagy and inhibit neuronal apoptosis [114]. Exo-miR-494 suppresses inflammation factors and cell apoptosis in the insulted region [52], while MiR-126 Exos enhance neurogenesis and inhibit apoptosis following SCI (spinal cord contusive injury) [80].

Additionally, Exos inhibit apoptosis via multiple signaling pathways. G protein-coupled receptor kinase 2 interacting protein 1 (GIT1)-BMSC-Exos alleviate neuronal apoptosis by promoting PI3K/AKT signaling pathway activation [77]. Li et al. [115] demonstrated that BMSCs-Exos efficiently triggered the upregulation of the Wnt/β-catenin signaling cascade, resulting in a significant decrease of Bax, cleaved caspase-3, and cleaved caspase-9. hUC-MSC-derived Exos reduced apoptosis via the Bcl-2/Bax pathway, facilitated the low-density lipoprotein receptor-related protein 6/Wnt/β-Catenin signaling cascade, and enhanced the level of c-myc and Cyclin D1 in damaged lesions after SCI [116]. Moreover, cellular damage can be mitigated by enhancing autophagy by administering treatments targeting the constituents of the lesion [117]. BMSC-Exos can inhibit cell apoptosis by activating autophagy by enhancing the autophagy-related proteins microtubule-associated protein 1 A/1B-light chain 3 IIB and beclin-1 expression, enabling autophagosome formation and promoting the potential efficacy of locomotor recovery in rats with SCI (spinal cord contusive injury) [118].

Overall, Exos exert antiapoptotic effects in various pathological conditions mainly through the PTEN/PI3K/AKT/mTOR, Wnt/β-catenin, JNK/c-Jun, JAK/STAT, Bcl-2/Bax, and caspase signaling pathways to inhibit endoplasmic reticulum (ER) stress and promote autophagy (Table 4) [119, 120].

Table 4 Anti-apoptosis effect following exos treatment in SCI animal models

Regulating inflammation

Excessive neuroinflammation impedes neuronal regeneration, thereby contributing to the poor prognosis of patients with SCI [41, 127]. Severe neuroinflammation hinders the ability of axonal regeneration in the lesion site to rebuild connections with neighboring neurons, which leads to long-lasting neurological deficits [61, 128]. Notably, the inhibitory effect of inflammatory and scarring activities observed following the application of progenitor cells in SCI are mediated by their secreted Exos [129].

Exos are paracrine factors released by progenitor cells, which inhibit dysregulated neuroinflammatory cascades. Exos transport and discharge anti-inflammatory molecules such as berberine, miR-181c, LncGm37494, and insulin growth factor-1 (IGF-1) [44, 76, 108, 130]. These molecules can reduce the level of reactive oxygen species (ROS) and inflammatory cytokines at the lesion site of the spinal cord tissue during the initial stages of secondary damage [79, 129, 131]. Additionally, exosomal miRNAs can be exchanged between different immune cells and repress gene expression, with such Exo-mediated intercellular communication potentially influencing immune cell maturation [24, 131]. Exosomal miRNAs regulate target cells and may be crucial for modulating biological processes [41, 132]. MiR-544-modified BMSC-Exos markedly suppress the generation of the inflammatory cytokines interleukin 1α (IL-1α), tumor necrosis factor-alpha (TNF-α), IL-17β, and IL-36β at lesion site of spinal cord tissues following SCI (spinal cord contusive injury) [133]. BMSC-Exo-miR-494 effectively suppresses inflammation and apoptosis in the affected region [52]. Furthermore, BMSC-derived exosomal miR-9-5p has been reported to enhance BBB scores at days 1, 3, 7, 14, and 28 post-treatment compared with the sham group by promoting fibroblast growth factor 2 expression by downregulating histone deacetylase 5-mediated deacetylation, thereby ameliorating inflammation and ER stress (Fig. 6A) [125]. Exos can also act as safe and efficient siRNA delivery vectors that can traverse the BSCB to convey biological genetic information. Exo-siRNA, which can specifically silence the connective tissue growth factor gene, significantly quenches inflammatory response and hinders neural cell apoptosis along with A1 astrocyte activation and glial scar deposition [124].

Fig. 6

Exos Extracted From MSCs Mitigate Inflammation Following SCI. A) Exos derived from BMSCs release miR-9-5p and mitigate neuronal inflammation following SCI. Reproduced with permission [125]. Copyright 2022, Molecular Immunology. B) HMSC-derived Exos immobilized in hydrogel can eliminate ROS and inflammatory factors, promoting nerve repair in SCI. Reproduced with permission [134]. Copyright 2020, Nano Letters. C) EXO-C@P accelerates locomotor function recovery through M2 macrophage polarization and injury volume restriction of SCI. Reproduced with permission [135]. Copyright 2021, Materials Science & Engineering C, Materials for Biological Applications. SCI spinal cord injury; MSC mesenchymal stem cell; BMSC bone marrow mesenchymal stem cells; HMSC human mesenchymal cell

Recent studies have further demonstrated that exosomal miRNAs inhibit both canonical and noncanonical inflammatory signaling pathways. BMSC-Exos can suppress the apoptosis and inflammatory response after injury and promote locomotor recovery by downregulating the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)/NF-κB signaling pathway [136]. MiR-181c in BMSC-Exos inhibits PTEN and the NF-κB expression on the decrease of I kappa B kinase alpha/beta (IKKα/β) phosphorylation and p65 expression in microglia nuclei, thereby mediating inflammation [108]. BMSC-Exos exert a protective effect in SCI (right semicircular spinal cord severed model) by suppressing the production and release of complement mRNA, as well as SCI-activated NF-κB (as indicated by significantly downregulated levels of p-p65 and p-IκBα) by binding to microglia [137]. MSC-Exos promote hind limb function recovery, and miR-145-5p expression is increased at the lesion site of SCI, leading to the suppression of the TLR4/NF-κB pathway and thereby suppressing the inflammatory reaction [138]. Liu et al. [79] and Wang et al. [122] clarified that MSCs-Exos treatment significantly improved the BBB score and that MSC-Exos reduced SCI (spinal cord contusive injury)-induced neurotoxic reactive A1 astrocyte activation following traumatic SCI. Wang et al. [122] also found that A1 astrocyte diminish was likely induced by suppressing the nuclear translocation of NFκB p65; the BBB score in the MSC-Exo treatment group was 14.450 ± 0.411. Hypo-Exos from MSCs mediate microglial polarization via enriched miR-216a-5p to modulate TLR4/NF-κB/PI3K/AKT signaling cascades. Hypoxia Exos from MSCs (HExos) enrich miR-216a-5p, which can modulate the TLR4/NF-κB/PI3K/AKT signaling pathway and mediate microglial polarization [139]. The overexpression of miR-544 in BMSC-Exos reduces inflammation following SCI (spinal cord contusive injury) [133].

NSC-derived Exos formed in the presence of IGF-1 upregulate miR-219a-2-3 expression to inhibit the yin yang 1/NF-κB pathway, thereby inhibiting inflammation and promoting neuroprotective effects following SCI (spinal cord contusive injury) [76]. Human epidural adipose tissue-derived MSCs can reverse thrombospondin 4 (THBS4) and B-cell lymphoma 3 (Bcl3) levels in SCI (clip compression injury); specifically, THBS4 supports local vascular inflammation, while BCL3 is aggregated at the nucleus and controls the activity of NF-κB in gene transcription [140]. Exos derived from long non-coding RNA (lncRNA) tectonic family member 2-modified MSCs attenuate inflammation via the miR-329-3p/IGF1R axis [123]. MSC-Exo lncGm36569, which acts as both an inhibitor and mimic of miR-5627-5p, inhibits neural cell ferroptosis via the miR-5627-5p/ferroptosis suppressor protein 1 axis [53].

Exos secreted by immune cells other than MSCs can also inhibit inflammation. Schwann cell-derived Exos (SCDEs) can enhance normal function restoration in mice following SCI by reducing the accumulation of chondroitin sulfate proteoglycan (CSPG). This is achieved by boosting TLR2 levels of astrocytes via the NF-κB/PI3K signaling cascade [43]. Peripheral macrophage (PM)-Exos activate microglial autophagy by downregulating the PI3K/AKT/mTOR signaling cascade [40]. MiR-126-3p originated from hypoxia-preconditioned VSC 4.1 neuron-derived Exos alleviate the hypersensitivity to pain caused by infrared radiation by restoring miR-126-3p expression in the affected site after SCI. This, in turn, regulates the activity of the PIK3R2-mediated PI3K and NF-κB pathways [141, 142].

Additionally, studies have explored different aspects of traditional mechanical research, including Exo application methods, modifications, and cell phagocytosis. Romanelli et al. [143] reported that intralesional application of Exos secreted by human umbilical cord mesenchymal stromal cells was more potent than intravenous administration regarding the restriction of the inflammatory process and glial scarring following SCI (spinal cord contusive injury). HMSC-derived Exos loaded on peptide-modified adhesive hydrogel (Exo-pGel) effectively mitigate inflammation and oxidation (Fig. 6B) [134]. EXO-C@Ps, incorporating a CAQK peptide to convey CRISPR/Cas9 components with the ability to edit the genome to upregulate soluble TNF receptor-1 (sTNFR1) at the lesion site, neutralize TNF-α and quench the inflammatory process activated by TNF-α (Fig. 6C) [135]. MF-NVs efficiently target ischemic and inflammatory organs and attenuate apoptosis and inflammation [91]. Finally, BMSC-Exos increase the expression of collagenous structure receptors (MARCOs) on macrophages, resulting in improved phagocytosis of engulfed myelin debris (Fig. 7) [75].

Fig. 7

BMSC-Exo Hydrogel Promotes the Macrophage Phagocytosis Effect after SCI. A) BMSC-Exo administration mixed with hydrogel upregulates MARCO, which in turn enhances the ability of macrophage to clear myelin debris that promotes the regeneration of axons following SCI. B) Hematoxylin & eosin (H&E) staining shows that BMSC-Exo-Hydrogel diminishes the SCI area following SCI in vivo. C) Immunofluorescence images show that BMSC-Exo-Hydrogel improves axon growth following SCI in vivo. Reproduced with permission [75]. Copyright 2021, Frontiers in Cell and Developmental Biology. SCI spinal cord injury; BMSC bone marrow mesenchymal stem cells

Additionally, pyroptosis, an innate immune response, is regulated by Exos in patients with SCI. M2 microglial Exos (M2-Exos) rich in miR-672-5p can downregulate the absent in melanoma 2 (AIM2)/apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC)/caspase-1 signaling pathway by blocking AIM2 activity, thereby preventing neuronal pyroptosis [39]. Tregs target NF-κB-activating protein via exosomal miR-709 to reduce microglia pyroptosis [41].

Macrophage/microglia activity constitutes an important factor in adjusting inflammatory responses in SCI. Modified macrophages release various Exos to regulate the inflammation response (Fig. 8A) [25]. Targeted intervention in SCI involves inhibiting the recruitment and proliferation of macrophages and preventing macrophage polarization. These processes contribute significantly to tissue regeneration and homeostasis maintenance [144, 145]. Exos derived from various cell types can suppress the inflammation process in the spinal tissue microenvironment after SCI and prevent M1 cells and reactive astrocyte activation. Additionally, Exos can facilitate microglia polarization to the M2 type, which has anti-inflammatory properties. Furthermore, Exos can extend the duration of M2 cell stay in the spinal cord [39, 42, 45, 146,147,148]. MSC-derived Exos specifically promote M2 polarization at the injury site after SCI (spinal cord contusive injury) [146] and HUC-MSC-Exos trigger the polarization of BMDMs to the M2 phenotype [149]. Dental pulp stem cell-derived Exos can decrease M1 polarization via the ROS-mitogen-activated protein kinase (MAPK)-NF-κB-P65 signaling pathway in SCI (spinal cord contusive injury) treatment; at 28 days post-SCI, the Exo-treated group presented with greater BMS scores (phosphate-buffered saline vs. Exos: 2.333 ± 1.155 vs. 4.667 ± 0.577) [147]. MiR-222-3P upregulation in endothelial progenitor cell Exos decreases proinflammatory macrophages and increases anti-inflammatory macrophages by activating the suppressor of the cytokine signaling 3 (SOCS3)/JAK2/STAT3 pathway; consequently, the ERC-Exos group showed better BMS scores [150]. BMSC-Exo-enriched miR-125a downregulates interferon regulatory factor 5 expression to promote M2-phenotype polarization [126]. LncRNA-Gm37494 overexpressed in hypoxia-pretreated ADSCs inhibits miR-130b-3p and enhances peroxisome proliferator-activated receptor gamma expression to promote microglial M1/M2 polarization, which suppresses inflammation by inhibiting the STAT/NF-κB pathways [130]. Exos produced from MSCs replicate the effects of a single MSC infusion on many factors, such as the enhanced expression of M2 macrophage markers [102]. HExos enriched in miR-216a-5p promote M2 polarization via the TLR4/NF-κB/PI3K/AKT signaling cascades [139]. BMSC-derived exosomal microRNA-124-3p ameliorates SCI (spinal cord ischemia injury) by inhibiting ER to nucleus signaling 1 and promoting M2 polarization [151]. Berberine-loaded M2 macrophage-derived Exos (Exos-Ber) induce macrophage/microglia phenotype polarization, consequently decreasing the amount of inflammatory cytokines TNF-α, IL-1β, and IL-6 (Fig. 8B) [44]. M2 macrophage-derived Exos inhibit the inflammatory response through macrophage polarization via the miR-23a-3p/PTEN/PI3K/AKT network [45]. MFG-E8, the main component of SCDEs, upregulates M2 polarization via the SOCS3/STAT3 signaling cascade [42]. Nevertheless, PM-Exos promote M2 polarization (Fig. 8C) [40]. Additionally, neural tissue-like electroconductive hydrogels carrying BMSC-Exos promote microglial M2 polarization via the NF-κB pathway and promote axonal regeneration via the PTEN/PI3K/AKT/mTOR pathway (Fig. 9) [82]. Moreover, Exos derived from Schwann cells loaded on hydrogel (NFs@MP-HAh@Exo) suppressed the inflammation process through M2 polarization and promoted neurons survival after SCI via the TLR4/NF-κB, MAPK, and AKT/mTOR pathways (Fig. 10) [121].

To attain anti-inflammatory effects, Exos inhibit complement mRNA synthesis and release, neuronal and microglial pyroptosis, neuronal cell ferroptosis, and excessive accumulation of lipid peroxides and suppress the activation of astrocytes to the A1 type. Exos also promote microglial autophagy, macrophage polarization, and myelin debris phagocytosis. Finally, the expression of proinflammatory cytokines is decreased. These numerous anti-inflammatory effects are achieved mainly by modulating the TLR4/MyD88/NF-kB signaling pathway, which regulates the inflammation process by stimulating ROS secretion and proinflammatory cytokines, including TNF-γ, IL-1, and interferon-γ, thereby eliciting secondary neurotoxicity effects [152]. In summary, Exos and their cargo can modulate diverse important molecular signaling pathways to mitigate the pathological microenvironment following SCI (Table 5).

Table 5 Anti-inflammatory effect following exo treatment in SCI Animal models

Fig. 8

Exos Inhibit Inflammation and Accelerate Macrophage Polarization to M2. A) The path from Exos derived from macrophage drug formulations to patient application. Reproduced with permission [25]. Copyright 2015, Journal of Controlled Release. B) M2 macrophage-derived Exos loaded with berberine quench inflammatory factors. Reproduced with permission [44]. Copyright 2021, Acta Biomaterialia. C) Macrophage-derived Exos accelerate microglial polarization to the anti-inflammation type by enhancing autophagy. Reproduced with permission [40]. Copyright 2021, International Journal of Biological Sciences

Fig. 9

Exo-loaded Electroconductive Hydrogel Exerts Potent Protective Effects Following SCI. A) Exo-loaded electroconductive hydrogel creates a favorable environment for neurogenesis, axonal regeneration, and inflammation inhibition. B) Exo-loaded electroconductive hydrogel accelerating dorsal root ganglia grown on the hydrogel. Scale bars: 100 μm. C) Neuronal and oligodendrocyte differentiation of NSCs is enhanced, while astrocyte differentiation is inhibited; moreover, axon outgrowth is increased via the PTEN/PI3K/AKT/mTOR pathway. Microglia M2 polarization is promoted by the NF-𝜅B pathway. Reproduced with permission [82]. Copyright 2022, Advanced Science. NSC neural stem cell; SCI spinal cord injury

Fig. 10

Nanofibers Containing MP (Methylprednisolone) and Exos Loaded on Hydrogel (NFs@MP-HAh@Exo) Inhibit Neuronal Apoptosis and the Inflammatory Reaction. A) NFs@MP-HAh@Exo regulate macrophage polarization. B) Immunofluorescence and hematoxylin & eosin stain images show that NFs@MP-HAh@Exo promote shrinkage of the SCI cavity in the injured lesion area after SCI. C) Immunofluorescence images show that NFs@MP-HAh@Exo promote axonal regeneration after SCI. D) Immunofluorescence images show that NFs@MP-HAh@Exo alleviate inflammation after SCI. Reproduced with permission [121]. Copyright 2023, ACS Nano. SCI spinal cord injury

Conclusions and future prospects

Exos have attracted significant interest as cell-free therapies owing to their fascinating biological properties. Increasing evidence from preclinical studies has confirmed the neuroprotective properties of Exos. On administration, Exos can specifically target and accumulate at spinal cord lesion sites and accelerate locomotor functional recovery. These beneficial effects have been mainly attributed to neurogenesis, angiogenesis, preservation of the BSCB, and anti-apoptotic and anti-inflammatory effects.

A thorough understanding of the potential mechanisms of action of Exos will facilitate their clinical application. Currently, Exos have successfully made the journey to being effectively applied in several Phase I trials [153,154,155]. However, several challenges remain regarding the establishment of the optimal application of Exos for SCI treatment. For example, several aspects are inconsistent between studies including the Exos isolation methods, application dosage, route of delivery, and treatment time points (Table S1). Additionally, SCI models have been developed using different methods (contusion injury, crush injury, clip compression injury, circular and semicircular spinal cord severed injury, and ischemia injury); moreover, the parameters of contusion and crush injuries vary, as well as SCI animal models, including both rats and mice (Table S2).

Improving the yield and purity of Exos is the most important priority since it remains the main bottleneck limiting their practical application. Although ultracentrifugation is considered the “gold standard” and is widely used in the field [25], this technology presents various limitations, including the simultaneous isolation of contaminants that are not exosomal in nature, limited reproducibility, low yield of RNA, potential damage to Exos, and insufficient capacity to process a large number of samples, making it unsuitable for clinical applications [156]. Establishing the application dosage and delivery method is also important, as these vary widely among studies. Additionally, the rapid clearance of Exos by host cells, their short half-life in vivo, and inefficient drug delivery to target tissues continue to impede Exos aggregation in the SCI area [155, 157, 158]. Combining the inherent advantages of Exos with a targeted medication has emerged as a new and potentially transformative therapeutic strategy that could significantly impact the future of SCI treatment. The remaining strategic challenges include the selection of a therapeutic agent, methods for loading cargo into Exos, enhancement of Exos stability, tissue targeting, and effective delivery of cargo to recipient cells that can utilize inherent Exo properties, such as immune modulation, regeneration promotion, and pathogen suppression [155]. Hence, it is imperative to optimize and enhance the Exo-loading capacity and techniques for improving targeting [159]. Particularly, culture conditions, including the cell type, passage number, number of cells seeded to initiate culture, and medium composition, influence not only Exos but also cargo levels [160]. Finally, a standardized animal model remains to be developed as current contusion parameters for the NYU-III weight-drop apparatus are discrepant between studies regarding height and weight, while animal model heterogeneity causes inconsistencies in SCI severity. Overall, these factors significantly influence the therapeutic effect, with the inter-study variability rendering the final curative results difficult to interpret.

In summary, although Exo-based therapeutics have been successful in numerous trials, some obstacles remain to be overcome before Exos can be tested clinically on a larger scale. Determining how Exos target specific cells and understanding the distinct physiological roles of different Exo subtypes will help facilitate the progress of Exos in the field of drug delivery.