Stem cell derived exosomes: Emerging cell-free therapeutics for neurodegenerative disorders

Abstract

Neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis represent a significant global health challenge with limited therapeutic options. In recent years, stem cell-based therapies have shown promise in promoting neural repair and modulating disease progression. However, concerns related to immune rejection, tumorigenicity, and ethical considerations have limited their clinical application. As an alternative, exosomes derived from stem cells have emerged as a novel, acellular therapeutic strategy. These nanoscale extracellular vesicles carry a rich cargo of proteins, lipids, and nucleic acids, capable of modulating neuroinflammation, promoting neuroprotection, and enhancing tissue repair. Their ability to cross the blood-brain barrier and their low immunogenicity make them especially attractive for treating central nervous system disorders. This review highlights the therapeutic potential of stem cell-derived exosomes in the management of neurodegenerative diseases, discussing their mechanisms of action, current research progress, and future clinical applications. The development of exosome-based therapies marks a significant step toward safe, effective, and cell-free neurodegeneration.

Key Words: Neurodegenerative diseases; Alzheimer’s disease; Parkinson’s disease; Stroke; Exosomes; Acellular therapy

Core Tip: Neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis, are progressive and irreversible conditions marked by neuronal loss, misfolded protein accumulation, and severe cognitive or motor decline. Current therapies cannot halt neuronal death, and stem cell transplantation is limited by the blood-brain barrier. Exosomes, nanoscale vesicles secreted by cells, overcome this limitation by naturally crossing the blood-brain barrier, carrying proteins, nucleic acids, and lipids with neuroprotective potential. Their role in diagnosis, biomarker discovery, and targeted drug delivery highlights exosomes as promising acellular therapeutics for neurodegenerative disorders, with growing clinical interest and translational potential.

INTRODUCTION

Neurodegenerative disorders are defined by the gradual degeneration and loss of neuronal cells, which are normally never regenerated due to the intrinsic limitations of endogenous regeneration within the central nervous system (CNS). The common neurodegenerative diseases include stroke, Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis[1]. These disorders collectively impose an increasing global health burden due to aging populations and the lack of disease-modifying therapies. Degeneration of neurons affects several brain regions, including the cortical and hippocampal areas in AD and the striatal regions in PD. Age is recognized as the primary risk factor for numerous neurological disorders[2]. While the origin of numerous diseases remains unidentified, an association of genetic predispositions and environmental variables can contribute to their onset[3]. The primary cause is the accumulation of misfolded proteins, resulting in the degeneration of neuronal cells in different brain regions, according to the physiological and pathological alterations associated with each disease[4]. The severity of symptoms in these disorders varies according to the damaged region of the brain. Neurodegenerative disorders are predominantly diagnosed based on clinical manifestations, supported by multifactorial assessments, including clinical history, cognitive evaluations, medical examinations, imaging studies, and cerebrospinal fluid biomarker analysis[5]. The early diagnosis is challenging due to the absence of effective peripheral markers. Unfortunately, there is currently no treatment that can stop or halt the permanent death of neurons. The protracted nature and substantial treatment expenses of chronic diseases provide significant challenges for patients and healthcare community, necessitating immediate intervention to discover effective solutions for this global issue[6].

Stem cell-based therapy is a promising approach for neurodegenerative diseases due to its potential for cellular regeneration, tissue repair, and neuroprotection. This therapy can replace damaged or dying neurons with healthy ones, restoring brain function and slowing disease progression. However, the primary obstacle to harnessing the therapeutic potential of these cells is the blood-brain barrier (BBB)[6,7]. The BBB, which is comprised of endothelial cells, astrocyte end-feet, and pericytes, is a diffusion barrier that is vital to the proper functioning of the CNS. It prevents the majority of blood molecules from penetrating the brain[8,9]. Cell-based therapies for the brain require overcoming the BBB, which restricts molecules larger than 400 Da. To bypass the BBB, various chemical methods (such as osmotic disruption, liposomes, nanoparticles (NPs), and cell-penetrating peptides) or physical methods (including microinjection, convection-enhanced delivery, focused ultrasound, and electroporation) must be used, which can be invasive and potentially harmful. This presents a major problem because these methods can have unintended effects, such as lowering cerebral blood flow levels, which worsens symptoms by impacting neuroinflammation and metabolism, and breaking the tight junctions of the BBB, which allows toxic substances and metabolic waste to invade. Accordingly, recent therapeutic strategies increasingly combine exosome delivery with targeting or BBB-bypass technologies. Delivery of engineered exosomes along with focused-ultrasound BBB opening, have substantially improved brain bioavailability in preclinical models[8,10].

Exosomes, nanoscale extracellular vesicles secreted by stem cells, have emerged as a promising acellular alternative. They mediate cell-to-cell communication by transferring bioactive cargo such as proteins, lipids, and RNAs that regulate inflammation, oxidative stress, and apoptosis. Mechanistically, exosome biogenesis follows either an endosomal sorting complex transport (ESCRT)-dependent pathway regulated by proteins such as apoptosis-linked gene 2-interacting protein X, tumor susceptibility gene 101, and vacuolar protein sorting 4 - or an ESCRT-independent pathway, driven by tetraspanins (CD9, CD63, CD81) and ceramide synthesis. These molecular routes not only determine vesicle formation but also control cargo selection, as RNA-binding proteins (e.g., hnRNPA2B1, YBX1) and lipid raft domains dictate which microRNAs (miRNAs), mRNAs, and signaling lipids are incorporated. This selective packaging is essential for therapeutic potency and reproducibility, influencing the exosome’s interaction with recipient cells and its stability in circulation. Exosome interaction with the BBB is a rapidly evolving area of research. While unmodified exosomes show limited passive diffusion (< 1% of systemically administered doses reach the brain), they exhibit several intrinsic features that enable partial BBB penetration. Exosome research in neurodegenerative diseases helps with early disease diagnosis and detection and offers novel approaches to disease treatment[8]. The transfer of exosomes can induce phenotypic alterations in receptor cells. Exosomes have a significant role in nerve inflammation. The generation and secretion of exosomes are primarily influenced by the features of both the parent cell and the recipient cell. Their ability to transport and fuse exosomes and connect with target cells for function depends on the presence of certain hallmark proteins on their surface, which can interact with other proteins like integrin. Hence, exosomes are highly targeted and specialized, with distinct contents and biological functions[8,11]. Brain-derived exosomes are present in body fluids, such as blood, which may be readily collected, and their contents may indicate the pathophysiological course of brain diseases[12,13]. The paracrine factors released by these exosomes can pass through the BBB more easily than those released by stem cells. The effects of these secreted paracrine factors are comparable to, or even identical to, those of their source cell[14]. Its peripheral availability, capacity to traverse the BBB, and enhanced targeting expand its potential as a biomarker for neurodegenerative diseases, hence demonstrating its utility in the diagnosis of such conditions. Its potential extends to its usage as a drug carrier in the treatment of neurodegenerative disorders including amyloid-β (Aβ) in AD, α-synuclein (α-syn) in PD and MS[15-17]. This review discusses exosome synthesis and isolation, the mechanisms behind exosome-mediated neuroregeneration, and the potential of exosomes as a therapy for patients of neurodegenerative disorders. This review also discusses the challenges and future potential of exosome therapy for clinical applications in neurodegenerative disorders applications.

NEURODEGENERATIVE DISEASES: BRIEF OVERVIEW

Neurodegenerative disorders are a class of slowly developing, irreversible diseases that are characterized by the loss of neurons and resulting atrophy of certain brain regions. Cognitive decline, severe motor impairment, reduced social functioning, and substantial problems with regular tasks are hallmarks of all neurodegenerative disorders. A mix of hereditary and environmental variables accounts for most neurodegenerative disorders. This complicates the prediction of the individual who will develop the disease. Age is the strongest indicator of risk for many neurodegenerative disorders[1]. It is possible to categorize neurodegenerative disorders based on the main genetic aberration, the location of neurodegeneration in the body, and the main clinical symptoms. A few examples of primary clinical traits are stroke, AD, dementia, PD, and Huntington’s disease. Brain regions affected by neurodegeneration include the frontotemporal cortex, the spinal cord, the ataxia of the cerebellum, and spinal muscular atrophy. Prion disease and synucleinopathies are the main molecular abnormalities[18]. The clinical manifestation was the primary focus of earlier classifications, but with the increasing biochemical understanding of neurodegenerative disorders, the attention has switched to the underlying pathological mechanisms. However, there is frequent overlap among neurodegenerative disorders, which are frequently depicted as a distinct entity. The diagnosis accuracy for any of the neurodegenerative diseases is poor. The neurological processes cannot be studied with pathological investigation, despite it being the gold standard for a broad range of disorders. The key to understanding disparities between clinical and pathological diagnosis is studying disease heterogeneity at autopsy. The development of biomarkers for the diagnosis of various diseases and the monitoring of disease progression in clinical trials is an ongoing endeavours, making this a crucial concept[18]. Known neurodegenerative disorders have been more common in recent decades. This growth is mainly related to two factors: First, longer life expectancy, which means that people have a higher chance of developing a condition at some point in their lives; and second, better earlier diagnosis, which means that healthcare systems are aware of more instances. Due to the growing number of patients and the necessity of a holistic approach, stringent requirements for medical data systems and the machine learning algorithms used by these systems are essential[1]. Treatments for neurodegenerative diseases focus on reducing the disease’s course, enhancing quality of life of patients, and dealing with associated problems. There is currently no cure for these conditions. Novel therapies for neurodegenerative diseases are a hot topic of current research. A new method of treatment involves regenerating damaged neurons using stem cells. The potential for stem cell transplantation to treat neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and others is exciting[19]. The unique properties of this treatment, as shown by this novel paradigm, include neuroprotection, neurodegeneration, remyelination, decreased inflammation of the nervous system, and functional recovery. However, stem cells cannot pass through the BBB due to the above mentioned problems. Despite progress, the exact method of using stem cells to repair damaged nerves remains unknown. However, researchers have identified exosomes, tiny components of stem cell secretions, as potential crucial factors in nerve repair[6]. Due to their small size, exosomes can cross the BBB. Remarkably, the beneficial effects of exosomes are similar to, or even identical to, those of the stem cells themselves. This discovery opens up new possibilities for treating neurological diseases using exosome-based therapies[6].

EXOSOMES COMPOSITION

Exosomes contain an aqueous core surrounded by a lipid bilayer. Their amphiphilic properties allow for the incorporation of hydrophilic and lipophilic compounds, rendering them significant in drug delivery[20]. Exosomes cargo possess a diverse composition that varies according to their origin and physiological condition. Table 1 outlines the compositions of each component of the exosomal structure. Exosomes are abundant in particular proteins, lipids, enzymes, and genetic components that contribute to intercellular communication and carry signaling molecules to both local and distant sites. Exosome membranes typically include various lipid derivatives, including phosphatidylserine, sphingomyelin, ceramides, and cholesterol. Proteins like cytoskeletal elements, tetraspanins, enzymes, lysosomal proteins, GTPases, annexins, and flotillin are found in exosome composition analysis. Other proteins included antigen presentation molecules, major histocompatibility complex class I and II, and intercellular adhesion molecule-1[21]. Exosomes include heat shock proteins (HSP) like HSP90 and HSP70 that help in antigen presentation. Furthermore, exosomes contain a substantial array of proteins known as tetraspanins. These transmembrane proteins have a major role in the formation of complex molecular structures because they can interact with a variety of other proteins/molecules, including integrins and major histocompatibility complex molecules. Exosomes are abundant in apoptosis-linked gene 2-interacting protein X and tumor susceptibility gene 101, which are essential for their synthesis via the ESCRT. Furthermore, tetraspanin proteins such as CD9, CD37, CD63, CD81, and CD82 have been identified as participating in an ESCRT-independent pathway. Typical exosome indicators are proteins that are unique to exosomes and not found in other types of vesicles. Moreover, in addition to the proteins commonly found in exosomes, some proteins are unique to the particular biological origin. Exosomes originating from intestinal epithelial cells include certain proteins, including C26, syntaxin 3, and A33, which are contingent upon the orientation of epithelial membrane for secretion, either basolateral or apical. Additionally, other examples of cell-specific exosomes encompass α4β1 on platelets and reticulocytes that express P-selectin. Furthermore, exosomes carry a wide variety of genetic material, including DNA, mRNA, and other kinds of non-coding RNAs, including miRNAs, circular RNAs, and long non-coding RNAs. Long non-coding RNAs are important in regulating cell differentiation and modifying the cell cycle, while circular RNAs are hypothesized to act as miRNA suppressors, competing with them to influence gene expression. Therapeutic claims in neurodegeneration increasingly depend on identifying which cargo classes drive benefit. Across AD/PD/MS/stroke models, neuroprotective effects repeatedly map to miRNAs regulating autophagy and lysosomal clearance, synaptic scaffolding and plasticity, microglial polarization, and oxidative stress responses, as well as proteins involved in heat-shock defense and trophic signaling. Because these cargos shift with donor-cell stress, aging, hypoxia, or inflammatory priming, cargo profiling is required to explain efficacy differences and to design reproducible, disease-specific exosome products[22].

Table 1 Components of exosomes structure.

Serial number	Structure	Compositions	Functions
1	Cytoskeletal elements	Actin, tubulin, cofilin, talin, vimentin	Structural support, vesicle formation, transport
2	Lysosomal protein	Lamp2b	Degradation, protein sorting, membrane trafficking
3	Exosome membrane	Sphingomyelin, phosphatidylserine, cholesterol, and ceramides	Encloses exosomal content, interacts with target cells
4	Tetraspanin family	CD63, CD9, CD37, CD81, and CD82	Membrane organization, protein interactions, cell adhesion
5	HSPs	HSP70, HSP90, HSP20, HSP27, HSP60, HSC70	Protein folding, stress response, cell protection
6	Intercellular adhesion molecule	ICAM-1, integrins, p-selectin, lactadhesion	Cell-cell interactions, signaling, immune responses
7	Fusion and membrane transport proteins	GTPases, flotillin, annexins, Rabs, dynamin, syntaxin	Vesicle fusion, content release, membrane trafficking
8	Transmembrane proteins	CD13, LAM1/2, PGRL	Signaling, cell-cell interactions, membrane anchoring
9	Immuno-regulator molecules	CD80, CD86	Modulate immune responses, tolerance, inflammation
10	Antigen presentation	MHC class I and II molecules	Activate immune cells, antigen display
11	Nucleic acids	mRNA, DNA, and non-coding RNAs	Genetic information transfer, gene regulation
12	Enzymes	Glycosidases, GAPDH, nitric oxide synthase, catalase, phosphatases, lipases, pgk1, ATPase	Catalyze biochemical reactions, metabolic processes
13	Growth factors and cytokines	TRAIL, TNF-α, TGF-β	Cell growth, differentiation, signaling
14	ESCRT-dependent exosomal biogenesis	ALIX, TSG101, clathrin	Exosome formation, sorting, release

Lamp: Lysosomal associated membrane protein; CD: Cluster of differentiation; HSP: Heat shock proteins; HSC70: Heat shock cognate protein 70; ICAM: Intercellular adhesion molecule; MHC: Major histocompatibility complex; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; pgk1: Phosphoglycerate kinase 1; TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand; TNF-α: Tumor necrosis factor α; TGF-β: Transforming growth factor β; ALIX: Apoptosis-linked gene 2-interacting protein X; TSG101: Tumor susceptibility gene 101.

EXOSOME BIOGENESIS AND PATHOLOGICAL MECHANISMS IN NEUROBIOLOGY

Exosome biogenesis is the process by which exosomes are produced, from the formation of early endosomes to the release of mature exosomes into the extracellular space (Figure 1). There are various mechanisms that contribute to exosome biogenesis. One route is the production of exosomes by multivesicular bodies (MVBs) within endosomes. Another pathway is the formation of microvesicles (MVs) through the plasma membrane. Inward membrane budding allows early endosomes to mature in MVBs and eventually produce intraluminal vesicles after endocytosis. MVBs can fuse with lysosomes or by docking at the cell periphery and secreting exosomes. Additional vesicle types include apoptotic bodies, migrasomes, and MVs. Apoptotic bodies are released during cell death through membrane budding, migrasomes are formed from retraction fibers, and vesicles are formed by fusing the outer autophagosome membrane with late-endosomes[23].

Figure 1 Exosomes biogenesis. Exosomes fuse with the plasma membrane after being generated within multivesicular endosomes during intraluminal vesicle production and are subsequently released through exocytosis. In an alternative mechanism, lysosome fusion, the multivesicular endosome merges with a lysosome to undergo degradation rather than exosome discharge. Ectocytosis causes microvesicles to form directly from the plasma membrane.

Exosomes can contribute to mechanisms previously associated with the psychopathology of neurological diseases, including neuroinflammation, neurogenesis, plasticity, and epigenetic control (Table 2). Biogenesis pathways are a controllable source of therapeutic performance. ESCRT-biased vesicles and tetraspanin/ceramide-biased vesicles differ in membrane identity, targeting ligands, and cargo loading logic, which changes CNS uptake routes and immune effects. In practice, this means that donor-cell selection and priming state determine whether an exosome batch is neuroprotective, immunosuppressive, or biologically inert. Standardizing or engineering biogenesis therefore becomes essential for potency consistency and regulatory translation. It has been suggested that the contents of exosomes in the cerebrospinal fluid may reflect changes occurring in the brain because of their ability to cross the BBB. Thus, exosomes originating from neurons could be useful as possible biomarkers for neurodegenerative disorders. Numerous researchers have demonstrated that exosomal release from the CNS is modulated by synaptic glutamatergic activity and calcium influx[24]. The regulated release of brain exosomes has been reported. Nonetheless, the comprehensive mechanism remains unclear. In axons, MVBs are present in greater abundance than in the soma or dendrites. The process of preferred compartmentalization remains unknown. The presence of these enhanced zones suggests evidence to the importance of synaptic exosomes and their tightly controlled release[25].

Table 2 Neuroregenerative effects of exosomes.

Mechanisms	Functions	Effects
Neurotransmitter release	Exosomes release neurotransmitters, promoting neuronal communication	Enhanced synaptic plasticity, cell survival
Cellular communication	Exosomes facilitate communication between neurons, oligodendrocytes, and microglia	Regulation of cellular processes, neuroprotection
Epigenetic control	Exosomes influence gene expression, regulating neurogenesis and neuroinflammation	Modulation of neuroregenerative processes
Synaptic plasticity	Exosomes promote synaptic strengthening, neuronal adaptation	Improved cognitive function, memory
Neuroprotection	Exosomes transfer protective signals, enhancing cell survival	Reduced neuroinflammation, oxidative stress
Calcium influx regulation	Exosomes modulate calcium influx, regulating neuronal excitability	Maintenance of neuronal homeostasis
Serotonin-mediated release	Exosomes released via serotonin pathways, influencing mood regulation	Modulation of mood, cognitive function
Microglia activation	Exosomes from microglia regulate neuroinflammation, immune responses	Neuroprotection, reduced inflammation
Oligodendrocyte support	Enhance neuronal survival, myelination	Improved neuronal regeneration
Blood-brain barrier crossing	Facilitate neuroregeneration	Access to central nervous system for therapeutic interventions

The neuronal cell in the brain can release exosomes with the help of serotonin. Bipolar disorder, schizophrenia, and anxiety have been hypothesized to be caused by dysregulated serotonin pathways. The release of exosomes from microglia can be controlled by serotonin, and as neurological problems frequently impair serotonin, these disorders may also affect the release of exosomes from microglia. Cellular communication and neurotransmitter release have a significant role in psychopathology. Given their significance in the regulation of cellular communication and its regulation through neurotransmitters, it is crucial to understand the function that exosomes play in the etiopathogenesis of neurological diseases[24,25].

Neuronal communication is possible through the release and absorption of exosomes through pinocytosis or phagocytosis. It has been demonstrated that a subpopulation of neuron-internalized exosomes can be released alongside the endogenous exosomes of the recipient neuron, thus promoting extended connections. These findings demonstrate that exosomes can promote communication within the cell and have the capability to transmit signals, even though their fate is still uncertain. Additionally, communication between neurons through exosomes plays a crucial role in significant processes, such as synaptic plasticity. The release of neurotransmitters may trigger the secretion of oligodendrocyte exosomes, while neurons can internalize these exosomes and utilize their cargo. The internalization of exosomal products from oligodendrocytes can exert significant pressure and enhance cell survival, providing cellular protection in the brain and facilitating their capacity to traverse the BBB. Recent discoveries about the properties of exosome signals in the brain are connected to current physiological and pathological understandings of mental diseases. Following the initial finding that the transport of exosomal cargo into recipient cells has functional implications, numerous investigations have been conducted on this mechanism concerning cell-to-cell communication in both disease states and healthy conditions[24-26]. Figure 2 gives an overview of exosomes biogenesis and their mechanisms of action in neurobiology.

Figure 2 Exosomes mechanism of action. This flow chart illustrates the complex mechanisms of exosomes in neurobiology, including their biogenesis, uptake, and effects on neuroprotection, neurodegeneration, neuroregeneration, and immunomodulation.

EXOSOMES ISOLATION AND CHARACTERIZATION

The true diagnostic potential of exosomes for neurodegenerative disorders requires isolation methods that can distinguish between exosomes subpopulations in the peripheral circulation and exosomes originating from the brain, such as astrocytic, neuronal, and oligodendroglial exosomes. It is extremely difficult to detect and analyze the content of brain-derived exosomes from blood samples due to the extremely low concentration of these exosomes in blood circulation. Therefore, the efficacy and dependability of exosomes detection technologies, as well as their accuracy and detection limitations, can be enhanced by creating feasible isolation procedures. New diagnostic techniques that clinical laboratories worldwide could use have been the focus of multiple scientific endeavours. Considerations such as simplicity, low cost, and high quality are crucial. A significant difficulty is the standardization of a methodology for clinical application in diagnostic laboratories worldwide, as most of the exosome separation techniques currently in use are labor-intensive, time-consuming, and need specialized and costly equipment. Isolation strategies determine not only yield but also biological interpretability and clinical feasibility. Low-purity preparations (e.g., precipitation-based methods) co-isolate soluble proteins and lipoproteins that can mimic or mask true exosome effects, confound mechanism claims, and introduce safety risks. In contrast, higher-purity approaches preserve vesicle integrity and are increasingly favored for scalable clinical pipelines. Under Minimal Information for Studies of Extracellular Vesicles 2023, method selection must be justified based on downstream purpose (mechanistic study vs therapeutic development) and accompanied by defined characterization and release criteria. Currently, there are a number of methods available for obtaining exosomes from human samples[11]. Table 3 displays the pros and cons of each isolation method.

Table 3 Comparison of exosomes isolation methods.

Isolation method	Purity	Pros	Cons
Ultracentrifugation	Low	Widely used, simple protocol	Time-consuming, multi-step process, high contamination risk
Size exclusion chromatography	High	Fast, simple and low-cost	Subtyping involves significant sample sizes and another procedure
Immunoaffinity-based approaches	High	Does not require special equipment, enrich cell-specific exosomes by targeted surface markers	Relies on high-cost antibodies, time consuming
Polymer-based precipitation	Low-to-medium	Low-cost method, easy protocol	Contamination risk, high cost, time consuming
Microfluidic devices	Low	Does not require special equipment, cost-effective	Design complexity
Nanotechnology	High	Commercially obtainable nanowires	High cost, restricts sample size

Ultracentrifugation

Ultracentrifugation is a popular technique for exosomes and is regarded as the gold standard. It is frequently employed to separate exosomes from other parts of biological material, like apoptotic bodies, because sedimentation rates are different. Preparative centrifugation and analytical ultracentrifugation are the two primary types of ultracentrifuges, which rotate at high speeds (between 100000 × g and 120000 × g). Analytical ultracentrifugation are optic systems-equipped, high-speed ultracentrifuges that can see particle fractionation and determine characteristics including mass, shape, and sedimentation rate in solution. Cells, viruses, proteins, nucleic acids, and exosomes are among the biological particles that are frequently purified using the preparative centrifugation process. Isolating small particles from large ones is the main idea behind this method, which involves gradually increasing the duration of centrifugation and applied centrifugal force. Based on their rate of sedimentation, the sample’s particles are distributed throughout the centrifuge tube as the centrifugation field continues. Body fluid or cell culture media samples are sequentially centrifuged at escalating rates to eliminate necrotic cells, apoptotic bodies, debris, and shedding vesicles. A final high-speed centrifugation (> 100000 × g) is essential to extract tiny extracellular vesicles predominantly composed of exosomes owing to their low density. The primary drawback of this technology is the extended duration necessary to extract the exosomes. Ultracentrifugation is a time-consuming, multi-hour process that is expensive and not commonly available, particularly in clinical labs, for isolating exosomes. The potential for low yields is another disadvantage of this technique, which requires repeated ultracentrifugation steps. The poor yield of exosomes specific to brain cells (e.g., neuronal exosomes ≤ 1% of the total blood exosomes) makes ultracentrifugation less useful. Ultracentrifugation has been used in conjunction with other procedures, including microfiltration and sucrose density gradients, to obtain a relatively pure population of exosomes. Improved isolated exosome quality is a result of these additional processes, but the output is still unsatisfactory[11,13,27].

Size-based isolation

Size exclusion chromatography (SEC) and differential filtering are two standard isolation methods for separating exosomes according to size. Differential filtration is employed to isolate exosomes from cell culture media and serum according to molecular weight and size. This process consists of exosome filtration stages. A 0.22 μm membrane filter is employed to eliminate big extracellular vesicles, cells, and cellular debris. Dialysis eliminates unbound proteins with a standard membrane size of 500 kDa. Finally, the sample is subjected to filtration using a 100 nm membrane filter. This method requires no highly specialized apparatus and substantially reduces processing time from around 16 hours for ultracentrifugation approach to a few minutes. SEC is a conventional method that uses a porous gel bead phase and a stationary phase to control the flow of particles of varying sizes through the column. The advantage of SEC is its relative speed and cost-effectiveness, as it preserves exosome integrity by avoiding significant shear forces, unlike ultracentrifugation. One disadvantage of this method is its poor throughput and suboptimal yields of exosomes[11,13,27].

However, high-quality exosomes can now be obtained using the SEC process. This method categorizes vesicles based on their sizes. The first mobile phase is biofluid, while the stationary phase is a porous gel filtration polymer. The stationary phase achieves differential elution by isolating larger particles initially, then smaller ones, and finally, proteins that are not attached to the membrane. This is a one-step isolation system that takes about 20 minutes to process each sample[28]. The isolation of relatively pure exosomes is highly efficient with SEC, primarily due to the reduction of high-density lipoproteins and contaminating plasma protein. Another benefit of SEC is that it enhances the integrity of separated exosomes by employing gravity instead of mechanical force for isolation[29]. A primary drawback of this approach is its inability to distinguish between exosomes and MVs of similar dimensions. A combination of immunocapture approaches is required for the isolation of exosomes that are specific to subtypes. Isolating significant quantities of subtype-specific exosomes necessitates considerable amounts (volumes) of starting materials[30].

These approaches represent substantial advancements beyond traditional magnetic bead capture, greatly enhancing the isolation efficiency of extracellular vesicles (exosomes), including exosomes. A multifaceted approach is necessary to separate specific exosomes. Numerous laboratories and research centers globally are employing the abovementioned methodologies. Nonetheless, standardized methodologies suitable for routine application in diagnostic laboratories remain absent[11,13,27].

Immunoaffinity-based approaches

The surface of exosomes is covered with many receptors and proteins. The unique ability of exosomes to bind to specific proteins (antigens) and antibodies, as well as to bind to specific ligands, presents an opportunity to create highly targeted methods for isolating exosomes. This realization led to the development of methods for isolating and purifying exosomes based on immunoaffinity. The selection of proteins for effective isolation depends on various criteria, including the cell of origin, and surface orientation. A number of transmembrane proteins found in exosomes, including annexin, CD9, CD81, CD82, CD171, RAB5, and the epithelial cell adhesion molecule, can be employed as particular markers to identify and isolate particular exosome populations with high purity. Magnetic bead immune separation, chromatographic stationary phase separation, and enzyme-linked immunosorbent separation are three subtypes of immunoaffinity-based methods that differ in the coated antibody substrates used. One frequently employed technique for the isolation and quantification of exosomes is the enzyme-linked immunosorbent assay, which utilizes biomarkers that are present on the surface. These peptides on the membrane can help isolate exosomes by precisely interacting with antibodies that target specific cargo substrates. Extending this technology to utilize non-covalent interactions will enable a more precise disassembly of exosomes from the magnetic beads, producing pure exosomes[31].

Immunoaffinity based methods can also be employed to separate cell-specific exosomes, like those produced from the brain, and from blood samples by targeting surface markers. Isolating exosomes, which are tiny extracellular vesicles, from neurons typically, involves the use of neural cell adhesion molecules and L1 cell adhesion molecules. Glutamate aspartate transporter has been used for exosomes derived from astrocytes, whereas 2’,3’-cyclic nucleotide 3’-phosphodiesterase has been used to enrich oligodendroglial exosomes. Magnetic bead-based isolation kits are an example of the newer, more innovative methods and commercial kits developed recently that rely on the antibody affinity binding of exosomes protein markers[32]. Exosomes are isolated using magnetic NPs, specifically iron oxide NPs, coupled with CD9 antibodies. The exosomes-NP complexes are subsequently collected through the application of a strong magnetic field produced by affixing a permanent magnet to the microchannel walls[32].

Producing pure and homogeneous exosome yields is an advantage of this method. However, this method is intentionally not designed to capture subpopulations that could be biologically significant but do not express the surface marker of interest. Selecting the appropriate surface marker is also challenging due to the presence of multiple post-translationally modified forms (e.g., cleavage products) in body fluids, which are not exclusive to brain cells. Moreover, CNS-derived exosomes constitute a minor portion of the total exosomes in the bloodstream. Therefore, a highly sensitive detection method is essential for the development of exosome-based diagnostics. A microfluidic technology effectively isolates exosomes from entire blood samples. Furthermore, functionalizing iron oxide NPs with anti-L1 cell adhesion molecules antibody rather than anti-CD9 yields a valuable resource for researchers focused on isolating neuronal-derived small extracellular vesicles from biological specimens[11,13,27].

Polymer-based precipitation

The variable solubility of exosomes and other components in solutions containing precipitants is the basis for exosome isolation using the polymer precipitation method. The solubility properties of the sample components can be changed by adding a water-soluble precipitant to the sample solution, which creates a high-viscosity environment. Exosomes are hydrophobic vesicles composed of a lipid bilayer. When precipitants are added to a system, it dehydrates the fluid, makes exosomes less soluble in water, and creates cloud-like or particle-like formations. Commercial kits, such as commercial polymer-based isolation kits, including Exo-spinTM (Cambridge Bioscience, United Kingdom), Total Exosome Isolation (ThermoFisher Scientific, MA, United States), and ExoQuick^® (System Biosciences, CA, United States), for exosome precipitation have become extensively utilized as a streamlined, one-step approach for researchers. The ExoQuick system is one of the exosomes isolation kits that are aiming to simplify and speed up separating electrolytes from biological fluids. This kit facilitates the precipitation of exosomes into a pellet through a polymeric mixture, subsequently processed through a purification column to diminish impurities like albumin, and immunoglobulin G. This technique is simple and rapid, necessitating only fundamental laboratory apparatus. However, low-medium purity and the production of large aggregates are drawbacks of this approach. Furthermore, the ExoQuick kit is costly and only permits the examination of a limited number of samples, which places a significant financial strain on clinics[11,13,27].

Microfluidic devices

Microfluidics involves the manipulation of fluid dynamics at microscopic sizes. Various microfluidic devices have been developed to enhance the efficacy of exosome isolation techniques. Diverse sorting techniques can be utilized in these devices, contingent upon the physical and biological characteristics of the exosomes. In addition to conventional methods such as size, density, and immunoaffinity, sophisticated sorting techniques including electrophoretic, electromagnetic, and sonic manipulations may be deployed. Advancements in microfluidics technology have led to substantial decreases in sample volume, reagent use, and isolation time[11,13,27]. The scalability, standardization, and validation of contemporary microfluidic devices are limited, despite their significant developments. Moreover, inadequate sample pretreatments, insufficient yield, or low specificity may hinder subsequent analysis[33].

Nanotechnology

No single exosome isolation approach has been perfected. The choice of an acceptable isolation technique is contingent upon the sample type, subsequent application, and the requisite level of purity. Research is currently being conducted to enhance separation and ultrasensitive detection to improve the diagnosis of neurodegenerative diseases through exosomes. Nanomaterial-based methods have demonstrated potential as an effective means to isolate cell-specific derived exosomes, such as brain-derived exosomes from biological samples, because NPs can be functionalized with ligands, including DNA, enzymes, proteins, and antibodies. The use of the nanotechnology toolset in addition to the previously discussed techniques offers new perspectives on the pathophysiology of exosomes as well as new prospects for improved diagnostic techniques[30,34]. A new study has introduced a novel and ultrasensitive technique that enhances the capture effectiveness of cell-specific exosomes by about threefold compared to traditional approaches. This approach utilizes antibody-coated magnetic nano-wires, allowing for the conjugation of exosome-specific antibodies, hence enabling cell-specific exosome isolation[20]. Additionally, a method integrating membrane-based exosome separation with streptavidin-modified iron oxide NPs has been employed for the swift and effective isolation of MVs[35]. A novel methodology termed ExoCounter has also been developed to quantify exosomes originating from human cells or serum by nanobead-labeled exosomes on an optical disc[36]. Nemati et al[37] isolated tumor-derived exosomes more recently using magnetic nanoplatforms (magnetic nanowires, nanorods, and magnetosomes). They demonstrated that magnetic nanowires outperformed magnetosomes and rods in terms of efficiency[37]. However, nanotechnology-based isolation techniques require high costs and small sample sizes.

EXOGENOUS DELIVERY STRATEGIES FOR EXOSOMES

Exosome-based therapies offer immense promise for the treatment of neurodegenerative diseases; however, an effective delivery system is essential to maximize their therapeutic potential. Several exogenous delivery strategies are being explored to overcome the challenges of delivering exosomes to the brain and targeted tissues. One promising method is intranasal administration, a non-invasive delivery route that provides direct access to the brain by bypassing the BBB. This method leverages the nasal cavity’s close proximity to the brain and its rich vascularization, which allows exosomes to be absorbed into the CNS more efficiently. Intranasal delivery has demonstrated particular potential in treating neurodegenerative diseases like AD and PD by enhancing exosome bioavailability in brain tissue. It offers a convenient and patient-friendly alternative to invasive methods, making it an attractive option for clinical applications. Another widely studied route is intravenous injection, which is a common and clinically approved method for systemic drug delivery. While intravenous administration allows for easy delivery and distribution of exosomes throughout the body, it faces significant limitations due to the difficulty in crossing the BBB. The vast majority of intravenously administered exosomes are typically distributed to peripheral organs, such as the liver and spleen, with minimal brain uptake. To overcome this barrier, several modifications have been proposed, such as the use of targeting ligands that can direct exosomes to specific brain regions or enhance their ability to cross the BBB. Additionally, techniques like focused ultrasound are being explored to temporarily open the BBB, allowing exosomes to enter the brain more effectively. These strategies hold the potential to significantly improve the efficacy of intravenous exosome delivery for neurological conditions. Localized delivery through biomaterials, such as hydrogels, presents another promising approach for exosome administration. Hydrogels are utilized as carriers for exosomes, which offer controlled release at targeted sites, such as injured brain regions or areas of neurodegeneration. This method ensures that exosomes are delivered directly to the site of action, providing sustained release over an extended period. Localized delivery not only maximizes the therapeutic effects at the target site but also minimizes systemic side effects and off-target distribution. Hydrogels and other biomaterials are particularly beneficial for tissue regeneration and repair, making them ideal for use in neurodegenerative disease models that require precise and sustained treatment. These exogenous delivery strategies provide innovative solutions to the challenges of exosome therapy. In addition, these strategies enhance its potential for clinical applications in the treatment of neurodegenerative disorders[38-40].

CLINICAL APPLICATIONS OF EXOSOMES IN NEUROLOGICAL DISORDERS

The complex cargo of neural exosomes includes nucleic acids and proteins. Neural exosomes are involved in immunological control, communication, bioenergetics, tissue regeneration, and metabolism, among many other biological processes. Their intercellular trafficking can influence endocrine or pancreatic cellular processes. Due to their diagnostic applications, presence in numerous body fluids, and minimally invasive nature, exosomes produced from various stem cells including mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), and embryonic stem cells (ESCs), are regarded as optimal candidates for treatment of neurodegenerative disorders[41]. MSCs are multipotent stem cells isolated from tissues such as bone marrow, adipose tissue, and umbilical cord. Known for their paracrine effects through exosome secretion, MSC-derived exosomes demonstrate neuroprotective, immunomodulatory, and tissue repair properties in neurodegenerative models. However, their clinical use is limited by variability in exosome yields based on donor factors, including age and health status. iPSCs are reprogrammed somatic cells that regain pluripotency, allowing differentiation into any cell type, including neurons. iPSC-derived exosomes are versatile and can be genetically engineered for enhanced therapeutic effects. They hold promise for neuroprotection, especially in Alzheimer’s and Parkinson’s, but iPSC generation is complex and costly, with concerns about tumorigenicity. NSCs, sourced from the brain or induced from other stem cells, produce exosomes that carry neurotrophic factors beneficial for neurodegenerative diseases. While NSC-derived exosomes help with neuroinflammation and synaptic plasticity, scalability remains limited due to challenges in obtaining and expanding NSCs. ESCs are pluripotent and produce exosomes with a broad range of therapeutic cargos for neurodegenerative diseases. However, their use raises tumorigenicity risks and ethical concerns due to sourcing from embryos. Researchers are evaluating exosomal therapy as a promising candidate for the effective treatment of neurological illnesses, since it plays a crucial role in activating regenerative processes in affected individuals[24].

Exosomes have demonstrated encouraging results in the therapy of several diseases in preclinical research, including MS, AD, PD, and stroke (Figure 3). Several clinical investigations are exploring the potential therapeutic effects of stem cell-derived exosomes in three clinical trials. As of November 14, 2024, a search of the ClinicalTrials.gov database showed eight clinical trials investigating the potential of exosomes for the treatment of neurodegenerative disorders. Neurodegenerative disorders were the keyword set for the diseases search and “exosome” or stem cell derived exosome for the intervention/treatment search.

Figure 3 Exosome therapeutic applications in neurodegenerative diseases. Exosomes improve cognitive function in Alzheimer’s disease, improve motor function in Parkinson’s diseases, enhance recovery in stroke, and reduce disease progression in multiple sclerosis.

Exosomes in AD

AD is the most recognized etiology of dementia, predominantly influencing the senior demographic. This neurodegenerative disorder is an irreversible and gradual condition that impairs cognitive function, personality, mental health, and behaviour. The three main signs of AD include amyloid plaques, which are the buildup of beta-amyloid peptide outside of cells, neurofibrillary tangles caused by the intracellular deposition of hyperphosphorylated tau protein, and chronic neuroinflammation. These three features are considered by numerous researchers to be the most convincing scientific explanations for the pathological features of AD. Abnormal protein accumulations within and outside nerve cells result in neurocommunication problems and the degeneration of certain neurons. The early stages of AD are supposedly caused by the accumulation of Aβ peptide in neurons, which is the primary component in amyloid plaques. Multiple studies indicate that the primary locus of Aβ synthesis in neurons is MVBs. Upon entry into the lysosomal lumen, the intraluminal vesicles that make up MVBs can either be destroyed or released into the extracellular matrix as exosomes by fusing with the plasma membrane. These findings underscore the significance of exosomes in the progression of AD via the transmission of Aβ deposits[6].

Despite numerous preclinical studies indicating that exosomes are promising candidates for the treatment of various neurodegenerative illnesses[42,43], their efficacy as therapeutic agents is significantly constrained by the low quantity secreted by stem cells. Exosomes produced by culture media usually contain less than one microgram of exosomal protein per milliliter of media. Cells subjected to stressful situations reportedly augment the quantity of intracellular multivesicular structures and enhance exosome secretion. Heat shock (HS) before cell separation is one method to improve the poor yield from cultured cells. Exosomal concentration and diameter are significantly increased in NSCs exposed to 42 °C for 3 hours compared to non-HS (NHS) cells. The concentration of exosomes formed from HS is thirteen times greater than that of exosomes obtained from NHS. While NHS-derived exosomes include a wider variety of proteins, HS-derived exosomes have a number of important biological functions, such as regulating cell death and DNA damage, suggesting they may have therapeutic use. In contrast to non-HS-derived exosomes, cells treated with HS-derived exosomes offer better neuroprotection against cell death caused by hydrogen peroxide and neurotoxicity caused by Aβ. When Aβ-induced cellular death and oxidative stress were tested, it was shown that exosomes made by HS completely reduced both outcomes[44]. This research demonstrates encouraging outcomes in employing HS to improve exosome formation and modify their cargo composition while preserving therapeutic effectiveness against Aβ caused neurotoxicity and oxidative stress[45,46]. An AD mouse model showed considerable improvements in spatial learning and memory ability when administered exosomes derived from human umbilical cord MSCs systemically. These improvements were compared to the control group in the Morris water maze. This work found that exosome-treated animals had a lower Aβ plaque load and a lower level of activated microglia, lending credence to the idea that exosomes have an anti-neuroinflammatory function in AD[47]. In addition to their ability to reduce neuroinflammation, exosomes also reduce oxidative stress, which is another feature of neurodegenerative disorders[48].

Exosomes in PD

PD is a progressive and chronic neurodegenerative disorder that primarily affects dopaminergic neurons. Alongside the degeneration of dopaminergic neurons, the development of Lewy bodies resulting from the accumulation of α-syn protein in the neuronal cytoplasm is a significant characteristic of PD pathogenesis. Like other neurological disorders, there is currently no effective treatment to impede the progression of PD, and existing standard therapies, such as levodopa, a direct precursor of dopamine, are solely employed to alleviate symptoms. To enhance efficiency and mitigate negative effects, levodopa is frequently combined with other pharmacological treatments for PD. Surgical intervention to administer electrical impulses to neurons (deep brain stimulation) is a successful albeit complicated and expensive treatment for alleviating symptoms of PD. Recent evaluations have assessed the efficacy and efficiency of nonpharmaceutical therapeutic modalities for PD, including gene therapy, stem cell therapy, miRNAs, and exosomes produced from these cells[6].

In PD, dopamine-loaded exosomes augment cerebral dopamine levels by over 15 times. The administration of human umbilical cord derived MSCs to a mouse model of PD demonstrated amelioration of behavioral symptoms, reduction in neuronal death, and an elevation in dopamine levels in the brain[45]. This study demonstrated that blood exosomes can be infused with dopamine, traverse the BBB, and transport dopamine to the brain, namely to the striatum and substantia nigra, regions associated with PD. In a progressive PD paradigm, exosomes generated from MSCs were demonstrated to mitigate cognitive impairment linked to altered neuronal cholesterol metabolism. Furthermore, using a cellular model of PD, exosomes produced by bone marrow MSCs showed anti-inflammatory and anti-oxidative stress characteristics[39]. One intriguing investigation is priming MSCs with α-syn to find out if the MSCs exhibit different neuroprotection in PD. Another study pre-treated MSCs with α-syn and evaluated the impact of these pre-treated MSCs on the autophagy and survival of dopaminergic neurons co-cultured with the MSCs[49]. Their findings indicated that priming MSCs with α-syn provides improved neuroprotection by augmenting stemness in the stem cells and enhancing autophagy in PD mice. The increased therapeutic efficacy and disease specificity of modified MSC-derived exosomes should lead to their increased utilization in research[49].

Exosomes in stroke disease

Stroke is a significant neurological condition characterized by a high incidence of disability and mortality globally, impairing the proper functioning of brain regions. Significant strokes are ischemic strokes. An ischemic stroke occurs when blood flow is obstructed in the artery supplying oxygenated blood to the brain. Blood clots frequently induce obstructions that lead to ischemic strokes. The two most effective therapies for acute ischemic stroke, according to the current guidelines set by the Food and Drug Administration (FDA), are endovascular thrombectomy and intravenous tissue plasminogen activator. Due to restricted access to these medications, large numbers of people remain untreated, with potential treatment occurring through neurorehabilitation and endogenous neuroregeneration mechanisms. Researchers are exploring the use of exosomes produced from stem cells as a novel strategy aimed at treating this condition[43]. Consequently, numerous in vitro and preclinical experiments have been conducted utilizing extracellular vesicles produced from stem cells in this domain[6]. The first intravenous injection of MSC-derived exosomes was carried out in a rat stroke model in 2013, which was followed by functional recovery and neuronal remodeling. Exosomes may increase neurite remodeling, synaptic plasticity, neurogenesis, and angiogenesis on ischemic boundary zones in this rat model of stroke, according to Xin et al[50]. One study used a pig ischemic stroke model to show the ability of exosomes produced from human NSCs to facilitate recovery[51]. The neuroprotective capabilities of NSCs derived exosomes were shown to enhance behaviour and mobility in stroked pigs by eliminating intracranial hemorrhage and decreasing the volume of cerebral lesions and brain edema in comparison to control pigs. NSCs derived exosome can enhance functional, tissue and cellular recovery in a middle-aged thromboembolic stroke model via modulating the immune response. Exosomes administered intravenously improved white matter integrity in rats following subcortical stroke by promoting axonal sprouting, remyelination, and oligodendrogenesis[51].

Exosomes produced by stem cells have the ability to improve results in age-related disorders, such as Alzheimer’s and stroke, by targeting miRNAs that may be overexpressed. These miRNAs are involved in cellular and molecular processes such as cellular senescence, telomere length, and circadian rhythm[52]. Neuroprotective miRNAs such as, miR-184, miR-210, miR-133b, and the miR-17-92 cluster were altered by exosomes in multiple investigations[53,54]. In a rat model, injecting exosomes from miR133b-overexpressing MSCs into an occluded area of the middle cerebral artery improved neuronal plasticity and functional recovery by reducing phosphatase and tensin homolog gene levels and activating downstream phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling after glycogen synthase kinase-3β was inactivated[53]. The inhibitory effects on proliferation of immune cells including T, B, and natural killer cells have been demonstrated by these biomolecules, in addition to the neuroprotective and nerve regeneration properties of stem cell-derived exosomes. Stroke mice were shown to have caused post-ischemic neurological recovery with the use of bone marrow MSC exosomes[55]. Additionally, in a model of acute ischemic stroke, it was found that exosomes isolated from human placental MSCs that expressed angiotensin-converting enzyme 2 enhanced the neuroregenerative effects following a stroke. In addition to regaining neurological function, it is possible that these exosomes could mitigate the harmful effects of an ischemic stroke[56]. Likewise, an animal study indicated that exosomes produced by bone marrow MSCs improved neurological recovery by reducing leukocyte infiltration into the brain. This suggests that these exosomes may mitigate neurological impairments and protect against ischemia[57]. Another study discovered that MSC derived exosomes induce neurogenesis and angiogenesis through their endogenous cargo, specifically miRNA-210 and miRNA-184[58].

Deng et al[58] found that in a mouse model of middle cerebral artery blockage, higher levels of miR-126 in MSC exosomes can lower neuronal apoptosis and considerably raise levels of tumor necrosis factor α, interleukin (IL)-1β, and IL-6. Administration of exosomes produced from primary mouse NSCs or human iPSC-derived cardiomyocytes intravenously alleviates experimental stroke disease symptoms and lesions, such as reduced infarct volume. This was because these exosomes can maintain astrocyte function and have neuroprotective properties. The study was conducted in mice models with focal cerebral ischemia. A rat model of ischemic stroke with middle cerebral artery occlusion showed that MSC exosomes combined with rosuvastatin could decrease infarct volume, increase functional relief, and protect neurons from further damage[59].

In addition, small exosomes produced from human iPSC derived MSCs may control angiogenesis by blocking signal transducer and activator of transcription 3-dependent autophagy following an ischemic stroke in mice. The best time to administer exosomes following the start of the disease is an essential consideration for their usage in stroke treatment. However, research reveals that exosomes work best when administered shortly after a stroke begins. Intravenous administration of MSC-derived exosomes within the first twenty-four hours after a stroke may enhance functional recovery and neurite remodeling. Injection of these identical exosomes three days later reduces bleeding and BBB leakage while improving white matter remodeling[60]. Using both in vivo and in vitro models of ischemic stroke, Sun et al[61] assessed the anti-ischemic effects of two stem cell-derived exosome types, namely NSC. In vitro ischemic damage was caused by oxygen-glucose depletion in primary astrocyte or neuronal cultures of mice. Astrocytes exposed to oxygen-glucose depletion were considerably protected by exosomes generated from NSCs. The results show that stem cells based on a cell-free therapy method are effective in treating stroke-related disability[61].

Exosome in MS disease

MS is a chronic disease that occurs when the body’s immune system attacks the protective coating on nerve cells in the brain and spinal cord. This damage, called demyelination, can slow or stop nerve signals, which can lead to a variety of symptoms including vision problems, muscle numbness and weakness, loss of balance and coordination, and difficult walking. Inflammatory demyelination of the CNS’s grey and white matter is produced by MS, the most frequent non-traumatic neurological impairment in young people, especially women. Pathological indicators of this diverse disease include demyelination, inflammation, oligodendrocyte loss, reactive gliosis, axonal and neuron degeneration, and typical lesion alterations of the BBB. On the other hand, most people agree that the main method MS causes inflammation and degeneration is by activating pro-inflammatory type 1 T helper (Th1) cells in the periphery and targeting the myelin sheath in the CNS by penetrating the BBB. Neurological damage in MS can manifest in a variety of ways, although the condition is typically classified into four basic forms by the National Multiple Sclerosis Society: Relapsing-remitting MS, secondary progressive MS, and primary progressive MS. Secondary progressive MS develops in over 80% of MS patients from relapsing-remitting MS. Current treatments for MS mostly involve immunomodulatory and immunosuppressive meds, which increase the likelihood of infection and malignancy. In the 1990s, interferon-γ (IFN-γ) was used as a first-line medication in the treatment of MS, marking the beginning of alternative disease modifying treatments (DMTs)[6]. There are currently six MS drugs that have been approved by the FDA in parenteral formulations. These drugs include IFNs, immunosuppressants, corticosteroids, glatiramer acetate, sphingosine-1-phosphate receptor modulators, monoclonal antibodies, and sphingosine-1-phosphate receptor modulators. These drugs target the immune system at different levels and work in different ways to reduce the frequency and intensity of attacks in relapsing MS patients and slow down the disease progression. Although DMT medications have a positive effect on relapsing MS by reducing relapse frequency, they do not help much with progressive MS or axonal damage. Even in really effective situations, the danger of major side effects, such as cardiomyopathy, limits continuation of treatment, and DMT’s efficacy, tolerability, and safety range from moderate to high[62,63].

Stem cell therapy for MS is often called an immune reconstitution therapy since it removes the immune system to help it self-renew[64]. The production of HLA-G, which prevents the death of natural killer cells in reaction to IFN-γ, the primary inflammatory mediator linked to the development of MS, is the primary cause of the immunomodulatory actions of stem cells. Stem cells have anti-inflammatory and paracrine neuroprotective and properties which could halt the degeneration of neurons and axons[65]. Impaired regulatory T cell function, which is essential for modulating the Th cells balance (Th1/Th2), is a pathogenic feature of MS. Inducing T regulatory responses and switching from Th1 to Th2 is crucial for MSCs to function clinically in immune response modulation of autoimmune disorders like MS[66]. According to research conducted by Clark et al[67], exosomes produced from placenta-derived MSCs (PMSCs) can mimic the therapeutic effects of therapy alone in the experimental autoimmune encephalomyelitis (EAE) model when administered at high dosages. Proteomic analysis of PMSC-derived exosomes showed the presence of hepatocyte growth factor and vascular endothelial growth factor. Inducing the regulatory T cells, PMSCs controlled the immune system through production of high quantities of these substances. This finding demonstrated that in the EAE mouse model, PMSC-derived exosomes produced immunomodulatory responses that were on par with PMSC treatments and induced myelin regeneration[67]. Similarly, MSC-derived exosomes produced from human adipose tissue administered intravenously improve EAE scores by lowering pro-inflammatory cytokine production, blocking immune cell infiltration and suppression, and improving overall score[68].

Microglial polarization towards the M2 phenotype is another new approach to eliciting immunological tolerance in MS patients. One type of resident macrophage in the CNS is the microglia, which can be quickly activated by microenvironments to differentiate into either proinflammatory cytokines (M1) that cause damage to the CNS or anti-inflammatory cytokines (M2) that promote tissue regeneration. It has been postulated that a change towards a pro-inflammatory M1 phenotype and an imbalance of M1/M2 macrophages in the early stages of MS contribute significantly to CNS tissue damage. Therefore, it is thought that reducing neurological symptoms of MS patients may be possible by promoting microglial polarization towards the M2 phenotype[69]. Li et al[70] investigated the effects of bone marrow MSC paracrine mechanisms, namely exosome mediation, on microglial polarization and motor function improvement in an EAE mouse model. Researchers found that exosomes produced from bone marrow MSC altered microglia polarization towards an M2-phenotype reduced demyelination and inflammation of the CNS and improved neural behavioral scores in the EAE mouse model, after comparing the untreated and treated groups. They also demonstrated that MSC exosome therapy greatly decreased M1-associated cytokines (IL-12 and tumor necrosis factor-α) while increasing M2-associated cytokines, such as IL-10[70].

Because exosomes can pass the BBB, they can carry drugs to the brain of people with MS. In many instances, the target specificity of exosomes can be enhanced by attaching various functional groups, such aptamers and antibodies to their surface. One example is to combat MS using surface-functionalized MSC-derived exosomes with an anti-myelin aptamer (LJM-3064). Previous investigations have established that LJM-3064 has a high potential to protein myelin and induce remyelination, in addition to its capacity to traverse the BBB. Through the immunomodulation impact of the MSC exosome and the remyelination effects of the LJM-3064 aptamer, the results demonstrated that the co-delivery of these two agents reduced the severity of sickness symptoms in C57BL/6 mice. The exosome not only has an anti-inflammatory impact, but it also improves the remyelination effect of the LJM-3064 aptamer in this system. So far, these data suggest that stem cell generated exosomes will form the basis of future MS treatment strategies, due to their safety, capacity to penetrate the BBB, and drug cargo capacity[71].

LIMITATIONS AND FUTURE PERSPECTIVES

The FDA has not yet authorized any exosome products for use in humans. Exosomes have been the subject of several small-scale phase I clinical trials conducted by various academic institutions and hospitals in the past several years. Researchers have concentrated on finding solutions to the problems that arise during pharmaceutical production, such as issues with scalability, consistency from batch to batch, compliance with good manufacturing practices standards, formulation, storage, quality controls, market access, relative expenses, value for money, and overall expenditures. A significant challenge in the creation of therapeutic exosomes has been generating them at industrial scale, even though there are numerous examples of therapeutic exosomes at laboratory scale (Figure 4). In order to achieve uniformity from batch to batch, it is recommended that the cell source used to produce exosomes should be homogeneous. Since heterologous primary cells differ both within and across donors, this can be difficult to achieve. Thus, it will be beneficial to use exosomes in future clinical applications from a source that has a clearly defined identity, is homogeneous, stable, and scalable, which all contribute to optimal production and high potency. One potential approach is to use clonally pure human ESC derived progenitor cells to generate more exosomes. A manufacturing process that follows good manufacturing practices requires the development of product release criteria for the final exosome product, in-process testing, quality control release procedures, a standard operating procedure, and cell sources for large-scale production. Scalable processes integrating tangential flow filtration plus size-exclusion chromatography, nuclease treatment, sterile single-use systems, and standardized potency assays are now being validated for clinical-grade production. Future trials should harmonize dosing units (particle number vs protein mass), incorporate biodistribution readouts, and follow emerging regulatory guidance for exosomes-based biologics to ensure safety, reproducibility, and commercial scalability. When planning the clinical use of exosomes in the future, it is important to address toxicity and safety in stroke preclinical models. Potential short- and long-term toxicity, bioactivity, and the persistence of reported effects should be considered when assessing the safety of exosomes. Adverse events such stroke, respiratory distress, convulsions, and renal failure must be carefully studied in animals after exosome administration in order to do the necessary preclinical study prior to entering the clinic[72].

Figure 4 Limitations and future perspectives of exosomes. The figure represents the limitations and future perspectives of exosomes in therapeutic application of neurodegenerative diseases. FDA: Food and Drug Administration; GMP: Good manufacturing practices.

Exosome research is still in its infancy, but there are compelling arguments from other domains that point to the possibility that it may shed light on the processes and mechanisms behind mental diseases. The function of exosomes in cell-to-cell communication is the focus of current exosome research. The main translational barriers are delivery efficiency, product consistency, and regulatory readiness. Although exosomes naturally cross the BBB, systemic dosing still yields low brain accumulation; thus, next-generation approaches focus on engineered brain-targeting ligands, optimized cargo loading, and hybrid exosome platforms to enhance neural selectivity and stability. Intranasal administration has emerged as a scalable, patient-friendly route with strong nose-to-brain biodistribution and therapeutic benefit in AD and PD models. In parallel, focused ultrasound-mediated BBB opening enables region-specific enhancement of exosome uptake, offering a controllable adjunct for deep brain targets. The process of exosomes bidirectional transport via the BBB still needs more research. Future prospects should also center on exosomal psychiatry because patients may show peripheral physiological alterations in mental illnesses, exosomes extracted from the CNS have great biomarking potential[24].

Despite the recent increase in exosomal research, there is still a lot we do not know about these tiny extracellular vesicles and how they affect disease. Exosomes, for example, must be produced in greater yields in future clinical trials without compromising their therapeutic efficacy. Do exosomes from the same cell line have different diameters is an essential question to ask. Furthermore, it is critical to determine whether size variety affects exosome content. To understand how various exosome cargos, affect their therapeutic efficacy, further research is required. In addition, for exosomes to reach disease states after systemic delivery, it is essential to enhance their transportability to the target organ. Lastly, understanding exosome targeting and removal mechanisms is crucial[73].

Strong nose-to-brain biodistribution and therapeutic benefit in AD and PD models. In parallel, focused ultrasound-mediated BBB opening enables region-specific enhancement of exosome uptake, offering a controllable adjunct for deep brain targets. The process of exosomes bidirectional transport via the BBB still needs more research. Future prospects should also center on exosomal psychiatry because patients may show peripheral physiological alterations in mental illnesses, exosomes extracted from the CNS have great biomarking potential[24].

Despite the recent increase in exosomal research, there is still a lot we do not know about these tiny extracellular vesicles and how they affect disease. Exosomes, for example, must be produced in greater yields in future clinical trials without compromising their therapeutic efficacy. Do exosomes from the same cell line have different diameters is an essential question to ask. Furthermore, it is critical to determine whether size variety affects exosome content. To understand how various exosome cargos, affect their therapeutic efficacy, further research is required. In addition, for exosomes to reach disease states after systemic delivery, it is essential to enhance their transportability to the target organ. Lastly, understanding exosome targeting and removal mechanisms is crucial[73].

CONCLUSION

Neurodegenerative disorders are common and serious brain conditions that lack adequate treatment. One promising new approach to treating neurodegenerative illnesses is stem cell treatment, which has the potential to regenerate and restore function to damaged axons and myelin. However, stem cells therapy has certain limitations. One promising new approach to avoid these problems is the use of exosomes in the treatment of neurodegenerative diseases. The advantages of exosomes over cellular therapies include their lack of tumorigenicity, low immunogenicity, simplicity of isolation, and the possibility of “off the shelf” storage. There have been no reported cases of immune response or rejection with exosomes derived from stem cells, and they have a high safety profile in addition to therapeutic properties similar to their parent cells. However, a systematic approach to address the above-mentioned limitations and purify and characterize functional exosomes is necessary in order to fully harness the potential of exosomes. Exosome engineering for optimal neurological outcomes after neurodegenerative disorders may still be a mystery, but future research may shed light on the best ways to design such vehicles.