Mesenchymal stem cell-derived extracellular vesicles as next generation drug delivery platforms

Abstract

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have become potential next-generation drug delivery systems that combine the biological effectiveness of cell-based therapies with the reliability and controllability of acellular approaches. MSC-EVs are nanoscale vesicles enclosed by membranes that can carry proteins, lipids and nucleic acids, facilitating directed intercellular communication and therapeutic regulation. This review offers an extensive summary of the biological foundation of MSC-EVs, their advantages compared to traditional nanocarriers and the mechanisms that regulate targeted delivery to diseased microenvironments. We methodically examine present methods for therapeutic cargo loading, encompassing both passive and active techniques, genetic and biogenic modifications of parental mesenchymal stem cells, along with surface functionalization. Recent developments in hybrid and stimuli-responsive extracellular vesicle platforms that improve stability, targeting accuracy, and controlled release are also emphasized. Additionally, we outline preclinical and upcoming clinical uses of MSC-EVs in cancer, cardiovascular, ischemic and various other diseased conditions. Ultimately, we thoroughly assess the translational hurdles associated with large-scale production, standardization, storage, safety and regulatory processes while highlighting prospects for upcoming clinical advancements. In summary, this review highlights the transformative capability of MSC-EVs as adaptable and biologically smart drug delivery systems, while emphasizing the need for aligned technological, regulatory and clinical progress to facilitate their effective conversion into authorized therapies.

Key Words: Mesenchymal stem cell-derived extracellular vesicles; Regenerative medicine; Drug delivery; Disease therapy; Translational nanomedicine

Core Tip: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can be modified to transport therapeutic agents like small drug molecules, RNA or proteins via various loading techniques. Engineered MSC-EVs facilitate focused delivery to affected tissues such as tumors, cardiovascular damage, neurological issues, inflammatory conditions and wound areas through receptor-mediated absorption and microenvironment-responsive pathways. The inherent biocompatibility, targeting ability and adaptability of MSC-EVs establish them as potential platforms for precision drug delivery and regenerative therapies.

INTRODUCTION

Extracellular vesicles (EVs) are a heterogeneous group of nanoscale lipid-bound particles that are released by almost all cell types and act as key mediators of pathophysiological processes[1]. They have emerged as promising natural carriers for therapeutic delivery owing to their intrinsic role in intercellular communication and their ability to transport proteins, lipids and nucleic acids to respective recipient cells and tissues. EVs combine biological targeting motifs with lipid bilayer membrane that protects cargo from degradation and can traverse biological barriers that restrict many synthetic carriers, thereby making them potential candidates for next generation drug delivery systems[2,3]. Among all EV sources, those derived from mesenchymal stem cells (MSCs) i.e., MSCs-derived EVs (MSC-EVs) occupy a central position. MSCs are extensively studied for their regeneration and immunomodulatory roles in various therapeutics studies[4-6]. Most importantly, several therapeutic effects that were previously ascribed to MSCs are now understood to be mediated, at least to some extent, by their secreted EVs. MSC-EVs also inherit surface proteins, lipids and functional cargos from their parental cells. These features allow them to recapitulate effective MSC activities, including anti-inflammatory, pro-regenerative and anti-apoptotic activities[7,8]. Importantly, they achieve these effects while avoiding the potential risks associated with cell transplantation, such as ectopic engraftment or malignant transformation. Due to these properties, MSC-EVs serve dual functions, acting as natural therapeutic agents and as engineerable vehicles for the delivery of small molecules, RNAs, proteins and even gene-editing cargos[9-11].

Compared with already established nanocarriers like liposomes and polymeric nanoparticles, MSC-EVs have emerged as biologically derived delivery systems with great potential to interact with recipient cells via native membrane proteins and signaling molecules. Rather than acting as pure synthetic carriers, MSC-EVs integrate biological functionality with delivery capacity, allowing for the modulation of immune responses, tissue repair, and intercellular signaling[12,13]. These features position MSC-EVs as promising candidates for next-generation drug delivery. Detailed comparisons with conventional nanocarriers are discussed in later sections of this review.

MSC-EVs can be obtained from clinically relevant cell sources like bone marrow, adipose tissue, and umbilical cord, and can be engineered at either the parental cell level or after isolation to optimize cargo delivery and therapeutic targeting[14,15]. Despite their promise, the clinical translation of MSC-EVs needs addressing key challenges related to scalability, reproducibility and regulatory standardization[16,17]. Methodological advances in MSC-EV technology, spanning sophisticated isolation techniques, surface modification, and enhanced payload loading, have accelerated preclinical validation across cardiovascular, neurodegenerative, inflammatory and cancer models. Consequently, pioneering clinical trials are currently underway to assess the safety, feasibility and therapeutic potential of these agents[18,19]. Also, while promising, these early translational efforts also highlight the remaining gaps that include the scalable good manufacturing practice (GMP) processes, validate potency biomarkers and carefully designed clinical endpoints that are tailored to EV-specific mechanisms of action. A systematic assessment of biological rationale, engineering strategies and methodology, comparative advantages, as well as translational obstructions is timely to guide and manage the development of MSC-EVs as a reliable, next generation drug delivery system[3,20].

This review consolidates existing evidence on MSC-EV biology and engineering, assesses cargo- loading and targeting techniques, juxtaposes MSC-EV platforms with traditional nanocarriers and delineates regulatory, manufacturing and clinical routes necessary to achieve their therapeutic promise. By outlining existing achievements and significant challenges, we seek to provide a pragmatic guide for researchers striving to translate MSC-EVs into clinically significant drug delivery systems. In accordance with current EV research guidelines, specifically the Minimal Information for Studies of Extracellular Vesicles 2018 recommendations, terminology in this review has been standardized to maintain clarity and precision[21]. The term EV is broadly applied unless particular subtypes like exosomes or small EVs are specifically identified according to recognized characterization standards, such as size distribution, biogenesis mechanisms, or molecular markers. In cases where the literature reviewed lacks adequate subtype details, results are presented with the general term EVs to prevent exaggerating conclusions specific to subtypes.

BIOLOGICAL BASIS OF MSC-EVS

MSC-EVs are membrane-bound nanoparticles derived from MSCs that facilitate intercellular communication and recapitulate many paracrine effects of their parental cells[22,23]. Understanding the biological composition, intrinsic activities and in vivo behavior of MSC-EVs is necessary, especially when considering them as drug delivery platforms, as their endogenous cargo, surface molecules and biodistribution collectively determine safety, targeting and therapeutic outcomes[10,24].

Characteristics and composition of MSC-EVs

MSC-EVs possess structural and molecular characteristics that directly impact their effectiveness as therapeutic delivery systems. These vesicles usually vary from 30-1000 nm in size, including subtypes like exosomes (approximately 30-150 nm), microvesicles (100-1000 nm) and apoptotic bodies (> 1000 nm) that have distinct biogenesis and functional characteristics[25-27]. The size distribution of EV populations is crucial for drug delivery considerations, as it affects biodistribution, efficiency of cellular uptake, and capacity for cargo loading. At the molecular scale, MSC-EVs comprise proteins, lipids, and nucleic acids that facilitate intercellular communication and therapeutic signaling. Typical surface proteins like tetraspanins (CD9, CD63, CD81) and adhesion molecules aid in recognition by recipient cells and improve internalization efficacy. Lipid elements such as sphingolipids and phosphatidylserine (PS) enhance membrane stability and protect the enclosed therapeutic agents from enzymatic breakdown during circulation[10,28-31]. Crucially, the composition of MSC-EVs directly influences their drug delivery efficacy, encompassing targeting ability, circulation longevity, and intracellular release of therapeutic agents. Grasping these characteristics is crucial for enhancing MSC-EV design for practical drug delivery purposes[15]. Collectively, these features provide the structural and functional basis for engineering MSC-EVs as therapeutic carriers. Therefore, it is important to understand EV biology for their application in drug delivery optimization and clinical translation.

Advantages over conventional nanocarriers

MSC-EVs possess various inherent characteristics that make them good candidates as next-generation drug carriers compared with numerous traditional synthetic systems, such as liposomes, polymeric nanoparticles, and lipid nanoparticles[15,31]. A major advantage is their natural biocompatibility, as EVs originate from endogenous cellular membranes, thereby minimizing immunogenicity and reducing toxicity compared to synthetic materials[32,33]. Another important characteristic is the presence of biological targeting and membrane proteins that include integrins and adhesion molecules, allowing biological recognition and enhanced cellular uptake. These membrane-associated molecules facilitate receptor-mediated internalization, which further helps improve intracellular delivery efficiency[34,35]. MSC-EVs also showed cargo protection and functional transfer ability, as their lipid bilayer protects nucleic acids and proteins from enzymatic degradation in circulation. This advantage is particularly important for RNA-based therapeutics where stability remains a critical limitation of synthetic systems[36,37]. Additionally, EVs demonstrate an ability to traverse biological barriers, including endothelial and epithelial barriers, which remain a major challenge for many synthetic nanocarriers. Studies have reported improved transport across physiological barriers like the blood-brain barrier when using EV-based systems[38-41]. These attributes can translate into better and improved safety and efficacy for certain payloads, for instance, nucleic acids and proteins, when harnessed in a proper way. However, these advantages are accompanied by limitations. EV heterogeneity, limited native loading capacity for certain drugs, and challenges in purification or isolation scalability continue to pose major restrictions compared with highly regulated synthetic platforms, which can achieve high yield with narrow polydispersity and precise surface chemistry. Consequently, the prevailing agreement is that EVs and synthetic nanoparticles are complementary to each other. EVs may be preferable in cases that involve biological signaling, immune evasion or barrier traversal, while synthetic carriers retain the benefits for efficient high-dose small-molecule delivery and consistent manufacturing quality[42-44].

Quantitative comparison with synthetic nanocarriers

While MSC-EVs show numerous qualitative advantages compared to synthetic nanocarriers, quantitative assessments are vital to support assertions concerning enhanced targeting and decreased immunogenicity. Various direct biodistribution studies using fluorescently labeled EVs have demonstrated multi-fold higher accumulation (approximately 3-5-fold) in specific organs and tumor tissues, depending on the cellular origin and disease model. This preferential localization has been attributed to integrin-mediated recognition and enhanced cellular uptake mechanisms that regulate organ-specific targeting and adhesion processes[45,46].

In murine models, the delivery of small interfering RNA (siRNA) via EVs resulted in quantifiable gene knockdown in brain tissues after systemic administration, while similar synthetic carriers showed notably lower efficiency in crossing the blood-brain barrier[47]. Immunogenicity comparisons indicate that EV-based systems typically trigger less complement activation and cytokine release than synthetic nanoparticles with polyethylene glycol (PEG). In controlled in vivo studies, EVs prompted slight elevations in pro-inflammatory cytokines like tumor necrosis factor α and interleukin-6 (IL-6) compared to synthetic nanoparticle formulations given at the same doses[15,48].

Nonetheless, it is crucial to highlight that synthetic nanocarriers often surpass EVs in their ability to load drugs, with polymeric nanoparticles and liposomes typically reaching loading efficiencies of over 60%-90%, compared with typical EV loading efficiencies, which vary from 5%-30% based on method and cargo type[49,50]. These results emphasize that although MSC-EVs possess distinct biological benefits, their effectiveness is context-specific and should be assessed in relation to therapeutic goals.

Biodistribution properties

After systematic administration, MSC-EVs generally show a tendency to concentrate in organs linked to the mononuclear phagocyte system, especially the liver, spleen and lungs. This distribution pattern is similar to other various nanoparticle systems and is affected by opsonization processes, along with quick absorption by local macrophages[51,52]. Biodistribution is heavily impacted by various experimental and biological factors like the administration route, EV surface composition, labeling technique used, and the physiological condition of the recipient cells or tissues[53,54]. Lipophilic dye-based labeling methods can create artifacts by transferring dye molecules to non-vesicular elements, emphasizing the need for thorough methodological validation[53,55]. Diseases conditions like inflammation, isolation, or tumor development can change biodistribution patterns by impacting vascular permeability and tissue accessibility. Disease-associated alterations establish the groundwork for increased EV accumulation in damaged or inflamed tissues, providing the physiological basis for therapeutic targeting strategies explored in subsequent sections[13,56-58]. The biogenesis pathways and cargo composition of MSC-EVs and their functional consequences are illustrated in Figure 1.

Figure 1 Biogenesis and composition of mesenchymal stem cell-derived extracellular vesicles. A: Extracellular vesicle biogenesis pathways showing the formation of exosomes via multivesicular bodies and microvesicles via plasma membrane budding; B: Major cargo components which together influence signaling and gene expression in recipient cells; C: Functional consequences relevant to therapeutic delivery. EV: Extracellular vesicle; MVBs: Multivesicular bodies; ILVs: Intraluminal vesicles; ESCRT: Endosomal sorting complex required for transport; miRNA: MicroRNA.

STRATEGIES FOR THERAPEUTIC CARGO LOADING

A pivotal technical barrier to clinical translation is efficient and reproducible loading of therapeutic cargos into MSC-EVs[59,60]. Loading therapeutic payloads into MSC-EVs needs to balance three competing aims: High cargo payload per vesicle, preservation of EV structural and functional integrity, and process scalability and reproducibility. Contemporary approaches fall into four practical categories: (1) Passive exogenous approaches; (2) Active physical/chemical methods; (3) Genetic and biogenic engineering of parent MSCs; and (4) Surface level functionalization that indirectly influences delivery and retention processes[61-63].

Passive loading approaches

Passive loading methods primarily rely on spontaneous portioning of cargo into EV membranes or lumens and are most effective for the small hydrophobic molecules. Incubation of EVs with drugs is the simplest approach but produces low encapsulation. A comparative experimental study reported incubation efficiencies of approximately 4.9% ± 2% for model cargos (incubation) vs higher and better efficiencies for active methods. Lipophilic drugs, such as paclitaxel and doxorubicin, partition better than the hydrophilic molecules; but even for these, passive incubation rarely exceeds the single digit percent encapsulation without an auxiliary treatment approach[49,60,64-66]. Moreover, detergent-assisted permeabilization, such as saponin, increases passive uptake by transiently forming pores in the EV bilayer[49,67]. In addition, saponin treatment has been reported to enhance loading up to ~11-fold over passive incubation for certain small hydrophilic cargos, but careful removal of residual detergent is important. Furthermore, there is a risk of membrane alteration and hemolytic toxicity if not adequately purified[60,68]. Passive methods are widely used for hydrophobic drugs and proof-of-concept research, as these methods preserve EV surface proteins and demand minimal equipment. However, low loading yields limit their utility for potent but high-dose small-molecules, as well as for nucleic acid-based therapeutics that require higher copy numbers per vesicle[50].

Active loading techniques

Active or post-isolation loading techniques generally use physical forces or chemical treatment to transiently disrupt EV membranes and force cargo into the lumen region, typically achieving substantially greater encapsulation than the passive incubation approach[69,70]. A systematic comparison found the following mean encapsulation efficiencies for model small molecules: Sonication ~26.1% ± 2%, extrusion ~22.2% ± 2%, freeze-thaw ~14.7% ± 1.1% and incubation ~4.9% ± 2%. Sonication and extrusion reliably achieve the greatest loading for small drugs, yet there is a risk of modifying EV size distribution, surface protein composition and zeta potential, potentially altering biodistribution and biological activity[49,65]. Electroporation is commonly utilized for loading nucleic acids like siRNA/microRNA (miRNA), as it temporarily disrupts membranes, thereby facilitating the entry of polyanionic substances into the vesicles[47,71]. Nevertheless, thorough methodological investigations revealed that electroporation often leads to siRNA aggregation, causing misleading co-sedimentation with EV fractions and producing inflated loading values[72,73]. The quantitative re-analysis suggested much lower actual encapsulation than previously reported. As a result, electroporation suggests an urgency of optimized buffers, controls for nucleases and additional quantification methods, such as RNase protection assays and density gradients, to verify genuine luminal loading[72]. Sonication-loaded exosomes containing paclitaxel demonstrate the functional improvements from active loading approaches. Sonication achieved significant payload recovery, and exosomes containing paclitaxel showed over 50-fold greater cytotoxicity in drug-resistant cell lines expressing P-glycoprotein compared with the free drug, due to the effective intracellular delivery and evasion of the efflux pumps[74]. These functional enhancements indicate that increased encapsulation along with suitable vesicle characteristics can lead to significant pharmacodynamic improvements.

Genetic and biogenic engineering of parent MSCs

Biogenic approaches leverage sorting of cells to encapsulate cargo during EV formation and represents the most dependable method to acquire EVs with enhanced nucleic acid and protein loads per vesicle. Two prevalent strategies are: (1) Enhancing the expression of target RNA/protein in parental MSCs; and (2) Sorting on the basis of fusion proteins[60,75]. For instance, MSCs infused with miR-126 mimics produced exosomes that contain extremely high levels of miR-126, which facilitated angiogenic and neuroprotective effects both in vitro and in vivo; research highlights a several-fold increase in target miRNA with EVs compared with naïve cells, which leads to related functional consequences[76,77]. In addition, biogenic methods generally produce significantly greater copy numbers per vesicle for RNAs as compared with those obtained by external post-isolation techniques, while also preventing aggregation artifacts during the electroporation process[50,78]. Limitations can include the requirement for a reliable expression system, potential regulatory complexity when using genetically modified cells, and batch variability related to cell culture conditions.

Surface engineering and ligand functionalization

Surface engineering does not directly transport cargo but significantly influences delivery efficiency by altering circulation half-life, cellular absorption and organ targeting[53,79]. Two most common employed strategies include post-isolation chemical conjugation (lipid-PEG-ligand attachment or click chemistry) in order to link targeting peptides or antibodies and parental cell modification to exhibit targeting components or stealth signals like CD47 on released EVs[80,81]. PEGylation or lipid-PEG incorporation can diminish rapid hepatic clearance and enhance tumor retention in pre-clinical imaging studies, whereas the parental expression of CD47 or other “don’t eat me” signals has been noted to prolong circulation time by nearly threefold in certain animal models[81,82]. Nevertheless, surface alterations must maintain EV functional proteins that are essential for uptake; also, excessive modification might obscure natural targeting motifs or trigger the adaptive immune responses after multiple administrations[65,83]. The major engineering approaches for therapeutic cargo loading using MSC-EVs are schematically depicted in Figure 2.

Figure 2 Engineering strategies for therapeutic cargo loading in mesenchymal stem cell-derived extracellular vesicles. A: Passive loading methods allow diffusion of small hydrophobic drugs into minimal structure disruption; B: Active loading techniques enhance cargo encapsulation through transient membrane permeabilization; C: Genetic and biogenic modification of parental mesenchymal stem cells allow selective incorporation of therapeutic molecules during extracellular vesicle formation; D: Surface engineering using targeting ligands, polyethylene glycol chains, or antibodies improve circulation duration, targeting precision, cellular uptake and tissue specific delivery. EV: Extracellular vesicle; siRNA: Small interfering RNA; MSC: Mesenchymal stem cell; PEG: Polyethylene glycol.

Comparative evaluation of cargo-loading strategies

While various cargo-loading methods have been developed for MSC-EVs, their practical effectiveness differs greatly based on therapeutic application and translational viability. A comparative evaluation of these strategies emphasizes important trade-offs among loading efficiency, vesicle integrity, scalability, and regulatory compatibility[59,60]. Passive loading techniques, such as drug incorporation through incubation, are commonly used because of their ease of use and ability to maintain EV structural integrity. Nevertheless, these methods generally produce low encapsulation efficiencies, frequently under 10%, restricting their use for high-dose therapies or nucleic acid transport[49,50]. Active loading techniques like electroporation, sonication, extrusion, and freeze-thaw cycles result in significantly improved encapsulation efficiencies, typically between 10%-30%, influenced by cargo type and processing conditions[49,67]. Even with enhanced loading performance, these techniques can change vesicle shape, disturb membrane proteins, and create aggregation artifacts, especially in nucleic acid delivery applications[72,84]. The genetic and biogenic modification of parental MSCs represents one of the most effective methods for loading nucleic acids and proteins into EVs, as cargo molecules are integrated during the vesicle formation process. This approach provides enhanced cargo specificity and biological compatibility yet adds extra regulatory complexity, owing to the genetic alteration of source cells[50,85]. Surface engineering approaches boost delivery effectiveness by optimizing circulation duration and tissue specificity instead of augmenting the cargo volume itself. While these methods show enhanced targeting effectiveness in preclinical models, their clinical viability relies on preserving EV stability and preventing unintended immune activation[86]. From a translational perspective, genetic engineering and hybrid loading techniques seem the most promising for nucleic acid delivery, while passive loading methods continue to be effective for small hydrophobic drugs. Nevertheless, no individual approach presently satisfies all clinical needs, and hybrid methods that integrate various techniques are being investigated more frequently to enhance therapeutic efficacy. Table 1 provides a comparative summary of MSC-EV cargo loading strategies.

Table 1 Comparative summary of mesenchymal stem cell-derived extracellular vesicle cargo loading strategies.

Loading strategy	Typical efficiency	Advantages	Limitations	Clinical feasibility	Representative applications	Ref.
Passive loading (incubation)	Approximately 5%-10%	Simple, preserves EV integrity	Low loading efficiency	Moderate (small molecules)	Paclitaxel loading	[49]
Electroporation	Approximately 15%-25%	Effective for nucleic acids	RNA aggregation risk	Moderate	siRNA delivery	[72]
Sonication	Approximately 20%-30%	High encapsulation efficiency	Membrane damage risk	Moderate	Chemotherapy drugs	[49]
Extrusion	Approximately 15%-25%	Uniform vesicle formation	Structural alteration	Moderate	Drug loading	[84]
Genetic engineering	High cargo specificity	Stable loading	Regulatory complexity	High potential	miRNA delivery	[85]
Surface functionalization	Not direct loading	Improved targeting	Immune response risk	High potential	Targeted therapy	[83]

EV: Extracellular vesicle; siRNA: Small interfering RNA; miRNA: MicroRNA.

Alongside direct cargo loading and surface level functionalization approaches, new technologies are increasingly aimed at combining MSC-EVs with synthetic material and responsive systems to improve delivery effectiveness. These sophisticated engineering platforms like hybrid nanovesicles, scaffolds made from biomaterials, and stimuli responsive EV systems, enhance the functional abilities of traditional EV engineering methods, and increase cargo capacity, stability and regulated release characteristics[48,87]. The subsequent section examines sophisticated hybrid and engineered EV platforms that signify the forthcoming phase in the development of MSC-EV based delivery technologies.

ADVANCED HYBRID AND ENGINEERED EV PLATFORMS

The engineering techniques outlined in the previous section primarily emphasize cargo loading and surface modification of native EVs, but recent advances have broadened these methods to include multicomponent and responsive delivery systems. These advanced systems seek to address the fundamental constraints of native EVs, including limited cargo capacity, rapid systemic clearance, and restricted control over release kinetics[87-89]. Hybrid and engineered EV platforms combine biological vesicles with synthetic nanomaterials, biomaterials, or externally responsive components to improve structural stability, targeting accuracy, and therapeutic effectiveness. These methods signify a logical advancement from fundamental EV alteration techniques to versatile delivery systems that can meet intricate therapeutic needs[15,48]. These platforms consist of membrane-fused EV-liposome hybrids, biomaterial-integrated EV reservoirs, and responsive delivery systems that facilitate controlled and localized therapeutic release[87,90]. Collectively, these advanced systems demonstrate the evolution of MSC-EV engineering from single-modification approaches to comprehensive therapeutic platforms, offering enhanced flexibility for clinical applications and specific disease uses.

Although advanced engineering techniques greatly enhance the efficiency and stability of cargo delivery, the overall therapeutic effectiveness ultimately relies on the capacity of engineered MSC-EVs to specifically engage with intended tissues. Thus, understanding the biological processes that control EV targeting and cellular absorption is essential for translating engineered delivery systems into clinically effective treatments. The next section explores the molecular mechanisms that facilitate targeted delivery.

EV-liposome hybrid nanovesicles

Membrane-fusion-based EV-liposome hybrids, also known as membrane-fused-based hybrid exosomes are formed by combining EV membranes with synthetic liposomes, resulting in vesicles that maintain and regulate EV surface proteins while providing benefits from the advantageous payload potential and versatile surface chemistry of liposomes[90]. There are several fabrication techniques available; incubating directly at physiological temperature, such as 37 °C, for about 6-12 hours can achieve high fusion efficiency with relatively low processing complexity. Method papers reporting fusion yields usually indicate a change in average particle size approximately 10-40 nm based on initial materials along with the presence of EV markers and liposomal lipids, confirmed by western blot and lipid tracer co-localization[91,92].

Quantitatively, hybrids assembled through sonication or extrusion have shown greater drug loading compared with native EVs. Lv et al[93] developed a thermosensitive exosome-liposome hybrid that simultaneously delivered docetaxel and granulocyte-macrophage colony-stimulating factor; these hybrids exhibited better drug encapsulation and a significantly improved therapeutic index in metastatic peritoneal cancer models as compared to either parent vehicle, resulting in notable tumor suppression and survival advantage in a mouse model. Likewise, Xie et al’s[94] study on exosome-liposome hybrids showed enhanced stability and targeting. These hybrids maintain exosomal surface proteins and allow for the inclusion of cationic or PEGylated lipids to adjust charge and circulation duration, resulting in greater tumor accumulation in rodent models as compared to naïve exosomes or liposomes by themselves. These studies often indicate multiple times higher efficacy in payload delivery rather than a single universal encapsulation rate as efficiency significantly varies based on liposome composition and fusion approaches[94]. Translationally, EV-liposome hybrids provide two major advantages: (1) Adjustable surface chemistry to optimize pharmacokinetic nature; and (2) Scalable liposome production processes that can be integrated with EV isolation. Limitations encompass possible dilution of EV-specific surface markers and batch heterogeneity that are caused by fusion and regulatory intricacies associated with merging biological and synthetic elements[91,95].

EV-biomaterial and hydrogel integration

The integration of MSC-EVs into biomaterial scaffolds or hydrogels is a progressively validated method to facilitate localized, prolonged release and to spatially restrict EV effects for the tissue regeneration process. Crosslinked hydrogels like gelatin methacrylate, chitosan and genipin-crosslinked matrices serve as reservoirs that hinder diffusion and protect EVs from rapid removal; documented release profiles differ based on mesh size and crosslink density but generally prolong EV availability from hours to days or even weeks[12,87]. For instance, exosome hydrogels crosslinked with genipin in skin wound models showed prolonged local release of exosomes and enhanced healing parameters. Also, injectable hydrogels have demonstrated total exosome release that lasts for 7-14 days, in contrast to over 70% release within 24 hours for free exosomes in solution[96,97]. Moreover, hydrogels facilitate multimodal approaches. The addition of inorganic fillers, adhesive peptides or enzymatically degradable linkers allows for on-demand release that responds to the local microenvironment[98,99]. Multiple preclinical studies on bone, cartilages and skin regeneration show statistically significant enhancements in angiogenesis, collagen deposition and functional recovery compared to standard bolus EV injections. In clinical settings, injectable in situ-forming hydrogel systems is very attractive, as they allow single-site administration with prolonged local dosing and minimized systemic exposure[100,101].

Stimuli-responsive and smart EV systems

‘Smart’ EV systems incorporate internal (pH, redox, enzyme) or external (heat, light, magnetic field, ultrasound) responsiveness to manage and regulate cargo release, improve endosomal escape or enable site-targeted accumulation[15,48]. Strategies encompass: (1) Embellishing EVs or hybrids with thermo-sensitive lipids that experience phase transition during mild hyperthermia; (2) Magnetic tagging facilitating magnetic targeting and magnetically induced heating; and (3) Enzyme-cleavable linkers incorporated in hydrogel matrices that preferentially release EVs in protease-abundant injured tissues[93,102]. The thermosensitive hybrid developed by Lv et al[93] exemplifies a situation where mild hyperthermia prompted the release of payload and combined with CD47 expression to lower clearance and enhanced tumor uptake and effectiveness in vivo. Quantitative improvements for stimuli-responsive systems vary with context: Thermally activated hybrids can produce over twofold increases in local drug levels after triggering, as compared to untriggered controls, while magnetic guidance research frequently indicates multiple-fold enhancements in local accumulation relative to passive delivery[103,104]. Nevertheless, integrating stimuli responsiveness adds manufacturing complexities and regulatory barriers. Therefore, these systems are presently best viewed as targeted solutions for high-required indications[93,102]. Table 2 summarizes the major engineering strategies, hybridization and translational progress in drug delivery systems utilizing MSC-EVs[9,97,105-117].

Table 2 Mesenchymal stem cell-derived extracellular vesicles as platforms for drug delivery.

Engineering/delivery strategy	Therapeutic cargo	Target disease/application	Delivery route	Major advantages over synthetic nanoparticles	Challenges	Ref.
Native MSC-EV therapy	Endogenous miRNAs, proteins	Cardiovascular, renal, liver injury	Intravenous	Natural biocompatibility. Low immunogenicity	Biodistribution to liver and spleen	[9,105]
Surface engineered exosomes	Targeting ligands	Tissue-specific targeting	Systemic	Enhanced homing and target specificity	Manufacturing scalability	[106,107]
CRISPR/Cas9 delivery via MSC-EVs	CRISPR/Cas9 gene-editing components	Gene editing applications	Experimental systemic delivery	Ability to cross biological barriers	Cargo loading efficiency	[108,109]
KRAS G12D siRNA-loaded exosomes (iExosomes)	siRNA	Metastatic pancreatic cancer	Intravenous (clinical trial)	Tumor-specific gene silencing	Standardization and dose control	NCT03608631[110]
MSC-exosomes loaded hydrogel	Exosomes-containing growth factors	Full thickness cutaneous wound healing and skin regeneration	Local/topical application	Sustained release, improved stability and retention enhanced wound closure, angiogenesis	Direct injection of exosomes, need for suitable carrier system, limited prior studies on hydrogel encapsulated exosomes	[97]
Bioreactor-based MSC culture	Bulk EV production	Clinical translation	GMP-scale	Increased yield	Phenotypic variability	[111,112]
MSC-EV spray formulation	Regenerative factors	Myocardial infarction (large animals)	Topical/local cardiac	Minimally invasive administration	Stability and storage validation	[113-117]
MSC-EVs vs synthetic nanoparticles comparison	Endogenous therapeutic cargo	Broad therapeutic use	Multiple routes	Reduced toxicity, better barrier penetration	Heterogeneity and pharmacokinetics	[108]

MSC-EV: Mesenchymal stem cell-derived extracellular vesicle; miRNA: MicroRNA; siRNA: Small interfering RNA; GMP: Good manufacturing practice.

Despite the notable enhancement in delivery efficiency and cargo stability offered by advanced engineering platforms, the therapeutic effectiveness still hinges on accurate interactions between EVs and target tissues. Grasping how designed EV systems identify, localize and function within specific microenvironments is consequently crucial. The next section addresses the molecular mechanisms that regulate EV targeting, such as receptor-ligand interactions, tissue homing signals, and processes that modulate the microenvironment.

MECHANISMS OF TARGETED DELIVERY

Targeted delivery by MSC-EVs is regulated by a combination of passive pathophysiological cues and active molecular interactions with recipient tissues[53,54]. In contrast to inert nanocarriers, MSC-EVs utilize intrinsic homing signals, receptor-ligand interactions, and immunomodulatory feedback mechanisms to attain selective accumulation and effective activity in diseased microenvironments, especially in inflamed and tumor tissues[46,118,119]. The targeted mechanisms described in this section apply to both native and engineered EV platforms discussed in previous sections.

EV targeting of inflamed or tumor tissues involves multiple stages influenced by: (1) Chemotactic gradients; (2) Vascular adhesion/permeability; and (3) Specific surface interactions with local stromal or immune cells[120]. Chemokine and chemokine receptor pairs, particularly C-X-C motif ligand 12/C-X-C receptor 4 are very important for the movement of leukocytes and MSCs, and inflamed tissues increase C-X-C motif ligand 12 and similar signals, which may also boost EV retention and functional absorption at sites of injury[121,122]. Tissue inflammation boosts endothelial permeability and the adhesion molecule expression, such as vascular adhesion molecule-1 and intercellular adhesion molecule-1, thereby promoting EV extravasation and local tumor buildup. Furthermore, tumor-derived EVs take advantage of this by promoting vascular permeability, thereby generating a positive feedback loop for niche conditioning[45,123]. The quantitative data suggests that the modification of certain EV surface features impacts the homing. Hoshino et al[45] demonstrated that silencing of exosomal integrin beta4 decreased lung uptake by over 3-fold 24 hours post-systemic injection and likewise diminished lung metastasis in mouse models, highlighting the casual involvement of exosomal integrins in organ targeting.

Receptor-ligand interactions

Direct docking and uptake of EVs entail a complex “surfaceome” composed of tetraspanins, integrins, immunoglobulin superfamily membranes, extracellular matrix (ECM) proteins and proteoglycans that interact with specific receptors on recipient cells[124,125]. PS present on the exofacial leaflets of EVs serve as an important recognition marker. PS is recognized by T-cell immunoglobulin and mucin domain-containing molecule 4, stabilin-2 and RAGE on phagocytes and endothelial cells, whereas the PS-bridging proteins like milk fat globule-epidermal growth factor 8/growth arrest-specific gene 6 connect EVs to integrins or TAM receptors to facilitate uptake and anti-inflammatory signaling[126,127]. Heparan sulfate proteoglycans facilitate the internalization of various types of EVs, experimentally inhibiting heparan sulfate proteoglycans or competing with heparin reduces EV absorption in vitro blood brain barrier models, thereby emphasizing the significance of glycan-lectin interactions for tropism and transcytosis[72]. Integrin-ECM recognition additionally influences tissues specificity: Tumor exosomal integrins α₆β₄/α₆β₁ relate to lung tropism while α_vβ₅ is linked with liver tropism, and blocking integrins reduces both EV uptake and subsequent niche conditioning[45].

Immune modulation and microenvironment remodeling

In addition to the simplest delivery, MSC-EVs actively reshape target microenvironments by transporting bioactive cargo and by interacting with surface receptors to influence immune cell characteristics[24,128]. MSC-EVs promote anti-inflammatory macrophage polarization through various mechanisms, like the transfer of miR-223/miR-146a, the induction of adenosine production that signals through A2A/A2B receptors, and the delivery of enzymes or proteins that activate signal transducers and activators of transcription 3 and additional anti-inflammatory pathways; research shows consistent increases in IL-10 and decreases in tumor necrosis factor α/IL-6 following EV treatment across several models[129,130]. MSC-EVs can modify the ECM and stromal behavior by providing matrix-modifying enzymes or fibronectin/integrin complexes that alter cell adhesion and attract bone marrow-derived cells, thus reinforcing a reparative niche; multiple in vivo models indicate enhanced angiogenesis and collagen deposition post-EV administration compared to controls[126,131]. Therapeutically, these synergistic actions (targeting and immunomodulation) allow MSC-EVs to preferentially accumulate in diseased areas and transform hostile inflammatory environments into favorable, pro-repair conditions, an effect consistently shown in models of lung injury, myocardial infarction and wound healing[128,132].

A comprehensive understanding of targeting mechanisms establishes the basis for designing MSC-EV-based treatments tailored for particular disease contexts. These mechanistic understandings have facilitated the development of treatment approaches for a variety of pathological conditions. The next section emphasizes the key therapeutic uses of MSC-EVs validated in preclinical and clinical research.

THERAPEUTIC APPLICATIONS

MSC-EVs have been extensively studied in various disease models due to their ability to deliver cargo while also possessing inherent immunomodulatory and regenerative properties[17,133]. The following section will highlight the most compelling preclinical evidence and new clinical findings.

Cancer therapy

MSC-EVs have been assessed for their roles as natural regulators of tumor dynamics and as modified agents for drug delivery[87,134]. MSC-derived exosomes loaded with paclitaxel showed significantly improved anti-tumor effects in models of multidrug-resistant cancer[135,136]. A previous study found that paclitaxel enclosed in exosomes boosted cytotoxicity in resistant cells expressing P-glycoprotein by over 50 times compared to the free drug, ascribed to improved intracellular accumulation and evasion of efflux pumps[74,136]. Exosome-mediated siRNA transport to tumors has shown effective gene-silencing in vivo. Studies have revealed that specifically directed exosomes carried siRNA throughout the body, resulting in quantifiable gene suppression in murine tissues, offering proof-of-concept for the transport of nucleic acids across biological barriers[47]. Moreover, MSC-EVs designed to transport tumor-suppressive miRNAs have shown anti-cancer effects. For instance, miR-124-enriched exosomes decreased glioma growth and migration by inhibiting the signal transducers and activators of transcription 3 pathway, resulting in a notable decrease in tumor volume in xenograft models[137,138]. Alternative approaches utilize MSC-derived exosome-mimetics or co-culture to generate paclitaxel-encapsulated vesicles that diminish tumor load in orthotopic models[139].

Ischemic and cardiovascular diseases

Preclinical research consistently demonstrated that MSC-EVs decrease infarct size, reduce apoptosis and enhance cardiac functions following myocardial ischemic reperfusion in both rodent and large animals. Various groups attribute advantages to EV-derived miRNAs, such as miR-21, miR-210 and miR-126, and pro-angiogenic proteins[140,141]. For instance, EVs rich in miR-21 reduced cardiomyocyte apoptosis and decreased infarct size in mouse models, whereas MSCs overexpressing protein kinase B released EVs that promoted angiogenesis and enhanced the ejection fraction post-myocardial infarction[142,143]. Quantitatively, several rodent studies demonstrated a 20%-40% decrease in infarct size and corresponding enhancement in left ventricular fraction shortening or ejection fraction compared with controls at 2-4 weeks after treatment, although the effect size differs based on model, dosage and administration routes[142]. The mechanistic outcomes consist of reduced cleaved caspase-3, enhanced vacuum densities and changes in inflammatory cell infiltrates. Also, EV hydrogel delivery has prolonged local retention and amplified functional recovery in numerous studies[12,144]. These promising clinical results have inspired initial translational strategies. However, the clinical trials in cardiovascular groups are still limited and need uniform potential tests and scalable EV production[145].

Inflammatory and autoimmune disorders

MSC-EVs exhibit strong immunomodulatory effects in several inflammatory disease models like colitis, rheumatoid arthritis, sepsis, graft-vs-host disease[146,147]. In colitis models, induced by dextran sulfate sodium or 2,4,6-trinitrobenzenesulfonic acid treatment, MSC-EV treatment lowers histologic damage scores, reduces pro-inflammatory cytokines and restores epithelial integrity; certain studies show an improvement of approximately 40%-60% in disease activity index compared with vehicle treatment[148-150]. Moreover, in autoimmune arthritis models, MSC-EVs containing miR-150-5p or miR-140-3p diminished synovial inflammation, inhibited vascular endothelial growth factor/matrix metalloproteinase expression and lowered clinical arthritis scores, frequently reversing paw swelling and histological cartilage damage compared with controls[151,152]. Numerous small human studies and case reports have investigated the safety of EV preparations for inflammatory conditions, indicating acceptable short-term safety but lacking adequately powered efficacy data; larger randomized trials with specified EV products are essential[153].

Neurological and neurodegenerative conditions

The capability of MSC-EVs to traverse or to be transported through the blood-brain barrier via intranasal or intracerebral methods has spurred extensive research in models of stroke, traumatic brain injury, Alzheimer’s disease and Parkinson’s disease[70,154]. Comprehensive pre-clinical meta-analyses indicate that EV therapy decreases infarct volume and enhances functional scores following ischemic stroke[155,156]. In Alzheimer’s disease models, intranasal MSC-EVs decreased amyloid accumulation and neuroinflammation, as well as enhanced cognitive function in mice, while modified EVs carrying neuroprotective miRNAs or growth factors mitigated the loss of dopaminergic neurons in Parkinson’s disease models[157,158]. Translation challenges persist, dose adjustment, targeting precision, and ongoing functional advantages in larger animal models, but the preclinical evidence is robust.

Tissue regeneration and wound healing

MSC-EVs promote wound healing, enhance angiogenesis, regulate and monitor inflammation and promote collagen remodeling in cutaneous musculoskeletal and periodontal models[12,159]. In diabetic wound models, hydrogels or scaffolds with EVs offered prolonged release and resulted in rapid closure rate, e.g., 30%-60% faster re-epithelialization and increased capillary density compared to bolus EVs or controls[160,161]. Mechanisms involve the transfer of pro-angiogenic miRNAs (miR-126, miR-210), activation of Notch signaling and protein kinase B/hypoxia-inducible factor-1alpha, along with stimulation of ECM synthesis and proliferation in dermal fibroblasts; bioengineered delivery methods like microneedles, collagen sponges or hydrogels reliably enhanced local retention and functional repair outcomes compared to free EV injections[162,163]. Table 3 summarizes the mechanistic pathways and therapeutic impacts of MSC-EVs across diverse disease conditions[164-175].

Table 3 Therapeutic and mechanistic roles of mesenchymal stem cell-derived extracellular vesicles relevant to drug delivery applications.

Disease condition	MSC source	Major EV cargo	Mechanistic pathways modulated	Therapeutic outcome	Experimental model	Ref.
Acute myocardial infarction	Bone marrow MSC-EVs	miR-125b	Suppression of cardiomyocyte apoptosis	↑ Cardiac repair	Mouse	[164]
Myocardial infarction	Bone Marrow MSC-EVs	miR-146a-5p	IL-1 receptor-associated kinase 1/NF-κB signaling	↓ Inflammation, ↑ cardiac function	Rat	[165]
Myocardial ischemia/reperfusion	Bone marrow-MSC-EVs	miR-29c	Phosphatase and tensin homolog/protein kinase B/mammalian target of rapamycin pathway	↓ Autophagy, ↓ infarct size	Mouse	[166]
Acute kidney injury	MSC-EVs	mRNA associated with mesenchymal phenotype	Renal tubular cell survival pathways	↑ Renal recovery	Mouse	[167]
Liver fibrosis	MSC-EVs	Anti-fibrotic mediators	Decreased fibrosis signaling	↓ Reduced liver fibrosis	Mouse	[168]
Osteoarthritis	Bone marrow-MSCs	Regulatory miRNAs	Inhibition of NF-κB signaling, downregulation of cyclooxygenase-2 and inflammatory cytokines	↑ Chondrocyte proliferation and matrix production	In vitro (human cartilage tissues)	[169]
Rheumatoid arthritis	Bone marrow-MSCs-EVs	miR-34a	Cyclin I-activated Ataxia telangiectasia mutated/Ataxia telangiectasia and Rad3-related/p53 pathway	↓ Synovial inflammation	Rat	[170]
Type 1 diabetes mellitus	Adipose-MSCs	Immunomodulatory factors	Increased IL-10 and transforming growth factor-β. Decreased IL-17 and interferon-γ	↑ Glycemic control, ↑ immune balance	Mouse	[171]
Cerebral ischemic/reperfusion injury	Bone marrow-MSCs-EVs	Long non-coding RNA KLF3-AS1	Sirtuin-1 stabilization, miR-206/USP22 axis	↓ Cerebral infarction, ↑ neurological function	Mouse	[172]
Traumatic brain injury	Umbilical cord-MSC-EVs	miRNAs	Suppression of neuronal apoptosis and microglial activation	↑ Neurological function	Rat	[173]
Bacterial pneumonia	Bone marrow-MSC-EVs	Paracrine mediators	Increased alveolar fluid clearance. Reduced bacterial load	Attenuated lung injury	Mouse	[174]
Myocardial ischemia/reperfusion injury	MSC-EVs	Pro-angiogenic factors	Activation of protein kinase B and glycogen synthase kinase-3β pathways	↓ Oxidative stress, ↑ angiogenesis	Mouse	[175]

MSC: Mesenchymal stem cell; EV: Extracellular vesicle; IL: Interleukin; NF-κB: Nuclear factor-kappa B; MSC-EV: Mesenchymal stem cell-derived extracellular vesicle.

TRANSLATIONAL CHALLENGES AND OPPORTUNITIES

The clinical application of MSC-EVs as therapeutic platforms shows great potential, but significant challenges related to the manufacturing, regulatory standards, product quality and clinical assessment remain[48,176]. While studies have consistently shown effectiveness across various disease models, translating MSC-EVs into clinical use necessitates strong strategies to address challenges that are distinct or more significant than those faced by traditional biologics or synthetic nanoparticles[177-179]. The major translational challenges and mitigation strategies for MSC-EV clinical development are provided in Table 4[21,177,180-183].

Table 4 Translational bottlenecks and mitigation approaches.

Translational challenge	Impact on clinical translation	Proposed mitigation strategy	Ref.
Batch heterogeneity	Variable efficacy	Standardized MSC banking	[180]
Donor variability	Product heterogeneity	Defined donor selection criteria	[21]
Low scalability	Limited clinical production	Bioreactor + TFF systems-based manufacture	[181]
Stability issues	Cold-chain dependence	Lyophilization with trehalose	[182]
Regulatory ambiguity	Delayed approval	Early regulator	[183]
Potency assay standardization	Difficulty assessing therapeutic activities	Functional bioassay validation	[180]
Comparability after process change	Regulatory approval risk	Analytical comparability testing	[177]
Storage stability limitations	Reduced shelf-life	Cryopreservation optimization	[182]
Cost effectiveness	Manufacturing feasibility concerns	Process automation	[181]

MSC: Mesenchymal stem cell; TFF: Tangential flow filtration.

Manufacturing, standardization and scalability

A crucial translational hurdle for MSC-EV therapies is the consistent, high-yield production that maintains the critical quality attributes while ensuring compliance with GMP[48,176]. Suspension bioreactors and hollow-fiber systems have emerged as the primary platforms for scaling up, as they enhance cell density and conditioned media volumes, potentially significantly increasing EV yield per culture compared to traditional 2D flasks; single-use stirred-tank bioreactors allow for closed, GMP-compliant operation while minimizing contamination risks[180]. Downstream isolation continues to balance purity, yield and scalability. Ultracentrifugation is a standard in research but lacks scalability and consistency; tangential flow filtration combined with size-exclusion chromatography is preferred for clinical production, as it allows concentration, buffer exchange and removal of soluble proteins at liter scales with satisfactory recoveries[180,181,184].

Furthermore, the standardization of critical quality attributes is crucial yet unresolved. Regulatory bodies and industry groups suggest employing orthogonal identity assays such as particle concentration through nanoparticle tracking analysis/flow, tetraspanin profiling via western blotting or enzyme-linked immunosorbent assay, luciferase immunoprecipitation system and RNA signatures, along with potency assays associated with the proposed mechanism of action[21,176]. Proposed release criteria in recent GMP pilot initiatives encompass particle to protein ratio limits, no presence of exogenous DNA, endotoxic thresholds, sterility, all according to pharmacopeial approaches and validated potency ranges among lots[180,181]. Analytical capability presents another limitation. Single vesicle techniques such as nano flow cytometry, single particle interferometric reflectance imaging yield essential insights into heterogeneity and subpopulations, yet they are not standard release assays; investment in strong, robust and validated analytics is necessary to ensure consistency during process changes and scaling[185].

GMP manufacturing challenges

The large-scale production of MSC-EVs under GMP conditions continues to be one of the major limitations to clinical application. Laboratory-scale production typically uses small culture systems, whereas clinical manufacturing demands scalable platforms that can produce uniform EV yields while maintaining biological activity[48,176]. Bioreactor-driven MSC culture systems, such as hollow-fiber bioreactors and stirred-tank setups, have emerged as effective options to enhance scalability. These systems facilitate enhanced cell density and continuous production of conditioned media, leading to significantly greater EV yields than traditional 2D culture techniques[180]. Nonetheless, shifting to GMP manufacturing introduced additional challenges, such as the requirement for closed-system processing, contamination management, validated purification processes, and uniform release testing procedures. Isolation methods like tangential flow filtration paired with size-exclusion chromatography are becoming more popular for clinical-scale purification because of their consistency and ability to scale up[181,184]. Even with these advances, producing GMP-grade EVs in a cost-effective and reproducible manner continues to be a significant challenge, especially for treatments that require large dosage quantities.

Safety, immunogenicity and regulatory considerations

EVs exist at the crossroad of biologics, cell therapies, and nanoparticle drug regulations, requiring developers to maneuver through a combined regulatory environment[8,186]. Safety information from preliminary clinical trials and systematic reviews suggest an overall positive short-term safety profile for EV products, with combined rates of serious adverse events noted to be low (< 1%) in mixed-indication datasets; nevertheless, the variability in trials, small sample sizes and differing product definitions restrict conclusions regarding uncommon or delayed events[183]. Primary safety issues encompass unintended biodistribution, immunogenicity due to host cell proteins or new surface modifications, as well as the distribution of replication-competent viruses or oncogenic DNA[48,176]. Recent reviews on immunogenicity indicate that EVs are less capable of stimulating the immune response compared with intact cells, but may trigger complement activation or provoke anti-PEG/anti-ligand responses when chemically altered; thus, preclinical studies incorporate complement activation assays like CH50, cytokine profiles and repeated dose toxicology in two species to evaluate immunotoxicity[183,187]. Furthermore, the regulatory classification, such as biological vs drug vs combination product, hinges on the intended claim. Unmodified, allogeneic MSC-EVs for immunomodulation are usually governed as biological medicinal products, whereas engineered EVs with drug payloads usually fall under the device regulations/combination in certain jurisdictions. Proactively engaging with the regulators to address chemistry, manufacturing and controls, potent assays and non-clinical packages is advisable and hastened approvals in various other advanced areas of biologics[181].

Regulatory considerations for EV-based therapeutics

The regulatory classification of MSC-EV therapeutics is intricate because of their mixed qualities, which integrate biological, cellular, and nanotechnology aspects[15,176]. In numerous regions, EV-derived products are classified as biological medicinal products or advanced therapy medicinal products based on their origin, alteration status, and intended medical use. Regulatory bodies like the United States Food and Drug Administration and the European Medicines Agency mandate thorough documentation of production methods, quality assurance procedures, and safety evaluations before granting clinical approval[48,176]. These specifications encompass: (1) Showcase of product uniformity across different batches; (2) Validation of potency tests; (3) Studies on toxicology and biodistribution; and (4) Data on long-term stability.

Moreover, regulatory directives are increasingly highlighting the importance of early engagement with regulatory bodies to establish acceptable manufacturing and characterization criteria for EV-based therapies[183,186]. As EV therapeutics advance toward commercialization, clear regulatory frameworks are anticipated to develop, yet ongoing harmonization of global standards is crucial for broad clinical acceptance.

Storage, stability and quality control

Long-term storage and distribution remain unaddressed practical challenges. Storing liquids at -80 °C is commonly practiced, and EV integrity is maintained for months in various studies, although the reliance on cold-chain logistics increases costs and logistical challenges. Lyophilization or free-drying method using optimized lyoprotectants such as trehalose and sucrose have demonstrated potential[188,189]. Additionally, various studies indicate maintenance of particle counts and bioactivity for months at refrigeration or room temperature following lyophilization and reconstitution. However, optimization is cargo- and matrix-dependent, along with some functional loss that has been noted without precise formulation and management[182,190]. The selection of storage buffers, concentration of cryoprotectant and type of container material significantly affect aggregation, future and loss of surface proteins. Moreover, the standardization stability protocols (both accelerate and real-time) linked to specific potency metrices are necessary to regulate submissions[182,190].

Quality control and potency assays

Implementing strong quality control and potency assays is crucial for obtaining regulatory approval for MSC-EV-derived therapeutics[21,176]. In contrast to traditional small-molecule drugs, EVs possess biological complexity and require multi-faceted characterization to ensure their identity, purity, and functionality[21,48]. Recommended quality control parameters encompass[21,176]: (1) Concentration of particles and distribution of size via nanoparticle tracking analysis; (2) Expression of surface markers on EVs like CD9, CD63, and CD81; (3) Evaluation of sterility, endotoxin concentrations, and residual host-cell DNA; and (4) Determining protein-to-particle ratios.

Alongside physicochemical characterization, potency assays should demonstrate the mechanism of action of the EV product. For instance, assays for angiogenesis, immune modulation, or measurements of target gene knockdown can be utilized to illustrate biological activity[21,180]. The development of standardized potency assays continues to be an unresolved challenge, as functional differences among EV batches could influence therapeutic effectiveness.

Clinical trial landscape and future directions

The number of registered EV clinical trials has have significantly increased, covering indications from pulmonary disease to dermatology and oncology. Nevertheless, many studies are still in early phases, single arm and utilize varied products[48,176]. Systematic reviews of completed trials indicate satisfactory short-term safety but very limited strong efficacy results thus far, mainly because of small sample sizes and endpoint choices[183]. Various opportunities exist to accelerate the translational process including: (1) Agreement on potency assays linked to mechanisms that can be used across different laboratories; (2) Collaborations between public and private sectors to create reference materials and conduct interlaboratory proficiency evaluations; (3) Flexible trial designs facilitating dose-finding with biomarker-driven endpoints; and (4) Combined manufacturing analytical system that support real-time release assessments[21,48]. Projects that showcase scalable GMP processes alongside validated potency tests and a defined regulatory pathway are expected to spur significant trials; industry cases currently entering late-stage testing set a precedent and draw investment[180,181,191]. A consolidated overview of regulatory and quality needs guiding MSC-EV translation is presented in Table 5[21,180-182,192-195].

Table 5 Regulatory and quality characteristics required for clinical translation of mesenchymal stem cell-derived extracellular vesicles pharmaceutical products.

Quality characteristics	Regulatory expectation	Analytical method	Clinical relevance	Ref.
Identity	EV marker confirmation	CD63/CD81, WB, ELISA	Ensures correct product characterization and prevents misidentification of EV preparations	[21]
Potency	Mechanism-linked assay	Angiogenesis, T-cell suppression	Demonstrates therapeutic efficacy and supports dose selection	[180]
Purity	Removal of proteins/DNA	SEC, TFF, PicoGreen	Prevents contamination-related toxicity and improves product safety	[181]
Sterility	GMP requirement	USP <71>: Sterility tests	Required to ensure patient safety during systemic administration	[192]
Stability	Shelf-life	Freeze-thaw, lyophilization	Enables long-term storage and consistent therapeutic dosing	[182]
Process comparability	Manufacturing consistency	CMC validation	Required when manufacturing changes occur during scale-up or optimization	[193]
GMP manufacturing	Quality assurance	Process validation	Ensures consistent large-scale production suitable for clinical trials	[194,195]

EV: Extracellular vesicle; WB: Western blotting; ELISA: Enzyme-linked immunosorbent assay; SEC: Size-exclusion chromatography; TFF: Tangential flow filtration; GMP: Good manufacturing practice; USP: United States Pharmacopeia; CMC: Chemistry, manufacturing and controls.

Overall, MSC-EVs offer an intriguing therapeutic potential, yet to achieve clinical scale, a synchronized progress in production, analysis, safety evaluation and regulatory planning must be implemented. Moreover, funding for standardized assays, GMP-compliant processes and meticulously designed clinical trials, alongside clear reporting of both the negative and positive outcomes, will influence whether MSC-EVs transition from promising biologics to licensed medications. Figure 3 outlines the translational flow and major challenges associated with bringing MSC-EV therapies to the clinic.

Figure 3 Translational flow and key challenges in clinical development of mesenchymal stem cell-derived extracellular vesicles therapeutics. Schematic representation of the essential phases in the clinical translation of mesenchymal stem cell-derived extracellular vesicles including mesenchymal stem cell expansion, scalable extracellular vesicle production, purification using techniques like tangential flow filtration and size-exclusion chromatography, and quality control assessment. Key translational challenges are also emphasized as essential factors affecting successful application in clinical setting. MSC: Mesenchymal stem cell; EV: Extracellular vesicle; TFF: Tangential flow filtration; SEC: Size-exclusion chromatography.

Critical evaluation of engineering readiness for clinical translation

Despite significant advancements in developing MSC-EVs for therapeutic delivery, existing technologies still fall short of completely addressing various translational barriers. For instance, advanced systems like CRISPR/Cas9-loaded MSC-EVs have shown proof-of-concept viability; however, practical constraints still pose significant challenges. The loading efficiency for large macromolecular systems like CRISPR components remains relatively low, often under 10%-15%, even when using electroporation or techniques for engineering parental cells[196,197]. Moreover, mass production of genetically altered MSC-EVs faces regulatory issues due to inconsistencies in transfection efficiency, shifts in cellular phenotype, and biosafety concerns linked to gene-editing elements[108]. Additionally, numerous sophisticated EV engineering methodologies are still fine-tuned at laboratory levels and have not shown reliable performance in accordance with GMP standards[48,176]. In the absence of strong scalability and reproducibility, these technologies cannot yet be deemed clinically advanced platforms. Consequently, while the complexity of engineering has greatly increased, further validation studies focusing on scalability, cost-effectiveness, and adherence to regulations are crucial prior to broad clinical application.

MSC-EV heterogeneity as a platform-level barrier

A fundamental limitation impacting the advance of MSC-EVs as standardized drug delivery platforms is their natural biological heterogeneity. Variability arises from distinctions in MSC donor sources, tissue origins, culture conditions, passage number, and isolation methods. These factors greatly affect EV size distribution, cargo composition, and functional characteristics, resulting in variability between batches that complicate regulatory approval procedures[21]. Comparative research has shown that EVs sourced from umbilical cord, adipose tissue, and bone marrow MSCs possess unique molecular signatures and therapeutic impacts, despite being generated in comparable environments. For instance, proteomic analysis has shown significant differences in growth factor levels and immunomodulatory substances among MSC sources, indicating that establishing a universal EV “platform” is still difficult[15,17]. Addressing this heterogeneity will likely require standardized donor selection criteria, established culture protocols, and validated isolation processes that can yield consistent EV preparations. In the absence of this harmonization, attaining regulatory consistency and reproducibility at the platform level will continue to pose a significant obstacle to clinical translation.

Batch-to-batch variability and product consistency

Variability between batches poses a significant obstacle for the clinical advancement of MSC-EV therapeutics. Differences in donor traits, cell culture environments, passage numbers, and isolation methods can greatly affect EV composition, functional efficacy, and therapeutic outcomes. Research has shown that even slight modifications in culture media formulation or oxygen levels can influence EV cargo characteristics, such as miRNA and protein composition. This variability hinders reproducibility between manufacturing batches and possesses challenges for regulatory approval, which necessitates uniform product quality and anticipated biological activity[21,27,48].

Standardized MSC banking systems, established cultural protocols, and automated production platforms are being developed to reduce variability. Moreover, enforcing stringent in-process controls and validated release standards is crucial for maintaining batch reproducibility and therapeutic reliability. Lacking dependable management of batch variability, MSC-EV therapies could encounter considerable regulatory challenges, especially during late-stage clinical trials[48,176].

Clinical feasibility of loading technologies

Even with substantial progress in EV cargo-loading methods, only a limited number of these techniques show adequate reliability for clinical application. Techniques that involve intricate equipment, like electroporation or extrusion, can face difficulties in ensuring uniformity in extensive production systems[59]. Conversely, passive loading techniques are more scalable but are not efficient for macromolecular therapeutics[60,69]. Emerging hybrid approaches that integrate genetic engineering with physical loading methods show promise in addressing individual constraints[48,60]. Nonetheless, validation in GMP conditions is still restricted, and standardized protocols for large-scale EV production are yet to be finalized. Thus, clinical translation will probably rely on choosing loading techniques that ensure efficiency, reproducibility, and regulatory alignment instead of solely prioritizing loading efficiency[15].

CONCLUSION

MSC-EVs have become an intriguing category of biologically inspired nanotherapeutics that bridges cell-based therapies with synthetic drug-delivery systems. Growing evidence from various disease models shows that MSC-EVs can mimic essential paracrine functions of their originating cells while providing advantages in safety, manufacturing and regulation. Their inherent biocompatibility, ability to transport intricate molecular payloads, and ability to dynamically engage with disease-related microenvironments emphasize their increasing significance as innovative therapeutic platforms.

This review highlights how developments in cargo-loading techniques, surface engineering, hybrid EV technologies and stimuli-responsive designs are enhancing the functional versatility of MSC-EVs. Equally important, mechanistic understanding of targeted delivery, including localization to inflamed or tumor tissues, receptor-ligand interactions and microenvironment modifications, offers a logical foundation for disease-specific implementation and enhancement. Preclinical evidence in oncology, cardiovascular and ischemic diseases, inflammatory and autoimmune conditions, neurological disorders and tissue regeneration consistently endorses the therapeutic potential of MSC-EVs, though efficacy and mechanisms are contextually dependent.

Despite these advances, the transition to everyday clinical application is limited by unresolved challenges in large-scale production, standardization, scalability and regulatory conformity. Tackling these challenges demands coordinated quality control systems, better potency tests integrated into the mechanism of action and smartly designed clinical trials that progress from safety to clear efficacy outcomes. Future progress will require the incorporation of GMP-compliant production technologies, enhanced analytics and flexible clinical trial design, supported by collaborative academic, industrial and regulatory agencies, will be crucial. Through these coordinated advancements, MSC-EVs are set to transition from experimental biologics to clinically approved, precision therapeutic options that can tackle existing unmet medical requirements.