Extracellular vesicle therapeutics for cardiac regeneration: Overcoming standardization hurdles

Abstract

A study by ShamsEldeen et al published in the recent issue of World Journal of Stem Cells, the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles (EVs) and moderate exercise was evaluated in a rat model of myocardial infarction. The results revealed that these two interventions exert a synergistic effect to facilitate functional recovery of the injured myocardium. The established therapeutic potential of EVs across multiple diseases, combined with the evidence from this study, underscores the inevitable progression of EV-based therapies toward clinical translation. This article therefore focuses on the core challenges hindering the clinical translation of EV-based therapies: The production and isolation of EVs in sufficient quantities with high purity; the establishment of a multidimensional quality control system; the accurate prediction of therapeutic efficacy; the guarantee of safe and effective clinical application; and the establishment of a proactive defense system against potential risks. These challenges are pressing practical issues that require immediate resolution. Addressing these challenges will require leveraging preclinical evidence to establish a robust, standardized system for EVs - encompassing production, quality control, potency assessment, clinical translation, and risk mitigation - to facilitate their streamlined transition into clinical practice.

Key Words: Mesenchymal stem cells; Extracellular vesicles; Exercise training; Myocardial infarction; Combination therapy; Clinical translation; Cardiac regeneration

Core Tip: This article systematically summarizes the findings and mechanisms elucidated by ShamsEldeen et al, based on current preclinical evidence, outlines the key challenges and countermeasures in translating extracellular vesicle - based therapies into clinical practice - paving the way for future clinical applications that may benefit a broader patient population, including but not limited to those with cardiac diseases. Furthermore, it provides a preliminary outlook on strategies for optimizing therapeutic efficacy, scaling up production, and expanding into diverse disease areas.

This editorial refers to “Mesenchymal stem-derived exosomes enhance therapeutic benefits of exercise in isoproterenol-induced myocardial ischemia: Targeting ERK and Akt/mTOR signaling” by ShamsEldeen et al, 2025; https://doi.org/10.4252/wjsc.v17.i10.109862.

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INTRODUCTION

In accordance with the minimal information for studies of extracellular vesicles (EVs) guidelines, the term “EVs” is used herein as a generic descriptor for the heterogeneous vesicle populations utilized therapeutically, whose precise biogenetic pathways may not be fully defined. The specific term “exosomes” is reserved for instances where original research definitively confirms their exosomal nature[1]. Cardiovascular disease remains the leading cause of global morbidity and mortality, affecting individuals across all age groups, regions, and populations. As the primary threat to human health, it imposes considerable economic burdens on individuals and nations alike. According to World Health Organization data, cardiovascular diseases accounted for approximately 17.9 million global deaths in 2021, representing 32% of all annual mortality[2]. According to the Global Burden of Disease 2025 study, cardiovascular diseases resulted in 437 million disability-adjusted life years globally in 2023, a 40% increase from the 320 million disability-adjusted life years recorded in 1990, thus remaining the leading cause of global disability and mortality[3]. Ischemic heart disease, intracerebral hemorrhage, ischemic stroke, and hypertensive heart disease collectively account for over 70% of the total cardiovascular disease burden, emerging as the core subtypes underpinning its global impact[3]. Notably, the incidence of cardiovascular diseases has surged in low- and middle-income countries in recent years, with a distinct trend toward earlier onset among younger populations[4]. Concurrently, these conditions have emerged as a major contributor to the global economic burden, whose widespread and growing impact poses significant challenges to global economic systems.

Current mainstream interventions for cardiovascular disease include pharmacotherapy, percutaneous coronary intervention, and coronary artery bypass grafting[5]. While these approaches effectively alleviate symptoms and modestly improve long-term prognosis, they have considerable limitations. Pharmacological treatments fail to reverse established structural damage or promote tissue regeneration. Although interventional procedures rapidly restore blood flow, they carry risks of restenosis and stent thrombosis, with limited efficacy in diffuse vascular disease or end-stage heart failure. Additionally, surgical procedures - characterized by significant trauma and prolonged recovery - are often poorly tolerated by elderly patients with multiple comorbidities[6].

Thus, there is an urgent need for the development of definitive therapies for cardiovascular disease. In recent years, innovative strategies such as cell transplantation, gene therapy, and regenerative medicine have emerged. Among these, mesenchymal stem cells (MSCs) have attracted considerable attention owing to their paracrine activity, with distinct advantages including relative ease of isolation from tissues such as bone marrow and adipose tissue, low immunogenicity, and multilineage differentiation potential[7]. Emerging evidence indicates that MSCs secrete a range of bioactive factors, including EVs, cytokines, and growth factors, which primarily modulate inflammation, promote tissue repair, and inhibit apoptosis[8]. Among these secretory products, EVs serve as pivotal mediators of intercellular communication, functioning as critical messengers by delivering bioactive molecules such as microRNAs (miRNAs), proteins, and lipids to exert central regulatory roles in physiological and pathological processes[9]. Specifically, EVs bidirectionally regulate apoptosis to maintain cellular homeostasis[10], suppress fibroblast activation, and promote extracellular matrix (ECM) degradation to ameliorate fibrosis[11]; they also precisely balance inflammatory responses through context-dependent release of anti- or pro-inflammatory molecules[12]. Furthermore, EVs directly stimulate angiogenesis and indirectly optimize the vascular microenvironment via transfer of angiogenic factors[13]. Their regulatory functions are determined by the parental cell origin and molecular cargo, positioning EVs as both therapeutic targets and promising delivery systems in disease treatment[14]. Emerging evidence highlights the considerable potential of EVs in the diagnosis and treatment of diverse diseases - including cardiovascular[15], respiratory[16], metabolic[17], and orthopedic disorders[18] - underscoring their broad application prospects that merit further investigation. A 2025 study by ShamsEldeen et al[19] published in the recent issue of World Journal of Stem Cells systematically evaluated the synergistic therapeutic effects of MSC-derived EVs (MSC-EVs) combined with moderate exercise training. Their work provides compelling new evidence for elucidating the cooperative mechanisms of EVs in cardiac repair, while also highlighting the translational gap between proof-of-concept research and clinical product development. This article will systematically outline these standardization challenges and delineate a roadmap toward an era of standardized, reproducible EV-based therapeutics.

SYNERGISTIC THERAPEUTIC MECHANISM

In this study, ShamsEldeen et al[19] established a rat model of myocardial ischemia using isoproterenol and divided the subjects into four groups: Sham, exosome monotherapy, exercise-only, and combination therapy. The results clearly indicated that the combination of MSC-EVs with exercise training demonstrated a pronounced improvement in cardiac function, characterized by a significantly enhanced left ventricular ejection fraction, a markedly reduced fibrosis extent, and an increased capillary density (Figure 1). The underlying mechanisms may involve five key aspects.

Figure 1 Combination of exercise and mesenchymal stem cell-derived extracellular vesicles contributes to attenuated cardiac hypertrophy via dual pathway modulation. ↑ : Increased expression/activation; ↓: Decreased expression/inhibition; ★: Fine-tuned regulation; Color code: Blue - exercise; green - EVs; purple – synergy. MSC-EVs: Mesenchymal stem cell-derived extracellular vesicles; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; GSH: Glutathione; EV: Extracellular vesicle; ERK: Extracellular signal-regulated kinase; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; SERCA2a: Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; MMP-9: Matrix metalloproteinase 9; ECM: Extracellular matrix; CHP: Cell-penetrating peptide; CEP: Cell-entering peptide; ROS: Reactive oxygen species.

First, suppression of core signaling pathways. The combined intervention of MSC-EVs and exercise significantly downregulates the hyperactivation of both extracellular signal-regulated kinase (ERK) and protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathways. This dual suppression effectively curbs the pathological vicious cycle of hypertrophy, fibrosis, and inflammatory responses driven by aberrant pathway activation, laying a molecular foundation for the restoration of cardiac function and structure.

Second, improved calcium cycling and enhanced contractile function. The combined strategy directly ameliorates cardiomyocyte calcium handling dysfunction by upregulating sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression via dual mechanisms: It inhibits ERK-mediated negative regulation of SERCA2a transcription and indirectly enhances SERCA2a synthesis through Akt/mTOR pathway modulation. Restored SERCA2a pump activity accelerates cytosolic calcium reuptake, significantly improving myocardial relaxation and contractility - ultimately translating to enhanced cardiac functional parameters such as stroke volume.

Third, antifibrotic mechanisms in ECM remodeling. The combined application of MSC-EVs and exercise reduces matrix metalloproteinase 9 (MMP9) expression, effectively suppressing pathological ECM remodeling. This effect is mediated by their inhibition of the Akt/mTOR and ERK signaling pathways - key regulators of MMP9 expression. Furthermore, a strong inverse correlation was observed between SERCA2a and MMP9 levels, suggesting that improved calcium homeostasis independently contributes to antifibrotic effects. Collectively, these mechanisms synergistically preserve myocardial structural integrity.

Fourth, regulation of oxidative stress and inflammatory responses. The combined therapy exerts potent antioxidant and anti-inflammatory effects, significantly reducing cardiac tissue levels of malondialdehyde, tumor necrosis factor-α, and interleukin-6 (IL-6) while concurrently elevating reduced glutathione content. Mechanistically, MSC-EVs directly deliver bioactive support to the injured myocardium, whereas exercise training enhances intrinsic cardiac defense mechanisms. This synergistic interaction improves the local myocardial microenvironment and mitigates direct cellular damage induced by oxidative stress and inflammation.

Fifth, cellular preservation and angiogenesis promotion. The combined regimen effectively reduces cardiomyocyte apoptosis by suppressing the apoptotic protein caspase-3, while concurrent upregulation of CD31-positive microvessel density indicates active angiogenesis in ischemic myocardial regions. Mechanistically, MSC-EVs deliver pro-angiogenic factors, and exercise exerts critical physiological shear stress on vascular walls - synergistically stimulating collateral vessel formation. This coordinated approach enhances myocardial perfusion and establishes a favorable microenvironment for cell survival and tissue repair.

STANDARDIZATION CHALLENGES AND STRATEGIC RESPONSES

ShamsEldeen et al’s preclinical study[19] in rat models clearly demonstrates significant synergistic therapeutic effects of combination therapy, highlighting the considerable potential of EV-based therapeutics. While these findings hold promise for future human applications, a substantial translational gap remains between efficacy in animal models and clinical implementation - animal study results cannot be directly extrapolated to humans, whose pathophysiology is more complex. Thus, systematic resolution of key standardization challenges [e.g., production, quality control (QC), functional assessment, and clinical validation; Figure 2] is a prerequisite to clinical exploration of this potential. Addressing these issues demands a fundamental paradigm shift, spanning from biological mechanism research to clinical trial design.

Figure 2 Standardized workflow and key decision points for clinical-grade extracellular vesicles in cardiac repair (depicts the four core components of clinical-grade extracellular vesicle therapy. Upstream cellular programming and production; rigorous characterization and quality control to define critical quality attributes; preclinical efficacy prediction and patient stratification for cohort selection; clinical translation with real-time monitoring and comprehensive safety evaluation to ensure effective treatment). EVs: Extracellular vesicles; SEC: Size exclusion chromatography; TFF: Tangential flow filtration; miRNA: MicroRNA; ITGA4: Integrin subunit alpha 4; CQAs: Critical Quality Attributes; hiPSC-CMs: Human induced pluripotent stem cell-derived cardiomyocytes; IL-6: Interleukin-6; sST2: Soluble suppression of tumorigenicity-2; PET: Positron emission tomography; NIRF: Near-infrared fluorescence.

Challenge 1: Production process bottlenecks

While EV-based therapeutics have shown efficacy in animal models, their clinical translation hinges on overcoming a key bottleneck: Scaling production from laboratory scale to Good Manufacturing Practice-compliant industrial levels. The inherently low secretion efficiency of native EVs requires massive cell expansion to achieve therapeutic doses (typically hundreds of micrograms to milligrams), translating to substantial cell culture demands and prolonged timelines[20] - representing the first major hurdle to clinical translation.

Regarding cell culture: MSCs and other cells used for EV production exhibit inter-donor variability. This inherent heterogeneity leads to substantial batch-to-batch differences in EV composition and functionality, which can compromise therapeutic consistency and predictability. Furthermore, EV yield, quality, and biological activity are highly sensitive to complex culture conditions - establishing a stable, reproducible cell culture system thus presents significant challenges[21], highlighting the need for further investigation in this area.

Regarding isolation and purification: Ultracentrifugation (UC) remains the most widely used laboratory method for EV isolation and is often regarded as the “gold standard”[22,23]. However, this approach has critical limitations: Low recovery rates (resulting in substantial EV loss), prolonged processing times, and exposure to high shear forces - factors that compromise EV structural integrity and bioactivity, directly impacting the batch-to-batch consistency of therapeutic potency. Furthermore, UC co-sediments protein aggregates and non-vesicular contaminants, leading to insufficient product purity and potential uncontrolled population biases due to differential sedimentation efficiency. Consequently, UC exhibits significant shortcomings in scalability, reproducibility, and control of critical quality attributes, making it unsuitable for therapeutic product manufacturing standards[22,23].

Alternative isolation methods include size exclusion chromatography (SEC)[24], tangential flow filtration (TFF)[25,26], anion exchange chromatography[27], and microfluidic technologies[28]. Among these, SEC is a highly compatible chromatographic technique that efficiently removes soluble protein contaminants, yielding exceptionally pure EV preparations while offering considerable potential to enhance batch-to-batch consistency. However, its limited sample loading capacity restricts large-volume processing efficiency, presenting a scalability bottleneck[24]. TFF offers notable scalability advantages, easily handling large-volume samples and serving as an ideal platform for continuous, large-scale production - though its purity limitations often require combination with SEC or other methods[25,26]. Microfluidic technology provides superior purity and resolution, alongside potential for high integration and automation that significantly reduces manual intervention and enhances process control. Current constraints include limited chip throughput and high equipment costs[28].

Each purification method has distinct advantages and limitations (Table 1), with no single technique achieving an optimal balance across recovery, purity, bioactivity, and throughput. Thus, orchestrated integration of these technologies is needed to develop integrated manufacturing processes. For instance, TFF can be used for rapid concentration of large-volume samples, followed by refined purification via SEC to obtain high-purity final preparations. Such hybrid approaches fully leverage the unique strengths of individual methods, establishing a foundational framework for standardized EV manufacturing. Further advances will be required to translate these concepts toward integrated, closed-system, and scalable manufacturing processes.

Table 1 Comparative analysis of major extracellular vesicles isolation methods for clinical translation.

Method	Recovery yield	Purity	Throughput/processable volume	Time/cost	Impact on bioactivity	Preservation of subpopulations
UC	Low (+)	Moderate (co-isolates protein aggregates & non-exosomal vesicles)	Low/difficult to scale (+)	Time: High; cost: Low (-)	Potential vesicle damage & aggregation due to high g-forces (-)	Poor (differential sedimentation may bias populations) (-)
SEC	Moderate (++)	High (effective removal of soluble proteins & contaminants) (++)	Moderate/challenging for large volumes (++)	Time: Moderate; cost: Moderate (++)	Gentle, maintains structural integrity & function (++)	Good (separation by hydrodynamic size, can preserve heterogeneity) (++)
TFF	High (+++)	Moderate (can require combination with SEC for high purity) (++)	High/highly scalable (handles large volumes) (+++)	Time: Low; cost: Moderate (+++)	Gentle, suitable for labile biologics (+++)	Good (size-based, less shear damage than UC) (++)
AEC	High (+++)	High (binds negatively charged vesicles, effective impurity removal) (+++)	High/scalable (+++)	Time: Low; cost: Moderate (+++)	Depends on elution conditions (salt/pH may affect activity) (+)	Moderate (may select for specific surface charge populations) (+)
Microfluidic technology	Variable (device-dependent) (+) to (++)	High (precise manipulation & sorting) (+++)	Low/currently limited by chip design (+)	Time: Rapid processing; cost: High per-device (research stage) (+)	Generally gentle (+++)	Promising for specific subpopulation isolation (precise sorting) (+++)

+++: Excellent/high; ++: Good/moderate; +: Fair/Low; -: Poor/negative impact. This semi-quantitative assessment integrates data from referenced studies and current field consensus. The optimal method choice often involves combining techniques (e.g., tangential flow filtration + size exclusion chromatography) to balance yield, purity, and bioactivity. UC: Ultracentrifugation; SEC: Size exclusion chromatography; TFF: Tangential flow filtration; AEC: Anion exchange chromatography.

Challenge 2: Characterization and QC bottlenecks

The heterogeneity and ill-defined nature of EVs used in animal studies preclude their direct translation to human applications. Ensuring safety and batch-to-batch consistency requires establishing rigorous characterization and QC systems that surpass laboratory standards.

Conventional metrics - such as particle size and concentration assessed via nanoparticle tracking analysis - are insufficient for EV definition, as they cannot distinguish vesicles from non-vesicular particles (e.g., lipoprotein aggregates) nor reflect molecular composition, biological activity, or therapeutic potency[29]. Defining EVs requires characterization beyond standard nanoparticle tracking analysis. Advanced techniques - including nanoscale flow cytometry, transmission electron microscopy, and atomic force microscopy - enable comprehensive physical profiling. Nanoscale flow cytometry simultaneously analyzes individual particle size and surface markers (CD9/CD63/CD81), providing immunophenotype-based absolute quantification and subpopulation analysis that serves as a critical bridge between physical attributes and molecular identity[30]. Transmission electron microscopy remains the gold standard for confirming vesicular ultrastructure, revealing the characteristic cup-shaped morphology that validates typical vesicle architecture[31]. Atomic force microscopy probes mechanical properties (stiffness, viscoelasticity) that may directly influence cellular interactions and tissue penetration. Collectively, these multidimensional physical characterizations establish reliable “identity signatures” for EVs[32].

Comprehensive molecular profiling is essential to elucidate the functional capacity and therapeutic potential of EVs. Specifically, proteomics, miRNA sequencing, and lipidomics delineate the molecular composition of EVs - fundamental for understanding their mechanistic actions and ensuring batch-to-batch consistency[33]. Proteomic analysis not only validates sample purity by detecting canonical markers (CD9, CD63, CD81) but also systematically identifies functional proteins (e.g., growth factors, cytokines, membrane receptors)[1,34]. The composition and proportional distribution of these functional proteins directly govern EV biological activity and dictate therapeutic potency. Many core functions of EVs rely on their regulatory miRNA cargo[35]. Sequencing-based miRNA profiling enables systematic mapping of vesicular content, facilitating identification of therapeutically relevant miRNA species. These profiles not only pinpoint candidate therapeutic molecules but also provide functional validation frameworks for vesicle activity[36]. Lipidomics complements biological characterization by analyzing the lipid bilayer - governing membrane stability, fusion capacity, and targeting specificity[37]. Integrated multi-omics analysis enables comprehensive EV qualification. Ultimately, overcoming characterization and QC bottlenecks requires establishing comprehensive quality attribute databases that correlate molecular signatures with functional potency. Table 2 summarizes principal methodologies employed for the systematic assessment of critical quality attributes of EVs.

Table 2 Testing technologies for critical quality attributes of extracellular vesicles.

Attribute category	Critical quality attributes	Example testing technologies
Physical characterization	Particle size distribution & concentration	NTA
	Single-particle sizing & surface markers (e.g., CD9, CD63, CD81)	Nanoscale flow cytometry
	Morphology/structure	TEM
	Mechanical properties (e.g., stiffness, viscoelasticity)	AFM
Molecular profiling	Protein composition (including marker & functional proteins)	Proteomics
	miRNA composition & profile	miRNA sequencing
	Lipid composition	Lipidomics
Functional potency	Cell survival/anti-apoptotic capacity	Functional cell-based assays (e.g., caspase-3 activity)
	Calcium handling capacity	Functional cell-based assays (e.g., calcium imaging)
	Mitochondrial function	Functional cell-based assays (e.g., mitochondrial membrane potential measurement)

NTA: Nanoparticle tracking analysis; TEM: Transmission electron microscopy; AFM: Atomic force microscopy; miRNA: MicroRNA.

Challenge 3: Potency assessment bottlenecks

Functional improvements observed in animal models do not directly translate to human therapeutic efficacy. Consequently, developing function-driven potency assessment systems that better mimic human disease physiology is crucial for accurately predicting clinical value. Conventional metrics - such as protein concentration and particle counts - no longer suffice. As highly heterogeneous vesicles[38], EV therapeutic efficacy depends on intrinsic capabilities and functional efficiency rather than quantity, necessitating a fundamental shift from physical quantification to function-driven evaluation.

First, human cardiomyocyte/cardiac progenitor cell models can be established to systematically evaluate core functional parameters, including cell viability/anti-apoptotic capacity, calcium handling, mitochondrial function, cellular uptake efficiency, and pro-angiogenic potential. Cell viability/anti-apoptotic capacity reflects the ability of EVs to enhance cell survival[39]. Calcium handling assesses EV potential to improve myocardial electrophysiological function and contractility[40]. Mitochondrial membrane potential measurements evaluate EV restorative effects on cellular energy metabolism[41]. Cellular uptake efficiency is a prerequisite for EV functionality, directly determining whether their cargo can be successfully delivered to exert intracellular effects[42]. This can be quantified by co-incubating target cells with lipophilic fluorescent dye-labeled EVs (e.g., PKH67 or DiD), followed by confocal microscopy or flow cytometry to assess internalization capacity[43]. Pro-angiogenic capacity directly links EV molecular characteristics to therapeutic goals such as ameliorating myocardial ischemia[44]. Tube formation assays using human umbilical vein endothelial cells provide visual assessment of EV-induced vascular network formation, corroborating in vivo findings like increased CD31-positive microvascular density. However, human cardiomyocyte/cardiac progenitor cell models remain simplified representations of the complex cardiac microenvironment. To more accurately predict clinical efficacy, further evaluation using advanced biomimetic models that closely resemble human physiology is essential. Promising platforms for such assessment include cardiac organoids and heart-on-a-chip systems.

Cardiac organoids - 3D microtissues derived from pluripotent stem cells - enable evaluation of EV effects on tissue-level contractile recovery, structural remodeling, and electrophysiological coupling, offering a more physiologically relevant system for assessing EV therapeutic potential. Heart-on-a-chip platforms allow real-time, non-invasive monitoring of key functional parameters (e.g., beat force, frequency, conduction velocity) in cardiac cells post-EV intervention, significantly enhancing the precision and predictive value of functional assessments. In summary, developing evaluation systems directly aligned with clinical applications is crucial for establishing the therapeutic value of EV-based products.

Challenge 4: Bottlenecks in clinical translation

Interspecies differences in immune and metabolic pathways between animals and humans limit the direct applicability of conventional clinical trial designs to human studies. To effectively and safely translate preclinical efficacy into clinical applications, reforming clinical translation strategies is essential. First, a key advancement involves shifting from traditional trial frameworks to flexible adaptive designs, where trial parameters - such as dosing regimens or sample size - can be modified based on interim analyses after trial initiation. This approach significantly enhances research efficiency and success rates while reducing time and financial investment.

Second, precise patient stratification is essential, as therapeutic efficacy depends on both drug-related factors and individual pathological states. In cardiac disease patients, accurate stratification requires integrating multi-omics data[45] (e.g., proteomic, transcriptomic, metabolomic profiles) to evaluate key biological processes: Inflammatory activation (e.g., interleukin-6, tumor necrosis factor-α), fibrotic signaling (e.g., galectin-3, MMP9), myocardial stress and injury (e.g., brain naturetic peptide, soluble fms-like tyrosine kinase-1), oxidative stress levels (e.g., myeloperoxidase, malondialdehyde), and endogenous repair potential (e.g., vascular endothelial growth factor A, miR-126). Advanced computational methods like machine learning can then reduce the dimensionality of these high-dimensional datasets and identify underlying patterns. This enables classifying patients into distinct molecular subtypes[46] - such as immune-inflammatory, fibroproliferative, or metabolic dysregulation phenotypes - facilitating tailored therapies based on predicted treatment responses.

Finally, molecular imaging techniques[47] - radionuclide labeling, near-infrared fluorescence imaging, and superparamagnetic iron oxide nanoparticles - enable non-invasive, real-time in vivo tracking of EVs. These methods provide direct evidence to optimize dosing regimens, delivery routes, treatment timing, and targeting efficiency. In summary, successful clinical translation of EV-based therapies requires moving beyond conventional approaches. It demands integrating adaptive trial designs, biomarker-guided stratification, and molecular imaging-based pharmacokinetics to achieve truly precision medicine.

Challenge 5: Mitigating potential risks

Despite the considerable therapeutic potential of EVs, their inherent biological properties may confer not only therapeutic benefits but also pathogenic risks. Several unpredictable challenges require careful consideration, including inherent EV-related risks, immunogenicity and procoagulant potential, safety profiles following engineering modifications, and arrhythmogenic/oxidative stress thresholds when combined with physical exercise.

First, inherent EV-associated risks. EVs exhibit considerable compositional complexity, carrying the risk of uncontrolled biological effects and tumor promotion - manifested as exacerbated local inflammation, aberrant fibrosis, or stimulation of latent microlesions. Mitigation strategies include rigorous QC of parent cell status, screening final products for carcinogenicity and pro-fibrotic signaling, and conducting functional validation[48]. EV in vivo distribution remains difficult to predict. Accumulation of substantial EV quantities in non-target tissues (e.g., liver, brain, or other healthy organs) may lead to hepatic dysfunction, disrupted neural signaling, or other unforeseen consequences. Countermeasures involve using molecular imaging during preclinical and clinical stages to precisely map real-time biodistribution[49], complemented by comprehensive histopathological examination and blood biochemical analysis of human organs. EV immunomodulatory effects display dual potential. Identical EV batches may exert pro-inflammatory or excessive immunosuppressive effects in different patients, or even in the same patient across disease stages[50]. Recommended approaches include baseline immune profiling, post-infusion immune monitoring during clinical trials, and incorporating immunophenotyping into product release criteria[51]. EVs possess potent signaling capabilities. Overdosing or an excessively narrow therapeutic window may disrupt normal metabolic and signaling pathways in target cells, potentially inducing toxicity. Preventive measures include establishing a safe therapeutic window during preclinical studies, implementing conservative dose-escalation protocols in early-phase trials, and conducting comprehensive safety monitoring[52].

Second, the immunogenicity and procoagulant risk of EVs, which are interrelated and influenced by multiple factors, represent critical safety concerns in clinical translation[53]. Immunogenic risks are typically triggered by low donor-recipient matching, repeated administration, abnormal donor cell status, or suboptimal preparation/storage conditions[54]. Procoagulant risks may arise from EVs carrying procoagulant surface components, immunocoagulative cross-activation, preparation/QC defects, or recipient-specific variations[55]. Adverse outcomes from these risks exhibit a gradient from subclinical abnormalities to life-threatening events, depending on risk severity, individual susceptibility, and timeliness of intervention. Moreover, immunocoagulative cross-activation often leads to synergistic effects, amplifying tissue damage. A comprehensive risk assessment combining in vivo monitoring and in vitro testing is therefore essential. Evaluation methods include: Using mixed lymphocyte reactions to assess the impact of allogeneic EVs on recipient T-cell proliferation[56]; detecting macrophage activation markers (e.g., CD86) to evaluate immune cell responses in vitro[57]; quantifying EV surface expression of major histocompatibility complex (MHC) molecules and immune checkpoint proteins [e.g., programmed death ligand-1 (PD-L1)] via flow cytometry or western blot to assess immunostimulatory potential[58]; conducting in vivo immune monitoring by tracking EV biodistribution and clearance rates, measuring serum anti-donor EV antibody titers, and profiling inflammatory cytokines[12,59,60]; evaluating coagulation function in vitro via thromboelastography, coagulation time assays, or thrombin generation tests[61]; quantifying specific biomarkers such as tissue factor (TF, CD142) and phosphatidylserine on EV surfaces[62]. These assessments enable preliminary risk prediction, significantly reducing the incidence of adverse outcomes. Corresponding prevention and mitigation strategies should be established, including: Donor screening or optimization: Prioritizing MHC-matched donors or pooling EVs from multiple donors[56]; genetic engineering: Knocking out the β2-microglobulin gene in donor cells to reduce MHC-I expression[63], or modifying EV surfaces with “immune-evasive” molecules such as CD47 or PD-L1 to inhibit macrophage phagocytosis and T-cell activation[64]; immunomodulatory adjunct therapy: Short-term, low-dose immunosuppressants during low-infection-risk periods to mitigate acute immune reactions, or combining EVs with immune-modulating cargo to specifically suppress rejection[65]; process optimization: Improving EV purity through multiple washing steps or size-exclusion chromatography to minimize non-specific immune activation by impurities[66]; QC: Incorporating procoagulant activity and TF expression into EV product specifications, and selecting batches with low procoagulant potential[67]; administration regimen optimization: Using low-dose, slow infusion to avoid transient high intravascular EV concentrations and reduce thrombosis risk; anticoagulation intervention: Co-administering low-molecular-weight heparin during clinical infusion, or engineering EVs to block TF - coagulation factor binding sites[68]; source-level risk reduction: Selecting donor cells with low TF expression, or removing highly procoagulant large EVs (e.g., apoptotic bodies) during isolation[69]. These preclinical intervention strategies must be rigorously implemented to minimize risk occurrence. Should risks materialize, corresponding management - such as drug discontinuation, symptomatic treatment, anticoagulation, thrombolysis, immunomodulator combination therapy, emergency intervention, or resuscitation - should be administered based on risk severity. Throughout EV administration, continuous monitoring is mandatory according to the following scheme: Pre-administration: Immune markers (HLA typing, anti-donor antibody titers) and coagulation parameters (prothrombin time, activated partial thromboplastin time, D-dimer, TF activity); during administration: Vital signs (body temperature, heart rate, blood pressure) and immediate indicators (oxygen saturation, signs of intravenous line inflammation); post-administration follow-up: Short-term (within 1 week): Complete blood count, inflammatory cytokines, coagulation function; long-term (1-3 months): Antibody titers, thrombotic imaging studies[70].

Third, safety evaluation of engineered EVs. The engineering process - whether physical, chemical, or genetic - may introduce new risks, such as structural damage, parental cell perturbation, or enhanced immunogenicity. Comprehensive safety assessment is therefore critical for the clinical translation of engineered EVs. Immunological safety is evaluated by measuring serum levels of specific antibodies (immunoglobulin G, immunoglobulin M), lymphocyte proliferation, and cytokine profiles to assess potential antibody-mediated immune rejection and immune cell activation[71]. Long-term animal studies dynamically monitor changes in immune cell subsets and antibody titers to evaluate immune memory formation and chronic immunotoxicity[72]. Biocompatibility assessment examines the effects of engineered EVs at varying concentrations on target and normal cells, evaluating viability, functional integrity, apoptosis, and necrosis to rule out modification-induced cytotoxicity[73]. Targeting specificity and off-target risk are validated using in vivo imaging and tissue fluorescence quantification to confirm targeting efficiency and exclude abnormal accumulation in non-target organs[74]. Long-term safety evaluation focuses on accumulation-related toxicity and potential carcinogenicity. Chronic animal studies quantify persistent EV residues in organs, while techniques such as soft agar colony formation assays and genomic sequencing assess abnormal proliferation and mutations potentially induced by pro-proliferative factors or gene-editing tools[53]. Production process safety requires standardized management of potential impurities (e.g., cellular debris, culture medium components, chemical modification reagents) to mitigate associated risks[75]. Therefore, future applications of EVs therapy in the context of exercise will require precise evaluation of the therapeutic dosage window and individualized regimens to avoid ineffective or excessive responses, ultimately enabling safe and effective cardiac protection.

Fourth, the arrhythmogenic/oxidative stress threshold during combined EV and exercise therapy. Research indicates that combining EVs with exercise modulates the oxidative stress threshold for arrhythmogenesis, primarily via EV-mediated intercellular communication and exercise-induced remodeling of redox system balance[76]. Moderate-intensity endurance exercise (e.g., at 70% of maximum heart rate) effectively stimulates the release of cardioprotective EVs. These act as signaling mediators that enhance cardiomyocyte resistance to oxidative stress through multiple mechanisms: Direct delivery of antioxidant enzymes, activation of intracellular defense systems like the nuclear factor erythroid-2-related factor 2 pathway, and modulation of pro-survival and mitochondrial repair signaling cascades (e.g., Akt and ERK). Collectively, these actions elevate the oxidative stress tolerance threshold[77,78]. Furthermore, bioactive molecules carried by EVs improve myocardial microcirculation and stabilize cardiomyocyte membrane potential. This reduces the risk of exercise-induced arrhythmias from oxygen supply-demand mismatch or ionic imbalance, demonstrating significant cardioprotective effects[79].

In summary, proactive prediction and mitigation of potential risks are necessary. Moving forward, establishing a robust risk prevention framework will be essential to ensure patient safety, maximize therapeutic benefits, and minimize potential harms in this promising therapeutic paradigm.

FUTURE PERSPECTIVES

The future development of EV-based therapies will focus on optimizing efficacy, scaling production, minimizing risks, and reducing costs. Engineering strategies represent a promising approach to enhance therapeutic performance, primarily through two key avenues.

First, cargo loading - the selective incorporation of bioactive molecules into EVs to augment their therapeutic effects. This can be achieved via post-loading methods (e.g., electroporation, sonication, incubation), where isolated EVs are loaded with functional molecules[80]; or pre-loading strategies, wherein parental cells are genetically modified to stably overexpress specific miRNAs, avoiding physicochemical damage to EVs[81]. Efficacy assessment should compare engineered vs native EVs both in vitro (using functional potency assays) and in vivo (employing myocardial infarction models evaluated by echocardiographic parameters such as ejection fraction and fibrosis extent).

Second, surface modification - the functionalization of EV membranes with homing peptides or ligands via genetic engineering or chemical conjugation to enable targeted accumulation in cardiac lesions[82]. Genetic engineering fuses genes encoding cardiac-homing peptides with EV membrane protein genes, leading to secretion of targeted EVs from modified parent cells[83]. Chemical conjugation utilizes click chemistry or hydrophobic interactions to link synthetic homing peptides directly to membrane lipids or proteins of isolated EVs[84]. Efficacy evaluation should prioritize molecular imaging to quantify targeting efficiency, followed by in vivo validation using the aforementioned cardiac functional readouts[85]. While engineering strategies significantly enhance EV performance, they may also introduce new risks - such as EV integrity loss during chemical loading or altered parental cell physiology from genetic modification[86]. Whether these changes compromise efficacy or introduce adverse effects requires further systematic investigation.

In the pursuit of scalable production, bioinspired synthetic EV-mimetic nanovesicles have emerged as a promising alternative. This technology enables in vitro artificial assembly of nanoscale vesicles, with its key advantage lying in selectively retaining only the therapeutically relevant components of natural EVs - such as specific membrane proteins, lipids, and nucleic acids - while excluding non-essential or potentially harmful biomolecules[87]. For industrial-scale manufacturing, the bottom-up assembly approach represents an ideal pathway[88]. This method offers fully definable and customizable composition, effectively overcoming the inherent heterogeneity and batch-to-batch variability of natural EVs. Beyond compositional consistency, it also presents potential safety advantages. However, significant challenges remain before clinical translation is feasible. These include accurately recapitulating the complex composition and functionality of natural EV membranes, and maintaining consistent product quality across manufacturing batches.

Accordingly, continuous optimization of the entire bioprocess for native EV production is essential, serving as a critical complement to biomimetic synthesis technologies. Yield and functional consistency can be enhanced by refining cell culture systems - including the development of defined media, scalable controllable bioreactors, and preconditioning strategies to boost EV secretion. Future integration of these cell culture optimization approaches with the aforementioned intelligently consolidated separation and purification strategies is poised to significantly improve the productivity of native EVs.

Notwithstanding the considerable clinical potential of advanced technologies such as engineered EVs, biomimetic nanovesicles, and organoid models in potency assessment, their successful translation into clinical practice remains beset with numerous challenges. From a manufacturing perspective, the processes for generating specific EVs via engineered cell modification or in vitro assembly of biomimetic particles are significantly more complex than those for small-molecule drugs. To achieve industrial-scale production compliant with Good Manufacturing Practice, a series of critical issues must be rigorously addressed, including real-time monitoring of critical quality attributes, process control to reduce product heterogeneity, and ensuring consistency from cell bank establishment to final formulation. Similarly, organoid cultures are highly susceptible to variations induced by ECM materials, cell sources, and culture conditions, leading to batch-to-batch inconsistencies that limit their application as standardized potency assay methods in industrial settings. From a regulatory standpoint, these innovative products currently lack well-defined evaluation pathways. Therefore, future advancement hinges not only on technological innovation but also on the simultaneous progress in three key areas: Scalable manufacturing processes, standardization of complex biological models, and proactive regulatory science. Only through such integrated efforts can these promising cutting-edge technologies be reliably translated into therapies that benefit patients.

For any therapeutic modality intended for clinical application - including EV-based therapies - cost-effectiveness is a critical determinant. Overall costs must therefore be considered throughout the complex processes of production, QC, and treatment strategy optimization. Consequently, research and development efforts should incorporate systematic evaluation and management of both production expenses and patient affordability.

CONCLUSION

A pivotal 2025 study by ShamsEldeen et al[19] in the World Journal of Stem Cells demonstrated that combined MSC-EVs and moderate exercise treatment exerts a synergistic (“1 + 1 > 2”) therapeutic effect in a rat cardiac disease model. These findings provide novel insights and experimental evidence for preclinical cardiac research, while further highlighting the considerable potential of EV-based therapies in translational medicine. Concurrently, this study underscores the significant challenges in bridging preclinical research and clinical application. Success requires overcoming a series of unique obstacles beyond mere extrapolation of animal data.

Key challenges in the preclinical stage include production: Conventional UC, limited by low yield and lengthy processing, necessitates developing integrated, optimized manufacturing processes; QC: Reliance solely on particle size and concentration is insufficient - comprehensive quality assessment requires establishing a multidimensional profile using nanoscale flow cytometry and multi-omics analyses; potency assessment: Shifting from physical quantification to a function-based evaluation system is critical, as therapeutic value depends on functional capacity rather than just particle number. For clinical translation, primary challenges involve trial design: The complex biology of EVs demands adaptive clinical trial designs, precise patient stratification, and real-time in vivo tracking via molecular imaging to maximize efficacy and minimize adverse events; risk mitigation: A robust, multi-layered risk defense system is essential to address both known risk factors and potential unknown variables.

In summary, the path to clinical application of EV therapies remains protracted. Future progress hinges on interdisciplinary collaboration across medicine, biology, engineering, pharmacology, analytical chemistry, data science, and regulatory science. A systematic approach to standardizing processes - from production and QC to defining clinical endpoints - is imperative to translate EV-based “drugs” from conceptual promise to practical reality, ultimately benefiting patients worldwide.