Targeting Mitochondrial Dynamics via EV Delivery in Regenerative Cardiology: Mechanistic and Therapeutic Perspectives

Evidence in recent preclinical models underscores that EVs derived from mesenchymal stem cells (MSCs), cardiac progenitors, iPSC-derived cardiomyocytes (iPSC-CMs), and other regenerative cell types [6,10,11] can attenuate ischemic and toxic injury by delivering mitochondrial-repair cargo. For example, EVs carrying ATP synthase subunits and electron-transport–complex proteins restore ATP production and improve oxidative phosphorylation (OCR) in hypoxic cardiomyocytes. EV-mediated activation of mitophagy via PINK1/Parkin also reduces accumulation of depolarized mitochondria and mitochondrial ROS during reperfusion injury [12,13,14]. iPSC-CM-derived EVs (including mitochondria-rich EVs or mitoEVs) have been shown to transfer mtDNA, TFAM, and OXPHOS components. These transfers transiently reconstitute ΔΨm, raise ATP/ADP ratios, and normalize calcium handling in injured cardiomyocytes, thereby limiting apoptotic signaling (BAX/BCL-2 balance) and caspase activation [2,12]. EVs modulate mitochondrial dynamics via regulators such as DRP1 and OPA1, and deliver miRNAs that fine-tune fusion/fission balance and mitophagy [13,15]. Despite promising functional readouts, substantial gaps persist: specificity of EV tropism to cardiomyocytes in vivo, quantitative assays for mitochondrial integration and long-term functional persistence of transferred organelles, the inflammatory risk posed by extracellular mtDNA/mitoDAMPs, and the urgent need for standardized EV isolation, characterization, dosing, and biodistribution protocols to enable reproducible translation [2,12,13,14,15].

This review synthesizes biomolecular mechanisms by which EVs modulate mitochondrial dynamics in cardiac regeneration, surveys recent therapeutic preclinical evidence, and identifies translational barriers and opportunities. By integrating insights from molecular biology, EV engineering, and cardiac pathophysiology, we outline how EV-based mitochondrial targeting may emerge as a natural evolution beyond traditional stem cell therapies, becoming a clinically viable strategy for myocardial repair.

Beyond canonical dynamics, mitochondria undergo structural and metabolic remodeling that critically shapes cardiomyocyte adaptation to stress. Remodeling encompasses cristae reshaping, supercomplex assembly, and shifts in substrate utilization, collectively optimizing oxidative phosphorylation efficiency. Inner-membrane fusion protein OPA1 mediates cristae architecture [20], while the MICOS complex regulates cristae junction stability and electron transport chain (ETC) supercomplex organization [21]. EVs, particularly from iPSC-derived cardiomyocytes (iPSC-CMs), can promote metabolic maturation by enhancing oxidative metabolism over glycolysis, likely via delivery of proteins and miRNAs that fine-tune ETC assembly and mitochondrial substrate preference. Integrating remodeling into the EV–mitochondrial axis expands the mechanistic framework: EVs not only restore structural integrity and dynamics but also recalibrate metabolic and bioenergetic competence in stressed cardiomyocytes [21].

Structural remodeling of mitochondria not only restores organelle integrity but also drives functional adaptation across the cardiac cellular ecosystem. EV-mediated interventions can recalibrate cardiomyocyte metabolism, promote substrate flexibility, and enhance immunometabolic crosstalk with resident fibroblasts and immune cells. By integrating remodeling with systems-level considerations, EVs act as modulators of networked bioenergetics, coordinating oxidative phosphorylation, ROS buffering, and inflammatory signaling to optimize cardiac repair. This perspective frames remodeling as an active driver of therapeutic benefit rather than a mechanistic footnote, highlighting the interplay between structural dynamics, metabolic reprogramming, and multicellular functional integration.

EVs—including small exosomes (50–150 nm), larger microvesicles, and mitochondrial-derived vesicles (MDVs)—carry complex cargo (mtDNA, mitochondrial proteins such as TFAM and components of the electron transport chain, cardiolipin, mitochondrial RNAs, miRNAs and proteins that regulate fission/fusion) and can affect intercellular modulation of mitochondrial quality control. EVs may deliver intact mitochondrial fragments or functional mitochondria (mitoEVs/M-EVs) that transiently restore respiration, or transfer regulatory small RNAs that alter expression or post-translational modification of DRP1, MFN2 or OPA1 in recipient cardiomyocytes [7,22].

Mechanistically, EV uptake by cardiomyocytes occurs via endocytosis, membrane fusion or receptor-mediated internalization; subsequent release of mitochondrial cargo into the cytosol or direct mitochondrial fusion can replenish ATP production and reduce ROS, while EV-delivered miRNAs can downregulate pro-fission signaling (e.g., suppress DRP1 expression) or upregulate mitofusins and biogenesis programs (PGC-1α pathway). Preclinical studies demonstrate that MSC-derived and iPSC-derived EVs stabilize mtDNA, improve ΔΨm, reduce ROS and activate PINK1/Parkin-dependent quality control—effects amplified when EVs are engineered for mitochondrial targeting or loaded with specific miRNAs/proteins [23,24,25]. Key regulators are summarized in Table 1.

In addition to revitalization, extracellular vesicles play an emerging role in mitochondrial quality control through a process known as transmitophagy—the intercellular transfer and degradation of damaged mitochondria. This mechanism complements traditional mitophagy by allowing cells to outsource mitochondrial clearance via vesicular pathways, thereby preserving tissue homeostasis under stress conditions [17]. Integrating transmitophagy into the framework of mitochondrial EV biology highlights a broader function of EVs beyond metabolic rescue—extending to quality control and mitochondrial turnover across tissues.

iPSC-CM-derived EVs represent a convergence of stem cell therapy and organelle-level mitochondrial transfer, offering a dual modality for myocardial repair. Beyond providing paracrine signals that enhance cardiomyocyte maturation and survival, these EVs deliver functional mitochondrial components, including ETC proteins, mtDNA, and metabolically active mitochondria, thereby complementing canonical metabolic maturation strategies in iPSC-CMs [2,3]. Notably, our work demonstrates that iPSC-CM EVs can accelerate oxidative metabolism, modulate fusion/fission dynamics, and rescue ΔΨm in stressed cardiomyocytes, filling a critical gap between conventional iPSC-derived cell therapy and direct mitochondrial replacement approaches. These findings suggest a translationally tractable route for precision EV therapy that leverages both network-level signaling and direct bioenergetic restoration, establishing iPSC-CM EVs as a mechanistic bridge from preclinical studies to potential clinical interventions [12,13,14,15]. These findings [2,3,12,13,14,15], led by our group, establish a foundational mechanistic framework that uniquely positions iPSC-CM EVs as a translational bridge between preclinical studies and clinical applications.

Most cardiac-focused studies use EVs derived from MSCs, induced pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac progenitor cells, or cardiac fibroblasts. MSC-EVs commonly carry mitochondria-protective microRNAs (e.g., miR-21, miR-222), mitochondrial proteins (ATP synthase subunits), and mitochondrial DNA fragments that correlate with preserved oxidative phosphorylation (OXPHOS) and reduced ROS in recipient cardiomyocytes [24,34]. Notably, "mito-EVs"—EVs containing intact or partial mitochondria—have been isolated from iPSC–cardiomyocyte cultures and shown to restore mitochondrial respiration in injured cardiomyocytes in vitro and improve function in large-animal MI models [24,35].

Studies have employed intravenous, intracoronary, and intramyocardial delivery routes; larger mitochondria-rich EVs (L-EVs) appear more likely to fuse directly with cardiomyocyte membranes, whereas small EVs (sEVs/exosomes) act via endocytosis and cargo release into endolysosomal or cytosolic compartments. Cargo engineering (miRNA loading, peptide-targeting moieties, or surface display of cardiotropic ligands) has improved myocardial uptake and mitochondrial targeting in rodent models [36,37].

Mechanistic readouts consistently report modulation of canonical mitochondrial regulators: decreased DRP1 phosphorylation (reduced fission), increased MFN1/MFN2/OPA1 expression (enhanced fusion), activation of PINK1–Parkin-mediated mitophagy where appropriate, restoration of mitochondrial membrane potential (ΔΨm), improved complex I–IV respiration, and lowered mitochondrial ROS. These molecular shifts translate to improved ATP content, preserved calcium buffering, reduced cardiomyocyte apoptosis, and smaller infarct sizes in ischemia–reperfusion and chronic heart-failure models [7,10].

EV cargo exerts effects through: (1) direct mitochondrial donation/fusion, (2) delivery of regulatory miRNAs that downregulate fission mediators or upregulate fusion/biogenesis genes (PGC-1α, NRF1), (3) transfer of mitochondrial proteins that stabilize electron transport chain complexes, and (4) modulating recipient cell mitophagy and inflammatory signaling. Recent omics and imaging studies corroborate functional mitochondrial transfer and bioenergetic rescue [5,38].

Translation is nascent: reproducible large-animal efficacy exists (porcine MI models) but human trials specifically examining mito-targeting EVs are limited; safety, immunogenicity, and standardized manufacturing remain unresolved. Heterogeneity in EV isolation, inconsistent dosing metrics (particle vs. protein vs. functional titers), and inadequate in vivo tracking impede cross-study comparability. Key gaps include quantitative assays for mitochondrial transfer, long-term fate of donor mitochondria, and off-target effects (immune activation, oncogenic risk) [39,40].

The translation of EV-based mitochondrial therapies from preclinical models into human studies is accelerating but remains at an early phase. Systematic evaluations of registered clinical trials indicate an increasing number of EV-related interventional studies across therapeutic areas, and early-phase cardiac-focused trials have begun to appear. Notably, a contemporary systematic review of EV clinical trials documented a rapid expansion of EV interventional studies and highlights the predominance of phase I/II designs focused on safety and feasibility. In the diovascular domain specifically, several early-phase and first-in-human studies now evaluate MSC- or stem cell-derived EVs administered by intravenous or intracoronary routes; at least one protocol evaluating intravenous MSC-derived EVs in cardiac indications is registered (ClinicalTrials.gov NCT06002841 [41]), signifying a move from preclinical large-animal validation toward human testing. These trials are typically designed to address (a) safety and immunogenicity, (b) biodistribution and persistence, and (c) early efficacy signals (cardiac biomarkers, imaging endpoints). These endpoints reflect the critical translational questions for mitochondrial-targeted EVs: whether mitochondrial cargo (mtDNA, TFAM, ETC proteins, intact mitochondria) persists, integrates functionally into host mitochondria, and improves organ-level bioenergetics without provoking detrimental inflammatory responses to extracellular mitochondrial components (e.g., mitoDAMPs).

Table 2 summarizes representative preclinical and early clinical studies that directly evaluated EV-mediated mitochondrial outcomes in cardiac contexts or that are registered to test EV therapeutics in cardiac patients. The table highlights key mechanistic endpoints (ΔΨm, ATP production, OCR, PINK1/Parkin activation), delivery routes, and principal translational limitations.

The principal translational bottlenecks for EV-based mitochondrial therapies are regulatory classification, potency assays, and scalable GMP manufacture. Regulatory agencies (FDA/EMA) currently consider EV therapeutics in contexts that span biologics, cell-derived products, and drug-delivery platforms; this creates ambiguity for potency definitions and comparability criteria. EVs carrying mitochondrial cargo raise unique safety concerns (extracellular mtDNA and mitoDAMPs) that can elicit innate immune activation via cGAS–STING/TLR9 pathways; thus, preclinical packages must include sensitive assays for immune activation and for potential horizontal transfer of mtDNA variants.

From a manufacturing standpoint, scalable production of mitochondria-enriched EV subpopulations (mito-EVs/L-EVs) remains technically demanding. Standardization requires (i) source cell banking under GMP, (ii) scalable culture systems (bioreactors), (iii) robust fractionation (TFF + SEC) to separate small EVs from L-EV/mito-EV fractions, and (iv) validated potency assays tailored to mitochondrial rescue (e.g., donor EV ATP-generating capacity, recipient ΔΨm rescue potency, or OCR-based functional assays). Recent progress in tangential flow filtration and multi-omic EV characterization provide feasible paths forward, but consensus potency endpoints remain to be defined.

Collectively, preclinical evidence supports EV-mediated modulation of mitochondrial dynamics as a mechanistically plausible and therapeutically promising strategy for myocardial repair. Rapid progress in engineered EVs and improved characterization methods make clinical translation feasible—provided that rigorous standardization, biodistribution profiling, and safety studies are prioritized [36,46].

Engineered EVs are routinely loaded with mitochondria-protective proteins (e.g., SIRT3, ATP5A1), mitochondria-stabilizing small proteins/peptides, mito-protective miRNAs, and enzymatic antioxidants. These cargos directly restore bioenergetics and redox balance in injured cardiomyocytes. Recent reports demonstrate nanoscale exosomes co-loaded with SIRT3 and metabolic modulators that increase mitochondrial respiration (OCR), raise complex I–IV activity, and reduce mitochondrial ROS and mPTP opening after ischemia–reperfusion injury. Engineered surface ligands (peptide motifs targeting cardiomyocyte troponin I or ischemia-exposed integrins) enhance myocardial tropism and EV uptake via receptor-mediated endocytosis or fusion, increasing intramitochondrial rescue efficiency [10,47]. Translational status: mostly preclinical; cardiac-specific tropism strategies moving toward early large-animal studies.

Larger EV subtypes (mitoEVs, M-EVs) can carry entire mitochondria or mitochondrial fragments (mtDNA, OXPHOS complexes). Transfer of these cargoes restores ATP production, ΔΨm, and respiratory chain integrity in recipient cardiomyocytes. Preclinical large-animal models show autologous M-EVs reduce infarct size and improve ejection fraction by reconstituting respiratory complexes and increasing ATP/ADP ratios in peri-infarct tissue. Mechanistically, transferred mitochondria integrate into host mitochondrial networks and promote fusion/fission recalibration via MFN2/OPA1 upregulation and DRP1 modulation [24,35]. Translational status: advanced preclinical; autologous human M-EV studies planned.

EVs shuttle miRNAs that regulate key dynamics regulators (e.g., miR-499/-30 family affecting DRP1 phosphorylation, miR-21/-214 influencing PINK1/Parkin signaling). Recent work demonstrates EV-mediated mitochondrial import of small RNAs, enabling functional modulation of fusion/fission proteins and downstream mitophagy/apoptosis pathways (caspase-9, BAX/BCL2 balance). High-throughput barcoding of sEVs loaded with gRNA allows pooled screening of EV cargo effects on recipient mitochondrial phenotypes [48,49]. Translational status: preclinical; mechanistic proof-of-concept established.

EVs have been engineered to deliver CRISPR/Cas RNPs and base editors targeting nuclear-encoded mitochondrial regulators (e.g., DRP1 phosphorylation sites, MFN2 expression) or nuclear genes that indirectly regulate mitochondrial quality control. Encapsulation strategies protect RNPs during circulation and enable endosomal escape in cardiomyocytes. These approaches have been validated in noncardiac models and are now adapted for cardiac mitochondrial targets [50,51]. Translational status: early-stage preclinical.

EV-mediated mitochondrial signaling in oncology and neurodegeneration governs metabolic rewiring, immune-metabolic crosstalk, and senescence. These insights are now repurposed for heart failure: EV-delivered mito-signals modulate immune cell phenotype (macrophage polarization), influence fibroblast activation, and alter cardiomyocyte substrate preference (fatty acid vs. glucose oxidation). Integration with immunometabolism enables combination strategies (EV + immunomodulator) to foster reparative inflammation resolution [5,11].

While current studies elucidate detailed molecular mechanisms of EV-mediated mitochondrial modulation, a systems-level framework is needed to unify these insights and guide translational impact [52]. EVs should be conceptualized not merely as cargo carriers but as network modulators orchestrating bioenergetics across multiple cardiac cell types. Integration of metabolomics (ATP/ADP ratio, ROS flux, NAD+/NADH balance), single-cell and spatial omics (differential EV responses in cardiomyocytes, fibroblasts, immune cells), and network modeling offers a holistic understanding of EV-mediated cardiac repair [52,53]. This framework positions EVs as precision tools in systems medicine, linking mitochondrial bioenergetics, immunometabolism, and tissue remodeling to create a unifying paradigm for regenerative cardiology. Multi-omic integration can guide EV engineering, dosing, and patient-specific therapeutics, accelerating clinical translation [54,55,56].

Schematic illustration of how mitochondria-enriched extracellular vesicles (EVs) restore cardiomyocyte function. The figure summarizes the major EV subtypes (small EVs, large EVs, and mitochondria-derived EVs), their representative cargos, and the downstream remodeling effects that enhance mitochondrial structure, oxidative phosphorylation efficiency, and overall cardiac repair.

Extracellular vesicles represent a multiscale mitochondrial delivery system encompassing small EVs (exosomes), large EVs (microvesicles), and mitochondria-derived EVs released under cellular stress (Figure 2). Their cargos—ranging from miRNAs and mitochondrial DNA to respiratory-chain proteins—collectively mediate mitochondrial repair and bioenergetic recovery in recipient cells. Following uptake, recipient mitochondria undergo cristae remodeling, stabilization of respiratory supercomplexes, and metabolic reprogramming that optimize oxidative phosphorylation and ATP generation. These remodeling events translate into improved cellular bioenergetics, reduced oxidative stress, and enhanced cardiomyocyte survival.

1. EV Types (The Delivery System): The figure depicts three primary types of EVs involved in mitochondrial communication and therapy: a. sEV (small EV): Generally referred to as exosomes, these are typically 30–150 nm in size. They are critical carriers of soluble factors and genetic material (miRNAs, proteins) that regulate mitochondrial function remotely, b. L-EV (Large EV): Also known as microvesicles or microparticles, these are larger (100–1000 nm+) and can carry entire organelle fragments, making them capable of transferring substantial mitochondrial components, c. mitoEV (Mitochondria-derived EV): A specialized subset, often released under cellular stress, that is specifically enriched with mitochondrial components (lipids, proteins, mtDNA fragments), acting as a highly targeted repair vehicle for damaged mitochondria. 2. Cargo (The Therapeutic Payload): These EVs carry a variety of molecular signals that mediate the therapeutic effect upon internalization by recipient cells: a. miRNAs (microRNAs): Non-coding RNAs that act as master regulators by targeting mRNA transcripts essential for mitochondrial biogenesis, dynamics, and the expression of Electron Transport Chain (ETC) subunits. b. Mitochondrial Fragments (Mitochondrial DNA/Lipids): Direct structural and functional components of the mitochondria that can be integrated into the recipient cell's existing mitochondrial pool, providing raw materials for repair and replenishment of damaged organelles, c. Proteins: These include enzymes, transcription factors, and key components of the ETC (e.g., Complex I or III subunits) that are directly incorporated into the recipient cell's mitochondria to immediately enhance or stabilize function. 3. Remodeling Effects (Recipient Mitochondrial Changes): Upon cargo delivery, the recipient cell's mitochondria undergo structural and functional reorganization: a. Cristae: Remodeling of the inner mitochondrial membrane structure, often resulting in an increase in cristae density and complexity, which directly correlates with enhanced surface area for oxidative phosphorylation (OXPHOS) and increased ATP production capacity, b. Supercomplexes: The stable assembly and stabilization of respiratory supercomplexes (e.g., the Respirasome, involving Complexes I, III, and IV). This organizational structure optimizes electron transfer efficiency, reduces electron leakage, and minimizes the production of reactive oxygen species (ROS), c. Substrate Shifts (e.g., FAO, Glycolysis): Metabolic reprogramming where the cell's preference for energy substrate changes. This can involve an improved capacity for Fatty Acid Oxidation (FAO), a shift toward optimized OXPHOS, or a switch toward glycolysis (Warburg effect) based on the specific tissue context and injury state. 4. Downstream Impact (Therapeutic Outcomes): The successful remodeling of recipient mitochondria translates into systemic and tissue-specific therapeutic benefits: a. Bioenergetics (ATP Production): The fundamental outcome is the restoration of cellular energy homeostasis, characterized by increased total ATP production, higher mitochondrial membrane potential (∆ψ m), and overall improved cell viability, b. Immunometabolism (Polarization): The influence of mitochondrial state on immune cell function. EV therapy can shift inflammatory immune cells (e.g., M1 macrophages) toward a reparative, anti-inflammatory phenotype (e.g., M2 polarization), modulating the local inflammatory environment, c. Cardiac Regeneration (Angiogenesis, Myocyte Proliferation): Specific to cardiovascular repair, the improved bioenergetics supports crucial healing processes, including stimulating angiogenesis (new blood vessel formation) and promoting the survival and proliferation of recipient cardiomyocytes (myocyte proliferation), leading to improved tissue function after injury.

Key hurdles remain: EV heterogeneity, inconsistent isolation methods (ultracentrifugation, size-exclusion, tangential flow filtration), lack of quantitative metrics for mitochondrial cargo per vesicle, and incomplete biodistribution data. Emerging solutions include standardized multi-omic EV characterization workflows, CRISPR barcoding for in vivo tracking, and cardiomyocyte-targeted ligands to improve therapeutic index. Harmonized preclinical models and agreed reporting standards are essential to accelerate safe clinical translation [41,57].

Despite the promising potential of extracellular vesicles (EVs) in targeting mitochondrial dynamics for cardiac repair, several technical, biological, and ethical challenges impede their clinical translation.

The isolation and characterization of EVs remain inconsistent due to the lack of standardized protocols, leading to variability in purity and yield. This heterogeneity complicates the assessment of their mitochondrial targeting efficiency and therapeutic efficacy. Moreover, tracking EVs in vivo poses significant challenges; current labeling techniques often suffer from low sensitivity and potential interference with EV function, hindering accurate biodistribution studies. Additionally, quantifying the precise mitochondrial effects of EVs is difficult due to the complex interplay of signaling pathways and the dynamic nature of mitochondrial processes. Advanced imaging and biochemical assays are required to dissect these interactions at a molecular level.

The specificity of EV-mediated delivery to target mitochondria is influenced by the source of EVs, their cargo content, and surface markers. While engineered EVs show promise in enhancing targeting precision, their design must consider the complex extracellular environment and potential interactions with non-target cells. Furthermore, the biodistribution of EVs is affected by systemic clearance mechanisms, including uptake by the mononuclear phagocyte system, which can limit their therapeutic reach. Understanding the kinetics of EV circulation and tissue penetration is crucial for optimizing their delivery to myocardial tissues.

The clinical application of EVs raises several ethical and safety issues. The potential for EVs to transfer oncogenic material or induce immune responses necessitates thorough evaluation of their long-term effects. Off-target delivery and unintended cellular uptake could lead to adverse outcomes, including inflammation or fibrosis. Rigorous preclinical and clinical studies are essential to assess the safety profile of EV-based therapies and establish guidelines for their use in regenerative cardiology. In parallel, regulatory frameworks established by agencies such as the FDA and EMA provide guidance on EV characterization, safety evaluation, and clinical trial design, ensuring that ethical and biosafety considerations are rigorously addressed [57,58].

Addressing these challenges requires interdisciplinary approaches combining molecular biology, bioengineering, and clinical expertise. Advancements in EV isolation techniques, cargo engineering, and in vivo tracking methods are pivotal to overcoming these barriers. Furthermore, comprehensive safety evaluations and ethical considerations must guide the development of EV-based mitochondrial therapies to ensure their efficacy and patient safety [59,60]. Table 3 summarizes the major technical, biological, and ethical challenges of EV-mediated mitochondrial modulation alongside proposed strategies to address them.

A prerequisite for clinical translation is the establishment of robust manufacturing and regulatory frameworks. GMP-grade EV production has been demonstrated using bioreactor-based expansion of MSCs and iPSCs, coupled with scalable isolation techniques such as tangential flow filtration and size-exclusion chromatography [57,58,61,62]. Regulatory agencies (FDA, EMA) emphasize standardization of characterization metrics (particle number, protein content, potency assays), long-term safety evaluation, and reproducibility across batches [63].

For mitochondrial-targeted EVs specifically, additional checkpoints are required: biodistribution studies, mitochondrial cargo quantification, and assessment of off-target effects, including potential transfer of oncogenic or pro-inflammatory mtDNA. Several early-phase clinical studies using EVs for cardiac or neurological indications provide proof-of-concept for safety and feasibility [64,65], yet no trial has explicitly focused on EV-mediated mitochondrial repair. Building this translational bridge requires integrated pipelines combining GMP production, validated mitochondrial potency assays, and regulatory harmonization across agencies, enabling safe and reproducible clinical translation.

A structured roadmap bridging academia, biotech, and pharma is essential to accelerate adoption of EV-mediated mitochondrial therapies. Key elements include: GMP-grade production: scalable bioreactor expansion and standardized isolation. Potency assays: designed for mitochondrial rescue (bioenergetic restoration, uptake, functional readouts). Safety and regulatory checkpoints: biodistribution, immunogenicity, and long-term off-target effect monitoring. Integration with biotech/pharma pipelines: enhanced drug delivery, engineered EV cargo for mitochondrial modulation, and alignment with regulatory requirements.

Translational note: combinatorial strategies, such as EVs + small molecules or EVs + immunomodulators, may enhance repair efficiency and accelerate clinical impact. Collectively, these steps ensure reproducible, safe, and effective applications of mitochondrial-targeted EV therapies.

Patient-specific genotypes, including mtDNA variants and nuclear-encoded mitochondrial genes, should inform EV cargo selection. Tailored approaches aim to maximize efficacy and minimize variability in treatment response.

Uniform protocols for EV preparation, characterization, and application are imperative. Standardization will facilitate cross-study comparisons and enhance reproducibility. International guidelines should harmonize methodologies, ensuring consistency and reliability in EV-based research and therapies.

The complexity of mitochondrial dynamics and EV biology necessitates collaboration across disciplines. Bioengineers can design EVs with specific targeting capabilities; systems biologists can model EV–mitochondrial interactions; cardiac surgeons and clinicians are critical to translating laboratory findings into clinically feasible interventions.