Nanoscale Delivery of MicroRNAs via Extracellular Vesicles: Mechanisms Potential in Modulating Cellular Senescence

Abstract

Senescence refers to the gradual decline in function and physiological integrity of organisms over time, signifying an irreversible natural process of life. Senescence is associated with abnormal alterations in intercellular communication, where signaling molecules exchanged between cells can either accelerate or impede this process. In the rapidly evolving field of nanomedicine, extracellular vesicles (EVs) have emerged as natural nanoscale delivery vehicles that facilitate the highly efficient transfer of diverse cargoes, including microRNAs (miRNAs), DNA, lipids, and proteins. Compared to synthetic nanocarriers, EVs possess superior biocompatibility and the unique ability to cross biological barriers. MiRNAs encapsulated within these endogenous nanoparticles have demonstrated the ability to regulate several aging processes both in vivo and in vitro. Notably, although there are still some challenges regarding the clinical translation of EV-derived miRNAs from stem cells, macrophages, and the circulatory system, their potential for nanotherapeutic applications in the field of anti-aging remains undeniable.Here, we comprehensively summarize the regulatory functions of EV-delivered miRNAs during the aging process and propose novel bio-inspired nanomedicine strategies for the anti-aging applications of EVs.

Graphical Abstract

Introduction

Senescence is an inescapable natural rule of existence, notwithstanding the significant endeavours of scientists to prolong human longevity and health span. As an irreversible and progressive physiopathological process, senescence can have significant implications for accelerating cell renewal, regulating tissue as well as organ growth, and maintaining homeostasis in the internal environment.¹

In order to better understand the process of senescence, López-Otín et al² provided a summary of nine markers of senescence, including genomic instability, telomere wear and tear, epigenetic alterations, loss of protein homeostasis, nutrient sensing dysregulation, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication In recent years, autophagy and chronic inflammation have also come to be recognized as a feature of senescence.³

Although aging significantly influences organismal regeneration, disease resistance, and homeostasis, its excessive accumulation might result in diminished tissue and cellular capabilities.⁴ This phenomenon is accompanied by a significant increase in the risk of various diseases associated with the aging process, including neurodegenerative diseases (eg, Alzheimer’s disease,⁵ Parkinson’s disease),⁶ cardiovascular diseases,⁷ and immune system disorders.⁸ Aging poses a serious challenge to human longevity and healthy aging.

EVs function encompasses not only its involvement in the senescence process but also its capacity to regulate the development of senescence-related diseases. The underlying mechanism by which EVs fulfill these functions is through the unique intercellular communication pathways that they employ. The therapeutic efficacy of EVs derived from both mesenchymal stem cells (MSCs)⁹ and endothelial colony-forming cells (ECFCs)¹⁰ has been demonstrated in the context of atherosclerosis and plaque development.

From a nanomedicine perspective, EVs are naturally occurring lipid-bilayer nanoparticles (typically 30 to 1000 nm in diameter) that can be released by the majority of cells, irrespective of physiological or pathological states.¹¹ As endogenous nanocarriers, EVs offer significant advantages over artificial liposomes or polymeric nanoparticles: they exhibit excellent biocompatibility, low immunogenicity, and inherent targeting capabilities, and can penetrate stringent physiological barriers such as the blood-brain barrier; However, numerous challenges associated with them cannot be overlooked, such as quality instability resulting from large-scale production, high heterogeneity caused by certain isolation methods, low efficiency of tissue-targeted delivery, and the need for long-term safety assessments.

As a heterogeneous group of cell-derived membrane structures, they can be categorized into two subtypes depending on their biogenesis. The first subtype consists of microvesicles, extracellular bodies, and microparticles that are shed directly from the plasma membrane. The second subtype consists of exosome that are released through the endosomal system.¹² Exosomes have the same topology as cells,¹³ and their biogenesis commences with endocytosis and plasma membrane invagination. The process of endocytosis gives rise to the formation of early-sorting endosomes (ESEs), which subsequently evolve into multivesicular vesicles (MVBs) subsequent to the influence of organelles or the fusion of these endosomes with one another. Thereafter, MVBs mature into late-sorting endosomes (LSEs). The process of membrane remodeling, including the sorting and enrichment of cargo molecules, the invagination and outgrowth of MVB membranes, and the generation of intraluminal vesicles (ILVs), is crucial for the subsequent fusion of ILVs with the cytoplasmic membrane, leading to the release of ILVs in the form of exosome release (Figure 1). ^14–17 This process is usually mediated by three mechanisms: the endosomal transport essential sorting complex (ESCRT)-dependent mechanism, the specific lipid molecule (ceramide, phosphatidic acid, sphingosine-1-phosphate, etc.) driven mechanism, and the four-pass transmembrane protein family driven mechanism,¹⁸ but the study of the synergistic regulatory effects of the above three types of mechanisms needs to be further investigated. In clinical studies of EVs, it is difficult to define subgroups by examining biogenesis, so the International Society for Extracellular Vesicles proposed another classification based on physical characteristics.¹⁹ EVs with diameters less than 200 nm are referred to as small EVs (sEVs), while those with diameters greater than 200 nm are referred to as medium or large EVs (M/L EVs). For ease of description, they are collectively referred to as EVs in the following section (Figure 1).

Figure 1.

EVs mainly include exosomes and microvesicles. Exosomes originate from the endosomal pathway, involving early-sorting endosomes (ESEs), multivesicular bodies (MVBs), and their fusion with the plasma membrane for release. In contrast, microvesicles are generated by direct outward budding of the plasma membrane. EVs carry diverse bioactive cargos, including miRNAs, proteins, and lipids, and mediate intercellular communication.

Initially recognized only as waste products shed by cells in biological fluids, a growing number of studies have demonstrated that EVs are not only capable of and involved in a variety of physiological roles, but also modulate a wide range of disease processes, including cellular homogenization, infection transmission, cancer development, and cardiovascular disease.²⁰ This is attributed to the unique function of EVs: mediating intercellular communication through molecular cargo delivery and biological signaling.²¹ Cells act by delivering EVs to target cells via autocrine, paracrine or endocrine secretion.^11,22 The key components of the effect are usually the cargoes - nucleic acids, proteins, lipids - that are loaded for the EVs by their secreting cells.²³ These cargoes can induce a variety of phenotypic responses upon uptake and transfer by target cells.²⁴ For instance, extravasated EVs (EVs containing high mobility group protein B1 [HMGB1]) have been observed to produce pro-inflammatory cytokines and chemokines. This phenomenon occurs through the binding of EVs to neutrophil Toll-like receptor 4 (TLR4), which subsequently activates neutrophils.^25,26

Nucleic acids, a major cargo of EVs, play critical roles in various physiological processes involving EVs. EVs contain a diverse array of nucleic acids, including but not limited to miRNAs, messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), ribosomal RNAs (rRNAs), and other types of nucleic acids. Sequencing analysis has revealed that miRNAs constitute the predominant class of small RNA molecules within EVs.²⁷ miRNA is a non-coding RNA composed of 18 to 25 nucleotides. It is initially transcribed into a primary-miRNA (pri-miRNA) form. Subsequently, it is transcribed into the nucleus by RNA polymerase II/III. Then, it is cleaved by Drosha and its cofactors into pre-miRNA. Finally, it is translocated into the cytoplasm, where it is processed by Dicer into a mature microRNA product.^28–30 The function of miRNA is to regulate gene expression by targeting mRNAs³¹ (Figure 2). The lipid-like bilayer structure of EVs enables the stabilization of miRNAs in enzymatic lysis within body fluids, circumventing immune rejection, facilitating the exchange of information between cells,³² and thereby underpinning the diverse physiological functions of EVs. Although numerous studies have demonstrated that miRNAs mediated by EVs are involved in aging-related signaling pathways, the mechanisms underlying the roles of miRNAs across different cell types, aging models, and disease contexts have not yet been fully integrated. Furthermore, from the perspective of nanomedicine, the mechanistic relationship between EVs-miRNA transfer and aging-related phenotypes has not yet been systematically evaluated. Therefore, this study aims to provide a comprehensive review of the mechanisms by which EV-miRNAs regulate aging, with a particular focus on the roles and mechanisms of action of different types of EV-miRNAs.The results of this study will hopefully serve as a foundation for further research on EVs.

Figure 2.

miRNAs are transcribed as pri-miRNAs in the nucleus, processed into pre-miRNAs by Drosha, exported to the cytoplasm, and cleaved by Dicer into mature miRNAs. These miRNAs are selectively packaged into EVs and released extracellularly, reaching recipient cells via autocrine, paracrine, or endocrine pathways. In target cells, miRNAs bind to the 3′UTR of mRNAs, leading to translational repression or degradation, thereby regulating senescence-associated signaling pathways.

Extracellular Vesicle miRNAs Accelerate Senescence Process

It is both unreasonable and unscientific to describe the role of EVs in senescence in absolute terms. In their role as a medium for communication, it is the cargo loaded in EVs that typically exerts its influence, rather than the EVs themselves. However, it should be noted that different cargoes often fulfill distinct functions. MiRNAs in EVs fulfill a multifaceted role in the senescence process. They can be utilized as a biomarker, reflecting the state of senescence and propagating the microenvironment of senescence. Furthermore, they can be employed as a therapeutic means to decelerate the senescence process.

Cellular Senescence

Cells are the bearers of life functions, and cellular senescence is one of the most significant processes of aging. The process of cellular senescence is initiated when a cell sustains damage to its DNA, telomeres, or oncogenes.³³ This results in the activation of a permanent proliferative cycle arrest, which effectively prevents the development of tumors and serves as the body’s intrinsic defense mechanism against cancer.³⁴ However, the release of a variety of senescence- and inflammation-related substances by senescent cells, such as senescence-associated secreted phenotypes (SASPs), has been demonstrated to perturb the extracellular environment and propagate the senescence state, inducing a variety of adverse effects.³⁵ In recent years, EVs have been shown to be an important contributor to SASPs, which are capable of propagating the senescence microenvironment and leading to senescence after being released by senescent cells into the extracellular environment.^36,37 Nucleic acids, a component of EVs, have been demonstrated to play a pivotal role in this process.^38,39 The release of EVs by aged keratinocytes that carry miRNA-30a has been demonstrated to interfere with the proliferation cycle of young cells. This interference leads to a reduction in the healing speed of skin wounds and the induction of cellular senescence.⁴⁰ In a similar vein, Fulzele et al⁴¹ discovered that levels of miR-34a-5p in muscle-derived circulating EVs were positively associated with age. These findings indicated that the presence of these EVs in the circulation led to a decline in the viability of bone marrow mesenchymal stem cells (BMSCs), a halt in their cell cycle progression, and an acceleration in the senescence of these cells, thereby creating a vicious cycle. Lee et al⁴² isolated and characterized EVs from non-senescent human dermal fibroblasts (HDFs) and senescent HDFs. Their findings revealed that miR-10a, miR-30c, and miR-451a were overexpressed in senescent HDF-EVs. In vitro experiments revealed that the promotion of HDF senescence by miR-10a, miR-30c, and miR-451a is associated with the regulation of reactive oxygen species cluster (ROS) production and mitochondrial autophagy. This study establishes these microRNAs as senescence factors in senescent HDF-EVs. The activation of the p53/p21CIP1 and p16INK4A/pRb pathways results in the arrest of the cell cycle, leading to cellular senescence.⁴³ The deletion of the anti-aging molecules Sirtuin-1 and Sirtuin-6 plays a pivotal role in this process.^44,45 In the event of oxidative stress, a decrease in phosphatase tension protein homologs (PTEN) will activate the p38-MAPK and PI3K-mTOR signaling pathways. The cargoes of the miR-34a and 570, contained within secreted EVs from lung cells, are regulated by this pathway, and they effectively reduce the expression of sirtuin-1 (SIRT1) and SIRT6. This reduction in expression, consequently, promotes the process of cellular senescence and the local expansion of senescence within the lung.⁴⁶

Genomic Instability

Genes are the fundamental components responsible for the storage of genetic information. As the cornerstone of life’s activities, genomic instability has been a central driver of senescence for an extended period. Genomes are perpetually exposed to DNA damage sources, including exogenous factors such as These include UV light, X-rays, compounds present in food, water, and air, as well as endogenous factors such as ROS, aldehydes, and advanced glycosylation end products (AGEs). Spontaneous reaction (hydrolysis) damage is another potential source of genomic instability.⁴⁷ While most of the damage is removed by the repair system, some remains due to faulty repair methods or inability to repair, among other factors. Accumulation of these factors can lead to genomic instability and the subsequent progression of senescence.⁴⁸ The effects of EVs on genome destabilization are complex and multidimensional. Intuitively, EVs have the capacity to induce direct DNA damage. For instance, the levels of DNA methylation, a crucial epigenetic mark, exhibit a marked decline over time. In a model consisting of senescent human umbilical vein endothelial cells (HUVECs), senescent cells release EVs carrying miR-21-5p and miR-217, which target the expression of DNA methyltransferase 1 and Sirtuin-1 in normal cells. While increasing their markers of senescence and acquiring a senescent phenotype, these EVs also lead to partial de methylation, thereby impairing genomic stability.⁴⁹ Veitch et al⁵⁰ discovered that cargo miR-30d-5p and miR-30e-5p, loaded by circulating EVs derived from senescence cardiac endothelial cells, enhanced cardiac oxidative stress and increased the expression of the DNA damage marker phosphorylated H2AX. Furthermore, EVs containing BCR-ABL1 mRNA, released from the human leukemia cell line K562, have been shown to upregulate BCR-ABL1 expression in BM-MSC in in vitro experiments. This process has been observed to enhance TGF-β1 secretion, resulting in DNA damage and subsequent malignant transformation.⁵¹ In addition to this, EVs contribute to genomic destabilization by interfering with the efficiency of the scavenging system. Human immortalized bronchial epithelial cell-derived EVs irradiated with 2 Gorey contain miR-1246, which can act as a transfer messenger to promote DNA damage by directly targeting the 3’UTR to down-regulate the expression of the LIG4 gene and reduce the efficiency of non-homologous end joining.⁵²

Immunity & Inflammation

Inflammatory senescence is closely related to immune senescence. Immune system senescence leads to immune cell dysfunction, resulting in an ineffective elimination of senescent cells and pro-inflammatory factors. Consequently, inflammation levels escalate, and the gradual accumulation of low-level chronic inflammation further exacerbates senescence, driving a vicious cycle.⁵³ EVs play a pivotal role in this process as mediators of intercellular communication. Telomeric repeat-containing RNA (TERRA) is a class of long-stranded non-coding RNAs transcribed from telomeric repeat sequences, which has been shown to inhibit telomerase activity⁵⁴ and is associated with senescence. cfTERRA is its cell-free form. In addition, Wang et al⁵⁵ have demonstrated that EVs containing cfTERRA can stimulate the production of inflammatory cytokines, such as TNF-α, IL-6, and CXCR4, enhance innate immune response signaling, and promote inflammatory senescence when incubated with immune response cells. Furthermore, EVs have been demonstrated to play a pivotal role in the polarization process of macrophages, thereby inducing inflammatory senescence. The activation state of macrophages dictates their categorization into two distinct groups: M1/M2-like macrophages. M1 macrophages predominantly exert pro-inflammatory effects.^56,57 Recent studies have demonstrated that miR-199a-5p of Human Kidney-2 cell-derived EVs, stimulated by human serum albumin (HSA), induces M1-like macrophage polarization by targeting the Klotho/TLR4 pathway.⁵⁸ Inflammatory Periodontal ligament stem cells -derived EVs with cargo miR-143-3p target and inhibit PI3Kγ expression by inhibiting PI3K/AKT signaling and activating the NF-κB signaling pathway thereby promoting M1-like macrophage polarization.⁵⁹ In a rat model of acute pancreatitis, miR-183-5p in EVs derived from adenoalveolar cells contributes to the polarization of macrophages toward M1 and exacerbates inflammation-related injury by down-regulating the expression of FoxO1 and inducing the release of pro-inflammatory cytokines.⁶⁰ In a renal tubulointerstitial inflammation model, Lv et al⁶¹ found that EVs mediated communication between tubular epithelial cells (TECs) and macrophages. The cargo miRNA-19b-3p was internalized by macrophages, and it directly targeted NF-κB/ SOCS-1, leading to polarization of the M1 phenotype. The extant literature suggests that EVs have the capacity to induce the polarization of monocytes to an M1 state, thereby amplifying the inflammatory response. Furthermore, EVs have been shown to modulate the immune response by transporting cytokines or other pro-inflammatory mediators. These mediators can directly act on target organs and influence inflammatory senescence.^62,63 In morphine-stimulated astrocyte-derived EVs, miRNA-23a is directly taken up by pericytes of the blood-brain barrier (BBB), resulting in a decrease in PTEN expression and subsequent migration of pericytes. Pericytes, integral components of the BBB, undergo migration, thereby modulating the function of the BBB, resulting in the influx of peripheral monocytes into the central nervous system (CNS)and the subsequent triggering of neuroinflammation.^64,65 Inflammation-stimulated neutrophil-derived EVs carrying miR-142-3p and miR-451 have been shown to impede the activation of ERK1/2 and eNOS-mediated signaling, while concomitantly up-regulating the expression of the inflammatory factors IL-6, IL-8, CXCL10, and CXCL11 following their internalization by endothelial cells (ECs), which leads to severe injury.⁶⁶ Research has shown that EVs produced from steatotic hepatocytes, which contain miR-1, activate the NF-κB pathway in ECs. Moreover, these EVs have demonstrated the capacity to suppress Kruppel-like factor 4 expression and augment the secretion of pro-inflammatory factors. Collectively, these effects foster endothelial inflammation and senescence, which contribute to the pathogenesis of atherosclerosis.⁶⁷ Tsukamoto et al⁶⁸ found that the expression of miRNA-129 is increased in senescent EVs present in the circulatory system. They further elucidated mir-129 mediates a negative feedback loop of NF-κB inhibition to compensate for the hyperinflammatory state associated with senescence. It has been demonstrated that miR-30b-5p is present in elevated concentrations within EVs derived from senescent cells. Following the internalization of these EVs by macrophages, a decrease in SIRT1 expression is observed, along with the activation of the NF-κB pathway. This, in turn, results in enhanced release of pro-inflammatory cytokines, stimulation of immune cells within the senescence microenvironment, and induction of chronic inflammation. This series of events contributes to the alteration of the inflammatory balance.⁶⁹ MSC-derived EVs carrying miR-21-5p have been shown to significantly increase SASP and inflammatory expression in recipient cells via Syndecan-1 (SDC1). SDC1 is a cell-surface acetylheparan sulfate proteoglycan that acts as a signaling “pivot” in the ECM adhesion site to exert pro-inflammatory effects.⁷⁰ Of particular significance is the bidirectional relationship between the network of EVs and inflammatory senescence. It has been established that inflammatory environments can also stimulate the secretion of EVs,^71,72 thereby progressively exacerbating the degree of senescence within a vicious circle network.

Mitochondrial Dysfunction

Mitochondria, the energy factories of the cell, are responsible for the production of most of the energy required by the cell to maintain normal life activities. Mitochondria also play a pivotal role in numerous critical biochemical processes within the cell, such as the regulation of the cell cycle and cell growth.^73,74 However, mitochondrial dysfunction may result in impaired energy metabolism, which can trigger a series of deleterious processes, including decreased respiratory chain enzyme activity, decreased mitochondrial membrane potential, decreased ATP synthesis, disruption of intracellular calcium homeostasis, impaired fatty acid β-oxidation, increased oxidative stress, and oxidative damage of mitochondrial DNA (mtDNA). These events can ultimately lead to cellular senescence and even cell death.^75–77 The proteome responsible for mitochondrial functions is jointly encoded by the nucleus and mtDNA, suggesting that communication between mitochondria and the nucleus is essential for its function.⁷⁸ As intercellular communication mediators, EVs play a role in this process as well. In the initial phase of acute myocardial infarction, miR-503 is found to be enriched in ECs-EVs, where it has been observed to bind directly to peroxisome proliferator-activated receptor gamma coactivator-1β (PGC-1β) and the mitochondrial deacetylase SIRT3. This results in the initiation of the PGC-1β/NRF-1 and SIRT3/PDH/ATP synthase communication axes, leading to the disruption of ETC complex-related genes and the deepening of PDH and ATP synthase acetylation, thereby impairing their activity. Finally, this results in mitochondrial dysfunction, which in turn leads to cardiomyocyte senescence and death.⁷⁹ It has been proposed that certain sources of EVs have the capacity to disrupt the energetic network of recipient cells. For instance, HeLa cells exposed to EVs from young and old individuals exhibited varied responses, with the former exhibiting enhanced and the latter exhibiting diminished oxygen consumption rates.⁸⁰ Comparable outcomes have been noted in clinical models of idiopathic pulmonary fibrosis (IPF): exosomes originating from lung fibroblasts that express miR-23b-3p and miR-494-3p inhibited SIRT3 expression in lung epithelial cells, resulting in the disruption of the mitochondrial energy network, increased levels of ROS, cellular damage, and ensuing cellular senescence.⁸¹ Concurrently, EVs miRNAs function as a rapid conduit between the inflammatory milieu and mitochondrial dysfunction. After 24 hours of exposure to TNF-α, significant increases in the levels of miR-34a and miR-146a were observed in EVs derived from hippocampal cells. These increases in miR-34a and miR-146a levels resulted in the stimulation of mitochondrial respiration and an increase in ROS. These effects were accompanied by severe proton leakage in recipient cells, leading to mitochondrial DNA damage and dysfunction.⁸² This, in turn, results in a further increase in ROS in a vicious feedback loop,⁸³ ultimately enabling EVs to induce stress and even senescence changes in neighboring cells. In addition to neighboring cells, EVs carrying miRNAs have the capacity to propagate a senescence environment at the distal end. Specifically, The study identified that miR-326-3p, detected in EVs from aged adipose tissue in diabetic model mice, inhibits the production of Rictor in cardiomyocytes. This suppression hinders energy metabolism, resulting in a reduction of mitochondrial membrane potential, an elevation of mitochondrial superoxide, and compromised mitochondrial respiratory activity. This ultimately results in mitochondrial dysfunction and initiates the dissemination of the aging microenvironment to distant areas of the heart.⁸⁴

Stem Cell Depletion

A decline in tissue regeneration and an impaired ability to repair tissue damage following injury are closely associated with cellular senescence. It is noteworthy that diverse tissues and organs possess distinct renewal and repair mechanisms, with stem cells playing a pivotal role in this regulatory system. A decline in the number and functionality of stem cells, coupled with the inability of the new cells generated through differentiation to fully replace and repair damaged or senescent cells, results in the gradual accumulation of senescent cells. Ultimately, this leads to the manifestation of a senescent phenotype in tissues.⁸⁵ EVs derived from aged bone marrow mesenchymal fluid have been observed to induce an augmented expression of senescence-associated β-galactosidase (SA-β-gal) in BMSCs and to impede cell proliferation, thereby inducing cellular senescence. Furthermore, these EVs have been observed to target heme oxygenase-1 via the miR-183 cluster (which includes miR-96, −182, and −183), thereby impeding osteogenic differentiation.⁸⁶ The process of osteogenic differentiation of BMSCs was found to be inhibited by EVs released from radiation-activated fibroblasts. These EVs mediated the delivery of miR-23a, and the elevated miR-23a targeted the CXCR4/CXCL12 axis in BMSCs, thereby disrupting their differentiation potential.⁸⁷ Furthermore, the levels of miR-31 were augmented in circulating EVs from elderly and osteoporotic patients. Among them, senescence ECs-derived EVs facilitate the internalization of overexpressed miRNA-31 by MSCs. The function of miRNA-31 is to inhibit osteogenic differentiation by knocking down its target, frizzled-like receptor-3.⁸⁸ Notably, obesity has also been observed to induce alterations in the microRNA profile of macrophage-secreted extracellular vesicles. Researchers have demonstrated the critical role of miRNA-140 in determining the fate of osteogenic and adipogenic differentiation of skeletal stem/progenitor cells. The miRNA-140 has been found to lead to the transformation of skeletal stem/progenitor cell differentiation by targeting the peroxisome proliferator-activated receptor α-adipocyte lipid transfer protein axis. This transformation is accompanied by a reduction in bone mass, deterioration of bone microstructure, and an increase in the number of adipocytes, ultimately leading to structural and functional deterioration of skeletal tissues in lean mice.⁸⁹ It has been found that EVs are capable of selectively loading miRNAs, which allows them to determine the direction of stem cell differentiation. In some cases, this process can even abrogate the differentiation potential of stem cells and mediate the senescence process in aging and related disease environments.

Telomere Abrasion

In eukaryotic chromosomes, there are multiple repetitive non-coding DNA segments known as telomeres at the extremities. These structures are imperative for the preservation of cellular and chromosomal functions. Conventional DNA polymerases are incapable of fully replicating the extremities of linear DNA.⁹⁰ As cells continue to divide, telomeres at the ends of chromosomes undergo a process of gradual loss, leading to telomere shortening. This, in turn, contributes to the onset of cell senescence and, ultimately, cell death.⁹¹ In a model of chronic kidney disease, Yin et al⁹² discovered that the transfer of macrophages expressing miRNA-155 via EVs promoted the senescence of renal TECs, which resulted in renal fibrosis. The internalization of EVs loaded with mir-155 by TECs results in the targeting of TRF1 and subsequent downregulation of its expression, leading to telomere dysfunction. In addition to this, in the life process regarding telomeres, researchers have discovered that there is a reverse transcriptase that restores the length of telomeres, known as telomerase. Telomerase can reverse transcribes the repetitive units that synthesize the telomeres at the ends of chromosomes.⁹³ Maintenance of telomerase activity is imperative for the retardation of cellular senescence and the preservation of cellular function. In a separate study, the cargoes of miRNAs in radiation cell-derived EVs were demonstrated to reversibly reduce telomerase activity in recipient cells, thereby mediating bystander effects to promote cellular senescence.⁹⁴

It can be observed that EVs profoundly participate in the aging process across multiple hierarchical levels, including genomic instability, cellular senescence, immune regulation, and inflammaging, through selective packaging of distinct miRNAs and mediating their transfer and subsequent internalization by recipient cells, whether under physiological homeostasis or pathological conditions.

Extracellular Vesicle miRNAs Decelerate the Senescence Process

The genetic cargo of EVs generally reflects the physiological state of their parental cells. However, EVs exhibit distinct miRNA profiles compared to their cellular origins, with specific miRNA subsets being selectively enriched as intercellular communication mediators to enable potent and targeted signaling. Scientific exploration of this selective packaging mechanism has revealed the remarkable potential of EV-associated miRNAs in anti-aging interventions. EVs derived from diverse cellular sources—including mesenchymal stem cells, macrophages, adipocytes, and circulatory system-derived cells—demonstrate senescence-delaying effects through miRNA-mediated regulatory pathways (Figure 3).

Figure 3.

EVs carrying miRNA payloads from various cellular sources are transported to recipient cells, where they regulate multiple aging-related signaling pathways, influence key features of aging, and ultimately help improve tissue homeostasis, delay biological aging, and extend healthy lifespan.

Stem Cell-Derived EVs

As primordial units of biological systems, stem cells inherently oppose aging due to their defining biological capacities—multipotent differentiation, robust self-renewal,⁸⁵ and central roles in tissue regeneration. While substantial evidence documents stem cells’ anti-senescence properties^95,96 and therapeutic applications in niche domains, clinical translation remains constrained by immunogenicity concerns, tumorigenic potential, and bioethical challenges. The use of stem cell-derived EVs has been demonstrated to circumvent undesirable effects while preserving the anti-senescence effects in which miRNAs play a pivotal role.

Mechanistically, CCN2 has been reported to accelerate cellular senescence by activating the PI3K/AKT signaling cascade.^97,98 Intriguingly, miR-15b-5p and miR-290a-5p—identified as enriched miRNA species in embryonic stem cell-derived EVs (ESC-EVs)—functionally antagonize CCN2-dependent pro-senescence signaling through sequence-specific silencing of CCN2 mRNA. This molecular intervention disrupts pathological AKT hyperactivation, thereby conferring senescence resistance in recipient cells. The efficacy of the treatment was further substantiated through the administration of injections into aged mice.⁹⁹ Previous studies have confirmed that treatment with MSC-EVs can markedly upregulate regenerative and angiogenic markers, restore endometrial thickness, glandular density, and vascular structure;¹⁰⁰ and significantly enhance the migration and proliferation capabilities of aged HUVECs, while ameliorating mitochondrial dysfunction and normalizing ROS overproduction. Mechanistic interrogation identified miR-146a as the principal bioactive mediator, which retards senescence progression across multiple subcellular compartments through SRC kinase dephosphorylation—achieved via suppression of SRC activation—thereby attenuating oxidative stress-induced DNA damage and curbing pathological ROS amplification cascades.¹⁰¹ Furthermore, MSC-EVs deliver miR-132-3p to ECs, mechanistically activating the Ras/PI3K/Akt/eNOS signaling axis. This pathway orchestration attenuates oxidative stress by suppressing ROS overproduction while concurrently preserving intercellular tight junction proteins to maintain vascular barrier integrity, collectively counteracting age-related vascular dysfunction.¹⁰² EVs derived from healthy MSCs facilitate the intercellular transfer of miR-302b, which transcriptionally upregulates HIF-1α to concurrently suppress SA-β-gal activity and downregulate pluripotency factors OCT4/KLF4. This molecular reprogramming restores oxidative phosphorylation efficiency and revitalizes dysfunctional senescent stem cells, thereby mitigating stem cell exhaustion during organismal aging.¹⁰³ Xue et al¹⁰⁴ demonstrated that miRNAs enriched in MSC-EVs drive macrophage M2 polarization by targeting the p38 MAPK/NF-κB signaling axis. This immunomodulatory reprogramming suppresses pro-inflammatory cytokine cascades at both local and systemic levels, while concurrently activating tissue-reparative transcriptional programs to enhance damage resolution. Similarly, endothelial progenitor cell (EPC)-derived EVs enhance macrophage functional plasticity through transfer of miR-222-3p, which activates the SOCS3/JAK2/STAT3 pathway. This molecular cascade upregulates anti-inflammatory markers, drives M2 polarization, and amplifies cellular proliferation and tissue regeneration through paracrine mechanisms,¹⁰⁵ collectively ameliorating inflammatory burden across pathological contexts.¹⁰⁶ Indeed, MSC-EVs have been demonstrated to not only mediate macrophage polarization, but also to directly participate in the intercellular communication of CD4+ T cells. By carrying microRNA-21 targeting PTEN, the PTEN/PI3K-Nrf2 axis is activated, thereby increasing the antioxidant level of CD4+ T cells and preventing cellular senescence.¹⁰⁷ Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UMSC-EVs) have been shown to deliver miRNA-675 to senescent cardiomyocytes. In the target cells, the mechanism of action of miR-675 involves the targeting of TGF-β1 and the subsequent inhibition of the TGF-β1/pSMAD/p21 signaling axis. This results in the down-regulation of SA-β-gal activity, a decrease in TGF-β1 expression, and, ultimately, the restoration of the proliferative capacity of senescence cardiomyocytes through the regulation of cell cycle re-entry.¹⁰⁸ In a separate investigation, miR-146a-5p and miR-21-5p were also identified as enriched miRNA species within UMSC-EVs.¹⁰⁹ Following intraovarian delivery, these miRNAs exhibited specific accumulation in primordial oocytes, where they stimulated PI3K/mTOR pathway activation. This molecular intervention restored both the quality and quantity of oocytes, ultimately enhancing fertility outcomes in aged female murine models through coordinated reversal of oxidative proteostatic collapse and mitochondrial dysfunction. In a study on murine rejuvenation, young adipose-derived mesenchymal stem cell extracellular vesicles (ADSC-EVs) demonstrated remarkable efficacy in attenuating age-associated functional decline across multiple domains—including motor coordination, locomotor performance, endurance capacity, grip strength, and fur regeneration. Systemic administration of ADSC-EVs significantly ameliorated frailty indices while reprogramming the metabolomic profile to resemble youthful patterns. Molecular profiling revealed concurrent reversal of senescence-associated markers and mitigation of pathological elevations in oxidative stress parameters and pro-inflammatory cytokines, collectively resetting the organismal aging trajectory at cellular and systemic levels. In vitro experiments on mouse adult myoblasts have demonstrated analogous effects. The in-depth study revealed that the delivery of miRNAs, including miR-125b-5p, miR-let7c-5p, and miR-214-3p, by ADSC-EVs is a significant factor in the anti-senescence effects observed.¹¹⁰

Although cell cycle arrest has traditionally been considered irreversible, emerging evidence suggests that senescence in certain cell types may exhibit dynamic progression or even plasticity.^111,112 This paradigm shift expands anti-aging therapeutic strategies beyond conventional senolytic (eliminating senescent cells) and senomorphic (suppressing SASP) approaches, positioning cellular rejuvenation—defined as the reversal of proliferative arrest through cell cycle re-entry—as a novel third modality. Such senescence reprogramming strategies aim to epigenetically reset transcriptional networks governing DNA damage response and metabolic homeostasis, thereby restoring replicative competence to anti-aging. Research has demonstrated that miR-302b is present in high concentrations within the EVs derived from HESC-EVs. Through the delivery mechanism of HESC-EVs, miR-302b exhibits a direct targeting of Cdkn1a and Ccng2 in senescent cells, leading to the suppression of their transcriptional expression and reactivate cyclin-dependent kinase /cyclin complexes. Finally overcoming cell cycle arrest and reversing senescence phenotypes associated with proliferative quiescence. In vivo experiments demonstrated that miR-302b restored tissue and organ function, as well as down-regulated age-related inflammation levels in senescence mice, thereby prolonging the healthy lifespan of these animals.¹¹³ UCMSC-EVs encapsulate functional miRNAs, notably miR-26a-5p and miR-381-3p, which orchestrate chondrocyte rejuvenation by modulating the p53 signaling axis in senescent chondrocytes. This targeted suppression of p53-mediated senescence cascades restores extracellular matrix synthesis and mitigates cartilage degradation, thereby therapeutically ameliorating age-related osteoarthritis through dual regulation of cellular senescence and inflammatory microenvironment remodeling.¹¹⁴

Macrophage-Derived EVs

The immune system, as a fundamental life-sustaining system, is indispensable for organismal self-regulation, and its progressive functional decline represents a core hallmark of aging.¹¹⁵ Macrophages, serving as central effectors of the innate immune system, play pivotal and multifaceted roles in aging dynamics. Beyond mediating senescent cell clearance, they critically modulate systemic aging trajectories through their regulatory control over inflammatory cascades, tissue regenerative processes, and metabolic homeostasis. Macrophage polarization states, classified as M1 or M2, have been shown to exert diametrically opposed regulatory roles in aging-related processes. Mechanistically, M1-EVs exacerbate pro-inflammatory responses via mTOR pathway hyperactivation, amplifying inflammaging through NF-κB-dependent cytokine production.¹¹⁶ Conversely, M2-EVs have been shown to attenuate pathological inflammation by suppressing the PI3K/mTOR signaling axis, thereby restoring tissue homeostasis and demonstrating therapeutic efficacy across diverse inflammatory models.¹¹⁷ Intraovarian administration of M2-EVs in aged murine models significantly downregulated pro-inflammatory mediators (TNF-α, IL-6, iNOS, IL-1β) while markedly enhancing anti-inflammatory markers (IL-10, Arg-1). The inflammation of the ovary was mitigated by the suppression of the pro-senescence inflammatory microenvironment through immunomodulatory reprogramming. Mechanistic validation revealed that M2-EVs execute their anti-senescence effects via shuttling specific miRNAs, particularly miR-99a-5p, which suppresses mTOR signaling through direct targeting of mTOR, thereby restoring follicular reserve and oocyte quality in geriatric ovarian tissue.¹¹⁸

Circulatory System-Derived EVs

At the cellular level, aging is principally driven by cell-autonomous processes including telomere attrition, genomic instability, mitochondrial dysfunction, proteostatic collapse, and stem cell exhaustion.² However, in multicellular organisms such as humans, systemic alterations in circulatory network-mediated intercellular/tissue/organ crosstalk emerge as equally critical determinants of aging trajectories,¹¹⁹ highlighting the hierarchical integration of molecular damage and organism-level communication breakdown in senescence progression. The rejuvenation of aged animals through circulatory coupling in heterochronic parabiosis models provides empirical validation that blood harbors youth-restorative systemic factors. A growing body of evidence now identifies blood-borne EVs as pivotal mediators of this phenomenon, with their cargo of senescence-modulating miRNAs demonstrating tissue-reparative and age-reversing capacities across multiple organ systems.^120,121 Chen et al¹²¹ demonstrated that plasma-derived EVs from young mice exert potent rejuvenative effects in aged murine models. Treated aged mice exhibited a significantly reduced frailty index, attenuated bone loss, and enhanced functional capacities across multiple physiological domains, including cognitive performance, cardiac output, locomotor endurance, energy metabolism, spermatogenic integrity, and fertility rates. At the molecular level, a triad of senescence-antagonizing miRNAs (miR-144-3p, miR-149-5p, and miR-455-3p) transcriptionally upregulate PGC-1α by directly targeting its transcriptional repressors, thereby rescuing age-associated bioenergetic decline through enhanced mitochondrial biogenesis and oxidative phosphorylation efficiency. EVs from sedentary or acutely exercised young mice have been demonstrated to demonstrate therapeutic efficacy against age-associated hepatic fibrosis. This effect is mediated through the targeted enrichment of miR-30c-5p in senescent livers, which restores hepatic triglyceride homeostasis and suppresses the pathological activation of hepatic stellate cells, thereby attenuating collagen deposition and fibrotic remodeling.¹²²

Beyond the demonstrated anti-senescence effects of young murine blood-derived EVs, Liu et al¹²³ identified that EVs isolated from serum of healthy males aged 18–22 years counteract Doxorubicin-induced senescence in the rat cardiomyocyte cell line H9C2. Mechanistically, the EVs facilitate the selective transfer of miRNA-34a, which upregulates the Protein Phosphatase 1 Nuclear Targeting Subunit through post-transcriptional modulation. This results in the attenuation of DNA damage response hyperactivation and the preservation of mitochondrial membrane integrity, collectively rescuing cardiac cellular senescence. A separate study identified a comparable enrichment of miRNA-17-3p in human serum-EVs and demonstrated its role in promoting H9C2 proliferation by suppressing tissue inhibitor of metalloproteinase 3 expression.¹²⁴ Notably, even within immature circulatory systems, EVs exhibit potent anti-senescence properties. Administration of human umbilical cord blood-derived EVs (UCB-EVs) to aged murine models significantly rescued senile bone loss, as evidenced by enhanced trabecular bone volume and cortical thickness via micro-CT. UCB-EVs orchestrated skeletal rejuvenation by stimulating osteogenic differentiation and suppressing osteoclast activity.¹²⁵ Subsequent studies identified that miR-3960 shuttled by UCB-EVs targets BMSCs, where it epigenetically silences Homeobox A2 (HOXA2) — a transcriptional repressor of Runx2 — through direct 3’UTR binding. This HOXA2 inhibition stimulates Runx2-driven osteoblastogenesis, thereby reversing age-associated osteopenia and restoring bone mineralization capacity in geriatric mice.¹²⁶

Other-Derived EVs

The integrity of the intercellular communication network between ECs and EPCs is pivotal in modulating vascular aging. EC-EVs serve as critical mediators of this network, with miR-214 identified as a key cargo enriched in EC-EVs. The miR-214 transfer via EC-EVs silences the expression of the Ataxia-Telangiectasia Mutated in recipient cells, thereby decelerating senescence-associated vascular dysfunction.¹²⁷ Human bronchial epithelial cell-derived EVs (HBEC-EVs) harbor a senescence-modulatory miRNA consortium — including miR-26a, miR-26b, miR-141, miR-200a, miR-16, and miR-148 — that coordinately suppress WNT5A and WNT10B expression. Mechanistically, HBEC-EVs mitigate pulmonary epithelial cell senescence during fibrotic remodeling by delivering these miRNAs to inhibit both canonical (β-catenin/TCF4-dependent) and non-canonical (Ca²⁺/NFAT-mediated) WNT signaling axes.¹²⁸ As secretory cells of the CNS, astrocytes (ASTs) provide critical support for synapse formation, and AST-EVs exhibited effects similar to those of ASTs. In an experimental model of sevoflurane-induced neurocognitive impairment in aged mice, the administration of AST-EVs has been shown to mitigate senescent cognitive decline by means of targeted delivery of microRNA-26a-5p to the neurons of the hippocampus. This microRNA has been found to bind to the 3’UTR of Neural Cell Adhesion Molecule (NCAM), thereby initiating a process of crosstalk between NCAM and the GSK3-β pathway. This interaction activates the AKT/GSK3-β/CRMP2 signaling cascade, which concurrently suppresses caspase-3-mediated neuronal apoptosis and enhances dendritic arborization via microtubule stabilization.¹²⁹

Beyond animal-derived EVs, emerging evidence reveals that EVs from diverse biological kingdoms harbor anti-aging potential. Intriguingly, plant-derived miRNAs exhibit a unique 2′-O-methylation modification at their terminal nucleotides — a critical biosynthetic step absent in mammalian miRNAs. This chemical armoring confers exceptional stability against periodate oxidation, allowing plant miRNAs to evade oxidative degradation in animal systems. Such biochemical resilience enables their cross-kingland regulatory capacity to persistently silence senescence-associated genes (eg, mTOR, NF-κB) through sequence-specific targeting, thereby orchestrating conserved longevity pathways in recipient organisms. Phellinus linteus (PL), a basidiomycete fungus with well-documented medicinal properties, was initially characterized by Japanese researchers for its potent antitumor activity.¹³⁰ Recent investigations have identified PL-EVs as novel dermatoprotective agents capable of counteracting UV-induced skin photoaging. PL-EVs significantly ameliorate cutaneous oxidative stress by reducing lipid peroxidation and enhancing endogenous antioxidant defenses. Crucially, the fungal-specific microRNA miR-CM1, identified as the principal molecular cargo of PL-EVs, directly binds to the 3’UTR of monooxygenase Mical2 — a redox-sensitive regulator of actin cytoskeleton dynamics. This miR-CM1-Mical2 axis suppresses ROS-generating NADPH oxidase 1 membrane translocation, thereby restoring epidermal redox homeostasis and attenuating SASP in UV-irradiated dermal fibroblasts¹³¹ (Table 1).

Table 1.

Anti-Senescence Effects of Extracellular Vesicle MicroRNAs

Sources	Cargo(s)	Experimental Model	Molecular Target	Senescence Endpoint	Mechanism(s) and Effect(s)	Ref.
Microglial	miR-124-3p	Aged mice	–	TNF-α, IL-1β, IL-6, IL-10; cell cycle protein D1	Reducing pro-inflammatory factors, cell cycle protein D1 and increasing anti-inflammatory factors through microglia polarization thereby alleviating cognitive deficits in the hippocampus.	[132]
C2C12	miR-27a-3p	MC3T3-E1	APC	ALP	Promoting osteogenic differentiation by decreasing APC expression and thereby activating the β-catenin pathway	[133]
MSC	miR-105-5p	Rats with intervertebral disc degeneration, NP cell	Sirt6	SA-β-gal, P16, CCK-8, MMP-3, ADAMTS-4	miR-105-5p activates the Sirt6 pathway and restores the viability of senescent myeloid cells by down-regulating the level of cAMP-specific hydrolase PDE4D	[134]
Fibroblasts	miR-125b	Aged mice and Dermal fibroblast	Sirt7	Cell proliferation, Cell cycle	miR-125b activates the TGF-β1 signaling pathway by inhibiting Sirt7 expression and accelerates myofibroblast differentiation and wound healing in aged mice	[135]
hADSC	miR-381-3p, miR-122-5p, miR-143-3p, miR-206	Human articular chondrocytes (HAC)	–	SA-β-gal, TNF-α, IL-6, MMP-13	hADSC-EVs inhibit HAC inflammation by down-regulating the expression of SASPs such as degradative enzymes, inflammatory cytokines and degenerative cartilage markers	[136]
EPCs	miR-17-5p	EC cell	PTEN, PI3K, Akt	SA-β-gal, P16, ROS	Phosphorylation of PI3K and Akt and down-regulation of PTEN expression activate miR-17-5p/PTEN/PI3K/Akt signaling pathway to inhibit cell senescence	[137]
UCMSC	miR-21-5p	Natural ovarian aged mice	PTEN, Bax	EDU, CCK-8	Inhibition of PTEN expression and apoptosis delays ovarian senescence	[138]
ADSC	miR-1246	Human skin fibroblasts	GSK3β	MTT, ROS, DNA damage, MMP-1	miR-1246 slows down UVB-induced skin photoaging by targeting GSK3β, thereby activating autophagy, attenuating apoptosis and ROS accumulation, DNA damage, and MMP-1 secretion.	[139]
Melatonin-treated vascular smooth muscle cells	miR-204, miR-211	5/6 Nephrectomy plus high phosphate diet-treated mice	BMP2	CCK8, SA-β-gal, RUNX2, ALP	Targeting RUNX2, BMP2 functionally inhibits differentiation and senescence of bone progenitor cells	[140]
Apt19s and miR-376b-5p-modified double-engineered BMSC-EVs	miR-376b-5p	Aged rats	Camsap1	CCK-8, ALP, SA-β-gal, SATB2, P53, P21, P16	Inhibition of Camsap1 expression effectively enhances osteogenic activity and alleviates senescence-related phenotypes	[141]
Intestinal cells treated with Pseudomonas fragilis	miR-1246	Aged mice	SKP2	CCK-8, ROS, DNA copy number, GSH, 8-OHDG	miR-1246 attenuates reproductive senescence by targeting S-phase kinase-associated protein 2, thereby suppressing ubiquitination-dependent degradation of p62/SQSTM1 and stabilizing its protein levels.	[142]
Human umbilical cord blood-derived mononuclear cells	miR-124-3p	Mice with Parkinson’s disease	–	PI, Ki-67, CCK-8, BrdU	Induction of neuronal differentiation and protection of N27 dopaminergic cells from 6-hydroxydopamine-induced toxic damage	[143]
ADSC	miR-204	Rats with diabetic nephropathy	CIDEC m6A	SOD, GSH-PX, CAT, MDA, ATP, ROS	Targeting METTL7A and thereby inhibiting CIDEC m6A methylation expression attenuates oxidative stress-induced mitochondrial dysfunction	[144]
Sr pre-processed SMSC-derived EVs	miR-143-3p	Temporomandibular joint osteoarthritis rats	Mfsd8	CCK-8, EDU, IL-1β	Targeting Mfsd8 reduces susceptibility to iron death and restores MDA levels in CC and attenuates inflammatory effects	[145]
Young bone marrow stem cell antigen 1-positive cells	miR-150-5p	Aged mice	MEKK3, JNK	IL-6, TNF-α, GAPDH	mir-150-5p reduces microglia polarization by inhibiting mitogen-activated protein kinase 3 /JNK/c-Jun axis and downregulating IL-6 and TNF-α	[146]
ADSC	miR-378	HaCat cells	Caspase-3	Transwell, EDU	Targeting caspase-3 ameliorates oxidative stress injury	[147]

Conclusions and Perspectives

Aging represents an integral biological continuum between vitality and mortality, a process through which scientific endeavors have focused on modulating this continuum to compress morbidity and extend healthspan. Historically, EVs were largely dismissed as cellular debris with negligible biological utility. However, contemporary research leveraging the systematic delineation of aging hallmarks (eg, genomic instability, loss of proteostasis) and advancements in geroscience has redefined EVs as critical mediators of intercellular senescence crosstalk. Their capacity to shuttle senescence-modulating cargos (miRNAs, SASP regulators) positions EVs as emerging biotherapeutic platforms for counteracting age-associated pathophysiological decline. The advent of next-generation sequencing technologies has enabled systematic profiling of EVs-associated miRNAs, revealing their evolutionarily conserved roles in orchestrating intercellular aging dialogues. In this review, we systematically delineate the progeronic roles of EVs-encapsulated miRNAs through the lens of canonical aging hallmarks. MicroRNAs in EVs are deeply ingrained in every aspect of the senescence process, whether they are mediating mitochondrial dysfunction, telomere wear and tear, and genomic instability to drive cellular senescence, or they are exacerbating systemic levels of immunoinflammation and causing a deepening of the senescence cycle. Conversely, from a therapeutic development perspective, we methodologically stratify EVs-associated miRNAs by their cellular origins to evaluate source-specific geroprotective efficacy. EVs derived from diverse biological sources — including stem cells, macrophages, and the circulatory system — have demonstrated conserved geroprotective effects through their miRNA cargoes. These origin-specific EVs-encapsulated miRNAs counteract hallmark aging processes by modulating senescence-associated pathways, collectively establishing EVs as multi-source therapeutic platforms for targeted senescence interception. The nascent field of EVs biology remains constrained by critical knowledge gaps, particularly regarding the spatiotemporal regulation of EVs biogenesis and the molecular logic governing miRNA sorting specificity and cargo-loading mechanisms. Decoding these fundamental processes — such as the RAB27A/TSG101-mediated exocytosis machinery and hnRNPA2B1-dependent miRNA methylation sorting — could illuminate novel senescence-associated pathways amenable to therapeutic interception. Furthermore, the isolation of EVs from new sources could also be beneficial. Some studies have indicated that differences in the source of EVs may result in EVs that exhibit divergent effects on senescence.^148–150 The current paucity of high-resolution in vivo tracking methodologies severely constrains our understanding of EVs biodistribution dynamics and receptor cell-type specificity — a critical knowledge gap that introduces substantial risk in clinical translation. Uncontrolled EVs tropism may precipitate off-target biodistribution events. Such aberrant trafficking could paradoxically exacerbate senescence-associated secretory phenotype propagation rather than conferring geroprotection. Recent advancements have demonstrated that EVs derived from youthful murine models and healthy cell lineages exhibit potent geroprotective effects, primarily mediated by their miRNA cargoes in preclinical studies.^121,151 Nonetheless, the excessive reliance on rodent models and in vitro systems does not accurately reflect the intricate pathophysiological characteristics of human aging, especially the nonlinear interactions of multi-organ senescence. Future research must prioritise longitudinal evaluations of EV treatment in human organoid aging platforms and non-human primate models to address this translational gap. This paradigm shift towards human-relevant validation frameworks is crucial for mitigating risks in clinical translation while preserving adherence to the primary objective of geroscience – extending healthspan through evolutionarily conserved aging modulation. While this review emphasizes the regulatory mechanisms of EVs-encapsulated miRNAs in aging processes, the clinical translation of these natural nanotherapeutics faces several nanotechnological challenges. Future research in nanomedicine must prioritize the development of standardized protocols for the large-scale isolation, purification, and characterization of EVs to ensure batch-to-batch consistency. Furthermore, integrating bioengineering strategies—such as surface modification of EVs with specific ligands for enhanced tissue-targeted delivery, and the optimization of miRNA loading efficiencies via electroporation or sonication—will be crucial for maximizing their geroprotective efficacy.^120,152,153 This functional complexity necessitates a holistic understanding of EV cargo hierarchies. We posit that With ongoing advancements in the modification and standardized preparation of extracellular vesicles, the identification of their contents, and rigorous clinical evaluation techniques, extracellular vesicle-based nanomedicines hold promise as important therapeutic tools for promoting healthy aging and alleviating age-related diseases. Nevertheless, numerous translational challenges must still be overcome before these therapies can be widely implemented in clinical practice.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32473109); the Science and Technology Department of Sichuan Province (2024YFFK0146); the Sichuan Agricultural University Research and Training Program Project (2025254X).

Abbreviations

EVs, Extracellular vesicles; miRNA, MicroRNA; MSCs, mesenchymal stem cells; ECFCs, endothelial colony-forming cells; ESEs, early-sorting endosomes; MVBs, multivesicular vesicles; LSEs, late-sorting endosomes; ILVs, intraluminal vesicles; ESCRT, endosomal transport essential sorting complex; TLR4, Toll-like receptor 4; mRNAs, messenger RNAs; lncRNAs, long non-coding RNAs; rRNAs, ribosomal RNAs; pri-miRNA, primary-miRNA; SASPs, senescence-associated secreted phenotypes; BMSCs, bone marrow mesenchymal stem cells; HDFs, human dermal fibroblasts; ROS, reactive oxygen species; PTEN, phosphatase tension protein homologs; SIRT1, sirtuin-1; AGEs, advanced glycosylation end products; HUVECs, human umbilical vein endothelial cells; DNMT1, DNA methyltransferase 1; TERRA, Telomeric repeat-containing RNA; HAS, human serum albumin; BBB, blood-brain barrier; ECs, endothelial cells; SDC1, Syndecan-1; mtDNA, mitochondrial DNA; PGC-1β, peroxisome proliferator-activated receptor gamma coactivator-1β; IPF, idiopathic pulmonary fibrosis; SA-β-gal, senescence-associated β-galactosidase; TECs, tubular epithelial cells; ESC, embryonic stem cell; EPC, endothelial progenitor cell; UMSC, Umbilical cord mesenchymal stem cell; UCB, human umbilical cord blood; HOXA2, Homeobox A2; HBEC, Human bronchial epithelial cell; ASTs, astrocytes; NCAM, Neural Cell Adhesion Molecule; PL, Phellinus linteus; ALP, Alkaline Phosphatase; MMP, Matrix Metalloproteinases; ADAMTS-4, A Disintegrin and Metalloproteinase with Thrombospondin Motifs-4; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDU, 5-Ethynyl-2′-deoxyuridine; BrdU, 5-Bromo-2′-deoxyuridine; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

Data Sharing Statement

No datasets were generated or analysed during the current study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.