Development of extracellular vesicles in diagnostics and therapeutics: From extracellular vesicles to precision medicine.

1. INTRODUCTION
Extracellular vesicles (EVs) are membrane-bound vesicles released by cells that carry biologically active molecules and deliver them to recipient cells.1 EVs are produced by most cell types and are present in body fluids such as blood. Historically, they were believed to be nonsignificant cellular debris despite containing bioactive molecules.2 Advances in isolation and purification techniques have revealed the critical roles of EVs in various physiological and pathological processes. EVs participate in cell-to-cell communication,3 immune regulation,4 tissue repair,5 and disease progression, including playing a role in cancer,6 immune responses,4 neurodegenerative diseases,7 and cardiovascular disorders.8
EVs exhibit substantial diversity in size, composition, and biosynthetic pathways, with these forming the basis for the classification of these vesicles. Table 1 summarizes the types of EVs and their biogenesis, functions, isolation methods, and surface markers.1,2,6,9–28
Because EVs have high stability and are able to evade immune attacks, they have attracted considerable research interest as having potential applications in diagnostics and therapy. Advances in separation techniques, including centrifugation, immunoprecipitation, and electrophoresis, have further expanded their use. EVs carry molecular markers that reflect their cell of origin, and therefore, they are valuable, noninvasive biomarkers for disease diagnosis. Additionally, EVs possess mechanisms that protect their molecular cargo, which supports their potential use as delivery vehicles for therapeutic agents directed toward specific target cells.29
EVs are increasingly recognized as crucial components of precision medicine because of their potential as biomarkers and therapeutics.30 Analysis of EV cargo can provide insights into the underlying mechanisms of various diseases, facilitate the identification of novel biomarkers, and support the development of targeted treatments tailored to individual patient profiles, which can advance the field of precision medicine.

2. STRUCTURE OF EVs
EVs are small membranous structures released by cells into the extracellular environment. They are composed of a plasma membrane, surface proteins, and internal cargo (Fig. 1). The main structural characteristics of EVs are as follows.

2.1. Plasma membrane
EVs contain a lipid bilayer membrane similar to the plasma membrane of their parent cells. This membrane is enriched with cholesterol, sphingolipids, and phosphatidylserine,31 providing stability and functionality and protecting internal cargo from environmental factors.

2.2. Surface proteins
The EV membrane carries surface proteins that vary on the basis of the vesicle’s parent cell. These proteins act as markers and functional molecules, including receptors, integrins, and adhesion proteins that facilitate interaction with target cells.32 Tetraspanin domains play critical roles in vesicle formation and cell targeting.14 Additionally, some EVs contain major histocompatibility complex molecules involved in immune responses.11

2.3. Internal contents
EVs contain bioactive molecules such as DNA, RNA, lipids, and proteins. These molecules remain stable within the vesicle, and they can be transferred to recipient cells, where they mediate processes such as cell regeneration, proliferation, differentiation, apoptosis, immune regulation, and angiogenesis.17
EVs carry receptors, bioactive lipids, proteins, and nucleic acids such as mRNA and microRNA. EVs bind to target cells, delivering mRNA for protein synthesis and microRNA for protein expression regulation, thereby affecting cellular function.26 Proteins contained within EVs also play key roles in their biological functions.

3. THE CLASSIFICATION OF EVs
Typical EVs can be categorized into exosomes, microvesicles, and apoptotic bodies.
Exosomes, which are typically 40 to 150 nm in size, originate in multivesicular bodies (MVBs) formed through endocytosis. These MVBs contain intraluminal vesicles and release exosomes when they fuse with the plasma membrane, delivering their contents to target cells.9
Microvesicles, which range from 150 to 1000 nm, are formed by direct budding of the cell membrane. They facilitate intercellular communication by transferring biomolecules to specific recipient cells.15
Apoptotic bodies emerge during programmed cell death. Dying cells undergo apoptosis release vesicles containing fragmented nuclei and cellular debris, which are eventually cleared through phagocytosis.2
In addition to these traditional categories, several emerging EV subtypes have been identified, including autophagic EVs, stress EVs, and matrix vesicles.1
Autophagic EVs, which range in size from 40 to 1000 nm, are formed during autophagy and originate from autophagosomes. These vesicles either fuse with lysosomes to degrade unnecessary cellular components or with the plasma membrane to release their contents.22 Their wide size range reflects the diversity of their origins and the variability of their cargo.21
Stress EVs, sized between 40 and 1000 nm, are released when cells experience stressors such as hypoxia and inflammation. These vesicles are shed from the membrane or produced through autophagic processes and play crucial roles in stress adaptation and disease mechanisms.
Matrix vesicles, which are 40 to 1000 nm in size, are secreted by cells involved in tissue remodeling, including osteoblasts, endothelial cells, and fibroblasts.25 These vesicles participate in calcification, extracellular matrix remodeling,24,25 and processes related to cancer progression in tumor microenvironments.33
Nonvesicular particles, ranging from 10 to 100 nm in size, do not contain a lipid bilayer but are enriched in proteins and other macromolecules. These particles arise from mechanisms such as protein aggregation and lipoprotein secretion,28 although their biological relevance remains under investigation.

4. BIOLOGICAL FUNCTIONS OF EVs
EVs, which vary in origin and molecular content, participate in multiple biological processes, including cellular communication, homeostasis, and disease progression.

4.1. Cellular communication
Cell communication was previously understood to occur through direct cell–cell contact or receptor-mediated signaling. However, research has revealed that EVs function as carriers of bioactive molecules that mediate intercellular communication.10 Through this mechanism, cells exchange information, regulate growth and differentiation, and coordinate responses to external stimuli. In pathological states such as diabetes, endothelial dysfunction increases the secretion of EVs.34 These vesicles transmit signaling molecules and interact with endothelial nitric oxide synthase, enabling endothelial cells to respond to shear stress and inflammation.35

4.2. Immune regulation
EVs regulate immune responses by affecting antigen presentation and modulating immune cell activation, proliferation, and differentiation. Depending on their cargo and interactions with target cells, EVs can stimulate or suppress immune responses.4 EVs released by antigen-presenting cells carry antigens and other signaling molecules, participating in immune responses.11

4.3. Tissue repair and regeneration
EVs transport bioactive molecules that regulate cell proliferation, inflammation, regeneration, and tissue repair.17 They reduce cell apoptosis and tissue inflammation while promoting proliferation, survival, and angiogenesis in both in vitro and in vivo models.36

4.4. Angiogenesis
EVs regulate angiogenesis by either stimulating or inhibiting the process, depending on their molecular composition. Endothelial cell–derived EVs promote angiogenesis by transferring microRNAs, proteins, and lipids or by activating proangiogenic signaling pathways. By contrast, EVs can suppress angiogenesis through endocytosis-mediated mechanisms and by increasing oxidative stress.17

4.5. Disease pathogenesis and progression
EVs are involved in the pathogenesis and progression of various diseases, including cancer, neurodegenerative disorders, cardiovascular diseases, and infectious diseases. Cells subjected to stressors such as hypoxia, DNA damage, and infections release EVs containing bioactive molecules.37 Because EVs protect their cargo from degradation, they can transfer pathogenic molecules from diseased to healthy cells. In neurodegenerative models, EV accumulation and aggregation promote disease pathogenesis, indicating they are potential biomarkers.7
Several studies have examined the relationship between EVs and tumors. Tumor-derived EVs spread pathogenic molecules and regulate blood and lymphatic endothelial cells, promoting tumor growth, angiogenesis, and metastasis.38 They also affect dendritic cells and natural killer cells, enabling tumors to evade immune mechanisms.1 Furthermore, tumor cells expel chemotherapeutic drugs through EVs, facilitating the spread of drug resistance signals to neighboring cells.39

5. FROM EVs TO PRECISION MEDICINE: CLINICAL APPLICATIONS OF EVs
The properties and essential functions of EVs enable their use in disease diagnosis, targeted therapy, immune regulation, and precision oncology, establishing EVs as powerful tools in precision medicine.

5.1. Biomarkers for disease diagnosis and prognosis
EVs carry molecular signatures that reflect their cellular origin and pathological state, and therefore, they are valuable biomarkers for noninvasive disease diagnosis, prognosis, and monitoring through biological fluids such as blood, urine, and cerebrospinal fluid.40 The concept of the “liquid biopsy,” which incorporates circulating tumor cells, circulating tumor DNA, and EVs has emerged as a revolutionary alternative to traditional tissue biopsy for evaluating cancer progression.41 EVs appear early in disease and remain throughout all stages, and their structural stability preserves molecular integrity, which indicates that they have utility in noninvasive cancer diagnostics.38
A study demonstrated that hepatocellular carcinoma (HCC)–specific EVs provide greater diagnostic accuracy than conventional α-fetoprotein assays do.42 In glioblastoma multiforme, blood-based detection of EVs enriched with heat shock protein 70 and calcitonin receptor protein demonstrated potential for enabling early diagnosis.6 Plasma EVs containing elevated phosphorylated tau and Aβ42 can also be used to identify Alzheimer’s disease up to 4 years before symptom onset, with this validated in a Johns Hopkins cohort.43 In cardiovascular disease, EVs expressing specific microRNAs are independent predictors of acute myocardial infarction and enable early detection, particularly in patients with negative troponin findings or those presenting within 3 hours of chest pain onset.44

5.2. Therapeutic delivery vehicles
Because of their structural stability, EVs are efficient delivery systems for therapeutic agents.
Engineered EVs loaded with doxorubicin reduce systemic toxicity and enhance drug incorporation and apoptosis in retinoblastoma cells, indicating they hold potential for use in localized cancer treatment.45 In addition, exosome-based delivery of α-synuclein (α-Syn) small interfering RNAs was reported to effectively reduce α-Syn levels and halt Parkinson’s disease progression in a mouse model, highlighting their potential in treating neurodegenerative diseases.46

5.3. Immune modulation
EVs derived from immune cells carry molecules and antigens that can modulate immune responses, thereby stimulating targeted immune reactions.2,11,19
Notably, EV-based cancer vaccines have been developed, with attenuated tumor-derived EVs used to stimulate dendritic cells and initiate an immune response against cancer cells. These vaccines can be personalized and do not promote tumor growth, and they hold potential as adjuvant therapies to reduce the risk of cancer recurrence.47 EVs with immunosuppressive properties are also being explored for the treatment of autoimmune diseases. For example, DC-derived exosomes expressing Fas ligand demonstrated anti-inflammatory effects in a rheumatoid arthritis rat model.48 Although these findings are promising, further research is required to evaluate the long-term safety and therapeutic stability of EV-based immunotherapies in humans.

5.4. Drug resistance and precision oncology
EVs transfer drug resistance molecules between cells, which can affect treatment outcomes and help with personalizing cancer therapies by enabling monitoring of disease progression and early identification of drug resistance. The data presented in Table 2 illustrate how EVs interact with tumor cells, modulate growth, suppress immune function, and support precision medicine applications, with the table providing an overview of their effects on recipient cells and tumor progression.19,38,47,49–55
In head and neck squamous cell carcinoma, CD73 plays a critical role in immune suppression. CD73-bearing EVs promote adenosine production, which supports immune evasion.54 These EVs also polarize macrophages toward an immunosuppressive phenotype and reduce the number of CD8+ T cells, facilitating the development of tumor metastasis and an immunocompromised microenvironment in lymph nodes. Furthermore, CD73-bearing EVs reduce the effectiveness of anti-PD-1 therapy by enhancing immunosuppression and upregulating immune checkpoint markers such as PD-1 and PD-L1.52 As a key mediator of immune suppression, CD73-bearing EVs are a predictor of poor prognosis and resistance to anti-PD-1 therapy and therefore represent a potential target for combination immunotherapy.
In a mouse model of oral squamous cell carcinoma, laminin-332-enriched EVs were reported to promote lymphatic endothelial cell (LEC) migration and tube formation, which are essential steps in tumor metastasis. These findings indicate that laminin-332-enriched EVs may serve as noninvasive biomarkers for lymph node metastasis. Furthermore, antilaminin-332 antibodies reduced EV-driven LEC activity. Blocking EV uptake through integrin α3 inhibition may be a therapeutic strategy.51
Phosphatidylserine-bearing EVs released from ovarian tumors suppress T-cell activation by inducing signaling arrest. Blocking phosphatidylserine with antiphosphatidylserine antibodies or Annexin V reduces this inhibitory effect, and depleting phosphatidylserine-bearing EVs restores T-cell function.19 These findings indicate that targeting phosphatidylserine-bearing EVs may counteract tumor-induced immunosuppression and enhance immunotherapy for ovarian cancer. Additionally, peptide-conjugated EVs encapsulating vinorelbine have been engineered to target lung cancer cells that overexpress epidermal growth factor receptor. These EVs demonstrated efficient drug uptake and strong tumoricidal activity in preclinical studies, with minimal toxicity to normal tissues.50 Together, these results indicate that EV-based delivery systems can improve drug targeting, reduce adverse effects, and present a promising therapeutic approach to lung cancer.
EVs offer substantial potential in precision medicine because they carry disease-specific molecules, facilitate intercellular communication, and modulate immune responses. Their applications in diagnostics, targeted therapy, and disease monitoring can support the development of personalized approaches to health care, providing opportunities for earlier detection, more precise treatment, and improved patient outcomes.
Several challenges limit the clinical application of EVs. One major challenge is the difficulty in distinguishing disease-derived EVs from normal EVs, which is essential for accurate detection.42 Current therapeutic applications also lack standardized methodologies and sufficient clinical evidence to confirm their effectiveness.29 EVs may additionally contain oncogenic molecules,47 indicating a need for differentiation between engineered and tumor-derived EVs. The absence of consistent protocols for evaluating EV-based vaccines and immune variability further hinders their assessment.53 Engineered EVs are also susceptible to enzymatic degradation or uptake by unintended cells.50,56 Finally, the need for specialized equipment increases the cost and processing time associated with large-scale EV production and analysis, creating additional barriers to clinical use.29
Further research is required to distinguish disease-derived EVs, optimize isolation techniques to improve yield and purity, and differentiate engineered EVs from tumor-derived EVs. Additionally, standardized protocols for assessing vaccine efficacy, strategies for producing targeted EVs for drug delivery,56 and cost-effective methods for large-scale EV production are required to achieve successful clinical applications.

6. CONCLUSION
EVs are crucial in advancing personalized medicine because of their roles in disease diagnosis, therapy, and cellular communication. Their ability to carry stable biomolecules creates opportunities for noninvasive diagnostics and targeted drug delivery. Although substantial progress has been made in understanding and applying EVs, several challenges remain, including a need to refine isolation techniques and to address safety concerns. With continued research and innovation, EVs have the potential to transform personalized health care by enabling more precise and effective treatments.