ApoEVs are distinguished from exosomes and microvesicles by their origin from distinct cellular processes specifically, ApoEVs are generated during the late stages of apoptosis as the result of a complicated process of membrane blebbing and fragmentation of cells (1, 16, 17). In general, ApoEVs are larger compared to exosomes and microvesicles, ranging from 50 nm to several micrometers in diameter (1).

The most recent studied indicate that ApoEVs production is not merely a random cell fragmentation, but it is driven by a tightly coordinated process. Specific molecular pathways, such as the activation of Rho-associated protein kinase 1 (ROCK1) and the formation of membrane protrusions known as apoptopodia. Inhibition of Pannexin 1 (PANX1) channel has also been shown to influence the release of ApoEVs by modulating this string-like membrane structure (18).

We suggest a new classification of ApoEV biogenesis pathways based on new data: Large membrane bleb formation with cellular contents, actin-myosin contraction, and ROCK1 activation characterize the Classical Membrane Blebbing Pathway (19, 20). The development of beaded apoptopodia with the shedding of smaller and more homogeneous ApoEVs containing different cargo profiles is described as the Apoptopodia-Mediated Pathway (14, 21). And, PANX1-Regulated Pathway This pathway is defined by the regulation of nuclear content incorporation into ApoEVs via PANX1 channels (22, 23).

The size range heterogeneity and cargo content variability seen between studies may be explained by this classification, which offers a more general explanation for ApoEV heterogeneity.

ApoEVs contain nuclei debris, and structurally recognizable organelles and other cellular components such as proteins, RNA, and DNA, as shown by microscopy. Based on the cell type and apoptotic triggers, their nuclear and mitochondrial content varies and can be used to distinguish them, Figure 1, Table 2.

The composition of ApoEVs further differs from that of other subtypes of EVs, including differences in surface markers expression. Besides the Major Histocompatibility Complexes I and II (MHC I, II), which are present on ApoEVs as well as other types of extracellular vesicles, ApoEVs are specifically enriched in complement proteins, and thrombospondin (TSP). Additionally, phosphatidylserine (PS) externalization onto ApoEV surfaces is a hallmark feature that serves as a phagocytic cell recognition signal (24).

Therefore, ApoEVs functional characteristics, cellular origin, and cargo composition, a comprehensive classification scheme is reported in Table 2. This framework provides a basis for understanding the heterogeneity of ApoEVs and their potential diagnostic applications. Type II and Type III ApoEVs may reflect inflammatory processes and dysregulated cell signaling in neurological disorders, whereas Type I ApoEVs, characterized by a high DNA content, may be particularly valuable for detecting genetic alterations in cancer.

The complex cell events that take place during apoptosis induce a characteristic alteration of the cellular proteome that is faithfully reflected in the cargo of ApoEVs (1, 5). These vesicles contain structural proteins which mirror the cell state and anatomical site at the time of death, intracellular enzymes that help define the parental original cell type (5), and key regulators of the apoptotic signaling pathway (1).

Comparative proteomic analyses demonstrate that ApoEVs and extracellular vesicles released by viable cells (EVs) possess distinct surface-marker repertoires. While CEVs are enriched in CD9, Programmed Cell Death 6-interacting protein (PDCD6IP or ALIX), Ras-related protein (RAB7), and Sphingosine-1-phosphate receptors 2 (S1PR2), ApoEVs display higher levels of CD63, Lysosomal-associated membrane protein-1 (LAMP1), Heat shock protein70 (HSP70), and Sphingosine-1-phosphate receptors (S1PR1, S1PR3, S1PR4) (25).

These differences have important diagnostic implications. For instance, membrane proteins uniquely abundant on ApoEVs can serve as indicators of tissue injury from specific cell types, facilitating the monitoring of organ- or cell-targeted pathologies (1, 5). For example, vimentin, which is highly expressed on apoptotic T cells, has been detected at elevated levels in the serum of patients with rheumatoid arthritis, myocarditis, and systemic lupus erythematosus (SLE) probably deriving from T cell ApoEVs (26).

In one illustrative example, shotgun proteomics of ApoEVs derived from biliary epithelial cells identified eleven proteins, including Annexin A6, heat shock protein 6 (HSP6), and LDL receptor- related proteins, are specific to biliary epithelial cell ApoEVs and not found in EVs shed by healthy control cells. A possible role in autoimmune response is suggested by the interactions of these proteins with immunological pathways including NF-kB, ERK, and Notch signaling (27).

It is worth noting that ApoEV proteomic profiles vary with the cell type, the nature of the pro-apoptotic stimuli, and the surrounding pathological milieu. This heterogeneity represents a challenge for biomarker standardization but also offers the opportunity to develop highly specific and personalized diagnostic assays provided that candidate markers are validated across different patient cohorts.

New therapeutic avenues have been opened by the finding that ApoEVs carry DNA and RNA (1). Key genetic changes characteristic of diseases such as cancer chromosomal rearrangements, deletions, or mutations can be detected in DNA isolated from ApoEVs (3, 28).

ApoEV-derived DNA offers several advantages as biomarker source. Firstly, it is very stable under a range of storage conditions, making it suitable for clinical workflows with delayed sample processing. making it suitable for clinical workflows with delayed sample processing. Secondly, it enables non-invasive way of detection of tumor specific mutations and can support minimal residual disease monitoring, cancer diagnosis, and assessment of treatment response (3).

The miRNA and mRNA cargo profile of ApoEVs reveal insight into the gene expression regulation of apoptosis and has been investigated as a biomarker source across a number of disorders (1, 29). For instance, in mouse models, endothelial cell-derived ApoEVs enriched in miR-126 have been shown to slow the formation of atherosclerotic plaque by recruiting Sca-1+ progenitor stem cells and preserving endothelial integrity (30).

The fragmentation pattern of nucleic acids occurring during the apoptotic process and carried by ApoEVs may be an important biomarker feature. Indeed, the well-known nucleosome-sized DNA fragments with a visible peak at around 150–200 bp have been demonstrated to be present also in plasma ApoEVs (31). Numerous neurological disorders, such as multiple sclerosis, ischemic stroke, and Parkinson’s disease, exhibit this fragmentation pattern, reflecting a shared mechanism of DNA packaging in ApoEVs which may serve as a monitorable biomarker. Additionally, as reported in Table 3, heterogeneity exists among the different types of nucleic acids within ApoEVApoEVs cargo, increasing their applications in diagnostics of diseases. Although each class offers distinct advantages, there are also certain technological issues that need to be resolved prior to their use in therapy applications.

Recent developments suggest new avenues for the generation of biomarkers by exploiting the natural DNA packaging mechanisms in ApoEVs through endogenous proteases. In apoptotic cells and, by extension, in ApoEVs, proteases like matrix metalloproteinase-2 (MMP-2) and caspase-3 (Casp-3) regulate DNA assembly, and this process is reflected in the DNA content of ApoEVs. By monitoring these enzymes as endogenous stimuli can leverage this natural mechanism to develop biomarkers (32). These strategies can find application in metastatic monitoring, response to treatment monitoring, and disease diagnosis. For example, one promising method for monitoring metastases is the controlled assembly of DNA in ApoEVs through protease activity (e.g., MMP-2 and caspase-3). ApoEVs can produce dynamic biomarkers that represent tumor aggressiveness, metastatic spread, and treatment response by using these enzymes as endogenous stimuli. With liquid biopsies, this method allows for non-invasive, real-time tracking of disease progression, which may enhance early detection and individualized treatment plans.

This analogy demonstrates ApoEVs’ heterogeneity in nucleic acid cargo and their applications in diagnostics of diseases. Although there are distinct benefits to each class, there are also certain technological issues that need to be resolved prior to their use in therapy applications.

Given ApoEV heterogeneity in size, density, and composition, their isolation poses significant technical challenges. Some of the recent isolation techniques include differential centrifugation, density gradient centrifugation, filtration, immune-affinity capture, and microfluidic platforms. The purity, yield, and integrity of isolated ApoEVs depend on the strengths and limitations of each isolation technique as it demonstrated in Table 4.

A variety of methods, each with distinct advantages and limitations, are used in the isolation of ApoEVs. Although differential centrifugation is easy to use and accessible, its clinical utility is limited by the low-to-medium purity it produces due to contamination risks (33). Density gradient centrifugation improves purity, but it is time-consuming and has a low throughput (34). Although size exclusion chromatography (SEC) offers high purity and preserved vesicle integrity by separating vesicles based on size, it has low resolution for particles of similar sizes and necessitates large sample volumes (35). Filtration devices such as Exodiscs (36) offer a quick and scalable method, but can cause vesicle deformation and clogging (37). Immunoaffinity capture is expensive and marker-dependent, nonetheless it offers high specificity for subpopulations (38). In the case of microfluidic devices, they lack throughput and need validation, but microfluidic devices allow automation and minimal sample use (39). Notably, apoptopodia isolation in conjunction with PANX1 inhibition shows promise as an experimental technique, providing high yield and homogeneity but requiring standardization (40, 41). While microfluidics and PANX1 inhibition demonstrate innovation in clinical translation, SEC and immunoaffinity stand out for purity; however, scalability and reproducibility continue to be major obstacles. Depending on the sample type, downstream analysis, and the working conditions, researchers should select the appropriate separation technique (42).

No one technique is best for every application, as illustrated by this thorough review. The research or clinical query, needed purity and yield, and resources available must all be taken into consideration when selecting a method of isolation. One might find a combination of methods to be the best compromise of convenience, yield, and purity for clinical use.

ApoEVs must be accurately detected and characterized in order to be used as biomarkers. Currently used techniques include electron microscopy, molecular assays (such as qPCR), flow cytometry, imaging flow cytometry, and nanoparticle tracking analysis. Although the sensitivity, specificity, and technical requirements of each approach vary, they all provide unique insights into ApoEV characteristics.

Among the different techniques, flow cytometry is the most frequently used for ApoEV analysis due to its ability to simultaneously analyze multiple markers through multiparametric analysis (43). The detection of small ApoEVs is diffiicult as well as the separation from background noise using standard flow cytometry. Standard flow cytometry typically struggles to detect particles below 200–300 nm due to limitations in light scattering sensitivity. However, dedicated small-particle or high-resolution flow cytometers have been developed, enabling detection of particles as small as 100 nm, including small extracellular vesicles like ApoEVs. Moreover, integrating flow cytometry and microscopy, imaging flow cytometry addresses some of these disadvantages and enables the visual confirmation of events (44).

While standard nanoparticle tracking analysis (NTA) can be helpful for determining particle size and concentration, it does not offer information on marker expression (45). Additionally, electron microscopy (EM) provides high-resolution imaging of ApoEV morphology, its low throughput and limited suitability for routine clinical application constrains its practical use (46). Artificial Intelligence (AI)-assisted video microscopy, which use deep convolutional neural networks to automatically identify apoptotic bodies and cells in label-free time-lapse microscopy, is one example of a recent development in detection techniques (47).This method provides high-throughput analysis with little sample preparation, although it needs to be validated in various cell types and disease scenarios to pass the checklist of “EV-TRACK: transparent reporting and centralizing knowledge in EVs research” (48). pros and cons of each detection method in the case of sensitivity and specificity is demonstrated in Table 5.

Several new technologies are being promising for increased sensitivity, specificity, and clinical value in the fast-moving field of ApoEVs detection and isolation. These include:

ApoEVs have very significant clinical potential in cancer diagnostics. ApoEVs harboring tumor-specific biomarkers are released into the bloodstream by cancer cells going through apoptosis, either naturally or in response to treatment (28, 56). These biomarkers offer important insights about the nature of tumors, the effectiveness of treatments, and the course of the disease.

EVs-derived biomarkers have shown diagnostic potential across various types of cancer. For instance, It can be possible that EpCAM-positive ApoEVs indicate colorectal cancer (57) CD138-positive microparticles assist in diagnosing multiple myeloma (58) KRAS-mutated EVs-DNA supports early detection of pancreatic cancer (59) miRNA signatures in plasma vesicles help monitor Hodgkin lymphoma treatment response (60) TrpC5-EVs are linked to chemoresistance in metastatic breast cancer (61) and urinary EVs included ApoEVs serve as protein biomarkers for prostate cancer diagnosis and prognosis (62, 63).

Nevertheless, a careful review of the literature identifies a number of shortcomings and inconsistencies in the state of the field. Results may be skewed by the fact that many studies do not differentiate ApoEVs from other EV subtypes. Furthermore, there is significant variation in the specificity of suggested biomarkers among research, with some exhibiting overlap between cancer and non-cancer illnesses. It sounds some of the biomarkers that mentioned as cancer biomarkers can be presented in healthy ApoEVs and EVs too. Additionally, majority of papers have not drawn a line between EVs and ApoEVs.

We must contrast ApoEVs with well-established methods like circulating tumor cells (CTCs), cell-free DNA (cfDNA), and traditional protein biomarkers in order to assess their potential as cancer biomarkers.

ApoEVs differ from other liquid biopsy components in both their advantages and disadvantages. ApoEVs come from apoptotic cancer cells and are not as stable CTCs, which come from intact cancer cells but are uncommon in very early-stage disease and technically difficult to isolate. CTCs need a large number of samples (64). Moreover, in comparison to the full payload (DNA, RNA, proteins) of ApoEVs, this durability stands in contrast to the fragility of cfDNA and the moderate half-life of cfDNA, which, although important, offers limited molecular information (DNA-only) (65).

ApoEVs exhibit greater promise for early identification than both conventional protein markers (typically raised only in advanced disease) and CTCs (sometimes undetected in early stages). However, because of its wider cellular origins, cfDNA may more accurately reflect clonal diversity than ApoEVs, which only partially capture tumor heterogeneity (66). Although ApoEV profiling is technically less resource-intensive than CTC isolation, which necessitates specialist platforms like CellSearch^®, it is still more difficult than protein marker assays (67).

Although their multi-analyte cargo (such as tumor-specific RNAs + surface proteins) offers unique diagnostic relevance, ApoEVs clinically lag behind CTCs and cfDNA in validation (68). A brief comparison between the analysis of cancer biomarker was described in Table 6. This comparison draws attention to a number of possible benefits of ApoEVs, such as their early detection potential, stability, and rich information content. In contrast to well-established biomarkers, ApoEVs also have limitations in terms of clinical validation and technical complexity.

The emerging field of ApoEVs as a potential source of cancer biomarkers is highly promising but not yet fully established. Indeed, several disputes and contradictory results still persist on the following aspects. The area of ApoEVs as a possible source of cancer biomarkers is still in its infancy. The origin of circulating DNA is a major topic of controversy. While some studies contend that ApoEVs are primarily responsible for tumor-derived circulating DNA, others counter that the majority of circulating tumor DNA is non-vesicular, possibly as a result of variations in isolation methods and the cancer types studied (69). Furthermore, there is still debate on the specificity of ApoEVs markers since while some studies emphasize their great sensitivity for detecting cancer, other studies show substantial overlap with inflammatory and other disorders, casting doubt on the accuracy of the diagnosis (1).Uncertainty surrounds the association between ApoEVs and treatment response as well (70). The necessity for bigger, rigorously standardized trials is further highlighted by the fact that standardization issues, such as inconsistent isolation and characterization techniques, make it difficult to draw firm conclusions regarding the clinical utility of ApoEVs in cancer detection.

Altogether, these open questions emphasize that future research should address such inconsistencies by ensuring strict clinical validation, thorough characterization of ApoEVs subtypes, and improved separation methods for their isolation and analysis.

As a result of its poor prognosis and the critical need for early detection, pancreatic cancer represents a particularly compelling application for ApoEVs-based biomarkers. In vitro studies have shown that pancreatic cancer cells treated with gemcitabine release increased levels of ApoEVs, offering a window for therapy monitoring. Moreover, ApoEVs-derived DNA can reveal KRAS mutations, present in over 90% of pancreatic ductal adenocarcinomas, thereby enabling early detection and monitoring of minimal residual disease would be feasible. However, it is still diffiicult to distinguish tumor-derived ApoEVs and normal cell-derived ApoEVs undergoing apoptosis within the inflammatory milieu of pancreatic cancer. The detection of pancreatic cancer-derived ApoEVs has been improved by recent advances in microfluidic technologies. For example, impedance cytometry measurement of ApoEVs can assess drug sensitivity of pancreatic tumor cell lines in a label-free manner, illustrating a promising approach for real-time monitoring therapy response that could be translated to the clinics (71).

While these developments look promising, some problems need to be solved before ApoEV- based strategies can be implemented for pancreatic cancer. These include increasing specificity to discriminate between benign and malignant conditions, improving sensitivity to detect early stages disease, and establishing standardized protocols for sample collection and handling.

Since the blood-brain barrier and the Accessibility to the involved tissues are restricted, neurodegenerative diseases present unique problems in diagnosis. ApoEVs provide a potential method of non-invasively tracking disease progression and neuronal damage. The potential of ApoEVs as biomarkers for the disease conditions multiple sclerosis (MS), Parkinson’s disease (PD), and Alzheimer’s disease (AD) has been under investigation by several investigations.

Pathogenic proteins in neutrally derived blood exosomes and EVs in Alzheimer’s disease have been shown to identify preclinical AD (72). Amyloid-42 (A-42), phosphorylated tau (P-T181-tau, P-S396-tau), and dysfunctionally phosphorylated type 1 insulin receptor substrate (IRS-1) are only a few of them (72, 73). A less invasive option to cerebrospinal fluid testing is the identification of these markers in blood-derived ApoEVs.

Similarly, plasma levels of neuron-and glia-derived ApoEVs correlate with infarct size and patient functional outcome in ischemic stroke, suggesting that ApoEVs cane serve as apoptosis markers in vivo, with prognostic value in neurological disease (74).

Close inspection does identify a number of flaws in the present study. Numerous obstacles restrict the potential of neural-derived EVs, such as ApoEVs, as biomarkers for neurodegenerative disorders. Because different isolation strategies produce different EVs populations, methodological variation makes cross-study comparisons more difficult (75, 76). Furthermore, there are still problems with specificity because the neuronal markers that are now employed to identify brain-derived ApoEVs in peripheral blood might not be selective enough, requiring additional research. Their potential for staging neurodegenerative disorders is hampered by the lack of evidence linking EVs levels with disease development and the incomplete understanding of the temporal dynamics of EVs release (77). Additionally, interpretation can be complicated by confounding factors that can change ApoEVs profiles independently of neurodegeneration, such as comorbidities (e.g., cardiovascular disorders) frequent in neurodegenerative patients (78).

We contrast ApoEVs with well-established biomarkers including cerebrospinal fluid (CSF) analysis, neuroimaging, and traditional blood biomarkers in order to assess their potential in the diagnosis of neurodegenerative diseases. A comparison of different methods are applied were summarized in the Table 7.

ApoEVs offer a viable, but unproven, substitute for recognized indicators of neurodegenerative diseases. EVs detection provides a less intrusive blood-based method than costly neuroimaging approaches or invasive cerebrospinal fluid (CSF) analysis, which requires lumbar puncture (79). EVs exhibit medium-high specificity with the distinct benefit of having direct cellular contents that may more accurately reflect disease mechanisms, whereas CSF biomarkers show very high specificity for neurodegeneration and neuroimaging offers excellent structural assessment (77). EVs may provide high sensitivity for early identification, potentially outperforming traditional blood biomarkers, which often exhibit low-medium sensitivity, according to preliminary data (80). In contrast to the known differential diagnostic capabilities of CSF analysis and sophisticated neuroimaging, their capacity to differentiate between various neurodegenerative illnesses is still being studied (81). In contrast to more straightforward, low-cost blood tests, EVs analysis is still technically complex and medium-to-highly expensive, but it provides more direct pathological information than traditional serum indicators (82). EVs-based diagnoses need more standardization and extensive validation studies prior to routine clinical application, whereas CSF analysis and neuroimaging have broad clinical validation (83).

According to this comparison, ApoEVs may be able to supplement current strategies by providing a minimally invasive technique that has the potential to be highly sensitive for early detection and direct reflection of disease causes. To prove their specificity and capacity to differentiate between various neurodegenerative diseases, more research is necessary. As majority research does not differentiate between EVs and ApoEVs.

More recent studies indicate that ApoEVs may be involved in the pathogenesis of neurodegenerative disorders by enabling the disease transmission by transferring toxic proteins from one cell to another. Indeed, Tau protein-carrying ApoEVs can drive tau pathology transmission throughout the brain in Alzheimer’s disease (84, 85). Similarly, ApoEVs carrying α-synuclein can drive Lewy body pathology transmission in Parkinson’s disease. These processes indicate that ApoEVs have active roles in disease etiology rather than being a mere consequence of neurodegeneration (86–88).

ApoEVs’ immunomodulatory function also promotes neuroinflammation, a shared aspect of neurodegenerative conditions. Based on the cargo and target cells, ApoEVs can stimulate or inhibit inflammatory responses that can influence the severity and progression of the disease (89–92).

These mechanistic findings imply that, beside play a role as biomarkers, ApoEVs have a potential in neurodegenerative illnesses as therapeutic targets. Potential approaches could be directed to modulate their effect by modifying ApoEV synthesis, cargo loading, or absorption.

Due to the urgent need for early biomarkers and the diffiiculties associated with obtaining brain tissue, Alzheimer’s disease represents a compelling study case for the application of ApoEV. Blood- based ApoEVs secreted by neurons can be identified in preclinical phases of disease (92).

According to Fiandaca et al., a pathogenic protein profile of blood exosomes derived from neutrally is able to consistently differentiate between AD patients and frontotemporal dementia patients and healthy controls (72). P-T181-tau, P-S396-tau, and Aβ1-42, recognized markers of AD pathology, were included in the biomarker panel.

In addition, Kapogiannis et al. demonstrated that exosomes from neurons that bore dysfunctionally phosphorylated type 1 insulin receptor substrate (IRS-1) were able to forecast the development of AD up to ten years before its onset (73). This evidence implies that ApoEVs-based monitoring has the potential to interventions prior irreversible effects of neurodegeneration.

These promising findings highlight the significant potential of ApoEVs, which could be fully realized through ongoing improvements in isolation specificity, standardized protocols, and longitudinal validation to track biomarker dynamics over time. Integrating ApoEV-based diagnostics with established modalities such as neuroimaging and genetic risk assessment may further increase diagnostic precision and accelerate their adoption in clinical settings.

Despite the considerable promise of ApoEV-based biomarkers, several technical and biological sources of variability still need to vibe addressed to ensure reliable clinical translation. Pre-analytical factors such as sample handling, blood collection tubes, processing delays, freeze-thaw cycles and storage conditions can all affects ApoEVs integrity and recovery, while heterogeneity of isolation methods may yield preparations with different purity, yield, and representation of particle subpopulations. Moreover, biological variables-, including circadian rhythm, exercise, diet, and the intrinsic heterogeneity of diseases like cancer or neurological disorders-, further complicate consistent biomarker measurement. ApoEV pro- files can be all affected by disease stages, subtypes, and patient factors. To overcome these challenges, a comprehensive standardization is essential. This include, the adoption of rigorous standard operating procedures for sample collection and processing, the development of reference materials for method validation, the integration of robust; quality control procedures through- out the analytical process, and the implementation of normalization strategies to account for biological variability (93).

Several factors are being taken into account in the process of ApoEVs-based diagnostics regulation:

According to the FDA’s Bioanalytical Method Validation Guidance (2018) and MISEV2018 guidelines, development of ApoEVs as clinical diagnostics necessitates careful navigation of regulatory benchmarks, starting with analytical validation to demonstrate method sensitivity, specificity, accuracy, and precision in ApoEV isolation and characterization (13). Following models such as the EMA’s Guideline on Clinical Evaluation of Diagnostics (2017) and neuronal EV studies in Alzheimer’s disease (72), clinical validation must then demonstrate diagnostic accuracy and predictive value through multicenter trials. With guidelines from the NIH Extracellular RNA Communication Consortium for EV characterization, strict quality control procedures including pre-analytical to analytical stages are essential (94).The area also needs universal reference materials, which are being addressed by programs like NIST’s RM 8640 and EV-TRACK (48). Moreover, regulatory systems such as the Food and Drug Administration(FDA) and European Medicines Agency (EMA) require analytical validity through method validation studies, clinical validity through prospective trials, and demonstrated clinical utility through outcome studies (95). As demonstrated by consortia like ISEV’s Rigorous Science initiative (93), which together seek to standardize and validate ApoEV-based diagnostic approaches, successful translation will ultimately depend on cooperative efforts across academia, industry, and regulatory bodies.