Application Of Extracellular Vesicles Research Articles

Recent Advances in Microfluidic-Based Extracellular Vesicle Analysis

Extracellular vesicles (EVs) serve as vital messengers, facilitating communication between cells, and exhibit tremendous potential in the diagnosis and treatment of diseases. However, conventional EV isolation methods are labor-intensive, and they harvest EVs with low purity and compromised recovery. In addition, the drawbacks, such as the limited sensitivity and specificity of traditional EV analysis methods, hinder the application of EVs in clinical use. Therefore, it is urgent to develop effective and standardized methods for isolating and detecting EVs. Microfluidics technology is a powerful and rapidly developing technology that has been introduced as a potential solution for the above bottlenecks. It holds the advantages of high integration, short analysis time, and low consumption of samples and reagents. In this review, we summarize the traditional techniques alongside microfluidic-based methodologies for the isolation and detection of EVs. We emphasize the distinct advantages of microfluidic technology in enhancing the capture efficiency and precise targeting of extracellular vesicles (EVs). We also explore its analytical role in targeted detection. Furthermore, this review highlights the transformative impact of microfluidic technology on EV analysis, with the potential to achieve automated and high-throughput EV detection in clinical samples.

Identification and validation of the surface proteins FIBG, PDGF-β, and TGF-β on serum extracellular vesicles for non-invasive detection of colorectal cancer: experimental study.

The absence of non-invasive biomarkers for the early diagnosis of colorectal cancer (CRC) has contributed to poor prognosis. Extracellular vesicles (EVs) have emerged as promising candidates for cancer monitoring using liquid biopsy. However, the complexity of EVs isolation procedures and absence of clear targets for detecting serum-derived EVs have hindered the clinical application of EVs in early CRC diagnosis. In the discovery phase, we conducted a comprehensive 4D-DIA proteomic analysis of serum-derived EVs samples from 37 individuals, performing an initial screening of EVs surface proteins. In the technical validation phase, we developed an extraction-free CRC-EVArray microarray to assess the expression of these potential EVs surface proteins in a multicenter study comprising 404 individuals. In the application phase, we evaluated the diagnostic efficacy of the CRC-EVArray model based on machine-learning algorithms. Through 4D-DIA proteomic analysis, we identified 7 potential EVs surface proteins showing significantly differential expression in CRC patients compared to healthy controls. Utilizing our developed high-throughput CRC-EVArray microarray, we further confirmed the differential expression of 3 EVs surface proteins, FIBG, PDGF-β and TGF-β, in a large sample population. Moreover, we established an optimal CRC-EVArray model using the NNET algorithm, demonstrating superior diagnostic efficacy with an AUC of 0.882 in the train set and 0.937 in the test set. Additionally, we predicted the functions and potential origins of these EVs-derived proteins through a series of multi-omics approaches. Our systematic exploration of surface protein expression profiles on serum-derived EVs has identified FIBG, PDGF-β, and TGF-β as novel diagnostic biomarkers for CRC. And the development of CRC-EVArray diagnostic model based on these findings provided an effective tool for the large-scale CRC screening, thus facilitating its translation into clinical practice.

Microbial extracellular vesicles contribute to antimicrobial resistance.

With the escalating global antimicrobial resistance crisis, there is an urgent need for innovative strategies against drug-resistant microbes. Accumulating evidence indicates microbial extracellular vesicles (EVs) contribute to antimicrobial resistance. Therefore, comprehensively elucidating the roles and mechanisms of microbial EVs in conferring resistance could provide new perspectives and avenues for novel antimicrobial approaches. In this review, we systematically examine current research on antimicrobial resistance involving bacterial, fungal, and parasitic EVs, delineating the mechanisms whereby microbial EVs promote resistance. Finally, we discuss the application of bacterial EVs in antimicrobial therapy.

Optimization of protocols for blood-derived extracellular vesicles for studies in liver diseases

Aim: Extracellular vesicles (EVs) have gained significant attention for their diagnostic and therapeutic potential in various diseases, including liver disorders. This study focuses on optimizing the isolation and characterization of small EVs from plasma and serum samples in patients with liver diseases, aiming to advance our understanding and potential clinical applications of EVs. Methods: Blood samples were collected from patients with end-stage liver disease (ESLD) enlisted in the TransplantLines Cohort and Biobank Study, and healthy donors. We employed differential ultracentrifugation (DUC) to evaluate three distinct protocols: a 3-step DUC, a washing step omitted [samples without washing (WW)], and a contaminant-depleted plasma (CDP) protocol. RNA isolation methodologies were compared, involving the use of TRI-reagent or the commercial AllPrep DNA/RNA kit. Further insights into EV composition were obtained through proteomic analyses, comparing samples subjected to traditional cell lysis (L) with those processed without lysis (NL). Results: We successfully isolated EVs from both plasma and serum samples as confirmed by the presence of specific EV markers, including CD9, CD63, CD81, and tumor susceptibility gene 101 (TSG-101). While some contaminants remained, such as albumin and lipoproteins, the protocol selected to continue EVs analysis was the 3-step protocol. Transmission electron microscopy (TEM) and nanotracking analysis (NTA) further confirmed EVs presence. RNA extraction was achieved using TRI-reagent, but not with the commercial kit highlighting the importance of selecting an appropriate method for RNA isolation. Finally, proteomics analysis showed that lysed samples were significantly more enriched in proteins compared to non-lysed samples, although protein variability was still present in both groups. Conclusions: Optimizing EV isolation techniques is essential for harnessing their potential in liver disease diagnosis and therapy. Further refinement of purification methods, a deep characterization of our cohort and understanding the variability and cargo within EVs will be crucial for future biomarker discovery and therapeutic applications in liver-related diseases.

Immunosuppressive microvesicles-mimetic derived from tolerant dendritic cells to target T-lymphocytes for inflammation diseases therapy

The utilization of extracellular vesicles (EV) in immunotherapy, aiming at suppressing peripheral immune cells responsible for inflammation, has demonstrated significant efficacy in treating various inflammatory diseases. However, the clinical application of EV has faced challenges due to their inadequate targeting ability. In addition, most of the circulating EV would be cleared by the liver, resulting in a short biological half-life after systemic administration. Inspired by the natural microvesicles (MV, as a subset of large size EV) are originated and shed from the plasma membrane, we developed the immunosuppressive MV-mimetic (MVM) from endotoxin tolerant dendritic cells (DC) by a straightforward and effective extrusion approach, in which DC surface proteins were inherited for providing the homing ability to the spleen, while αCD3 antibodies were conjugated to the MVM membranes for specific targeting of T cells. The engineered MVM carried a large number of bioactive cargos from the parental cells, which exhibited a remarkable ability to promote the induction of regulatory T cells (Treg) and polarization of anti-inflammatory M2 macrophages. Mechanistically, the elevated Treg level by MVM was mediated due to the upregulation of miR-155-3p. Furthermore, it was observed that systemic and local immunosuppression was induced by MVM in models of sepsis and rheumatoid arthritis through the improvement of Treg and M2 macrophages. These findings reveal a promising cell-free strategy for managing inflammatory responses to infections or tissue injury, thereby maintaining immune homeostasis.

Codetection of Proteins and RNAs on Extracellular Vesicles for Pancreatic Cancer Early Diagnosis.

Tumor-derived extracellular vesicles (EVs) carry tumor-specific proteins and RNAs, thus becoming prevalent targets for early cancer diagnosis. However, low expression of EV cargos and insufficient diagnostic power of individual biomarkers hindered EVs application in clinical practice. Herein, we propose a multiplex Codetection platform of proteins and RNAs (Co-PAR) for EVs. Co-PAR adopted a pair of antibody-DNA probes to recognize the same target protein, which in turn formed a double-stranded DNA. Thus, the target protein could be quantified by detecting the double-stranded DNA via qPCR. Meanwhile, qRT-PCR simultaneously quantified the target RNAs. Thus, with a regular qPCR instrument, Co-PAR enabled the codetection of multiplex proteins and RNAs, with the sensitivity of 102 EVs/μL (targeting CD63) and 1 EV/μL (targeting snRNA U6). We analyzed the coexpressions of three protein markers (CD63, GPC-1, HER2) and three RNA markers (snRNA U6, GPC-1 mRNA, miR-10b) on EVs from three pancreatic cell lines and 30 human plasma samples using Co-PAR. The diagnostic accuracy of the 6-biomarker combination reached 92.9%, which was at least 6.2% higher than that of 3-biomarker combinations and at least 13.5% higher than that of 6 single biomarkers. Co-PAR, as a multiparameter detection platform for EVs, has great potential in early disease diagnosis.

Unlocking the Potential of Extracellular Vesicles as the Next Generation Therapy: Challenges and Opportunities.

Mesenchymal stem cells (MSCs) have undergone extensive investigation for their potential therapeutic applications, primarily attributed to their paracrine activity. Recently, researchers have been exploring the therapeutic potential of extracellular vesicles (EVs) released by MSCs. MEDLINE/PubMed and Google scholar databases were used for the selection of literature. The keywords used were mesenchymal stem cells, extracellular vesicles, clinical application of EVs and challenges EVs production. These EVs have demonstrated robust capabilities in transporting intracellular cargo, playing a critical role in facilitating cell-to-cell communication by carrying functional molecules, including proteins, RNA species, DNAs, and lipids. Utilizing EVs as an alternative to stem cells offers several benefits, such as improved safety, reduced immunogenicity, and the ability to traverse biological barriers. Consequently, EVs have emerged as an increasingly attractive option for clinical use. From this perspective, this review delves into the advantages and challenges associated with employing MSC-EVs in clinical settings, with a specific focus on their potential in treating conditions like lung diseases, cancer, and autoimmune disorders.

Extracellular vesicle‐based biomarker in head and neck cancer: prospects and challenges

AbstractHead and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. However, very few established diagnostic biomarkers for HNSCC have been universally applied in clinical practice. Recently, much attention has been paid to extracellular vesicles (EVs) regarding their roles as cancer biomarkers because EVs carry plentiful cargoes, including lipids, proteins, nucleic acids, and metabolites. In HNSCC, several molecules carried by EVs, which are derived from peripheral blood and saliva, have been implicated to be effective in cancer detection, staging, treatment planning, response monitoring, and prognosis prediction. Although several EV molecules have been identified to be significantly correlated with a set of clinical‐pathological parameters of HNSCC, several key limitations need to be resolved before the clinical application of EVs as carriers of biomarkers in HNSCC. In this review, we discuss current knowledge in the literature regarding EV‐based biomarkers in HNSCC, emphasizing current limitations of their clinical applications.

Exosomes derived from tumor adjacent fibroblasts efficiently target pancreatic tumors

The application of extracellular vesicles, particularly exosomes (EXs), is rapidly expanding in the field of medicine, owing to their remarkable properties as natural carriers of biological cargo. This study investigates utilization of exosomes derived from stromal cells of tumor adjacent normal tissues (NAF-EXs) for personalized medicine, which can be derived at the time of diagnosis by endoscopic ultrasound. Herein, we show that exosomes (EXs) derived from NAFs demonstrate differential bio-physical characteristics, efficient cellular internalization, drug loading efficiency, pancreatic tumor targeting and delivery of payloads. NAF-derived EXs (NAF-EXs) were used for loading ormeloxifene (ORM), a potent anti-cancer and desmoplasia inhibitor as a model drug. We found that ORM maintains normal fibroblast cell phenotype and renders them incompatible to be triggered for a CAF-like phenotype, which may be due to regulation of Ca2+ influx in fibroblast cells. NAF-EXs-ORM effectively blocked oncogenic signaling pathways involved in desmoplasia and epithelial mesenchymal transition (EMT) and repressed tumor growth in xenograft mouse model. In conclusion, our data suggests preferential tropism of NAF-EXs for PDAC tumors, thus imply feasibility of developing a novel personalized medicine for PDAC patients using autologous NAF-EXs for improved therapeutic outcome of anti-cancer drugs. Additionally, it provides the opportunity of utilizing this biological scaffold for effective therapeutics in combination with standard therapeutic regimen.

Systems Approaches to Cell Culture-Derived Extracellular Vesicles for Acute Kidney Injury Therapy: Prospects and Challenges.

Acute kidney injury (AKI) is a heterogeneous syndrome, comprising diverse etiologies of kidney insults that result in high mortality and morbidity if not well managed. Although great efforts have been made to investigate underlying pathogenic mechanisms of AKI, there are limited therapeutic strategies available. Extracellular vesicles (EV) are membrane-bound vesicles secreted by various cell types, which can serve as cell-free therapy through transfer of bioactive molecules. In this review, we first overview the AKI syndrome and EV biology, with a particular focus on the technical aspects and therapeutic application of cell culture-derived EVs. Second, we illustrate how multi-omic approaches to EV miRNA, protein, and genomic cargo analysis can yield new insights into their mechanisms of action and address unresolved questions in the field. We then summarize major experimental evidence regarding the therapeutic potential of EVs in AKI, which we subdivide into stem cell and non-stem cell-derived EVs. Finally, we highlight the challenges and opportunities related to the clinical translation of animal studies into human patients.

Most recent advances and applications of extracellular vesicles in tackling neurological challenges.

Over the past few decades, there has been a notable increase in the global burden of central nervous system (CNS) diseases. Despite advances in technology and therapeutic options, neurological and neurodegenerative disorders persist as significant challenges in treatment and cure. Recently, there has been a remarkable surge of interest in extracellular vesicles (EVs) as pivotal mediators of intercellular communication. As carriers of molecular cargo, EVs demonstrate the ability to traverse the blood-brain barrier, enabling bidirectional communication. As a result, they have garnered attention as potential biomarkers and therapeutic agents, whether in their natural form or after being engineered for use in the CNS. This review article aims to provide a comprehensive introduction to EVs, encompassing various aspects such as their diverse isolation methods, characterization, handling, storage, and different routes for EV administration. Additionally, it underscores the recent advances in their potential applications in neurodegenerative disorder therapeutics. By exploring their unique capabilities, this study sheds light on the promising future of EVs in clinical research. It considers the inherent challenges and limitations of these emerging applications while incorporating the most recent updates in the field.

The application of extracellular vesicles in orthopedic diseases

The application of extracellular vesicles in orthopedic diseases

Detecting and exploring kidney-derived extracellular vesicles in plasma.

Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

Emerging role of mesenchymal stem cells-derived extracellular vesicles in vascular dementia.

Vascular dementia (VD) is a prevalent cognitive disorder among the elderly. Its pathological mechanism encompasses neuronal damage, synaptic dysfunction, vascular abnormalities, neuroinflammation, and oxidative stress, among others. In recent years, extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have garnered significant attention as an emerging therapeutic strategy. Current research indicates that MSC-derived extracellular vesicles (MSC-EVs) play a pivotal role in both the diagnosis and treatment of VD. Thus, this article delves into the recent advancements of MSC-EVs in VD, discussing the mechanisms by which EVs influence the pathophysiological processes of VD. These mechanisms form the theoretical foundation for their neuroprotective effect in VD treatment. Additionally, the article highlights the potential applications of EVs in VD diagnosis. In conclusion, MSC-EVs present a promising innovative treatment strategy for VD. With rigorous research and ongoing innovation, this concept can transition into practical clinical treatment, providing more effective options for VD patients.

Extracellular Vesicles for Drug Delivery and Theranostics In Vivo.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanopouches generated by all cells and are abundant in various body fluids. Depending on the parent and recipient cells, EVs exchange diverse constituents including nucleic acids, proteins, carbohydrates, and metabolites. Morphologically, EVs suffer from low zeta potentials and short circulation times, but they also offer low intrinsic immunogenicity and inherent stability. Some crucial factors for the effective clinical application of EVs include controlling immune system clearance, achieving the large-scale production of EVs with efficient quality control, and determining the dominant mechanism of the in vivo action of EVs. In this Perspective, we shed light on how these intriguing nano-objects are utilized in cellular imaging and drug delivery for disease therapeutics. We also discuss potential strategies for overcoming the associated limitations.

Inactivation of the NLRP3 inflammasome mediates exosome-based prevention of atrial fibrillation.

Rationale: Extracellular vesicles (EVs) from human explant-derived cells injected directly into the atria wall muscle at the time of open chest surgery reduce atrial fibrosis, atrial inflammation, and atrial fibrillation (AF) in a rat model of sterile pericarditis. Albeit a promising solution to prevent postoperative AF, the mechanism(s) underlying this effect are unknown and it is not clear if this benefit is dependent on EV dose. Methods: To determine the dose-efficacy relationship of EVs from human explant-derived cells in a rat model of sterile pericarditis. Increasing doses of EVs (106, 107, 108 or 109) or vehicle control were injected into the atria of middle-age male Sprague-Dawley rats at the time of talc application. A sham control group was included to demonstrate background inducibility. Three days after surgery, all rats underwent invasive electrophysiological testing prior to sacrifice. Results: Pericarditis increased the likelihood of inducing AF (p<0.05 vs. sham). All doses decreased the probability of inducing AF with maximal effects seen after treatment with the highest dose (109, p<0.05 vs. vehicle). Pericarditis increased atrial fibrosis while EV treatment limited the effect of pericarditis on atrial fibrosis with maximal effects seen after treatment with 108 or 109 EVs. Increasing EV dose was associated with progressive decreases in pro-inflammatory cytokine content, inflammatory cell infiltration, and oxidative stress. EVs decreased NLRP3 (NACHT, LRR, and PYD domains-containing protein-3) inflammasome activation though a direct effect on resident atrial fibroblasts and macrophages. This suppressive effect was exclusive to EVs produced by heart-derived cells as application of EVs from bone marrow or umbilical cords did not alter NLRP3 activity. Conclusions: Intramyocardial injection of incremental doses of EVs at the time of open chest surgery led to progressive reductions in atrial fibrosis and inflammatory markers. These effects combined to render atria resistant to the pro-arrhythmic effects of pericarditis which is mechanistically related to suppression of the NLRP3 inflammasome.

Extracellular vesicles in chronic kidney disease: diagnostic and therapeutic roles.

Chronic kidney disease (CKD) is a progressive disorder characterized by structural and functional changes in the kidneys, providing a global health challenge with significant impacts on mortality rates. Extracellular vesicles (EVs), are vital in the physiological and pathological processes associated with CKD. They have been shown to modulate key pathways involved in renal injury, including inflammation, fibrosis, apoptosis, and oxidative stress. Currently, the application research of EVs in the diagnosis and treatment of CKD is highly prevalent. However, there is currently a lack of standardized guidelines for their application, and various methodologies have advantages and limitations. Consequently, we present an comprehensive summary elucidating the multifaceted involvement of EVs in both physiological and pathological aspects in CKD. Furthermore, we explore their potential as biomarkers and diverse therapeutic roles in CKD. This review provides an overview of the current state of research on application of EVs in the diagnosis and therapeutic management of CKD.

Unraveling the Multifaceted Roles of Extracellular Vesicles: Insights into Biology, Pharmacology, and Pharmaceutical Applications for Drug Delivery.

Extracellular vesicles (EVs) are nanoparticles released from various cell types that have emerged as powerful new therapeutic option for a variety of diseases. EVs are involved in the transmission of biological signals between cells and in the regulation of a variety of biological processes, highlighting them as potential novel targets/platforms for therapeutics intervention and/or delivery. Therefore, it is necessary to investigate new aspects of EVs' biogenesis, biodistribution, metabolism, and excretion as well as safety/compatibility of both unmodified and engineered EVs upon administration in different pharmaceutical dosage forms and delivery systems. In this review, we summarize the current knowledge of essential physiological and pathological roles of EVs in different organs and organ systems. We provide an overview regarding application of EVs as therapeutic targets, therapeutics, and drug delivery platforms. We also explore various approaches implemented over the years to improve the dosage of specific EV products for different administration routes.

Potential Use of Extracellular Vesicles in the Treatment of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain, and several studies have evaluated the efficacy of extracellular vesicles (EVs) in the treatment of IVDD. The databases PubMed, Embase, and Cochrane Library were systematically searched from inception to the end of 2022 to identify studies investigating the therapeutic potential of cell-derived EVs for IVDD treatment. The following outcome measures were utilized: magnetic resonance imaging (MRI) Pfirrmann grading system, disc height index (DHI), histological grading, and apoptosis rate. A comprehensive meta-analysis was conducted, including a total of 13 articles comprising 19 studies involving 218 experimental animals. Comparative analysis between normal cell-derived EVs and placebo revealed significant reductions in MRI grade, increased DHI values, decreased nucleus pulposus cell apoptosis rates, and improved tissue grades. These findings collectively demonstrate the effective inhibition of IVDD through the application of EVs derived from cells. In conclusion, this study provides an updated synthesis of evidence supporting the efficacy of EVs as a promising therapeutic approach for IVDD treatment.

Adipose Tissue-Derived Extracellular Vesicles: A Promising Biomarker and Therapeutic Strategy for Metabolic Disorders.

Adipose tissue plays an important role in systemic energy metabolism, and its dysfunction can lead to severe metabolic disorders. Various cells in adipose tissue communicate with each other to maintain metabolic homeostasis. Extracellular vesicles (EVs) are recognized as novel medium for remote intercellular communication by transferring various bioactive molecules from parental cells to distant target cells. Increasing evidence suggests that the endocrine functions of adipose tissue and even the metabolic homeostasis are largely affected by different cell-derived EVs, such as insulin signaling, lipolysis, and metabolically triggered inflammation regulations. Here, we provide an overview focused on the role of EVs released by different cell types of adipose tissue in metabolic diseases and their possible molecular mechanisms and highlight the potential applications of EVs as biomarkers and therapeutic targets. Moreover, the current EVs-based therapeutic strategies have also been discussed. This trial is registered with NCT05475418.