Exosome prospects in the diagnosis and treatment of non-alcoholic fatty liver disease.

The growing prevalence of NAFLD and its global health burden have provoked considerable research on possible diagnostic and therapeutic options for NAFLD. Although various pathophysiological mechanisms and genetic factors have been identified to be associated with NAFLD, its treatment remains challenging. In recent years, exosomes have attracted widespread attention for their role in metabolic dysfunctions and their efficacy as pathological biomarkers. Exosomes have also shown tremendous potential in treating a variety of disorders. With increasing evidence supporting the significant role of exosomes in NAFLD pathogenesis, their theragnostic potential has become a point of interest in NAFLD. Expectedly, exosome-based treatment strategies have shown promise in the prevention and amelioration of NAFLD in preclinical studies. However, there are still serious challenges in preparing, standardizing, and applying exosome-based therapies as a routine clinical option that should be overcome. Due to the great potential of this novel theragnostic agent in NAFLD, further investigations on their safety, clinical efficacy, and application standardization are highly recommended.

Shedding Light on Cellular Secrets: A Review of Advanced Optical Biosensing Techniques for Detecting Extracellular Vesicles with a Special Focus on Cancer Diagnosis.

In the relentless pursuit of innovative diagnostic tools for cancer, this review illuminates the cutting-edge realm of extracellular vesicles (EVs) and their biomolecular cargo detection through advanced optical biosensing techniques with a primary emphasis on their significance in cancer diagnosis. From the sophisticated domain of nanomaterials to the precision of surface plasmon resonance, we herein examine the diverse universe of optical biosensors, emphasizing their specified applications in cancer diagnosis. Exploring and understanding the details of EVs, we present innovative applications of enhancing and blending signals, going beyond the limits to sharpen our ability to sense and distinguish with greater sensitivity and specificity. Our special focus on cancer diagnosis underscores the transformative potential of optical biosensors in early detection and personalized medicine. This review aims to help guide researchers, clinicians, and enthusiasts into the captivating domain where light meets cellular secrets, creating innovative opportunities in cancer diagnostics.

Extracellular vesicles (EVs)' journey in recipient cells: from recognition to cargo release. / èƒžå¤–å›Šæ³¡åœ¨å—ä½“ç»†èƒžä¸­çš„æ— ç¨‹ï¼šä»Žè¯†åˆ«åˆ°è´§ç‰©é‡Šæ”¾.

Extracellular vesicles (EVs) are nano-sized bilayer vesicles that are shed or secreted by virtually every cell type. A variety of biomolecules, including proteins, lipids, coding and non-coding RNAs, and mitochondrial DNA, can be selectively encapsulated into EVs and delivered to nearby and distant recipient cells, leading to alterations in the recipient cells, suggesting that EVs play an important role in intercellular communication. EVs play effective roles in physiology and pathology and could be used as diagnostic and therapeutic tools. At present, although the mechanisms of exosome biogenesis and secretion in donor cells are well understood, the molecular mechanism of EV recognition and uptake by recipient cells is still unclear. This review summarizes the current understanding of the molecular mechanisms of EVs' biological journey in recipient cells, from recognition to uptake and cargo release. Furthermore, we highlight how EVs escape endolysosomal degradation after uptake and thus release cargo, which is crucial for studies applying EVs as drug-targeted delivery vehicles. Knowledge of the cellular processes that govern EV uptake is important to shed light on the functions of EVs as well as on related clinical applications.

Roseburia intestinalis-derived extracellular vesicles ameliorate colitis by modulating intestinal barrier, microbiome, and inflammatory responses.

Inflammatory bowel disease (IBD) is a chronic disorder characterized by recurrent gastrointestinal inflammation, lacking a precise aetiology and definitive cure. The gut microbiome is vital in preventing and treating IBD due to its various physiological functions. In the interplay between the gut microbiome and human health, extracellular vesicles secreted by gut bacteria (BEVs) are key mediators. Herein, we explore the role of Roseburia intestinalis (R)-derived EVs (R-EVs) as potent anti-inflammatory mediators in treating dextran sulfate sodium-induced colitis. R was selected as an optimal BEV producer for IBD treatment through ANCOM analysis. R-EVs with a 76 nm diameter were isolated from R using a tangential flow filtration system. Orally administered R-EVs effectively accumulated in inflamed colonic tissues and increased the abundance of Bifidobacterium on microbial changes, inhibiting colonic inflammation and prompting intestinal recovery. Due to the presence of Ile-Pro-Ile in the vesicular structure, R-EVs reduced the DPP4 activity in inflamed colonic tissue and increased the active GLP-1, thereby downregulating the NFκB and STAT3 via the PI3K pathway. Our results shed light on the impact of BEVs on intestinal recovery and gut microbiome alteration in treating IBD.

Extracellular vesicle-liposome hybrids via membrane fusion using cell-penetrating peptide-conjugated lipids.

Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.

Extracellular vesicles and macrophages in tumor microenvironment: Impact on cervical cancer.

Cervical cancer is a serious threat to women's health. Extracellular vesicles exist in most body fluids for communication between organisms, having different effects on the occurrence, development, angiogenesis, and metastasis of cervical cancer, and are expected to become new targets for treatment. Macrophages are natural immune systems closely linked to the development of cervical cancer. In recent years, an increasing number of studies have confirmed the role of extracellular vesicles and macrophages in the gynecologic tumor environment. This article reviews the mechanism of action and application prospects of extracellular vesicles and macrophages in the cervical cancer microenvironment. In addition, the relationship between extracellular vesicles and macrophages from different sources is described, which provides ideas for the diagnosis and treatment of cervical cancer.

Embarking on life's blueprint: investigating the crucial involvement of extracellular vesicles in embryo development.

Extracellular vesicles (EVs) contain biological molecules, such as proteins, lipids, and diverse nucleic acids, which alter various physiological and pathological processes in recipient cells. This review examines the current understanding of EVs in terms of their biological characteristics, effects on embryonic development, and potential therapeutic value in treating reproductive disorders. EVs play a crucial role in early embryonic development, from fertilization to the pre-implantation stage, as well as during gastrulation, cell differentiation, and organogenesis. In the pre-implantation period, EVs interact with maternal reproductive tissue and promote implantation receptivity, whereas during gastrulation, they regulate cell differentiation and contribute to tissue formation and maintenance. Abnormal bioactive molecules in EVs are associated with developmental disorders; therefore, EVs can serve as biomarkers. In addition, EVs are potential therapeutic agents that can deliver genetic material to targeted tissues or organs. The findings of this review highlight the potential role of EVs in intercellular signaling during embryonic development. This will help advance assisted reproductive technologies and therapies to overcome infertility and developmental disorders.

Extracellular vesicles as the next-generation modulators of pharmacokinetics and pharmacodynamics of medications and their potential as adjuvant therapeutics.

BACKGROUND AND MAIN BODY: Pharmacokinetics (PK) and pharmacodynamics (PD) are central concepts to guide the dosage and administration of drug therapies and are essential to consider for both healthcare professionals and researchers in therapeutic planning and drug discovery. PK/PD properties of a drug significantly influence variability in response to treatment, including therapeutic failure or excessive medication-related harm. Furthermore, suboptimal PK properties constitute a significant barrier to further development for some candidate treatments in drug discovery. This article describes how extracellular vesicles (EVs) affect different aspects of PK and PD of medications and their potential to modulate PK and PD properties to address problematic PK/PD profiles of drugs. We reviewed EVs' intrinsic effects on cell behaviours and medication responses. We also described how surface and cargo modifications can enhance EV functionalities and enable them as adjuvants to optimise the PK/PD profile of conventional medications. Furthermore, we demonstrated that various bioengineering strategies can be used to modify the properties of EVs, hence enhancing their potential to modulate PK and PD profile of medications. CONCLUSION: This review uncovers the critical role of EVs in PK and PD modulation and motivates further research and the development of assays to unfold EVs' full potential in solving PK and PD-related problems. However, while we have shown that EVs play a vital role in modulating PK and PD properties of medications, we postulated that it is essential to define the context of use when designing and utilising EVs in pharmaceutical and medical applications. HIGHLIGHTS: Existing solutions for pharmacokinetics and pharmacodynamics modulation are limited. Extracellular vesicles can optimise pharmacokinetics as a drug delivery vehicle. Biogenesis and administration of extracellular vesicles can signal cell response. The pharmaceutical potential of extracellular vesicles can be enhanced by surface and cargo bioengineering. When using extracellular vesicles as modulators of pharmacokinetics and pharmacodynamics, the 'context of use' must be considered.

Extracellular vesicles in anti-tumor drug resistance: Mechanisms and therapeutic prospects.

Drug resistance presents a significant challenge to achieving positive clinical outcomes in anti-tumor therapy. Prior research has illuminated reasons behind drug resistance, including increased drug efflux, alterations in drug targets, and abnormal activation of oncogenic pathways. However, there's a need for deeper investigation into the impact of drug-resistant cells on parental tumor cells and intricate crosstalk between tumor cells and the malignant tumor microenvironment (TME). Recent studies on extracellular vesicles (EVs) have provided valuable insights. EVs are membrane-bound particles secreted by all cells, mediating cell-to-cell communication. They contain functional cargoes like DNA, RNA, lipids, proteins, and metabolites from mother cells, delivered to other cells. Notably, EVs are increasingly recognized as regulators in the resistance to anti-cancer drugs. This review aims to summarize the mechanisms of EV-mediated anti-tumor drug resistance, covering therapeutic approaches like chemotherapy, targeted therapy, immunotherapy and even radiotherapy. Detecting EV-based biomarkers to predict drug resistance assists in bypassing anti-tumor drug resistance. Additionally, targeted inhibition of EV biogenesis and secretion emerges as a promising approach to counter drug resistance. We highlight the importance of conducting in-depth mechanistic research on EVs, their cargoes, and functional approaches specifically focusing on EV subpopulations. These efforts will significantly advance the development of strategies to overcome drug resistance in anti-tumor therapy.

The Core-Shell Microneedle with Probiotic Extracellular Vesicles for Infected Wound Healing and Microbial Homeostasis Restoration.

Wound healing is a dynamic process involving the timely transition of organized phases. However, infected wounds often experience prolonged inflammation due to microbial overload. Thus, addressing the viable treatment needs across different healing stages is a critical challenge in wound management. Herein, a novel core-shell microneedle (CSMN) patch is designed for the sequential delivery of tannic acid-magnesium (TA-Mg) complexes and extracellular vesicles from Lactobacillus druckerii (LDEVs). Upon application to infected sites, CSMN@TA-Mg/LDEV releases TA-Mg first to counteract pathogenic overload and reduce reactive oxygen species (ROS), aiding the transition to proliferative phase. Subsequently, the sustained release of LDEVs enhances the activities of keratinocytes and fibroblasts, promotes vascularization, and modulates the collagen deposition. Notably, dynamic track of microbial composition demonstrates that CSMN@TA-Mg/LDEV can both inhibit the aggressive pathogen and increase the microbial diversity at wound sites. Functional analysis further highlights the potential of CSMN@TA-Mg/LDEV in facilitating wound healing and skin barrier restoration. Moreover, it is confirmed that CSMN@TA-Mg/LDEV can accelerate wound closure and improve post-recovery skin quality in the murine infected wound. Conclusively, this innovative CSMN patch offers a rapid and high-quality alternative treatment for infected wounds and emphasizes the significance of microbial homeostasis.

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.

Methods for the Characterization of the Colloidal Properties of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are extracellular vesicles secreted by either Gram-positive or Gram-negative bacteria. These BMVs typically possess a diameter between 20 and 250 nm. Due to their size, when these BMVs are suspended in another medium, they could be constituents of a colloidal system. It has been hypothesized that investigating BMVs as colloidal particles could help characterize BMV interactions with other environmentally relevant surfaces. Developing a more thorough understanding of BMV interactions with other surfaces would be critical for developing predictive models of their environmental fate. However, this bio-colloidal perspective has been largely overlooked for BMVs, despite the wealth of methods and expertise available to characterize colloidal particles. A particular strength of taking a more colloid-centric approach to BMV characterization is the potential to quantify a particle's attachment efficiency (α). These values describe the likelihood of attachment during particle-particle or particle-surface interactions, especially those interactions which are governed by physicochemical interactions (such as those described by DLVO and xDLVO theory). Elucidating the influence of physical and electrochemical properties on these attachment efficiency values could give insights into the primary factors driving interactions between BMVs and other surfaces. This chapter details methods for the characterization of BMVs as colloids, beginning with size and surface charge (i.e., electrophoretic mobility/zeta potential) measurements. Afterward, this chapter will address experimental design, especially column experiments, targeted for BMV investigation and the determination of α values.

Neutrophils exposed to a cholesterol metabolite secrete extracellular vesicles that promote epithelial-mesenchymal transition and stemness in breast cancer cells.

Small extracellular vesicles (sEVs) are emerging as critical mediators of intercellular communication in the tumor microenvironment (TME). Here, we investigate the mechanisms by which sEVs derived from neutrophils treated with the cholesterol metabolite, 27-hydroxycholesterol (27HC), influence breast cancer progression. sEVs released from 27HC treated neutrophils enhance epithelial-mesenchymal transition (EMT) and stem-like properties in breast cancer cells, resulting in loss of adherence, increased migratory capacity and resistance to cytotoxic chemotherapy. Decreased microRNAs (miRs) within the sEVs resulted in activation of the WNT/ß-catenin signaling pathway in recipient cells and suggest that this may be a predominant pathway for stem-like phenotype and EMT. Our findings underscore a novel mechanism by which 27HC-modulated neutrophils contribute to breast cancer pathophysiology through EV-mediated intercellular communication, suggesting potential therapeutic targets in cancer treatment.

Extracellular vesicles miR-574-5p and miR-181a-5p as prognostic markers in NSCLC patients treated with nivolumab.

Immune checkpoint inhibitors (ICIs) have revolutionized the management of advanced non-small cell lung cancer (NSCLC), although patient survival is still unsatisfactory. Accurate predictive markers capable of personalizing the treatment of patients with NSCLC are still lacking. Circulating extracellular vesicles involved in cell-to-cell communications through miRNAs (EV-miRs) transfer are promising markers. Plasma from 245 patients with advanced NSCLC who received nivolumab as second-line therapy was collected and analyzed. EV-miRnome was profiled on 174/245 patients by microarray platform, and selected EV-miRs were validated by qPCR. A prognostic model combining EV-miR and clinical variables was built using stepwise Cox regression analysis and tested on an independent patient cohort (71/245). EV-PD-L1 gene copy number was assessed by digital PCR. For 54 patients with disease control, EV-miR changes at best response versus baseline were investigated by microarray and validated by qPCR. EV-miRNome profiling at baseline identified two EV-miRs (miR-181a-5p and miR-574-5p) that, combined with performance status, are capable of discriminating patients unlikely from those that are likely to benefit from immunotherapy (median overall survival of 4 months or higher than 9 months, respectively). EV-PD-L1 digital evaluation reported higher baseline copy number in patients at increased risk of mortality, without improving the prognostic score. Best response EV-miRNome profiling selected six deregulated EV-miRs (miR19a-3p, miR-20a-5p, miR-142-3p, miR-1260a, miR-1260b, and miR-5100) in responding patients. Their longitudinal monitoring highlighted a significant downmodulation already in the first treatment cycles, which lasted more than 6 months. Our results demonstrate that EV-miRs are promising prognostic markers for NSCLC patients treated with nivolumab.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicle miR-208a-3p Alleviating Spinal Cord Injury via Regulating the Biological Function of Spinal Cord Neurons.

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague-Dawley (SD) rats were used to construct SCI model in vivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway.

A novel extracellular vesicle paradigm for the treatment of COVID-19 induced acute respiratory distress syndrome (ARDS).

Efficacy of mesenchymal stem cells (MSCs) for treatment of acute respiratory distress syndrome (ARDS) suggests bioactive bone marrow MSC extracellular vesicles (BM-MSC EVs) may be effective. A patient with severe COVID-19 associated ARDS who was presumed to expire was treated with a BM-MSC EV preparation (14 doses over two months) as a rescue treatment for refractory COVID ARDS. Near complete reversal of lung inflammation and fibrosis (per computed tomography), near complete restoration of mobility, hospital discharge (3 months) with resumption of normal activities of daily living (one year) and return to work occurred. No adverse events occurred despite repeated dosing of investigational product, highlighting safety of this potential therapy for ARDS.

Hybrid hair follicle stem cell extracellular vesicles co-delivering finasteride and gold nanoparticles for androgenetic alopecia treatment.

Androgenetic alopecia (AGA) is a non-fatal disease prevalent worldwide. However, mixed efficacy has been observed among different therapies for hair regrowth in AGA patients. Thus, a nano-platform with synergistic treatments based on a hybrid extracellular vesicle encapsulating gold nanoparticles (AuNPs) and finasteride (Hybrid/Au@Fi) was constructed through membrane fusion between hair follicle stem cell (HFSC)-derived extracellular vesicles and liposomes. These hybrid vesicles (HVs) not only fuel hair regrowth by providing cellular signals in extracellular vesicles, but also improve storage stability, follicle retention, and drug encapsulation efficiency (EE%) for finasteride inhibiting 5α-reductase, and nano-size AuNPs that simulate low-level laser therapy (LLLT) with similar photothermal effects in vitro. The EE% of finasteride in these HVs reached 45.33%. The dual administration of these extracellular vesicles and finasteride showed a strong synergistic effect on HFSCs in vitro. In an AGA mouse model, once-daily topical Hybrid/Au@Fi (115.07 ± 0.32 nm, -7.50 ± 1.68 mV) gel led to a faster transition of hair follicles (HFs) from the catagen to the anagen, increased hair regrowth coverage, and higher quality of regrowth hair, compared to once-daily 5% minoxidil treatment. Compared to topical minoxidil, the multifaceted synergistic therapy of Hybrid/Au@Fi through topical administration offers a new option for intractable AGA patients with low side effects.

Amniotic fluid-derived small extracellular vesicles for predicting postnatal severe outcome of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a life-threatening condition with high morbidity and mortality rates. The survival rate of neonates with severe CDH is reportedly only 10%-15%. However, prenatal prediction of severe cases is difficult, and the discovery of new predictive markers is an urgent issue. In this study, we focused on microRNAs (miRNAs) in amniotic fluid-derived small EVs (AF-sEVs). We identified four miRNAs (hsa-miR-127-3p, hsa-miR-363-3p, hsa-miR-493-5p, and hsa-miR-615-3p) with AUC > 0.8 to classify good prognosis group and poor prognosis group in human study. The AUC for hsa-miR-127-3p and hsa-miR-615-3p, for predicting the poor prognosis, were 0.93 and 0.91, respectively. In addition, in the in vivo study, the miRNA profiles of the lung tissues of CDH rats were different from those of control rats. Additionally, two elevated miRNAs (rno-miR-215-5p and rno-miR-148a-3p) in the lung tissues of CDH rats were increased in the AF-sEVs of CDH rats. Our results suggest that severe CDH neonates can be predicted prenatally with high accuracy using miRNAs contained in AF-sEVs. Furthermore, miRNA profile changes in AF-sEVs reflected the lung status in CDH. Our findings may contribute to the development of advanced perinatal care for patients with CDH.