Extracellular vesicles (EVs) have emerged as highly promising natural nanomedicines and nanocarriers, holding transformative potential for the treatment of various diseases. However, the lack of rapid and comprehensive characterization techniques for EV preparation analysis, coupled with the absence of efficient quality control methods, significantly hinders process optimization and large-scale production. To address these challenges, we developed a label-free clustering analysis (LFCA) platform that integrates nano-flow cytometry for particle size distribution analysis with a clustering algorithm to deconvolute EV subpopulations and distinguish them from impurities. This platform enables the rapid quantification of EV component distribution and composition within 5 min using minimal sample input. Leveraging the high-throughput capabilities of LFCA, we established a cascaded workflow incorporating a microcarrier-based 3D culture system, a custom tangential flow filtration device, and multimodal size exclusion chromatography for EV preparation from adipose mesenchymal stem cells. This approach achieves a 4-fold increase in EV yield compared to ultracentrifugation while maintaining comparable purity and preserving EV integrity. Critically, the resulting EVs exhibited enhanced functional potency in pro-angiogenic and anti-inflammatory assays, confirming the clinical relevance of our optimized production system. These advancements provide a scalable solution for EV production, paving the way for clinical applications.

Aim: High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Methods: Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 113 and 155 nm) by using light-scatter-based detection thresholds [side scatter (SSC), forward scatter (FSC), violet side scatter (VSSC)] across a concentration range from 106 to 109 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter-based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding. Results: Upon qualitative and quantitative analysis, we observed that instruments differed in sensitivity, the NF could detect 68 nm SiNPs, while both IF and CF were able to detect down to 91 nm SiNPs when using a scatter-based threshold, which was improved by using FSC and VSSC compared to SSC, respectively. We show that the NF required a higher sample concentration to ensure optimal detection, while IF and CF benefited from more diluted samples. Next, we defined a single particle detection range measuring fluorescent rEV and demonstrated that fluorescence-based detection improved the detection of particles of interest due to lower background interference. Conclusion: We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.

What is already known about this topic?:The Staphylococcus aureus (S. aureus) sequence type 8 (ST8) clone has emerged as a dominant epidemic clone strongly associated with community-associated methicillin-resistant S. aureus (CA-MRSA) infections worldwide. However, documentation of this clone in China remains sparse. What is added by this report?:This report documents the first staphylococcal food poisoning outbreak in Zhejiang Province, China, caused by methicillin-susceptible S. aureus (MSSA) of the ST8 (CC8)-spa t024 lineage harboring enterotoxin genes sea, seq, and sek. Whole-genome phylogenetic analysis revealed that the outbreak strains belonged to a distinct evolutionary subclade (Clade II.1). These strains demonstrated close genetic relatedness to previously reported European lineages. What are the implications for public health practice?:The identification of the ST8-spa t024 MSSA strain carrying sea, seq, and sek genes underscores the substantial public health risk in China. This finding highlights the emerging threat of foodborne illnesses caused by novel or under-recognized S. aureus ST8 strains and emphasizes the urgent need for enhanced surveillance of CA-S. aureus among food handlers to prevent future outbreaks.

Cases of scarlet fever have increased since 2011 across China. However, genomic epidemiological knowledge of Streptococcus pyogenes, the causative agent, is limited. Here we present a longitudinal analysis of S. pyogenes isolates (n = 1,029) across emm1 and emm12 genotypes collected from eight provinces across China between 1993 and 2024. Genomic data integrated with national scarlet fever incidence data confirmed emm12 and emm1 as dominant genotypes underlying five incidence peaks and disease resurgence in 2024. Phylogenetic analysis showed independent evolution of these genotypes in China compared to global epidemic lineages. Four emm12 clades were present in China before 2011 but were replaced by a single clade, Clade II, by 2020. A dominant emm1 clade, M1china, distinct from global lineages and the M1UK lineage, represents >98% of emm1 cases in China. Sub-clade expansion coincides with carriage of integrative conjugative elements containing macrolide and tetracycline resistance genes and virulence gene-encoding prophage. Ongoing maintenance of these elements in emm1 and emm12 populations likely underlies the resurgence of scarlet fever in China.

Persisters represent a transient, antibiotic-tolerant subpopulation within isogenic bacterial populations, contributing to infection relapses. However, the mechanisms driving persister formation and resuscitation remain elusive. Here, we developed nano-flow cytometry (nFCM)-based methods for single-cell quantification of toxin (T) RelE and antitoxin (A) RelB levels, as well as for monitoring persister states through cell wall growth. We demonstrate that bacteria elevate the T/A ratio through two distinct TA expression modalities to withstand bacteriostatic antibiotic challenge, with T/A = 1.0 as a critical threshold. Intriguingly, single-cell resuscitation dynamics revealed that subinhibitory antibiotic exposure promotes entry into a deeper dormant state characterized by elevated T/A ratios, underscoring the importance of maximizing therapeutic antibiotic concentrations. Crucially, we uncovered a triphasic detoxification process during resuscitation where progressive toxin depletion drives T/A ratio reduction to a critical proliferation-permissive threshold. Proteomic profiling unveiled that persisters with high RelE production have increased transmembrane transporter levels linked to stress response and drug efflux. Our findings offer pivotal molecular insights underlying persister transitions and underscore the need for high-throughput, single-cell analysis of these heterogeneity phenotypes.

The discrepancy between the genotypic and phenotypic expression of enterotoxins in S. aureus had long been a significant challenge in toxin detection. However, the accurate and rapid application of Raman spectroscopy for the genotypic and phenotypic characterisation of S. aureus enterotoxins remains problematic. To address this, the present study utilised a single-cell Raman spectra database from 31 S. aureus isolates, acquired via a Raman laser tweezer system. When combined with convolutional neural network analysis, this approach achieved an average accuracy of 99.71% for identifying single-gene toxin types and 99.44% for multi-gene toxin types, with an average phenotypic identification accuracy of 98.71%. Notably, the phenotypic identification accuracy for the three strains carrying the sea and seb genes reached 100%, and the validation accuracy using unknown genotypes and phenotypes exceeded 85%. Furthermore, the CNN analysis identified characteristic spectral peaks for S. aureus enterotoxin genotypes at 1663–1665 cm−1, 1570 cm−1, and 1117–1119 cm−1, corresponding to protein α-helices, guanine, and nucleic acid backbones respectively. Representative peaks for the phenotype were found at 1302–1314 cm−1 and 912–923 cm−1, corresponding to proteins/lipids and polysaccharides, respectively. Representative peaks for different virulence phenotypes carrying multiple enterotoxin genes were located at 1074–1076 cm−1, 1253–1255 cm−1, 1326 cm−1, and 1327 cm−1, corresponding to proteins, nucleic acids, and lipids, respectively. Furthermore, metabolomic analysis of three S. aureus strains (sea+seb+, sea+seb−, sea−seb+) revealed metabolic differences in fatty acids, purines, phenylalanine, and aspartic acid, consistent with the corresponding distinct Raman spectral peaks (1458, 1179, 1406–1409 cm−1). Thus, this study employed S. aureus as a proof-of-concept, establishing for the first time a method combining Raman laser tweezers with convolutional neural networks for identifying S. aureus enterotoxin genotypes and phenotypes. It clarified the Raman spectral differential peaks and their corresponding biomarkers among five classical enterotoxin genotypes and phenotypic strains, providing a novel approach for accurate toxin typing and virulence characterisation.

Mitochondrial adenosine triphosphate (mitoATP) serves as the primary bioenergetic currency for oxidative phosphorylation (OXPHOS)-driven malignancies, yet its precise organelle-level quantification remains challenging due to mitochondrial heterogeneity and cytosolic interference. Herein, we report MitoATP-nFCM, a nano-flow cytometry platform enabling single-mitochondrion ATP measurement via simultaneous fluorescence and side scatter detection. We uncover 1.7-1.9-fold higher ATP levels in isolated mitochondria from breast (MCF-7, MDA-MB-231) and colon (HCT-15, HCT-116) cancer cells than in their normal counterparts. Single-organelle analysis further reveals coordinated metabolic reprogramming in cancer mitochondria, featuring elevated membrane potential, increased ATP synthase expression, and reduced hexokinase 2 levels, demonstrating their OXPHOS-dominant bioenergetic phenotype that contrasts with classical Warburg-effect expectations. Furthermore, we establish a screening strategy to identify highly potent cancer-selective inhibitors targeting mitochondrial metabolism. We find that bedaquiline (ATP synthase inhibitor) outperforms oligomycin A in specificity, VLX600 (electron transport chain inhibitor) shows superior selectivity to rotenone/metformin, and CPI-613 (tricarboxylic acid cycle blocker) surpasses other glutaminase inhibitors. MitoATP-nFCM establishes a quantitative single-organelle platform that profiles elevated mitoATP levels in cancer cells and enables precision screening of OXPHOS-targeting inhibitors.

Spectral nano-flow cytometry enables multiparameter characterization of nanoscale biological particles,such as viruses and extracellular vesicles,at single-particle level.However,its performance is limited by the conventional photomultiplier tube(PMT)-triggered electron-multiplying charge-coupled device(EMCCD)exposure strategy,which suffers from a high lower-size detection limit and significant background noise.To address these limitations,a high-sensitivity spectral nano-flow cytometry system based on a field-programmable gate array(FPGA)-controlled single photon counting avalanche photodiode(APD)triggering mechanism was developed in this work.Key innovations included replacing the PMT with a high-quantum-efficiency APD combined with optical spatial filtering to substantially suppress background noise,implementing an FPGA-based high-speed signal processing chain that achieved precise identification and synchronous triggering of APD pulse signals within a 0.2-30 MHz frequency range using equal-precision measurement and threshold comparison algorithms,and optimizing the spectral detection module to enhance fluorescence collection efficiency and spectral resolution.Experimental results demonstrated that the system clearly distinguished a mixture of fluorescent silica beads with molecules of equivalent soluble fluorochrome(MESF)values of 574 and 1438.When detecting Di-8-ANEPPS-labeled red blood cell-derived extracellular vesicles(RBC EVs),the false-trigger rate was reduced from>70%(with PMT triggering)to<10%,with clear baseline separation between target peaks and noise.Furthermore,in experiments involving dual-labeled RBC EVs(Alexa Fluor 488(AF488)/XFD488 Hydroxylamine(XDF488)and Di-8-ANEPPS),the system successfully achieved decoupling of overlapping fluorescence spectra at single-particle level and accurately quantified the fluorescence contribution of each dye.This work provided key technological support for high-throughput,high-precision spectral analysis of weakly fluorescent nanoscale biological particles.

High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, namely the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 114 and 155 nm) by using light-scatter based detection thresholds (SSC, FSC, VSSC) across a concentration range from 10 6 to 10 9 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding for their qualitative and quantitative analysis. We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.

Surface functionalization is an effective approach for enhancing the cancer-targeting efficiency of extracellular vesicles (EVs). However, the lack of direct comparisons between functionalization strategies has hindered the rational design of EV delivery systems. To address this gap, we developed a nano-flow cytometry-based methodology to quantitatively evaluate ligand conjugation and its relationship to targeting efficiency across three representative post‑production EV engineering strategies: lipid modification, protein modification, and membrane insertion. All three strategies achieved high conjugation efficiencies (>90%) with ligand densities ranging from several to tens of ligands per 100 nm2 under optimized conditions. Beyond ligand density, functionalization strategies resulted in varying degrees of ligand clustering and reduced accessibility of endogenous cell-binding proteins on EVs, such as MFGE8, leading to differences in targeting performance. For milk-derived EVs, lipid modification achieved the highest ligand density, the most uniform conjugation, and minimal disruption to surface protein accessibility, yielding superior cancer-targeting efficiency. These findings highlight the importance of precise quantification of ligand conjugation performance and provide a robust methodology for optimizing surface functionalization to advance EV-based drug delivery.

Iron, particularly redox-active ferrous ions (Fe2+), is essential for biological processes. Despite their pivotal roles, analysis of Fe2+ ions within individual extracellular vesicles (EVs) has been hindered by the ultralow Fe2+ content and substantial heterogeneity of EVs. To address this, we developed a novel approach by integrating an Fe2+-specific fluorescent chemosensor (Ac-FluNox) with nano-flow cytometry (nFCM) for precise single-EV Fe2+ mapping. Method specificity to Fe2+ was validated via Fe2+-loaded liposomal models at the single-particle level. Comprehensive profiling of Fe2+ distributions in HT-1080-derived EVs under varying ferroptotic stress conditions revealed the striking heterogeneity in Fe2+ loading among EVs and a strong positive correlation between EV Fe2+ levels and their parental cells. Notably, we identified an EV-mediated Fe2+ export mechanism that functionally parallels to ferroportin (FPN)-dependent iron efflux, suggesting EVs may serve as a compensatory iron-release pathway during FPN inhibition. The nFCM platform achieved superior detection sensitivity with high throughput (up to 104 particles per min), providing a powerful analytical tool for investigating EV heterogeneity and Fe2+-mediated regulatory networks in iron homeostasis and ferroptosis-related pathologies.

What is already known about this topic?:Staphylococcus aureus (S. aureus) represents a clinically significant pathogen and serves as a common causative agent of foodborne intoxication. The S. aureus strain ST59 constitutes the predominant clone associated with both community-associated methicillin-resistant S. aureus (CA-MRSA) and hospital-associated MRSA (HA-MRSA) infections. However, staphylococcal food poisoning (SFP) outbreaks attributed to ST59 MRSA have been documented in only a limited number of Chinese cities through retrospective investigations. What is added by this report?:This report documents the first recorded outbreak of staphylococcal food poisoning (SFP) in Henan Province, which was attributed to the ST59-spa t441-SCCmec IVa CA-MRSA strain producing enterotoxins A and B. The confirmed source of the outbreak was contamination of donkey and goose meat with S. aureus enterotoxins A and B. Additionally, comprehensive genomic analysis identified multiple virulence genes and antibiotic resistance genes within the outbreak-related strains. What are the implications for public health practice?:The identification of foodborne clones of ST59 CA-MRSA in this outbreak underscores the prevalence and transmission risks associated with this hypervirulent lineage. These findings highlight the critical need to strengthen surveillance measures for CA-MRSA among food industry workers and implement enhanced food safety protocols.

This study sought to develop a method to accurately and quantitatively measure the staphylococcal enterotoxin genes sea and seb in milk samples using duplex droplet digital polymerase chain reaction (ddPCR). Specific primers and probes were designed for sea and seb. By optimizing the concentrations of primers and probes and the annealing temperature, a duplex ddPCR detection system was established, and the specificity and sensitivity of the method were evaluated. Standard curves were generated using plasmid DNA, pure cultures, and milk samples spiked with Staphylococcus aureus, and a high correlation coefficient (R2 = 0.99) was achieved within the ranges of 1 × 101-1 × 105 copies/μL, 2 × 103-2 × 107 cfu/mL, and 2 × 103-2 × 107 cfu/mL, respectively, for these samples. Using the gradient dilution method with pure cultures and milk samples spiked with S. aureus, the limit of detection (LOD) was 2 × 103 cfu/mL using primers targeting both enterotoxin genes. The test results exhibited good accuracy and repeatability, with three parallel repetitions revealing intra-assay and inter-assay coefficient of variations of <10% and <20%, respectively. When milk samples were spiked with S. aureus at concentrations of 2 × 101 and 2 × 102 cfu/mL, both sea and seb could be detected during the fourth and fifth hours of pre-enrichment, respectively. This study successfully established a duplex ddPCR detection system with high sensitivity and specificity for the quantitative detection of sea and seb in milk samples, thereby permitting the accurate detection of S. aureus directly in milk specimens.

Extracellular vesicles (EVs) are heterogeneous lipid membrane-bound nanoparticles that carry diverse biomolecular cargos, including proteins, nucleic acids, lipids, and metabolites. Serving as fundamental mediators of intercellular communication in both physiological and pathological contexts, EVs have become a central focus in basic and translational research. Their participation in diverse biological processes-from immune regulation and tissue homeostasis to cancer progression and neurodegenerative diseases-highlights their dual functional roles. However, the intrinsic heterogeneity of EVs poses significant challenges for accurate characterization using conventional ensemble-averaging methods. Nano-flow cytometry (nFCM) represents a transformative technological advancement for single-particle EV analysis, offering high sensitivity, throughput, and multiparametric detection capabilities. This review first examines recent progress in nFCM instrumentation that improves sensitivity and resolution. We then summarize key applications of nFCM in uncovering previously inaccessible biophysical and biochemical characteristics of EVs at the single-particle level. Finally, we discuss current limitations hindering broader implementation and outline future directions to advance the field.

Streptococcus pyogenes infections such as scarlet fever have increased in China since 2011. The genomic drivers of this increase are not known. The temporal clonal shifts within dominant emm12 and emm1 populations are unknown due to limited sampling. Based on a large longitudinal strain collection, we aimed to describe the molecular epidemiological characteristics and population structure of dominant S. pyogenesemm types circulating in China between 1993–2020.Here, we analyzed genomes of 945 China emm12 and emm1 isolates encompassing 1993–2020. The genomic data was integrated with national data on notifications of scarlet fever in the same period. The bacterial population structure and genomic features were analyzed to characterize the dynamics of circulating lineages during this period. We found there are five incidence peaks during 1993–2020 with S. pyogenes emm12 and emm1 being the dominant genotypes. The two genotypes driving scarlet fever in China have evolved independently of the global epidemic lineages. Four emm12 clades were evident prior to 2011, with a single clade replacing other emm12 populations by 2020. One dominant emm1 clade (termed M1china) represents > 98% of clinical cases in China since the 1980s, and diverged into three subclades that can be clearly distinguished from global epidemic lineages. Compared to clinical strains from the 1990s, selection and expansion of emm12 and emm1 sub-clades coincides with high carriage of drug resistance genes for macrolides and virulence-encoding prophage. The ongoing selection of multi-drug resistant mobile elements and prophage in both emm1 and emm12 populations underlie the resurgence of scarlet fever since 2011 in China. The first detection in China of a genotype M1UK isolate in 2018 warrants additional enhanced surveillance.

Subcellular chemical mapping of endogenous biomolecules without labeling remains a pivotal challenge in life sciences, constrained by the limited lateral resolution and detection sensitivity of existing techniques. Herein, we report the development of tapered fiber projection laser desorption/ionization mass spectrometry (TFPLDI-MS), a new platform enabling nanoscale single-cell mass spectrometry imaging (SC-MSI). By integrating tapered fiber laser delivery with a telescopic projection system, this method achieves 570 nm lateral resolution, 400 nm imaging resolution, and a 6 amol detection limit, even at a 100 mm working distance. Exploiting enhanced ion yields from plasmonic nanoparticles, we demonstrate simultaneous nanoscale mapping of diverse exogenous drugs and endogenous phospholipids within individual cells with 400 nm pixel sizes, outperforming available laser-based SC-MSI techniques in imaging resolution, detectable analyte numbers at nanoscale sampling amounts, and operational flexibility of fiber-based sampling systems. Through spatially resolved lipidomics of HeLa cells at subcellular resolution, our TFPLDI-MSI system reveals drug-specific lipid alterations during apoptosis induced by 5-fluorouracil, paclitaxel, and cisplatin, elucidating heterogeneous therapeutic responses at the single-cell level. Crucially, the TFP system's contamination-free operation, indefinite operational lifespan, and modular design establish the TFPLDI-MS as a transformative tool for next-generation SC-MSI techniques, bridging the gaps between nanoscale chemical imaging and biomedical applications.

Staphylococcal enterotoxin-like W (SElW) is a novel, widely prevalent enterotoxin-like protein that functions as a classical staphylococcal superantigen (SAg) and has been shown to exacerbate infections caused by the S. aureus epidemic clone CC398. However, the genetic distribution and amino acid polymorphisms, biological and antitumor activity, and T cell receptor (TCR) binding sites of SElW in S. aureus strains prevalent in China have not been investigated. The carrier rate and distribution of selw were determined by PCR, the stability and antitumor activity of recombinant SElW (rSElW) protein were evaluated. The superantigen activity of the five mutants (Y18A, N19A, W55A, C88A, and C98A) was compared to that of wild-type SElW (WT-rSElW) to assess the role of these sites in mediating TCR binding. The selw gene was detected in all (986/986, 100%) dominant clonal lineages of S. aureus and most strains (69.1%, 56/81) had a full-length selw open reading frame with a sequence identity of 90.5%. rSElW was heat-stable but not resistant to pepsin and trypsin digestion. Additionally, rSElW significantly inhibited the proliferation of MCF-7 and AGS, but not A549 in vitro. The rSElW mutants C88A and C98A markedly reduced T cell proliferation and IL-2, IFN-γ and TNF-α secretion compared to WT-rSElW. rSElW is a highly prevalent SAg that binds to the TCR via C98 and C88, which may serve as novel therapeutic targets for S. aureus infections and its application in anti-tumor activity needs to be further evaluated in vivo.

BACKGROUND Helicobacter pylori (H. pylori ) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA -NT and cagA -NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA -NT and pAdtrack/cagA -NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA- NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA- NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA- NT and pAdtrack/cagA- NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA- NT and pAdtrack/cagA- NE at different times after transfection. CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori .

In the past decade, there has been an explosive growth in studies of extracellular vesicles (EVs), nanoscale membrane-bound vesicles (30–1000 nm) secreted by cells to mediate intercellular communication. Given their abundance in body fluids, excellent biocompatibility, and good stability, EVs are considered as promising diagnostic biomarkers and therapeutic agents. Nevertheless, the small size and large heterogeneity of EVs, diversity of EV isolation and characterization techniques, and lack of quality controls have led to controversial results and misconceptions. To overcome these barriers to rigorous studies, numerous international committees and research groups have aimed to establish criteria and guidance for EV studies. In this chapter, the guidelines and minimal requirements for general EV research, sample collection and processing, EV isolation and characterization, and EV clinical research are summarized. We believe that these guidelines will help researchers, particularly those new to the EV field, to conduct reliable and repeatable EV research.

Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell-derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface-displayed SAs to interact with IAV through multivalent HA-SA interactions. This high-avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose-dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti-IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti-IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics.