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One decade of intense research has shown that protein and nucleic acid disease markers in physiological fluids are enriched in nanocarriers like extracellular vesicles (EVs), supermeres and exomeres. The nanocarriers from diseased cells are low in abundance (10^2 to 10^6 per ml) and are extremely heterogeneous with different subsets carrying distinct biomarker cargoes. Single-nanocarrier colocalization statistics can shed light on the tumor heterogeneity, the tissue origin and the metastatic state. Consequently, large library assays of multiple markers on each EV, without extensive pretreatment that compromises sensitivity, is the holy grail of Nanocarrier Diagnostics. It may lead to healthcare transformations like an annual blood test for pan-cancer screening, thus reducing cancer mortality rate by as much as 80% with early diagnostics. There are myriads of technical challenges to his new diagnostic platform: interference from dispersed proteins, low sensitivity of existing technologies and number of targets detectable per nanocarrier etc. My lab has recently developed an array of nanocarrier sensing technologies to meet these challenges with new concepts like multivalent capture, charge-base sensing without Debye screening, size-based sensing with Surface Acoustic Waves, EV enzyme activity assay, quantification with non-Gaussian Brownian rotation, selectivity-enhancing ionic strength polarization, continuous isoelectric fractionation, electrokinetic signals without non-specific electron transfer etc. The most mature of these technologies is now being commercialized by Aopia Biosciences (aopiabio.com) as an EV purification instrument (NanoEx) for biomarker proteomics and for therapeutic EV. I will review the other technologies that are currently being developed into products. All publications can be found on my group webpage: www.nd.edu/~changlab

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Bacteria and their viruses (bacteriophages, or phages) play important roles in marine, freshwater, and terrestrial ecosystems. The closely related unicellular cyanobacterial genera Prochlorococcus and Synechococcus are the most abundant photosynthetic cells in the oceans. Together, they are estimated to fix 12 Gt of carbon per year, contributing to about 25% of the net marine primary production.

Cyanobacterial viruses (cyanophages) are lytic double-stranded DNA viruses that infect ~15% of marine cyanobacteria on a daily basis.

Prochlorococcus cells have been shown to secrete EVs that contain lipids, proteins, DNA, and RNA. Because Prochlorococcus EVs are highly abundant in the oceans and support the growth of heterotrophic bacteria, it was hypothesized that Prochlorococcus EVs play important roles in the flow of genetic material, energy, and nutrients in marine microbial communities. Interestingly, transmission electron microscopy (TEM) revealed direct interactions between Prochlorococcus EVs and cyanophages, suggesting that EVs possess cyanophage receptors. Although EVs secreted by E. coli and Vibrio cholerae are known to inhibit phage infection, whether Prochlorococcus EVs can inhibit cyanophage infection of Prochlorococcus cells remains to be tested.

Our results indicate that certain proteins are specifically present in EVs to interact with phages, which may lead to the discovery of novel antiphage defense systems in cyanobacterial EVs.

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Extracellular vesicles, specifically exosomes, play a crucial role in various biological processes, including cell-to-cell communication and intercellular delivery. Additionally, exosomes are regarded as liquid biopsy biomarkers for diagnostic applications. However, their submicron size presents challenges in isolating exosomes from other cell-derived extracellular vesicles (EVs). In this talk, we will explore microfluidic devices designed to separate EVs using electrokinetic methods.

Osteoarthritis (OA) is a debilitating and irreversible joint disease marked by progressive cartilage degradation, osteophyte formation, subchondral bone remodeling, and synovial inflammation. Despite its high global prevalence, effective and safe disease-modifying osteoarthritis drugs (DMOADs) remain elusive. In this study, we present a novel dual-action therapeutic platform comprising a bioactive peptide that promotes chondrogenic differentiation and primes mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) for OA treatment.

To ensure clinical-grade EV production, peptide-primed MSC-EVs were isolated using an advanced purification workflow combining Asymmetric Nanopore Membrane (ANM) technology with tangential flow filtration (TFF). The ANM platform enables size-based, charge-selective, and pressure-stable separation of EVs from protein contaminants and debris, thereby preserving EV bioactivity while ensuring purity and scalability. This process complies with and surpasses the minimal criteria defined by the International Society for Extracellular Vesicles (ISEV) for clinical application. Therapeutic efficacy was validated through a series of in vitro and in vivo studies, including chondrogenic induction assays, biodistribution analysis in murine OA models, and cartilage regeneration assessment in a rat model of anterior cruciate ligament transection (ACLT)-induced OA. Mechanistically, the peptide activates receptor X–mediated signaling pathways in adipose-derived stem cells (ADSCs), significantly upregulating key chondrogenic genes such as SOX9, collagen type II, and COMP. Intra-articular administration of the peptide alone demonstrated chondroprotective effects. More importantly, EVs derived from peptide-primed MSCs (peptide-MSC-EVs) exhibited robust regenerative capacity—enhancing ECM synthesis, restoring chondrogenic gene expression, and inhibiting matrix-degrading enzymes like MMP13. In addition, AlphaFold2-Multimer–based structural modeling identified critical bioactive subregions within the peptide responsible for its superior chondrogenic function.

Collectively, these findings establish a strong preclinical foundation for a next-generation, cell-free DMOAD strategy. The integration of ANM-TFF technology for high-yield, clinical-grade EV manufacturing further supports the translational advancement of this platform toward Good Manufacturing Practice (GMP) compliance, investigational new drug (IND) filing, and future early-phase clinical trials for osteoarthritis.

Mesenchymal stromal cell-derived exosomes (MSC-EVs) are gaining recognition as powerful modulators of disease, not by targeting individual symptoms, but by restoring immune balance at the source. This talk explores the emerging view that immune modulation is the central mechanism driving the broad therapeutic potential of MSC exosomes across diverse conditions—from inflammation and fibrosis to tissue injury and autoimmunity. I will highlight key immunologic pathways influenced by MSC-EVs, including macrophage polarization, T cell regulation, and inhibition of neutrophil and complement activity. By reframing MSC exosomes as immune reprogramming agents, this session provides a unifying lens to understand their pleiotropic effects and positions them as next-generation, cell-free biologics with cross-disease relevance.

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A challenge with exosome-based diagnostics and therapeutics that poses a barrier to their translation into clinical practice is the low number of exosomes that can be isolated. Various methods have therefore been developed to increase exosome yield, although they are not without considerable limitations, not least the heterogeneity in the exosomal population that is obtained. We demonstrate that exposing to just several minutes of low-level high frequency (10 MHz) insults in the form of nanometer-amplitude acoustic waves is capable of effectively stimulating exosomal production and secretion. Given the very high post-stimulation retention in cell viabilities (>95%), it is possible to subject the same population of cells to repeated stimulation cycles to maintain lipodome and proteome homogeneity. In particular, we show the possibility of achieving eight- to ten-fold increases in homogeneous exosome production over 7 stimulation cycles, equivalent to approximately 2-fold/hr. In addition, we also explicate the calcium-dependent mechanistic pathway that governs the cells’ response to the high frequency mechano-stimulation, and show that such an intricate ability to modulate second messenger signalling can facilitate the possibility of directing downstream fates across different cell types.

Extracellular vesicles (EVs) are critical mediators of intercellular communication, facilitating the transfer of bioactive molecules between diverse cell types. These membrane-bound particles, secreted by various cells, play a pivotal role in modulating the phenotypes and signalling pathways of the recipient cells. Our study investigates the complex mechanisms by which EV-mediated communication influences the development and progression of hepatocellular carcinoma (HCC). We demonstrate that a conducive tumor microenvironment significantly contributes to HCC tumorigenesis and metastasis. Clinically, our findings reveal notable differences in the protein cargo profiles of circulating EVs between healthy individuals and HCC patients. These distinct protein signatures hold promise as non-invasive diagnostic biomarkers. Additionally, targeting specific EV-associated proteins presents a potential therapeutic strategy to inhibit HCC progression. This research underscores the potential of EVs as both diagnostic tools and therapeutic targets in HCC management.

Urogenital diseases affect people’s life, particularly for the most common prostate cancer (PCa). Clear differentiation of high-grade and clinically insignificant PCa is critical for clinical decision-making. Here, we developed a proprietary urinary exosome isolation approach (EVLatch) and established a facile diagnostic workflow. We discovered that EEF1A1 levels, abundantly expressed on urinary exosomes, positively correlate to urinary exosome counts irrespective of source and collection time and demonstrated that EEF1A1 enables in-assay quantification of urinary exosomes. Importantly, a prostate cancer urinary EVLatch-based artificial intelligence diagnostics (PURE-AID) classification system utilizing PCA3, HOXC6, and DLX1 as targets with SPDEF for reference and EEF1A1 for quality checking, trained on 271 patients, achieved an area under the receiver operating characteristic curve (AUROC) of 0.76 in the test set of 351 patients. Combination of PURE-AID with prostate-specific antigen (PSA) and age increases AUROC to 0.80 and reduces 54.3% of unnecessary biopsies with 86.8% sensitivity. Our study provides a new classification system for differentiating high-grade PCa in a workflow- and patient-friendly manner.

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Extracellular vesicles are a fascinating new class of therapeutics with the potential to transform the treatment of a wide spectrum of diseases with high unmet medical needs. Several key characteristics include low or no immunogenicity, ease of crossing cell membranes, and ability to communicate between cells. Herein, we focus on developing a precision-engineered exosome platform technology that can carry defined sets of effector molecules, such as mRNA, miRNA, and shRNA, that exert their effects through defined mechanisms of action. Our extracellular vesicles manufacturing platform is a silicon wafer-based electroporation technology that facilitates the massive production of exosomes and carries effector molecules secreted by producer cells. Extracellular vesicles-based cancer therapies and various disease treatments will be reported. Our goal is to develop our extracellular vesicles as an off-the-shelf product with preserved bioactivity. Together, we aim to pave the way towards engineering extracellular vesicles-based biologics with a novel manufacturing approach for pressing unmet medical needs.

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Extracellular vesicles (EVs) carry molecular cargoes that reflect the physiological and pathological states of their cells of origin. We profile EV RNA and protein cargoes from blood and brain-related samples to identify biomarkers for neurological disorders and HIV infection. Our findings reveal disease-associated signatures linked to inflammation, synaptic function, and viral host interactions. These results highlight the potential of EVs as accessible biomarkers for diagnosis and disease monitoring.

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Extracellular vesicles (EVs) serve as critical mediators of intercellular communication in health and disease, yet their intrinsic heterogeneity challenges conventional analytical methods. Nano-flow cytometry (nFCM) emerges as a transformative technology, enabling high-sensitivity, high-throughput single-particle analysis to uncover previously inaccessible EV characteristics at unprecedented resolution. This presentation highlights key applications of nFCM in discovering cancer biomarkers for clinical diagnosis and optimizing therapeutic EV production—demonstrated through a novel label-free clustering analysis platform that integrates nFCM to achieve scalable, high-purity EV manufacturing with 4-fold increased yield compared to traditional methods. By bridging fundamental research with translational applications, nFCM is significantly advancing our understanding of EV biology and expanding their therapeutic potential.

Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions.

Alzheimer’s disease (AD) is marked by progressive amyloid-β (Aβ) accumulation, primarily driven by β-secretase (BACE1) activity. However, the mechanisms sustaining Aβ overproduction remain unclear. Here, we show that circulating extracellular vesicles (EVs) from APP/PS1 mice (APPEVs) promote neuronal Aβ generation. Compared to wild-type EVs (WTEVs), APPEVs are activating the JAK2–STAT1 pathway in neurons, leading to BACE1 upregulation and enhanced β-cleavage of APP in lipid rafts. Furthermore, proteomic analysis identified complement C1q as a key cargo in APPEVs responsible for this signaling activation. Intravenously injected APPEVs crossed the blood-brain barrier without disrupting tight junctions, induced neuronal BACE1 expression, and elevated brain Aβ levels in vivo. Blocking C1q effectively suppressed these effects. Our findings reveal a peripheral-to-central mechanism in which circulating EVs enriched in C1q drive neuronal Aβ production through JAK2–STAT1–BACE1 signaling, contributing to AD progression.

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Extracellular vesicles (EVs) are increasingly recognized as critical non-cellular components of the immune system. Our recent studies identified that EVs secreted by injured renal cells trigger inflammation by transferring pro-inflammatory signals to innate immune cells. In turn, these activated innate immune cells release EVs that promote T cell activation through co-stimulatory signaling. Single-EV analysis has advanced our ability to characterize EV subsets, enabling a more precise understanding of their biological functions. Additionally, urinary EV profiling from the prospective of single EV has facilitated the identification of EV biomarkers associated with immune regulation in renal injury.

Our research focuses on the methodological development and translational application of bacterial extracellular vesicles (BEVs). We have established strategies to distinguish BEVs derived from Gram-negative and Gram-positive bacteria, enabling their detection in circulation for infection diagnosis and antimicrobial resistance profiling. In addition, we demonstrated the therapeutic potential of Akkermansia muciniphila-derived BEVs in liver cancer, and explored probiotic BEVs for immune modulation. Our work highlights the diagnostic and therapeutic value of BEVs and provides a foundation for their clinical application in infectious and metabolic diseases.

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Background: Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are increasingly recognized as promising therapeutics for central nervous system (CNS) disorders. Hypoxia preconditioning enhances their regenerative and immunomodulatory functions. This study evaluated the safety and therapeutic efficacy of hypoxia-conditioned MSCs and their EVs in two distinct CNS models: Alzheimer’s disease (AD) and spinal cord injury (SCI).
Methods: In the AD arm, β-amyloid–induced rats received intrathecal hypoxic MSCs, intrathecal EVs, or intranasal EVs. In the SCI arm, clip-compression rats were treated with three intrathecal doses of hypoxic umbilical cord MSCs (1 × 10⁶ cells) or EVs (300 µg protein, ~5 × 10⁹ particles). Safety was assessed by clinical signs, hematological/biochemical indices, and histology. Cognitive and motor outcomes were measured using Morris Water Maze, Y-maze, Novel Object Recognition (AD), and BBB score, Rotarod, sensory and bladder function (SCI). Tissue analyses included microglial activation in AD and cavitation/inflammatory infiltration in SCI.
Results: Both hypoxic MSCs and EVs were safe in AD and SCI models. In AD, MSCs and EVs delivered both via intrathecal or intravenous routes significantly improved spatial learning, working and recognition memory, while suppressing microglial activation. EVs also demonstrated efficacy via intranasal delivery, suggesting a minimally invasive route for diffuse neurodegeneration. In SCI, both MSCs and EVs enhanced locomotor recovery, motor coordination, and bladder function, while reducing cavitation and inflammation; however, MSCs provided more sustained hindlimb motor improvement through 28 days.
Conclusion: Hypoxia-conditioned MSCs and their EVs exhibit robust safety and therapeutic efficacy across neurodegenerative and traumatic CNS models. While MSCs deliver longer-lasting motor recovery in SCI, EVs offer logistical advantages—particularly via non-invasive intranasal administration—making them attractive for AD. Together, these findings support hypoxic MSC-EVs as a versatile, cell-free therapeutic platform adaptable to multiple CNS disorders, especially when cell-based therapy is not feasible.