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The ZetaView® X40 introduces next-generation advances in Nanoparticle Tracking Analysis (NTA) and Fluorescence-NTA (F-NTA), combining enhanced optics, fluidics, and analysis algorithms for highly precise size and novel concentration measurements. A major innovation is the introduction of fluorescent antibodies from Particle Metrix, specifically targeting the tetraspanins CD9, CD63, and CD81. Optimized fluorophore conjugations and dedicated staining protocols ensure robust and reproducible detection of extracellular vesicle (EV) subpopulations with minimized background interference.

Biological application examples illustrate these methodological benefits: EVs isolated from human plasma show consistent scatter-based sizing and concentration, while fluorescence tracking enables reliable identification of specific EV subfractions. Additional analyses of virus-like particles confirm the system’s versatility for diverse biological nanoparticles.

To complement the presentation, a hands-on session with the ZetaView X40 will be offered. Participants are encouraged to bring their own samples for live demonstration and data collection, providing direct experience with the instrument’s workflow and performance. Together, the ZetaView X40 and new F-NTA antibodies establish a robust, flexible platform for nanoparticle characterization, advancing NTA and F-NTA as methodological standards in research.

Technological advances in optical imaging and sensing have enabled new ways to visualize and characterize the complex and dynamic processes in biology and medicine. Label-free multimodal multiphoton imaging is used to image and optically characterize physical and biomolecular properties of extracellular vesicles (EVs). Real-time imaging visualizes the spatiotemporal distribution of EVs in situ in tissue microenvironments, in biofluids, or even in vivo. Optical signatures identify cancer-associated EVs and with AI analysis, demonstrate correlations to disease aggressiveness. This platform technology is based on Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy. Co-registered images are collected from two- and three-photon excited autofluorescence intensities/lifetimes of NAD(P)H and FAD, which reflect EV and parent cell redox state. Second/third harmonic generation provided structural imaging of collagen and lipid/aqueous interfaces. Label-free imaging and characterization of EVs in cell cultures, animal models, and from tissues, serum, and urine from human breast cancer and healthy subjects was performed. Optical signatures revealed changes in EV properties based on metabolic states (redox ratios). Increased metabolic activity in human breast tumors was reflected in EV signatures from label-free images of the tissue collected intraoperatively using a portable imaging system. AI analysis of multimodal images was applied for spatial biology visualization of the tissue microenvironment and association of EVs to various cell types and vasculature. Label-free characterization of EVs from urine, serum, and tissue of human breast cancer subjects showed trends indicative of the presence and aggressiveness of the disease. Label-free multimodal nonlinear imaging/characterization of EVs represents a platform technology. With spatiotemporal imaging and single-EV resolution, new investigations into the dynamics, distribution, and trafficking of EVs is possible to elucidate biological processes and generate new optical biomarkers for clinical diagnosis and treatment monitoring.

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Breast cancer continues to be the second leading most cause of cancer-related mortality in women. Thus, new biological insights and therapeutic targets are required. Elevated circulating cholesterol is associated with a poor prognosis while patients taking cholesterol lowering medication (statins) have an increased recurrence free and overall survival time. We have found that while cholesterol does have effects on breast tumors, its metabolite, 27-hydroxycholesterol (27HC) is a major mediator of these effects. 27HC works in several different ways. (1) In estrogen receptor positive cancer, 27HC activates this receptor to promote proliferation. (2) 27HC works through the liver x receptor (LXR) in myeloid immune cells to suppress T cell activity. (3) 27HC induces extracellular vesicle (EV) biogenesis in myeloid cells. These EVs independently stimulate tumor growth and metastasis, in part through inducing a stem-like phenotype in cancer cells. This talk will focus on how 27HC increases EV biogenesis, and how those EVs influence tumor pathophysiology. Collectively, this work forms the mechanistic basis of how cholesterol supports tumor growth and progression.

While extracellular vesicles (EVs) have shown significant promise over the past decade in diagnostic, mechanistic, and therapeutic applications, several technical challenges exist which continue to limit their clinical translation, including their scalable production, selective isolation, and efficient characterization. In this talk, I will highlight several approaches to overcoming or mitigating these issues including EV production using commercially available bioreactors, nanoplasmonic sensors combined with machine learning to classify EVs based on their Raman spectra, and a new approach called light-induced extracellular vesicle and particle adsorption (LEVA) for micropatterning EVs at subcellular resolution in various applications. In combination, we demonstrate the incredible potential of applying micro-nanofabrication strategies to create platform technologies that will advance EV research.

EXODUS is an automatic exosome isolation system with high yield and high purity from a variety of biological samples. This innovative approach combines negative pressure oscillation with double-coupled ultrasonic harmonic oscillation. EXODUS offers versatile label-free solutions to extracellular vesicle isolation with rapid processing time and reproducible results. It has demonstrated great potentials in nanoparticle purification, biomarker detection and diagnositcs, innovative drug delivery and therapeutics in biomedical research and clinical translation.

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Prof. Marni Boppart will discuss the lab’s recent efforts to develop novel plasma- and stem cell-derived EVs for rejuvenating skeletal muscle mass in older adults. These preclinical studies provide the foundational basis for future clinical trials.

Skeletal muscle is increasingly recognized as an active secretory organ that releases nanoscale exosomes capable of supporting brain homeostasis and repair, establishing a dynamic muscle-brain axis. We hypothesized that neuronal innervation and associated bioelectrical signaling are key regulators of muscle-derived secretory activity. To investigate this, we developed an in vitro model of engineered skeletal muscle tissue innervated by stem cell-derived neurons, precisely modulated through defined chemical and mechanical cues. This presentation will highlight how neuronal innervation supports the muscle secretome, particularly the molecular composition of exosomes, as revealed by whole RNA sequencing. Furthermore, we will present the functional implications of neuron-regulated exosomal cargo on brain cell behavior by applying these vesicles to a neural-glial tissue model. We will demonstrate changes in neuronal architecture and electrophysiological network activity, providing insight into cross-organ bioelectrical communication. Altogether, this study establishes a novel platform for dissecting and harnessing exosome-mediated signaling pathways central to muscle-brain communication and bioelectronic medicine.

Extracellular vesicles (EVs) are key nanoscale messengers, yet their behavior in complex tissue environments is not well understood from a biophysical perspective. This talk discusses how EVs navigate dense extracellular matrices, with transport governed by matrix viscoelasticity and enhanced by aquaporin-mediated deformability. It also examines how EVs interact with target cells to trigger signaling, particularly under shear stress. Bridging soft matter physics and therapeutic development, this work highlights the mechanical and biological roles of EVs in tissue regeneration.

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EV research is rapidly accelerating as the field more fully understands their biological functions and their utility as disease biomarkers. New technologies and tools are needed to facilitate rapid, simple, rapid, selective, and sensitive detection of EVs with specific characteristics, while also quantifying their nonheterogeneous, dynamically changing molecular cargo and surface proteins. This talk will summarize our team’s efforts to utilize DNA origami nanostructures that include designer DNA Nets and hand-like DNA NanoGrippers to selectively capture intact virions and other nano-objects by binding with outer proteins, and subsequent ability to digitally count them in a label-free fashion by Photonic Resonator Interferometric Scattering Microscopy (PRISM). Using high video frame rates, we observe persistent “wiggling” characteristics of captured and tethered nano-objects that provides very high signal-to-noise and detection limits in the range of 100-1000 objects/ml. Our approach paves a path towards a rapid, simple, and highly sensitive alternative to conventional approaches for EV enrichment and characterization. Meanwhile, detection of specific proteins displayed on EV outer surfaces or carried within EVs as cargo can be used to link EVs in the circulation with their tissue of origin, but are especially challenging to detect due to their low concentration. Simple, rapid, and ultrasensitive detection of EV-related protein biomarkers are of interest to measure longitudinal concentration changes. Recently we have been exploring how “proximity” assays for protein targets can be adapted to provide a nonenzymatic mechanism for signal amplification while also providing simplicity and sensitivity that is superior to existing methods that utilize enzymatic amplification. Recently, we demonstrated a novel approach called Proximity Initiated Nucleic Acid Target Amplification (PINATA) that is capable of protein detection at room temperature in a single step, using a small portable detection instrument. The PINATA assay implements amplification using toehold-mediated strand displacement reactions combined with digital detection of individual gold nanoparticle tags for ultrasensitive, rapid protein quantification.

We are developing screening tests consisting of novel integrated systems for process automation, biomarkers specific to early disease onset, and assays for the early detection of cancer. The commonality in these tests is that they consist of microfluidic devices made from plastics via injection molding that can be integrated to nanofluidic devices via a fluidic motherboard. The hardware for our tests can be mass produced at low-cost to facilitate bench-to-bedside transition and point-of-care testing (PoCT) that is typically required for large scale population screening. The assays use liquid biopsy markers as the input, which can be secured in a minimally invasive manner appropriate for screening tests. Recently we have generated a strategy to produce plastic nanofluidic devices, which provides unique opportunities for single-entity analyses in a label-free fashion, which can also be injection molded. In this presentation, we will discuss a specific application of our screening test, which consists of the analysis of EVs for the early detection of ovarian cancer. Ovarian cancer is the 5th most deadly cancer for women in the US and has a 46% 5-y survival rate. Unfortunately, ~85% of cases are diagnosed at a late stage of disease providing poor outcomes for these patients. Therefore, new strategies for early detection are required. The screening test we will discuss consists of a microfluidic chip for EV affinity selection, which possesses a high density array of pillars surface-decorated with antibodies, to efficiently select EVs. This chip can be integrated to a nanofluidic chip for the label-free enumeration of EVs to determine elevated levels of EVs in the plasma of patients suspected of having high grade serous ovarian cancer. Unique EV-associated surface proteins were discovered for selection of ovarian cancer EVs specifically for the early detection of disease – these markers consist of surface antigens associated with the fallopian tubes where the cancer originates. The selected EVs can be photolytically released from the capture surface and counted using a nano-Coulter Counter chip (nCC), which consisted of in-plane nanopores. Both steps of the screening test described here were carried out using a microfluidic and nanofluidic chip integrated to a control motherboard for automating sample processing with results produced within 20 min.

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Extracellular vesicles (EVs) comprise a broad class of cell derived nanoparticles that play important roles in natural processes and may be harnessed as therapeutic delivery vehicles. Realizing this potential requires technologies for modifying EV composition and function in a manner that confers specific delivery properties, as well as analytical capabilities for guiding such bioengineering. This talk will present work my lab has led to characterize and ultimately modulate EV-mediated delivery of cargo ranging from Cas9 RNPs to AAV vector capsids. It will highlight our new experimental characterization tools that enable rapid screening and unambiguous assessment of functional delivery. I will also place this work in the context of the broader field of mammalian synthetic biology.

Ceramide generated by neutral sphingomyelinase 2 (nSMase2) was initially proposed to be essential for ESCRT-independent EV biogenesis. However, recent studies have challenged the specificity of nSMase2 in this process. Rather than revisiting ceramide’s role in EV formation, we shifted our focus to a more compelling question: how ceramide defines the function and signaling potential of EVs. Our data show that ceramide is enriched in EVs with pathological function in Alzheimer’s disease (AD). Ceramide can be produced by either nSMase2 or acid sphingomyelinase (aSMase), with activation triggered by Aβ, oxidative stress, or proinflammatory cytokines in a sex-specific manner. These ceramide-rich EVs (CREVs) are secreted by astrocytes and selectively target neuronal mitochondria. I will present new studies from our laboratory that elucidate the pathological function of CREVs and explore therapeutic strategies aimed at inhibiting ceramide generation in AD.

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Early identification of Krabbe disease through newborn screening is essential to initiate timely therapies that can slow disease progression. This study evaluates the efficacy of a transient neonatal enzyme replacement therapy (ERT) using extracellular vesicles (EVs) to deliver galactosylceramidase (GALC) to the brain. HeLa cells were engineered to overexpress GALC, and EVs containing GALC were administered via a single intrathecal injection to GALC-deficient Twitcher mice shortly after birth. While this intervention did not extend overall survival, it significantly reduced cortical psychosine accumulation and brain inflammation. Notably, four days post-treatment, microglial cells exhibited increased expression of anti-inflammatory markers IL-10 and TREM2. These findings suggest that early EV-based ERT may be considered as an adjunct strategy to reduce neuroinflammation when administered with hematopoietic stem cell transplantation or gene therapy.

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Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) hold promises as cell free therapeutics. However, clinical translation remains hindered by donor-to-donor variability, limited scalability, and lack of precise control over EV cargo. In contrast, autologous EVs from patient biofluids such as plasma, saliva, or urine are readily accessible and well-suited for point-of-care applications. Yet, these vesicles lack the immunomodulatory payloads characteristic of MSC-EVs. We hypothesize that autologous EVs can be transformed into potent therapeutic carriers by engineering them with functional cargo such as CRISPR-Cas9 ribonucleoprotein (RNP) complexes. A key bottleneck is loading cargo into EVs without compromising their structural or functional integrity. To overcome this, we developed EVOLVE (Engineering of Vesicles by Optimal Loading via Endovesiclosis)—a modular, force-free platform that efficiently loads functional cargo, such as CRISPR-Cas9 ribonucleoproteins, into autologous plasma-derived EVs without compromising their integrity. EVOLVE leverages guanidinium-functionalized nanoparticles for cargo delivery and enables built-in imaging capabilities through nanoparticle incorporation. In a murine acute lung injury model, EVOLVE-engineered EVs carrying CRISPR RNPs targeting NLRP3 achieved 90% survival and reduced inflammation, outperforming MSC-EVs. This approach represents a scalable, clinically adaptable strategy for next-generation EV-based nanotherapeutics.

Neurodegenerative diseases such as Parkinson’s disease (PD) pose major diagnostic challenges due to asymptomatic or non-specific early stages. Extracellular vesicles (EVs) have emerged as promising biomarkers, carrying disease-relevant biomolecules on their surface or within their lumen that provide insights into disease progression. This study presents a label-free surface plasmon resonance (SPR)-based biosensing platform for selective detection and molecular profiling of PD-associated EVs. In vitro PD models were established by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA) to mimic early and late stages, alongside untreated controls. The custom-built SPR biosensor was functionalized with antibodies against CD81, NCAM, and multiple α-synuclein species to enable EV capture and profiling. Using conformation- and phosphorylation-specific α-synuclein antibodies, the platform sensitively discriminated EV subpopulations and revealed stage-dependent enrichment of membrane-associated aggregated α-synuclein. Analysis of lysed EVs further confirmed elevated intravesicular aggregated α-synuclein, demonstrating its presence both on membranes and within vesicles. Assay sensitivity was enhanced by increasing antibody surface coverage and incorporating Alix as an internal reference, with the α-synuclein/Alix ratio progressively increasing with disease stage. Overall, this SPR platform enables sensitive and quantitative profiling of both EV surface and cargo biomarkers, offering strong potential for early diagnosis and mechanistic studies in neurodegenerative diseases.

Retinal ischemia damages retinal cells, leading to vision loss. Extracellular vesicles (EVs) derived from dental pulp stem cells have neuroprotective functions attributed to microRNA (miRs). We profiled miRs within the EVs of inflammatory-preconditioned DPSCs and overexpressed miR-22-3p in DPSC EVs due to its neuroprotective actions, termed functionally engineered EVs (FEE-22). We hypothesize that FEE-22 treatment will prevent loss of function after retinal ischemia. We transfected DPSCs with a lentiviral system containing miR-22-3p and GFP marker to generate FEE-22. We confirmed the overexpression of miR-22 in DPSCs and EVs using RT-qPCR. We determined the functional efficiency of FEE-22 in a rodent model via electroretinography (ERG) responses of retinal cells at baseline, day 7, 14, and 28. ERGs include a complete field intensity to measure inner retinal cells, flicker response to assess photoreceptors, and pattern ERG (PERGs) for RGCs. The left eye served as a positive non-ischemic control for each rat. A two-way ANOVA was used for statistical analysis.FEE-22 treatment improved flicker amplitude starting at day 14 and b-wave amplitude over time. FEE-22 conserved PERG amplitude.FEE-22 preserved retinal function vs PBS. This effect was widespread in the retina, as reflected by improved flicker amplitude, b-waves, and PERGs responses.