Conference Registration, Materials Pick-Up, Coffee and Networking

Michael Graner, Professor, Dept of Neurosurgery, University of Colorado Anschutz School of Medicine -- Conference Co-Chairperson

Introduction to the EV, LNP 2025 Conference -- Topics Covered and Synergies Across Disciplines

Noah Malmstadt, Professor, Mork Family Dept. of Chemical Engineering & Materials Science, University of Southern California -- Conference Co-Chairperson

Introduction to LNPs -- Why are LNPs Gaining Attention and a Survey of Applications

Session Sub-Title: Extracellular Vesicles 2025 -- Progress in Research, Diagnostics & Therapeutics Development

Chairperson: Professor Michael Graner, University of Colorado - Anschutz Medical Center

Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, USA

Label-Free Detection and Identification of Single Protein Molecules Harvested from Extracellular Vesicles

Extracellular vesicles contain a wide range of different types of molecular cargo, such as lipids, carbohydrates, nucleic acids, and proteins. However, due to their small size (~150 nm for exosomes), the number of molecules is limited. For example, there can be ~20,000 protein molecules per EV vesicle. Because the proteome is estimated to consist of >100,000 different proteins, there are less than a single copy of a particular protein per EV particle. As such, specialized techniques have been developed to search for these rare proteins, such as OLink, which use the proximity ligation assay and quantitative PCR to detect these rare events. Challenges include the complicated and long workflow and the use of expensive antibodies. To address this issue, we are developing a nanotechnology to quantify and identify single protein molecules based on peptide fingerprinting.

Resistive Pulse Sensing (RPS) is a label-free and single-molecule detection approach that requires simple instrumentation to implement and as such, can be mobilized to be integrated into in vitro diagnostic assays for not only detecting but identifying key disease-associated biomarkers with high analytical sensitivity, such as proteins. Thus, RPS is a logical choice for coupling with liquid biopsy markers for the precision management of a variety of diseases due to the significant mass limits imposed on any assay in which a liquid biopsy marker is used. We have developed a unique measurement modality and sensor technology (dual in-plane nanopore sensor) that couples RPS to nanoscale electrophoresis. Not only does the sensor generate the typical RPS measurement parameters, but also the molecular-dependent electrophoretic mobility, which we call the time-of-flight (ToF). The RPS parameters coupled with the ToF and machine learning lead to high detection efficiency and classification accuracy of single molecules harvested from liquid biopsy markers, including proteins. Our devices, which are made from plastics via high-scale production modalities (injection molding), consist of channels with dimensions ranging from 1 to 100 nm (effective diameter) that are 10’s of microns in length.

In this talk, I will discuss the operational parameters and unique applications of our dual in-plane nanopore sensor for peptide fingerprinting of single protein molecules. The technology I will discuss involves the use of an immobilized nanoscale enzymatic reactor (INER) containing a proteolytic enzyme to digest the input protein molecule into peptides that generates a fingerprint of the protein by the nanoscale electropherogram pattern produced. Operational characteristics of this sensor for peptide fingerprinting will be shared.

Banishree Saha, Associate Director, Early Clinical Development, Takeda Pharmaceuticals, United States of America

Exosomes as Biomarkers: A New Frontier in Medical Diagnostics

Exosomes represent a promising frontier in medical diagnostics, offering unique capabilities as biomarkers for various diseases, including liver disease. These nano-sized vesicles, secreted by cells, carry proteins, lipids, and nucleic acids that reflect the physiological status of their parent cells. Emerging studies underscore their role in intercellular communication and their potential in disease detection and monitoring. This talk will explore recent advancements in exosome isolation and characterization techniques and their application in diagnosing cancer, neurological disorders, infectious diseases, and liver disease. We will discuss the challenges and future directions for integrating exosome-based diagnostics into clinical practice, aiming to enhance precision medicine through non-invasive and early detection methods.

Mid-Morning Coffee Break and Networking with Exhibitors

Sven Kreutel, CEO, Particle Metrix, Inc., USA and Germany

Technology Spotlight Presentation

Andrew Godwin, Professor and Division Director, Deputy Director, KU Cancer Center, University of Kansas Medical Center, USA

Title to be Confirmed

Johnny Zhuang, Business Development & Product Application Scientist, EXODUS BIO, United States of America

Technology Spotlight Presentation

Networking Lunch in the Atrium

Session Sub-Title: EV-based Therapeutics -- Status Update, ca. 2025

Damien Pearse, Professor, Department of Neurological Surgery; The John M. and Jocelyn H.K. Watkins Distinguished Chair in Cell Therapies, University of Miami Miller School of Medicine, USA

Title to be Confirmed

Mousumi Ghosh, Research Associate Professor, The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, United States of Americ

Investigating the Therapeutic Potential of Modified Microglial Exosomes in Spinal Cord Injury Repair

Traumatic spinal cord injury (SCI) results in severe, long-term disability and high mortality, with limited effective treatments to mitigate secondary tissue damage or restore function. Although cell transplantation shows promise in experimental SCI, numerous challenges have restricted its clinical application. Recently, exosomes-nanoscale vesicles secreted by all cell types-have gained attention as a potential therapeutic approach for CNS injuries and diseases due to their exceptional bioavailability, safety, and stability. Exosomes carry bioactive molecules, including miRNAs and proteins, that promote cell survival, axon regeneration, and anti-inflammatory effects.

Microglia, the CNS's innate immune cells, play a crucial role in SCI repair, but their exosomal contributions remain underexplored. In this study, we evaluated the neuroprotective and pro-regenerative effects of phenotypically modulated microglia-derived exosomal vesicles (pmMGEVs), preconditioned using a combination of anti-inflammatory cytokines and small molecule modulators. In vitro, pmMGEVs were readily taken up by neurons, significantly enhancing neuronal survival and growth on inhibitory substrates such as chondroitin sulfate proteoglycan. Systemic administration of pmMGEVs in clinically relevant murine models of moderate SCI improved locomotor function compared to naïve MGEVs.

Multi-omics analysis of pmMGEV cargo, compared to naïve MGEVs, identified specific miRNAs and transcription factors likely driving these therapeutic effects. These findings suggest that pmMGEVs represent a promising, cell-free therapeutic approach for enhancing SCI repair and functional recovery.

Natasha Sosanya, Research Scientist, US Army Institute of Surgical Research (USAISR)

Identifying Stress-Exacerbated Thermal-Injury Induced Pain Biomarkers

Using a model of Combat and Operational Stress Reaction (COSR), our lab recently showed that exposure to an unpredictable combat stress (UPCS) procedure prior to a thermal injury (TI) increases pain sensitivity in male rats. Additionally, our lab has recently shown that circulating extracellular vesicle-microRNAs (EV-miRNAs), which normally function to suppress inflammation, were down-regulated in a male rat model of neuropathic pain. In this current study, female and male rats were exposed to UPCS followed by TI and then evaluated for changes in circulating EV-miRNAs.

Adult female and male Sprague Dawley rats were exposed to the UPCS procedure for either 2 or 4 weeks. Groups consisted of the following: non-stress (NS), stress (S), NS+TI, and S+TI. Mechanical sensitivity was measured, and plasma was collected at relevant timepoints to include baseline, during UPCS exposure, and after TI. EV-miRNA isolation was then performed, followed by small RNA sequencing and subsequent data analysis.

UPCS exposure alone resulted in mechanical allodynia in both males and female rats at specific time points. Thermal-injury induction occurring at peak UPCS resulted in increased mechanical allodynia in the injured hind paw compared to thermal-injury alone. Differential expression of the EV-miRNAs was observed between the NS and S groups as well as between NS+TI and S+TI groups. Consistent differences in EV-miRNAs are detectable in both COSR as well as during the development of mechanical sensitivity. Therefore, EV-miRNAs may potentially serve as key regulators, biomarkers, and targets in the treatment of COSR and thermal-injury induced mechanical sensitivity.

Mid-Afternoon Coffee Break and Networking in the Exhibit Hall

Unchained Labs Technology Spotlight Presentation

Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands

Plasma Extracellular Vesicles in Reducing Hospital Referrals for Chest Pain Patients

Chest pain induced by exercise or stress is very common in USA and EU with approximately 500,000 GP visits/year in the Netherlands. Based on a better safe than sorry strategy, the GP refers about half of these patients suspected for obstructive coronary artery disease for a coronary CT scan to the hospital. After the scan, only 20% of male and 10% of female patients have indeed obstructive coronary artery disease. To increase the efficiency and reduce costs and time to scan, we developed a blood test based on plasma extracellular vesicle (EV) proteins that can, with an AUC of 0.8, determine which chest pain patients have obstructive coronary disease or not. To withhold chest pain patients from referral for a CT scan, however, an AUC 0.8 is not sufficient. For this, we now investigate the combination of microfluidic EV isolation with existing proteomics technologies of OLINK and EvoSep One LCMSMS and clinical parameters to safely exclude chest pain patients for referral for CT-scan.

Hsueh-Chia Chang, Bayer Professor of Chemical and Biomolecular Engineering, University of Notre Dame, United States of America

Large-Scale Isolation and Detection of Nanocarriers Biomarkers with Nanotechnologies: Parsing Heterogeneity

Intense research in the last 2 decades have shown that nucleic acid and protein biomarkers are secreted from parent cells as cargoes of nanocarriers like lipoproteins, exosomes (or small extracellular vesicles sEVs) and ribonucleoproteins. These nanocarriers range from 10 nm to 1 micron in size and are extremely heterogeneous. It is now clear that the heterogeneity is due to the different biogenesis pathways of the nanocarriers and hence each nanocarrier variant contains cell-specific and disease-specific biomarkers. I will summarize the results from my lab on the fractionation of these nanocarriers and on profiling of their colocalized protein and miRNA cargoes. Due to the nanodimension of the carriers, we utilize low-cost nanoporous membrane technologies with nanopores of comparable size. The goal is to develop a pan-disease screening platform for the annual blood test, after all the biomarkers are discovered. I will report preliminary data with clinical samples from 100-patient cohorts. These technologies were also used for nanocarrier biomarker discovery, in conjunction with NanoString, NGS and proteomic/lipidomic mass spectrometry. The large-volume data offered by massively parallelized versions of our technologies are ideal for regression studies with Machine Learning. Some of these technologies are being commercialized by Aopia Biosciences, a company I co-founded.

Networking Reception with Beer & Wine

Close of Day 1 of the Conference

Morning Coffee and Networking in the Exhibit Hall

Noah Malmstadt, Professor, Mork Family Dept. of Chemical Engineering & Materials Science, University of Southern California, USA -- Conference Co-Chairperson

LNPs -- A Deep Dive

Owen Fenton, Assistant Professor at the University of North Carolina at Chapel Hill, United States of America

Investigating the Role of Hypoxia on Lipid Nanoparticle Mediated mRNA Delivery

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy and mechanism driven study that details how hypoxia impacts the cellular response to messenger RNA Lipid Nanoparticle therapies. In brief, our work provides a comparative analysis as to how various states of oxygenation impact lipid nanoparticle-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different lipid nanoparticle chemistries, 3 different cell lines, 4 different oxygenation ratios, and 6 different reoxygenation conditions. In brief, our results demonstrate that hypoxia decreases the efficacy of mRNA Lipid Nanoparticles by upwards of 80%. Further, our results also suggest that these efficacy decreases correlate with decreased intracellular ATP levels in hypoxic cells. Taken collectively, our results suggest that cellular and tissue oxygen levels may be an important factor to consider when developing messenger RNA Lipid Nanoparticle based therapies.

Briana L. Simms, Assistant Professor of Chemistry, University of Cincinnati, United States of America

Design, Synthesis, and Characterization of Structurally Tunable Lipid Nanoparticles (LNPs) for Transdermal Delivery

Lipid nanoparticles (LNPs) have emerged in the literature as a viable vehicle for therapeutic delivery, gaining significant popularity over the last 30 years. LNPs are composed of a mixture of biocompatible lipids, which results in variance in nanoparticle properties such as size, surface charge density, or rigidity. This heterogeneity within the formulation can decrease targeting efficiency in drug delivery applications. To address these challenges, we have developed a library of structurally tunable lipid nanoparticles (stLNPs) formed by the self-assembly of a single synthetic lipid with modifiable features. This results in uniform physical (i.e. size and morphology) and mechanical (i.e. rigidity, flexibility, and bilayer morphology) properties across the nanoparticle formulation and allows for the specific selection of LNP properties ideal for the application. With a long-term goal of advancing these materials towards clinical application for targeted transdermal delivery, we have designed and characterized synthetic lipids comprised of a poly amidoamine (PAMAM) dendron chemically linked to an acyl tail group. We have incorporated tunable features that afford nanoparticles with a range of properties. To assess the feasibility of these materials in transdermal delivery, we have treated human skin with the stLNPs and evaluated their impact on skin physiology and penetration depth. The work described herein will provide a framework for the strategic design and synthesis of LNPs for targeted drug delivery. This work will also allow us to better understand how small structural changes to materials can influence the final fate of LNPs in vivo.

Mid-Morning Coffee Break and Networking in the Exhibit Hall

Bowen Li, Assistant Professor, Canada Research Chair in RNA Vaccines and Therapeutics, University of Toronto, Canada

AI-Guided Development of Lipid Nanoparticles for mRNA Delivery

The ability to transfect selective cell types within the targeted tissue in vivo is critical for potential therapeutic applications of mRNA. Although great advances have been made in mRNA vaccines, the ideal chemical and formulation composition of lipid nanoparticles (LNPs) for extra-hepatic delivery of nucleic acids are largely unknown. The traditional development of new lipids and formulations has been challenging, given the complexity of biological systems. In this talk, I will introduce an AI-guided high-throughput platform where thousands of chemically diverse libraries of lipid-like materials can be rapidly synthesized using multicomponent reactions and formulated into LNPs, which can then be screened for tissue- or cell-specific gene delivery. This platform technology increases the diversity of synthetic material structures and facilitates the identification of structure-function relationships.

Brian Feng, CEO, Osem Fluidics, United States of America

Advancement of Mixing Methods for Assembly of Nucleic Acid-Lipid Nanoparticles

The mixing of nucleic acid and lipid nanoparticles has evolved from simple manual pipette mixing to microfluidics, offering increased control over mixing rate, time, and flow conditions for the assembly of lipid nanoparticles. Although microfluidics is the gold standard for assembling LNPs, there are improvements to current methods that can increase the adoption of microfluidics. In this talk, we will introduce a novel microfluidics format for producing desirable lipid nanoparticles that makes it accessible to all researchers worldwide.

Andreas Möller, Professor, Director, JC STEM Lab of Personalized Cancer Medicine, Chinese University of Hong Kong

Title to be Confirmed

Networking Lunch

Chukwumaobim Nwokwu, Assistant Professor of Biochemistry, Florida Gulf Coast University, United States of America

Liposomes as a Successful, Safe and Sustainable Drug Delivery Platform for Kaempferol in the Treatment of Glioblastoma

Conventional chemotherapeutics present problems from poor pharmacokinetic profile to toxicity to healthy tissues, which have hampered their clinical application. Nano-pharmaceutical formulations using lipid-based and polymeric materials have shown great promise in circumventing these drawbacks, because of their biocompatibility, biodegradability, targeted delivery potential, and versatile drug-loading capacity. The putative anticancer compound, kaempferol was encapsulated in liposomes (Lip-K), and its cytotoxic and apoptotic effects against human glioblastoma cells (LN-229) and healthy human astrocytes were investigated. Cytomorphological and biochemical evaluations confirmed apoptotic indices, which continued unabated even in the presence of free radical scavengers. Overall, the results suggest that liposome-encapsulated kaempferol offers an effective and relatively safe cancer treatment option, with the promise of overcoming antioxidant-induced drug resistance. Unlike most cancer therapeutics, the ability of liposomes to cross the blood-brain barrier sheds light on the potential use of liposomal kaempferol to treat glioblastoma.

My Mahoney, Professor, Department of Pharmacology, Physiology, and Cancer Biology, Thomas Jefferson University, United States of America

Therapeutic potential of EVs in IVIG

Extracellular vesicles (EVs) play a critical factor in intercellular communication and immune regulation. Intravenous (IVIg) immunoglobulin (Ig) replacement therapy boosts the immune system in patients with antibody deficiencies or suppresses inflammation in autoimmune, infectious, and chronic inflammatory diseases. Here, we suggest a novel mechanism of immunomodulation through IVIg EV-associated cytokines. EVs were isolated from IVIg and unprocessed human plasma (UHP) from healthy donors. Bead-based multiplex analysis of known EV surface proteins by flow cytometry showed that IVIg EVs displayed significantly reduced platelet markers and enriched stemness markers compared to UHP. Luminex multiplex cytokine immune profiling revealed that the levels of 22 pro-inflammatory cytokines were dramatically reduced in IVIg EVs compared to UHP EVs. Interestingly, high levels of the pro-inflammatory cytokine IFN-γ were detected in IVIg EV-rich fractions. Here, we demonstrate that IFN-γ binds to EVs, most likely through circulating anti-IFN-γ antibodies, blocking IFN-γ-induced JAK/STAT1 activation. In summary, IVIg EVs can function as decoys to suppress inflammatory signaling pathways.

Natalia Tumidajski, Principal Scientist & GMP Site Head, ExoCel Bio, Pennsylvania Biotechnology Center, United States of America

Space Microgravity and Hypoxia Conditions as a Novel Approach to Enhance Regenerative Potency of MSC-derived Exosomes in Specialized 3D Cell Culture System

Research in microgravity is emerging as a new approach in studying biological systems and designing better treatment options. Recent studies in space have shown that microgravity transforms the behavior of stem cells and has unparallel effects on their growth, proliferation, and secretion of bioactive molecules such as extracellular vesicles. Regardless of continuous advancements in stem cell-based therapies, there are still significant challenges to ensure reproducibility, stability, and maximized potency for biomedical applications. Universal 2D cell culture system fails to mimic a natural environment for stem cells limiting their full capabilities including reduced stemness and deficient cell to cell interactions. Our approach was to create a natural niche for mesenchymal stem cells where they can cultivate their full biological potential and release the most potent exosomes- significant meditators in cell-to-cell communication via enriched cargo of proteins, lipids, and mRNAs. In this study, using placental MSCs we generated 3D cell models- spheroids with 5th dimensional technology and hydrogel-based organoids. They were then cultured in bioreactors in specialized 3D systems using microgravity, hypoxia, and rotational speed as the main conditions to mimic unique space environment. Upon isolation of exosomes from conditioned media the comprehensive characterization was conducted to validate their enhanced biological activity and regenerative potential. This completely novel and innovative study could ignite a cutting-edge revolution for regenerative applications such as wound healing, tissue engineering, and organ regeneration.

Speaker Details to be Confirmed

Mid-Afternoon Coffee Break and Networking with the Exhibitors

Speaker Details to be Confirmed

Speaker Details to be Confirmed

Bringing EVs and Space Summit Together

Chaired by Dr. Kristin Kopperud and Dr. Mike Roberts -- International Space Station (ISS) - National Laboratory

**Presentations and Panel Discussion in this Session Unify EV and Space (LEO Microgravity) Research**