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Nano and Micro Technologies

Biomolecular Sensing and Imaging I

Light-induced Extracellular Vesicle Adsorption

Friday, October 25, 2024
4:45 PM - 5:00 PM EST
Location: Room 316

Introduction: The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix (ECM) and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a disruptive and versatile EV micropatterning technique that will rapidly advance the study of ECM- and surface-bound EVs and other particles.

Materials and

Methods: LEVA occurs when UV illumination is selectively applied to PLL/PEG-functionalized surfaces using a digital micromirror device (DMD), creating positively charged regions on the surface with highly predictable EV binding affinity based on the grayscale values of input templates (Fig 1A-C). LEVA’s versatility is demonstrated using commercial GFP-EV standards, E. coli outer membrane vesicles (OMVs), and EVs from U-87 MG glioblastoma bioreactors to create 2D micropatterns of the EVs on surfaces with different geometric shapes and gradients. The binding kinetics of LEVA were characterized using time-lapse total internal reflective fluorescence microscopy (TIRFM) and supercomputer-enabled COMSOL Multiphysics simulations for both small and large EVs. Following initial optimization, several applications were tested, including single EV characterization by fluorescence colocalization using TIRFM, glioblastoma cell migration on migrasome-mimetic trails, and neutrophil activation and swarming by OMVs using time-lapse fluorescence microscopy. All EVs were characterized according to MISEV guidelines, including NTA, TEM, zeta potential, and immunoblotting.

Results, Conclusions, and Discussions: Time-lapse TIRFM and COMSOL simulations demonstrated slower adsorption kinetics of large EVs compared to small EVs due to a combination of Brownian motion and charge (Fig 2A-H). Successful LEVA patterning with consistent 2 µm resolution enabled: (1) characterization of single EVs by TIRFM colocalization of fluorescent probes for CD63 and microRNA miR-21 (2) observation of U-87 MG single cell migration behavior on “1D” U-87 MG migrasome-mimetic trails, and (3) characterization of OMV micropattern geometry effect on localized peripheral blood-derived neutrophil swarming (Fig 3A-G). LEVA’s scalability, high-resolution, speed, and versatility will enable rapid advancements in the study of ECM- and surface-bound EVs and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.

Acknowledgements (Optional): This work was supported by The Ohio State University Center for Cancer Engineering-Curing Cancer Through Research in Engineering Sciences, the National Institutes of Health grants: K99EB033857, UG3/UH3TR002884, U18TR003807, and the LEGACY Postdoctoral Scholars Program at Ohio State. SM would like to thank Professor Angela Brown at Lehigh University for encouraging her interest in OMV research. We would also like to thank our healthy donors whose blood donations enabled the neutrophil studies.