Browsing by Author "Jacobson, Stephen C."

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Characterization of Extracellular Vesicles by Resistive-Pulse Sensing on In-Plane Multipore Nanofluidic Devices
    (American Chemical Society, 2023) Young, Tanner W.; Kappler, Michael P.; Hockaden, Natasha M.; Carpenter, Richard L.; Jacobson, Stephen C.; Biochemistry and Molecular Biology, School of Medicine
    Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min-1. The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.
  • Thumbnail Image
    Item
    Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing
    (ACS, 2020-09) Zhou, Xiaoju; Brown, Brooke A.; Siegel, Amanda P.; El Masry, Mohamed S.; Zeng, Xuyao; Song, Woran; Das, Amitava; Khandelwal, Puneet; Clark, Andrew; Singh, Kanhaiya; Guda, Poornachander R.; Gorain, Mahadeo; Timsina, Lava; Xuan, Yi; Jacobson, Stephen C.; Novotny, Milos V.; Roy, Sashwati; Agarwal, Mangilal; Lee, Robert J.; Sen, Chandan K.; Clemmer, David E.; Ghatak, Subhadip; Surgery, School of Medicine
    Bidirectional cell–cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte–macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.