• About
    • Login
    View Item 
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of Institutional RepositoryCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    New Technologies for Mechanism Elucidation and High Throughput Screening of Endosome Disruption by Carriers Designed for Intracellular Biologic Drug Delivery

    Kilchrist, Kameron V
    : https://etd.library.vanderbilt.edu/etd-07222019-144923
    http://hdl.handle.net/1803/15474
    : 2019-07-23

    Abstract

    Since the FDA approved the first recombinant protein 37 years ago, drug developers have harnessed biologics to treat human disease. Despite tremendous progress in extracellular biologic development, there are only eight FDA-approved intracellular-acting biologic medicines. Intracellular biologics must overcome additional cellular barriers: they must enter the cell via endocytosis and escape the endosome to avoid lysosomal degradation. Here, we elucidate the molecular mechanisms of cellular uptake and endosomal escape for a promising preclinical nanomedicine designed to address intimal hyperplasia, a leading complication following coronary artery bypass graft treatments. We uncover a mechanism of non-specific interaction between the polymer carrier, poly(propylacrylic acid) (PPAA) and the cellular membrane of vascular smooth muscle cells, which triggers macropinocytosis, leading to a 49-fold increase in intracellular accumulation within 30 minutes over free peptide. Further, a novel kinetic assay based on Galectin8-YFP (Gal8-YFP) reveals significant endosomal disruption within 30 minutes. We next developed and validated the powerful Gal8-YFP assay into a high throughput format for screening of libraries of carriers / formulation for endosome escape and intracellular delivery. We show that Gal8-YFP is both high throughput and also a superior predictor of intracellular bioactivity over traditional screening assays, such as cellular uptake, lysotracker colocalization, and hemolysis. Finally, we show that the technique is amenable to in vivo screening of endosome disrupting carriers in an orthotopic breast cancer model. Finally, we engineer two novel endosome disruption assays based on split firefly luciferase. Following endosomal disruption, the two halves of luciferase form a functional enzyme to produce significant increases in luminescence following addition of D-luciferin substrate in cells treated with endosome disrupting PPAA. This novel luminescence-based assay requires no image processing and is a “turn on” assay more amenable to in vivo applications than Gal8-YFP. The combined data give new fundamental mechanistic insights into existing biologic intracellular delivery technologies. This works also establishes new endosome disruption screening tools that are higher throughput, more predictive of bioactivity, and facile to incorporate into drug and nanomedicine discovery pipelines.
    Show full item record

    Files in this item

    Icon
    Name:
    201907_Kilchrist_KV_Doctoral_D ...
    Size:
    10.28Mb
    Format:
    PDF
    View/Open

    This item appears in the following collection(s):

    • Electronic Theses and Dissertations

    Connect with Vanderbilt Libraries

    Your Vanderbilt

    • Alumni
    • Current Students
    • Faculty & Staff
    • International Students
    • Media
    • Parents & Family
    • Prospective Students
    • Researchers
    • Sports Fans
    • Visitors & Neighbors

    Support the Jean and Alexander Heard Libraries

    Support the Library...Give Now

    Gifts to the Libraries support the learning and research needs of the entire Vanderbilt community. Learn more about giving to the Libraries.

    Become a Friend of the Libraries

    Quick Links

    • Hours
    • About
    • Employment
    • Staff Directory
    • Accessibility Services
    • Contact
    • Vanderbilt Home
    • Privacy Policy