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Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis

Deighan, Clayton J
http://rave.ohiolink.edu/etdc/view?acc_num=osu1440158128

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Chemical Engineering.
In recent years there has been a tremendous increase in the number of investigations regarding the nanoscale vesicles passed between cells in the body collectively known at extracellular vesicles (EVs). The present consensus within the community is that these nanoparticles are critical regulators of signaling between noncontacting cells. They have been demonstrated to carry functional cargos including nucleic acids and proteins which can modulate activity in recipient cells. Extracellular vesicles have been proposed as an ideal delivery vehicle for new therapeutic modes such as RNA interference. In spite of the tremendous body of research there is little agreement on what exactly constitutes an EV, the challenges of separating EVs for use in the clinic, and the influence of current isolation methods on downstream results. Herein the basic biology and numerous methods for isolating and detecting EVs are reviewed and discussed. In the context of potential therapeutic applications the recovery of EVs by a traditional ultracentrifugation isolation and reagent precipitation is investigated using a particle balance approach. The losses and variability inherent to the isolation protocols are quantified and compared. The productivity of the selected HEK-293T cell line for producing EVs in culture is characterized and the potential obstacles to realization of EV therapeutics at scale are analyzed. Separately the fluorescence detection mode of Nanoparticle Tracking Analysis (NTA) is characterized to determine the number of flourophores which must be entrained in a single nanoparticle for tracking. This work is needed in the field because while the detection of EVs by light scattering NTA is a common technique, there is little data regarding the sensitivity of f-NTA. Using a model system of cationic lipoplex nanoparticles formed with Cy3 labeled DNA the number of fluorophores incorporated in successfully tracked particles is measured and an estimation of the lower limit of sensitivity is calculated. This result will help others in the field better plan experiments for biomarker identification on a single EV basis.
Jeffery Chalmers (Advisor)
Michael Paulaitis (Committee Member)
David Wood (Committee Member)
David Tomasko (Committee Member)
122 p.
Chemical Engineering
extracellular vesicles, exosomes, microvesicles, nanoparticle tracking analysis,

Recommended Citations

Citations

  • Deighan, C. J. (2015). Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1440158128

    APA Style (7th edition)

  • Deighan, Clayton. Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1440158128.

    MLA Style (8th edition)

  • Deighan, Clayton. "Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1440158128

    Chicago Manual of Style (17th edition)

osu1440158128
876
© 2015, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.