Abstract

Extracellular vesicles (EVs) are membrane vesicles secreted by cells and distributed widely in all biofluids. Extracellular vesicles can modulate the biological activities of the recipient cells. Due to their role in intercellular communication, they are receiving attention for therapeutic and diagnostic applications. The first step to better understand EVs and to utilize them as therapeutic and diagnostic tools is to purify them from a variety of biofluids. Membranes have been extensively used for purification of different biological species from biological fluids. As the first aim, a novel microfluidic system, termed as tangential flow for analyte capture (TFAC) was developed to isolate nanoparticles and EVs using ultrathin nanomembranes. Ultrathin nanomembranes were found well-suited for TFAC system when compared with conventional thickness membranes. TFAC also proved feasible for capturing of EVs from undiluted plasma.

Fluorescent labeling of EVs has been employed for studying uptake and biodistribution of EVs. However, far too little attention has been paid to the effect of the fluorescent labeling on the size of EVs. In the second aim, the effect of PKH labeling, the most commonly used dye, on the size of EVs was systematically evaluated by nanoparticle tracking analysis (NTA). PKH labeling did not preserve the size of EVs and caused a size increase in all the PKH labeling conditions tested. The observed size shift may alter the uptake and biodistribution of EVs, suggesting that PKH labeling is not a reliable technique.

Precise quantification and characterization of EVs is an important step towards utilizing them as therapeutic and diagnostic tools. EVs have been analyzed using bulk techniques such as western blot which is challenging due to the heterogeneity of EVs. Therefore, a robust and well-established technique for quantification and characterization of individual EVs is required. As the third aim, the efficacy of a virus detection technology for EVs was evaluated. Virus Counter 3100 (VC3100) is a fluorescence-based technique with similar principles as flow cytometry and was purpose-built for detection of small nanoparticles such as viruses. Due to the similarity in size and density of viruses and EVs in many biofluids, it was hypothesized that the VC3100 could detect EVs similarly to flow cytometry characterization of cells. Fluorescently labeled EVs from different sources were successfully quantified by the VC3100. Furthermore, VC3100 was also used to determine the expression level of target protein markers. Therefore, VC3100 is a powerful technique for precise quantification and protein profiling of EVs.

Publication Date

9-25-2020

Document Type

Dissertation

Student Type

Graduate

Degree Name

Microsystems Engineering (Ph.D.)

Department, Program, or Center

Microsystems Engineering (KGCOE)

Advisor

Thomas Gaborski

Advisor/Committee Member

Blanca Lapizco-Encinas

Advisor/Committee Member

Michael Schertzer

Campus

RIT – Main Campus

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