Date of Award
Doctor of Philosophy (PhD)
R. Kenneth Marcus
Leah B. Casabianca
Exosomes are vesicles secreted by cells having a size range from 30-150 nm and carrying genetic materials that are important for intercellular functions. Besides, mounting evidence suggests that tumor cells secrete more exosomes than normal cells making it important to quantify exosomes as well. Thus, it is important to be able to efficiently isolate and quantify exosomes for potential use in clinical diagnostics, as well as to develop a deeper understanding of their role in intercellular processes. Apart from exosomes, Lentivirus is another bioparticle that has drawn great attention from researchers. Lentivirus is a genus of retroviruses with a size range of 80–100 nm in diameter and is increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Traditional methods for exosome and lentiviruses isolation and quantification are time-consuming, have low purity yields (i.e., high protein carryover), and may cause exosome aggregation. Therefore, a better isolation and quantification method for exosomes and lentiviruses is required.In the last two decades, Marcus and coworkers have successfully employed capillary-channeled polymer (C-CP) fibers as the stationary phase for protein and IgG separations via reversed-phase (RP), ion exchange (IEX), hydrophobic interaction (HIC), and affinity chromatography. In this study, we have used the C-CP column for the study on isolation and quantification of exosomes from human urine, blood plasma, and cell culture media, and that of lentivirus from cell culture media. To improve the isolation quality, different parameters, such as flow rates, column lengths, modifiers (acetonitrile and glycerol), separation modes (linear-gradient and step gradient), were optimized. The process has been verified via analytical instruments such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR), were used for characterizing the exosomes and lentiviruses. In addition, a rapid, low-cost, and efficient reversed-phase HPLC method for protein separation of proteins of wide-ranging molecular weights was developed with novel polypropylene Y-shaped (trilobal) (PPY) C-CP column. In this study, we have shown the capability of using the C-CP column for the separation of exosome, lentivirus, and proteins with the optimized parameters.
Huang, Sisi, "Rapid Isolation and Quantification of Exosome and Lentivirus by Hydrophobic Interaction Chromatography on a Polyester Capillary-Channeled Polymer Fiber Stationary Phase" (2021). All Dissertations. 2880.