Analytical Approaches to Study Vesicular and Exosomal Release

Abstract: Vesicles, as a special type of entity nanometers in size, are crucial for survival of multicellular organisms. Intracellular vesicles mainly contain specific signaling molecules, transmitters, and modulators whereas extracellular vesicles (EVs) are bioactive organelles carrying a wide range of proteins, genetic materials, and other molecules. Secretion from vesicles is essential to manipulate many biological pathways and intercellular communication. Understanding the regulatory mechanisms of vesicular secretion is mandatory for uncovering the pathologies of neurological disorders and developing related pharmaceuticals. Several electrochemical techniques have been proposed and developed to unravel vesicular neurotransmitters at single cell and subcellular levels. These methodologies provide high spatiotemporal resolution and sensitivity while enabling direct quantification of electroactive molecules from individual vesicles. Single cell amperometry (SCA) can be employed to determine the number of signaling transmitters being released during an exocytosis event. Vesicle impact electrochemical cytometry (VIEC) and intracellular vesicle impact electrochemical cytometry (IVIEC) are two methods that allow the quantification of the number of signaling molecules stored inside single vesicles from isolated or intracellular vesicles, respectively. In this thesis work, vesicular structure as well as their content and release have been investigated. In paper I, open carbon nanopipettes (CNPs) with different radii between 50 and 600 nm were employed to quantify vesicular content in isolated vesicles of adrenal chromaffin cells by VIEC. Paper II was continuation of work from paper I, the mechanistic study of L-DOPA was carried out by using IVIEC with various sized CNPs. SCA was introduced to capture the dynamic release of single exosomes from a single living cell in paper III. In paper IV, the combination of electrochemistry and mass spectrometry was applied to investigate the effects of ketamine on dopamine storage and exocytosis, as well as the alterations of cellular lipid composition. Generally, electrochemical methods have been considered as a powerful tool to understand structures of vesicles and their biological functions.