In-depth characterization of extracellular vesicles for their use as disease biomarkers and next-generation nanovectors in the precision medicine era

Resumen

Extracellular vesicles (EVs) comprise a heterogeneous group of ubiquitous nanoparticles naturally secreted by all types of cells. These tiny particles serve as mediators for the transport of different molecules, assisting the cellular cross-talk between cells of the same and different organisms. EVs exhibit outstanding properties as carriers, including the ability to protect their cargo, cross biological barriers, low immunogenicity, and extended circulation times compared to synthetic nanoparticles. Furthermore, some EVs have shown affinity for specific tissues and targeted delivery. These exceptional properties have brought these vesicles to the forefront as a tool with great potential to reduce toxicity and maximize therapeutic efficacy of multiple drugs. Moreover, EV composition mirrors that of the progenitor cell, providing valuable information on cell type and physiological state. This feature, together with the fact that small fractions of EVs produced by different tissues can be found in organic fluids, positions EVs as promising, highly accurate, and readily accessible biomarkers, as well as a valuable tool for understanding the mechanisms underlying different processes involved in human health and disease. The objective of this doctoral thesis is to contribute to the advancement of EVs as both nanocarriers and biomarkers for biomedical purposes. The work is divided into two parts: the study of EVs as nanocarriers (Part I) and as biomarkers (Part II), with five chapters, four of them corresponding to published or forthcoming articles consolidating the knowledge in this rapidly evolving field. The current reliance on cell line cultures for obtaining EVs presents significant challenges, including low yields, high associated costs, and safety concerns, which represent major drawbacks hindering their use as nanotransporters. To overcome these challenges, in Part I, Chapter 1, we proposed a novel approach utilizing fermented food by-products (BPs) as an alternative and cost-effective source of biocompatible EVs. We employed various scalable methods and successfully isolated EVs from different BPs (BP-EVs). Ultrastructural and systems biology-based characterization of the BP-EVs revealed a high abundance of exosomes and the presence of proteins and lipids with excellent signaling abilities. Besides, in vitro cytotoxicity and in vivo distribution studies showed absence of cytotoxicity and excellent biodistribution in mice after oral administration, including ability to reach the brain. Levering their capacity to reach the central nervous system, we conducted a Systematic Review in Chapter 2 to explore the effectiveness of plant-derived nootropics as potential cargoes and selected the extract bacoside A (BacA) from Bacopa monnieri to load the BP-EVs. In Chapter 3, we described the methodology and efficiency of loading BP-EVs with BacA. The innovative use of food BPs as a source of EVs opens up exciting opportunities for the future development of therapeutic interventions. In fact, ongoing research is currently active (September 2023) involving a European patent application and two running projects. The second part of the thesis focusses on the utilization of EVs as valuable tools for understanding disease and exploring their potential as biomarkers. In Part II, Chapter 1, an in-depth analysis of the protein composition of brain EVs in schizophrenia was conducted. This investigation revealed unique molecular blueprints associated with this mental illness, providing valuable insight into the involvement of EVs in an altered brain connectome. Part II, Chapter 2, delved into and additional layer of information carried by EVs, specifically investigating the presence of ureido protein modifications (uPMs) in the proteome of seminal EVs from non-normozoospermic (non-NZ) men. This study unveiled abnormal profiles of uPMs, with increased homocitrullination and specific downregulation of citrullination in EVs from non-NZ men. These novel findings hold promise to contribute to a deeper understanding of the intricate role of these minuscule particles in human physiology. The research conducted in this thesis may help to pave the way for significant diagnostic and therapeutic advancements, contributing to the utilization of EVs as versatile tools for disease research and treatment, with the potential to revolutionize the landscape of medicine