**2. Biology of extracellular vesicles**

The mechanism of EV internalization in the target cell may be mediated by multiple mechanisms, EV-surface contact molecule interaction to boost juxtracrine downstream signaling, fuse with the membrane to deposit payloads into the cytosol, or are taken up by phagocytosis, macropinocytosis, or receptor-mediated endocytosis is unclear [1, 5]. EVs play a crucial role in facilitating the cell-cell environment under normal and pathological conditions and in the pathogenesis of many diseases [3]. This section reviews the relationship between EV composition and interactions with biological membranes before delivering EV cargo to the target cells and stimuliinduced EV release. This section presents EVs biogenesis, different cargo loading mechanisms of EVs, their release, and the role of G Proteins. This book also discusses the scientific advances that have made it feasible to address the existing bottlenecks associated with the isolation and characterization of EV subsets from body fluids. The roadmap for effective immunocapture and molecular characterization is presented along with the review on immunoaffinity-based techniques for separating specific EV subsets from plasma and biofluids.

Exosomes play an important role in cell-cell communication, signal transduction, immune response in normal and disease backgrounds. Their possible use as diagnostic and prognostic biomarkers and the leverage to use them as therapeutic carrier vehicles have sparked tremendous clinical interest. The main constituents of exosomes are proteins, nucleic acids, and metabolites, as summarized in **Figure 1**. Among the highly enriched proteins in exosomes, the tetraspanins (CD9, CD63, CD81, CD82) help exosome-cell fusion, while the Heat shock proteins (HSP70, HSP90) are involved in stress response and antigen-binding and presentation, and other proteins (Alix, TSG101) are involved in exosome release. Some of these proteins are involved in exosome biosynthesis (Alix, flotillin, and TSG101), whereas others are considered exosomal biomarkers (e.g., TSG101, HSP70, CD81, and CD63) (**Figure 1**).

An intriguing question is how the cellular cargo is selectively sorted in the exosomes? This section of the book addresses the EV biogenesis, cargo loading mechanisms, their release, and the role of G Proteins (**Figure 2**). Another critical aspect of EV biology is how they are taken up by recipient cells? Whether EVs naturally cross biological barriers or need genetic modifications? This section highlights several critical areas, including the interplay of EVs with biological membranes, EV target cell internalization mechanism, the relationship between EV composition and interactions with biological membranes, and stimuli-induced EV release. It is also essential to understand the biophysical aspect of cellular vesicles' morphology and formation mechanisms discussed in this section. **Figure 1** elaborates exosome's hallmarks and shows different isolation techniques, including size exclusion chromatography, polymeric precipitation, electrokinetic microarray chip, ultracentrifugation-based, immunoaffinity capture, and sucrose density gradient.

This book also specially emphasizes the bottlenecks associated with the isolation and characterization of EV subsets from plasma, thereby limiting a better understanding of their biological significance. A chapter reviews the immunoaffinity-based techniques for separating specific EV subsets from plasma and presents a roadmap for effective immunocapture and molecular characterization. This section also discusses other popular techniques of exosome production *in vitro* and suggests the challenges of *in vivo* physiological or pathological characterization of exosomes. The usual sources of exosomes are bodily fluids (plasma, saliva, urine, etc.), somatic cells

(cardiomyocytes, fibroblast, pneumocyte, etc.), stem cells or pluripotent cells, and tumor or diseased cells (**Figure 1**). Red blood cell (RBC) contamination presents a significant challenge in EV isolation from urine for the non-invasive source of disease biomarkers. This section presents an Innovative method describing the removal of RBCs contamination from the urine fraction. This section elaborates the scientific advances that have made it feasible to characterize and engineer EVs, leading to their use as tools in biomarker discovery and disease diagnosis, prognosis, therapeutic application, and theranostics [6]. The potential of liquid biopsy is significant and can be essential for both diagnosis and therapy monitoring [7]. Blood and saliva EVs may assist achieve this without needing tissue samples.
