Molecular engineering of extracellular vesicles for drug delivery: Strategies, challenges, and perspectives.

Extracellular vesicles (EVs) have emerged as promising therapeutic carriers due to their inherent biocompatibility and intercellular communication capabilities. However, the clinical translation of naturally secreted/naïve, EVs is hindered by several key challenges, including limited loading capacity of therapeutic cargo, poor targetability, off-target accumulation, and rapid systemic clearance. Naïve EVs are confined to delivering biomolecules incorporated during their biogenesis, often necessitating high doses to achieve therapeutic efficacy. Addressing these limitations through molecular engineering is critical to advance EV-based therapeutics. Innovative engineering approaches, including endogenous cargo loading during EV biogenesis, modifications post-EV isolation, and in vivo engineering of EVs, have expanded the functional versatility of EVs beyond their natural capabilities. This review systematically explores the landscape of molecular engineering strategies designed to enhance the therapeutic performance of EVs. We discuss approaches aimed at increasing the quantity and quality of innate EV cargo, efficient methods for loading exogenous small and macromolecular drugs, techniques for expressing or conjugating targeting ligands on EV surfaces for prolonging circulation time and for achieving cell/tissue specific targeting, and EV labeling approaches for tracking the delivery of either intact EVs or EV components. This review also highlights emerging strategies for in vivo engineering of EVs to generate functional EVs for therapeutic applications. In addition, the review addresses the major challenges associated with EV engineering and offers future perspectives to guide continued innovation. By integrating molecular engineering with EV biology, this review highlights transformative strategies that are advancing EVs as drug delivery platforms. The insights provided aim to catalyze the next generation of engineered EVs, pushing the boundaries of their clinical potential for treating diverse pathologies.