Designing nature's nanocarriers: advances and challenges in functionalizing bacterial extracellular vesicles.

Bacterial extracellular vesicles (bEVs) are nanoscale, membrane-bound particles naturally secreted by bacteria and are increasingly being explored as therapeutic carriers. Their small size, ability to encapsulate and protect cargo, inherent bioactivity, and compatibility make them attractive candidates for therapeutic development. However, precise and reproducible functionalization remains a central challenge for clinical translation. Post-isolation physical and chemical modification methods enable cargo loading and surface conjugation but are often limited by membrane disruption, heterogeneous functionalization, and scale-up constraints. In contrast, genetic engineering approaches program vesicle composition during biogenesis, enabling controllable luminal loading, surface display, and hypervesiculation. In this review, we examine luminal targeting strategies (secretion pathway-mediated loading, lipoprotein anchoring, and membrane scaffold fusions), surface display platforms (ClyA, Lpp-OmpA hybrids, autotransporters, and ice nucleation protein), and genetic yield-enhancement mechanisms. Finally, we outline key translational considerations to advance bEVs toward clinically viable, programmable delivery systems.