Macropinocytosis and Fast Endophilin-Mediated Endocytosis Mediate Absorption of Garlic Chive-Derived Vesicle-like Nanoparticles in Human Intestinal Epithelial Cells

Dietary extracellular vesicles (EVs) or vesicle-like nanoparticles (VLNs) have been shown to exert beneficial functions in a wide range of diseases such as cancer, colitis, and metabolic diseases. They have also been used as natural carriers for medications. Despite the promising translational potential of dietary EVs or VLNs, the molecular mechanisms of their absorption in the gastrointestinal tract are not well understood. In this study, we investigated the absorption mechanisms of garlic chive-derived VLNs (GC-VLNs) using C57BL/6J mice and a human intestinal epithelial cell line, Caco-2 cells. We found that orally administered GC-VLNs crossed the epithelial layer of the small intestine and entered the underlying lamina propria. GC-VLNs were taken up and transported across the fully differentiated Caco-2 epithelial monolayer. Proteins and lipids, but not RNAs, in GC-VLNs mediated their uptake by Caco-2 cells. Chemical inhibitor treatments demonstrated that macropinocytosis and fast endophilin-mediated endocytosis (FEME) played key roles in the internalization of GC-VLNs. On the other hand, clathrin-coated pit-mediated endocytosis and clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endocytic compartment endocytosis did not contribute to GC-VLN uptake. Activation of macropinocytosis and FEME using their specific activators promoted the internalization of GC-VLNs. In addition, genetic manipulation of key molecules in macropinocytosis and FEME confirmed the important engagement of these two specific endocytic pathways in GC-VLN absorption by human intestinal epithelial cells. Our study has provided proof-of-principle evidence to advance our understanding of the absorption mechanism of GC-VLNs, which would be the key to further manipulation and engineering of these nanoparticles to improve their delivery efficiency as therapeutic modalities or drug carriers.