HBV Envelope Protein-Bearing Vesicles Show Preferential Uptake in Hepatocyte-Derived Cells

Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 > HepG2 > Huh7 > non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells.