[Preparation of HLYVSPW peptide-displaying outer membrane vesicles and validation of their in vitro targeting effects on triple-negative breast cancer cells].

Bacterial outer membrane vesicles (OMVs) are readily amenable to engineering but naturally lack active targeting ability. In this study, the HLYVSPW peptide was displayed on the surface of OMVs using cytolysin A (ClyA) to construct Toll-like receptor 2 (TLR2)-targeted engineered OMVs (HLYV-OMVs), and their in vitro targeted uptake by triple-negative breast cancer cells was evaluated. HLYV-OMVs were prepared by ultracentrifugation and characterized by transmission electron microscopy, dynamic light scattering, zeta potential analysis, and proteinase K protection assays. TLR2-high-expressing MDA-MB-231 cells, TLR2-knockdown MDA-MB-231 cells (231-siTLR2), and MCF-10A cells were used to evaluate cell viability, cellular uptake, and batch-to-batch reproducibility. HLYV-OMVs exhibited a typical bilayer vesicular morphology with particle sizes of approximately 56-64 nm, and the HLYVSPW peptide was successfully displayed on the vesicle surface. Within the tested dose and exposure ranges, HLYV-OMVs did not cause an obvious decrease in cell viability. Their uptake by MDA-MB-231 cells was higher than that by TLR2-knockdown cells and MCF-10A cells and increased in a dose-related manner. Particle size and cellular uptake results showed a certain degree of reproducibility across batches. These findings indicate that HLYV-OMVs exhibit a TLR2 expression-associated uptake advantage in vitro and may provide an experimental basis for developing targeted delivery platforms for triple-negative breast cancer.