Violacein-Loaded Outer Membrane Vesicles from <i>Salmonella enterica</i> Exhibit Potent Anti-Melanoma Activity <i>in Vitro</i> and <i>in Vivo</i>

Violacein exhibits antitumor activity, indicating potential for future clinical application. However, an efficient delivery system is required for the clinical use of this hydrophobic compound. Effective delivery systems can enhance the solubility and bioavailability of hydrophobic compounds like violacein, facilitating its clinical application for antitumor therapy. Recent studies have demonstrated that outer membrane vesicles (OMVs) can serve as nanocarriers. This article constitutes the first report to present both <i>in vivo</i> and <i>in vitro</i> investigations of OMVs derived from a hypervesiculating mutant of <i>Salmonella enterica</i> Typhimurium as a delivery vehicle for violacein. In this study, <i>S. enterica</i> Typhimurium Δ<i>tolRA</i> (with a hypervesiculated phenotype) was transformed with a plasmid encoding the violacein biosynthesis operon. OMVs and violacein-loaded OMVs were isolated, characterized, and used in the treatment of murine melanoma. We assessed the cytotoxic effect of these violacein-loaded OMVs in both two-dimensional (2D) and three-dimensional (3D) cell cultures. Violacein-loaded OMVs reduced melanoma cell viability (IC₅₀: 9.30 × 10⁸ vesicles/mL) and delivered violacein in melanoma cells. Additionally, tumor regression was associated with treating tumor-bearing mice with violacein-loaded OMVs or nonviolacein-loaded OMVs (5 × 10⁹ vesicles/mouse). The antitumor response was linked to the accumulation of M1-type macrophages in the tumor microenvironment and the overexpression of mRNA for antitumor mediators Inducible Nitric Oxide Synthase, Tumor Necrosis Factor-alpha, and Interleukin-6 (iNOS, TNF-α, and IL-6). Our results suggest that OMVs can act as nanocarriers for highly hydrophobic agents and induce antitumor responses to eliminate tumors.