GPC3 and CD133-targeted peptide dual modification enhances the therapeutic effect of doxorubicin carried by OMVs on hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a high mortality rate because of the limitations of conventional chemotherapies (e.g., doxorubicin, DOX), including poor tumor targeting, systemic toxicity, and chemoresistance. Bacterial outer membrane vesicles (OMVs) are promising drug carriers but have limitations like nonspecific phagocytosis and weak targeting. In this study, a dual-targeted nanosystem ((GPC3 + CD133)^T-OMVs@DOX) was developed using GPC3/CD133 peptide-modified OMVs loaded with DOX to enhance HCC-specific targeting, optimize drug release, and reduce off-target toxicity. Compared with single-targeted or unmodified OMVs, dual-targeted (GPC3 + CD133)^T-OMVs significantly increased binding/internalization in GPC3/CD133-coexpressing Huh-7 cells (<i>p</i> < 0.05). DOX loading yielded high LE (∼81%), with a pH-responsive release of 40% at pH 7.4 vs. 80% at pH 5.0 over 48 h. Sparstolonin B (SsnB), a TLR2/4 inhibitor, reduced macrophage phagocytosis by ∼60%, prolonging the duration of DOX-loaded OMVs in circulation. In vivo, the dual-targeted system inhibited tumor growth by 75%, outperforming free DOX (40%) and single-targeted OMVs-DOX (55%). Mice treated with the OMVs maintained a stable weight, whereas those given free DOX showed ∼15% weight loss. Histology revealed minimal organ damage in the dual-targeted group vs. severe cardiomyocyte injury with free DOX. In conclusion, the dual-targeted OMV system enhances tumor specificity via cooperative GPC3/CD133 recognition, optimizes pH-responsive release, and reduces nonspecific clearance by modulating macrophages. These features improve antitumor efficacy and mitigate toxicity, positioning OMV-based nanocarriers as promising platforms for precision HCC therapy.