An in situ-forming injectable silk hydrogel with a cocktail of chemotherapeutic drugs simultaneously targeting cancer cells and cancer stem cells

Abstract

Breast cancer recurrence and therapeutic resistance are primarily driven by breast cancer stem cells (bCSCs), which are poorly eliminated by conventional chemotherapy. To address this limitation, we developed an in situ-forming injectable silk hydrogel for sustained dual delivery of doxorubicin (DOX) and salinomycin (SAL), enabling concurrent targeting of bulk tumor cells and bCSCs. The hydrogel forms under physiological conditions without the need for UV irradiation or toxic initiators and exhibits tunable mechanical and degradation properties. An optimized 8% hydrogel exhibited controlled release behavior of DOX and SAL, characterized by a limited initial burst followed by sustained release for up to 30 days. In vitro viability assays demonstrated that DOX or SAL alone reduced cancer cell viability by approximately 45–55%, whereas the dual-drug loaded hydrogel (SilkVS-DOX + SAL) achieved a cytotoxicity of roughly 90%. Mammosphere formation assays demonstrated a marked reduction in both mammosphere number and size, indicating potent inhibition of bCSC self-renewal. Consistently, expression of bCSC-associated stemness markers was reduced by approximately three-fold relative to free-drug treatments. Flow cytometric analysis further confirmed enhanced induction of apoptosis within the bCSC-enriched population following treatment with SilkVS-DOX + SAL. In vivo studies using 4T1 tumor-bearing mice demonstrated that the localized, sustained release of DOX and SAL from the injectable silk hydrogel synergistically suppressed tumor growth, while significantly reducing systemic toxicity compared to individual drug administration. Overall, the in situ silk injectable hydrogel with sustained dual-drug release effectively eliminates both bulk tumor cells and bCSCs, making this injectable silk hydrogel a promising strategy for reducing breast cancer stem cells, which may reduce the chances of recurrence and enhance therapeutic outcomes.