A multiple-responsive nano-prodrug combining chemo-differentiation-cancer stem cell inhibition toward triple-negative breast cancer.

To overcome therapy resistance driven by breast cancer stem cells (BCSCs) and the systemic toxicity of conventional chemotherapy, we engineered a multi-stimuli-responsive nanoprodrug (DT/PAC@AI NPs) implementing a "chemo-differentiation-cancer stem cell inhibition" strategy. This system co-delivers irinotecan (IRI), all-trans retinoic acid (ATRA), and CPUL119 via (1) a hypoxia-responsive prodrug (PAC: PEG2k-Azo-CPUL119) and (2) a pH/esterase-responsive conjugate (AI: ATRA-IRI). The targeted NPs (90-130 nm), modified with DSPE-PEG2k-Try for LAT1-mediated uptake, demonstrated tumor microenvironment-triggered drug release: rapid PAC release under hypoxia and enhanced AI release at pH 5.0/esterase. In vitro, DT/PAC@AI NPs showed 20% higher cellular uptake and potent cytotoxicity against MDA-MB-231 cells (IC50 = 4.77 ± 0.32 µM vs. free drugs: IRI, 23.17 µM; CPUL119, 9.27 µM; ATRA > 50 µM), inducing 3.7-fold more apoptosis (32.6% vs. 8.7%). Critically, they reduced CD44+/CD24- BCSCs by 13.7% and inhibited tumor sphere formation by 87.6%. In vivo, they achieved optimal tumor suppression and BCSC elimination in xenografts with negligible systemic toxicity. This nanoprodrug platform offers straightforward synthesis, high drug-loading capacity, and potent dual-action efficacy against bulk tumor cells and BCSCs, presenting a promising advanced breast cancer therapy.