Dual-trigger hyaluronic acid nanoprodrug incorporating a 2-nitrobenzenesulfonyl linker for CD44-targeted and glutathione-responsive drug delivery.

Selective intracellular activation of anticancer agents while minimizing toxicity toward non-target tissues remains a major challenge in drug delivery systems (DDSs) and prodrug design. In this study, we designed a hyaluronic acid-based prodrug system, HA-Ns-Dox, that integrates receptor-mediated cellular uptake with intracellular glutathione (GSH)-responsive activation. A doxorubicin (Dox) prodrug modified with a 2-nitrobenzenesulfonyl (Ns) group was synthesized and subsequently conjugated to hyaluronic acid (HA) via click chemistry to generate HA-Ns-Dox with both CD44-targeting capability and GSH-responsive activation properties. HA-Ns-Dox formed stable nanosized assemblies in aqueous solution and exhibited high stability under low-GSH conditions mimicking extracellular environments, whereas concentration-dependent activation of Dox was observed under high-GSH conditions mimicking intracellular reductive environments. In vitro studies demonstrated that HA-Ns-Dox was selectively internalized into the CD44-high human breast cancer cell line MDA-MB-231, accompanied by intracellular prodrug activation and nuclear accumulation of Dox. In contrast, cellular uptake and activation were minimal in the CD44-low cell line MCF-7, and cytotoxicity was significantly suppressed even at high concentrations. Cytotoxicity studies further revealed that HA-Ns-Dox exhibited significant antiproliferative activity in CD44-high cells while substantially reducing toxicity toward non-target cells. Collectively, these findings demonstrate the potential of combining CD44-targeted delivery with GSH-responsive prodrug activation to improve cancer cell selectivity.