Multifunctional Smart Injectable Triblock Copolymer Hydrogel for Highly Efficient Dual Drug−Gene Delivery in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) remains one of the most aggressive breast cancer subtypes, lacking targeted therapies and exhibiting elevated levels of intracellular Cu²⁺ ions. Smart multifunctional nanocarriers capable of dual drug-gene delivery offer a promising strategy for improved TNBC management. In this study, we present an injectable, triple-stimuli-responsive polymeric nanocarrier engineered via reversible addition-fragmentation chain-transfer (RAFT) polymerization for targeted TNBC theranostics. The amphiphilic triblock copolymer integrates three functional components: a photochromic spiropyran (SP) unit enabling light responsiveness, a pH-sensitive cationic hydroxyethyl methacrylate-glycine (HEMA-Gly) segment facilitating nucleic acid complexation, and a temperature-responsive N-isopropylacrylamide (NIPAAM) unit enabling thermally triggered behavior. Notably, the nanocarrier shows strong fluorescence quenching in the presence of Cu²⁺ ionsoverexpressed in TNBCsupporting selective cellular recognition. Doxorubicin (DOX) loading and release studies demonstrated controlled, sustained release under mildly acidic conditions mimicking the tumor microenvironment. <i>In vitro</i> evaluations using MDA-MB-231 TNBC cells confirmed the platform's multifunctionality. Cytotoxicity assays indicated biocompatibility, while fluorescence imaging revealed efficient cellular uptake of DOX-loaded micelles. Furthermore, the system modulated cell cycle progression and significantly enhanced intracellular and mitochondrial reactive oxygen species (ROS) production, indicating the potential to induce ROS-mediated apoptosis. Gene delivery experiments showed high transfection efficiency of nucleic acid-loaded micelles, attributed to the cationic HEMA-Gly block. Additionally, intracellular quenching studies confirmed the polymer's selective sensitivity toward TNBC-associated Cu²⁺ ions. Overall, the RAFT-synthesized triblock copolymer represents a versatile theranostic platform capable of simultaneous drug release, gene transfection, ROS induction, and Cu²⁺-guided TNBC detection. Its targeted responsiveness and multifunctional performance highlight its promise for advanced TNBC diagnostics and combination therapy.