A pH-responsive dual-drug nanoplatform for stromal remodeling and enhanced chemotherapy via MMP3/TGF-β inhibition

The dense, fibrotic extracellular matrix (ECM) generated by cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) presents a formidable physical barrier that severely limits the penetration and efficacy of chemotherapeutic agents. This study aimed to design and validate a pH-responsive, dual-drug nanosystem (RPAE-QM) capable of overcoming this stromal resistance by coordinated delivery of a stromal-modulating agent and a potent cytotoxic payload. The RPAE-QM nanosystem was constructed to co-encapsulate the stromal-modulating agent quercetin (Que) and the chemotherapeutic drug DM1. RPAE-QM exhibited significant pH-responsive drug release, leading to strong synergistic cytotoxicity and superior tumor destruction capability in 3D spheroid models. Mechanistic investigation provided a definitive explanation for this efficacy. Molecular docking and molecular dynamics simulations predicted that Que. has high affinity and exceptional kinetic stability when binding to MMP-3. Subsequent experiments confirmed the downstream consequences of this interaction: treatment with Que. caused dose-dependent inhibition of both MMP-3 and TGF-<i>β</i> ₁ secretion from CAFs. Moreover, this was accompanied by a significant, concentration-dependent reduction in the phosphorylation of Smad2/3, a key downstream effector of the TGF-<i>β</i> signaling pathway. The RPAE-QM nanosystem provides an effective dual-action strategy, simultaneously addressing the stromal barrier while delivering a potent cytotoxic agent. Mechanistically, our findings indicate that Que. suppresses the pro-fibrotic MMP3/TGF-<i>β</i>/Smad signaling axis in our CAF model. This work therefore introduces a dual-action therapeutic concept, offering a mechanistically-defined approach to disrupt stromal barriers and improve drug efficacy at the pre-clinical proof-of-concept stage.