“NO” Means Yes: Unlocking the Therapeutic Synergy of Nitric Oxide (NO) and Chlorambucil (Cbl) via Photoresponsive Sequential Delivery in a Triple-Negative Breast Cancer 3D Spheroidal Platform with Transcriptomic Insights

Nitric oxide (NO) is a multifunctional signaling molecule in oncology, influencing tumor progression, apoptosis, and immune responses. In contrast, chlorambucil (Cbl), a DNA-alkylating chemotherapeutic, induces cytotoxicity through DNA damage. Here, we report a photoresponsive nanoparticle platform for sequential codelivery of NO and Cbl, where NO is released within 10 min of irradiation, followed by Cbl release within 30 min. The nanoplatform employs a coumarin-4-ylmethyl photoremovable protecting group (PRPG) to achieve light-triggered spatiotemporal release of both agents. Using 2D cultures and 3D triple-negative breast cancer (TNBC) spheroids, we observed that NO-assisted chemotherapy markedly improved tumor inhibition and apoptosis compared to Cbl alone. Transcriptomic profiling (RNA-seq) revealed modulation of oncogenic pathways (PI3K/AKT, NF-κB, MAPK, EMT) with upregulation of tumor suppressors (TP53, CASP10, TIMP1) and downregulation of oncogenes (BCL2, MMP9, VEGFC). Additionally, suppression of IL-6, TNF-α, and ECM remodeling factors indicated reduced metastatic potential. This strategy highlights NO-mediated synergy as a promising approach to overcome chemoresistance in TNBC.