Glutathione Responsive iNOS Inhibiting Polymeric Prodrug for Targeted Inhibition of Angiogenesis

Background: Inducible nitric oxide synthase (iNOS) is a key driver of aberrant angiogenesis in inflammatory conditions and cancer, making it an attractive therapeutic target. Nevertheless, its function can be affected by the complex immune responses and tumor microenvironment (TME). Hence, combinatorial treatment approaches that simultaneously target iNOS and immune-modulatory signaling are strongly recommended for cancer therapy. Moreover, the current iNOS inhibitors are limited by poor pharmacokinetics and a lack of selectivity. Results: To address these challenges, we developed a glutathione (GSH)-responsive iNOS-inhibiting polymeric prodrug (GRIP) decorated with betamethasone succinate (NPBeS). These dual-function nanoparticles (NPBeS) remain stable under physiological conditions but selectively release their payload in response to elevated GSH levels, a hallmark of the TME. Only upon activation by GSH, NPBeS inhibits iNOS, as evidenced by suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. NPBeS also normalized vascular endothelial growth factor (VEGF)-mediated tube formation in HUVECs and 3T3-L1 fibroblast cell migration, and angiogenesis in the CAM assay, demonstrating its anti-angiogenic activity. Importantly, GRIP did not impair acetylcholine (ACh)-induced vasodilation in rat aorta, even at elevated concentrations, indicating preservation of eNOS function. Conclusions: This is the first report of a GSH-responsive polymeric prodrug system that leverages intracellular GSH for both controlled release of anionic therapeutic agents and in situ synthesis of an iNOS antagonist. Through these two complementary pathways, the system enables targeted, sustained anti-angiogenic effects and promotes vascular normalization. This dual-function platform holds strong potential for the treatment of cancer-associated angiogenesis. Graphical Abstract: