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Life sciences · Journal article
Nano Letters · November 3, 2026
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Abstract Nanoparticle-based combination therapy suffers poor spatiotemporal coordination from mismatched drug release. Herein, we report a poly(l-glutamic acid) (PLG) nanosystem that exploits distinct ester hydrolysis kinetics of a vascular-disrupting agent (combretastatin A4, CA4) and a hypoxia-responsive prodrug (imidazoquinoline azide, IMDQ-N3) for spatiotemporal coordination, while incorporating antigen-capturing moieties to enhance immune responses. Importantly, to overcome the limitations of conventional labeling methods, which only track the nanocarrier backbone and are prone to false-positive results, we developed a label-free, dual-extraction LC-MS/MS workflow to distinguish free from conjugated drugs, revealing the in vivo fate and providing direct quantitative validation of our kinetic design at the drug level. Our results show that free drugs accounted for only 4.6% and 0.2% of total circulating drugs, markedly reducing toxicity. The optimized formulation achieved spatiotemporally coordinated differential release and activation within tumors. This synergistic kinetic design and novel analytics provide a design paradigm and validation tool for PLG-based combination therapy.