Carbon Monoxide-Releasing Nanoreactors Transiently Modulate TLR4 Signaling to Resolve Inflammation

Abstract Uncontrolled inflammation drives diverse acute and chronic diseases, yet effective suppression without compromising host defense remains a major therapeutic challenge. Although carbon monoxide (CO) is an endogenous gasotransmitter with well-established anti-inflammatory activity, its clinical translation has been limited by the lack of delivery systems capable of controlling dose, release kinetics, and tissue localization. Here, we report a metal-free micellar nanoreactor that implements a nanoconfined cascade between endoperoxides (EPOs) and 3-hydroxyflavone (3-HF) derivatives for sustained CO generation. Thermal decomposition of EPOs continuously produces singlet oxygen (1O2), which subsequently oxidizes adjacent CO precursors to release CO with programmable kinetics under physiological conditions. Systematic molecular screening revealed that the cascade kinetics are governed by the structures of the EPOs as well as aromatic-ring extension and chalcogen substitution within the 3-HF scaffold, enabling oxidation rates to be tuned over 3 orders of magnitude. Relative to an equivalent mixture of separately assembled micelles, coassembly accelerated S-HF oxidation and enhanced anti-inflammatory activity, consistent with more efficient capture of the short-lived 1O2 intermediate within the micellar core. Sustained intracellular CO generation transiently decreased surface-detectable TLR4/MD-2, attenuated downstream inflammatory signaling and cytokine amplification, and promoted macrophage repolarization, while TLR4/MD-2 levels subsequently returned toward baseline. The nanoreactors alleviated inflammation and tissue injury in mouse models of APAP-induced acute liver injury, LPS-induced endotoxemia, and allergic airway inflammation. These findings establish an autonomous, nanoconfined reaction cascade for sustained gasotransmitter delivery and transient modulation of pathological inflammation.