Injectable anaerobic hydrogel loading Clostridium butyricum for local hydrogen therapy against colorectal cancer

Hydrogen (H₂), a non-toxic therapeutic gas, exerts antitumor effects by disrupting intracellular redox homeostasis and inducing mitochondrial dysfunction. The high diffusibility of H₂, as well as its storage difficulties, hinders its sustained release at tumor sites. Thus, the therapeutic effect remains limited. Clostridium butyricum exhibits great potential for cancer treatment attributing to its inherent capability of continuous H₂ generation. However, its obligate anaerobic nature prevents survival under aerobic conditions, such as the well-vascularized tumor regions with high oxygen levels. To address this challenge, we synthesized a hydrogel with anaerobic interior, based on the oxygen-depleting reaction between laccase and vanillin. As a delivery vehicle for Clostridium butyricum, this hydrogel (CB@Anaerobic-Gel) maintained its viability under aerobic conditions. As a result, the encapsulated bacteria can produce H₂ and metabolites throughout tumor regions-including both hypoxic zones and well-vascularized areas. In vitro studies confirmed that H₂ released from CB@Anaerobic-Gel disrupts redox homeostasis and suppresses mitochondrial respiration in colon cancer cells. These effects synergized with metabolites from C. butyricum to induce tumor cell death. In vivo experiments showed that intratumoral injection of CB@Anaerobic-Gel significantly suppressed tumor growth. Therefore, this method offers a novel approach for tumor hydrogen therapy and a versatile platform for oxygen-sensitive therapeutics.