Macrophages, as a critical component of the immune system, play a central role in maintaining tissue homeostasis, defending against pathogenic microorganisms, and modulating inflammatory responses [[1], [2], [3]]. Their functional status is primarily dictated by their polarization state, which is broadly categorized into two major phenotypes: M1 (classically activated) and M2 (alternatively activated). M1 macrophages predominantly secrete pro-inflammatory cytokines and are involved in host defense and the initiation of acute inflammatory responses. In contrast, M2 macrophages mainly produce anti-inflammatory mediators and are associated with tissue remodeling, immune regulation, and resolution of inflammation [[4], [5], [6], [7], [8]]. Accumulating evidence indicates that hydrogen sulfide (H2S), as an endogenous gaseous signaling molecule, plays a pivotal role in regulating macrophage polarization, offering promising therapeutic potential for the treatment of various inflammation-associated diseases [[9], [10], [11], [12], [13], [14]].

With regard to the suppression of M1 polarization, H2S primarily exerts its effects through S-sulfhydration, a reversible post-translational modification that alters the activity, stability, and molecular interactions of target proteins [15]. By modifying key transcription factors such as NF-κB and STAT1, which are central to inflammatory signaling pathways, H2S inhibits their activation and subsequently suppresses the expression of downstream pro-inflammatory mediators, including IL-6, TNF-α, and iNOS [12,16,17]. This mechanism has been substantiated in multiple experimental models of inflammation. For example, in a rat model of myocardial infarction, administration of exogenous H2S donors significantly reduced serum levels of pro-inflammatory cytokines and attenuated tissue damage, demonstrating robust anti-inflammatory efficacy [[18], [19], [20], [21], [22], [23]].

In promoting M2 polarization, H2S activates key signaling cascades such as AMPK and PI3K/Akt, thereby facilitating the transition of macrophages toward an anti-inflammatory phenotype [[24], [25], [26], [27], [28]]. Activation of these pathways leads to the upregulation of M2-specific markers, including Arg1 (arginase 1), IL-10, and CD206, which collectively enhance the capacity of macrophages for tissue regeneration and immune modulation. In experimental models of colitis, H2S treatment has been shown to markedly ameliorate intestinal inflammation and promote mucosal healing, accompanied by an increased presence of M2 macrophages [[29], [30], [31], [32], [33], [34]]. Furthermore, studies on diabetic wound healing have demonstrated that H2S accelerates tissue repair through M2 polarization, underscoring its therapeutic relevance in chronic inflammatory conditions. Therefore, the development of H2S-releasing agents with targeted delivery to macrophages represents a promising area of research [[35], [36], [37], [38], [39]]. It is also important to note that the regulatory effects of H2S on macrophage polarization exhibit both concentration- and time-dependent characteristics. At low concentrations, H2S typically exerts anti-inflammatory and cytoprotective effects; however, at higher concentrations, it may trigger oxidative stress and cytotoxicity [10,12,[40], [41], [42], [43], [44]]. We measured the time-dependent fluorescence intensity profiles at hydroxyl radical concentrations of 5 μM, 50 μM, and 100 μM (Fig. S29) With the increase in hydroxyl radical concentration, both the fluorescence intensity and the fluorescence rate increased. Consequently, in the context of clinical translation, precise control over the release kinetics and dosage of H2S is essential to harness its therapeutic benefits effectively.

Our previous research found that designing hydrogen sulfide donors with reactive oxygen species (ROS) response characteristics can achieve controlled release of hydrogen sulfide based on the ROS level in the cellular microenvironment and further exert tissue repair effects [[45], [46], [47], [48]]. Among various ROS, Hydroxyl radicals are typically generated through secondary reactions, with the most prominent pathway being the metal ion-catalyzed Fenton reaction involving hydrogen peroxide. In contrast to hydrogen peroxide, which serves dual roles in cellular signaling and oxidative toxicity, hydroxyl radicals are predominantly detrimental in biological systems and exhibit significantly higher reactivity and destructive potential. They represent key cytotoxic species responsible for oxidative damage to critical biomolecules—including proteins, DNA, and lipids—leading to cell dysfunction, senescence, apoptosis, and the pathogenesis of various diseases under conditions of oxidative stress. Their extreme reactivity results in nonselective attacks via hydrogen abstraction or radical addition mechanisms [[49], [50], [51], [52]]. Under normal physiological conditions, the concentration of hydroxyl radicals is extremely low; however, in pathological states, it may increase to the micromolar level. Fluorescence probe technology has a detection range spanning from micromolar to nanomolar concentrations, making it widely applicable for in vivo detection of hydroxyl radicals. [[53], [54], [55]]. As cells lack dedicated enzymatic systems for hydroxyl radical detoxification, scavenging relies primarily on low-molecular-weight antioxidants such as glutathione [[56], [57], [58], [59], [60]].

Therefore, the development of donor molecules capable of releasing hydrogen sulfide in response to hydroxyl radicals holds substantial biological significance and therapeutic potential, based on the above research foundation, we designed and synthesized a new type of hydrogen sulfide donor with macrophage-targeting ability and hydroxyl radical response characteristics.

As shown in the schematic diagram, this hydrogen sulfide donor introduces a folic acid structure, which can specifically recognize and bind to the highly expressed folate receptors on the surface of macrophages, thereby significantly enhancing the targeted delivery efficiency of the hydrogen sulfide donor molecules to polarized macrophages [[61], [62], [63], [64], [65]]. Subsequently, due to the significantly elevated level of ROS (especially hydroxyl radicals) in polarized macrophages, it can trigger the oxidative cleavage reaction of the hydrogen sulfide donor prodrug molecules and release biologically active hydrogen sulfide molecules under the catalytic action of carbonic anhydrase in vivo. This targeted-released hydrogen sulfide can precisely exert antioxidant and anti-inflammatory effects within macrophages, thereby effectively intervening and treating various inflammation-related tissue injuries.