Challenge

Photodynamic therapy (PDT) has exhibited substantial advantages for its spatiotemporal selectivity, minimal invasiveness, and low biotoxicity in cancer therapy. Meanwhile, single-atom catalysts (SACs) have recently received enormous interest for unique electronic structures, such as well-defined and precisely situated metal centers, identical coordination environments, tailorable compositions, and versatile functionalities. However, the therapeutic efficiency of SACs is still challenged by the difficulties of efficient and controllable delivery into tumors while maintaining high catalytic activities across biological systems. Thus, an improved SACs-based strategy is required for cancer-specific PDT with maximized therapeutic efficacy and minimal side effects on normal tissues.

In situ synthesis of nanomedicines inside specific cells has been aroused for chemotherapy, overcoming random distributions of nanomedicines in healthy tissue to minimize side effects. However, in situ synthesis of SACs is still challenged by the difficulties of specific release of metals and ligands within the cancer sites. Moreover, the complex Tumor Microenvironment (TME), with variable acidic environments, hypoxic conditions, and redox species, would lead to poor stabilities and unpredictable side reactions during the in situ synthesis of SACs.

Particularly, the therapeutic efficiency of ROS-dependent PDT is significantly influenced by tumor hypoxia, limiting the generation of therapeutic ROS reagents without sufficient O₂. Alternatively, Type-I PDT could relieve the dependence on intracellular O₂ by generating ·OH through the Fenton-like oxidation of H₂O₂. However, it was still limited by the insufficient endogenous H₂O₂ at tumor sites. Therefore, generating adequate ROS species independent of endogenous O₂ and H₂O₂ would considerably increase the PDT efficiency in TME.

Approach

To address the challenge of the in situ synthesis of SACs, high stability nanomedicine precursors and high photocatalytic activity synthesized SACs are required. Herein, a nanomedicine precursor of 2D/2D C₃N₄-MnO₂ is prepared, which is lowly toxic to normal tissues. Responded to the upregulated GSH in TME, the in situ release of C₃N₄ and Mn²⁺ from C₃N₄-MnO₂ is initiated. Subsequently, atomically dispersed Mn²⁺ is captured by C₃N₄, facilitating the in situ synthesis of C₃N₄-Mn SACs within TME (Figure 1).

In situ synthesized C₃N₄-Mn SACs can induce the generation of hydroxyl radicals (·OH) under red irradiation (660 nm). The red shift of the absorption also facilitates the red light-irradiated applications (660 nm) with greater penetration depth than that of white light in PDT. Meanwhile, the water-splitting process to generate ·OH avoid the limitations of tumor hypoxia and limited hydrogen peroxide (H₂O₂) in the tumor on the PDT effect.

Figure 1. Schematic diagram of the in situ synthesis of C₃N₄-Mn SACs for PDT.

Results

We first focused on the in situ synthesis process and confirmed the generation and single atom structure of C₃N₄-Mn SACs by various characterizations. Subsequently, we found that the obtained C₃N₄-Mn SACs can catalyze the generation of ·OH under red irradiation, even in the absence of oxygen and H₂O₂. Demonstrated by mass spectrometry, we confirmed that the oxygen of ·OH comes from water and the ·OH was generated via a water splitting process. The legend-to-metal charge transfer (LMCT) process under irradiation and subsequent water splitting process was further demonstrated by a series of experiments and calculations. In brief, the LMCT process induced the generation of stable charge separated state of C₃N₄-Mn SACs under irradiation. Subsequently, the holes on N induced a hydrogen transfer process, further leading to the homolysis of O-H bond in water and the generation of ·OH. On the other hand, the H on C₃N₄-Mn SACs can reduce the intracellular pyruvate acid (the important energy molecule) to lactate acid, further enhancing the effectiveness of PDT (Figure 2).

Figure 2. The LMCT-based photocatalytic generation of ·OH by water splitting over C₃N₄-Mn. A photoexcited charge reparation state was formed to produce ·OH via O-H homolysis.

Furthermore, the of lipid peroxidation (LPO) process induced by ·OH generated under irradiation was monitored by mass spectrometry. As resulted, with the increase of irradiation time, lipids were oxidized to form lipid peroxides, which were further oxidized into cytotoxic small molecules (such as MDA and 4-HNE). The intermediates in the LPO process were captured and monitored by mass spectrometry within a 30 min irradiation (Figure 3). Besides, the corresponding in situ synthesis process, water splitting process, and the LPO process have been validated by in vitro experiments, which facilitates the efficient in vivo cancer therapy.

Figure 3. Examination of LPO process on LA by C₃N₄-Mn SACs under red-light irradiation.

Highlights

We have designed a method for the in situ synthesis of SACs for tumor selective therapy. The nano medicine precursors (C₃N₄-MnO₂) was in situ reduced by the upregulated GSH in TME, and the single atom nanomedicine (C₃N₄-Mn SACs) was in situ synthesis in tumor for efficient PDT, which greatly avoid the side effects on the healthy tissues.

Under red light irradiation, the photoinduced charge-separation state in C₃N₄-Mn SACs is constructed via a LMCT process. Through a hole-driven homolysis of O-H bond, C₃N₄-Mn SACs could efficiently induce the O₂-independent water splitting for efficient and selective ·OH generation. This minimizes the limitations of TME hypoxia or H₂O₂, facilitated the efficient Type-I PDT.

Based on mass spectrometry characterizations, the intermediates were determined and monitored, facilitating detailed mechanism examinations of ·OH generation by water splitting and the subsequent LPO process. As demonstrated, ROS in PDT were generated from water splitting, which initiated the generation of toxic small molecules (MDA, 4-HNE) for cancer therapy via LPO processes.

For more detail on the experiments and results, please read our paper: https://doi.org/10.1038/s41467-024-46987-1