Cancer is one of the most serious diseases threatening human health. The tumor microenvironment (TME) in solid tumors are often featured with low extracellular pH, hypoxia, high hydrogen peroxide (H2O2) and glutathione (GSH) levels [[1], [2], [3], [4]], abnormal vasculature and inflammation [5,6]. Photodynamic therapy (PDT), which relies on the generation of cytotoxic reactive oxygen species (ROS) by light irradiating photosensitizers (PSs) in the presence of oxygen (O2), has emerged as a noninvasive therapy against various types of cancers [[7], [8], [9]]. However, the efficiency of PDT is severely hindered by the hypoxic TME, and the O2 consumption during PDT further aggravate the hypoxic TME [10]. Chemodynamic therapy (CDT) is another promising tumor therapeutic method [[11], [12], [13]] by utilizing metal-mediated Fenton/Fenton-like reactions or Haber-Weiss reactions to convert H2O2 into highly reactive hydroxyl radical (•OH) and O2. CDT not only can lead to dysregulation of redox homeostasis in cancer cells but also has potential for alleviating the hypoxic TME to potentiate O2-consuming therapeutic strategies. However, high GSH levels in cancer cells can act as a protector against oxidative stress [14], and thereby maintaining the redox homeostasis disturbed by PDT and CDT. Ferroptosis is a newly discovered iron-dependent mode of cell death, which is different from apoptosis, necrosis, and autophagy [15]. Interestingly, many Fenton reaction inducers can lead to the occurrence of ferroptosis by depleting GSH in the TME [16] so that can conquer the impediments of CDT or PDT. Therefore, design a nanoreactor that could integrate TME modulation, PDT, CDT and ferroptosis induction together will obtain satisfactory multi-pronged synergistic therapeutic effects.

Various nanoenzymes with oxidase (OXD)-like, peroxidase (POD)-like and catalase (CAT)-like catalytic activities have been used with CDT or PDT, to trigger ROS generation and ferroptosis to achieve highly efficient antitumor effects [17]. The most investigated nanomaterials targeting ferroptosis are iron-based inorganic materials such as iron oxide nanoparticles, iron-doped nanomaterial and iron-organic frameworks [18]. To achieve the combination of PDT/CDT/ferroptosis, these multimodal therapy systems generally require tedious synthesis and complicated materials, which hinders their clinical translation. Additionally, the inorganic materials are always challenged by their long-term tissue retention in living body [19,20]. More importantly, these multimodal therapy systems mostly use extra delivery systems. The addition of extra nanomaterials might introduce uncertain toxicity to humans. Therefore, the development of a clinically transformable nanoenzyme with ferroptosis induction ability, combined CDT and PDT, and low toxicity is highly desirable.

Chlorin e6 (Ce6), a second-generation photosensitizer approved by the U.S. Food and Drug Administration (FDA), has been widely used in PDT because of its low toxicity to normal tissues and strong fluorescence imaging ability [21]. Considering construction of multimodal therapeutic systems with potential clinical translation, Ce6 and the ferroptosis inducer erastin self-assembled supramolecular Ce6-erastin nanodrug was developed for ferroptosis-promoted PDT by promoting ROS production [22]. However, this system could not modulate TME to exert CDT. Hemin, which is the catalytic center of hemoglobin with high bioavailability and ignorable cumulative intoxication, has been approved by the FDA since 1983 [23,24]. It possesses high biological catalytic activity to induce the generation of O2 and ROS, including superoxide anions (•O2−) and highly toxic •OH. Therefore, hemin and Ce6 were reported to self-assemble into a multifunctional nanoparticle to achieve the combination of PDT/CDT/ferroptosis [25]. Nevertheless, hemin and Ce6 both exhibit hydrophobicity and are prone to aggregation in aqueous solution [26,27], making it challenging for practical clinical applications. Oleanolic acid (OA), a natural pentacyclic triterpenoid from plant with low toxicity, shows prominent anti-cancer effects in treating various cancers [[28], [29], [30]]. Besides, OA can downregulate the expression of hypoxia inducible factor-1α (HIF-1α), thus alleviating the tumor hypoxic microenvironment [31]. Another study reported that OA can activate ferroptosis in Hela cells by promoting acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression, thereby reducing the survival rate of Hela cells [32]. Furthermore, our previous research has shown that OA can promote NO synthesis and NO can further react with •O2− to produce peroxynitrite (ONOO−) [33], a kind of reactive nitrogen species (RNS) which is more toxic than •OH [34]. More importantly, OA has excellent self-assembly ability to form co-assembled nanodrugs, which not only could achieve enrichment at tumor site through enhanced permeability and retention (EPR) effect, but also enhance the anti-tumor effect of the entrapped drugs and avoid the introduced toxicity of extra carriers [[35], [36], [37]].

Based on the above fundamental research foundation, in this work, we aimed to construct a carrier-free nanoreactor named OCH self-assembled by OA, Ce6 and hemin for highly efficient multimodal synergistic cancer therapy via a simple co-assembly strategy (Scheme 1). After intravenous injection of OCH, OCH reaches the tumor site through the EPR effect, and then enters into cancer cells. The drugs can be released in the TME with weak acid and high GSH levels. Serving as a chemodynamic reagent and a ferroptosis inducer, the released hemin exhibited intrinsic catalytic activity, leading to the depletion of GSH and the generation of O2 and ROS. With the participation of OA, the more toxic RNS produced and the hypoxic TME was further alleviated to improve the photodynamic activity of Ce6 to produce more 1O2. At the same time, OA can also exert its own anti-cancer effect, and work jointly with hemin to induce the occurrence of ferroptosis. The preparation, characterization, and the physiochemical properties of OCH were investigated. The intracellular uptake, ROS generation, regulation of the related proteins, and the in vitro and in vivo synergistic therapeutic effects of OCH were studied in details in the following.