Sonodynamic therapy (SDT) is an emerging and promising cancer treatment modality, which employs ultrasound (US) to stimulate sonosensitizers, and then react with molecular oxygen or biological substrates to generate cytotoxic ROS to kill cancer cells [[1], [2], [3], [4]]. The outstanding strengths of SDT such as noninvasiveness, spatiotemporal controllability, and no resistance, particularly the deep tissue penetration capability of US, endow SDT with great potential for the therapy of deep-seated tumor or large solid tumor, which is precisely the Achilles' heel of phototherapies [[4], [5], [6], [7], [8]]. SDT thus has been drawing considerable attention in recent years. It is well known that molecular oxygen-mediated ROS generation is the primary pathway of sonodynamic action [9,10]. However, hypoxia is a common feature of solid tumors, resulting from the rapid proliferation of tumor cells and abnormal angiogenesis [11,12]. The treatment outcome of oxygen-dependent SDT is severely restricted in hypoxic tumor [13,14]. Moreover, hypoxia-inducible factors (HIFs) will be activated in hypoxia tumor microenviroment, which accelerate tumor growth, strengthen the aggressiveness of tumor, and promote tumor metastasis [13,[15], [16], [17]]. Clearly, overcoming tumor hypoxia is crucial for achieving optimal anticancer efficacy.

To date, the strategies for alleviating hypoxia mainly involve oxygen-carrying material, in situ oxygen generation, and reducing oxygen consumption during therapy [[18], [19], [20], [21]]. However, the efficiency of in situ oxygen generation is generally low due to the constraints of the biological environment, and reducing oxygen consumption such as nonoxygen-dependent therapy does not truly address the issue of tumor hypoxia [18]. An effective approach to alleviate tumor hypoxia involves the use of oxygen-carrying materials [[21], [22], [23]]. As an artificial blood substitute, perfluorocarbon (PFC) not only shows good biocompatibility, and high oxygen-carrying capacity, but also can undergo US-mediated phase change to release oxygen [[24], [25], [26]]. Therefore, PFC is an ideal oxygen-carrying material for SDT.

Sonosensitizers are the principal factor for SDT. Various sensitizers, including organic and inorganic sonosensitizers, as well as their hybrid have been reported [[27], [28], [29], [30]]. Owing to their superior biocompatibility relative to inorganic sonosensitizers, organic molecular sonosensitizers, often derived from photosensitizers, have been widely concerned [31,32]. However, these photosensitizers generally display low sonodynamic activity. Recently, we reported for the first time a phthalocyanine-artesunate (PcA) conjugate, which showed remarkably higher (about 60-fold) ROS generation efficiency in its aggregated form than its monomer form, namely aggregation-enhanced sonodynamic activity (AESA) effect. It also displayed ca. 10-fold higher ROS generation than the known sonosensitizer protoporphyrin IX did [33].

Inspired by the results, we elaborately constructed an oxygen-carrying, dual-modal imaging-guided versatile nanoliposomal sensitizer based on the phthalocyanine-artesunate (PcA) conjugate, denoted as PcA-PFO@FLPs, for SDT of hypoxic tumors (Scheme 1). Upon ultrasound (US) irradiation, PcA-PFO@FLPs could release oxygen and efficiently alleviate hypoxia of the mouse breast cancer 4 T1 cells with boosted intracellular ROS generation, which could induce apoptosis. Moreover, the robust ROS generation of PcA-PFO@FLPs could also lead to iron-free ferroptosis, demonstrated by the down-regulation of glutathione (GSH) and the excessive accumulation of lipid peroxidation (LPO). Additionally, in vivo fluorescence and photoacoustic (PA) imaging indicated that PcA-PFO@FLPs could be selectively retained in tumor tissue due to folic acid (FA), which could target the folate receptor (FR) overexpressed in tumor, modified on the liposomes. Consequently, the oxygen-carrying PcA-PFO@FLPs exhibited efficient sonodynamic anticancer efficacy against 4 T1 tumor-bearing mice. Importantly, it could be rapidly metabolized after US treatment, demonstrating great potential for clinical application. Therefore, this work presents a new paradigm of efficiently multifunctional nanosensitizers for imaging-guided SDT of hypoxic solid tumor.