With the rapid development of nanotechnology, there has been growing interest in new strategies for the treatment of tumors. [1] External non-invasive or minimally invasive treatments have advantages over traditional radiotherapy and chemotherapy. [[2], [3], [4]] Among these, photodynamic therapy (PDT) is a widely used non-invasive treatment, which has made a significant contribution to the clinical diagnosis and treatment of tumors. However, PDT is restricted by the penetration depth (<1 cm) into tissues, which limits the use of PDT treatment for deep tumors. [5] In contrast, ultrasound (US) has the advantages of being a non-invasive, deep-penetrating acoustic method which has been used successfully to improve access to deep tumors alongside other biomedical applications. [6] Sonodynamic therapy (SDT) utilizes low-intensity ultrasound to excite a sonosensitizer that in turn generates reactive oxygen species (ROS) via cavitation. ROS are small, oxygen-containing molecules including singlet oxygen (1O2) and hydroxyl radicals (•OH) which are used in SDT to kill tumor cells. [7,8] This has proven to be effective for the treatment of hypoxic tumors. However, some sonosensitizers have limited access to the complex tumor microenvironment (TME) which hinders the generation of ROS at tumor sites. [9,10] Thus, development of a therapeutic modality that can adjust the TME to enable sonosensitizers to effectively generate ROS is of benefit for advances in sonodynamic therapy.

Improvements in the efficacy of SDT can be achieved using rational adjustments to the TME. [11] Compared with normal cells, the TME has distinct properties including; vascular abnormalities, hypoxia, mild acidosis, and, elevated hydrogen peroxide expression. [[12], [13], [14]] Some of these characteristics act as a defense system formed by the TME that resists attacks by various anti-tumor agents. In particular, the hypoxic condition in the TME severely limits the efficacy of SDT, hindering the generation of adequate 1O2. [15] Fortunately, catalase activity can generate in-situ O2 from H2O2 present in the TME and thereby alleviate TME hypoxia to provide improvements in SDT. Overall, rational regulation of the TME plays an important role in enhancing the efficacy of SDT.

At present, sonosensitizers are generally divided into two types: those based on either organic or inorganic moieties. [16] Organic sonosensitizers have limitations, such as, insolubility or low solubility in aqueous media, low accumulation at the target tumor, and adverse phototoxicity effects. [[17], [18], [19], [20]] These limitations lead to unsatisfactory SDT effects. In contrast, the high chemical stability of inorganic sonosensitizers combined with low phototoxicity make these more suitable for SDT applications. [21,22] However, inorganic materials often lack the ability to target tumor sites. This has inspired the development of tumor-targeted carriers capable of delivering inorganic sonosensitizers for the current study. Recent reports using live microorganisms capable of targeting tumor sites (for example anaerobic or facultative anaerobic bacteria and even oncolytic viruses) suggest these bio-vehicles could be employed. [[23], [24], [25]] Furthermore, the unique TME found in solid tumors permits bacteria (e.g., Escherichia coli, attenuated Listeria, etc.) to effectively and selectively colonize tumor tissues. [26,23, [27], [28], [29]] Related nanomedicines, constructed exploiting the natural tropism of anaerobic or facultative anaerobic bacteria to target tumor sites, have showed performance in multimodal cancer therapy. [30,31] The combination of microorganisms and nanomaterials not only enables the accurate delivery of the microbial therapy, but integrates the stimuli-responsive function of nanomaterials. Specifically, development of new microbial nanomedicine that selectively targets the tumor sites, such that normal cells are unaffected by the treatment.

Activation of an anticancer immune response based on immunogenic cell death (ICD) is another effective way to target tumors. [32,33] It has been reported that cellular immunity can be elicited by chemotherapy or under the action of ROS-generating reaction leading to cell damage and ICD. [34,35] For instance, the cytotoxic ROS (e.g., •OH, 1O2 and O2•−) produced during SDT are essential in activating the intracellular ‘danger’ signaling pathways that govern ICD responses. [36] Moreover, endogenous ROS induced by Se NPs is highly desirable for an improved SDT efficacy, which subsequently strengthens the ICD effect within tumor cells. [[37], [38], [39]]

In view of the above considerations, a new sonosensitizer strategy is proposed and optimized here. We used engineered LI as a platform loaded with the modified sonosensitizer Au-RuO2 NPs as well as Se NPs as an optimized microbial sonosensitizer. The RuO2 NPs act as sonar sensitizing agents and this effect was further optimized in term of its’ performance by inclusion of Au NPs. Further modification, by attachment of Se NPs to LI together provided an additional enhancement of the sonosensitizer function of the assembled microbial sensitizer, [email protected] This microbial sonosensitizer can be successfully delivered to tumor tissue where it can generate O2in situ thereby alleviating tumor hypoxia. Subsequent US irradiation of cells treated with [email protected] can generate excess ROS, which in turn can act synergistically with the Se NP-induced endogenous ROS to induce ICD-mediated antitumor immunity. In addition, the activity of LI after US irradiation treatment was greatly reduced, which ensured its biosafety. This work provides a new strategy for realizing tumor-specific enhanced combination therapy under the guidance of PA imaging. (Scheme 1).