Almost 2.2 million women were diagnosed with breast cancer worldwide in 2021, and 0.7 million deaths were recorded[1]. As the most common malignant tumour in women, it shows high genetic heterogeneity. Among all breast cancer types, triple-negative breast cancer (TNBC) is the most invasive subtype and does not express oestrogen receptor, progesterone receptor or human epidermal growth factor receptor 2[2,3]. Although the general 5-year survival rate of breast cancer has improved greatly, most patients with TNBC and distant metastasis have a median survival time of only 13 months[3]. TNBC is not responsive to either endocrine therapy or molecularly targeted therapy because of its molecular phenotype. Therefore, conventional postoperative chemotherapy and radiotherapy are the main systemic treatments for TNBC and are often efficient solely during the initial stage[4]. For the more advanced stage of TNBC, novel agents such as PARP inhibitors and immunotherapy agents, have shown impressive results. However, the objective remission rate (ORR) in TNBC is only approximately 23% because of the lack of therapeutic targets[5]. The poor prognosis among metastatic TNBC patients generates an urgent need for the development of nonspecific new treatment modalities.

Photodynamic therapy (PDT) is a safe and minimally invasive treatment strategy. PDT uses light to activate photosensitizers, and the reactive oxygen species (ROS) generated during photochemical reactions cause cell death. PDT has emerged as a promising cancer treatment approach in recent years[6]. The inadequate penetration depth of light is the biggest limitation of PDT on large and deep-sited tumors. Intra-tumor light delivery (interstitial PDT, iPDT) was used to eliminate larger tumors, in iPDT, one or more laser fibers are inserted into tumors via needles, or placed in catheters[7]. IPDT has been employed in the treatment of many tumors such as prostate, pancreatic, head and neck cancer and glioma[8]. Sonodynamic treatment (SDT), on the other hand, uses ultrasound to activate sonosensitizers. Because ultrasound penetrates approximately 10 cm deep into tissue, SDT can be used to treat deep-seated tumours in a manner similar to PDT[9]. The combination of PDT and SDT, known as sono-photodynamic therapy (SPDT), benefits from both approaches. SPDT can obtain a better therapeutic effect with less sensitizers as well as lower light/ultrasound energy than PDT or SDT alone, thus further reducing side effects.

The sensitizers used in SPDT respond to both light and ultrasound, and they play an important role in the therapeutic effects of SPDT. Ce6 is a second-generation sensitizer with excellent antitumour activity when activated by light and/or ultrasound[10]. However, it is difficult to efficiently transport Ce6 into tumour tissues, thus negatively affecting the antitumour efficacy of SPDT. To address this problem, liposomes were employed in this study. Liposomes are lipid-based drug carriers that have been readily accepted as safe and compatible. Liposomes with an appropriate size can easily accumulate in malignant cells[11] because of the enhanced permeability and retention (EPR) effect. Therefore, we loaded Ce6 into liposomes to improve Ce6 accumulation in tumours.

In the SPDT process, sensitizers deliver energy from lasers or ultrasound to oxygen, and reactive oxygen species (ROS), such as singlet oxygen, hydroxyl radicals and lipid peroxides, are generated to damage tumour cells[12]. Thus, adequate oxygen in the tumour microenvironment is essential for SPDT efficacy[13]. Solid tumours are known to have a complex hypoxic microenvironment. The consumption of O2 by SPDT may further aggravate the oxygen deficit inside tumours, which would seriously interfere with SPDT antitumour efficacy[14,15]. To solve this problem, many strategies have been introduced, such as perfluorocarbon-based delivery methods, manganese oxide (MnO2) nanostructures, photosynthetic bacteria and PDT combined with oxygen therapy[16,17]. However, these oxygen production strategies highly depend on other microenvironmental factors, such as pH, H2O2 and enzyme activity. The resulting oxygen release could vary greatly because of heterogeneity inside tumours. To address this problem, we developed a smart O2 delivery system using oxygen microbubbles and low-intensity ultrasound. As an oxygen carrier, lipid microbubbles can respond to ultrasound and release oxygen as arranged[18]. Therefore, oxygen microbubbles (O2MB) are used in this study to reverse the hypoxia situation inside tumours and then increase tumour sensitivity to SPDT. On one hand, O2MB can directly increase oxygen content in tumors which can improve SPDT efficacy, on the other hand, the relief of tumor hypoxic microenvironment may affect tumor blood vessels[19] and tumor immune microenvironment[20], which is beneficial to long-term tumor suppression. Hence, O2MB is a promising carrier that can deliver oxygen to hypoxic tumour tissues.

In this study, an efficient SPDT strategy was established for TNBC treatment. As illustrated in Figure 1, we developed a microsystem of high singlet oxygen yield Ce6-liposomes combined with lipid microbubbles that carry oxygen (O2MB). Once the microsystem accumulated inside the tumour, low-intensity focused ultrasound was used to boost O2MB. The released oxygen from O2MB reversed hypoxic conditions in tumours, and ultrasound and light were subsequently used to activate Ce6-liposomes, ultimately significantly enhancing the antitumour effect of Lipo-Ce6-SPDT. This combined strategy can obtain superior anti-TNBC effects both in vitro and in vivo.