IR780 is a lipophilic heptamethine cyanine agent with lipophilic properties that has been clinically employed in FL/PA imaging-guided procedures (Li et al., 2018). IR780 exhibits an inherent ability to target tumor cells and mitochondria, allowing for deep tumor penetration without the need for additional mitochondria-targeting moieties due to its lipophilic cationic property (Wang et al., 2014). Additionally, IR780 demonstrates significant SDT properties (Chen et al., 2017); however, it is characterized by limited water solubility and poor photostability. Despite these limitations, the combination of IR780 with FL/PA imaging approaches holds promise for imaging-guided SDT in deep tumors (Cheng et al., 2015).

In 2019, Zhang et al. (2019) developed IR780-loaded nanodroplets (IR780-NDs) with a well-designed core-shell structure (core: perfluoropentane (PFP); shell: IR780), enabling a US-mediated multifunctional IR780-NDs SDT system with deep penetration, mitochondrial targeting, and simultaneous FL/PA/US imaging abilities (Figure 2). Under US irradiation (3 min, 650 kHz, 2.4 W/cm²), IR780-NDs penetrated from the tumor surface to its core through the acoustic droplet vaporization (ADV) effect of PFP (Mura et al., 2013; Ho et al., 2017), demonstrating excellent depth of penetration. In comparison to NDs (without IR780), IR780-loaded NDs selectively accumulated in and were activated by mitochondria (0.87 vs. −0.15 Pearson coefficient value, respectively). This targeted accumulation can induce oxidative stress through US-mediated ROS production and resulted in apoptosis of 4T1 cells (<40% vs. >90% cell viability, respectively, at 121 μg/mL). Moreover, the efficacy of IR-780 NDs was evaluated in 4T1 tumor mouse models, demonstrating enhanced SDT, including FL/US/PA imaging abilities, likely attributed to the mitochondria-targeted SDT effect in cancer cells and amplified by the unique ADV effect of PFP.

Immunogenic cell death (ICD) is a form of cell death that stimulates the release of tumor-specific antigens and danger-associated molecular patterns (DAMPs), triggering a systemic antigen-mediated antitumor immune response (Krysko et al., 2012). Recent studies have suggested that ICD has the potential to enhance the efficacy of SDT (Yue et al., 2019; Li X. et al., 2022). In 2021, Ren et al. (2021) employed mitochondrial-targeting liposomal nanoparticles (MLipRIR NPs) to combine FL/PA multimodal imaging and SDT, reinforced by ICD (Figure 3). The NPs were developed by encapsulating R162 (glutamate dehydrogenase 1 inhibitor) and IR780 within a mitochondria-targetable lipid bilayer, efficiently entering the 4T1 cell and specifically targeting the mitochondria through a TPP moiety modification on the NPs surface. Under US irradiation, IR780 released from MLipRIR NPs generated high levels of ROS, impeding mitochondrial respiration and leading to local glutathione (GSH) consumption. In addition, the co-released R162 inhibited glutamate dehydrogenase 1 (GDH1) by interfering with the anaplerotic use of glutamine in the mitochondrial TCA cycle (Jin et al., 2015). These effects induced ferroptosis of cancer cells through lipid peroxide accumulation, effectively activating ICD. MLipRIR NPs also demonstrated superior US-mediated cytotoxicity in 4T1 cells (16.4% cell viability; R162 concentration = 1 μg/mL) relative to LipRIR NPs without TPP moiety modification (27.6% cell viability; R162 concentration = 1 μg/mL) under US irradiation (1 min, 1.0 MHz, 1.5 W/cm², 50% duty cycle). By leveraging the bimodal imaging capabilities of MLipRIR NPs, in vivo FL/PA multimodal imaging was successfully conducted in the 4T1 mouse models. Furthermore, studies in 4T1 mouse models showed that MLipRIR NPs efficiently reduced tumor size and mitigated severe side effects of cancer treatment on other tissues.

Ji et al. (2021) developed mitochondria-targeted formulations (PIH-NO) by encapsulating IR780 in a human serum albumin-based NO donor (HSA-NO) and an oxygen-carrier perfluorodecalin (FDC), a type of perfluorocarbon (PFC) (Figure 4). PIH-NO accumulated in the 4T1 cell mitochondria (PC = 0.76), leading to mitochondrial dysfunction, relief of hypoxia, and amplified ICD through ROS production, enhanced by the explosive release of NO and O₂ upon US irradiation. Notably, significant cytotoxicity was observed in the PIH-NO (30% cell viability; equivalent IR780 concentration = 4 μg/mL) compared to PIH (without NO donor) (50% cell viability; equivalent IR780 concentration = 4 μg/mL) and IH (without NO donor and oxygen-carrier) (70% cell viability; equivalent IR780 concentration = 4 μg/mL) under US irradiation conditions (1 min, 1.0 MHz, 1.5 W/cm², 50% duty cycle). Furthermore, observations in 4T1 mouse models indicated that PIH-NO effectively reduced tumor size and significantly decreased the number of pulmonary metastatic nodules. Hence, this study demonstrated the potential of gas transportation to amplify mitochondrial SDT, enhancing its therapeutic efficacy, inducing anticancer immune responses and preventing metastasis.

Shi et al. (2023) developed a novel approach for mitochondrial SDT using adipocyte-derived organelle LDs as carrier agents with an inherent affinity for mitochondria (Figure 5). The developed LDs (CPI-613/IR780@LDs), contained IR780 and CPI-613, a FDA-approved TCA cycle inhibitor. By synergistically inhibiting the mitochondrial TCA cycle and enhancing ROS production at the tumor site, the combination of mitochondrial SDT and CPI-613 enabled efficient mitochondrial SDT in Panc02 tumors. Additionally, combining mitochondrial SDT and anti-PD-1-mediated immunotherapy reinforced the immune response in Panc02 tumors. CPI-613/IR780@LDs demonstrated significantly increased cell cytotoxicity, overcoming the limited efficacy of SDT induced by the hypoxia in the tumor microenvironment (TME) after US irradiation (1 min, 1 MHz, 0.5 W/cm², 50% duty cycle) in both normoxic and hypoxic Panc02 cells (<40% cell viability of both normoxic and hypoxic Panc02 cells; CPI-613 concentration: 154.24 μmol/L, and IR780 concentration: 20 μmol/L). This approach successfully attenuated the progression of primary and metastatic tumors in Panc02-bearing mouse models.

The 5-aminolevulinic acid (ALA) is a powerful SDT agent that is converted to PpIX and delivers it to the heme synthetic root of cancer mitochondria. Previous studies have explored the use of ALA-based SDT for various cancers (Li et al., 2015; Wu et al., 2019).

Cancer cells rely on two main pathways for the energy production: mitochondrial aerobic oxidation and cytoplasmic anaerobic glycolysis (Fu et al., 2019a; Fu et al., 2019b). Mitochondria-targeted SDT inhibits the mitochondrial aerobic oxidation process by generating an excessive amount of ROS, thus impacting tumor cell growth. However, this approach alone may have limitations because tumor cells can still obtain energy through anaerobic glycolysis (Wang et al., 2017). To address this, Xie et al. (2017) demonstrated the synergistic effect of combining ALA, a precursor of PpIX, the glycolysis inhibitor 2-deoxyglucose (2-DG), and microbubbles (MBs) . Upon cell internalization, PpIX induced by ALA selectively accumulated in the mitochondria of the HepG2 cells. Furthermore, US irradiation (5 min, 3 MHz, 2 W/cm², 60% duty rate) resulted in a potentiated therapeutic outcome with the combination system compared to both the ALA combined with 2-DG and MB (33.2%; 2-DG concentration = 2 mmol/L) and the ALA combined with 2-DG alone (49.2%: 2-DG concentration = 2 mmol/L). This study demonstrated that SDT enhanced with the glycolytic inhibitor 2-DG induces cell death in HepG2 cells, and MB enhances the outcome of SDT. However, further research is needed to investigate SDT application in vivo.

HMME, a derivative of PpIX, is commonly used as a photodynamic agent. However, it can also be activated by US irradiation to effectively generate ROS for SDT (Tian et al., 2009). Due to its hydrophobic nature, HMME is typically loaded into liposomes.

Chen M. et al. (2017) developed a mitochondria-targeting liposomal formulation of HMME modified with TPP (HMME-Lipo-TPP) and investigated its efficacy for SDT (Figure 6). The formulation specifically targets mitochondria in MCF-7 cells and rapidly releases HMME upon US irradiation (3 min, 1 MHz, 0.5 W/cm²), leading to the production of ROS and induction of cell apoptosis. The cytotoxicity of HMME-Lipo-TPP in MCF-7 cells was superior to that of non-targeted controls (HMME-Lipo) under similar experimental conditions (∼32% vs. ∼44% cell viability, respectively, at 20 μg/mL). Although the in vitro characteristic of HMME-Lipo-TPP outperformed those of HMME-Lipo, further studies in vivo are required to validate its efficacy.

Ce6 is a second-generation SDT agent that has been widely used to overcome the limitations of conventional agents, such as limited tumor targeting ability and slow clearance from normal tissues (Klyta et al., 2011). However, Ce6 still has drawbacks, including inefficient intracellular accumulation and low tumor specificity, limiting its SDT efficacy.

Cao et al. (2023) used a mitochondria-targeted approach using Ce6-loaded extracellular vesicles (EVs) (EV (TPP-Ce6)) to create a biocompatible and efficient mitochondria-targeting SDT (Figure 7). EVs have demonstrated clinical applicability as effective carriers for delivering SDT agents to tumor cells (Kalluri et al., 2020; Moller, 2020). In this study, Ce6 was conjugated with a TPP ligand and encapsulated within EVs. This formulation significantly increased the mitochondrial uptake of Ce6 (0.58 for EV (TPP-Ce6) vs. 0.31 for Ce6 Mander’s overlap coefficient). Under US irradiation (1 min, 1 MHz, 0.3 W/cm²), EV (TPP-Ce6) exhibited higher sono-cytotoxicity in MCF-7 cells than Ce6 alone (∼30% for EV (TPP-Ce6) vs. ∼70% for TPP-Ce6 cell viability; TPP-Ce6 (or Ce6) concentration = 10 μM, EVs concentration = 4.96 × 10¹⁰ EVs/mL). Furthermore, the co-encapsulation of prooxidant piperlongumine (PL) into EV (TPP-Ce6) demonstrated the most effective tumor inhibition through the synergistic effect of chemo-SDT under similar experimental conditions (∼6.5% cell viability; TPP-Ce6 (or Ce6) concentration = 10 μM, PL concentration = 3 μg/mL, EVs concentration = 4.96 × 10¹⁰ EVs/mL). Animal studies in MCF-7 cell mouse models demonstrated that this system significantly reduced tumor growth and, importantly, minimized side effects on other tissues.

Zhang et al. (2020) reported a synergistic approach combining FL/PA bimodal imaging, mitochondria-targeted SDT, and starvation therapy using a combination of IR780 and HMME for FL/PA imaging and SDT and glucose oxidase (GOx) for starvation therapy (Figure 8). The system employed core/shell structured polylactic-co-glycolic acid (PLGA) nano-formulations (IHG@P), which consisted of GOx in the core and IR780 and HMME in the shell. This nano-formulation efficiently penetrated into 4T1 tumor cells, targeting the mitochondria, due to the inherent affinity of IR780 (PC = 0.806). As an adjuvant therapy, the GOx-mediated starvation therapy effectively suppressed cytosolic anaerobic glycolysis, limiting the energy supply of cancer cells and inducing tumor apoptosis. Under US irradiation (30 s, 2 W/cm²), the IHG@P group (8.48% Cell viability; PLGA concentration = 60 μg/mL) demonstrated the most significant 4TI cancer cell apoptosis compared to the groups receiving starvation therapy alone (∼68% cell viability; PLGA concentration = 60 μg/mL) or SDT therapy alone (∼58% cell viability; PLGA concentration = 60 μg/mL) groups. Additionally, IHG@P was used for in vivo FL/PA multifunctional imaging of 4T1 cell mouse models, showing similar synergistic effects of SDT and starvation therapy.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited treatment options and a high metastasis rate, posing considerable challenges for effective therapy (O’Reilly et al., 2015). To address this issue, Han et al. (2021) developed a novel SDT complex nano-micelle (PEG-IR780@Ce6) for the efficient treatment of TNBC. This nano-micelle combines two sono-sensitizing agents, PEGylated IR780 (PEG-IR780) and Ce6 (Figure 9) and exhibited enhanced uptake by MDA-MB-231 TNBC cells compared to PEG-IR780 or free Ce6 alone. The nano-micelle was efficiently delivered and accumulated in the mitochondria of MDA-MB-231 cells, resulting in ROS generation at the tumor site upon US irradiation. In vitro SDT experiments demonstrated the superior anticancer effects of PEG-IR780@Ce6 compared to PEG-IR780 or free Ce6 alone, highlighting its potential for sonodynamic TNBC therapy (3 min, 1 MHz, 0.6 W/cm², 50% duty rate) (35% for PEG-IR780@Ce6 vs. 85% for free Ce6 and 43% for PEG-IR780 cell viability, at 40 μg/mL). Furthermore, in vivo SDT results demonstrated that PEG-IR780@Ce6 effectively eradicated TNBC tumors, along with significant suppression of the invasion and migration, while exhibiting minimal side effects.

To achieve effective sonodynamic immunotherapy (SDIT), it is crucial to enhance the level of ICD induction of current SDT agent-based inducers (Zhang et al., 2018). Therefore, the development of a new SDT agent with the potential to promote high ROS production and with mitochondrial targeting ability is timely and necessary.

Li et al. (2022) introduced a novel SDT agent called TPP-conjugated gadolinium (III) porphyrinate (GdPorP), which offers several advantages including high-efficiency ROS production, active mitochondrial targeting, and good MR contrast (Figure 10). To enhance tumor-targeting affinity and biocompatibility, GdPorP was coated with MAL-PEG-b-PDPA, forming pH-responsive nanomicelles (RPGdP). Upon delivery to H22 cancer cells, GdPorP accumulated in the cancer cell mitochondria (PC = 0.93), leading to increased cell death and amplifying ICD. Significant cell cytotoxicity was observed using RPGdP compared to the TPP-free RPGd upon the US irradiation (1 min, 1 MHz, 1.5 W/cm²). To further enhance the in vivo SDIT efficacy of this system, the authors incorporated an indolamine 2,3-dioxygenase (IDO) inhibitor within the RPGdP, resulting in a nanopharmaceutical named RPGdPI. In H22 orthotopic liver tumor mouse models, RPGdPI effectively suppressed the growth of deep-seated primary tumors and induced significant antitumor effects by reversing the immunosuppressive tumor microenvironment.