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. 2026 Sep 1. Online ahead of print. doi: 10.1039/d6sc05519b

Counterion modulation of aza-BODIPY aggregation behaviour for boosted sonodynamic therapy

You Dou a,b,†, Guoyan Fang d,†, Junhua Zhang b,†, Yuting Wang c, Yuntao Li a, Sheng Qiu a,✉, Jun Li e,✉, Yao Sun c,f,✉, Junrong Li b,✉
PMCID: PMC13549310  PMID: 42707700

Abstract

Small-molecule based sonosensitizers hold great promise for deep-seated tumor therapy, yet their structure–activity relationships (SARs) remained largely unexplored, hindering their further biomedical applications. Herein, we propose a counterion-engineering strategy to modulate the aggregation behaviour of aza-BODIPY dyes, thereby achieving high-performance sonodynamic therapy (SDT). We demonstrated that Cl−-paired aza-BODIPY dye (BT-Cl) formed nanoparticles with the smallest hydrodynamic diameter and yielded the highest levels of reactive oxygen species (ROS). BT-Cl NPs also displayed potent sonodynamic cytotoxicity toward both 4T1 and U87-MG cells. Consequently, BT-Cl exhibited excellent tumor growth inhibition with minimal side effects. Overall, this work underscored the critical role of noncovalent ion-pairing interactions in governing small-molecule sonosensitizer aggregation and provided a facile approach for the rational design of high-efficiency sonosensitizers.

By leveraging noncovalent counterion pairing, this strategy deciphers the aggregation-dependent structure–activity relationship of aza-BODIPY sonosensitizers, enabling maximized ultrasound-triggered ROS generation.graphic file with name d6sc05519b-ga.webp

Introduction

Sonodynamic therapy (SDT) has emerged as a non-invasive cancer treatment modality that combines low-intensity ultrasound with sonosensitizers to generate cytotoxic reactive oxygen species (ROS) at targeted sites. This approach offered exceptional deep-tissue penetration while minimizing damage to surrounding healthy tissues.1–9 So far, small-molecule sonosensitizers have garnered significant attention as prime candidates for clinical translation, attributed to their well-defined molecular architecture, facile synthetic modification, favourable biosafety profiles, and tunable therapeutic performance.10 The current landscape of small-molecule sonosensitizers encompasses organic dyes, metal complexes, and natural products, including porphyrins, phthalocyanines, BODIPY dyes, cyanines, phenothiazines and metal–ligand complexes.11–26 Nevertheless, a comprehensive understanding of the structure–activity relationship (SAR) governing sonodynamic performance remains unclear, hindering their further applications.

Recently, a counterion-regulation strategy has been developed to improve the anti-fluorescence and anti-ROS quenching effects of small-molecule dyes.27–35 It was well established that the incorporation of bulky anions such as hexafluorophosphate (PF6−) and tetraphenylborate (PhB−) could significantly enhance the intersystem crossing (ISC) efficiency of photosensitizers while simultaneously suppressing aggregation-caused quenching (ACQ). For instance, pairing NIR-II heptamethine fluorophores with hydrophobic counterions resulted in markedly improved absorption bands and brightness.28 Similarly, a triphenylphosphine cation paired with a Cy5 dye displayed exceptional anti-fluorescence quenching and PDT effects.31 Moreover, counterion modulation has been shown to potentiate the photodynamic immunotherapy mediated by aggregation-induced emission dyes.32 Inspired by these pioneering examples, we hypothesized that extending this strategy to the design of high-performance sonosensitizers could yield significant benefits—an avenue that remained largely unexplored.

In this work, we proposed a counterion engineering strategy to precisely modulate the aggregation behavior of aza-BODIPY dyes, enabling high-performance sonodynamic therapy (Scheme 1). In vitro results revealed that the Cl−-paired aza-BODIPY derivative (BT-Cl) self-assembled into nanoparticles with the smallest hydrodynamic nanosize (∼230 nm) and yielded the highest ROS production upon US irradiation. Theoretical calculations further revealed that this minimized nanosize dimension facilitated the intersystem crossing (ISC) process, thereby substantially boosting the SDT efficacy. At the cellular level, BT-Cl NPs exhibited efficient ROS generation and potent sonodynamic cytotoxicity in both 4T1 and U87 cells. In vivo evaluations in a 4T1 tumor-bearing mouse model confirmed that BT-Cl nanoparticles significantly suppress tumor growth under US irradiation, with minimal side effects. This work establishes counterion engineering as a facile yet highly effective paradigm for the rational design of advanced sonosensitizers, which may be further extended to other ionic dyes and sonosensitizer scaffolds.

Scheme 1. (a) Schematic diagram of the design strategy with different anions. (b) The sonodynamic therapeutic mechanism based on BT-Cl.

Scheme 1

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Results and discussion

Design and synthesis of BTs

Initially, the parent dye was functionalized with tetraphenylethylene (TPE) and julolidine groups to suppress aggregation-caused quenching and enhance NIR-II emission,36–41 while platinum (Pt) units were introduced to facilitate the ISC process and promote reactive oxygen species (ROS) generation under ultrasound irradiation.

Based on this molecular framework, counterion pairing was introduced as a single external regulatory parameter. Through anion-exchange strategies, chloride (Cl−), trifluoromethanesulfonate (CF3SO3−), and perfluorinated tetraphenylborate (PhB−) were incorporated to afford BT-Cl, BT-CF3SO3, and BT-F5-TBP, respectively (Fig. 1a). Because the electronic chromophore remains unchanged, this design enables systematic investigation of how counterion size influences molecular interactions and photophysical behaviour. The resulting compounds were synthesized via an anion-exchange strategy with moderate yields and fully characterized by NMR spectroscopy and high-resolution mass spectrometry (HRMS) (SI, Fig. S1–S4).

Fig. 1. Characterization and sonodynamic performance of BT-based sonosensitizers with different counterions. (a) Molecular structures of BT with corresponding counterions. (b) Molecular volumes of the three counterions (Cl−, CF3SO3−, and F5-TPB−). (c) Normalized absorption spectra of BT-Cl, BT-CF3SO3, and BT-F5-TPB in DMF. (d–f) Fluorescence spectra of DCFH in the presence of BT-Cl (d), BT-CF3SO3 (e), and BT-F5-TPB (f) under ultrasound irradiation. (g) Heatmap summarizing ROS generation efficiency among the three sonosensitizers and DCFH over time. (h) DPBF absorbance changes reflecting 1O2 generation capacity of BT-Cl, BT-CF3SO3, BT-F5-TPB, and DPBF alone under ultrasound. (i) ESR measurements of BT-Cl, BT-CF3SO3, and BT-F5-TPB under ultrasound irradiation using TEMP as a 1O2 indicator. (j) ROS generation ability on BT-Cl at different penetration depths with ultrasound irradiation (1 W cm−2, 1.0 MHz, 50% cycle) for 5 min. (k) Sonostability assessment of BT-Cl and ICG for different times (0–30 min). (l) UV-vis absorption spectra of NADPH in the presence of BT-Cl under ultrasound irradiation.

Fig. 1

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Ultrasound-responsive performance of the BT compounds

To investigate the influence of counterion engineering on the sonodynamic behavior of the BT system, we first estimated the molecular volumes of the corresponding counterions using the Molinspiration molecular property calculator based on its group-contribution method (Fig. 1b). The calculated counterion volumes increased progressively in the order of Cl− (24.24 Å3) < CF3SO3− (80.64 Å3) < F5-TPB− (437.74 Å3). Such differences in size and steric characteristics were expected to influence intermolecular packing and electronic interactions, potentially affecting the photophysical properties and sonodynamic activity of the aza-BODIPY derivatives.29

To determine whether these structural variations were reflected in their optical properties, the photophysical properties of BT-Cl, BT-CF3SO3 and BT-F5-TBP were first examined in dichloromethane (DCM). As shown in Fig. 1c and S5, a gradual red shift in the near-infrared absorption band was observed as the counterion size increased from Cl− to CF3SO3− and F5TPB−, suggesting that variations in counterion size can modulate the electronic interactions within the BT chromophore.

We first examined the effect of counterion modulation on photoinduced ROS generation. Under 808 nm laser irradiation, the ROS-generating capacities followed the order BT-F5-TPB > BT-CF3SO3 > BT-Cl (Fig. S6). We next investigated whether these differences would influence sonodynamic ROS generation. Using 2,7-dichlorofluorescin (DCFH) as a fluorescent probe, BT-Cl exhibited the highest ROS generation efficiency among the three compounds (Fig. 1d–g). Upon ultrasound irradiation, the fluorescence intensity increased rapidly to 11.1 times its initial value within 5 min, whereas BT-CF3SO3 and BT-F5TPB produced only 5.48-fold and 4.61-fold enhancements, respectively. In comparison, the DCFH-only control displayed only a slight increase (1.72-fold). These results demonstrate that the nature of the counterion has a pronounced influence on sonodynamic ROS generation, with the small Cl− counterion being particularly favorable for enhancing SDT performance.

To further corroborate these conclusions, DPBF degradation and electron paramagnetic resonance (EPR) spectroscopy were employed to comprehensively assess ultrasound-triggered ROS generation. As shown in Fig. 1h and S7, the DPBF absorbance of BT-Cl decreased significantly upon US irradiation, with the normalized absorbance (A/A0) dropping to approximately 0.40 after 5 min. In parallel, EPR measurement was performed using TEMP as a spin-trapping agent. The BT-Cl + US group displayed the strongest TEMP signal, whereas progressively weaker signals were observed in the BT-CF3SO3 + US and BT-F5-TPB + US groups (Fig. 1i). Collectively, these results demonstrated that the Cl− counterion remarkably enhanced the sonodynamic ROS generation capability of BT molecules.

Considering its outstanding ROS production efficiency, BT-Cl was selected for further evaluation. We further compared the sonodynamic performance of BT-Cl with that of the commercial Ru(bpy)3Cl2 under identical conditions. As shown in Fig. S8, BT-Cl induced a substantially stronger DCFH fluorescence response, indicating its superior ROS-generating capability under ultrasound irradiation.

To further determine whether other ROS species were generated by BT-Cl under ultrasound irradiation, HPF and DHR123 were employed to detect ˙OH and O2˙−, respectively. Only slight increases in the fluorescence intensities of both probes were observed with the increase of ultrasound irradiation time, indicating the generation of small amounts of ˙OH and O2˙−. Collectively, these results confirmed that 1O2 was the predominant ROS generated by BT-Cl under ultrasound irradiation, whereas ˙OH and O2˙− made minor contributions (Fig. S9).

Ultrasound-triggered ROS generation remained clearly detectable at tissue-mimicking depths ranging from 0 to 9 cm, demonstrating the excellent tissue penetration capability of ultrasound and highlighting the potential of BT-Cl for applications in tissue-mimicking models (Fig. 1j). In addition, BT-Cl also exhibited substantially higher sonostability than indocyanine green (ICG) under continuous US irradiation (1 W cm−2, 1 MHz, 50% cycle) for 30 min, further supporting its potential as a durable sonosensitizer (Fig. 1k and S10).

Beyond ROS production, the well-established redox activity of Pt(ii) ions could endow BT-Cl with sonocatalytic activity toward intracellular reducing species.20 To this end, the sonocatalytic oxidation of NADPH by BT-Cl under ultrasound irradiation was evaluated (Fig. 1l and S11). In the presence of BT-Cl, the characteristic absorption peak of NADPH at approximately 333 nm gradually decreased with prolonged ultrasound irradiation, indicating efficient NADPH oxidation. Moreover, the calculated NADPH oxidation turnover frequency (TOF) of BT-Cl reached 8.4 h−1, demonstrating its effective sonocatalytic redox activity.

Collectively, these findings establish BT-Cl as the optimal sonosensitizer among the three BT complexes and reveal that counterion engineering provides an effective strategy for enhancing sonodynamic performance.

Theoretical calculations for BTs

To gain deeper insight into the origin of this counterion-dependent behavior, we next investigated the influence of different counterions on the aggregation characteristics and molecular packing of the BT system through Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and theoretical calculations.

First, the experiment of DLS demonstrated that the nano-size of BT-Cl (270.6 nm), BT-CF3SO3 (354.6 nm) and BT-F5-TBP (720.8 nm) is positively correlated with the volume of counterions. The TEM experiment (Fig. S12) further verified the distinct aggregate morphologies across different systems, consistent with the DLS measurements. To further elucidate the origin of the particle size discrepancy, we analyzed the intermolecular packing patterns of different systems. As displayed in Fig. 2a, adjacent molecules in BT-Cl exhibit short contact distances (approximately 3.44–3.49 Å), indicating that the small-sized Cl− ions imposed weak steric hindrance and facilitated tighter molecular packing. In contrast, the intermolecular distances gradually increased with the enlargement of anion volume for BT-CF3SO3 and BT-F5-TPB.

Fig. 2. Theoretical calculations and intermolecular packing analysis of BT-Cl, BT-CF3SO3, and BT-F5-TBP. (a) Calculated intermolecular distances for BT-Cl, BT-CF3SO3, and BT-F5-TBP. (b) Frontier molecular orbital distributions (HOMO and LUMO) of BT-Cl, BT-CF3SO3, and BT-F5-TBP obtained at the PBE0/def2-TZVP level based on the optimized geometries. The excitation energies of the triplet excited states were calculated using time-dependent density functional theory (TD-DFT) at the same level of theory. The corresponding energy gaps (ΔEgap and ΔEST) between singlet and triplet states are also presented in the figure.

Fig. 2

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Notably, BT-F5-TPB presented an intermolecular spacing of roughly 5.35 Å, demonstrating that the bulky F5-TPB− anions effectively hinder the proximity of adjacent chromophores and yield a looser overall packing structure. Further analysis of the electronic structures of these systems (Fig. 2b) revealed that the three compounds exhibited distinctly different excited-state energy level distributions. Among them, BT-Cl possesses the smallest singlet-triplet energy gap (ΔEST = 0.29 eV), while the ΔEST values of BT-CF3SO3 and BT-F5-TPB increase to 1.27 eV and 1.46 eV, respectively. These results suggest that the close π–π contacts and compact molecular packing of BT-Cl enhance intermolecular electronic coupling, thereby altering its excited-state energy-level distribution and resulting in a smaller ΔEST. Collectively, these findings indicate that the cationic BT scaffold acts as the direct ROS-generating unit, whereas the counterion modulates its sonodynamic activity by regulating molecular packing and excited-state energetics rather than generating ROS independently.

Cell uptake and SDT in vitro

Encouraged by the promising SDT performance of BT-Cl in solution, we conducted further experiments at the cellular level to explore its therapeutic efficacy. We encapsulated a free solution of BT-Cl into DSPE PEG5000 and then formed amphiphilic BT-Cl nanoparticles (BT-Cl NPs) with good biocompatibility and tumor-targeted potential. After preparation, we employed transmission electron microscopy (TEM) and dynamic light scattering (DLS) to characterize the morphology of BT-Cl NPs (Fig. S13). TEM images revealed well-defined spherical nanoparticles with an average diameter of approximately 230 nm. Consistently, DLS measurements showed a single peak corresponding to a hydration diameter of around 260 nm. Importantly, nanoparticle formulation produced little change in the ultrasound-induced DCFH fluorescence response of BT-Cl, demonstrating that its ROS-generating activity was well preserved after incorporation into DSPE-PEG5000 (Fig. S14).

Taking advantage of the intrinsic NIR-II fluorescence of BT-Cl, we next evaluated the cellular uptake behavior of the nanoparticles. After incubation with BT-Cl NPs, the fluorescence signal in 4T1 and U87 cells increased in a time-dependent manner and reached a maximum at 10 h in 4T1 cells and 9 h in U87 cells, indicating that BT-Cl NPs were taken up by the cells effectively (Fig. S16 and S17). To further elucidate the uptake pathway, 4T1 and U87 cells were incubated under different conditions, including low temperature and the presence of specific metabolic and endocytic inhibitors (Fig. S18 and S19). In both cell lines, the uptake of BT-Cl NPs was substantially suppressed at 4 °C and in the presence of 2-deoxy-d-glucose, suggesting an energy-dependent internalization process.

Moreover, methyl-β-cyclodextrin (M-βCD), an inhibitor of caveolae-mediated endocytosis, markedly reduced intracellular fluorescence, indicating that BT-Cl NPs are primarily internalized via a caveolae-dependent pathway.42–46

Given the pivotal role of reactive oxygen species (ROS) in therapeutic efficacy, intracellular ROS generation by BT-Cl NPs upon ultrasound or 808 nm laser irradiation was evaluated using DCFH-DA as a fluorescent probe. As shown in Fig. 3a, 4T1 cells treated with BT-Cl NPs under ultrasound irradiation exhibited markedly enhanced green fluorescence compared with the control, control + US and BT-Cl NPs groups, indicating a significant increase in intracellular ROS generation. A similar increase in fluorescence was observed in U87 cells treated with BT-Cl NPs under ultrasound irradiation (Fig. S20). Furthermore, pronounced ROS generation was detected in 4T1 cells following treatment with BT-Cl NPs and 808 nm laser irradiation (Fig. S21). Flow cytometric quantitative analysis of 4T1 cells further verified this trend, showing considerably higher ROS levels in the BT-Cl NPs + US group than in the other groups (Fig. 3b and c). Collectively, BT-Cl NPs efficiently generate intracellular ROS upon ultrasound irradiation in both 4T1 and U87 cells and also retain effective photoinduced ROS-generation capability in 4T1 cells. As shown in Fig. 3d, BT-Cl NPs exhibited negligible cytotoxicity toward 4T1 cells in the absence of ultrasound (US) irradiation, with cell viability remaining above 86% even at 50 µM, whereas US exposure markedly enhanced their cytotoxicity.

Fig. 3. In vitro cytotoxicity of BT-Cl NPs combined with ultrasound. (a) Fluorescence images of 4T1 cells stained with DCFH-DA after different treatments. Scale bar: 50 µm. (b and c) Flow cytometry histograms and corresponding MFI quantification of intracellular ROS levels in different groups. (d) Viability of 4T1 cells treated with different concentrations of BT-Cl NPs in the presence or absence of ultrasound (US). (e and f) Fluorescence images of 4T1 cells stained with Calcein-AM/PI and JC-1 following various treatments with or without US irradiation (1 W cm−2). Scale bar, 300 µm. (g and h) Flow cytometry analysis of Annexin V-FITC/PI double-stained cells within 4T1 cells after various treatments and corresponding statistical quantification. ****p < 0.0001.

Fig. 3

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A similar concentration-dependent sonodynamic response was observed in U87 cells, whose viability decreased to approximately 18% following treatment with 100 µM BT-Cl NPs and US irradiation (Fig. S22). These results demonstrate the effective sonodynamic cytotoxicity of BT-Cl NPs in both cell lines.

To further verify the therapeutic outcome, Calcein-AM/PI dual fluorescence staining was performed in 4T1 and U87 cells. Viable cells stained with Calcein-AM emit green fluorescence, whereas dead cells labelled with propidium iodide (PI) display red fluorescence. As shown in Fig. 3e, the control and US-only groups exhibited predominantly green fluorescence with a negligible red signal, revealing minimal cytotoxicity induced by ultrasound alone. Similarly, cells treated with BT-Cl NPs alone exhibited a fluorescence distribution comparable to that of the control groups, further validating their good biocompatibility and low dark toxicity. In contrast, the BT-Cl NPs + US group displayed drastically attenuated green fluorescence accompanied by a substantial increase in red fluorescence. The merged image was predominantly covered by red signals, confirming massive tumor cell death triggered by the combined treatment. A comparable staining pattern was observed in U87 cells, with pronounced red fluorescence appearing only after treatment with BT-Cl NPs and US irradiation (Fig. S23). These results further demonstrate the effective sonodynamic cytotoxicity of BT-Cl NPs in both 4T1 and U87 cells.

Considering that DNA is a putative target of Pt anticancer drugs, the interaction of BT-Cl with double-stranded ctDNA was examined by UV-vis absorption titration (Fig. S15). Analysis of the absorption titration data yielded an apparent binding constant (Kb = 4.97 × 102 M−1), confirming a measurable interaction between BT-Cl and ctDNA. The cell cycle progression was analyzed.47–50 We treated 4T1 cells with BT Cl NPs under US irradiation and counted the cells in different phases of the cell cycle using flow cytometry. The percentage of cells in the G2/M phase increased to 5.0%, indicating that BT Cl NPs combined with US irradiation inhibited the proliferation of cancer cells by blocking the cell cycle in the G2/M phase (Fig. S24). Given that mitochondria are highly susceptible to ROS-induced oxidative stress, mitochondrial membrane potential (ΔΨm) was subsequently evaluated in both 4T1 and U87 cells using JC-1 staining (Fig. 3f). In 4T1 cells, treatment with BT-Cl NPs under US irradiation caused a remarkable shift of JC-1 fluorescence from red (aggregated form, high membrane potential) to green (monomeric form, low membrane potential). A similar red-to-green fluorescence transition was observed in U87 cells following the combined treatment (Fig. S25), indicating mitochondrial dysfunction in both cell lines. Given the close correlation between mitochondrial dysfunction and cell apoptosis, Annexin V-FITC/PI double-staining flow cytometry was further applied to quantitatively assess the apoptosis of 4T1 and U87 cells. As shown in Fig. 3g and h, the apoptosis rate of 4T1 cells markedly increased from 1.94% in the control group to 45.4% after BT-Cl NPs + US treatment, demonstrating that ultrasound-activated BT-Cl NPs effectively induced tumor cell apoptosis. Consistently, BT-Cl NPs combined with US irradiation markedly increased apoptosis in U87 cells compared with the corresponding control groups (Fig. S26). Collectively, these results demonstrate that ultrasound-activated BT-Cl NPs induce mitochondrial depolarization and apoptosis in both 4T1 and U87 cells. To further verify whether this apoptotic process was mediated through caspase activation, 4T1 cells were pretreated with Z-VAD-FMK, a pan-caspase inhibitor. Notably, Z-VAD-FMK markedly rescued cell viability compared with the BT-Cl NPs + US group, indicating that the sonodynamically induced cell death was predominantly mediated by a caspase-dependent apoptotic pathway (Fig. S27). These results suggest that US-activated BT-Cl NPs can effectively trigger tumor cell apoptosis via a caspase-dependent apoptotic pathway.

Overall, these extensive results demonstrated that ultrasound-activated BT-Cl NPs induce ROS-mediated mitochondrial dysfunction, which is accompanied by caspase-dependent apoptotic cell death.

To delve deeper into the therapeutic mechanism of BT-Cl NP-mediated sonodynamic therapy, we performed transcriptome sequencing on 4T1 cells treated with PBS and the BT-Cl NPs + US. The heatmap revealed remarkable alterations in the gene expression profile of cells after BT-Cl NPs + US treatment, and distinct clustering separation was observed between the two groups. Clearly distributed upregulated genes (red) and downregulated genes (blue) indicated that BT-Cl NPs + US treatment triggered extensive transcriptional reprogramming (Fig. 4a). Venn diagram analysis further identified the differentially expressed genes between the two groups (Fig. 4b). A total of 10579 genes were co-expressed in both groups. In addition, 911 genes were uniquely expressed in the BT-Cl NPs + US group, while 1104 genes were specifically expressed in the PBS group. The volcano plot identified 5563 differentially expressed genes (DEGs), including 2202 significantly upregulated genes and 3361 significantly downregulated genes (Fig. 4c). The number of downregulated genes was notably higher than that of upregulated ones, suggesting that BT-Cl NPs + US treatment broadly suppressed cellular transcriptional activity, accompanied by the activation of cell death-related genes. These transcriptomic results corroborate the biological effect of inducing tumor cell apoptosis.

Fig. 4. Transcriptomic characterization of cellular responses to BT-Cl NPs + US treatment. (a) Heatmap of differentially expressed genes (DEGs) in the BT-Cl NPs + US and PBS groups. (b) Venn diagram showing the overlap of expressed genes between the two groups. (c) Volcano plot of DEGs between the two groups. Red and blue dots represent significantly upregulated and downregulated genes, respectively (|log2 FC| ≥ 1; padj ≤ 0.05). (d) GO enrichment analysis of DEGs. (e) KEGG pathway enrichment analysis of DEGs.

Fig. 4

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We next conducted Gene Ontology (GO) enrichment analysis to explore the biological functions of DEGs. These genes were mainly enriched in biological processes such as transcriptional regulation, metabolic process modulation and peptidase activity regulation (Fig. 4d). Notably, GO terms associated with cysteine-type peptidase (caspase) activity were significantly enriched. As the caspase family serves as key executors of apoptosis, this transcriptomic evidence further supports that BT-Cl NPs + US treatment activated the canonical apoptotic pathway, which was highly consistent with the foregoing cell apoptosis assays. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was carried out for DEGs. The results showed that the DEGs were primarily enriched in signaling pathways closely associated with cell proliferation, survival, and apoptosis, including the PI3K-AKT, MAPK, FoxO and Notch pathways, as well as focal adhesion (Fig. 4e). The PI3K-AKT and MAPK pathways are critical for regulating cell growth and survival, whereas the FoxO pathway participates in oxidative stress response and apoptosis modulation. The co-enrichment of these pathways indicated that BT-Cl NPs + US treatment disturbs pro-survival signals and amplifies apoptosis-related signal transduction, thereby facilitating programmed cell death in tumor cells.51 Collectively, transcriptomic analysis demonstrated that BT-Cl NPs + US treatment causes widespread gene expression reprogramming at the transcriptomic level and achieves excellent anti-tumor effects via inducing oxidative stress and activating caspase-associated apoptotic pathways.

SDT performance in vivo

Before evaluating the SDT effect in vivo, we first performed hemolysis assays to evaluate the in vivo biocompatibility of BT-Cl NPs (Fig. S28). No obvious hemolysis was triggered across the tested concentration range of 6.25–100 µM, demonstrating the favorable blood compatibility of BT-Cl NPs. Next, the in vivo antitumor effect of BT-Cl NPs under US irradiation was evaluated in 4T1 tumor-bearing mouse models. Briefly, 4T1 cells were implanted in mice, followed by a seven-day tumor-growth period. After that, BT-Cl NPs were administered via tail vein intravenous injection, followed by US irradiation once every two days (Fig. 5a and S29). The therapeutic efficacy was assessed after 14 days of treatment. To investigate in vivo biodistribution and tumor targeting behavior, NIR-II fluorescence imaging was conducted. A strong fluorescence signal was observed at the tumor site, indicating efficient accumulation of BT-Cl NPs, which can be driven by the enhanced permeability and retention (EPR) effect (Fig. 5b and S30). Ex vivo imaging also revealed appreciable fluorescence in the lung, liver, and spleen. The lung signal may reflect temporary entrapment of the nanoparticles during their passage through the dense pulmonary microvasculature, whereas their distribution in the liver and spleen is likely attributable to uptake by the mononuclear phagocyte system and subsequent elimination.52 The fluorescence intensity reached the maximum at 10 h post-injection and remained at the peak level without obvious decay up to 36 hours, indicating prolonged tumor retention and enabling NIR-II imaging-guided SDT (Fig. 5b and c). During the treatment period, BT-Cl NPs exhibited negligible systemic toxicity, as no body weight loss was observed in either the presence or absence of US irradiation. In contrast, the BT-Cl + US group exhibited significant tumor growth inhibition, with markedly reduced tumor volumes and weight compared with the PBS, PBS + US, and BT-Cl NPs groups without US, indicating a strong in vivo antitumor efficacy (Fig. 5d–f and S31).

Fig. 5. Antitumor performance induced by BT-CI NPs under ultrasound irradiation on 4T1 tumors. (a) Schematic therapeutic regimen of BT-Cl NPs in 4T1 tumor-bearing mice. (b and c) NIR-II fluorescence tracking and corresponding signal intensity analysis of BT-CI NPs in 4T1 tumor-bearing mice (n = 3). (d–f) Body weight variations, tumor growth curves, and photographs of excised tumors from different groups (*P < 0.05, one-way ANOVA). (g) H&E and TUNEL-stained tumor slices 14 days after the initial intravenous injection.

Fig. 5

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At the end of treatment, we performed Hematoxylin and Eosin (H&E) staining and TUNEL staining on tumor tissues from each group (Fig. 5g). Compared with the control group, the BT-Cl NPs + US group exhibited severe destruction of tumor tissue architecture, along with extensive apoptotic and necrotic regions. In agreement with this observation, TUNEL staining revealed the strongest apoptotic signals in this group. These results are consistent with in vitro ROS detection and cell apoptosis assays, confirming that BT-Cl NPs can effectively trigger tumor cell death under ultrasound irradiation. Importantly, H&E staining of major organs (heart, liver, spleen, lung and kidney) showed no obvious tissue damage or pathological abnormalities (Fig. S32), confirming the excellent biosafety of BT-Cl NPs in vivo. Overall, these in vivo results validate the potent antitumor efficacy of BT-Cl NPs under ultrasound irradiation and further confirm that sonodynamic therapy effectively induces tumor suppression through ROS-associated cellular damage leading to apoptosis, while maintaining favorable systemic safety.

Conclusion

In summary, we developed a series of aza-BODIPY-based sonosensitizers bearing counterions with different ionic volumes to regulate their sonodynamic performance. A clear dependence of ROS generation on counterion size was observed, where the Cl− paired derivative (BT-Cl) exhibited the highest ROS production under ultrasound irradiation. To elucidate this trend, combined DLS, TEM, and theoretical studies suggested that counterion size may influence intermolecular packing and electronic interactions, which are likely to affect ISC efficiency and consequently ROS generation. Based on these findings, BT-Cl was selected for further biological evaluation after formulation into nanoparticles. Under ultrasound irradiation, BT-Cl NPs induced ROS generation, mitochondrial depolarization, and apoptosis in both 4T1 and U87 cells, demonstrating potent sonodynamic activity. Finally, in vivo studies using a 4T1 tumor model confirmed that BT-Cl NPs significantly inhibited tumor growth under ultrasound irradiation while retaining favorable biosafety. Overall, this work provides a simple yet effective counterion engineering strategy to regulate the aggregation behaviour and enhance sonodynamic therapeutic performance, offering new insights into the function of noncovalent ion pairing in the rational design of sonosensitizers.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of “Central China Normal University” and approved by the Animal Ethics Committee of “Central China Normal University (Approval No. CCNU-IACUC-2023001)”.

Author contributions

Y. S., J. L., J. L. and S. Q. designed the research. Y. D., Y. W., J. Z., X. G., and Y. L. synthesized and characterized the compounds, performed the measurements, and analysed the data. All authors wrote and revised the manuscript.

Conflicts of interest

The authors declare no competing interests.

Supplementary Material

SC-OLF-D6SC05519B-s001

Acknowledgments

We acknowledge the support from the National Natural Science Foundation of China (U25A20592, 22474047 and 22022404), the China Postdoctoral Science Foundation (no. 2024M761096), the National Science Foundation of Hubei Province (2026AFB696), the Hubei Province Postdoctoral Innovation Talent Training Program (2025HBBSHCXB072), the Fundamental Research Funds for the Central Universities (2662025SYPY005), and the State Key Laboratory of Green Pesticide (qiankejiao[2023]114).

Data availability

All the data supporting this article have been included in the main text and the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6sc05519b.

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Supplementary Materials

SC-OLF-D6SC05519B-s001
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Data Availability Statement

All the data supporting this article have been included in the main text and the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6sc05519b.

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