NIR-II aggregation-induced emission sonosensitizer for pyroptosis induction in bladder cancer

Abstract

Sonodynamic therapy (SDT) represents a promising methodology that employs sonosensitizers in conjunction with low-intensity ultrasound for the eradication of malignant tumors, featuring precise treatment capabilities, deep tissue penetrability, and minimal side effects. Conventional sonosensitizers often face challenges such as aggregation-caused quenching (ACQ), which hampers the efficiency of reactive oxygen species (ROS) generation. In this study, we report a novel benzothiadiazole-based sonosensitizer derivative, BBTPA, exhibiting aggregation-induced emission (AIE) characteristics. By co-assembling BBTPA with the ROS-responsive polymer PMD and the amphiphilic polymer DSPE-PEG₂₀₀₀, we engineered BBTPA nanoparticles (NP^BBTPA). Upon ultrasound exposure, NP^BBTPA produces ROS efficiently, inducing mitochondrial damage and triggering pyroptotic cell death. Moreover, NP^BBTPA induces immunogenic cell death (ICD) under ultrasound stimulation, thus enhancing antitumor immune responses. This study extends the utility of AIE-based sonodynamic agents in efficient cancer therapy, holding promising prospects for bladder cancer treatment.

Graphical abstract

Highlights

• •A benzothiadiazole-based sonosensitizer with A-π-D-π-A conjugation shows AIE feature and strong NIR-II fluorescence.
• •NP^BBTPA nanoparticles enable efficient ROS generation under ultrasound and ROS-responsive dissociation.
• •Ultrasound-activated NP^BBTPA induces mitochondrial damage, triggers pyroptosis and immunogenic cell death (ICD).
• •In vivo studies reveal 70.5% tumor growth inhibition with 1.9-fold increased CD8⁺ T cell infiltration.
• •The integrated theranostic platform shows great potential for bladder cancer therapy.

1. Introduction

Bladder cancer, marked by high recurrence and mortality rates, continues to pose a significant global health challenge [1]. Although conventional treatment modalities like surgery, chemotherapy, and radiotherapy can be efficacious, they frequently entail severe side effects and exhibit limited effectiveness in advanced disease stages [2]. This highlights the critical necessity for novel therapeutic strategies that integrate high specificity and minimal invasiveness. Sonodynamic therapy (SDT) has emerged as a promising alternative owing to its unique advantages. SDT employs ultrasound, a non-invasive and deeply penetrating physical stimulus, to activate sonosensitizers and generate highly reactive oxygen species (ROS), thereby inducing tumor cell death [3]. Notably, SDT has been demonstrated to induce cell pyroptosis, a form of programmed cell death regulated by the gasdermin protein family. This mechanism triggers a robust inflammatory and immune response, which can synergistically augment the effectiveness of immunotherapy.

The therapeutic performance of SDT is critically dependent onthe properties of sonosensitizers, which must combine efficient ROS with excellent biocompatibility. Conventional sonosensitizers often encounter challenges associated with aggregation-induced quenching (ACQ), which suppresses ROS generation and limits therapeutic efficacy. To overcome this limitation, increasing attention has turned to aggregation-induced emission (AIE) materials [4]. Unlike ACQ materials, AIE systems exhibit minimal fluorescence in solution but become highly emissive upon aggregation, making them particularly advantageous for SDT applications [5]. Additionally, AIE materials typically exhibit robust photostability and biocompatibility, further augmenting their therapeutic potential. Their unique photophysical properties, combined with exceptional photostability and biocompatibility, not only enable efficient ROS generation but also support near-infrared (NIR) fluorescence imaging. This dual functionality enables real-time visualization of therapeutic responses, thus integrating diagnosis and treatment into a single platform—a capability that is especially valuable for precision oncology [6].

A significant advancement in AIE materials lies in their capability to emit within the second near-infrared window (NIR-II, 1000-1700 nm), offering distinct advantages over traditional imaging modalities [7]. NIR-II imaging offers deeper tissue penetration, superior spatial resolution, and diminished autofluorescence, facilitating precise tumor localization and real-time tracking of therapeutic agents [8]. Integration of NIR-II imaging functionalities into AIE-based sonosensitizers holds promise for achieving accurate tumor targeting, real-time assessment of therapeutic efficacy, and personalized optimization of treatment regimens. Integrating therapeutic and diagnostic functions into a single agent simplifies the treatment process, improves clinical outcomes, and may potentially lower overall healthcare costs.

In recent years, organic fluorophore sonosensitizers based on donor-acceptor-donor (D-A-D) scaffolds have been developed for sonodynamic cancer therapy. These sonosensitizers induce antitumor effects by generating intracellular ROS upon ultrasound (US) stimulation [9]. To investigate the effect of enlarging the D-A-D structured conjugated system on its properties, we synthesized three benzothiadiazole-based derivatives. Benzothiadiazole and Benzobisthiazole, known for their strong electron-accepting properties, are utilized in constructing efficient push-pull electronic systems [10]. The rigid structure of the thiophene ring reduces molecular distortion, promotes π-π stacking, and result in a high degree of π-electron conjugation, commonly serving as an electron donor in molecular structures. The single bonds connecting the benzene rings to nitrogen atoms in triphenylamine allow free rotation, forming a non-planar propeller-like structure. This characteristic imparts electron-donating capability to triphenylamine derivatives, along with outstanding hole transport properties, high photostability, and low ionization potentials. Finally, we synthesized a benzothiadiazole-based sonosensitizer, BBTPA, a D-π-A-π-D unit where benzobisthiadiazole serves as the electron acceptor, triphenylamine acts as the electron donor, and thiophene functions as the π-bridge. With AIE characteristics, it exhibits strong NIR-II fluorescence and efficient ROS generation under ultrasound excitation (Scheme 1A). BBTPA, PMD and DSPE-PEG₂₀₀₀ form NP^BBTPA through self-assembly in this work. It is worth noting that PMD polymer main chain and paired carboxylic acid in side chains contain ROS-sensitive thio-ketal bonds (Scheme 1B). NP^BBTPA selectively accumulates at tumor sites in bladder cancer mice and is effectively absorbed by cancer cells. Subsequently, the thio-ketal bonds in PMD can undergo rapid cleavage, triggered by excessive ROS in tumor cells, which results in polymer degradation and nanoparticle dissociation. After being incorporated into stimuli-responsive polymers to form NP^BBTPA, BBTPA can severely damage mitochondria under US irradiation through the generation of type I and type II ROS. More importantly, it promotes the activation of inflammatory caspases, inducing pyroptosis and immunogenic cell death (ICD). BBTPA demonstrates potent antitumor effects by inducing mitochondrial dysfunction, cell pyroptosis, and enhancing antitumor immune responses [11]. The unique combination of therapeutic efficacy and NIR-II imaging capabilities renders NP^BBTPA a promising candidate for bladder cancer diagnosis and treatment, thus advancing the frontiers of personalized medicine (Scheme 1C).

Scheme 1.

Schematic illustration of the NIR-II AIE acoustic sensitizer NP^BBTPA for cancer therapy. A) The chemical structures of BTPA, BBTA, and BBTPA and the properties of BBTA, BTPA and BBTPA were compared. B) The synthesis process of NP^BBTPA The antitumor mechanism of BBTPA nanoparticles. C) The antitumor mechanism of NP^BBTPA. NP^BBTPA + US can generate ¹O₂ and ·OH, inducing pyroptosis and ICD effects.

2. Results and discussion

2.1. Synthesis and characterization of BBTPA

Initially, Suzuki-Miyaura coupling reactions were conducted between 4- (diphenylamino) phenylboronic acid pinacol ester and either 4,7-dibromo-5,6-dinitrobenzo[c] [1,2,5]thiadiazole or 4,7-bis(5-bromothiophen-2-yl)-5,6-dinitro-2,1,3-benzothiadiazole to produce BTPA and BBTA, respectively. Subsequent denitration of BBTA using Zn/NH₄Cl reduction and thionylaniline-induced ring closure yielded the target compound BBTPA (Fig. 1A) [12]. All products were characterized by proton nuclear magnetic resonance (¹H NMR) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) (Figs. S1–S4), verifying the successful synthesis of BTPA, BBTA, and BBTPA. Subsequently, the photophysical properties of BTPA, BBTA, and BBTPA were tested by ultraviolet-visible (UV-VIS) absorption spectroscopy after dissolving them in tetrahydrofuran. The spectra indicated that BTPA had a peak absorption at 535 nm, while BBTA exhibited a red-shifted peak at 602 nm. Notably, BBTPA displayed a broader NIR absorption range (800-900 nm) with a peak at 856 nm (Fig. 1B), demonstrating the efficacy of molecular engineering in modulating photophysical properties [13]. Steady-state/transient fluorescence spectroscopy analysis revealed fluorescence peaks at 625 nm for BTPA, 712 nm for BBTA, and a strong emission in the 900-1400 nm range for BBTPA, with a peak at 1119 nm (Fig. 1C). This emission profile is likely attributed to the extended π-conjugation system of benzobisthiadiazole and the incorporated dithiophene, facilitating electron delocalization and resulting in a red-shifted and broadened absorption spectrum extending into the NIR region [14]. These characteristics position BBTPA as a potential candidate for NIR-II bioimaging.

Fig. 1.

Synthesis and characterization of BTPA, BBTA and BBTPA. A) Synthesis route of BTPA, BBTA and BBTPA. B) UV-VIS spectra of BTPA, BBTA and BBTPA in tetrahydrofuran. C) Fluorescence spectra of BTPA, BBTA and BBTPA. D) Relative maximum photoluminescence intensity (I/I₀) of BBTPA in the mixture of DMSO/toluene with kincreased toluene fractions. E) The calculated HOMO–LUMO distributions and the lowest-excited singlet–triplet splitting ΔE_ST values for click-activated BTPA, BBTA and BBTPA with the optimized molecular structure geometries.

To further evaluate its AIE characteristics, solvent-dependent fluorescence spectra of BBTPA were measured. In the good solvent DMSO, BBTPA exhibited weak fluorescence. However, its emission intensity increased markedly in a concentration-dependent manner upon gradual addition of the poor solvent toluene (Fig. S5), ultimately exhibiting a 3.25-fold enhancement (Fig. 1D). This outcome underscores the notable AIE characteristics of BBTPA, highlighting its potential for applications in aggregated states. Meanwhile, BBTPA exhibited a fluorescence quantum yield of 2.2%, which was significantly higher than that of the commercial fluorescent dye IR26 (0.5%).

Time-dependent density functional theory (DFT) calculations were conducted to explore the photophysical properties and ROS generation capacity of BTPA, BBTA, and BBTPA [15]. The optimized geometries derived from these calculations indicated that these molecules adopt three-dimensionally twisted conformations, which minimize intermolecular interactions like π–π stacking known to cause non-radiative decay in aggregated or solid states [16]. This conformation helps retain absorbed energy, facilitating fluorescence via radiative pathways or intersystem crossing (ISC) to the triplet state (T₁, the first excited triplet state), thereby potentially promoting ROS generation [17]. Calculated singlet-triplet energy gaps (ΔE_ST) of 0.34 eV (BTPA), 0.42 eV (BBTA), and 0.91 eV (BBTPA) indicate low ΔE_ST values that enhance ISC efficiency (Fig. 1E), thus improving ROS generation capacity. Moreover, the D-A-D framework promotes intramolecular charge transfer (ICT), enhancing the stability of the excited-state configuration and fluorescence efficiency. Increased conjugation length in molecules with extensive conjugated π-electron systems leads to enhanced electron delocalization and narrower HOMO-LUMO energy gaps, resulting in strong absorption and emission in the near-infrared region [18]. The electronic structures of the three compounds exhibit distinct differences, with HOMO-LUMO gaps of 2.3715 eV (BTPA), 2.1214 eV (BBTA), and 1.4104 eV (BBTPA), respectively. Notably, the significantly reduced HOMO-LUMO gap of BBTPA enables excellent NIR-II fluorescence properties while maintaining high ISC efficiency through optimized ICT processes. BBTPA, with its AIE characteristics, narrow-bandgap optical properties, and appropriate ΔE_ST value, shows promise as a sonodynamic therapeutic agent. Notably, its three-dimensionally twisted structure effectively balances fluorescence emission and ROS generation, thus rendering it a promising candidate for theranostic applications.

2.2. Preparation and characterization of NP^BBTPA

While BBTPA displays remarkable AIE properties, its limited water solubility may hinder its in vivo utility. To overcome this challenge, we synthesized a ROS-sensitive polymer (PMD) capable of self-assembling with BBTPA and DSPE-PEG₂₀₀₀ to form nanoparticles (NP^BBTPA) (Fig. 2A) [19]. The morphology of NP^BBTPA was assessed using transmission electron microscopy (TEM), revealing a uniform spherical structure with an average diameter of approximately 100 nm (Fig. 2B). Dynamic light scattering (DLS) measurements indicated an average hydrodynamic size of 125.6 nm and a polydispersity index of 0.11 (Fig. 2C), with a Zeta potential of −23.43 mV (Fig. 2D). These results collectively confirm the successful fabrication of spherical NP^BBTPA with uniform morphology and narrow size distribution via nanoprecipitation. Moreover, the stability of NP^BBTPA was evaluated, and it maintained a consistent particle size in phosphate-buffered saline (PBS) for 7 days (Fig. 2E).

Fig. 2.

Synthesis and characterization of NP^BBTPA. A) Schematic illustration of the fabrication process for NP^BBTPA. B) TEM image. C) Hydrodynamic radius and D) Zeta potential. E) DLS profiles of NP^BBTPA stored in PBS buffer for 7 days. F) UV–VIS spectra. G) Fluorescence spectra. H) ESR spectra of ⋅OH captured by the DMPO probe. I) ESR spectra of ¹O₂ trapped by the TEMP probe. J) The plot of ln (A_t/A₀) upon exposure to US irradiation. K) Time-dependent absorption spectra of DPBF with NP^BBTPA. L) GPC spectrum of PMD M) TEM images of NP^BBTPA + H₂O₂.

Subsequent UV-VIS spectroscopic analysis of NP^BBTPA's photophysical properties revealed a maximum absorption wavelength at 861 nm (Fig. 2F), consistent with BBTPA. Steady-state and transient fluorescence spectroscopy illustrated strong fluorescence emission in the 900∼1400 nm range, showcasing its potential for NIR-II bioimaging (Fig. 2G). Electron spin resonance (ESR) spectroscopy was employed to monitor the generation of singlet oxygen (¹O₂) and hydroxyl radicals (·OH) under ultrasound excitation. For ·OH detection, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) served as the trapping agent, and a characteristic quartet signal was detected in the NP^BBTPA + US group, confirming the presence of ·OH in the system. Notably, no obvious signals were detected in the non-US-treated groups, confirming that NP^BBTPA generates ROS exclusively upon US stimulation (Fig. 2H). For ¹O₂ detection, 2,2,6,6-tetramethylpiperidine (TEMP) was used as the trapping agent. After mixing TEMP with NP^BBTPA, the mixture was subjected to US irradiation at 1.0 MHz frequency, 1.5 W cm⁻² intensity, 50% duty cycle, and 2 min duration. A distinct triplet signal was observed in the NP^BBTPA + US group (Fig. 2I), indicating the generation of ¹O₂. To screen for optimal ultrasound parameters, we employed 1,3-diphenylisobenzofuran (DPBF) to assess the ROS generation capacity under different ultrasonic conditions. The experimental results demonstrated that the maximal ROS production was achieved at an ultrasound intensity of 1.5 W cm⁻² and a frequency of 1 MHz; additionally, the rate of ROS generation slowed down after 2 min of ultrasound irradiation (Fig. S6). Therefore, the ultrasound parameters of 1.5 W cm⁻², 1 MHz and 2 min were selected for all subsequent experiments. Moreover, the Type I ROS generation was again assessed using tetramethylbenzidine (TMB). The results showed that NP^BBTPA could generate ·OH upon ultrasound stimulation (Fig. S7). Type II ROS production was assessed using DPBF, revealing a degradation rate constant of 0.0016 s⁻¹ (Fig. 2J and K), indicative of ¹O₂ generation by NP^BBTPA under ultrasound stimulation [20]. The PMD molecular backbone contains a sulfur bond that is sensitive to reactive oxygen species (ROS). Notably, the material exhibits extremely low cytotoxicity [21] and its molecular weight has been determined to be 11,782 Da (Fig. 2L) [22]. Furthermore, related tests were conducted under simulated oxidative stress conditions (i.e., exposure to H₂O₂): dynamic light scattering (DLS) results showed a significant increase in average particle size (Fig. S9); transmission electron microscopy (TEM) characterization further confirmed that the nanoparticles had degraded into fragments (Fig. 2M). These results fully demonstrate the ROS-responsive dissociation properties of NPBBTPA and highlight its potential for controlled delivery in biological systems [23].

2.3. Cellular uptake of NP^BBTPA, reactive oxygen species generation capacity, and in vitro antitumor activity

Efficient cellular uptake of nanoparticles is crucial for their antitumor efficacy [24]. Leveraging prior evidence of NIR-II fluorescence emission by NP^BBTPA, we utilized its intrinsic fluorescence to investigate UMUC3 cell uptake [25]. Confocal laser scanning microscopy (CLSM) imaging illustrated robust red fluorescence in UMUC3 cells after 7 h of NP^BBTPA exposure (Fig. 3B), indicating successful internalization. Flow cytometry analysis further revealed a time-dependent uptake trend for NP^BBTPA (1 h, 4 h, 7 h) (Fig. 3C). Particularly, the mean fluorescence intensity after 4 h was 3.9 times higher than the control (Fig. 3D), suggesting that NP^BBTPA is internalized by UMUC3 cells likely via endocytosis. Subsequent assessment of NP^BBTPA's capacity to induce intracellular ROS employed the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Flow cytometry quantification showed that the fluorescence intensity in the NP^BBTPA combined with ultrasound (US) group was 1.6-fold higher than that in the NP^BBTPA-alone group (Fig. 3E and F). Correspondingly, CLSM observations revealed intense green fluorescence exclusively in the NP^BBTPA + US group, distinct from PBS, PBS + US, and NP^BBTPA-alone groups (Fig. 3G). These findings underscore the ability of NP^BBTPA, under ultrasound excitation (1.0 MHz, 1.5 W cm⁻², 50% duty cycle, 2 min), to effectively induce ROS production intracellularly.

Fig. 3.

Endocytosis, ROS production and cytotoxicity with NP^BBTPA upon ultrasound radiation (1.5 W cm⁻², 1 MHZ, 2 min). A) Schematic illustration of the intracellular uptake of NP^BBTPA and the subsequent ROS scavenging and 2D live/dead in UMUC3 cells. B) Representative CLSM images of UMUC3 cells treated with NP^BBTPA at 7 h. The cell nuclei were stained with DAPI (blue), and actin filaments (F-actin) were stained with phalloidin (green). C) Flow cytometry plot and D) quantitative analysis of intracellular uptake of NP^BBTPA in UMUC3 cells. E) The production levels of ROS in UMUC3 cells treated with PBS, PBS + US, NP^BBTPA, and NP^BBTPA + US, respectively, were measured by flow cytometry. F) Quantification of the ROS from (E). G) CLSM images demonstrated ROS production in UMUC3 cells following treatment with PBS, PBS + US, NP^BBTPA, and NP^BBTPA + US, respectively. Green fluorescence indicated the presence of ROS, and blue fluorescence marked the nuclei stained with DAPI. H) The survival rates of UMUC3 cells. I) The survival rates of MB49 cells. J) The anticancer efficacy of NP^BBTPA was demonstrated via live/dead cell assays imaged by CLSM. US refers to ultrasound radiation. Data are represented as mean ± SD (n = 3). Statistical significances between each pair of groups were analyzed using a one-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

To assess the in vitro antitumor efficacy of NP^BBTPA, MTT cytotoxicity assays were performed on three bladder cancer cell lines to evaluate the efficacy of NP^BBTPA with or without ultrasound stimulation. Results revealed that NP^BBTPA alone at 30.00 μg/mL exerted minimal cytotoxicity on UMUC3 cells (survival rate: 94.63%), whereas combined ultrasound treatment significantly decreased cell viability to 59.45% (Fig. 3H). The IC₅₀ values of NP^BBTPA + US for UMUC3, MB49 and T24 cells were 36.23 $\pm$ 5.13 μg/mL, 41.73 $\pm$ 4.70 μg/mL and 33.69 $\pm$ 2.55 μg/mL (Fig. 3I, Fig. S9). Importantly, NP^BBTPA exhibited negligible cytotoxicity in the absence of ultrasound activation across all cell lines, underscoring its favorable biosafety profile and highlighting that its cytotoxic effects are contingent upon ultrasound activation, indicative of sonodynamic toxicity. Subsequent live/dead cell staining using calcein AM and propidium iodide (PI) further confirmed the antitumor efficacy of NP^BBTPA (Fig. 3J). Cells treated with NP^BBTPA alone exhibited intense green fluorescence and weak red fluorescence, indicating low cytotoxicity without ultrasound activation. Conversely, the NP^BBTPA + US-treated group exhibited intense red fluorescence and diminished green fluorescence, signifying increased cell death. These results demonstrate that NP^BBTPA is efficiently internalized by cancer cells, and upon ultrasound activation, it induces intracellular ROS generation, ultimately leading to significant cancer cell death. Its cytotoxicity is strictly dependent on ultrasound activation.

2.4. NP^BBTPA induces mitochondrial damage and pyroptosis in vitro

Given the central role of mitochondria in regulating cell death pathways, we next investigated whether NP^BBTPA damages these organelles and the subsequent mechanisms leading to cytotoxicity. Colocalization analysis demonstrated a strong correlation (Pearson correlation coefficient = 0.89) between NP^BBTPA (green fluorescence) and the mitochondrial marker MitoTracker (red fluorescence), indicating predominant localization of NP^BBTPA to mitochondria (Fig. 4A, Fig. S10). The assessment of mitochondrial membrane potential alterations via JC-1 staining showed markedly enhanced green fluorescence in cells subjected to NP^BBTPA + US treatment (Fig. 4B).

Fig. 4.

Induced that NP^BBTPA damages mitochondrial to enhance cellular pyroptosis upon ultrasound radiation (1.5 W cm⁻², 1 MHZ, 2 min). A) Costained image of commercial mitochondria probe (MitoTracker) with NP^BBTPA and R value was calculated by PCC of colocalization finder (ImageJ). B) CLSM images of UMUC3 cells stained with JC-1 following various treatments. C) Flow cytometry plot and D) quantitative analysis of JC-1 of NP^BBTPA in UMUC3 cells. E) The TEM images of mitochondria in PBS and NP^BBTPA. F) Protein expression of GSDME, GSDME-N and Cleaved-Caspase3 in UMUC3 cells determined by Western blot upon various treatments. G) Direct visualization of pyroptosis in UMUC3 cells under a confocal microscope. Arrows indicate cells exhibiting pyroptotic morphology. H) Extracellular ATP concentration in the specified treatment groups. I) Proportions of released LDH in the specified treatment. US refers to ultrasound radiation. Data are presented as mean ± standard deviation (n = 3). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons (A, B, C, and D). ∗∗∗∗p < 0.0001.

Flow cytometry analysis showed that the red/green fluorescence ratio in the NP^BBTPA + US group was three times lower than that in the PBS group (Fig. 4C and D), suggesting a significant reduction in membrane potential following NP^BBTPA + US treatment. Consistently, TEM analysis showed normal mitochondrial morphology in the PBS group, while the NP^BBTPA + US-treated group exhibited characteristic signs of mitochondrial damage, including swelling, disruption of cristae, and even disappearance (Fig. 4E). Given the link between mitochondrial damage and pyroptosis [26], we investigated the underlying molecular mechanisms. Western blot analysis demonstrated a significant upregulation of cleaved caspase-3 and GSDME-N protein expression in the NP^BBTPA + US group (Fig. 4F). CLSM demonstrated typical pyroptotic features in dying cells from the NP^BBTPA + US group, such as cellular swelling, membrane blebbing, and eventual plasma membrane rupture (Fig. 4G) [27]. Furthermore, increased release of ATP and lactate dehydrogenase (LDH) in the NP^BBTPA + US group indicated robust induction of pyroptosis (Fig. 4H and I) [28]. ELISA results showed that cells in the NP^BBTPA + US group exhibited significantly increased release of inflammatory mediators (TNF-α, IL-6 and IL-18) compared with those in other treatment groups (Fig. S11–13). In conclusion, upon ultrasound activation, NP^BBTPA effectively induces mitochondrial damage and triggers ROS generation, resulting in severe mitochondrial dysfunction, which in turn activates cleaved caspase-3, leading to GSDME cleavage and initiation of the canonical pyroptotic pathway.

2.5. NP^BBTPA-induced ICD effect

ROS can effectively eliminate cancer cells by inducing ICD [22]. To investigate the potential of NP^BBTPA in combination with ultrasound for inducing ICD, we employed CLSM to analyze the subcellular localization of high-mobility group box 1 (HMGB1) in UMUC3 cells after different treatments (Fig. 5A). Our findings indicated minimal HMGB1 release in the PBS, PBS + US, and NP^BBTPA-alone groups, whereas NP^BBTPA + US treatment significantly enhanced HMGB1 efflux. This HMGB1 release can activate myeloid differentiation signaling pathways, thereby improving antigen processing and presentation [29]. Concomitantly, calreticulin (CRT) exhibited limited translocation in the PBS, PBS + US, and NP^BBTPA-alone groups, while NP^BBTPA + US treatment notably promoted CRT relocation from the endoplasmic reticulum to the cell membrane, as evidenced by strong green fluorescence on the cell surface (Fig. 5B). This observation was further quantitatively validated by flow cytometry, which showed a 2.1-fold elevation in fluorescence intensity when compared to the NP^BBTPA-alone group (Fig. 5C and D). These results highlight the ability of NP^BBTPA + US treatment to induce intracellular CRT translocation, facilitating macrophage-mediated tumor antigen presentation. Overall, these outcomes underscore the potential of NP^BBTPA combined with US to induce ICD.

Fig. 5.

Confirmation that NP^BBTPA upon ultrasound radiation contributes to immunogenic cell death in vitro (1.5 W cm⁻², 1 MHZ, 2 min). A) Representative CLSM images of the expression of HMGB1 and B) CRT in UMUC3 cells upon various treatments. C,D) Quantitative study of the exposure of CRT by flow cytometry. E) Schematic showing NP^BBTPA promotes DC maturation to amplify pyroptosis. F,G) Flow cytometry plot and quantitative analysis of the maturation of bone marrow dendritic cells (BMDCs). US refers to ultrasound. Data are represented as mean ± SD (n = 3). Statistic significances between every two groups were calculated by oneway ANOVA test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Furthermore, considering the critical role of dendritic cell (DC) maturation in antitumor immune responses, we also explored whether the induced ICD could further enhance DC activation [30]. Subsequently, we co-cultured MB49 cells treated with different modalities with bone marrow-derived dendritic cells (BMDCs) and monitored their maturation status via flow cytometry (Fig. 5E). Our results demonstrated that BMDCs co-cultured with NP^BBTPA + US-treated cells displayed the highest maturation rate (45.7%), representing a 1.8-fold increase compared to those co-cultured with NP^BBTPA-alone-treated MB49 cells (Fig. 5F and G). These collective findings illustrate that NP^BBTPA, upon US stimulation, effectively upregulates ICD biomarkers and enhances DC maturation through a mechanism involving mitochondrial damage and amplified caspase-3/GSDME-mediated pyroptosis [31].

2.6. RNA sequencing analysis

To further investigate the antitumor mechanism of NP^BBTPA, we performed whole-genome RNA sequencing on UMUC3 cells treated with PBS, NP^BBTPA, and NP^BBTPA + US [32]. Differential gene expression analysis showed that NP^BBTPA + US treatment significantly upregulated 1177 genes and downregulated 922 genes relative to the PBS control group (Fig. 6A). KEGG pathway enrichment analysis indicated that NP^BBTPA + US inhibited mitochondria-related metabolic pathways (e.g., cysteine and methionine metabolism, citrate cycle [TCA cycle], oxidative phosphorylation, pyruvate metabolism, cholesterol metabolism signaling pathway) as well as key energy metabolism pathways (e.g., glycolysis/gluconeogenesis) and the DNA replication signaling pathway (Fig. 6B and C). Additionally, NP^BBTPA + US treatment upregulated immunostimulatory factors in the tumor microenvironment, including IL-18, CXCL8, and IL-11 (Fig. 6D) [33]. These findings suggest that NP^BBTPA, when activated by ultrasound, induces cell death by disrupting mitochondrial function. Moreover, it reshapes the tumor immune microenvironment by promoting the release of immunostimulatory factors to alleviate immunosuppression, ultimately demonstrating potent antitumor effects. This discovery establishes a critical molecular basis for employing NP^BBTPA as an innovative combined sonodynamic-immunotherapy strategy.

Fig. 6.

Transcriptome analysis was conducted on UMUC3 cells treated with NP^BBTPA followed by ultrasonic irradiation (1.5 W cm⁻², 1 MHz, 2 min). A) Venn diagram of the identified differentially expressed genes. B) KEGG enrichment analysis of DEGs in UMUC3 cells treated with NP^BBTPA + US. C) GSEA showing positive enrichment of genes altered in NP^BBTPA + US-treated cells (data analyzed using the unmodified GSEA software package). D) Heat-map of gene expressions in cells treated with PBS, NP^BBTPA, and NP^BBTPA + US.

2.7. NIR-II fluorescence imaging and biosafety evaluation of NP^BBTPA

To assess the in vivo biodistribution and antitumor efficacy of NP^BBTPA, NIR-II imaging and therapeutic investigations were conducted in C57BL/6 mice bearing MB49 tumors (Fig. 7A) [34]. Following intravenous administration of NP^BBTPA, its biodistribution was monitored over 12 h using NIR light irradiation (808 nm, 1 W cm⁻², 2 min) (Fig. 7B). The data showed a gradual increase in fluorescence signals at the tumor site, reaching a peak at 2 h after injection (Fig. S14). Subsequent ex vivo imaging of excised tumors and major organs (heart, liver, spleen, lung, and kidney) confirmed significantly higher fluorescence intensity in tumor tissues compared to the heart and lung, with 1.5-fold and 1.1-fold higher fluorescence intensity than that in the heart and lung, respectively (Fig. 7C), demonstrating effective tumor targeting.

Fig. 7.

Biodistribution and anticancer efficacy of NP^BBTPA under ultrasound irradiation (1.5 W cm⁻², 1 MHz, 2 min) in a subcutaneous MB49 tumor model using C57BL mice. A) Schematic illustration of treatment schedule. B) Left: NIR-II fluorescence bio-imaging at various time points post i.v. injection of NP^BBTPA. right: NIR-II fluorescence intensity in the tumor and various organs at 12 h. C) Biodistribution of NP^BBTPA 12 h post-administration. D) Body weight changes in mice under various treatments. E) Tumor growth inhibition curves upon various treatments. F) Representative photograph and G) Corresponding tumor weight of the tumors 12 days after the treatment. H) up: H&E staining of the tumor tissues upon various treatments. down: TUNEL staining of the tumor tissues upon various treatments. US refers to ultrasound radiation. US refers to ultrasound radiation. Data are presented as mean ± standard deviation. Statistic significances between every two groups were calculated by oneway ANOVA test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

For efficacy evaluation, tumor-bearing mice received four treatment cycles as outlined in Fig. 7A, and their tumor volumes and body weights were monitored every two days. No significant body weight loss was observed in the NP^BBTPA + US group relative to PBS controls over 12 days, indicating good biocompatibility (Fig. 7D). Analysis of tumor growth inhibition revealed that neither ultrasound alone nor NP^BBTPA alone significantly affected tumor growth rates compared to the PBS control group. In contrast, the NP^BBTPA + US group exhibited a substantial tumor growth inhibition rate of 70.5% (Fig. 7E). Tumors were excised and weighed on day 12, with the NP^BBTPA + US group showing a significantly lower mean tumor weight (0.24 ± 0.08 g) compared to the PBS (0.68 ± 0.07 g), PBS + US (0.67 ± 0.09 g), and NP^BBTPA-alone (0.71 ± 0.08 g) groups (Fig. 7F and G), highlighting the potent tumor-suppressive effects of NP^BBTPA upon ultrasound stimulation.

To elucidate the underlying mechanism, histopathological analyses were performed on post-treatment tumor tissues. Hematoxylin and eosin (H&E) staining revealed extensive characteristic nuclear fragmentation in the NP^BBTPA + US group, while TUNEL staining detected abundant apoptotic cells (Fig. 7H) [35]. Collectively, these findings demonstrate robust tumor-targeted accumulation of NP^BBTPA and its strong sonodynamic antitumor efficacy, supporting its potential for clinical translation in cancer therapy.

2.8. NP^BBTPA + US releases DAMPs to induce immune responses

To comprehensively investigate the immunostimulatory effects of NP^BBTPA, we conducted systematic flow cytometry analysis on tumor, spleen, and lymph node tissues from treated mice (Fig. 8A) [36]. Dendritic cells (DCs), known as potent antigen-presenting cells in vivo, play a crucial role in antitumor immunity [37]. Subsequently, we assessed DC maturation in tumor and lymph node tissues of mice treated with NP^BBTPA under ultrasound stimulation using flow cytometry. The results revealed a significantly higher proportion of mature DCs (CD80⁺CD86⁺) in tumor tissues of the NP^BBTPA + US group (47.5%) compared to the PBS group (15.3%) and NP + group (18.0%) (Fig. 8B, Fig. S15). Similarly, in lymph node tissues, the proportion of mature DCs in the NP^BBTPA + US group (16.3%) was 2.5-fold and 2.3-fold higher than that in the PBS group (3.85%) and NP^BBTPA group (4.61%), respectively (Fig. 8C, Fig. S16). These findings suggest that NP^BBTPA under ultrasound stimulation notably enhances DC maturation in tumor and lymph node tissues, thereby boosting antitumor immune responses.

Fig. 8.

In vivo activation of the systemic antitumor immune response induced by NP^BBTPA under ultrasound exposure (1.5 W cm⁻², 1 MHz, 2 min). A) Schematic showing the evaluation of immune response after various treatments. B) The percentages of populations of mature DCs within MB49 tumors and C) DLN in each group are presented as histograms. D) The percentages of populations of NK cells within MB49 tumors in each group are presented as histograms. E) The ratio of M1 to M2 macrophages within MB49 tumors in each group are presented as histograms. F) representative flow cytometry scatter of CD8⁺ T cells and G) Quantitative analysis plot in MB49 tumors. H) representative flow cytometry scatter of CD8⁺ T cells and I) Quantitative analysis plot in spleen. J) up: Expression of Cleaved caspase-3 in MB49 tumors by immunohistochemistry staining. down: Immunofluorescence imaging of CD8⁺ T cell infiltration in MB49 tumor tissues following different treatments.

Further analysis of the tumor immune microenvironment (TME) revealed significant modulation of key immune subsets following NP^BBTPA + US treatment [38]. Activated NK cells (NK1.1⁺CD69⁺KLRG1⁺⁾ in tumor tissues in tumor tissues increased to 59.2%, compared to 19.6% in the NP^BBTPA-alone group (19.6%) (Fig. 8D, Fig. S17). Additionally, the tumor-associated macrophage (TAM) M1/M2 ratio shifted towards a pro-inflammatory phenotype (1:1.36) in the NP^BBTPA + US group, compared to 1:1.98 and 1:1.62 in the PBS and NP^BBTPA-alone groups, respectively (Fig. 8E, Fig. S18). These results indicate that NP^BBTPA under ultrasound activation not only enhances the cytotoxic activity of NK cells against tumors but also effectively alters the immunosuppressive microenvironment by promoting M1-polarized TAMs with antitumor properties, thus fostering an immune microenvironment conducive to tumor eradication.

Given the essential role of mature DCs in T cell activation [39], we evaluated the infiltration of CD8⁺ T cells (CD3⁺CD8⁺) in tumor tissues. Our results revealed a significantly higher proportion of CD8⁺ T cells in the tumor tissues of the NP^BBTPA + US treatment group (53.7%) compared to the PBS group (22.2%) and the NP^BBTPA group (28.8%) (Fig. 8F, G, Fig. S19). This trend was also observed in the spleen tissues, with the proportion of CD8⁺ T cells in the NP^BBTPA + US treatment group (33.5%) being 1.5-fold and 1.4-fold higher than that in the PBS group (17.7%) and the NP^BBTPA group (18.4%), respectively. These findings strongly suggest that NP^BBTPA combined with ultrasound stimulation enhances CD8⁺ T cell infiltration in both tumor and spleen tissues (Fig. 8H, I, Fig. S20).

Building upon the observed therapeutic effects, we further investigated the role of NP^BBTPA in activating pyroptosis in vivo and its impact on anti-tumor immunity. Immunofluorescence staining analysis showed that the NP^BBTPA + US treatment group exhibited a significant increase in cleaved caspase-3 expression in tumor tissues compared to the NP^BBTPA-alone group, indicating that ultrasound-activated NP^BBTPA effectively induces pyroptosis in vivo. Evaluation of immune effects revealed enhanced CD8⁺ T cell infiltration in tumor tissues of the NP^BBTPA + US treatment group, as evidenced by prominent red fluorescence signals (Fig. 8J). In conclusion, the results indicate that ultrasound-activated NP^BBTPA elicits tumor cell pyroptosis and potentiates adaptive immune responses, thereby promoting immune memory protection, suppressing tumor metastasis, and preventing recurrence.

3. Conclusion

In this study, we synthesized a novel sonosensitizer, BBTPA, featuring a benzothiadiazole core with an A-π-D-π-A conjugated structure. BBTPA exhibits AIE properties and sonodynamic activity. Notably, it emits strong fluorescence in the 900-1200 nm NIR-II region, rendering it a promising material for NIR-II bioimaging. By co-assembling BBTPA with mDSPE-PEG₂₀₀₀ and the ROS-responsive polymer PMD, we fabricated NP^BBTPA that can efficiently generate ROS upon ultrasound activation. This ROS production induces mitochondrial damage and triggers caspase-3/GSDME-mediated pyroptosis, thereby initiating robust ICD and subsequent DC maturation. In vivo experiments showed that NP^BBTPA combined with ultrasound irradiation achieved a tumor growth inhibition rate of 70.5%. Flow cytometry analysis further revealed a 1.9-fold increase in CD8⁺ T cell infiltration in tumor tissues compared to the control group, highlighting its potent immunostimulatory effect. Coupled with its excellent biocompatibility and safety profiles, NP^BBTPA provides a promising theranostic platform for bladder cancer treatment, integrating precise NIR-II imaging with effective sonodynamic immunotherapy.

CRediT authorship contribution statement

Meixin Shan: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Xinwei Wang: Formal analysis, Data curation, Conceptualization. Zhu Wang: Validation, Supervision, Software, Conceptualization. Chun Xu: Writing – review & editing, Writing – original draft, Supervision. Leijiao Li: Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation. Wenliang Li: Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation. Haihua Xiao: Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Wasilijiang Wahafu: Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis.

Ethics approval and consent to participate

All animal experiments were also approved by the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (approval #NCC2025A606) and were performed in compliance with institutional and national guidelines for the care and use of laboratory animals. Table of content graph created with BioRender.com.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article: