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Synergizing sono-piezo with exosome suppression using doping-engineered hydroxyapatite for potentiated tumor treatment through immunoactivation

Journal of Nanobiotechnology volume 23, Article number: 495 (2025) Cite this article

Abstract

Piezoelectric nanomaterials that generate reactive oxygen species (ROS) through piezoelectric polarization under mechanical stimulation have emerged as a promising cancer therapy platform. However, their potential is limited by poor piezoresponse, low catalytic efficiency, and the exacerbation of immunosuppression due to ROS-induced release of tumor-derived exosomes. In this study, we employed a doping-engineered strategy by incorporating manganese ions (Mn2⁺) into hydroxyapatite (HAP) to enhance its piezocatalytic performance, while combining exosome inhibition to achieve a synergistic improvement in tumor therapy. Mn2⁺-doped HAP was synthesized via a one-pot hydrothermal method and subsequently modified with a ROS-cleavable lipid, DSPE-TK-mPEG. During the modification process, the exosome inhibitor GW4869 was loaded, resulting in the formation of GW4869-loaded Mn2⁺-HAP-Lipid nanocomposites (abbreviated as GMHL). The introduction of Mn2+ significantly reduced the bandgap of HAP, thereby enhancing its piezoelectric catalytic activity to generate ROS under ultrasound (US) stimulation, which triggered the cleavage of ketone-thiol bond in DSPE-TK-mPEG and led to the efficient release of GW4869. In multiple tumor models, GMHL effectively retard tumor growth and inhibited the production of tumor-derived exosomal PD-L1 upon US stimulation, thereby triggering an anticancer immune response through modulation of the immunosuppressive tumor microenvironment.

Introduction

Malignant tumors remain one of the leading global public health challenges, with increasing rates of morbidity and mortality each year [1, 2]. While traditional cancer treatments such as surgery, chemotherapy, and radiotherapy have been widely used in clinical practice, they are often associated with limited therapeutic efficacy and significant side effects [3, 4]. Among the various therapeutic approaches, the efficient generation of ROS has emerged as a key strategy for tumor therapy [5,6,7]. Several catalytic therapies aimed at producing large quantities of ROS to induce tumor cell death have been explored, including photodynamic therapy (PDT), US-mediated sonodynamic therapy (SDT), and others [8,9,10,11]. Notably, ultrasound therapy has gained widespread clinical use due to its non-invasive nature, low energy attenuation, high tissue penetration, and minimal side effects [12, 13]. Piezoelectric materials, in particular, have proven to be effective sensitizers in ultrasound therapy by catalyzing the generation of ROS [14, 15]. The mechanical stress induced by ultrasound causes deformation of piezoelectric materials, creating an internal electric field that facilitates the separation of electron-hole pairs, ultimately driving ROS generation [16, 17]. A variety of piezoelectric materials have been studied for their role in tumor therapy, including barium titanate (BTO) [18, 19], zinc oxide (ZnO) [20], and hydroxyapatite (HAP) [21], among others. HAP, a biomimetic material found in bones and teeth, is known for its biocompatibility and stability, with applications in bone grafting, prosthetics, and drug delivery [22, 23]. However, its potential in piezoelectric catalytic tumor therapy is limited due to its wide bandgap and tendency for electron-hole recombination, reducing its piezoelectric catalytic efficiency.

In the past, various attempts have been made to improve the efficiency of piezoelectric catalysis, including the introduction of metal ions [24], the establishment of heterostructures [25, 26], and the creation of oxygen vacancies [27, 28]. Among these approaches, the introduction of metal cations (e.g., Fe3+, Mn2+, Cu2+) can enhance catalytic activity by generating doping energy levels, which facilitate carrier separation and prevent the recombination of electron-hole pairs [29]. Additionally, metal cation doping may lower the band gap of piezoelectric materials, making electron excitation more accessible [17]. However, the generation of ROS, particularly that induced by ultrasound, is often accompanied by the release of tumour-derived exosomes that express high levels of programmed death ligand 1 (PD-L1) [30,31,32]. The secreted exosomal PD-L1 can bind to PD-1 receptors on T cells, reducing T-cell activity at the tumor site and contributing to immunosuppression [33, 34]. Therefore, combining efficient ROS production with the suppression of exosome secretion may enhance immune activation, leading to more effective tumor treatment.

Herein, in this study, a Mn2+ doping HAP piezoelectric, loaded with exosome inhibitor GW4869 and then modified with a ROS-cleavable lipid, DSPE-TK-mPEG, was prepared (GMHL). Compared to conventional piezoelectric materials such as BaTiO3 or ZnO, Mn2⁺-doped HAP offers superior biosafety, biodegradability, and ease of synthesis. As a naturally occurring calcium phosphate material, HAP is highly biocompatible and degrades into non-toxic ions, making it more suitable for biomedical applications. Meanwhile, HAP nanoparticles can be easily synthesized via hydrothermal methods under mild conditions, providing better scalability and reproducibility than the more complex fabrication processes required for piezoelectric materials. Moreover, Mn2+ doping narrows HAP’s bandgap, significantly enhancing its piezoelectric catalytic activity and ROS generation under US stimulation. The generated ROS were capable of cleaving the ketone-thiol bond in DSPE-TK-mPEG, thereby releasing GW4869. Cell experiments demonstrated that under US stimulation, GMHL effectively induced intracellular ROS production, promoted cancer cell death, and concurrently inhibited US-triggered exosome secretion. Upon intravenous administration, GMHL exhibited significantly enhanced tumor accumulation. In multiple tumor models, GMHL, in combination with US stimulation, markedly suppressed tumor growth. Furthermore, GMHL with US stimulation inhibited the secretion of tumor-derived exosomal PD-L1, enhanced T-cell activity, and boosted antitumor immunity (Scheme 1) [35]. In summary, this study presents a doping-engineered piezoelectric nanoplatform based on biosafe and biodegradable Mn2⁺-doped HAP, which overcomes the limitations of traditional piezoelectric materials by integrating superior biological compatibility, enhanced catalytic activity, and a ROS-responsive drug release mechanism. Uniquely, GMHL integrates ROS-mediated tumor cell killing with a responsive exosome inhibition mechanism via the ROS-cleavable prodrug structure. Such a combination not only amplifies antitumor efficacy but also attenuates exosome-mediated immunosuppression, an often-overlooked consequence of ROS-based therapies. By integrating enhanced piezoelectric catalysis with exosome-targeted immunomodulation, this work establishes GMHL as a next-generation therapeutic strategy that addresses the limitations of conventional piezoelectric systems and advances toward clinical translation.

Scheme 1
scheme 1

A schematic illustrating the design of Mn2⁺-doped HAP nanorods loaded with an exosome inhibitor for ultrasound-activated piezoelectric therapy. Mn2⁺ doping, achieved via a one-pot hydrothermal method, enhances the piezocatalytic activity of HAP by reducing its bandgap, facilitating ROS generation under US stimulation. The Mn2⁺-HAP was further functionalized with a ROS-cleavable lipid, DSPE-TK-mPEG, and loaded with the exosome inhibitor GW4869, forming GMHL nanocomposites. Upon US activation, ROS-mediated lipid cleavage enables the controlled release of GW4869, effectively suppressing tumor growth and reducing tumor-derived exosomal PD-L1, thereby modulating the immunosuppressive tumor microenvironment and enhancing anticancer immunity

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Results and discussion

Preparation and characterization of MHL

The HAP and Mn2+ doped HAP nanorods (NRs) were first synthesized using a modified hydrothermal process (Fig. 1a) [36]. Transmission electron microscope (TEM) imaging showed that Mn2+ doping did not affect the morphology of HAP, which exhibited a uniform rod-like morphology (Fig. 1b, S1). TEM elemental mapping analysis displayed the uniform distribution of elements including Mn, Ca, P, and O in the Mn-HAP NRs (Fig. 1c). Consistently, X-ray photoelectron spectroscopy (XPS) results confirmed the existence of those elements, further validating the successful synthesis of Mn2+-doped HAP NRs (Fig. 1d&e). Subsequently, the crystal structure of Mn-HAP NRs was characterized by X-ray diffraction (XRD). As shown in Fig. 1f, the diffraction peaks of Mn-HAP NRs matched well with the standard pattern of hydroxyapatite (JCPDS #09-0432). The successful doping of Mn2+ also resulted in a color change of HAP NRs from the initial milky white to brown, which was further verified by the spectral shift observed in the UV-Vis-NIR absorption spectra (Fig. 1g). Then, to improve their stability in physiological solutions, the obtained Mn-HAP NRs were coated with lipids containing ROS-cleavable thioketal linker (abbreviated as MHL), as detailed in the experimental section. Dynamic light scattering results revealed that the modified MHL exhibited a narrow size distribution, with an average size of around 107.1 nm (Fig. 1h). After modification, the surface charge of the nanorods shifted from positive to negative, with the zeta potential changing from 8.11 mV to -3.58 mV (Fig. 1i). Fourier Transform infrared spectroscopy (FTIR) results revealed two prominent characteristic peaks at 2,889 cm− 1 and 1,108 cm− 1 on MHL NRs, corresponding to the stretching vibrations of C-H and C-O-C, respectively, indicating the existence of PEG (Fig. 1j). Moreover, the content of lipids on MHL by thermogravimetric analysis (TGA) was determined to be ~ 26.1% (Fig. 1k). Metal doping is recognized as an effective way to modulate the energy band structure of piezoelectrics to enhance their piezocatalytic performance. We then investigated the energy levels and electronic structures of Mn-doped HAP. As shown in Fig. 1l &m, the band gap energies were determined using the Kubelka-Munk function of UV-visible diffuse reflectance spectroscopy, yielding values of 3.90 eV for HAP and 1.85 eV for Mn-HAP. Such results indicate that Mn doping significantly reduces the band gap of HAP, favorable for the separation of electron-holes. Mechanically, Mn2⁺ doping introduces localized electronic states within the band structure of HAP, primarily due to the presence of partially filled 3d orbitals of Mn2⁺. These impurity states can hybridize with the native conduction and valence bands of HAP, thereby reducing the energy gap between them and effectively narrowing the bandgap. In addition, the substitution of Ca2⁺ (ionic radius: 1.00 Å) by Mn2+ (ionic radius: 0.83 Å) induces lattice distortion due to the size mismatch, which can further alter the crystal field environment and electronic structure of HAP, contributing to the bandgap modulation [37,38,39]. Meanwhile, by using XPS-based valence band spectra, the valence band (VB) of HAP and Mn-HAP were estimated to be 3.24 and 3.08 eV, respectively (Figure S2). Consequently, the conduction band (CB) of HAP and Mn-HAP were determined to be -0.66 eV and 1.23 eV, respectively, from the equation ECB = EVB - Eg. After that, the piezoelectric properties of Mn-HAP NRs were verified by piezoelectric responsive force microscopy (PFM). Mn-HAP exhibited distinct piezoelectric characteristics, as shown by phase images with a clear phase hysteresis loop and an amplitude-voltage hysteresis curve resembling a butterfly loop under a bias voltage ranging from − 10 V to + 10 V (Fig. 1n). Collectively, these results indicate that Mn2+ doping can effectively reduce the bandgap width of HAP NRs, and the resulting Mn-HAP NRs exhibit excellent piezoelectric properties (Fig. 1o).

Fig. 1
figure 1

Preparation and Characterization of MHL. (a) Schematic illustration of the preparation process for MHL NRs. (b) Typical TEM images and (c) elemental mapping of Mn-HAP NRs. (d) XPS spectrum of Mn-HAP NRs and (e) high resolution of Mn2p XPS spectrum. (f) XRD spectrum of Mn-HAP NRs. (g) UV-vis-NIR spectra of HAP and Mn-HAP NRs. Inset: Photographs of their ethanol solutions. (h) Average particle size and (i) Zeta potential of Mn-HAP and MHL NRs. (j) FTIR spectra of Mn-HAP and MHL NRs. (k) Thermogravimetric analysis results of Mn-HAP and MHL NRs. (l) The bandgap of HAP and (m) Mn-HAP NRs determined using Kubelka-Munk equation. (n) Phase and displacement response of Mn-HAP NRs as analyzed by piezoresponse force microscopy. (o) Schematic illustration of energy band structure of HAP and Mn-HAP NRs. Data were presented as mean ± SEM

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ROS generation and release of GW4869 from GMHL under US stimulation

To explore the US-driven piezoelectric catalytic ROS generation capacity of MHL, methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were used as probes to detect hydroxyl radical (·OH) and singlet oxygen (1O2) production, respectively (Fig. 2a). The blue color of MB mixed with MHL gradually faded with the increasing US irradiation time, and accompanied by a weakening of its characteristic absorption peak at 662 nm, indicating the effective production of ·OH (Fig. 2b&c). Furthermore, MHL exhibited better degradation rate to MB compared to HAP-Lipid (HL) under US conditions, indicating that Mn2+ doping enhances the ability of HAP to generate ·OH (Fig. 2d, S3). Then, we used DPBF to indicate the 1O2 generation. As expected, MHL exhibited US time-dependent DPBF degradation, and the degradation effect was superior to that of HL (Fig. 2e-g, S4). These results collectively verify the excellent piezoelectric catalytic ROS generation capacity of MHL.

US stimulation has been reported to promote exosome secretion, which may contribute to tumor immune evasion and reduce therapeutic efficacy. GW4869 is a well-established and specific inhibitor of neutral sphingomyelinase (nSMase), a key enzyme involved in exosome biogenesis and secretion. It has been widely applied in cancer therapy to block exosome-mediated processes such as angiogenesis and immune suppression [40,41,42]. In our study, GW4869 was encapsulated into the hydrophobic lipid layer of GMHL to achieve controlled inhibition of exosome release during US stimulation (Fig. 2h). The efficient loading of GW4869 was assessed by a UV-Vis-NIR spectrophotometer. As shown in Fig. 2i, GMHL displayed a distinct GW4869 characteristic absorbance peak at 360 nm, confirming its successful encapsulation with a loading efficiency of 87.7%. Afterwards, we carefully evaluated the US-induced ROS generation to trigger the cleavage of thioketal bond in DSPE-TK-mPEG, leading to the efficient release of GW4869. It was found that the average size of GMHL decreased from 226.1 nm to 109.2 nm upon being exposed to US irradiation (Figure S5), indicating US-triggered lipid layer dissociation. Then, the GMHL was subjected to US stimulation for 2 min at 20-minute intervals. After each stimulation, the supernatant containing the released GMHL was collected and analyzed using a UV-Vis-NIR spectrophotometer. As shown in Fig. 2j, in the absence of US, GMHL showed minimal GW4869 release. Novelty, when US was applied, a significant amount of GW4869 was released. After three rounds of US, the cumulative release of GW4869 reached 64.5%, demonstrating that the GMHL can effectively respond to the ROS to trigger the efficient release of GW4869. Therefore, all those results demonstrate that our GMHL has excellent piezoelectric ability to trigger ROS production, induce the dissociation of the lipid bilayer structure, and facilitate the release of GW4869 (Fig. 2k).

Fig. 2
figure 2

ROS generation and release of GW4869 from GMHL under US stimulation. (a) Scheme illustrating the detection of ROS generation using specific probes. (b) UV-Vis-NIR spectra showing the degradation of MB over time, indicating the generation of hydroxyl radicals induced by MHL under US stimulation. Inset: photographs of MB solutions incubated with MHL and exposed to US stimulation at various time intervals. (c) The corresponding MB degradation rate obtained from (b). (d) UV-vis-NIR spectra showing the degradation of MB after different treatments as indicated. (e) Time-dependent UV-Vis-NIR spectra showing the degradation of DPBF, indicating the generation of singlet oxygen induced by MHL under US stimulation. Inset: photographs of DPBF solutions incubated with MHL and exposed to US stimulation at various time intervals. (f) The corresponding DPBF degradation rate obtained from (e). (g) UV-Vis-NIR spectra showing the degradation of DPBF after different treatments as indicated. (h) Structural formula of the exosome inhibitor GW4869. (i) UV-Vis-NIR spectra of free GW4869, MHL and GMHL. (j) Time-dependent release of GW4869 from GMHL under US stimulation. (k) Mechanism of US-induced ROS generation to trigger the cleavage of thioketal bond in DSPE-TK-mPEG and lead to the release of GW4869 from GMHL. Data were presented as mean ± SEM. The p values were calculated by one-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001

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In vitro cell killing effect of GMHL-mediated piezoelectric catalysis

Inspired by the efficient ROS generation efficiency of GMHL, we evaluated their cell killing effects at the cellular level. We first investigated the cell endocytosis behavior of GMHL with 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate Salt (DiD) labeling via co-incubating with CT26 tumor cells, and traced it by confocal laser scanning microscopy (CLSM) imaging. As shown in Fig. 3a, the intracellular fluorescence signal of DiD gradually enhanced with the extension of incubation time, indicating that GMHL could be effectively internalized by CT26 cells in a time-dependent manner. Flow cytometry results further confirmed that the GMHL could be effectively endocytosed by CT26 cells (Fig. 3b&c). Then, the intracellular ROS generation was evaluated by using a commercial ROS probe, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), which could be rapidly oxidized by ROS into highly fluorescent 2′,7′ -dichlorofluorescein molecules. As shown by the CLSM results, the GHL plus US induced moderate intracellular ROS production under US simulation. Whereas, the ROS generation was almost negligible in the US, GHL and GMHL groups. Remarkably, the GMHL plus US stimulation exhibited most intense intracellular fluorescence signals, indicating the highly effective ROS production (Fig. 3e&f). Flow cytometry results were consistent with the above observations (Fig. 3d, S6). Numerous studies have demonstrated that piezoelectric effect can induce the activation of cell membrane calcium channels, which prompted the influx of Ca2+ and lead to Ca2+ overload [43]. Thereafter, the intracellular Ca2+ contents were assessed using the intracellular Ca2+ probe, Fluo-4 Acetoxymethyl Ester. It was observed that the CT26 cells treated with GHL and GMHL showed a moderately increased intracellular Ca2+, probably ascribing to the dissociation of HAP to release Ca2+. Excitingly, GMHL plus US stimulation treatment could greatly elevate the intracellular Ca2+ contents, higher than that of GHL plus US stimulation group (Fig. 3g&h). These results can be explained by the fact that the Ca2+ released from HAP, together with piezoelectric effect-induced Ca2+ influx synergistically amplifies intracellular Ca2+ accumulation.

Next, the impact of GMHL combined with US irradiation on mitochondrial damage was evaluated by monitoring the mitochondrial membrane potential using a JC-1 probe. As shown in Fig. 3i&j, CT26 cells treated with GMHL plus US irradiation exhibited the strongest green fluorescence and minimal red fluorescence, indicating severe mitochondrial damage primarily driven by intracellular ROS and Ca2⁺ overload. Followingly, the potential of GMHL combined with US irradiation to induce cell apoptosis was analyzed using the Annexin V-FITC/PI double-labeling kit. As shown in Fig. 3k&l, CT26 cells treated with GMHL plus US irradiation exhibited a remarkable apoptosis rate, with late apoptosis reaching 80.9%, significantly higher than 66.4% for GHL plus US treatment. Subsequently, the dead/live staining results further confirmed that GMHL plus US irradiation exerted the strongest cytotoxic effect on CT26 cells (Fig. 3m). Additionally, the cytotoxic effect of GMHL was assessed using the standard MTT assay. The results demonstrated that GMHL plus US irradiation achieved the highest cell-killing efficacy among all treatment groups in a concentration and US-duration dependent manner (Fig. 3n, S7). Therefore, these results collectively demonstrate that GMHL combined with US stimulation exerts an excellent killing effect on tumor cells, primarily through piezoelectric catalysis-induced intracellular ROS generation and Ca2⁺ overload (Fig. 3o).

Fig. 3
figure 3

In vitro cell killing effect of GMHL-mediated piezoelectric catalysis. (a) Confocal fluorescence images of CT26 cells incubated with DiD labeled GMHL for various time intervals. (b) Flow cytometry curves and (c) corresponding semi-quantitative results of CT26 cells incubated with DiD labeled GMHL for various time intervals. (d) Flow cytometry and (e) confocal fluorescence imaging showing the intracellular ROS production by a ROS probe, DCFH-DA, under different treatments. (f) Quantification analysis of intracellular ROS fluorescence in (e). (g) Confocal fluorescence images showing the intracellular Ca2+ levels stained by Fluo-4 AM and (h) corresponding quantitative analysis after different treatments. (i) Confocal fluorescence images showing the change of mitochondrial membrane potential in CT26 cells after different treatments for 24 h and (j) corresponding quantitative analysis. (k) Flow cytometry analysis of apoptosis and (l) corresponding statistical analysis. (m) Confocal fluorescence images of CT26 cells stained with calcein AM and PI after various treatments. (n) Relative cell viability of CT26 cells treated with different concentrations of GHL and GMHL with or without US irradiation (n = 5). (o) Scheme illustrating the mechanism of GMHL-mediated piezoelectric catalysis in inducing cell death under US stimulation. Data were presented as mean ± SEM. The p values were calculated by one-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001

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Exosome release Inhibition and ICD induce capacity of GMHL

Growing evidence suggests that US stimulation can promote the secretion of exosomes from tumor cells [30], which contain immune checkpoints such as PD-L1, contributing to immune evasion and the suppression of immune surveillance. Therefore, we evaluated the potential of our MHL loaded with the exosome inhibitor GW4869 to suppress exosome release during US exposure (Fig. 4a). The released exosomes after different treatments were collected using an exosome extraction kit and quantified by Bicinchoninic Acid (BCA) analysis of exosomal protein levels. The results demonstrated that tumor cells treated with MHL + US irradiation released a substantial amount of exosomes, showing higher exosomal protein content than the control group. However, when GW4869 was introduced to MHL, exosome secretion was effectively suppressed after US irradiation, as evidenced by a lower exosomal protein content compared to the control group (Fig. 4b). Then, we investigated the ability of GMHL combined with US irradiation to induce immunogenic cell death (ICD), characterized by the release of signaling molecules such as translocation of calreticulin (CRT), high mobility group box 1 (HMGB1), and adenosine triphosphate (ATP) (Fig. 4h). As shown in the Fig. 4c&d, CT26 cells treated with GMHL plus US irradiation exhibited the highest green fluorescence signal, significantly stronger than that observed in the other groups, indicating that this treatment enhances the translocation of CRT to the cell surface. At the same time, the GMHL plus US-treated group exhibited the lowest levels of intranuclear HMGB1 signaling compared to the other treatments (Fig. 4e&f), suggesting efficient extranuclear release of HMGB1. Subsequently, we also evaluated the extracellular release of ATP after various treatments. As shown in Fig. 4g, GMHL plus US treatment promoted the substantial release of ATP, with ATP content in the culture medium being 1.83 times higher than that of the control group. Furthermore, we evaluated the effect of GMHL in inducing DC maturation under US stimulation to activate subsequent immune responses (Fig. 4i). Flow cytometry results showed that GMHL plus US irradiation significantly upregulated the expression of co-stimulatory molecules CD80 and CD86, indicating effective DC maturation (Fig. 4j&k). Collectively, the results demonstrate that GMHL under US stimulation can release the exosome inhibitor GW4869 to inhibit tumor-derived exosome release, while simultaneously triggering ICD and promoting dendritic cell maturation, all of which contribute to the initiation of an anti-tumor immune response.

Fig. 4
figure 4

Exosome release inhibition and ICD induce capacity of GMHL. (a) Scheme showing the inhibition of tumor-derived exosome secretion by GMHL under US stimulations. (b) Determination of exosomal protein content in cell supernatants after different treatments by BCA kits. (c) Confocal fluorescence images of CT26 cells after different treatments and then stained with CRT antibodies. (d) Corresponding quantitative analysis results in (c). (e) Confocal fluorescence images of CT26 cells after different treatments and then stained with HMGB1 antibodies. (f) Corresponding quantitative analysis results in (e). (g) ATP release levels of CT26 cells after different treatments. (h) Schematic representation of GMHL-mediated ICD effects under US stimulation. (i) Schematic representation of the process of isolating immature dendritic cells from mouse femur and then adding to the CT26 cells pre-received with different treatments to evaluate the DC maturation. (j) Representative flow cytometry plots of maturated DC and (k) corresponding statistical analysis after various treatments. Data were presented as mean ± SEM. The p values were calculated by one-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001

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In vivo anti-tumor ability of GMHL on CT26 tumor model

Prior to conducting in vivo therapeutic experiments, we first investigated the pharmacokinetic behavior of GMHL through an in vivo optical imaging system. The results showed that intravenous (i.v.) injection of 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DIR)-labeled GMHL into CT26-bearing mice led to a gradual increase in fluorescence within the tumor, indicating that GMHL has excellent tumor accumulation capability, likely due to the enhanced permeability and retention (EPR) effect of the nanomaterials (Fig. 5a, S8). After 24 h post injection, the mice were humanely executed to obtain major organs and tumors for ex vivo imaging. As shown in Fig. 5b, the tumor exhibited the highest fluorescence among all organs, further confirming the high tumor accumulation of GMHL. Quantitative analysis revealed that GMHL was quantified to be 2.24% ID/g (percentage of injected dose per gram of tissue) after 24 h post-injection in the tumor, only lower than that in the liver and spleen (Fig. 5c). Inspired by the high tumor accumulation, we subsequently evaluated the therapeutic effect of GMHL with US irradiation on a CT26 tumor-bearing mouse model. In this experiment, a total of 25 mice were randomly divided into five groups (n = 5): (1) Control, (2) US, (3) MHL + US, (4) GMHL, (5) GMHL + US. Tumors were then subjected to US irradiation (30 kHz; 3.0 W cm–2; 5 min) 24 h after intravenous injection, and thereafter the tumor volume and body weight of mice were monitored every other day (Fig. 5d). It was observed that tumor growth in the MHL + US treatment group was moderately inhibited compared to the control group. Remarkably, the GMHL + US treatment showed the most significant effect on tumor growth inhibition, with significantly prolonged the survival time (Fig. 5e-g). Moreover, we found that those treatments had no significant side-effect on the body weight of mice in the tested dose, indicating their excellent biosafety (Fig. 5h). Hematoxylin and Eosin (H&E) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of tumor sections revealed significant histological changes and cell apoptosis, indicating the effective therapeutic efficacy of GMHL plus US irradiation (Fig. 5i).

Considering that US treatment-induced cell death is accompanied by the secretion of tumor cell-derived exosomes, which highly express PD-L1, these exosomes mediate the onset of immunosuppression and contribute to poor therapeutic outcomes [30]. Subsequently, we analyzed the expression levels of PD-L1 in the tumor after different treatments using immunofluorescence staining. As shown in Fig. 5j, the MHL + US treatment exhibited a high PD-L1 fluorescence signal, indicating that such piezoelectric catalysis-mediated cell death promotes PD-L1 expression. Notably, the GMHL + US treatment group showed the lowest fluorescence signal, suggesting that the introduction of GW4869 effectively inhibited the secretion of exosomes, which in turn, sensitized the therapeutic effect.

Fig. 5
figure 5

In vivo anti-tumor ability of GMHL on a CT26 tumor model. (a) In vivo fluorescence imaging of CT26 tumor-bearing mice after i.v. injection of DIR-labeled GMHL NRs. (b) Ex vivo fluorescence imaging of major organs and tumors in CT26 tumor-bearing mice taken at 24 h after i.v. injection of GMHL NRs. (c) Biodistribution profiles of GMHL in CT26 tumor-bearing mice taken at 24 h after i.v. injection of GMHL. (d) Scheme illustrating the experiment schedule of GMHL combined with US stimulation on a CT26 tumor model. (e) Individual tumor growth curves and (f) mean tumor growth curves of CT26 tumor-bearing mice after different treatments as indicated. (g) Survival curves of CT26 tumor-bearing mice after different treatments as indicated. (h) Average body weights of CT26 tumor-bearing mice after different treatments as indicated. (i) H&E and TUNEL stained tumor slices collected after different treatments as indicated. (j) Immunofluorescence images of PD-L1 stained tumor slices collected after different treatments as indicated. Data are presented as mean ± SEM

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Therapeutic effect and immunological evaluation of GMHL on a B16F10 melanoma tumor model

Encouraged by the excellent anti-tumor efficacy of GMHL-mediated piezoelectric catalysis in the CT26 tumor-bearing mouse model, we next selected a highly aggressive B16F10 melanoma tumor model, commonly used in clinical immunotherapy, to further assess the anti-tumor efficacy of such treatment. As shown in Fig. 6a, C57BL/6 mice bearing B16F10 melanoma tumor were randomly divided into five groups (n = 5): (1) control, (2) US, (3) MHL + US, (4) GMHL, (5) GMHL + US. Tumors were then treated with US irradiation (30 kHz; 3.0 W cm–2; 5 min) 24 h after intravenous injection of various agents, and thereafter the tumor volume of each mouse was monitored every other day. Although melanomas tumors exhibited faster growth rates due to their high aggressiveness, tumor growth was moderately inhibited in the MHL + US-treated group compared to the control. Interestingly, it was found that GMHL + US treatment significantly inhibited tumor growth and prolonged survival time in mice, consistent with the previous results observed in CT26 tumor-bearing mice (Fig. 6b-c, S9). In addition, there were no significant changes in the body weight of mice throughout the monitoring period, indicating that these treatments exhibited good biosafety (Fig. 6d).

Fig. 6
figure 6

Therapeutic effect and immunological evaluation of GMHL on a B16F10 melanoma tumor model. (a) Scheme illustrating the experiment schedule of GMHL combined with US stimulation on a B16F10 melanoma tumor model. (b) Mean tumor growth curves of B16F10 tumor-bearing mice after different treatments as indicated. (c) Survival curves of B16F10 tumor-bearing mice after different treatments as indicated. (d) The average body weights of B16F10 tumor-bearing mice after different treatments as indicated. (e) Flow cytometric analysis of DC maturation and (f) corresponding quantitative results after different treatments as indicated. (g) Flow cytometric analysis of the tumor-infiltrated CD8+ T cells and (h) corresponding quantitative results after different treatments as indicated. (i) Flow cytometric analysis of the intratumoral NK1.1+ cells and (j) corresponding quantitative results after different treatments as indicated. (k) The secretion levels of TNF-α, (l) IFN-γ, (m) IL-1β within tumors after different treatments as indicated. Data are presented as mean ± SEM. The p values were calculated by one-way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001

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Inspired by the superior therapeutic efficacy of GMHL-mediated piezoelectric catalysis on suppression of tumor growth, we next conducted a systematic investigation to elucidate its antitumor immunomodulatory mechanisms. To this end, twenty B16F10 melanoma-bearing C57BL/6 mice (n = 4) were subjected to identical treatment protocols as previously described. On day 5, the mice were sacrificed and their tumors were excised, mechanically dissociated and enzymatically digested to generate single-cell suspensions. Then, those cells were stained with the appropriate fluorochrome-conjugated antibodies following the standardized protocol, and analyzed via flow cytometry. Results in Fig. 6e, f, S10 revealed robust dendritic cell maturation in the MHL + US treated group, with further amplification observed in the GMHL + US treated group. Notably, GMHL + US treatment induced a 10.3-fold increase in total leukocyte (CD45+ cells) infiltration within tumor compared to controls (Figure S11, 12), indicative of enhanced immunogenicity. Meanwhile, we found that GMHL + US treatment also augmented intratumoral effector CD8+ T cell populations (CD3+CD8+), with a concomitant elevation in cytotoxic activity as evidenced by Granzyme B expression within this subset (Fig. 6g, h, S13, 14), confirming potentiation of adaptive antitumor immunity. In addition, innate antitumor immune engagement was evident through a 3.7-fold increase in tumor-infiltrating natural killer (NK) cells (CD45+NK1.1+CD3−) relative to controls (Fig. 6i, j, S15). It was also observed that the GMHL + US treatment could greatly enhance the intratumoral secretion of effector cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), as well as proinflammatory cytokine IL-1β (Fig. 6k-m). These results suggest that the synergistic combination of US-activated piezoelectric catalysis with exosome suppression using GMHL can alleviate tumor immunosuppression and restore the anti-tumor immune responses.

Conclusions

In summary, we successfully constructed a novel Mn2+-doped HAP piezoelectric nanorod capable of delivering the exosome inhibitor GW4869, with controlled release triggered by the ROS generated during piezoelectric catalysis (GMHL). The doping of Mn2+ into HAP effectively reduces the bandgap, enhancing piezoelectric catalysis under US stimulation and enabling efficient ROS generation, which triggers the cleavage of the ketone-thiol bond in DSPE-TK-mPEG, thereby releasing GW4869. GMHL demonstrated significant intracellular ROS generation, promoted cancer cell death, and effectively blocked US-induced exosome secretion. When administered intravenously, GMHL exhibited significantly enhanced tumor accumulation. In diverse tumor models, GMHL combined with US stimulation effectively inhibited tumor growth. Additionally, GMHL treatment inhibited the secretion of tumor-derived exosomal PD-L1, enhanced T-cell activity, and bolstered anti-tumor immunity. These results highlight the potential of GMHL as a dual-function therapeutic strategy, utilizing both piezoelectric catalysis and exosome inhibition to enhance anti-tumor efficacy and promote immune system activation, offering a promising approach for effective cancer treatment.

Experimental section

Chemicals and reagents

Anhydrous calcium chloride (CaCl2), Manganous chloride (MnCl2), and anhydrous sodium dihydrogen phosphate (NaH2PO4) were obtained from Sinopharm Chemical Reagent Co., Ltd., China. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)]5000 (DSPE-PEG5000), Phospholipid-ketothionein-methoxypolyethylene glycol (DSPE-TK-mPEG), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol were obtained from Chongqing Yusi Pharmaceutical Technology Co., Ltd., China. GW4869 was purchased from Yeasen Biotechnology Co., Ltd., China. Methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were purchased from J&K Scientific Co., Ltd., China. Antibodies against cell surface markers for flow cytometry were purchased from Biolegend. All ELISA kits were purchased from ThermoFisher Scientific (China).

Synthesis of Mn-HAP NRs

Mn-doped HAP nanorods were synthesized using a hydrothermal method. Briefly, 0.210 g of anhydrous CaCl2 (1.9 mmol) and 0.0197 g of MnCl2 (0.1 mmol) were dissolved in 10 mL of deionized water under magnetic stirring. Then, 10 mL of NaH2PO4 solution (1.2 mmol) was was slowly added dropwise into the mixed solution under continuous stirring. After 30 min of stirring at room temperature, the pH of the solution was adjusted to 12 using 1 M NaOH solution. The resulting mixture was then transferred to a 50 mL Teflon-lined stainless steel autoclave and maintained at 200 °C for 24 h. After cooling to room temperature, the resulting product was collected by centrifugation (10000 rpm, 5 min), washed three times with deionized water and ethanol, respectively, and finally dispersed in 30 mL of ethanol. The final Mn-HAP nanorod suspension was stored at 4 °C for further use. For comparison, undoped HAP nanorods were synthesized under the same conditions without the addition of MnCl2.

Synthesis of GMHL and MHL NRs

A two-step method was employed to modify the obtained Mn-HAP. First, 10 mg of sodium n-octanoate dissolved in ethanol was added to the Mn-HAP solution (2 mL, 5 mg mL-1) under ultrasonic conditions, followed by vigorous sonication for 20 min. The resulting mixture was then centrifuged and redispersed in chloroform. Subsequently, cholesterol (5 mg), DPPC (10 mg), DSPE-PEG-5000 (16 mg), DSPE-TK-mPEG (8 mg), and GW4869 (0.2 mg) were added and stirred overnight. Finally, the chloroform was evaporated, and the resulting GMHL product was dissolved in an aqueous solution and stored at 4 °C for future use. The loading efficiency of GW4869 was detected by UV-Vis-NIR spectrophotometer.

MHL was synthesized using the same procedure as GMHL, except without the addition of GW4869.

Characterization

The morphologies and elemental composition of Mn-HAP were determined by transmission electron microscope (TALOS 200X, Sigma) and X-ray photoelectron spectroscopy (Thermo Kalpha). The absorption of the samples was measured by UV-Vis-NIR spectrophotometer (Thermo GENESYS50). X-ray diffraction patterns of the samples were measured by X-ray diffractometer (Bruker D8 advance). The solid-state UV diffuse reflectance spectra of the samples were measured using a UV-Vis spectrophotometer and the band gap variation was calculated (Shimadzu UV3600). The particle size and zeta potential of the samples were measured using a Malvern Zetasizer (Zetasizer Pro). Fourier transform infrared spectroscope was used to measure the functional groups of the samples (INVENIOS).

Evaluation of ·OH generation

To investigate the ability of MHL to induce ·OH generation under US simulation, 7.5 µL of MB (1.5 mg mL− 1) was added to 1 mL of deionized water containing MHL (90 µg mL− 1). After US exposure (30 kHz, 4.0 W cm− 2) for different time durations, the absorbance of MB at 662 nm was measured using a UV-Vis-NIR spectrophotometer.

Evaluation of 1O2 generation

To investigate the ability of MHL to induce 1O2 generation under US simulation, 1 mL of MHL (90 µg mL⁻¹) was mixed with 20 µL of DPBF (1 mg mL− 1) and subjected to US exposure (30 kHz, 4.0 W cm− 2) for different time durations. The absorbance change of DPBF at 416 nm was then measured using a UV-Vis-NIR spectrophotometer.

Evaluation of GW4869 release from GMHL under US simulation

GMHL was subjected to US exposure (30 kHz, 3.0 W cm− 2) for 2 min at 20-minute intervals, with the process repeated three times. The supernatant was collected by centrifugation, and the GW4869 content in the supernatant was quantified using a UV-Vis-NIR spectrophotometer at 360 nm.

Cellular culture

Murine CT26 and B16F10 melanoma cells (Cell Bank, Shanghai Institutes for Biological Sciences, CAS) were cultured in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO2 humidified incubator.

Cellular uptake of GMHL

CT26 cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured for 24 h. Then, dye DiD labeled GMHL was added and co-incubated for different time durations (1, 2, and 4 h). Cellular uptake was assessed using CLSM and flow cytometry.

Detection of intracellular ROS generation

CT26 cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured for 24 h. The cells were then co-incubated with GMHL or GHL for 2 h, followed by US exposure (30 kHz, 3.0 W cm− 2, 2 min). After continuing to incubate for 2 h, the cells were incubated with DCFH-DA (10 µM) for 30 min. Nuclei were stained with DAPI, and fluorescence was observed using a CLSM.

Calcium influx assay

CT26 cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured for 24 h. Then, the cells were co-incubated with GMHL or GHL for 2 h, followed by ultrasound exposure (30 kHz, 3.0 W cm− 2, 2 min). After another 2 h of incubation, the cells were stained with Fluo-4 AM probe (0.5 µM) for 30 min. Following three PBS washes, the cells were counterstained with DAPI and observed under a CLSM.

Detection of mitochondrial dysfunction

To assess the impact of GMHL on mitochondrial integrity, CT26 cells were seeded in 12-well plates at a density of 2 × 105 cells/well and treated with GMHL or GHL, followed by US exposure (30 kHz, 3.0 W cm− 2, 2 min). After 24 h, the cells were stained with the JC-1 reagent and analyzed using CLSM to evaluate changes in mitochondrial membrane potential.

Evaluation of cell killing effect of GMHL

CT26 cells were plated in 24-well plates at a density of 1 × 105 cells/well and cultured for 24 h. The cells were then co-incubated with GMHL or GHL for 2 h, followed by US exposure (30 kHz, 3.0 W cm− 2, 2 min). After another 24 h incubation, the cell viability was measured by a standard MTT assay.

The Calcein/PI dead and live dual-staining was performed to visually assess cell viability.

Quantification of exosome secretion

Exosomes were extracted from the cell supernatants after different treatments using the exosome isolation kit according to the manufacturer’s protocol (Beyotime). Then, the extracted exosomes were lysed using the lysis solution, and the proteins in the exosomes were quantified using the BCA protein assay kit.

Detection of ICD induction ability of GMHL

CT26 cells were seeded in 12-well plates at a density of 2 × 105 cells/well and treated with GMHL or GHL, followed by US exposure (30 kHz, 3.0 W cm− 2, 2 min). After 2 h, the cells were permeabilized with 0.1% Triton X-100 and blocked with 5% fetal bovine serum to prevent nonspecific binding. The cells were then washed three times with PBS and incubated with primary antibodies against HMGB1 and CRT at 37 °C for 1 h. After 30 min of incubation with secondary antibodies, the cells were visualized using CLSM. The extracellular ATP released from CT26 cells after various treatments was detected using a commercial ATP assay kit (Beyotime).

Animal experiments

Female Balb/c and C57BL/6J mice (6–8 weeks) were purchased from Jinan Xingkang Biotechnology Co., Ltd. All animal experiments were performed according to the experimental protocol approved by the Animal Welfare and Research Ethics Committee of Shandong First Medical University (W202303030131). Subcutaneous CT26 and B16F10 tumor models were established by injecting 2 × 106 cells of the corresponding cell lines into the back of mice.

To assess the tumor enrichment ability of GMHL, DIR labeled GMHL was intravenously injected into CT26 tumor-bearing mice, and then the mice were imaged by an in vivo imaging system at different time points. 24 h post-injection, these mice were executed and their major organs and tumors were removed for ex vivo fluorescence imaging.

To assess the therapeutic efficacy of GMHL-mediated piezoelectric catalysis and exosome inhibition, CT26 tumor-bearing mice were randomly assigned to five groups: (1) control, (2) US, (3) MHL + US, (4) GMHL, and (5) GMHL + US. Each mouse was intravenously administered with 200 µL of the respective nanorods. At 24 h post-injection, the designated groups underwent US treatment (30 kHz, 3.0 W cm⁻2, 5 min). Tumor dimensions (length, L, and width, W) were measured every two days using digital calipers, and tumor volume was calculated using the formula: L × W2/2. The body weight of each mouse was also recorded. Additionally, at 24 h after treatment, tumors were harvested for H&E and TUNEL staining analysis, as well as for PD-L1 immunofluorescent staining.

To evaluate the antitumor effect of GMHL-mediated piezoelectric catalysis and exosome inhibition in suppressing the growth of highly malignant B16F10 melanoma tumors, 25 C57BL/6 mice bearing with B16F10 melanoma tumors were randomly assigned into five groups (n = 5): (1) control, (2) US, (3) MHL + US, (4) GMHL, and (5) GMHL + US. Then, those mice were treated as described previously in the CT26 tumor model. Furthermore, to evaluate the underlying immunological mechanisms of GMHL-mediated piezoelectric catalysis, another set of five groups of B16F10 tumor-bearing mice (n = 4) received the same treatments. On day 5 after various treatments, these mice were sacrificed, and their tumors were harvested and processed into single-cell suspensions using a standard homogenization procedure. Then, those cells were stained with antibodies for further flow cytometry analysis. The CD8+ T cells were labeled with anti-CD3-FITC (clone 17A2, catalog: 100204) and anti-CD8-APC-Cy7 (clone 16-10A1, catalog: 104721). Dendritic cells were stained with anti-CD11c-FITC (clone N418, catalog: 117306), anti-CD80-APC (clone 16-10A1, catalog: 104714), and anti-CD86-PE (clone GL-1, catalog: 105008). Moreover, the secreted cytokines including interleukin IL-1β, TNF-α, and IFN-γ in the tumor supernatant were analyzed via ELISA kits.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 8.0). Data are presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare differences among multiple groups. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.005.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 52302350 and 22307057), Shandong Provincial Natural Science Foundation (No. ZR2024QC370, ZR2021QE112), the Natural Science Foundation of Jiangsu Province (No. BK20220401) and Shandong Provincial Taishan Scholars Program (tsqn202312237).

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  1. Xinran Qu and Qin Fan contributed equally.

Authors and Affiliations

  1. Science and Technology Innovation Center, Shandong First Medical University& Shandong Academy of Medical Sciences, Jinan, Shandong, 250117, P. R. China

    Xinran Qu, Yingying Liu, Jinqiao Zhang, Boyu Yuan, Xianzhou Cai & Ziliang Dong

  2. Biomedical Sciences College, Shandong Medicinal Biotechnology Centre, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, 250117, P. R. China

    Xinran Qu & Yingying Liu

  3. State Key Laboratory of Flexible Electronics (LoFE) & Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, Nanjing, Jiangsu, 210000, P. R. China

    Qin Fan

  4. Department of Thoracic Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, P. R. China

    Rulin Zhuang

  5. Dongzhimen Hospital, Affiliated to Beijing University of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, 100007, P. R. China

    Luli Ji

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Contributions

X.Q., and Q.F. contributed equally to this work. X.Q. and Q.F. designed the study, conducted the investigation, developed the methodology, curated the data, performed formal analysis, and wrote the main manuscript text. J.Z., B.Y., X.C., and L.J. provided technical support, and assisted in data analysis. Y.L., R.Z., and Z.D. provided supervision, and revised the manuscript. All authors reviewed and approved the final manuscript.

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Correspondence to Yingying Liu, Rulin Zhuang or Ziliang Dong.

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Qu, X., Fan, Q., Liu, Y. et al. Synergizing sono-piezo with exosome suppression using doping-engineered hydroxyapatite for potentiated tumor treatment through immunoactivation. J Nanobiotechnol 23, 495 (2025). https://doi.org/10.1186/s12951-025-03564-y

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