Abstract Reactive oxygen species (ROS), generated by sonosensitizers, play a pivotal role in tumor cell apoptosis during sonodynamic therapy (SDT), particularly for tumors located deep within tissues. Nevertheless, conventional sonosensitizers present limitations including inadequate ROS generation, insufficient tumor-specific accumulation, and associated adverse effects, significantly restricting their clinical applicability. To address these limitations, novel drug-free multifunctional nanoparticles (designated HGMP NPs) were synthesized. These NPs consist of mesoporous polydopamine (MPDA)-loaded protoporphyrin IX (PpIX), further surface-modified with glucose oxidase (GOx) and hyaluronic acid (HA), to achieve integrated photothermal, sonodynamic, and starvation-based tumor therapy. Upon exposure to near-infrared (NIR) irradiation (808 nm) combined with ultrasound (US), HGMP NPs exhibited pronounced synergistic anticancer effects. Specifically, the photothermal effect triggered by NIR irradiation effectively enhanced local oxygen supply within tumor sites, thus significantly augmenting ROS production and improving the therapeutic outcomes of SDT. Concurrently, GOx-mediated glucose depletion induced tumor starvation and produced hydrogen peroxide (H[2]O[2]), further exacerbating oxidative stress within the tumor microenvironment. Transcriptomic analysis revealed that ROS and TNF signaling pathways represented key mechanisms underlying tumor elimination by this multimodal synergistic strategy. Real-time PCR analysis and ELISA assays further validated activation of the TNF signaling pathway. Importantly, this study first confirmed the high biocompatibility and biosafety of HGMP NPs via serum metabolomics, demonstrating no detectable systemic metabolic perturbations. Collectively, the prepared HGMP NPs provide a rational paradigm for synergistic anticancer therapy. These findings highlight the potential of HGMP NPs as an exceptionally safe and effective nanoplatform for cancer treatment, offering valuable insights into future developments in cancer nanomedicine. Graphical Abstract [52]graphic file with name 12951_2025_3705_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03705-3. Keywords: Drug-free nanoparticles, Cancer treatment, Synergistic therapy, ROS storms, Sonodynamic therapy Introduction Cancer poses a persistent global public health challenge, driven by highly complex biological mechanisms and signaling pathways [[53]1]. Colorectal cancer (CRC), in particular, remains a leading contributor to cancer-related morbidity and mortality worldwide [[54]2]. Currently available conventional therapies for cancer mainly encompass chemotherapy, surgical resection, and radiation therapy [[55]3, [56]4]. However, these traditional modalities generally lack precise tumor-targeting capability, resulting in systemic toxicity and suboptimal therapeutic outcomes [[57]5]. Therefore, developing effective, biocompatible therapeutic strategies with reduced toxicity is imperative. In this context, multimodal therapeutic approaches have increasingly gained attention due to their enhanced efficacy and reduced adverse effects [[58]6–[59]8]. Multimodal therapy integrates two or more treatments, such as surgery, chemotherapy [[60]9, [61]10], radiotherapy [[62]11, [63]12], gene therapy [[64]13, [65]14], photodynamic therapy (PDT) [[66]15, [67]16], photothermal therapy (PTT) [[68]17, [69]18], and immunotherapy [[70]19]. This strategy effectively reduces side effects and significantly enhances therapeutic outcomes [[71]20]. However, studies involving multimodal treatments with three or more modalities remain limited, highlighting an area for further exploration and innovation. PTT represents an attractive non-invasive therapeutic option characterized by high tumor selectivity and substantial therapeutic effectiveness [[72]21]. PTT utilizes photothermal agents (PTAs) capable of transforming absorbed light energy into heat, thus inducing localized hyperthermia to eradicate cancer cells [[73]22, [74]23]. Among various PTAs, polydopamine (PDA) demonstrates favorable biocompatibility, ease of preparation, and excellent NIR photothermal conversion capability, thus making it highly suitable for photothermal applications [[75]24, [76]25]. However, the suboptimal drug-loading capacity of PDA limits its broader application. Fortunately, MPDA NPs, a novel class of PDA-derived nanostructures, possess a large surface area, thereby enhancing drug-loading capacity and therapeutic efficacy [[77]26–[78]31]. Thus, MPDA NPs have become promising photothermal nanocarriers. Despite these advantages, PTT is constrained by the limited penetration depth of NIR light, resulting in incomplete tumor eradication and restricted clinical applications [[79]3, [80]32]. SDT is an emerging cancer treatment that utilizes US to activate sonosensitizers, generating singlet oxygen (^1O[2]) and other ROS to eliminate cancer cells [[81]33, [82]34]. Moreover, US has superior tissue penetration depth, good patient compliance, and minimal damage to normal tissues [[83]35, [84]36]. PpIX, with excellent biocompatibility, accumulation potential, and ease of metabolism, can be activated by both light and US. Upon activation, it consumes molecular oxygen to generate cytotoxic ROS that irreversibly destroy tumor cells and tissues [[85]37, [86]38]. However, hypoxia, a common characteristic of tumor microenvironments, severely hampers ROS production and weakens SDT efficacy [[87]39–[88]42]. Therefore, combination therapy is essential for enhancing SDT outcomes. Cancer cells exhibit abnormal metabolic behavior, consuming glucose more aggressively than normal cells to support their rapid proliferation [[89]43–[90]45]. Consequently, blocking glucose and nutrients in tumors has emerged as an effective antitumor strategy. GOx catalyzes glucose oxidation into gluconic acid and H[2]O[2], disrupting glucose metabolism and inhibiting tumor cell growth [[91]46, [92]47]. Thus, GOx-mediated starvation therapy (ST) is widely utilized due to its potent ability to reduce glucose levels in tumor tissues and inhibit tumor proliferation [[93]48]. However, glucose deprivation alone is often insufficient to induce significant apoptosis in cancer cells, underscoring the necessity of complementary therapeutic approaches. Given the limitations of monotherapies, the development of multifunctional therapeutic strategies has received increasing attention. It is hypothesized that a multifunctional nanoplatform integrating PTT, SDT, and ST could overcome the respective limitations of each modality and improve cancer treatment outcomes. In this study, we designed a novel multifunctional tumor-targeting nanoplatform, HA/GOx-MPDA@PpIX nanoparticles (HGMP NPs), using MPDA as the core carrier loaded with PpIX and coated with GOx and HA for synergistic antitumor therapy. Within the tumor microenvironment, GOx interrupts the energy supply of tumor cells and generates highly toxic ROS. ROS production is further enhanced through SDT, effectively destroying cancer cells. Moreover, the mild photothermal effect induced by PTT under 808 nm NIR irradiation increases oxygen availability at the tumor site, enhancing ROS generation [[94]33]. Additionally, SDT compensates for the shallow tissue penetration of PTT, enabling more complete tumor eradication. Importantly, the catalytic function of GOx remains stable across various local tumor temperatures, ensuring continuous glucose depletion and ROS production throughout the therapeutic process. Collectively, this multimodal synergistic approach combining PTT, SDT, and ST achieved a superadditive synergistic effect (1 + 1 + 1 > 3), maximizing therapeutic outcomes (Scheme [95]1). Our study provides a robust and promising therapeutic strategy, offering an effective and safe nanoplatform for cancer treatment. Scheme 1. [96]Scheme 1 [97]Open in a new tab Schematic Illustration of Synthesis and Cascaded Antitumor Mechanisms of HGMP NPs Materials and methods Materials Dopamine hydrochloride (DA) was obtained commercially from Macklin (Shanghai, China). Pluronic F127, reduced glutathione, 1,3-diphenylisobenzofuran (DPBF), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were supplied by Aladdin (Shanghai, China). Protoporphyrin IX (PpIX), glucose oxidase (GOx), and glucose were purchased from Yuanye (Shanghai, China). Methanol, formic acid, dichloromethane, and acetonitrile (HPLC grade) were acquired from Merck & Co. (Billerica, MA, USA). Preparation of HGMP NPs Briefly, hyaluronic acid (HA, 40 mg) was dissolved in 20 mL Tris buffer solution (pH adjusted to 7.0) at room temperature. Subsequently, MPDA@PpIX NPs (M@P NPs, 20 mg) and glucose oxidase (GOx, 10 mg) were introduced into the HA-containing solution, followed by continuous stirring at ambient temperature for 2 h. The obtained precipitate was purified by centrifugation (12000 rpm, 15 min, 4 ℃) and washed three times with ultrapure water. After freeze-drying, the resulting NPs were labeled as HGMP NPs. Characterization The nanoparticle morphology and size were assessed via transmission electron microscopy (TEM). Particle size distribution and zeta potential measurements were carried out using a Malvern Zetasizer (Nano ZS90X, Malvern, UK). Fourier transform infrared spectroscopy (FTIR, NICOLET iS10) was utilized to evaluate the chemical composition. Ultraviolet-visible (UV-vis) absorption spectra were recorded using an Agilent 8453 spectrophotometer (Agilent Technologies, USA). Photothermal effect and photothermal stability To investigate photothermal performance, HGMP NPs were exposed to irradiation with an 808 nm NIR laser at varying power intensities (0.5, 1.0, 1.5, 2.0, and 2.5 W/cm^2) for 10 min. Additionally, different concentrations of HGMP NPs (0, 50, 100, 200, and 400 µg/mL) were irradiated for 10 min at 808 nm. The photothermal stability of HGMP NPs was evaluated by performing five cycles of irradiation (808 nm, 1.0 W/cm^2, 10 min per cycle), each followed by natural cooling to room temperature. Generation of H[2]O[2] HGMP NPs and GOx were incubated with glucose solutions at various concentrations for 30 min. After incubation, optical density (OD) values at 405 nm were measured following the addition of the H[2]O[2] detection solution. In vitro cell-targeted uptake experiments MC-38 cells were seeded in 6-well plates for 12 h and then treated with MPDA, M@P, or HGMP NPs (200 µg/mL) for 4 h. Cells were then washed three times with PBS and fixed with 4% paraformaldehyde. Finally, cells were observed by OLYMPUS FV3000 microscope. To validate the targeting capability derived from HA-CD44 receptor affinity, cells were pre-incubated with free HA for 3 h prior to the introduction of NPs. In vitro cellular uptake assays under different conditions were performed as follows: MC-38 cells seeded in 6-well plates were treated with HGMP NPs or free PpIX (200 µg/mL) for 4 h, then exposed to 808 nm NIR laser (1.5 W/cm²) or US (1 MHz, 3.0 W/cm^2, duty cycle 50%) for 10 min. Cell nuclei were subsequently labeled with DAPI, and fluorescence imaging was conducted using a fluorescence microscope. Concentration-dependent cellular uptake studies were conducted by incubating MC-38 cells (pre-seeded in 6-well plates for 12 h) with various HGMP NP concentrations (0, 12.5, 25, 50, 100, and 200 µg/mL) for 4 h, after which cellular uptake was observed with an inverted fluorescence microscope. For flow cytometry analysis, MC-38 cells seeded in 6-well plates for 12 h were exposed to HGMP NPs for 2, 4, or 6 h. Subsequently, cells were detached by trypsinization, resuspended in phosphate-buffered saline (PBS), and subjected to flow cytometry. Data were processed using FlowJo software (version 10). In vitro cytotoxicity Cytotoxicity of M@P and HGMP NPs was assessed using the CCK-8 assay. Briefly, MC-38 cells were treated with varying concentrations of M@P or HGMP NPs for 24 h. Similarly, NIH-3T3 cells were incubated with HGMP NPs under identical conditions. Cell viability was then determined by measuring optical density at 450 nm. Apoptosis analysis To analyze apoptosis, MC-38 cells seeded into 6-well plates were exposed to either 100 µg/mL of M@P or HGMP NPs or left untreated for 4 h. Subsequently, the harvested cells were labeled with Annexin V-FITC (5 µL) and PI (5 µL) under dark conditions for 10 min. The apoptosis rates were quantified by flow cytometry. In vitro ROS detection Intracellular ROS levels were measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). MC-38 cells were classified into six experimental groups: (1) PBS alone, (2) PBS combined with US, (3) M@P, (4) M@P combined with US, (5) HGMP, and (6) HGMP combined with US. After incubation and PBS washing, cells underwent US irradiation at an intensity of 1.5 W/cm^2, followed by the addition of DCFH-DA and incubation for 30 min at 37 °C. Fluorescence microscopy was employed to detect intracellular fluorescence. Transcriptomic analysis Transcriptomic analysis was employed to explore gene expression alterations induced by multimodal treatments. MC-38 cells received one of the following treatments: PBS, HGMP, HGMP + NIR, HGMP + US, or HGMP + NIR + US. Subsequently, total RNA was extracted from digested cell samples using TransZol Up reagent and immediately snap-frozen in liquid nitrogen. High-throughput sequencing was carried out by Wuhan Metware Biotechnology Co., Ltd. Real-time PCR (RT-PCR) Real-time PCR analysis was conducted following an established protocol [[98]49]. Briefly, total RNA from MC-38 cells was isolated using TransZol Up reagent (TransGen Biotech, Shanghai, China) and transcribed into cDNA with SweScript All-in-One RT SuperMix (Servicebio, Wuhan, China). The 2^⁻ΔΔCt method was applied to calculate relative gene expression, using GAPDH as the internal control. Primer sequences used are detailed in Table [99]S1. Animal and establishment Female C57BL/6J mice (NO. 202372508) and female BALB/c mice (No. 110324251103698673) were procured from Cavens Biological Technology (Changzhou, Jiangsu, China). To establish the colon tumor model, MC-38 cells (1.0 × 10^7 cells) were injected subcutaneously into the right gluteal region of 5-week-old C57BL/6J mice. Likewise, to establish the liver tumor model, H22 cells (5 × 10^6 cells) were injected subcutaneously into the right gluteal region of 6-week-old BALB/c mice. All animal procedures adhered strictly to protocols outlined by the Guide for the Care and Use of Laboratory Animals at Guangxi University and were approved by the university’s Animal Experimental Ethics Committee (GXU-2024-001 and GXU-2025-263). In vivo thermal imaging When tumor sizes reached roughly 150 mm^3, mice were intravenously administered 100 µL of either M@P or HGMP NPs (10 mg/kg), while the control group received an equivalent volume of PBS. Twelve h after nanoparticle administration, tumor area was irradiated by NIR (808 nm, 1.5 W/cm^2) for 10 min. Thermal images and corresponding tumor area temperatures were recorded using an NIR thermal camera throughout the process every 2 min. In vivo assessment of antitumor effects Tumor-bearing C57BL/6J and BALB/c mice were randomly allocated into eight experimental groups: (1) PBS, (2) PBS + US + NIR, (3) M@P, (4) M@P + US + NIR, (5) HGMP, (6) HGMP + US, (7) HGMP + NIR, and (8) HGMP + US + NIR. PBS or NPs (M@P or HGMP, both at 10 mg/kg) were intravenously administered every four days for two cycles. Tumor size and mouse body weight were recorded every two days. After 14 days, all mice were euthanized, and tumor volumes were measured using a vernier caliper according to the following calculation: graphic file with name d33e542.gif 1 Here, V denotes the tumor volume, with length and width representing the longest and shortest diameters, respectively. Histopathological analysis of tumor tissue and vital organs was carried out by H&E staining. Tumor sections were also subjected to terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. Additionally, ROS levels in tumor sections were evaluated by staining with DCFH-DA, and cellular localization was visualized using DAPI labeling. Throughout the experiment, no tumors exceeded the Maximum permitted size of 2,000 mm^3. Metabolomic analysis Serum samples derived from tumor-bearing and healthy C57BL/6J mice were prepared following established protocols [[100]50]. Briefly, serum was combined with precooled methanol-water solution (3:1, v/v) and dichloromethane, then vortexed thoroughly and incubated on ice for 10 min. After centrifugation at 13,000 rpm for 15 min at 4 °C, the supernatant was collected and dried under vacuum conditions at ambient temperature. The residue was subsequently redissolved in 100 µL of acetonitrile-water mixture (3:1, v/v), vortexed again, and centrifuged to obtain the final supernatant for metabolomic profiling. Quality control (QC) samples underwent the same preparative procedures. Finally, untargeted metabolomic analysis was executed using a Waters ACQUITY UPLC I-Class PLUS System (Waters, Milford, MA; SN: L22BSP766G) integrated with a desorption electrospray ionization source. ELISA analysis To quantify intracellular tumor necrosis factor-α (TNF-α) and Caspase 3 levels, MC-38 cells were seeded in 6-well plates and incubated with M@P NPs or HGMP NPs (200 µg/mL) for 4 h, followed by exposure to an 808 nm NIR laser (1.5 W/cm²) or US (1 MHz, 3.0 W/cm², 50% duty cycle) for 10 min. After a further 48 h of incubation, cells were harvested, and TNF-α and Caspase 3 levels were determined using ELISA kits. For in vivo cytokine analysis, serum samples were collected from each group after 14 days of antitumor treatment. The concentrations of interleukin-6 (IL-6) and TNF-α were measured using ELISA kits in accordance with the manufacturer’s. Statistical analysis Experimental results are expressed as the mean ± standard deviation (SD). Statistical comparisons between two groups were assessed using Student’s t-test, with significance levels marked as follows: (*) for P < 0.05, (**) for P < 0.01, and (***) for P < 0.001. Results and discussion Characterization of NPs In this study, MPDA NPs were synthesized via a simple one-pot method using TMB and Pluronic F127 as organic templates in an alkaline environment [[101]51, [102]52]. MPDA NPs were then obtained by removing the organic templates using organic solvents. MPDA NPs contain abundant functional groups capable of forming covalent bonds, facilitating further modifications [[103]53, [104]54]. To construct the multifunctional nanoplatform, PpIX was encapsulated within the mesoporous cavities of MPDA NPs. Finally, HA and GOx were grafted onto the surface through electrostatic adsorption and Michael addition reactions to prepare HGMP NPs. The TEM images in Fig. [105]1A and Fig. S1A demonstrated that MPDA NPs had uniform particle size and good dispersion. The synthesized HGMP NPs exhibited a spherical morphology with uneven surfaces (Fig. [106]1B and S1B). After encapsulating PpIX, the particle size of M@P NPs increased to 263.33 ± 6.41 nm, while the final size further increased to 429.07 ± 4.90 nm following surface modification with HA and GOx. The successful synthesis of HGMP NPs was confirmed by TEM and dynamic light scattering (DLS) analyses (Fig. [107]1C). Changes in the zeta potential (Fig. [108]1D) further substantiated HGMP NP formation. Specifically, PpIX encapsulation reduced the zeta potential from −20.6 ± 1.14 mV to −26.8 ± 0.26 mV, and further surface modifications with GOx and HA resulted in additional reductions, confirming successful NP functionalization. Fig. 1. [109]Fig. 1 [110]Open in a new tab Characterization of nanoparticles (NPs). TEM images of (A) MPDA NPs and (B) HGMP NPs. (C) Size distribution analysis of MPDA, M@P, and HGMP NPs. (D) Zeta potential of MPDA NPs, M@P NPs, PpIX, and HGMP NPs. (E) Nitrogen adsorption-desorption isotherms and pore-size distribution of MPDA NPs. (F) Cumulative release profiles of PpIX at various time points (0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h) under pH 7.5 and pH 6.5 conditions (n = 3). (G) UV-vis absorbance spectra of GOx, MPDA NPs, M@P NPs, PpIX, HA, and HGMP NPs. (H) FTIR spectra and (I) XRD patterns of HGMP NPs, M@P NPs, PpIX, MPDA NPs, and DA NPs (scale bar: 200 nm) MPDA NPs exhibited a type-IV adsorption isotherm (Fig. [111]1E), confirming their mesoporous characteristics. Brunauer–Emmett–Teller (BET) surface analysis demonstrated an area of 11.89 m²/g and a mean pore diameter of 18 nm, indicating substantial potential for drug encapsulation (Fig. [112]1E). As shown in Fig. S2, the loading content (LC) of PpIX in HGMP NPs was 31.66%, with an encapsulation efficiency (EE) of 63.32%. GOx loading efficiency was determined to be 44.74% based on standard curves (Fig. S3). These findings collectively indicated that MPDA NPs exhibited excellent drug delivery potential, making them highly suitable for therapeutic applications. PpIX release behavior from HGMP NPs was evaluated at pH 7.4 and pH 6.5, simulating physiological conditions and the acidic tumor microenvironment (TME), respectively (Fig. [113]1F). Under the simulated acidic tumor environment (pH = 6.5), 78.3% of PpIX was released after 72 h of incubation, while higher than physiological condition (pH = 7.4) about 32.3%. These results indicated that PpIX release could be significantly enhanced in acidic tumor environment, favoring tumor-specific ROS generation while minimizing side effects on normal tissues (Fig. [114]1F). Successful nanoparticle synthesis was further validated by UV-vis spectroscopy. The characteristic absorption peaks for PpIX at approximately 405 nm and three minor peaks within the 500–600 nm spectral range were evident, matching closely the profiles observed for M@P and HGMP NPs. This confirmed effective PpIX encapsulation into the MPDA NPs. Furthermore, the peak around 290 nm specific to HGMP NPs signified successful GOx integration, attributed to a redshift from π-π stacking interactions between aromatic ring structures present in MPDA and GOx [[115]55]. Collectively, these UV-vis spectra affirmed the successful assembly of HGMP NPs (Fig. [116]1G). Additionally, FTIR spectroscopy corroborated the preparation of HGMP NPs. A distinct peak at approximately 1621 cm^−1 corresponded to N–H bending vibrations in aromatic rings, whereas the peak at 1093 cm^−1 signified the stretching vibration of the C–O bond. Characteristic PpIX peaks at 1629 cm^−1 and 1550 cm^−1 were indicative of N–H deformation vibrations. Correspondingly, absorption bands at 1623 cm^−1 and 1536 cm^−1 observed in the M@P NPs further validated the successful incorporation of PpIX within MPDA NPs. HGMP NPs exhibited distinct FTIR signals characteristic of HA and GOx, demonstrating successful surface modification (Fig. [117]1H). Furthermore, X-ray diffraction (XRD) analysis supported successful HGMP NP synthesis (Fig. [118]1I). Collectively, these results (Fig. [119]1A-I) confirmed the successful synthesis of HGMP NPs. NP stability is crucial for effective antitumor treatment. To evaluate this, HGMP NPs were incubated in water, phosphate-buffered saline (PBS, pH 7.4), and 10% fetal bovine serum (FBS) for 72 h. Under all conditions, the dynamic size of HGMP NPs remained stable, indicating their satisfactory stability (Fig. S4 and S5). Additionally, as shown in Fig. S6, particle sizes of MPDA and HGMP NPs exhibited negligible changes after 7 days of storage at either 25 °C or 4 °C, further indicating the stability of NPs during storage conditions. Moreover, we evaluated the structural robustness of HGMP NPs after US exposure via TEM and DLS analyses (Fig. S7). No notable alterations in morphological or size distribution were observed, confirming the structural robustness of HGMP NPs. Collectively, these results exhibited the fabulous stability of HGMP NPs, providing a solid basis for their safe and effective application in anticancer therapy. Assessment of photothermal performance Considering previously documented photothermal characteristics of MPDA NPs [[120]56], we thoroughly assessed the photothermal capabilities of HGMP NPs derived from MPDA. As presented in Fig. [121]2A, increasing HGMP NP concentrations under constant near-infrared (NIR) irradiation at 808 nm (1.5 W/cm^2) resulted in enhanced temperature elevation, highlighting a concentration-dependent photothermal response. Additionally, laser power influenced photothermal performance; with a constant HGMP NP concentration, higher laser intensities significantly increased temperature (Fig. [122]2B). Specifically, irradiation at 808 nm and 2.5 W/cm^2 rapidly elevated the temperature to 70.4 °C, whereas a lower intensity of 0.5 W/cm^2 produced only a modest increase of 20.4 °C. These observations, verified visually using infrared thermal imaging (Fig. S8A and B), confirmed the superior photothermal conversion efficiency of HGMP NPs (Fig. [123]2A and B). Fig. 2. [124]Fig. 2 [125]Open in a new tab Performance evaluation of HGMP NPs. (A) Concentration-dependent temperature changes in HGMP NPs suspension under 808 nm NIR irradiation (1.0 W/cm^2) (B) Temperature changes of HGMP NPs under 808 nm NIR irradiation at various power densities (0.5, 1.0, 1.5, 2.0, and 2.5 W/cm^2). (C) Heating and cooling curves for five cycles of HGMP NPs. (D) “On-off” temperature changes in HGMP NPs suspension under 808 nm NIR irradiation. (E) Linear regression analysis of the cooling process. (F) Oxidation rate of DPBF in MPDA NPs, H[2]O, and HGMP NPs under US irradiation, respectively. (G) pH value changes of glucose suspension upon the addition of HGMP NPs, GOx, or HGMP NPs suspension without glucose. (H) H[2]O[2] concentration changes resulting from the reaction between glucose and free GOx or GOx released from the HGMP NPs at various glucose concentrations. (I) Relative GSH content in HGMP NPs suspension subjected to different formulations. (NS: P > 0.05) Further investigation into HGMP NP stability involved repeated heating-cooling cycles. Temperature variations were minimal throughout five consecutive cycles, indicating robust stability and excellent reusability (Fig. [126]2C). Moreover, photothermal conversion efficiency was quantitatively evaluated and determined to be 41.6%, based on analysis of heating-cooling profiles (Fig. [127]2D) and corresponding linear regression data (Fig. [128]2E). These results collectively highlighted the outstanding photothermal performance, stability, and reusability of HGMP NPs. Assessment of sonodynamic and starvation performance PpIX is widely recognized as an effective sonosensitizer, capable of generating ^1O[2] upon US irradiation [[129]57]. To assess PpIX-mediated ^1O[2] generation, 1,3-diphenylisobenzopropylfuran (DPBF) was utilized as a probe. The degradation of DPBF, indicated by reduced UV-vis absorbance at 410 nm, served as evidence of ^1O[2] production. As shown in Fig. [130]2F, DPBF degradation was significantly faster in the HGMP NP suspension (200 µg/mL) compared to MPDA NPs and H₂O, confirming substantial ^1O[2] generation upon US irradiation. Moreover, to identify specific ROS species generated, electron paramagnetic resonance (EPR) probe 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was employed to detect the •OH. As shown in Fig. S9, no obvious •OH signals were detected in the group of MPDA and MPDA + US group. But a characteristic 1:2:2:1 quadruple signal occurred in HGMP + US group much stronger than HGMP group, indicating that •OH could be intensively generated by US-treated sonosensitizer PpIX (Fig. S9). These results indicated that HGMP NPs have significant potential for sonodynamic cancer therapy. GOx catalyzes glucose decomposition into H[2]O[2] and gluconic acid. To confirm GOx catalytic functionality within HGMP NPs, we analyzed the formation of these reaction products. As gluconic acid reduces pH [[131]41], pH measurements provided an indirect quantitative approach. As depicted in Fig. [132]2G, HGMP NP suspensions without glucose maintained stable pH values (~ 6.81). However, in the presence of glucose, a significant pH reduction was observed over time in both free GOx and HGMP NP groups, demonstrating effective catalytic glucose conversion into gluconic acid. These results confirmed that GOx catalytic activity remained intact after NP modification and that HGMP NPs exhibited strong catalytic capacity (Fig. [133]2G). Importantly, GOx activity remained stable even at elevated temperatures (approximately 60 °C; Fig. S10). Additionally, H[2]O[2] levels in HGMP NP suspensions were measured. As shown in Fig. [134]2H, increasing glucose concentrations led to markedly elevated H[2]O[2] production in both free GOx and HGMP NP groups, further confirming successful glucose-to-H[2]O[2] conversion. These findings suggested intact GOx activity within HGMP NPs, effectively blocking energy supply and generating toxic ROS for antitumor treatment (Fig. [135]2G and H). Glutathione (GSH), an abundant endogenous reductant in tumor cells, protects against peroxide and ROS-induced damage [[136]58]. To ensure sufficient ROS generation, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) was selected as a probe to detect GSH depletion. No significant change in relative GSH levels was observed in HGMP NP suspensions, with or without 808 nm NIR irradiation, in the absence of glucose. In contrast, in the presence of glucose, relative GSH levels significantly decreased, suggesting HGMP NPs effectively depleted GSH through starvation therapy (ST). Furthermore, combined treatment with US and NIR irradiation resulted in a more pronounced reduction in GSH levels, indicating enhanced ROS generation and increased GSH consumption (Fig. [137]2I). Collectively, HGMP NPs demonstrated favorable photothermal effects, sonodynamic activity, and GOx catalytic performance, underscoring their significant potential for cascaded synergistic PTT, SDT, and starvation therapy (ST) in cancer treatment. In vitro cellular experiments Intracellular uptake of NPs Before evaluating in vitro activity, cellular uptake of different NPs (MPDA, M@P, HGMP, and HA + HGMP) was analyzed using confocal laser scanning microscopy (CLSM) (Fig. [138]3A). As shown in Fig. [139]3A, HGMP NPs exhibited stronger fluorescence than M@P NPs, likely due to HA-mediated active targeting of CD44 receptors. To further confirm this targeting mechanism, cells were pretreated with free HA to block CD44 receptors [[140]59]. This pretreatment markedly reduced the fluorescence intensity, suggesting enhanced uptake of HGMP NPs was primarily due to HA targeting (Fig. [141]3A). Consistent with the fluorescence imaging results, this finding was further quantitatively validated by flow cytometry analysis (Fig. S11). Fig. 3. [142]Fig. 3 [143]Open in a new tab In vitro cellular experiments. (A) Fluorescence images of MC-38 cells incubated with MPDA, M@P, and HGMP NPs with or without HA pre-treatment (scale bar: 50 μm). (B) Relative cell viability of MC-38 cells with different concentrations of M@P and HGMP NPs after varied treatments. (C) FDA/PI live-dead cell staining to access apoptosis in MC-38 cells. (D) Intracellular ROS generation capacity illustrated in MC-38 cells under different treatments. (E) Intracellular ^1O[2] generation capacity illustrated of MC-38 cells under different treatments (scale bar: 50 μm). (NS: P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001) Subsequently, time-dependent uptake of HGMP NPs was evaluated (Fig. S12A). Fluorescence intensity progressively increased at 2, 4, and 6 h, indicating time-dependent internalization. Similarly, concentration-dependent uptake was observed when MC-38 cells were incubated with increasing HGMP NP concentrations (Fig. S12B). Upon 808 nm NIR or US irradiation, cellular uptake efficiency of HGMP NPs significantly increased (Fig. S12C). This enhancement might be attributed to increased membrane permeability from photothermal effects and vacuolization induced by US treatment, facilitating NP internalization. Prior studies suggest caveolae-mediated endocytosis as a predominant internalization pathway for MPDA-based NPs [[144]60–[145]65]. Flow cytometry analysis further confirmed efficient cellular internalization of HGMP NPs (Fig. S12D-F). Collectively, these results demonstrated successful tumor targeting and effective uptake of HGMP NPs by tumor cells (Fig. [146]3A and S12A-F). Cell growth Inhibition assay Cytotoxicity and cell growth inhibition induced by HGMP NPs were evaluated using CCK-8 assays, flow cytometry analysis, and live-dead cell staining (FDA: green fluorescence; PI: red fluorescence). Preliminary results demonstrated negligible cytotoxicity towards NIH-3T3 cells at concentrations up to 200 µg/mL, thus confirming excellent biocompatibility (Fig. S13). In contrast, viability of MC-38 cells decreased upon HGMP NP treatment, likely due to HA-mediated targeting and GOx-induced starvation therapy (Fig. [147]3B). Importantly, no significant cytotoxicity was observed in MC-38 cells treated only with 808 nm NIR or US irradiation (Fig. [148]3B). However, upon combined NIR and US irradiation, the cytotoxicity of M@P and HGMP NPs against MC-38 cells increased concentration-dependently, with HGMP NPs exhibiting superior efficacy (Fig. [149]3B). Notably, the HGMP + NIR + US group demonstrated less than 5% cell viability at 200 µg/mL, highlighting robust therapeutic effects achieved through synergistic PTT, SDT, and ST (Fig. [150]3B). The cytotoxic effects were further visualized using fluorescence microscopy after live-dead cell staining with FDA and PI. The control and M@P groups predominantly displayed green fluorescence, indicating viable cells. In contrast, the HGMP + US + NIR group exhibited intense red fluorescence, signifying extensive cell death (Fig. [151]3C). These observations aligned with CCK-8 assay results, further validating potent antitumor effects mediated by synergistic PTT, SDT, and ST (Fig. [152]3C). Flow cytometry experiments also confirmed enhanced cell death in the HGMP + NIR + US group (Fig. S14). Collectively, these results demonstrated superior synergistic cytotoxic effects of HGMP NPs compared to monotherapies, underscoring their significant potential for antitumor therapy (Fig. [153]3B and C). In vitro photothermal and sonodynamic evaluation After confirming effective uptake and cytotoxicity, the photothermal and sonodynamic performance of HGMP NPs in tumor cells was evaluated. First, photothermal performance of M@P and HGMP NPs was assessed using infrared thermal imaging. MC-38 cells treated with M@P or HGMP NPs exhibited substantial temperature increases under 808 nm NIR irradiation, with HGMP NPs showing a greater effect than M@P NPs. In contrast, MC-38 cells treated with PBS showed only slight temperature elevation (Fig. S15), confirming efficient cellular accumulation and superior photothermal properties of HGMP NPs. Next, intracellular ROS production was measured using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which emits green fluorescence upon ROS oxidation (Fig. S16A). Compared to PBS, PBS + US, and M@P groups, HGMP and M@P + US groups exhibited moderate fluorescence, indicating ROS generation upon US irradiation. The HGMP + US group displayed significantly stronger fluorescence, reflecting enhanced ROS production from the synergistic effects of ST and SDT (Fig. [154]3D). Besides, flow cytometry analysis (Fig. S17) was performed to quantitatively assess ROS generation in MC-38 cells, consistent with the fluorescence imaging results shown in Fig. [155]3D. Moreover, ROS production was assessed at multiple time points to investigate the dynamics of ROS generation. As shown in Fig. S18, the HGMP + US group exhibited the highest fluorescence intensity at 20 min, followed by a gradual decline from 20 to 120 min, reflecting the kinetic changes of ROS generation during SDT-mediated antitumor activity. Additionally, ^1O[2] generation was assessed using the ^1O[2] sensor green (SOSG) probe. Under US irradiation, ^1O[2] produced by HGMP NPs converted SOSG to fluorescent SOSG-EP (Fig. S16B). Fluorescence images showed the HGMP + US group had the highest fluorescence intensity, indicating substantial ^1O[2] generation (Fig. [156]3E). Quantitative analysis by ImageJ software further confirmed significantly higher fluorescence intensity in the HGMP + US group compared to other groups, emphasizing effective cellular targeting and robust ^1O[2] generation (Fig. S19). Collectively, these results indicated that HGMP NPs, upon exposure to 808 nm NIR and US irradiation, exhibited excellent photothermal performance and ROS generation, providing a foundation for effective multimodal cancer therapy. Potential mechanisms of HGMP + NIR + US mediated synergistic therapy To elucidate potential mechanisms underpinning synergistic therapeutic efficacy of HGMP NPs combined with NIR and US irradiation, mRNA sequencing analyses were performed on MC-38 cells. Principal component analysis (PCA, Fig. S20A) confirmed data consistency and reliability. Differentially expressed genes (DEGs) among various treatment groups relative to controls were visualized via Venn diagram (Fig. S20B). A volcano plot was used to illustrate the DEGs following treatment with HGMP combined with NIR and US irradiation. As depicted in Fig. [157]4A, compared to the PBS group, 197 genes were upregulated, and 250 genes were downregulated. The corresponding heat map (Fig. [158]4B) highlighted extensive gene expression modulation by HGMP, emphasizing the synergistic effects of PTT, SDT, and ST. Fig. 4. [159]Fig. 4 [160]Open in a new tab Transcriptome sequencing and RT-PCR analysis. (A) Volcano plots illustrating differentially expressed genes (DEGs) in MC-38 cells treated with HGMP + NIR + US irradiation compared to PBS. (B) Heat map depicting the expression profiles of DEGs in MC-38 cells following HGMP + NIR + US treatment and PBS treatments. (C) KEGG pathways enrichment analysis of DEGs identified in the HGMP + NIR + US treatment and PBS treatments. (D, E) Gene set enrichment analysis (GSEA) for biological pathways in the HGMP + NIR + US treatment and PBS treatments, based on the Gene Ontology (GO) database. Relative mRNA expression levels of TNF-α (F), TNFR1 (G), Caspase 8 (H), and Caspase 3 (I) after different treatments, detected by RT-PCR. (J) GO enrichment analysis for functional categories in the HGMP + NIR + US treatment and PBS treatments. (NS: P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001) Subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to identify critical signaling pathways associated with the observed responses. The KEGG enrichment map (Fig. [161]4C) revealed the top 20 significantly enriched pathways, notably apoptosis-related pathways such as the TNF signaling pathway. Additional Gene Set Enrichment Analysis (GSEA) based on KEGG annotations confirmed significant activation of these pathways (Fig. [162]4D and E), suggesting their crucial involvement in inducing apoptosis during combined treatment. Finally, RT-PCR analyses validated these findings by quantifying expression levels of essential genes within the TNF signaling pathway. As shown in Fig. [163]4F-I, expression levels of TNF-α, TNFR1, Caspase 8, and Caspase 3 significantly increased in the HGMP, HGMP + NIR, HGMP + US, and HGMP + NIR + US groups, confirming activation of the TNF signaling pathway. Notably, the HGMP + NIR + US group exhibited the most pronounced upregulation, supporting the multimodal synergistic antitumor effect mechanism. These findings collectively confirmed that activation of the TNF signaling pathway plays a critical role in enhancing the antitumor efficacy of HGMP NPs. Gene Ontology (GO) analysis was performed to elucidate functional categories of DEGs, classified into biological processes (BP), cellular components (CC), and molecular functions (MF). As illustrated in Fig. [164]4J, DEGs associated with biological processes were mainly involved in metabolic processes, cellular processes, and biological regulation. Cellular components predominantly included protein-containing complexes, and molecular functions mainly involved catalytic activity and binding. Furthermore, GSEA comparing the HGMP + NIR + US group to single-modality groups (HGMP + NIR or HGMP + US) revealed additional upregulated pathways (Fig. S20C and D), providing further evidence of synergistic effects. ELISA analysis further confirmed the critical role of the TNF signaling pathway in mediating antitumor mechanisms (Fig. S21A and B). In summary, these results indicated that the combination of HGMP NPs, NIR, and US irradiation induced significant apoptosis in MC-38 cells. The analysis also highlighted the critical role of ROS in synergistic therapy, emphasizing the prominent involvement of the TNF pathway. In vivo biodistribution and photothermal assessment Effective tumor accumulation of HGMP NPs is critical for precise therapeutic outcomes. Therefore, the biodistribution of these NPs was investigated in tumors using fluorescently labeled PpIX. Following intravenous administration of either M@P or HGMP NPs into mice bearing MC-38 tumors, nanoparticle accumulation was monitored at specified intervals. Fluorescence imaging showed a progressive enhancement in fluorescence signal at the tumor region over time, presumably attributable to the enhanced permeability and retention (EPR) effect. Importantly, HGMP NPs exhibited significantly higher fluorescence intensity compared to M@P NPs, reflecting improved tumor targeting facilitated by HA modification (Fig. [165]5A and S22). Fig. 5. [166]Fig. 5 [167]Open in a new tab In vivo biodistribution and photothermal assessment of HGMP NPs. (A) In vivo representative fluorescence imaging of PpIX fluorescence at different time points after intravenous injection of M@P NPs and HGMP NPs via the tail vein (n = 3). (B) Representative ex vivo fluorescence images of PpIX in harvested organs. (C) Real-time infrared thermal imaging and (D) corresponding photothermal heating curves of tumor sites under NIR irradiation (808 nm, 1.5 W/cm^2, 10 min), respectively. Subsequently, tumors along with Major organs were collected at 12 and 24 h post-injection for ex vivo fluorescence imaging. Consistent with in vivo imaging findings, ex vivo analysis revealed substantially stronger fluorescence signals in tumors from the HGMP group (Fig. [168]5B). Quantitative ex vivo biofluorescence analysis of major organs and tumor after 12 h post-administration further demonstrated the superior tumor-targeting accumulation of HGMP NPs (Fig. S23). Fluorescence intensity primarily appeared in kidneys and livers, suggesting these organs as primary sites for NP metabolism and elimination (Fig. [169]5B). Infrared thermal imaging was further employed to confirm tumor-specific enrichment and the photothermal efficacy of HGMP NPs. At 12 h post-injection, MC-38 tumor-bearing mice were exposed to 808 nm NIR irradiation for 10 min. Infrared thermal images were obtained (Fig. [170]5C), and temperature increases at the tumor site were recorded at 2-min intervals. The tumor temperature in the HGMP group significantly increased from approximately 30.76 ± 0.32 °C to 61.80 ± 0.30 °C, notably higher than temperatures observed in PBS and M@P groups under identical conditions (Fig. [171]5D). These findings confirmed that HGMP NPs exhibited favorable active targeting ability and provided robust photothermal effects in vivo. In vivo synergistic antitumor therapy Motivated by the promising in vitro anticancer effects, the synergistic antitumor efficacy of HGMP NPs in vivo was examined using an MC-38 tumor xenograft mouse model. The experimental procedure is depicted in Fig. [172]6A, where mice bearing subcutaneous MC-38 tumors were randomly allocated into eight treatment groups. Based on previous fluorescence imaging data, tumors were treated with 808 nm NIR irradiation (1.5 W/cm^2 for 10 min) or US (1 MHz, 3.0 W/cm^2, duty cycle 50%) for 10 min at 12 h after nanoparticle administration, specifically on days 2 and 8 of the treatment schedule. Fig. 6. [173]Fig. 6 [174]Open in a new tab In vivo antitumor efficacy of HGMP NPs. (A) Schematic illustration of the in vivo experiment procedure. (B) Tumor growth curves of MC-38 tumor-bearing mice over 14 days of treatment (n = 5). (C) Tumor inhibition rate calculated from tumors resected after 14 days of treatment (n = 5). (D) Representative photographs of tumors resected from mice in different treatment groups (n = 5). (E) H&E and (F) TUNEL staining of tumor tissues to evaluate antitumor outcome across treatment groups (scale bar: 100 μm). (G) DCFH-DA staining of tumor tissues to assess ROS levels after different treatments (scale bar: 100 μm). (NS: P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001) Tumor volumes and body weights were monitored every two days. As shown in Fig. [175]6B, tumor volumes in mice treated with PBS + NIR + US showed no significant differences from PBS or M@P groups, indicating minimal effects of M@P NPs and irradiation alone. Tumor growth was modestly inhibited in the HGMP group, possibly due to GOx-mediated starvation therapy. Tumors in HGMP + NIR or HGMP + US groups showed moderate inhibition (Fig. [176]6B), suggesting monotherapy was suboptimal. The highest tumor suppression (approximately 91.05 ± 3.12%) occurred in the HGMP + NIR + US group (Fig. [177]6C), attributed to enhanced tumor accumulation and the combined synergistic effects of PTT, SDT, and ST. At the end of treatment, mice were euthanized, and their vital organs and tumors were collected. Extracted tumor photographs (Fig. [178]6D) demonstrated similar inhibition trends among groups, confirming efficient HGMP NP accumulation and potent multimodal therapy (Fig. [179]6D). Additionally, hematoxylin-eosin (H&E) and TUNEL staining of tumor tissues (Fig. [180]6E and F) revealed minimal necrosis in PBS, PBS + NIR + US, and M@P groups, while the HGMP + NIR + US group exhibited extensive tissue damage and DNA fragmentation. ROS generation in tumor sections (DCFH-DA staining) further confirmed enhanced apoptosis in the HGMP + NIR + US group (Fig. [181]6G). Among H&E, TUNEL, and ROS staining results demonstrated that HGMP NPs induced strong tumor apoptosis via synergistic effects (Fig. [182]6E-G). Subsequently, to further investigate the universality of the HGMP NPs platform, H22 tumor-bearing mice were established and treated with the same treatment regimen as the MC-38 tumor xenograft model. Notably, the treatment yielded the most pronounced antitumor effects in the HGMP + NIR + US group, with a tumor inhibition rate was 81.1% (Fig. S24A and B). A more direct visualization of the antitumor effects of HGMP was provided by digital images (Fig. S24C). Throughout the experimental period, no obvious changes in body weight were observed in H22 tumor-bearing mice, indicating the biosafety of HGMP NPs (Fig. S24D). Histological analysis through H&E and TUNEL staining of tumor sections revealed considerable tissue necrosis and DNA fragmentation in the HGMP + NIR + US group, which were markedly more prominent compared to the other treatment groups (Figure S23E), further confirming the broad applicability of the HGMP NPs platform. Taken together, these findings underscore the potential of HGMP NPs as a robust and promising platform for cancer therapy (Fig. S24A-E). In vivo biosafety and biocompatibility evaluation Given the potential distribution of NPs to healthy tissues [[183]66], biosafety assessment is critical. Multiple approaches were employed to rigorously evaluate the safety of HGMP NPs. Metabolomics analysis was performed to investigate the impact on endogenous metabolism. Orthogonal partial least squares discriminant analysis (OPLS-DA) was initially employed to visualize group differences (Fig. [184]7A). A 200-iteration permutation test confirmed the reliability of the OPLS-DA model (Fig. [185]7B). Relationships among metabolites across HGMP, HGMP + US, HGMP + NIR, and HGMP + NIR + US groups were depicted using a Venn diagram (Fig. S25A). Differential metabolites between groups were illustrated in volcano plots (Fig. [186]7C-F and Fig. S25B). Differential metabolites in the HGMP + US group were predominantly upregulated, whereas those in the HGMP + NIR group were downregulated. Importantly, the metabolic profile of the HGMP + NIR + US group closely resembled the HGMP group, indicating that multimodal therapy mitigated metabolic disruptions caused by monotherapy. Heatmap analysis further corroborated this conclusion (Fig. [187]7G and Fig. S25C-G). Collectively, these findings demonstrated negligible metabolic impact of HGMP NPs under combined NIR and US treatment, confirming excellent biosafety. Fig. 7. [188]Fig. 7 [189]Open in a new tab Effects on metabolic homeostasis of HGMP NPs for antitumor therapy via serum metabolomic analysis. (A) OPLS-DA model diagrams showing the separation of metabolomic profiles between treatment and control groups. (B) Permutation test confirming the robustness and reliability of the OPLS-DA model. (C-F) Volcano map depicts highlighting differential metabolites between HGMP group vs. PBS group, HGMP + US group vs. PBS group, HGMP + NIR group vs. PBS group, and HGMP + NIR + US group vs. PBS group. (G) Heatmap of differential metabolites across treatment groups, illustrating metabolite variability and trends. To detect in vivo immunotoxicity, blood serum samples were also utilized to evaluate for TNF-α and IL-6 levels. The obtained results indicated that no significant change in TNF-α and IL-6 levels was observed after various treatments (Fig. S26A and B). This might due to immune response activated by HGMP NPs, inducing macrophages to clear NPs during the treatment period during the treatment period [[190]67, [191]68], ultimately resulting in no difference in the levels of TNF-α and IL-6 between the treatment group and the PBS group. Therefore, serum cytokine levels suggested that HGMP NPs did not trigger immunotoxicity after various treatments, demonstrating superb nanoparticle biocompatibility. Throughout the treatment duration, no notable body weight fluctuations occurred in MC-38 tumor-bearing mice, suggesting minimal systemic toxicity or adverse effects on overall mouse development (Fig. S27). Additionally, histological examination of key organs via H&E staining indicated no significant tissue injury in either experimental or control groups (Fig. S28), further confirming excellent nanoparticle biosafety. Furthermore, hepatic and renal toxicity was evaluated in MC-38 tumor-bearing mice following treatment. Liver and kidney weight indices showed no abnormalities (Fig. S29A and B). Biochemical markers of liver function (AST and ALT) and kidney function (BUN and CRE) were also measured (Fig. S29C-F). No marked disparity was observed between the treatment groups and the PBS control group, further supporting the biosafety of HGMP NPs (Fig. S29A-F). To evaluate the hemocompatibility of HGMP NPs, a hemolysis assay was performed (Fig. S30). Red blood cells were incubated with varying HGMP NP concentrations (10–250 µg/mL), with PBS and ultrapure water serving as negative and positive controls, respectively. Following incubation, hemolysis rates and images were documented. Prominent erythrocyte hemolysis was observed only in the positive control, whereas negligible hemolysis appeared in the negative control. Remarkably, even at the highest tested concentration (250 µg/mL), hemolysis rates induced by HGMP NPs remained below 5%, indicating superior hemocompatibility (Fig. S30). Collectively, these findings suggest that HGMP NPs exhibit minimal impact on systemic metabolism and possess favorable hemocompatibility. Importantly, synergistic treatment not only alleviated metabolic perturbations induced by monotherapies but also did not induce organ toxicity, supporting the excellent biosafety of HGMP NPs for potential clinical applications. To assess potential adverse effects of 808 nm NIR irradiation or US exposure on normal mouse tissues, histological analyses via H&E staining were conducted on the skin tissues of tumor-bearing mice subjected to the respective irradiation treatments. As shown in Fig. [192]8A, no significant changes or abnormal skin thickening occurred, confirming neither irradiation modality induced damage to healthy tissues. Fig. 8. Fig. 8 [193]Open in a new tab Biosafety evaluation of HGMP NPs via histopathological and blood biochemical analyses. (A) H&E staining of the skin from tumor-bearing MC-38 mice after different treatments (scale bar: 200 μm). (B) H&E staining imaging of major organs (heart, lung, liver, kidney, and spleen) from mice treated with HGMP NPs (scale bar: 50 μm). Furthermore, the systemic toxicity of HGMP NPs was evaluated in healthy C57BL/6J mice intravenously administered with HGMP NPs, followed by an observational period of 28 days. Histopathological assessments of major mouse organs revealed no detectable pathological abnormalities (Fig. [194]8B), thus further validating the biocompatibility and safety profile of HGMP NPs in vivo. Additionally, blood biochemical analysis was performed in mice treated with HGMP NPs. Results showed no apparent abnormalities in blood parameters at multiple time points, indicating that HGMP NPs did not induce short-term or long-term hematological toxicity (Fig. S31). Consequently, these findings confirmed the high biocompatibility and biosafety of HGMP NPs, highlighting their promise as an exceptionally safe and effective nanoplatform for multimodal antitumor therapy. Conclusion In summary, we successfully designed novel multifunctional NPs (HGMP NPs) that integrate PTT, SDT, and ST for synergistic antitumor therapy. HA-mediated active targeting of CD44 receptors facilitated tumor-specific accumulation of HGMP NPs. Upon 808 nm NIR and US irradiation, HGMP NPs demonstrated potent photothermal effects and sonodynamic performance. NIR-triggered PTT effectively ablated tumor cells while simultaneously enhancing SDT efficacy, further amplifying ROS production. Concurrently, GOx catalyzed glucose conversion to H[2]O[2], synergizing with SDT to induce a tumor-specific ROS storm, leading to cell apoptosis. Moreover, transcriptome sequencing identified the ROS and TNF signaling pathways as major mechanisms underlying tumor elimination. This was further corroborated by the upregulated expression of key TNF pathway genes validated by RT-PCR and ELISA assays. Notably, metabolomic analysis revealed exceptional biocompatibility of these drug-free NPs for the first time, indicating highly safe and effective cancer therapy. Collectively, our findings offer a promising synergistic approach, and this biocompatible nanoplatform is anticipated to have significant clinical translation potential for multimodal cancer therapy. Supplementary Information [195]Supplementary Material 1.^ (51.3MB, docx) Author contributions Mingsen Wen: Writing - review & editing, Writing - original draft, Visualization, Validation, Methodology, Formal analysis, Data curation. Hongwei Chen: Writing - review & editing, Writing - original draft, Visualization, Validation, Methodology, Formal analysis, Data curation. Song Xu: Data curation, Validation, Formal analysis. Shanyi Yang: Validation, Formal analysis. Xuan Guan: Validation. Xuancheng Wang: Data curation. Zhiyong She: Validation. Zhijuan Wei: Validation. Ying Tong: Data curation. Jichu Luo: Software. Qixuan Qin: Validation. Xueting Lin: Validation. Yuru Tan: Validation. Yanying Nong: Supervision, Funding acquisition. Qisong Zhang: Writing - review & editing, Supervision, Funding acquisition, Conceptualization. Funding This work was supported by the Guangxi Key R&D Program Projects (No. GuikeAB25069047); Natural Science Foundation of Guangxi (No. 2023GXNSFBA026255); Fund Project of Guangxi Zhuang Autonomous Region Administration of Traditional Chinese Medicine (No. GXZYA20240312). Data availability No datasets were generated or analysed during the current study. Declarations Ethics approval and consent to participate All animal experiments were conducted in accordance with the protocols approved by the Guide for the Care and Use of Laboratory Animals of Guangxi University and were approved by the Animal Experimental Ethics Committee of Guangxi University (Approval Number: GXU-2024-001 and GXU-2025-263). Consent for publication All authors agree to submit the manuscript for publication. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Mingsen Wen and Hongwei Chen contributed to the work equally. Contributor Information Yanying Nong, Email: yanyingnong@126.com. Qisong Zhang, Email: zhangqisong@gxu.edu.cn. References