Image-guided therapy of colorectal cancer using a zirconium coordinated nanosensitizer.

Introduction
Colorectal cancer (CRC) is a major global health burden [1]. In 2022, the International Agency for Research on Cancer (IARC) reported that CRC ranks third in global incidence and second in mortality among cancers [2]. Despite advances in surgery, chemotherapy, targeted therapy, and immunotherapy [3, 4], the prognosis of advanced CRC remains poor due to late diagnosis and high recurrence rates [5, 6]. There is thus an urgent need for more effective and precise therapeutic strategies.
Sonodynamic therapy (SDT) has emerged as a promising non-invasive therapeutic modality [7]. SDT uses low-intensity ultrasound (US) to activate sonosensitizers, generating reactive oxygen species (ROS) that induce immunogenic cell death (ICD) and promote the release of damage-associated molecular patterns (DAMPs), enhancing antitumor immunity [8–10]. Compared to PDT, SDT offers superior tissue penetration and tumor selectivity [11]. SDT presents unique advantages over conventional therapies, including high selectivity that minimizes systemic toxicity, the ability to induce multiple forms of programmed cell death that enhance immune recognition, and controllability that facilitates combination with immunotherapies such as PD-1/PD-L1 inhibitors to achieve synergistic effects [10, 12]. Nevertheless, its clinical translation remains limited by insufficient sonosensitizer efficiency, the hypoxic tumor microenvironment (TME), and challenges in drug delivery [13, 14].
Nanotechnology provides opportunities to overcome these limitations. Nanonosensitizers improve ROS generation, tumor accumulation, and TME modulation, thereby enhancing SDT efficacy [15, 16]. Their tunable structure enables integration with immunotherapies, increasing treatment precision and safety. Recent developments include nanoplatforms that enhance nuclear ROS accumulation, combine antibacterial and redox-regulating functions, or co-deliver immunomodulatory agents, highlighting the potential of nanonosensitizers for personalized CRC therapy [17–19].
Nanotechnology further improves drug delivery and imaging in oncology [20, 21]. Their high surface area, tunable size, and modifiable surfaces facilitate good tumor accumulation (possibly via the enhanced permeability and retention effect), increase cellular uptake, and enable controlled drug release, thereby prolonging therapeutic action and minimizing side effects [22–24]. Nevertheless, conventional NPs face challenges from the complex TME and show variable therapeutic outcomes. Metal-coordinated nanomaterials, combining metal ions with organic molecules or polymers, have emerged to address these challenges [25]. They not only combine the advantages from both metal and organic ligands, but also respond to specific TME features, improving tumor accumulation, retention, and therapeutic efficacy. Targeting ligands can also be incorporated to guide nanomaterials precisely to tumor cells while minimizing off-target effects [26, 27].
Based on this concept, we designed a zirconium (Zr)-based coordinated nanomaterial (Zr-HMME) for CRC SDT and imaging. Zr is biocompatible and its radioactive isotope 89Zr can be used seamlessly for PET imaging [28–31]. HMME, a second-generation sonosensitizer, reacts with Zr to generate ROS under US activation and also possesses fluorescence imaging capability [32, 33]. To improve pharmacokinetics and tumor targeting, the nanomaterial surface was modified with PEG, forming HP, which prolongs circulation and reduces toxicity [34, 35]. To further enhance specificity, linaclotide was conjugated to the nanomaterial (HP-LNC), targeting guanylyl cyclase C (GCC), which is overexpressed on CRC cells, including primary tumors and metastases [36–38]. GCC is restricted to normal intestinal epithelium but mislocalized during CRC progression, becoming a tumor-specific target. Its consistent expression in metastatic CRC while absent in normal lymph nodes makes GCC an ideal biomarker for detecting micrometastases and enabling receptor-mediated delivery, thus enhancing therapeutic specificity and efficacy [39–43].
In summary, HP-LNC is a multifunctional nanomaterial that combines GCC-targeted delivery, SDT efficacy, and PET/fluorescence imaging, providing a promising platform for precise CRC diagnosis and therapy with strong translational potential.

Materials and methods

Materials and reagents
HMME was purchased from Shanghai Xianhui Pharmaceutical Co., Ltd. Zirconium chloride (ZrCl4) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Aminopyrene (PY, Mw = 217) and Poly(acrylic acid) (PAA, Mw = 450,000) were supplied by Sigma-Aldrich. NH2-PEG-MAL (Mw = 5000) was sourced from Toyongbio (Shanghai, China). Linaclotide (Mw = 1526.74) was purchased from Gotopbio Co., Ltd. (Hangzhou, China). Methanol, ethanol, trimethylamine, N,N-dimethylformamide (DMF) and 1,3-diphenylisobenzofuran (DPBF) were acquired from Shanghai Macklin Biochemical Technology Co., Ltd. Fetal bovine serum (FBS) was received from ExCell Bio, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), phosphate buffered saline (1×PBS), penicillin (10,000 U/mL)/streptomycin (10,000 µg/mL) mixture and TrypLETM Express enzyme were obtained from Gibco. Cell counting kit-8 (CCK-8), reactive oxygen species detection kit, and one step TUNEL apoptosis assay kit were purchased from Beyotime Biotechnology (Shanghai, China). HMGB1 (10829-1-AP, 1:500), CRT (27298-1-AP, 1:5000), caspase 3 (82202-1-RR, 1:10000), and β-actin (66009-1-LG, 1:20000) antibodies were provided by Proteintech (Rosemont, IL).

Characterization and measurements
The morphology of the nanoparticles (NPs) was observed with scanning electron microscopy (SEM, ZEISS Gemini 360, Germany) and transmission electron microscopy (TEM, FEI Talos F200X, USA). The hydrodynamic particle size and potential of the samples were measured using dynamic light scattering (DLS, NanoBrook 90Plus PALS, Brookhaven). The UV–Vis-NIR absorption spectra of the nanoparticles were measured on a UV–Vis-NIR spectrometer (Lambda 365, Perkin-Elmer). X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA) was used to analyze the elemental composition and surface chemistry. TGA was used to investigate the decomposition process and composition of the nanosonosensitizer (TG, PerkinElmer STA 8000, USA). Protein expression levels were quantified using Western blotting (WB, Tanon 2500), and the results were visualized with an electrophoresis gel imaging system (GelView 6000Pro II).

Synthesis of HZ, HP, and HP-LNC nanosonosensitizer
The synthesis of HZ, HP, and HP-LNC nanosonosensitizer was performed under controlled conditions. To begin, 10 mg of HMME was dissolved in 20 mL of methanol/trimethylamine (100:1 v/v) under dark conditions with continuous stirring until completely dissolved. Then, a 10 mL methanol/DMF solution (17:3 v/v) containing 30 mg of zirconium tetrachloride (ZrCl₄) was slowly added to the HMME solution. The mixture was stirred at room temperature for 30 min, then subjected to sonication at 40 kHz for 8 h, with a low temperature maintained throughout the process. The resulting precipitates were collected by centrifugation (20,231×g, 20 min), washed three times with an ethanol/DMF solution (7:3 v/v), followed by three washes with water, and then resuspended in water. Next, 5 mg of Py-PAA-PEG-Mal in 5 mL of water was added to the aqueous suspension of 10 mg HZ (5 mL) and stirred for 20 min. The resulting mixture was then sonicated in an ice bath for 2 h. The excess Py-PAA-PEG-Mal was removed through repeated centrifugation (20,231×g, 15 min for at least two cycles) to obtain HP nanoparticles. To synthesize HP-LNC, linaclotide (3 mg, sequence: CCEYCCNPACTGCY) was activated in PBS with 5.4 mg of Traut’s Reagent (2-iminothiolane hydrochloride) under gentle stirring for 1 h. Subsequently, 9 mg of HP in 9 mL of water was added to the activated linaclotide solution, followed by sonication in an ice bath for 2 h. Finally, HP-LNC was collected by centrifugation (20,231×g, 15 min) and washed three times with water. One portion of the precipitate was dried at 60 °C, and the other was suspended in water for stock preparation. In the synthesis of 89Zr-HP-LNC, 200 µL of 0.1 M HCl was mixed with 74 MBq of 89Zr-oxalate (purchased from DC AMS Pharma, Nanjing, China), and the oxalate was fully evaporated by heating at 200 °C for 4 h. The resultant 89ZrCl4 was subsequently added to the reaction mixture, together with non-radioactive ZrCl4 as a radioactive precursor. All other reaction conditions were maintained as previously described.

US-induced 1O₂ generation
The generation of singlet oxygen (¹O₂) by HP conjugates under ultrasound (US) activation was evaluated using 1,3-diphenylisobenzofuran (DPBF) as a probe in a quantitative decolorization assay. Briefly, 75 µL of DPBF solution (2 mg/mL in DMSO) was added to 3 mL of HP aqueous suspension (20 µg/mL). The mixture was then exposed to US irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 10 min. Absorbance was recorded every minute during the irradiation to monitor ¹O₂ generation.

Interaction between cells and nanosonosensitizers
The interaction between T84 cells and HP-LNC was investigated using laser confocal scanning microscopy (LCSM) and flow cytometry to evaluate fluorescence uptake and endocytosis. T84 cells were seeded at a density of 1.5 × 10⁵ cells per dish and incubated overnight at 37 °C for LCSM analysis. The cells were then incubated with specific concentrations of HP and HP-LNC at 37 °C for 0.5 and 2 h. After incubation, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, and then washed again three times with PBS. The cells were subsequently stained with DAPI-containing antifade mounting medium for 15 min before being imaged using an LCSM. For flow cytometry analysis, T84 cells were plated in 6-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 h. The cells were then treated with sonosensitizers for different time points (0, 0.5, 2, 3, and 4 h). After digestion, the cells were resuspended in 200 µL PBS, passed through a cell strainer, centrifuged, and analyzed using a FACScan (Becton Dickinson).

Cytotoxicity test
The cytotoxicity of nanosonosensitizers against T84 cells was assessed by CCK-8 cell viability assays and Live/Dead cell staining under fluorescence microscopy. In brief, T84 cells (1 × 10⁴ cells/well) were seeded in 96-well plates and cultured overnight at 37 °C. The cells were exposed to a range of gradient concentrations of nanosonosensitizers for 24 h, followed by ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min. After an additional 6 h incubation, cell viability was evaluated using the CCK-8 assay. To evaluate the effects of SDT, live/dead cell staining was performed. T84 cells were cultured in 24-well plates for 24 h, followed by treatment with specific concentrations of HP and HP-LNC for an additional 24 h, then exposed to ultrasound for 2 min. Following an additional 6 h incubation, the cells were stained with calcein AM/PI according to the manufacturer’s protocol and observed under an inverted fluorescence microscope.

Detection of intracellular ROS
The generation of ROS was quantified using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe. T84 cells (2 × 10⁵) were seeded onto confocal microscopy dishes and cultured overnight at 37 °C. After the cells had adhered, the medium was replaced with fresh medium containing specific concentrations of HP and HP-LNC. Following 24 h of co-incubation, ROS generation was induced by ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min. The cells were then washed with PBS to remove the residual medium, followed by incubation with DCFH-DA (1 µM) for 30 min at 37 °C in the dark. After removing excess dye with PBS washes, the cells were stained with Hoechst 33,342 (5 µg/mL) for 10 min, and fluorescence imaging was performed using a confocal microscope.

TUNEL assay for the detection of apoptotic cells
The TUNEL assay was used to detect DNA fragmentation, a hallmark of early apoptosis. T84 cells were seeded in confocal dishes at a density of 2 × 10⁵ cells per dish and incubated at 37 °C for 24 h. The cells were incubated with specific concentrations of sonosensitizers (HP and HP-LNC) for 24 h, followed by ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min. After an additional 6 h incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100 for 5 min. The TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase (TdT) and fluorescently labeled dUTP, was then applied, and the cells were incubated at 37 °C for 1 h. After washing with PBS, the cells were counterstained with an antifade mounting medium containing DAPI for 15 min and observed under an LCSM to identify apoptotic cells.

Intracellular damps detection by Immunofluorescence
Immunofluorescence staining was performed to investigate the release of intracellular DAMPs, specifically the cell surface exposure of CRT and nuclear-to-cytoplasmic translocation of HMGB1. T84 cells were seeded in confocal dishes at a density of 2 × 10⁵ cells/dish and incubated at 37 °C for 24 h. The cells were subsequently treated with predefined concentrations of HP and HP-LNC for 24 h, followed by ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min. After an additional 6 h incubation, the cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100 for 5 min (this step was omitted for CRT staining). The cells were blocked with 1% BSA for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After washing with PBS, the cells were incubated with FITC-conjugated secondary antibodies for 2 h. Finally, nuclei were counterstained with an antifade mounting medium with DAPI for 15 min and the samples were imaged using an LCSM.

Immunoblotting
To assess the expression of key proteins, including pro-caspase 3 and HMGB1, Western blot analysis was conducted. T84 cells were plated in 6-well plates at a density of 3 × 10⁵ cells per well and cultured overnight at 37 °C. The cells were treated with predetermined concentrations of sonosensitizers for 24 h. After washing three times with PBS, the cells were subjected to ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min, followed by an additional 6 h incubation. Protein extraction was carried out by adding 150 µL of RIPA lysis buffer containing 1.5 µL PMSF and 1.5 µL phosphatase inhibitor to each well, followed by incubation on ice for 30 min. After centrifugation, the supernatants were collected, and protein concentrations were quantified using a BCA assay. The remaining samples were mixed with 5× loading buffer and denatured at 99 °C for 15 min. Protein loading volumes were adjusted according to the quantification results. The protein samples were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes, followed by blocking with 5% skim milk for 2 h. The membranes were incubated overnight at 4 °C with primary antibodies targeting pro-caspase 3, HMGB1, and β-actin. After washing with TBST three times, HRP-conjugated secondary antibodies were applied for 2 h at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) detection kit and imaged.

Release of ATP
T84 cells were cultured in 6-well plates (2 × 10⁵ cells/well) at 37 °C for 24 h. Following incubation, the cells were treated with various groups: Blank, US, HP + US, and HP-LNC + US, and incubated for an additional 24 h. After three washes with PBS, the cells were exposed to ultrasound irradiation (1 MHz, 1 W/cm², 50% duty cycle) for 2 min, followed by a further 6-h incubation. The ATP concentration in the supernatant was measured according to the manufacturer’s instructions (Beyotime Biotechnology).

Flow cytometric analysis of dendritic cell maturation
Bone marrow cells were harvested from the femurs and tibias of mice and cultured in RPMI-1640 medium supplemented with 10% FBS, recombinant murine GM-CSF (20 ng/mL), and IL-4 (10 ng/mL) to induce differentiation into dendritic cells (DCs). Half of the culture medium was replaced with fresh cytokine-containing medium on days 1, 3, and 5. On day 7, the immature DCs were co-cultured for 24 h with conditioned media collected from tumor cells subjected to different treatments. Following co-incubation, DCs were collected, washed, and stained with fluorophore-conjugated antibodies against CD11c, CD80, and CD86, followed by flow cytometric analysis to assess DC maturation.

Establishment of a subcutaneous colorectal cancer model
Female BALB/c-nu mice (7 weeks, ~ 20 g) were supplied from GemPharmatech Co., Ltd. (Nanjing, China). Animal experiments were conducted following the National Guidelines for the Care and Use of Laboratory Animals (China) and approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. Female BALB/c-nu mice, aged 6–7 weeks, were utilized for both subcutaneous and intravenous injection studies. For the establishment of subcutaneous tumor models, 100 µL of T84 cell suspension (6 × 107 cells/mL) was injected into the right dorsal flank of each mouse. Tumor growth was monitored, and treatments were initiated once the tumors reached a volume of approximately 90 mm³.

Pharmacokinetics and biodistribution
A subcutaneous tumor model was established in BALB/c-nu mice to evaluate the in vivo fluorescence imaging performance of HP-LNC. Once the tumor volume reached approximately 100 mm³, in vivo imaging and biodistribution studies were conducted. After intravenous injection of the nanomaterial, fluorescence imaging was performed at predefined time points (0, 10, 24, 48, 72 h) using a BLT AniView600 Multimodal Imaging system (Excitation/Emission: 745/820 nm, Biolight Biotechnology Co., Ltd., Guangzhou, China). At 72 h post-injection, the mice were sacrificed, and the major organs were harvested for fluorescence imaging analysis. Simultaneously, 0.2–0.4 MBq 89Zr-HP-LNC was administered to BALB/c-nu mice via tail vein injection. Blood samples were collected at predetermined time points (5, 15, and 30 min; 1, 2, 4, 8, 12, and 24 h post-injection) using capillary tubes from the eye’s venous plexus. The weight of each sample was recorded. Radioactivity was quantified using a gamma counter calibrated for the specific radionuclide. Each blood sample was placed in a dedicated counting tube, and counts per minute (CPM) were measured and recorded for further analysis.

Organ distributions and PET imaging of Zr-HMME nanosonosensitizers
To establish tumor models, T84 cells (100 µL, 6 × 107 cells/mL) were subcutaneously injected into the dorsal flank of BALB/c-nu mice. Tumor growth was monitored periodically, and imaging analysis commenced once the tumor diameter reached approximately 6 mm. PET scans were performed using a Super Nova 304 microPET/CT scanner (MAMMI). Each tumor-bearing mouse was intravenously administered 2–4 MBq of either 89Zr-HP-LNC and 89Zr-HP, suspended in 150 µL PBS. PET imaging was conducted at predefined time points post-injection, specifically at 0.5, 12, and 48 h. The image reconstruction was performed using the three-dimensional ordered subset expectation maximization (3D-OSEM) algorithm, without applying attenuation or scatter correction. To ensure the accuracy of the PET imaging, gamma counting was used to quantify the radioactivity distribution in the organs, confirming that the quantitative tracer uptake from the PET imaging was consistent with the in vivo radioactivity distribution in tumor-bearing mice. After the final PET scan at 48 h post-injection, the mice were sacrificed, and their tumors, blood, and major organs were harvested and weighed. The radioactivity of each sample was quantified with a Perkin-Elmer WIZARD2 gamma counter, and the results were expressed as %ID/g (mean ± standard deviation).

Evaluation of antitumor therapeutic efficacy in animal models
Antitumor therapy was initiated when the tumor volume reached approximately 90 mm³, with nanomaterial administered intravenously every two days. Mice were randomly assigned to four groups: Blank, Blank + US, HP + US, and HP-LNC + US, with four mice per group. Ultrasound (US) treatment was applied for 2 min, 24 h after each injection. Throughout the experiment, body weight and tumor volume were monitored regularly to assess therapeutic efficacy and potential side effects. Mice were euthanized when the tumor volume exceeded 500 mm³, and their tumors and major organs were collected for subsequent analysis. Tissue sections were prepared for histological examination. For the liver metastasis experiment, following the antitumor treatment described above, 2 × 10⁶ T84 cells were injected into the mice via the spleen. On day 18, the entire liver was harvested for photographic documentation and histopathological analysis.

Statistical analysis
All measurements and tests were performed in triplicate, with a minimum sample size of three per test to ensure statistical reliability. Statistical significance between experimental groups was assessed using an unpaired two-tailed Student’s t-test. A P-value of < 0.05 was considered statistically significant, corresponding to a 95% confidence interval.

Results and discussions

Synthesis and characterization of Zr-HMME nanoplatforms
The Zr-HMME (HZ) nanoconjugate was synthesized using a sonication method [44]. To enhance its structural stability and water solubility, pyrene-polyacrylic acid-polyethylene glycol-maleimide (Py-PAA-PEG-Mal) was synthesized and attached to the HZ through π-π interactions under sonication in an ice bath [45, 46]. Subsequently, linaclotide was conjugated to Zr-HMME-PEG (HP) via a thiol-maleimide reaction, exploiting the high specificity between the thiol groups on the peptide and the maleimide moieties on HP. This reaction was conducted under mild physiological conditions, ensuring efficient and stable linkage formation without compromising the bioactivity of linaclotide. The modification endowed the nanoconjugate, designated as HP-Linaclotide (HP-LNC), with tumor-targeting functionality, thereby enhancing its potential therapeutic efficacy (Scheme 1). The morphology and dispersion of the nanoconjugates were analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The images (Fig. 2A and B and S1a-b) demonstrated that HP-LNC and HP exhibited a nearly spherical shape with uniform dispersion and an average diameter of approximately 100 nm. Dynamic light scattering (DLS) measurements further confirmed the excellent dispersibility of both HZ and HP in aqueous solutions, with particle sizes of around 130 nm and 150 nm, respectively (Fig. S2). The larger particle size observed in aqueous suspension compared to the SEM and TEM images can be attributed to the extension of the PEG chains on the surface of the nanoconjugates and the adsorption of water molecules. Moreover, under similar conditions, the hydrated diameter of HP-LNC was approximately 185 nm, confirming the size discrepancy between dry and hydrated states (Fig. 2C). Zeta potential analysis revealed a notable shift in the surface charge of HZ from positive to negative following PEGylation (Fig. S3), providing clear evidence for the effective modification of Py-PAA-PEG-Mal on its surface. Inductively coupled plasma (ICP) analysis demonstrated a molar ratio of Zr to HMME of 293:50, indicating highly efficient complexation between Zr and HMME and supporting the successful formation of the Zr-HMME nanoconjugate. This combined data strongly supports the effective incorporation of both Py-PAA-PEG and linaclotide into the nanoconjugate, ensuring its intended functionality.

The fluorescence properties of HMME and HZ were analyzed using fluorescence spectroscopy, revealing significant alterations in the fluorescence characteristics of HZ compared to HMME. After synthesizing HZ, the fluorescence peaks of HMME at 623 nm and 678 nm completely disappeared, indicating a substantial reduction in fluorescence intensity at these wavelengths [47]. Notably, a red shift was observed, with a new peak emerging at 630 nm, albeit with significantly reduced fluorescence intensity (Fig. 2D). This shift can be attributed to the incorporation of zirconium, which enhances the molecular symmetry of the conjugate and reduces the Q band absorption peak [48]. The presence of the Q band in HZ suggests that it forms a complex through interactions between the functional groups of HMME and Zr, rather than exhibiting typical metalloporphyrin characteristics (Fig. S4). The UV-Vis spectra of HP and HP-LNC exhibited minimal changes when compared to HZ (Fig. 2E). We found that the significant shifts in the UV–vis absorption peaks after the modification of HZ with Py-PAA-PEG to form HP can be attributed to molecular structural changes, extension of the conjugated system through π–π interactions or hydrogen bonding, alterations in the surrounding environmental polarity, and possible electron transfer effects. These molecular interactions and environmental changes collectively modulate the electronic transitions of HZ, thereby leading to the observed spectral shifts. FTIR analysis provided further insight into the interaction between HMME and zirconium ions. In the FTIR spectrum of HZ, the disappearance of the carboxyl stretching vibration band at 1706 cm⁻¹ indicated the interaction between zirconium ions and the carboxyl group of HMME. Furthermore, the appearance of carboxylate salt bands at 1410 cm⁻¹ and 1577 cm⁻¹ provided additional evidence for this interaction. The N–H stretching bands observed at 3313 cm⁻¹ and 913 cm⁻¹ in HZ suggested that the N–H groups in the porphyrin ring did not coordinate with zirconium ions (Fig. 2F). In contrast, the FTIR spectrum of HP-LNC displayed a peak at 1640 cm⁻¹, corresponding to the C = O stretching vibration, which is typically associated with the secondary structure of peptide chains (Fig. S5).
Thermogravimetric analysis (TGA) of HZ revealed three distinct stages in its thermal degradation profile [49]. The initial weight loss between 0 and 200 °C was attributed to the removal of adsorbed or crystallization water, followed by a gradual mass loss from 200 to 300 °C. A sharp decline between 400 and 540 °C indicated the primary decomposition of HMME, while degradation stabilized beyond 540 °C, reflecting a limited reduction in organic content. The TGA curve indicated that the HMME content in HZ was approximately 27.71% (Fig. S6). Meanwhile, the TGA results indicate that Py-PAA-PEG constitutes about 7.55% of the HP composition (Fig. 2G). The X-Ray photoelectron spectroscopy (XPS) analysis confirmed the presence of multiple elements, including zirconium, in HZ, thereby validating the structural and compositional integrity of the synthesized material (Fig. 1H). The spectra further revealed that zirconium predominantly existed in its tetravalent state (Fig. S7).

1,3-Diphenylisobenzofuran (DPBF) is a commonly used probe for detecting ROS, particularly singlet oxygen (¹O₂), by monitoring the attenuation of absorbance at 410 nm [50]. Due to its photosensitive and photodegradable nature, all experiments were conducted in complete darkness to prevent light-induced interference. To address the spectral overlap between the absorption of HP around 400 nm and the characteristic absorption peak of DPBF at 410 nm (Fig. S8b), a high-concentration DPBF solution (2 mg/mL) was utilized. This modification enhanced the sensitivity for detecting absorbance changes, which decreased in a time-dependent manner in the “HP + US” group, confirming the generation of ¹O₂ under ultrasound treatment (Fig. S8a). In contrast, minimal spectral changes were observed in the control groups (“DPBF” and “DPBF + US”), demonstrating that ¹O₂ production is primarily driven by the synergistic interaction of HP and ultrasound (Fig. S8c and S8d). We performed the same DPBF assay for HP-LNC (Fig. 2I). Consistent with HP, the “HP-LNC + US” group exhibited a significant time-dependent decrease in DPBF absorbance, further confirming the efficient ROS generation capability of HP-LNC under ultrasound irradiation. To further support the generation of singlet oxygen by HP-LNC under ultrasound activation, we performed SOSG assays that revealed a gradual increase in fluorescence intensity over the course of ultrasound exposure (Fig. S9). These findings indirectly indicate the effective production of singlet oxygen, consistent with the expected sonodynamic behavior of HP-LNC nanomaterials.

Cellular uptake and cytotoxicity evaluation
To evaluate the cellular uptake of HP conjugates, T84 cells (GCC+) were incubated with HP and HP-LNC, each at a concentration of 100 µg/mL, for 0.5 h and 2 h, respectively. Confocal microscopy demonstrated a time-dependent increase in intracellular uptake for both sonosensitizers, with HP-LNC showing higher fluorescence intensity and faster intracellular localization compared to HP alone (Fig. 3A and B). This suggests that linaclotide enhances the targeting ability of HP-LNC [51]. Flow cytometry analysis further corroborated the uptake pattern, with endocytosis peaking at 2 h (Fig. 3C). The endocytosis of both HP and HP-LNC exhibited a time-dependent pattern, reaching a plateau at 3 h (Fig. S10). These findings highlight the superior tumor-targeting potential of HP-LNC, which can be attributed to its enhanced cellular uptake efficiency. To assess the biosafety of HZ nanoparticles before in vivo experiments, cytotoxicity assays were conducted on T84 and SW480 colorectal cancer cells, as well as HUVECs (human umbilical vein endothelial cells) (Fig. 3D).
The Cell Counting Kit-8 (CCK-8) assay revealed that after 24 h of incubation with HP-LNC nanoparticles (0–100 µg/mL), T84 cell viability showed a significant decrease at 100 µg/mL, while the viability of SW480 cells and HUVECs remained above 80%, demonstrating excellent biocompatibility. In contrast, T84 cells with HMME under the same conditions displayed significant concentration-dependent toxicity (Fig. S11). Notably, ultrasound treatment enhanced the activity of HMME within HP nanomaterials, improving their functional performance across a range of concentrations (Fig. 3E). Further analysis revealed that T84 cells incubated with HP-LNC at various concentrations for 24 h, followed by ultrasound exposure (1 MHz, 50% duty cycle, 1 W/cm², 2 min), showed a marked reduction in cell viability at higher concentrations. At 100 µg/mL, cell viability dropped to approximately 10% (Fig. 3F), with increased cytotoxicity observed at higher concentrations. This increase in cytotoxicity at higher concentrations underscores the potent tumor-targeting cytotoxicity of HP-LNC, with therapeutic efficacy closely linked to precise dosage control. Western blot analysis was performed to assess the expression levels of GCC (123 kDa) across different cell lines, with β-tubulin (55 kDa) serving as the loading control (Fig. S12a). The quantitative results demonstrated significant differences in GCC expression among the tested cells, correlating with their sensitivity to HP-LNC treatment (Fig. S12b).

Intracellular ROS and DNA fragmentation (TUNEL) analysis in cell damage
Intracellular ROS generation in T84 cells was assessed using the DCFH-DA probe [52], which emits green fluorescence upon oxidation to dichlorofluorescein (DCF) under oxidative stress. Minimal ROS production was observed in cells treated with ultrasound alone. In contrast, the “HP + US” and “HP-LNC + US” groups exhibited significantly increased ROS levels, as indicated by strong green fluorescence signals (Fig. 3G and H). These findings suggest that HP-LNC nanoparticles enhance ROS generation upon ultrasound activation, highlighting their potential as effective nanosonosensitizers for increasing oxidative stress in tumor cells. As shown in Fig. 3G, the ROS generation in the HP-LNC group under ultrasound exposure was markedly higher than that in the HP group, despite their comparable cellular uptake as demonstrated in Figs. 3A–C. This difference is likely due to the enhanced sonodynamic efficiency of HP-LNC, improved intracellular stability resulting from nanocarrier encapsulation, and its potential for optimized subcellular localization in ROS-sensitive organelles.
To further evaluate the pro-apoptotic effects of HP conjugates, a TUNEL apoptosis detection kit was used to label apoptotic cells, with red fluorescence indicating apoptotic cell death. Confocal microscopy imaging and mean fluorescence intensity (MFI) analysis (Fig. 3I and J) revealed that the “HP-LNC + US” treatment group exhibited the highest red fluorescence intensity, indicating the most significant apoptosis induction in T84 cancer cells. These results underscore the potential of combining HP-LNC with ultrasound to enhance sonodynamic therapy efficacy and optimize therapeutic strategies against CRC.

Live-dead staining assay
The cytotoxic effects of HP conjugates combined with ultrasound were further evaluated using a live/dead cell viability/cytotoxicity assay kit (Fig. 4A). Fluorescence microscopy analysis showed a significant reduction in the green-to-red fluorescence ratio (green indicating live cells and red indicating dead cells), reflecting increased cell death induced by HP and HP-LNC under ultrasound exposure [53]. Among the treatment groups, the HP-LNC + US group exhibited the most pronounced cytotoxic effects. These results suggest that the enhanced targeting specificity and improved ROS generation efficiency of HP-LNC, likely due to its superior intracellular localization and amplified oxidative stress, contribute to its superior ability to induce cell death.

Release of damps induced by SDT with Zr-HMME nanosonosensitizers
In the tumor microenvironment, ICD plays a crucial role by facilitating the release of DAMPs from dying tumor cells, thereby triggering antitumor immune responses. This process is fundamental to the immunological effects of ICD [54, 55]. To explore the molecular mechanisms of cell death induced by HP-LNC combined with ultrasound, Western blot analysis was performed to assess the expression of apoptosis-related proteins, such as pro-caspase 3 and HMGB1 (Fig. 4B and C). The analysis revealed a significant reduction in the expression of these proteins in the HP-LNC + US group compared to others, indicating the involvement of apoptotic pathways and ICD, potentially mediated by the CD91 receptor. HMGB1, a highly conserved nuclear protein involved in gene transcription and DNA repair, is typically localized within the nucleus. However, under stress or treatment conditions, it is released into the extracellular space, as observed in the HP-LNC + US group, suggesting enhanced apoptosis and ICD. Quantitative analysis of Western blot data showed significant differences between groups (Fig. S13a and S13b), reinforcing the role of HP-LNC in promoting apoptosis and ICD upon ultrasound activation.

ATP, as a pivotal energy molecule, is integral to a wide range of physiological and pathological processes. Variations in ATP levels can profoundly affect cellular functions, with reductions commonly observed during apoptosis, necrosis, or exposure to toxic environments. To evaluate ATP levels across various treatment groups, an ATP detection kit was utilized. The findings indicated a decrease in ATP levels in both the HP + US and HP-LNC + US groups. Notably, the HP-LNC + US group demonstrated the most significant reduction, a result primarily attributed to the targeted action of linaclotide (Fig. S14).
LCSM further confirmed the induction of ICD, showing a marked increase in CRT membrane expression in both the HP + US and HP-LNC + US treatment groups (Fig. 4D). Notably, the HP-LNC + US group exhibited the highest MFI (Fig. 4E). To further validate CRT expression, Western blot analysis was performed (Fig. S15). The results showed that CRT expression was significantly upregulated in the “HP + US” and “HP-LNC + US” groups compared to the control groups, consistent with the immunofluorescence results in Fig. 4D. In addition, HMGB1, a critical marker of ICD, was significantly released into the extracellular space following treatment with ultrasound-sensitive agents, with the HP-LNC + US group displaying the most pronounced HMGB1 release (Fig. 4F and G). This underscores its superior efficacy in promoting antitumor immune responses through CRT externalization and HMGB1 release. By contrast, both the LNC and LNC + US groups exhibited only faint HMGB1 and CRT signals (Fig. S16), indicating that linaclotide targeting alone or ultrasound activation without HMME loading was insufficient to induce robust DAMPs release. Overall, compared to HP, the HP-LNC + US treatment substantially enhanced DAMPs release and overexpression, primarily due to the anchoring effect of linaclotide. This treatment effectively promoted CRT translocation and HMGB1 release, facilitating dendritic cell (DC) maturation and enhancing the immune response. Consequently, immune suppression within the tumor microenvironment was alleviated, leading to improved tumor elimination. These findings highlight the significant potential of HP-LNC in promoting DAMPs release and activating ICD, underscoring its promise as an effective therapeutic strategy.
Bone marrow-derived dendritic cells (BMDCs) were incubated for 24 h with conditioned media collected from tumor cells subjected to different treatments (BLANK, US, HP + US, and HP-LNC + US). Flow cytometry was then performed to evaluate the expression of dendritic cell maturation markers CD11c, CD80, and CD86. Figure 4H presents representative flow cytometry plots showing CD80 and CD86 expression within CD11c positive cells in each treatment group, while Fig. S17 provides quantitative analysis of the proportions of CD80 and CD86 positive cells. The results demonstrated that conditioned media from the HP-LNC + US group significantly promoted dendritic cell maturation, as evidenced by markedly higher CD80 and CD86 expression compared to the other groups. These findings indicate that the combined treatment effectively activates the immune system by enhancing dendritic cell maturation, thereby contributing to its antitumor effect.

In vivo imaging of Zr-HMME nanosonosensitizers
Due to the strong background interference at the fluorescence imaging wavelength of HMME in the nanomaterial, Cy7 was incorporated to improve imaging sensitivity. The results indicate that HP-LNC accumulates in tumors more rapidly than HP, with the HP-LNC group demonstrating stronger and more sustained tumor uptake (Fig. S18 and S19). To investigate the in vivo metabolism and clearance of HP-LNC, quantitative fluorescence analyses of major organs including heart, liver, spleen, lungs, and kidneys were performed at multiple time points from 72 h to 13 days post-intravenous injection (Fig. S20a). The results indicated predominant accumulation of HP-LNC in the liver and spleen, consistent with uptake by the reticuloendothelial system (RES). Additionally, relatively high fluorescence signals were detected in the kidneys, likely due to renal excretion of free components released after partial dissociation of the nanomaterial. Fluorescence intensity in these organs decreased gradually over time, reflecting continuous metabolism and clearance (Fig. S20b). This biodistribution profile aligns with known clearance mechanisms of nanomaterials via major detoxification organs. These findings provide comprehensive insight into the pharmacokinetics of HP-LNC and support its favorable biocompatibility and safety profile. Notably, fluorescence intensity peaked between 24 and 48 h post-injection, and even at 72 h post-injection, HP-LNC remained significantly at the tumor site, suggesting prolonged retention. These findings underscore the superior tumor-targeting capability and extended retention of HP-LNC, positioning it as a promising fluorescence imaging probe for tumor detection and therapeutic applications.
The study involved the intravenous injection of 89Zr-labeled HP conjugates into tumor-bearing BALB/c-nu mice, with subsequent monitoring of their biodistribution and tumor accumulation using real-time PET imaging (Fig. 5). The 89Zr-HP-LNC group demonstrated significantly enhanced tumor accumulation kinetics, a result attributed to the incorporation of linaclotide. Notably, 89Zr-HP-LNC was detectable at the tumor site as early as 0.5 h post-injection, reached peak levels at 12 h, and maintained elevated concentrations long after 48 h. Tumor uptake was quantitatively assessed through region-of-interest (ROI) analysis, as depicted in Fig. 5B. These observations corroborated with fluorescence imaging results, which confirmed favorable tumor retention. Following the acquisition of the final PET images, at 48 h post-injection, the mice were euthanized, and major organs and tissues, including the heart, liver, spleen, lungs, kidneys, tumor, blood, muscles, bones, and brain, were harvested. The samples were weighed, and their radioactivity was quantified using γ-counting (Fig. 5C). The in vivo distribution of 89Zr was evaluated by calculating the percentage of injected dose per gram (%ID/g) for each organ or tissue. The results revealed that both 89Zr-HP and 89Zr-HP-LNC predominantly accumulated in the liver and spleen, with the highest radioactivity detected in the spleen. Aside from the tumor, liver, and spleen, some radioactivity was observed in bones at 48 h, which indicated the detachment of some zirconium from 89Zr-HP conjugates. Meanwhile, minimal radioactivity was observed in other tissues. Notably, the 89Zr-HP-LNC group exhibited significantly higher tumor accumulation (approximately 6.3 ± 2.1%ID/g, n = 4) compared to the 89Zr-HP group, reinforcing the conclusion that linaclotide incorporation enhances the tumor-targeting specificity of the nanomaterials.
Additionally, blood samples collected at various time points post-injection were analyzed for radioactivity, revealing that the blood half-life of 89Zr-HP-LNC and 89Zr-HP were approximately 2.4 ± 0.6 h and 1.2 ± 0.6 h, respectively (Fig. 5D). These findings suggest that linaclotide modification not only enhances tumor-targeting properties but also significantly extends the circulation time of the material in the bloodstream, while the detailed mechanisms will be explored in the future.

Antitumor efficacy of Zr-HMME nanoparticles
In this study, we employed BALB/c-nu mice as an animal model to evaluate the in vivo anti-tumor efficacy of a novel nanomaterial. T84 cells in the logarithmic growth phase were mixed with cell matrix gel at a 7:3 ratio under cold conditions to prepare a cell suspension. A volume of 100 µL (6 × 10⁶ cells) was subcutaneously injected into the right flank of each BALB/c-nu mouse. When the tumor volume reached approximately 90 mm³, the mice were randomly allocated into four groups (n = 4 per group): Control, US, HP + US, and HP-LNC + US. The mice were injected with different sonosensitizers via the tail vein and received ultrasound treatment 24 h later (Fig. 6A). Body weight and tumor volume were recorded every two days throughout the experiment. The results showed no significant differences in body weight among the groups (Fig. 6C); this demonstrates favorable biological safety in mice. On day 30, the mice were euthanized, and tumor sizes were measured. Notably, the HP-LNC + US group exhibited the smallest tumor volume, with a significant difference compared to the other groups (Fig. 6B). Tumor volume and weight decreased significantly in the HP + US and HP-LNC + US groups compared to the control and ultrasound-only groups. Notably, the reduction in tumor volume and weight was more pronounced in the HP-LNC + US group than in the HP + US group (Fig. 6D–H).
The TUNEL staining of tumor tissues revealed a significantly higher apoptosis rate in the HP-LNC + US and HP + US groups (Fig. 6I). Immunofluorescence analysis of tumor sections demonstrated that nuclear HMGB1 downregulation indicated immunogenic cell apoptosis, suggesting that HP-LNC + US facilitated immune response activation (Fig. 6J). Additionally, the upregulation of membrane-bound CRT further validated immunogenic cell apoptosis and indicated the release of DAMPs, a process also influenced by HP-LNC + US, and enhanced the immune response within the tumor microenvironment (Fig. 6K). Immunohistochemical analysis of Ki67, HMGB1, and CRT further confirmed the antitumor effects of HP-LNC + US. A significant reduction in Ki67 expression was observed after HP-LNC + US treatment, indicating suppressed tumor cell proliferation. Concurrently, the nuclear-to-cytoplasmic translocation of HMGB1 suggested the induction of immunogenic cell apoptosis. The increased membrane localization of CRT provided further evidence of immunogenic cell death, which may contribute to the activation of downstream immune responses. Meanwhile, HE staining results demonstrated that HP-LNC + US exhibited a strong inhibitory effect on tumor tissue (Fig. 6L). HE staining of major organs, including the heart, liver, spleen, lungs, and kidneys, revealed no significant pathological changes following HP-LNC or HP injection, indicating that the material exhibits excellent biocompatibility and safety (Fig. S21).

Inhibition of hepatic tumor metastasis by Zr-HMME nanomaterials
To further investigate the systemic immune response elicited by different treatments, we measured the serum levels of macrophage-associated cytokines in tumor-bearing mice across four groups: Blank, US, HP + US, and HP-LNC + US. The enzyme-linked immunosorbent assay (ELISA) results revealed that the HP-LNC + US group showed a significant increase in pro-inflammatory cytokines, such as TNF-α, IL-1β, and IFN-γ, which indicates a robust activation of innate immunity. In contrast, this group exhibited markedly decreased levels of anti-inflammatory cytokines IL-10 and IL-4, along.

with the immunosuppressive chemokine CCL5, compared to the other groups (Fig. 7A and F, S22). These alterations in cytokine profiles suggest that the combination of HP-LNC with ultrasound irradiation fosters a shift toward a pro-inflammatory, M1-like macrophage phenotype while inhibiting M2-like polarization. This immunomodulatory effect likely contributes to the observed enhancement in systemic antitumor immunity and provides mechanistic insight into the inhibition of liver metastasis following HP-LNC-based treatment.
The therapeutic efficacy of nanosonosensitizers was further assessed in a CRC liver metastasis model. The model was established by injecting 2 × 10⁶ T84 cells into the spleens of mice on day 0, thereby inducing liver metastasis. By day 8, the mice were randomly divided into two treatment groups (n = 6 per group): Ultrasound and HP-LNC + US. These groups were designed to evaluate the therapeutic effects of HP-LNC + US on tumor metastasis and assess its efficacy in inhibiting metastasis. Beginning on day 8, nanosonosensitizers were administered through tail vein injection, followed by ultrasound treatment at the liver site 24 h post-injection. Treatment was repeated every two days, with a total of three injections. On day 20, the mice were euthanized, and their livers were collected for analysis of metastatic lesions. The HP-LNC + US group exhibited a significant reduction in liver metastatic lesions compared to the US group (Fig. 7G and H and S23). This finding indicates that the combination of HP-LNC and ultrasound treatment effectively inhibits tumor growth and metastasis.

Conclusion
In summary, the HP-LNC nanoplatform developed in this study demonstrates significant potential as a multimodal therapeutic strategy for CRC. This platform integrates SDT with dual-modal imaging, utilizing the radioisotope 89Zr for PET and fluorescence imaging for precise tumor localization and real-time monitoring of treatment response. The incorporation of linaclotide enhances tumor-targeting specificity, while PEGylation improves the nanomaterial’s biocompatibility, stability, and extends its in vivo circulation time. Upon ultrasound activation, the platform generates ROS and induces ICD, leading to effective tumor suppression and activation of a robust anti-tumor immune response. This combined therapeutic and immunological effect underscores the potential of Zr-HMME-based nanoplatforms as an effective therapeutic strategy for CRC. Furthermore, the integration of diagnostic imaging capabilities with therapeutic intervention highlights the translational promise of this approach, offering valuable insights for future clinical applications in targeted therapy and molecular imaging, particularly in addressing the challenges associated with advanced and metastatic CRC.

Supplementary Information