A precise theranostic nanoplatform amplifies anti-tumor efficacy via copper ionophores and sonodynamic therapy
Xiaoqin Luo, Xiaojuan Wang, Sheng Li, Qing Chen, Jibin Song, Junqiang Chen
TL;DR
A new nanoplatform combines sonodynamic therapy and copper-induced cell death to effectively treat tumors while allowing real-time monitoring.
Contribution
The study introduces a novel nanoplatform that synergizes sonodynamic therapy and cuproptosis for enhanced tumor treatment and real-time imaging.
Findings
The nanoplatform increases intracellular ROS levels approximately 20-fold compared to controls.
It significantly enhances dendritic cell maturation by ~8-fold and increases CD4+ and CD8+ T cell infiltration.
The platform enables real-time tumor imaging via near-infrared fluorescence.
Abstract
Tumor heterogeneity and therapeutic resistance remain major challenges in cancer treatment. Sonodynamic therapy (SDT), a noninvasive therapeutic modality with deep tissue penetration capability, has shown considerable promise in tumor therapy. However, its efficacy is often limited by insufficient reactive oxygen species (ROS) generation and the lack of real-time treatment monitoring. Cuproptosis, a recently identified copper-dependent form of regulated cell death, is closely associated with mitochondrial metabolic dysfunction and offers new opportunities for synergistic cancer therapy. Herein, we report a multifunctional nanoplatform (ICCP NPs) that integrates SDT, cuproptosis induction, and near-infrared (NIR) fluorescence imaging for precise and visualized cancer theranostics. The nanoplatform utilizes CuS nanocarriers to co-encapsulate the sonosensitizer chlorin e6 (Ce6) and the…
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Taxonomy
TopicsNanoplatforms for cancer theranostics · Ultrasound and Hyperthermia Applications · Nanoparticle-Based Drug Delivery
Introduction
1
Cancer constitutes a critical global public health issue at present, posing a persistent and far-reaching threat to individual survival as well as societal advancement [1]. Despite the progress achieved through existing treatment approaches including surgery, radiotherapy, chemotherapy, and targeted therapies, persistently high rates of recurrence and metastasis continue to adversely affect patient survival and quality of life [2,3]. In light of this situation, exploring and creating new therapeutic approaches to substantially improve efficacy and reduce patient suffering is an urgent priority in medical research.
Sonodynamic therapy (SDT) is an emerging cancer treatment modality that primarily relies on ultrasound (US) to activate sonosensitizers enriched in tumor tissues. This activation drives the localized production of reactive oxygen species (ROS), thereby triggering substantial oxidative stress, ultimately leading to apoptosis of cancer cells [4,5]. In addition, immunogenic cell death (ICD) can be induced by SDT, leading to the release of damage-associated molecular patterns (DAMPS) and tumor-associated antigens, which promotes dendritic cell maturation and antigen presentation, thereby triggering tumor-specific T-cell immune responses and enhancing anti-tumor immunity [5,6]. Compared with conventional photodynamic therapy, SDT offers superior targeting precision, deeper tissue penetration (>10 cm) and stronger focusing capacity, thus exhibiting greater potential in cancer treatment [7,8]. Although SDT has promising prospects in oncology, it is still hampered by the tumor microenvironment. Particularly, overexpressed intracellular antioxidant molecules such as glutathione (GSH) can directly scavenge ROS generated by SDT, weaken the killing effect of oxidative stress on tumor cells, and thus severely impair the therapeutic efficacy of SDT [9]. Based on this, it is of great significance to enhance the SDT effect and elevate tumor oxidative stress through synergistic strategies to overcome current therapeutic bottlenecks.
Cuproptosis is an innovative form of cell death that has attracted significant interest in cancer therapeutics [[10], [11], [12]]. The excessive accumulation of intracellular copper ions is a prerequisite for initiating cuproptosis. These copper ions can specifically binding to lipoylated mitochondrial proteins, induce aberrant protein aggregation and the depletion of iron-sulfur cluster proteins, ultimately triggering severe proteotoxic stress and subsequently leading to tumor cell death [[13], [14], [15]]. Moreover, cuproptosis can induce ICD, which promotes immune cell infiltration and activates systemic anti-tumor immunity, offering a promising strategy for cancer immunotherapy [16,17]. However, the relatively low concentration of copper ions in tumor tissues makes it difficult to induce cuproptosis. Currently, copper ionophores such as disulfiram and elesclomol have been developed to enhance cuproptosis [18,19], yet these small-molecule drugs generally suffer from insufficient targeting selectivity and poor tumor retention [20]. In contrast, by taking advantage of their suitable size, nanocarriers enable the passive accumulation of copper ions in tumor tissues. As multifunctional nanocarriers integrating copper ion delivery and drug loading capabilities, CuS Nanoparticles (NPs) have been demonstrated to specifically respond to the acidic pH of the tumor microenvironment, enabling the controlled release of copper ions and thereby inducing cuproptosis [[21], [22], [23]]. Simultaneously, CuS NPs act as efficient chemodynamic therapy (CDT) agents by catalyzing Fenton-like reaction to produce hydroxyl radicals (·OH) and deplete overexpressed intracellular GSH, thus amplifying ROS-induced oxidative stress [24,25]. Research has confirmed that the synergistic therapeutic strategy combining SDT and cuproptosis can effectively overcome the limitations of single-modality treatments and has emerged as a cutting-edge research direction in the field of tumor precision therapy [[26], [27], [28], [29], [30]]. The integration of therapy and diagnosis represents an inevitable trend in the advancement of precision medicine. However, the application of nanocarriers in tumor therapy is hindered by the absence of effective real-time monitoring techniques—their accumulation levels, spatial distributions, and optimal intervention times are hard to assess accurately, making the entire treatment process more or less blind [31,32]. Consequently, incorporating fluorescence imaging capabilities into nanocarriers for real-time visual monitoring of drug delivery and enrichment has become an urgent research priority [[33], [34], [35]]. Among various imaging modalities, near-infrared (NIR) fluorescence imaging has emerged as a research hotspot in the field of tumor imaging in recent years, thanks to its unique advantages of excellent tissue penetration capability and minimal autofluorescence interference [36,37]. Notably, no relevant reports have been published regarding the NIR fluorescence imaging-guided synergistic therapeutic strategy that combines SDT and cuproptosis via CuS NPs as the core nanocarrier.
Herein, we developed a multifunctional nanoplatform (IR808/Ce6@CuS&DSPE-PEG_2000_, ICCP NPs) that integrates NIR imaging, SDT and cuproptosis for precise tumor therapy. The platform utilizes CuS NPs as a nanocarrier to co-load the sonosensitizer Ce6 and the NIR fluorescent dye IR808, with surface modification by DSPE-PEG_2000_ to enhance stability and biocompatibility. ICCP NPs passively accumulate in tumor tissues, enabling real-time tumor imaging via NIR fluorescence. Under the acidic tumor microenvironment, ICCP NPs gradually degrade and release Cu^2+^, which generates ROS via Fenton-like reactions, leading to oxidative stress. Meanwhile, excessive intracellular Cu^2+^ induces aggregation of lipoylated proteins and depletion of iron–sulfur cluster proteins, thereby triggering cuproptosis. Moreover, US-activated Ce6 generates massive ROS via the sonodynamic effect, further amplifying oxidative damage. Synergistic effects of SDT and cuproptosis promote ICD and activate anti-tumor immune responses (Scheme 1). Experimental findings confirm that ICCP NPs exert potent tumor suppression through this synergistic therapeutic mechanism. This work proposes a novel strategy for constructing imaging-guided synergistic nanoplatforms and shows promising potential for future applications.Scheme 1. Schematic illustration of the fabrication of the ICCP NPs nanoplatform and its synergistic anti-tumor mechanism. a) The stepwise synthesis of ICCP NPs. b) Functional mechanisms of ICCP NPs, combining anti-tumor effects and NIR imaging.Scheme 1
Materials and methods
2
Synthesis of IR808/Ce6@CuS&DSPE-PEG2000(ICCP NPs)
2.1
In the synthesis procedure, CuS NPs (5 mg) were dispersed in deionized water (5 mL). Under dark conditions, 5 mL of Ce6 solution (1 mg/mL) and 5 mL of IR808 solution (1 mg/mL) were incorporated sequentially, and the mixture was then subjected to continuous magnetic stirring for 24 h. After the reaction, the product was subjected to repeated centrifugation and washing to obtain the IR808/Ce6@CuS complex. Subsequently, IR808/Ce6@CuS (5 mg) and DSPE-PEG_2000_ (10 mg) were co-dissolved in ethanol (5 mL). After thorough mixing, the solution was rapidly injected into 10 mL of deionized water. The mixture was magnetically stirred to homogeneity, transferred to a rotary evaporator for full ethanol removal. The crude product was subjected to repeated washing with deionized water for the elimination of unbound impurities. The final purified nanoparticles (ICCP NPs) were obtained for subsequent studies.
Detection of •OH and 1O2
2.2
Methylene blue (MB) and 1,3-diphenylisobenzofuran (DPBF) were used to detect •OH and ^1^O_2_, respectively. Briefly, different concentrations (50-200 μg/mL) of CCP NPs were mixed with 1 mL of MB (8 μg/mL), with or without H_2_O_2_ (50 μM). The mixtures were then subjected to US treatment under fixed parameters (1 MHz, 1.5 W/cm^2^, 50% duty cycle) for varying durations. The dynamic changes in absorbance at 668 nm were recorded to assess the production of •OH. To evaluate the ultrasound (US)-triggered ^1^O_2_ generation efficiency, ICCP NPs at various concentrations (0–200 μg/mL) were dispersed in 1 mL of PBS and then mixed with 1 mL of DPBF (80 μg/mL). After stirring under light-protected conditions, the absorbance changes at 418 nm were recorded using an Ultraviolet-visible (UV-Vis) spectrophotometer. An equal volume of the above mixture was separately subjected to US irradiation (1 MHz, 1 W/cm^2^, 50% duty cycle) with irradiation durations set at 0, 2, 4, 6, 8 and 10 min. The absorbance changes at 418 nm were also recorded to analyze the degradation degree of DPBF.
GSH depletion capability of ICCP NPs
2.3
The GSH depletion capacity of ICCP NPs was evaluated by a GSH assay kit. Due to the interfering absorption peak of Ce6 around 404 nm, CuS NPs was utilized to investigate the GSH depletion effect of ICCP NPs. CuS NPs at different concentrations and 25uL of 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) solution were mixed, following the manufacturer's protocol. To conduct a quantitative analysis of the substance, a UV-Vis spectrophotometer was used to measure the specific absorbance at 412 nm. Under identical experimental conditions, the ability of CuS NPs to deplete GSH at different incubation times was also tested.
Cellular uptake
2.4
To detect the intracellular copper ion uptake of ICCP NPs, 4T1 cells were seeded in cell culture dishes at a density of 2 × 10^5^ cells per dish and co-incubated with 100 μg/mL ICCP NPs for 1, 2, 4 and 8 h, respectively. The cells were gently washed twice with PBS, after which Hoechst 33342 and CuproGreen staining solutions were added to the dishes successively, followed by further incubation for 20 min. After an additional three washes with PBS, the fluorescent signal of intracellular Cu^2+^ was detected using a confocal laser scanning microscope (CLSM).
Simultaneously, 4T1 cells were seeded in confocal dishes at a density of 4 × 10^5^ cells per dish and allowed to attach for 24 h. Cells were then treated with ICCP NPs and incubated for different time periods. At each designated time point, FITC-labeled LysoTracker was added for 30 min, followed by Hoechst staining of nuclei for 10 min. The intracellular distribution of Ce6 and its colocalization with lysosomes were observed at each interval using CLSM.
Cytotoxicity assessments
2.5
Cytotoxicity was evaluated using the CCK-8 assay. Briefly, 4T1 cells were seeded in 96-well plates (1 × 10^5^ cells/mL, 100 μL per well) and allowed to attach overnight. Cells were then subjected to different treatments with or without US irradiation (1.5 W/cm^2^, 1.0 MHz, 2 min, 50 % duty cycle) and incubated for 24 h. Thereafter, CCK-8 reagent was added to each well, followed by an appropriate incubation period. Finally, the absorbance of each well was measured at 450 nm using a microplate reader to assess cell viability.
The viability of 4T1 cells was evaluated using a Live/Dead assay with dual fluorescent staining. Cells were seeded in 24-well plates and cultured for 24 h. The cells were then treated under various conditions, with or without US irradiation, and incubated for another 24 h. Afterward, they were stained with Calcein-AM and PI for 30 min at 37 °C, and fluorescence imaging was performed to distinguish live and dead cells.
To further assess the spatial distribution of live and dead cells, 4T1 multicellular tumor spheroids (MCTS) were prepared. Briefly, a 1.5% agarose solution was added to a 96-well plate and allowed to solidify. 4T1 cells were then seeded on top of the agarose layer at 1 × 10^4^ cells/well and cultured for 24 - 48 h to form MCTS. Individual spheroids were carefully transferred to confocal dishes and stained with Calcein-AM and PI for 30 min. The three-dimensional distribution of live and dead cells within the spheroids was imaged using CLSM.
Intracellular GSH and ROS evaluation
2.6
4T1 cells were seeded in 6-well plates and exposed to the specified conditions for 24 h with or without US irradiation (1.5 W/cm^2^, 1.0 MHz, 2 min, 50 % duty cycle). Subsequently, the GSH content was measured by a GSH assay kit in accordance with the manufacturer's protocol, and the absorbance at 512 nm was taken at 512 nm with a microplate reader.
Meanwhile, to detect intracellular ROS, identically treated cells were incubated with the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) for 30 min at 37 °C. Following incubation, the cells were gently washed with PBS to remove excess probe. The fluorescence signals were observed and captured with a fluorescence microscope.
Change of mitochondrial membrane potential (MMP)
2.7
To investigate the changes in MMP, 4T1 cells were seeded in 24-well plates (4 × 10^4^ cells/well) and cultured overnight. After 24 h of different treatments with or without US irradiation, the cells were examined by the JC-1 kit. Finally, intracellular fluorescence changes were monitored by fluorescence microscopy.
Colony formation assay
2.8
To evaluate the long-term inhibitory effect of ICCP NPs on 4T1 cell proliferation, a colony formation assay was performed. Briefly, cells were plated in 6-well plates at 1000 cells/well and allowed to form sufficiently large colonies. The cells were treated under different conditions with or without US irradiation and cultured for another 48–72 h. After treatment, cells were sequentially washed with PBS, followed by fixation with 4% paraformaldehyde and staining with 0.2% crystal violet. Finally, the staining results were photographed for documentation.
Cellular immunofluorescence
2.9
4T1 cells were plated in confocal dishes and treated under various conditions for 24 h with or without US irradiation. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, cellular membranes were permeabilized with 1% Triton X-100 for 20 min, and blocked with 5% bovine serum albumin (BSA) for 20 min to minimize non-specific binding. Cells were then incubated overnight at 4 °C with primary rabbit polyclonal antibodies against DLAT (13426-1-AP, 1:200), FDX1 (12592-1-AP, 1:250), CRT (10292-1-AP, 1:200), and HMGB1 (10829-1-AP, 1:200) (Proteintech, Wuhan, China). After washing several times with PBS to remove unbound antibodies, the cells were incubated with FITC-labeled (A0423, green fluorescence) and Cy3-labeled (A0516, red fluorescence) Goat Anti-Rabbit IgG (H + L) secondary antibodies (1:200; Beyotime, Shanghai, China) for 1 h at room temperature under light-protected conditions. The cells were washed several times with PBS to remove unbound antibodies. Subsequently, cells were incubated with appropriate fluorescent secondary antibodies for 1 h at room temperature under light-protected conditions. Following several washes with PBS, nuclei were counterstained with DAPI for 10 min to allow visualization. Finally, immunofluorescence signals were visualized using a CLSM.
In vivo fluorescence imaging
2.10
To assess the in vivo biodistribution of ICCP NPs, unilateral and bilateral subcutaneous tumor models were established in mice using 4T1 breast cancer cells. Once the tumor volume of the mice attained 100 mm^3^, free IR88 and ICCP NPs were injected intravenously into the tumor-bearing mice through the tail vein. At the predetermined time points, in vivo fluorescence imaging was performed on the mice using the IVIS Spectrum system to intuitively observe the distribution characteristics and metabolic trends of the drug in vivo. Meanwhile, the mice were euthanized at 48 h and 96 h post-administration, respectively. Three mice were randomly selected from each group, and their tumor tissues as well as major organs including the heart, liver, spleen, lungs and kidneys were collected for further ex vivo imaging and subsequent related analyses.
Evaluation of the anti-tumor efficacy in the unilateral tumor model
2.11
Female BALB/c mice (6–8 weeks, 18-20g) were obtained from GemPharmatech Co., Ltd. (Jiangsu, China). To establish the tumor model, 2 × 10^6^ 4T1 cells were subcutaneously injected into the right inguinal region of each mouse. When the tumor volume reached approximately 100 mm^3^, the tumor-bearing mice were randomly assigned to six groups: (1) PBS, (2) PBS + US, (3) CP NPs + US, (4) CCP NPs + US, (5) ICCP NPs, (6) ICCP NPs + US (ICCP 6 mg/kg). Five mice were assigned to each group. The above six groups of mice were subjected to different drug treatment on days 0, 3, 6 and 9, respectively. US (1.0 MHz, 50% duty cycle, 1.5 W/cm^2^, 3 min) was applied to designated groups 24 h post-injection. During the treatment, the changes in body weight and tumor volume of the mice were monitored every other day.
After 12 days of treatment, mice were euthanized. Subsequently, blood samples, tumor tissues, and major organs including the heart, liver, spleen, lungs, and kidneys were then collected for analysis. The blood samples were subjected to hematological and biochemical analysis. The organ specimens from each group were processed via routine procedures (fixation, dehydration, and embedding) prior to hematoxylin-eosin (H&E) staining, so as to comprehensively evaluate the biosafety of the drug in vivo. To evaluate the anti-tumor efficacy, tumor tissues from each treatment group were collected for paraffin embedding and sectioning. Subsequently, H&E staining, TUNEL apoptosis assay, and Ki67 immunohistochemical staining were performed to detect the necrosis, apoptosis, and proliferation status of the tumor tissues. Furthermore, immunofluorescence staining was performed to detect the expression levels of DLAT, CRT, and HMGB1 in tumor tissues.
Investigation of immune activation in the unilateral tumor model
2.12
To evaluate immune cell infiltration within tumors, mice were euthanized after the 12-day treatment period, and tumor tissues were dissociated into single cells. For quantification of mature dendritic cells in tumor tissues, single-cell suspensions were incubated with anti-CD11c, -CD80, and -CD86 antibodies for 30 min at 4 °C in the dark, washed with 1% FBS-PBS, and analyzed by flow cytometry. Similarly, to assess T-cell infiltration, a parallel suspension was stained with anti-CD3, -CD4, and -CD8 antibodies using an identical protocol, followed by flow cytometric identification of CD4^+^ and CD8^+^ T cell populations.
Anti-tumor efficacy of ICCP NPs in bilateral tumor model
2.13
Female BALB/c mice (6–8 weeks, 18-20g) were obtained from GemPharmatech Co., Ltd. (Jiangsu, China). To establish the Bilateral tumor model, 2 × 10^6^ 4T1 cells were subcutaneously injected into the bilateral inguinal regions of mice. When the tumor volume reached approximately 100 mm^3^, the tumor-bearing mice were randomly assigned to three groups: (1) PBS (2) ICCP NPs, (6) ICCP NPs + US. After intravenous injection of I ICCP NPs for 24 h, the US irradiation was performed. Furthermore, during the treatment period, IVIS was employed to dynamically monitor the accumulation of the drug at the tumor sites of bilateral tumor-bearing mice after administration. During the treatment, the changes in body weight and tumor volume of the mice were monitored every other day. After 12 days of treatment, mice were euthanized.
Statistical analysis
2.14
All quantitative results were reported as mean ± standard deviation (SD). All statistical tests are performed by GraphPad Prism 10. For comparisons between two groups, two-tailed Student's t-test was used. For comparisons between three groups or more experiments, one-way or two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons. The following thresholds were used to denote statistical significance: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.
Results and discussion
3
Fabrication and verification of ICCP NPs
3.1
To enhance the anti-tumor efficacy of cuproptosis, we developed a multifunctional nanoplatform based on CuS NPs to achieve passive accumulation and release of Cu^2+^ at the tumor site. By co-loading the sonosensitizer Ce6 and the NIR dye IR808 onto CuS NPs, followed by surface modification with DSPE-PEG_2000_ to improve biocompatibility and prolong blood circulation, we successfully constructed a composite nanoplatform (ICCP NPs). These ICCP NPs integrate SDT, cuproptosis and NIR imaging, establishing a multifunctional theranostic system. For comparison, CCP NPs (Ce6-loaded CuS NPs with DSPE–PEG_2000_ surface modification) and CP NPs (CuS NPs modified solely with DSPE–PEG_2000_) were also prepared as control formulations. Fourier Transform Infrared (FTIR) analysis revealed the characteristic absorption peaks of DSPE-PEG_2000_, confirming its successful encapsulation on the surface of ICCPNPs (Fig. S1). According to the TEM images (Fig. 1a and b), CuS NPs and ICCP NPs exhibit a spherical morphology with relatively uniform sizes,the particle sizes were ≈80 nm and 90 nm, respectively. According to the nitrogen adsorption and desorption curves, the CuS NPs exhibit a hysteresis loop type (Fig. 1c), indicating the presence of a mesoporous structure and cavities that enable efficient drug loading. The hydrodynamic diameters of CuS NPs, CCP NPs and ICCP NPs determined by dynamic light scattering (DLS) were 171.4 ± 4.42 nm, 183.7 ± 8.73 nm and 183.9 ± 9.40 nm, with low polydispersity index (PDI) values and excellent dispersibility (Fig. 1d and Fig. S2). Since these nanoparticles have sizes below 200 nm, they can passively accumulate at tumor sites. Zeta potential measurements revealed a positive value of +5.01 ± 0.13 mV for CuS NPs (Fig. 1e), whereas CCP NPs and ICCP NPs exhibited negative zeta potentials of −6.63 ± 0.59 mV and −2.14 ± 0.43 mV. These observed shifts in zeta potential may stem from the presence of carboxyl functional groups within the Ce6 [28]. The alterations in zeta potential further confirm the successful preparation of CCP NPs and ICCP NPs. Energy dispersive spectroscopy (EDS) and mapping images verified that Cu, S, C, N and O were distributed within the ICCP NPs (Fig. 1f), conforming the successful synthesis of both CuS NPs and ICCP NPs. The successful co-loading of Ce6 and IR808 was confirmed by the UV-vis spectrum of ICCP NPs, which exhibited characteristic absorption peaks at 404 nm and 656 nm for Ce6, and at 776 nm for IR808 (Fig. 1g).Fig. 1. Synthesis and characterization of ICCP NPs. TEM images of a) CuS NPs and b) ICCP NPs. c) N2 adsorption-desorption isotherm and pore-size distribution of CuS NPs. d) Hydrodynamic diameter distribution of CuS NPs, CCP NPs, and ICCP NPs measured by DLS. e) Zeta potential measurements of CuS NPs, CCP NPs, and ICCP NPs. f) Elemental mapping results of ICCP NPs. g) UV-Vis absorption spectra for different materials. h) XRD analysis of CuS NPs and ICCP NPs. i-n) XPS survey and high-resolution spectra of ICCP NPs.Fig. 1
X-ray powder diffraction (XRD) analysis of ICCP NPs revealed that their diffraction peaks were essentially consistent with those of CuS NPs, matching the characteristic peaks of standard hexagonal-phase CuS microcrystals (JCPDS 06-0464), further verifying the successful preparation of ICCP NPs (Fig. 1h). X-ray photoelectron spectroscopy (XPS) was further applied to investigate the elemental composition of ICCP NPs and the valence states of Cu and S. Full-spectrum analysis confirmed the presence of Cu, S, C, N, and O in ICCP NPs (Fig. 1i–n), which is consistent with the results of EDS and elemental mapping. High-resolution XPS spectra showed that the binding energy peaks of Cu 2p_3_/2 and Cu 2p_1_/2 at 934.33 eV and 954.09 eV matched the characteristic peaks of Cu^2+^, while the binding energy peaks of S 2p_3_/2 and S 2p_1_/2 at 161.74 eV and 163.04 eV correspond to characteristic peaks of S^2−^, further confirming the successful synthesis of CuS NPs and ICCP NPs. Ce6 was efficiently encapsulated into CuS NPs with an encapsulation efficiency of 75.32 ± 2.84% and a corresponding loading capacity of 37.66 ± 1.42% (Fig. S3). Furthermore, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis revealed that ICCP NPs contained 30.94% Cu^2+^, which provides a sufficient source of Cu^2+^ to induce cuproptosis.
Sonodynamic/chemodynamic properties and cellular uptake
3.2
ROS are a class of highly reactive oxygen-derived chemical species, such as the superoxide anion (O_2_^−^), hydroxyl radical (•OH), and singlet oxygen (^1^O_2_) [38]. Therefore, methylene blue (MB) was employed as a probe to assess the capacity of ICCP NPs for mediating •OH production. As the absorption peak of IR808 overlaps with the UV absorption spectrum of MB degradation products, which might compromise the reliability of detection outcomes, CCP NPs were selected for this study to accurately evaluate ROS generation. MB degradation assays revealed that in the presence of H_2_O_2_ (Fig. 2a), a positive correlation existed between CCP NPs concentration and MB degradation rate, thereby confirming that Cu^2+^ can generate ·OH through a Fenton-like reaction [39,40]. Under US activation, CCP NPs also induced MB degradation, which indicated that CCP NPs are capable of promoting ROS generation upon US stimulation (Fig. 2b). When both H_2_O_2_ and US were applied simultaneously (Fig. 2c), MB degradation rate was significantly enhanced, indicating a significant increase in the production of ROS. Furthermore, 1,3-diphenylisobenzofuran (DPBF) was employed to monitor the generation of ^1^O_2_. The results illustrated that the characteristic absorption peak of DPBF showed no apparent variation with the gradual increase in ICCP NPs concentration in the absence of US irradiation. In contrast, the characteristic absorption peak of DPBF declined progressively with the prolonged duration of US irradiation, demonstrating that ICCP NPs are capable of generating ^1^O_2_ upon exposure to US irradiation (Fig. 2d and e). These findings confirm that the ICCP NPs can efficiently generate ROS through CDT and SDT, laying a critical foundation for subsequent tumor therapy.Fig. 2. Physicochemical properties of ICCP NPs and cellular uptake. a) Different concentrations of CCP NPs and H_2_O_2_ generate ·OH through a Fenton-like reaction. b) ROS generation by CCP NPs under US irradiation at various time points. c) ROS generation by CCP NPs and H_2_O_2_ under US irradiation at various time points. d)^1^O_2_ generation by ICCP NPs at different concentrations. e) ^1^O_2_ generation by ICCP NPs with different durations of US irradiation. f) GSH depleting capacity of CuS NPs at various concentrations. g) GSH depleting capacity of CuS NPs at various time points. h) pH-responsive release rate of Ce6. i) Uptake of copper ions in 4T1 cells following co-incubation with ICCP NPs for various durations. j) Assessment of colocalization between ICCP NPs and lysosomes at various time points. k) Penetration depth of ICCP NPs inside tumor spheroids.Fig. 2
GSH is a major endogenously overexpressed antioxidant in tumor cells, which is involved in scavenging ROS and maintaining cellular redox homeostasis [41,42].To assess the GSH depletion capability of ICCP NPs, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) was employed as the specific detection reagent. It should be noted that CuS NPs was selected to evaluate the GSH depletion capability of ICCP NPs due to the interference from the absorption peak of Ce6 around 404 nm. As shown in Fig. 2f and g, the reaction of DTNB with GSH yielded a distinct absorption peak at 412 nm, reflecting a high level of GSH within the system. However, following the addition of CuS NPs, the intensity of this characteristic peak exhibited a concentration-dependent gradual reduction with increasing Cu^2+^ levels, thereby confirming that an efficient redox reaction occurs between Cu^2+^ and GSH. Further time-dependent assays revealed a steady reduction in absorbance at 412 nm as co-incubation proceeded, which confirms the persistent and robust GSH-scavenging function of CuS NPs [43].
When exposed to pH 5.5, the architecture of ICCP NPs was completely disintegrated and disrupted, this phenomenon not only confirms the acid-responsive degradation property of ICCP NPs but also synchronously triggers the release of Cu^2+^, Ce6, and IR808 (Fig. S4). The total cumulative release of Ce6 reached 80% within 48 h (Fig. 2h). Owing to the intrinsic red fluorescence of Ce6, the cellular uptake of this component was investigated prior to evaluating the therapeutic efficacy of ICCP NPs. The intracellular red fluorescence intensity gradually increased with prolonged co-incubation times (1, 2, 4, and 6 h), indicating time-dependent cellular uptake of Ce6 (Fig. S5). Meanwhile, the release behavior of Cu^2+^ from ICCP NPs after co-incubation with 4T1 cells was examined using a copper ion probe. The results showed that the intracellular green fluorescence signal gradually intensified with extended co-incubation time (1, 2, 4, and 8 h), indicating that ICCP NPs undergo time-dependent degradation in the tumor microenvironment and continuously release a substantial amount of copper ions (Fig. 2i). To comprehensively investigate the subcellular distribution of ICCP NPs after cellular uptake, we performed a colocalization study using a green fluorescent lysosomal marker. The results indicated that within 1–4 h of co-incubation, the red fluorescence (Ce6) and green fluorescence (lysosomes) exhibited significant time-dependent colocalization, indicating that ICCP NPs were internalized into lysosomes by cells through endocytosis. After 8 h of co-incubation, the colocalization efficiency of the two fluorophores decreased significantly, suggesting lysosomal dysfunction and effective lysosomal escape of ICCP NPs (Fig. 2j). This result was further confirmed by the Pearson's correlation coefficient, which decreased from 0.94 ± 0.02 at 4 h to 0.51 ± 0.03 at 8 h (Fig. S6). Furthermore, a tumor spheroid model was established in this study to evaluate the penetration ability of ICCP NPs within tumor spheroids. As shown in Fig. 2k,the red fluorescence signal of free Ce6 was confined to the region of 30–45 μm in tumor spheroids and attenuated rapidly with increasing depth, whereas the fluorescence signal of ICCP NPs was stably distributed throughout the entire tumor spheroid from the surface layer to the deep region (30–105 μm). These findings indicate that the nanocarrier can effectively enhance the penetration efficiency of agents in tumor tissues.
The anticancer activity of ICCP NPs in vitro
3.3
Under the acidic tumor microenvironment, ICCP NPs undergo specific cleavage, releasing Cu^2+^ that catalyzes the conversion of overexpressed intracellular H_2_O_2_ into highly toxic ·OH, simultaneously depleting the overexpressed GSH [39]. Upon US activation, Ce6 efficiently generates ^1^O_2_ through SDT mechanisms. The integration of SDT and CDT induces a sharp increase in ROS, effectively causing oxidative damage to tumor cells. Concurrently, Cu^2+^ can be reduced to Cu ^+^ via GSH-mediated redox reaction, which consumes intracellular GSH. The generated Cu^+^ specifically targets lipoylated proteins in the mitochondrial tricarboxylic acid cycle (TCA), triggering their abnormal oligomerization and subsequent activation of cuproptosis [44]. Notably, the dynamic balance of intracellular Cu^+^/Cu^2+^ acts as a key intrinsic factor governing the synergistic anti-tumor effects of CDT, SDT, and cuproptosis, with its fluctuations closely linked to cellular redox homeostasis. Under oxidative stress conditions, progressive GSH depletion inherently limits the continuous reduction of Cu^2+^ to Cu^+^, establishing a redox-responsive copper cycling that ensures transient yet sufficient Cu^+^ generation to initiate cuproptosis, while Cu^2+^-mediated oxidative stress further amplifies tumor cell damage [10,45,46].
To examine the anti-cancer properties of ICCP NPs (Fig. 3a), the well-established 4T1 murine breast carcinoma model was utilized for experimental assessment. First, the biocompatibility of the developed nanoparticles was evaluated using the Cell Counting Kit-8 (CCK-8). The results showed that after treatment with CP NPs and ICCP NPs at concentrations ranging from 12.5 to 200 μg/mL, the viability of 4T1 cells remained above 80%, demonstrating the favorable biocompatibility and cellular safety of these nanoparticles (Fig. S7). Subsequently, the survival rate of 4T1 cells under different treatment conditions was assessed. The results revealed that under US irradiation, both the CCP NPs and ICCP NPs groups exhibited significant inhibitory effects on the growth of 4T1 cells (Fig. 3b). Excessive GSH serves as a critical antioxidant in the tumor microenvironment, scavenging and maintaining cellular redox homeostasis. Cu^2+^ can deplete intracellular GSH, thereby promoting ROS accumulation and exacerbating oxidative stress damage. In this study, we measured intracellular GSH levels following various treatments. Both CP NPs/US NPs and ICCP NPs effectively depleted GSH, primarily attributable to the catalytic activity of Cu^2+^ (Fig. 3c). In contrast, treatment with CCP NPs/US or ICCP NPs/US led to a more pronounced reduction in intracellular GSH, indicating that ROS further consumes reduced GSH. This dual mechanism synergistically enhances GSH depletion and amplifies ROS-induced oxidative stress.Fig. 3. Anti-cancer effect of ICCP NPs upon US irradiation. a) Schematic diagram of the anti-tumor mechanism of ICCP NPs upon US irradiation. b) Cell viability assessment after 24 h of incubation with distinct experimental treatments (n = 4). c) GSH content in 4T1 cells following exposure to diverse experimental treatments (n = 3). d)Assessment of intracellular ROS production following various treatments by DCFH-DA assay and e) Quantitative analysis of its fluorescence intensity (n = 3). f) JC-1 staining in 4T1 cells following various treatments. g) Representative Calcein-AM/PI staining images for viability assessment of 4T1 cells and multicellular tumor spheroids following various treatments. h)Colony formation of 4T1 cells following various treatments.Fig. 3
Subsequently, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was utilized to detect the intracellular ROS generation ability of ICCP NPs in tumor cells. As shown in Fig. 3d and e, CCP NPs/US and ICCP NPs/US induced the most intense green fluorescence, with an intensity approximately 20 times that of the control group, indicating robust intracellular ROS generation. The synergistic combination of SDT and CDT markedly enhanced ROS production, thereby inducing intense oxidative stress and ultimately achieving highly efficient tumor cell killing [6]. Since both intracellular Cu^2+^ overload and massive accumulation of ROS can induce mitochondrial membrane depolarization [47]. Therefore, this study employed the JC-1 detection kit to evaluate mitochondrial membrane depolarization in 4T1 cells under different treatments. The results (Fig. 3f) showed that the CP NPs/US and ICCP NPs exhibited weakened red fluorescence and enhanced green fluorescence, confirming damage to the mitochondrial membrane potential. Notably, the CCP NPs/US and ICCP NPs/US induced a more significant enhancement in green fluorescence. Furthermore, cytotoxicity assessed by Calcein-AM/PI staining revealed abundant dead cells in the CCP NPs/US and ICCP NPs/US (Fig. 3g, S8 and 9). Subsequently, the anti-proliferative effect of ICCP NPs were determined by a colony formation assay. The results indicated that, compared with other treatment groups, the CCP/US and ICCP/US significantly inhibited colony formation (Fig. 3h). Overall, these findings demonstrate that CCP NPs/ICCP NPs upon US irradiation have powerful anti-tumor efficacy, exhibiting remarkable abilities to induce apoptosis and inhibit cell proliferation.
The immunostimulatory effects of ICCP NPs in vitro
3.4
Cuproptosis is a process where copper ions are specifically bound to lipoylated TCA cycle enzymes [13]. This binding triggers the abnormal oligomerization of the key mitochondrial metabolic enzyme DLAT and simultaneously undermines the stability of iron-sulfur cluster proteins, thereby inducing their extensive degradation and loss [15,44]. This cascade of events induces severe proteotoxic stress, which mediates tumor cell death via a unique metabolism-dependent mechanism (Fig. 4a). Therefore, this study systematically examined the protein expression levels of DLAT and FDX1 in 4T1 cells under different treatment conditions. Immunofluorescence results showed that DLAT expression was relatively low in the CP NPs/US and ICCP NPs groups, whereas DLAT expression was significantly increased in the CCP NPs/US and ICCP NPs/US groups (Fig. 4b and Fig. S10). Furthermore, both immunofluorescence and Western blot analyses consistently indicated that FDX1 protein expression was significantly reduced in the CCP NPs/US and ICCP NPs/US groups (Fig. 4b,S11 and 12). These findings indicate that CCP NPs/US and ICCP NPs/US can effectively activate molecular pathways associated with cuproptosis.Fig. 4. The anti-tumor immune mechanism of ICCP NPs. a) Schematic illustration of cuproptosis mechanism. b) Immunofluorescence analysis of DLAT and FDX1 in 4T1 cells following various treatments. c) Schematic diagram illustrating the synergistic induction of ICD by SDT and cuproptosis. d) Immunofluorescence analysis of CRT and HMGB1 in 4T1 cells following various treatments. e) Quantitative analysis of CRT fluorescence intensity. f) ATP content in 4T1 cells following various treatments.Fig. 4
This study further investigated whether the combination of SDT and cuproptosis could synergistically induce ICD and promote the release of DAMPs (Fig. 4c). DAMPs include the cell surface exposure of calreticulin (CRT), translocation of high mobility group box 1 (HMGB1), and secretion of ATP [48]. The results demonstrated that 4T1 cells treated with CP NPs/US or ICCP NPs showed only slight CRT exposure, whereas 4T1 cells treated with CCP NPs/US and ICCP NPs/US induced significant CRT exposure on the cell membrane (Fig. 4d, e, and Fig. S13). Moreover, both CCP NPs/US and ICCP NPs/US exhibited extracellular translocation of HMGB1(Fig. 4d andFig. S14). Simultaneously, the highest level of ATP secretion was detected in these treatment groups (Fig. 4f). These findings demonstrate that the synergistic effect of SDT and cuproptosis can efficiently induce ICD in tumor cells and significantly promote the release of DAMPs. This process not only directly kills cancer cells but also triggers danger signaling pathways. Consequently, it transforms immunologically "cold" tumors into "hot" tumors, thereby laying a solid foundation for activating subsequent anti-tumor immune responses [6].
Anti-tumor efficacy of ICCP NPs in subcutaneous breast cancer model
3.5
To evaluate the in vivo anti-tumor efficacy of ICCP NPs and their ability to induce apoptosis and cuproptosis in tumor cells, we conducted studies using a subcutaneous breast cancer xenograft model. Prior to the in vivo experiments, we first confirmed the successful loading of IR808 onto ICCP NPs, which is essential for reliable NIR imaging and biodistribution analysis. Under identical centrifugation conditions, free IR808 dye remained in the supernatant without forming a pellet, whereas IR808-loaded ICCP NPs sedimented at the bottom of the Eppendorf tube, with NIR fluorescence detectable in the pellet (Fig. S15). Subsequently, ICCP NPs were administered to tumor-bearing mice via tail vein injection to assess their in vivo NIR imaging performance and biodistribution. As shown in Fig. 5a, NIR fluorescence signals in tumor tissues were detectable 4 h after ICCP NPs injection, gradually increasing and reaching a maximum at 48 h. This indicates that ICCP NPs passively accumulate in tumors via the enhanced permeability and retention (EPR) effect and providing a reference for optimizing the dosing schedule and intervals for subsequent in vivo treatments. In contrast, the free IR808 group exhibited negligible tumor fluorescence, suggesting limited accumulation and rapid clearance. Ex vivo imaging at 48 h further confirmed the efficient tumor accumulation of ICCP NPs (Fig. 5b). Collectively, these results demonstrate that ICCP NPs enable effective NIR imaging, laying a solid foundation for image-guided sonodynamic therapy and cuproptosis-based synergistic treatment.Fig. 5. The anti-tumor effect of ICCP NPs in vivo. a) Biodistribution of free IR808 and ICCP NPs in tumor-bearing mice. b) NIR imaging of harvested organs 48 h post-injection. c) Schematic diagram of the anti-tumor experiment in BALB/c mice. d) Body weight fluctuations in tumor-bearing mice following various treatments throughout the therapeutic period. e) Tumor volume growth curves in tumor-bearing mice from different treatments. f) Tumor histological assessment by H&E staining, TUNEL, Ki67 staining, and DLAT immunofluorescence following various treatments.Fig. 5
Subsequently, we further evaluated the in vivo anti-tumor efficacy of ICCP NPs. Throughout the study, all tumor-bearing mice were housed under controlled conditions and received four cycles of treatment via tail vein injection. Based on the NIR imaging results, the dosing interval was adjusted to once every three days to maximize tumor passive targeting and therapeutic efficacy. Ultrasound irradiation (1.0 MHz, 50% duty cycle, 1.5 W/cm^2^) was applied 24 h after each drug administration for a duration of 3 min. Parameters of 1.0 MHz and 1–2 W/cm^2^ can achieve an effective penetration depth of approximately 4 cm in murine tumor tissues [49], sufficient to cover the subcutaneous tumor models employed in this study. Moreover, 1.0 MHz and 1–2 W/cm^2^ has been widely reported in preclinical and clinical therapeutic ultrasound studies without causing significant adverse effects, supporting the translational feasibility of these settings [47,[49], [50], [51]]. Body weight and tumor volume were monitored throughout the experiment (Fig. 5c and d). Tumor volume monitoring showed that CP NPs/US and ICCP NPs only weakly inhibited tumor proliferation. In contrast, the CCP NPs/US and ICCP NPs/US exhibited the most potent suppression of tumor growth (Fig. 5e,Fig. S16), accompanied by minimal tumor volume (Figs. S17 and 18). Hematoxylin and eosin (H&E) staining results revealed extensive nuclear pyknosis, fragmentation, and dissolution in tumor tissues treated with CCP NPs/US and ICCP NPs/US (Fig. 5f). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) demonstrated that both CCP NPs/US and ICCP NPs/US induced widespread tumor cell apoptosis (Fig. 5f and Fig. S19). Ki67 staining further confirmed a significant reduction in Ki67-positive cells in the CCP NPs/US and ICCP NPs/US groups, indicating severe suppression of tumor cell proliferation (Fig. 5f). Furthermore, DLAT immunofluorescence analysis of tumor tissues across treatment groups showed prominent DLAT oligomerization in the CCP NPs/US and ICCP NPs/US groups (Fig. 5f and Fig. S20), demonstrating their significant capability to induce cuproptosis. These results demonstrated that both CCP NPs/US and ICCP NPs/US treatment regimens could not only effectively induce tumor cell apoptosis and markedly constrain cell proliferation, but also initiate the cuproptosis via triggering DLAT protein oligomerization, ultimately exerting a robust synergistic anti-tumor efficacy. Compared with previous studies [16,28,52], ICCP NPs exhibit several notable advantages. They achieve high copper loading for efficient cuproptosis induction, allow real-time monitoring of tumor accumulation via NIR imaging, and integrate SDT-triggered and CDT-mediated ROS generation within a single platform, enhancing anti-tumor efficacy while minimizing off-target effects. In addition, their design takes full advantage of passive tumor-targeting, controllable release, and multimodal therapeutic integration. These features collectively improve therapeutic efficiency and controllability, supporting the rational design of ICCP NPs and their potential for translational applications.
Evaluation of the efficacy of ICCP NPs in promoting anti-tumor immune response
3.6
ICD represents a critical component of cancer immunotherapy, which drives the release of DAMPs [47,53]. This process activates anti-tumor immune responses by promoting dendritic cell (DC) maturation, antigen presentation, cytotoxic T lymphocyte (CTL) activation, and cytokine secretion [54]. To explore the immune mechanism triggered by ICCP NPs under US irradiation, we conducted a series of experiments, with the corresponding working model depicted in Fig. 6a. Immunofluorescence results revealed that both CCP NPs/NPs US and ICCP NPs/US significantly enhanced CRT externalization to the cell membrane and translocation of HMGB1 (Fig. 6b, S21 and 22). After different treatments, the mature DC proportions in tumor tissues were 17.57 ± 1.53% and 14.90 ± 3.96% in the CP NPs/US and ICCP NPs groups, respectively (Fig. 6c and d). In contrast, the combination therapy of CCP NPs/US and ICCP NPs/US demonstrated enhanced immune activation capacity, significantly increasing the mature DC proportions to 34.33 ± 4.44% and 36.03 ± 2.76%, respectively, approximately 8-fold higher than that of the control group. Moreover, the synergistic treatments promoted the highest degree of T cell infiltration in tumor tissues, with CD4^+^ T cell proportions reaching 9.24 ± 1.22% and 9.36 ± 1.20%, and CD8^+^ T cell proportions reaching 7.60 ± 0.52% and 7.80 ± 0.86% in the CCP NPs/US and ICCP NPs/US groups, corresponding to approximately 5.5-fold and ∼6.5-fold increases compared with the control group (Fig. 6e–h). These results indicate that CCP NPs/US and ICCP NPs/US not only induce ICD but also robustly reverse the immunosuppressive tumor microenvironment, thereby enhancing adaptive anti-tumor immune responses and contributing to long-term tumor growth suppression.Fig. 6ICCP NPs mediated anti-tumor immunity in vivo. a) Schematic diagram llustrating the synergistic anti-tumor immune mechanism of ICCP NPs through SDT and cuproptosis. b) Immunofluorescence analysis of CRT and HMGB1 across different treatments. c) Assessment of mature DC proportions (CD80^+^/CD86^+^) in tumor tissues via flow cytometry and d) corresponding quantitative analysis. e) Assessment of CD4^+^ T cells proportions in tumor tissues via flow cytometry and f) corresponding quantitative analysis. g) Assessment of CD8^+^ T cells proportions in tumor tissues via flow cytometry and h) corresponding quantitative analysis.Fig. 6
Anti-tumor efficacy of ICCP NPs in bilateral tumor model
3.7
Distant metastasis represents the primary cause of tumor-associated mortality during disease progression [55,56]. Thus, an optimal therapeutic strategy should accomplish the dual clearance of primary tumors and metastatic lesions. Thus, we further established a bilateral tumor-bearing model to evaluate the anti-tumor efficacy of ICCP NPs via an immunotherapeutic regimen mediated by SDT and cuproptosis (Fig. 7a). In vivo imaging confirmed the sustained accumulation of ICCP NPs at the tumor site during treatment (Fig. 7b). Following 12 days of treatment, the tumor-bearing mice were euthanized, and tumor tissues were harvested for subsequent experimental analysis. As shown in Fig. 7c–h, when ICCP NPs were administered alone without US irradiation, they exerted a moderate inhibitory effect on the proliferation of both primary and distant tumors, yet their therapeutic activity was restricted. In contrast, the combined therapeutic strategy of ICCP NPs with US irradiation could trigger a potent synergistic effect, ultimately exerting maximal tumor-inhibitory efficacy against both primary and distant tumors. Furthermore, results from the macroscopic tumor morphology images revealed that both primary and distant tumors in the ICCP NPs/US group were significantly smaller than those in other groups (Figs. S23 and 24). Our results demonstrate that the combined SDT and cuproptosis strategy not only markedly suppresses primary tumor growth but also effectively elicits an abscopal effect, thereby further inhibiting the proliferation of distant tumors.Fig. 7. Anti-tumor efficacy of ICCP NPs in bilateral tumor models. a) Schematic Diagram of the Bilateral Tumor Therapy Procedure. b) Biodistribution of ICCP NPs in bilateral tumors during treatment. c) Tumor volume growth curves in primary tumors under different treatments. d) Dynamic changes in primary tumor volume during treatment. e) Tumor weight of primary tumors in different treatment groups. f) Tumor volume growth curves in distant tumors under different treatments. g) Dynamic changes in distant tumor volume during treatment. h) Tumor weight of distant tumors in different treatment groups.Fig. 7
Systemic safety evaluation of ICCP NPs
3.8
Having verified the robust anti-tumor activity of ICCP NPs, we performed a systematic evaluation of their biosafety. Serum biochemistry was examined in this study, including hematological indices and key indicators of hepatic and renal function. The analysis indicated no statistically significant differences in these indices among the various treatment groups of mice (Fig. 8a–c). Hemolysis assay further demonstrated that even at a high concentration of 150 μg/mL, ICCP NPs induced less than 4% hemolysis, indicating excellent hemocompatibility (Fig. 8d). Moreover, H&E staining of major organs revealed no obvious histopathological abnormalities in any of the treatment groups (Fig. 8e). These results indicate that ICCP NPs exhibit minimal systemic toxicity and a favorable biosafety.Fig. 8. Biocompatibility assessment of ICCP NPs. a) Hematological parameters of each group following different treatments. b) liver and c) kidney function assays following different treatments. d) Hemolysis assay of ICCP NPs at different concentrations. e) H&E staining of major organs following different treatments.Fig. 8
Conclusion
4
In conclusion, an intelligent anti-tumor theranostic platform (ICCP NPs) with integrated SDT, cuproptosis and NIR imaging functions was successfully established in this study. Precise tumor localization and real-time guidance during the treatment process are achieved by this platform, while ROS are efficiently generated via SDT, resulting in a significant enhancement of tumor cell killing efficiency. Tumor-specific release of Cu^2+^ further promotes ROS production through Fenton-like reactions and depletes overexpressed GSH in tumor cells, intensifying intracellular oxidative stress. Furthermore, cuproptosis is further induced by the sustained accumulation of Cu^2+^. The combined action of SDT and cuproptosis effectively activates ICD, remodels the tumor immune microenvironment, and initiates a systemic anti-tumor immune response. Consistent with these mechanisms, a series of experiments have confirmed that the ICCP NPs exhibits good biocompatibility and significant tumor suppression efficacy. Therefore, this therapeutic approach significantly elevates the precision and effectiveness of cancer therapy, underscoring its potential for further development as an innovative direction in cancer treatment.
Ethics approval statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of China–Japan Friendship Hospital (No. zryhyy21-23-02-01).
Funding statement
This work was financially supported by the 10.13039/501100001809National Natural Science Foundation of China (U22A20326), the Postdoctoral Fellowship Program Grade (C) of 10.13039/501100002858China Postdoctoral Science Foundation (GZC20250963) and 10.13039/501100007129Natural Science Foundation of Shandong Province (ZR2025LZL013).
CRediT authorship contribution statement
Xiaoqin Luo: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Xiaojuan Wang: Data curation, Formal analysis, Methodology, Software. Sheng Li: Data curation, Methodology, Software. Qing Chen: Funding acquisition, Supervision, Writing – review & editing. Jibin Song: Funding acquisition, Resources, Supervision, Writing – review & editing. Junqiang Chen: Funding acquisition, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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