The enhanced photothermal therapy against gastric cancer by mitochondria/STAT3-targeted nanoplatform with OXPHOS blocking

Abstract

Near-infrared (NIR) photothermal therapy (PTT) has provided an innovative modality for the ablation of gastric cancer (GC) with minimized damage to normal tissues. However, the upregulation of heat shock proteins (HSPs) and the abnormal vascularization at the tumor site, as well as the low specificity with the diffusional hindrance of therapeutic agents to cancer cells, severely hamper this mono-therapeutic strategy. To overcome these obstacles, we designed and prepared a mitochondria/STAT3-targeted nanoplatform (ATO/CR NPs), which is self-assembled by Drug Administration (FDA)-approved atorvaquinone (ATO) and NIR phototherapeutic agent CR to fight GC. The ATO/CR NPs exhibit enhanced PTT efficiency owing to the oxidative phosphorylation (OXPHOS) blocking in mitochondria for the downregulation of ATP and HSP based on ATO. More importantly, the ATO from ATO/CR NPs can also specifically target STAT3 in GC cells to restrain proliferation, inhibit angiogenesis, and promote apoptosis. Hence, the multimodal NIR ATO/CR NPs initiate accurate targeting of cancer cells, triggering serious mitochondrial dysfunction and cellular apoptosis to amplify the photo-ablation activity, which provides a promising strategy for GC treatment, warranting further preclinical exploration.

Graphical abstract

The mitochondria/STAT3-targeted ATO/CR NPs can specifically accumulate in tumor tissue through the EPR effect and active targeting STAT3 protein in gastric cancer cells, which will boost apoptosis, suppress proliferation, restrain angiogenesis activity, and block OXPHOS to decrease the ATP generation with HSPs inhibition for PTT promotion, providing a promising avenue in gastric cancer therapy for further preclinical investigation.

Highlights

• •ATO/CR NPs enable precise tumor targeting, inducing mitochondrial dysfunction and apoptosis in gastric cancer cells.
• •ATO/CR NPs blocks OXPHOS to reduce ATP and HSPs, inhibits STAT3/Bcl-2, blocks angiogenesis, and arrests cell cycle to enhance GC photo-ablation.
• •ATO/CR NPs exhibit excellent biocompatibility, low dark toxicity, strong phototoxicity, and efficient tumor ablation in vivo.

1. Introduction

Gastric cancer (GC) represents a heavy global health burden, based on its high mortality and incidence rate, which is the fifth most commonly diagnosed malignancy worldwide, with an undesired prognosis and 5-year survival rate of <10% [[1], [2], [3]]. Currently, surgery followed by adjuvant chemoradiotherapy is the standard-of-care treatment for GC in the clinic [4]. However, even about 60% of patients experience local relapse or distant metastasis, which has been a major clinical challenge [5]. Recently, although near-infrared (NIR) light-triggered photothermal therapy (PTT) has provided an innovative modality for the GC treatment with reduced toxic side effects, the hyperthermia-activated overexpression of intracellularly related heat-shock proteins (HSPs) can impede the phototherapeutic efficacy due to the increased heat tolerance of cancer cells [[6], [7], [8], [9]]. Therefore, novel strategies are urgent to optimize anticancer efficiency.

In recent years, nanotechnology has drawn significant attention to selectively passive-targeted nano-drug delivery platforms as a great potential approach via accumulating therapeutic agents into tumors by the enhanced permeability and retention (EPR) effect [[10], [11], [12], [13], [14], [15]]. Although conceptually inspiring, nano-drug precise accumulation encounters biological barriers of fast circulation in the bloodstream and massive diffusion hindrance, making it difficult to penetrate deep tumors [[16], [17], [18]]. Hence, the innovative active-targeted drug delivery strategy, which can initiate specific targeting to biological receptors, has been widely employed to maximize therapeutic efficiency [19]. Among various protein targets, the signal transducer and activator of transcription 3 (STAT3) is aberrantly hyperactivated in GC to promote the GC development with dual functions of signal transduction and transcriptional activation [20,21]. Notably, specific targeting of the abnormally activated STAT3 can promote cell apoptosis and inhibit angiogenesis, as well as suppress tumor invasion and metastasis, which is a potential tumor therapeutic strategy [[22], [23], [24], [25]]. Hence, there is an urgent need for novel STAT3 inhibitors that have been approved by the drug regulatory authority against GC in the clinic.

Significantly, it has been shown that atovaquone (ATO), as a U.S. FDA-approved agent developed in the 1990s, can inhibit STAT3 phosphorylation, which is an effective STAT3 inhibitor to restrain cellular proliferation for promoting cellular apoptosis, as well as downregulate the expression of VEGF for anti-angiogenesis at the tumor site [[26], [27], [28], [29], [30], [31]]. In addition, the oxidative phosphorylation (OXPHOS) metabolic pathway in mitochondria can produce ATP for cell survival in the mitochondrial inner membrane by electron transportation to transmembrane protein complexes, called the electron-transport chain (ETC), wherein nicotinamide adenine dinucleotide (NADH) and succinate are electron donors Scheme 1. Generally, OXPHOS inhibition can induce unstable mitochondrial morphology, decrease mitochondrial membrane potential, disturb mitochondrial respiration distribution, and reduce energy (ATP) generation with down-regulated HSPs [32]. Accordingly, OXPHOS blocking initiated by ATO has been regarded as an innovative cancer treatment strategy to reduce the oxygen consumption rate for ATP limitation with HSP inhibition and cell dysfunction [33]. However, the lipophilic property of ATO induces low bioavailability and poor biocompatibility with serious side effects when administered alone [34]. Thus, it is important to select a suitable drug carrier and conjugate ATO with other novel therapeutic agents for synergistic cancer therapy.

Scheme 1.

Graphical illustration of the antitumor activity and mechanism based on ATO/CR NPs to activate OXPHOS blocking for enhanced photothermal therapy enabled by mitochondria/STAT3 targeting. Insert: (i) OXPHOS blocking via ATO for ATP lessening in the mitochondrial inner membrane, called ETC. (ii) Molecular docking result of ATO and the STAT3 protein.

Croconaine (CR) dyes, as organic pseudooxocarbon molecules, are the donor-acceptor-donor type zwitterionic composites with planar π-conjugation feature, displaying intense NIR-absorption with desired molar extinction coefficient, low energy gap, desired fluorescence imaging (FLI) property, and excellent photothermal performance, which can be readily synthesized and further chemical-modified for imaging-guided cancer photo-ablation [35]. Besides, CR dyes present superior thermal/photostability and photobleaching resistance compared to traditional NIR dyes [36]. Moreover, the CR molecules can be directly compounded with other agents or easily self-assembled into nanoparticles based on the π-π stacking effect to improve drug stability and therapeutic effect [37]. Hence, the CR dyes are promising to extend the function of NIR dyes in theranostics. In addition, compared with inorganic photothermal materials such as precious metals, the organic small-molecule system used in this study features mild synthesis conditions and readily tunable structures. Moreover, the assembled composite nanodrugs maintain high photothermal ablation efficiency while enabling multiple endogenous synergistic mechanisms through rational molecular design, thereby enhancing therapeutic efficacy with specific molecular mechanism [[38], [39], [40], [41], [42]].

Given the combined concerns above, in this study, we developed a multifunctional nanomedicine (ATO/CR NPs) for the enhanced PTT effect to fight GC. Firstly, the conjugate CR possesses excellent fluorescence signal and NIR PTT effect, which can self-assemble with STAT3/OXPHOS inhibitor ATO into high molar extinction coefficient (4.45 × 10⁴ M⁻¹ cm⁻¹) and superb photothermal conversion efficiency (46.3%) ATO/CR NPs, displaying homogeneous size (102.5 nm). Meaningfully, the released ATO after the illumination would target STAT3 to decrease anti-apoptotic Bcl-2 level, restrain the angiogenesis activity, and impede cell cycle-related proteins (CDK4, CDK6, and Cyclin D1) for cancer suppression, as well as block OXPHOS by inhibiting the complex III of the ETC to inhibit the ATP production, thus restraining the expression of HSPs to promote cancer PTT. More importantly, the ATO/CR NPs exhibit superb biocompatibility, excellent phototoxicity, and low dark toxicity, initiating desired and enhanced cellular apoptosis in vitro. Accordingly, guided by fluorescence/photothermal imaging (FLI/PTI), the tumors in vivo can be completely ablated (Scheme 1).

2. Results and discussion

2.1. Synthesis, physicochemical characterization, and photothermal properties

The synthetic method of CR is shown in Fig. S1. Initially, ITI was obtained by the reaction of (4-Iodophenyl) hydrazine hydrochloride with 3-methyl-2-butanone. Subsequently, CR was synthesized by reacting ITI with coronatine, achieving a yield of 58%. The structures were proved via NMR and MALDI-TOF Mass spectrometry analysis (Fig. S2–S6). Fig. 1B shows that the ITI and CR are freely soluble in dichloromethane (DCM) with an orange-to-green color change, which results from a condensation reaction that forms an extended conjugated system and leads to a red shift in the UV-Vis absorption spectrum. In addition, CR can conveniently be assembled with ATO into ATO/CR nanoparticles (NPs, Fig. 1A) by the technology of reprecipitation, which proves that the acquired ATO/CR NPs can homogeneously disperse in water compared with CR molecular (in water), wherein the ATO/CR NPs dispersion is transparent, revealing the ATO/CR NPs display superb water dispersibility. The optical absorbance performances of ATO molecule, CR NPs, and ATO/CR NPs are λ_max at 253 nm, 792 nm, and 797 nm, in which ATO/CR NPs show wide and red-shift near-infrared (NIR) absorption bands ranging from 600 nm to 900 nm affiliated CR, as well as 253 nm absorbance from ATO, exhibiting the strong conjugated planarization and hydrophobic effect as well as π–π stacking interaction between CR and ATO molecules within the nanostructures (Fig. 1C). Besides, the molar extinction coefficients of the CR molecule and ATO/CR NPs at 787 nm and 797 nm were 1.62 × 10⁵ M⁻¹ cm⁻¹ and 4.45 × 10⁴ M⁻¹ cm⁻¹, respectively (Fig. S7A–S7B and Fig. 1D and 1E), exhibiting excellent light-absorbing capacity. The bright fluorescent emission of the CR molecule and final ATO/CR NPs is shown in Fig. S7C; the emission λ_max of ATO/CR NPs is at 891 nm, revealing the potential for bioimaging applications. All results confirm the NIR ATO/CR NPs agent is an outstanding candidate for synergistic tumor ablation.

Fig. 1.

Schematic structure, physicochemical, and photothermal characterization of ATO/CR NPs. (A) Graphical illustration of ATO/CR NPs preparation. (B) Pictures of ITI, CR in DCM, and CR, ATO/CR NPs in ddH₂O. (C) Normalized absorbance of ATO in DCM and CR NPs, ATO/CR NPs in ddH₂O. (D) Absorbance curves of ATO/CR NPs in ddH₂O. (E) Linear absorbance versus concentration from (D). (F) TEM image of ATO/CR NPs. (G) DLS size distribution of ATO/CR NPs. (H) Calculated the HOMO-LUMO energy gap of iodinated CR. (I, J) Photothermal curves of ATO/CR NPs dispersion in different conditions. (K) Photothermal curve of ATO/CR NPs (1 mL, 60 μM) during laser on and off (808 nm, 1.2 W/cm², control: water). (L) Heating and cooling curves of ATO/CR NPs (60 μM) and ICG for five cycles (808 nm, 1.2 W/cm²). (M) Size and PDI changes of ATO/CR NPs incubated at rt for 7 days. (N) Schematic illustration of the thermal imaging of ATO/CR NPs (60 μM) irradiated by laser over chicken breast tissues. (O) The temperature increases of ATO/CR NPs irradiated by different lasers across varying thicknesses of chicken breast tissue.

According to the transmission electron microscope (TEM) image, the ATO/CR nanosystem presents a homogeneous spherical morphology with superb dispersion (Fig. 1F). Besides, the dynamic light scattering (DLS) displays ATO/CR NPs exhibit an average size of 102.5 nm (PDI: 0.1866) with a narrow distribution and negative Zeta potential of −28.31 mV (Fig. 1G and Fig. S7D), wherein the small size and stable property of the nanocomposite can boost internalization at tumor site by the passive targeting effect with prolonged blood circulation, which will result in the synergistic tumor suppression.

The theoretical calculations indicate that donor-acceptor-donor structure with iodinated heavy atoms of CR can effectively reduce the electron distribution between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the molecular, inducing a lower energy gap of ΔE = 1.58 eV when compared that of brominated heavy atoms of CR (ΔE = 1.59 eV), which suggests that it will promote non-radiative transition to release heat (Fig. 1H and S8A). Herein the photothermal property of ATO/CR NPs was evaluated in Fig. 1I, which displays the temperature change processes of ATO/CR NPs dispersion under 808 nm laser illumination (1.2 W/cm²) with increasing concentrations (5, 10, 20, 40, and 60 μM), and the temperature increases are 9.7, 14.4, 19.5, 24.1, and 28.5 °C, respectively. Besides, the temperature of ATO/CR NPs (60 μM) individually increased by 9.5, 16.7, 19.5, 24.1, and 28.5 °C under 0.4, 0.6, 0.8, 1.0, and 1.2 W/cm² laser irradiation (Fig. 1J). The above results show excellent photothermal efficiency with obvious concentration and power-density dependences. Finally, the η (photothermal conversion efficiency) of ATO/CR NPs is 46.3% (Fig. 1K and S8B), which is considerably higher than some other phototherapeutic drugs [43,44].

The desired photostability is vital for the biomedical application of phototherapy mediators. Firstly, the temperature rises and the size of ATO/CR NPs after five alternate irradiations do not exhibit obvious changes, which proves that the superior stability of the agent when compared with traditional photothermal agent ICG (Fig. 1L and S8C). Moreover, the size, PDI, and dispersion color of ATO/CR NPs show no visible changes after storage in H₂O, RPMI 1640 medium, and PBS at room temperature for 7 days, nor after storage at 4 °C for 14 days (Fig. 1M and S8D-S8F), further demonstrating the excellent stability of the ATO/CR NPs. This superb photothermal efficiency and good stability of ATO/CR nano-prodrug make it a promising tumor photoablation candidate.

To simulate an in vivo situation, chicken breast tissue, which is homogeneous with low-fat content, is innovatively employed in a laser penetration assay through enhancing its thickness (0-6 mm) over ATO/CR NPs (Fig. 1N). It shows that higher temperatures were significantly tested with 808 nm laser illumination vs. 730 nm laser (1.2 W/cm²) (Fig. 1O). Herein, when heating ATO/CR NPs to 42 °C (irreversible damage temperature), the 808 nm laser penetrated a greater thickness of chicken breast tissue (estimated at 3 mm) than the 730 nm laser (estimated at 2 mm), which reveals that 808 nm laser can realize deeper tissue penetration and higher photothermal conversion efficiency, implying the potential of ATO/CR NPs for synergistic cancer therapy via NIR light irradiation.

2.2. The expression of STAT3 and survival prognosis in GC

To explore the expression of STAT3 and survival prognosis in GC, firstly, we compared 31 cancers and corresponding normal tissues on STAT3 expression levels based on Gene Expression Profiling Interactive Analysis (GEPIA) data, which exhibits a high level in gastric tumor tissue (Fig. 2A). Moreover, the Human Protein Atlas (HPA) database analyses about 20 types of tumor tissues and shows that STAT3 expression levels are tumor-specific and have high expression in the MKN45 GC subtype (Fig. 2B and 2C). Importantly, as displayed in Fig. 2D, the immunohistochemical data from the HPA database reveal a marked upregulation of STAT3 protein in GC tissues when compared with normal tissues, further offering robust evidence for its potential as a therapeutic target to fight GC.

Fig. 2.

STAT3 expression levels in normal and GC tissues. (A) STAT3 expression in human tumors vs. normal tissues from the GEPIA database (STAD: stomach adenocarcinoma). (B) STAT3 expression levels in 20 different tumor types based on the HPA database. (C) STAT3 expression levels in 42 different subtypes of GC based on the HPA database. (D) Representative immunohistochemical micrographs to detect the protein level of STAT3 in stomach and GC samples from the HPA database (scale bar: 100 μm).

2.3. Anticancer performance in vitro

Inspired by the excellent morphological and spectroscopic properties of ATO/CR NPs, the cytotoxicity and anticancer performance in vitro were assessed (Fig. 3A). Firstly, the MTT assay based on MKN45 GC cells was conducted to assess the drug toxicity. Fig. 3B exhibits that the cell viability meaningfully decreases in a dose-dependent manner and triggers an IC₅₀ (half-maximal inhibitory concentration) of 10.81 μM incubated by ATO/CR NPs when compared to the IC₅₀ of 13.50 μM initiated by CR NPs upon 808 nm laser (1.2 W/cm²) illumination, showing synergistic cell ablation effect. Significantly, the cell viability is 90.6% even at 18 μM concentration of CR NPs, revealing low dark-toxicity and superb biocompatibility, making it a promising candidate against cancer. Besides, the cytotoxicity of ATO NPs and ATO/CR NPs without laser irradiation was explored to assess the cytotoxicity for MKN45 GC cells, and a slight decrease was observed after coincubation with high concentrations of them, showing cell apoptosis caused by ATO (Fig. 3C). Moreover, a hemolytic assay was preliminarily conducted to evaluate the in vivo toxicity of ATO/CR NPs [45]. The hemolysis rates induced by ATO/CR NPs (5-120 μM) are consistently well below the 5% safety threshold and significantly lower than the positive control (H₂O), further revealing the superb biocompatibility of the therapeutic NPs (Fig. 3D).

Fig. 3.

Cytotoxicity of ATO/CR NPs. (A) Schematic illustration of enhanced antitumor effect based on ATO/CR NPs-mediated PTT. (B, C) MTT assay of MKN45 GC cells in different conditions (808 nm laser, 1.2 W/cm²). The P values were calculated using one-way ANOVA; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. (D) Hemolysis tests using fresh mice's red blood cells (Inserted images: bottom image is erythrocytes and ATO/CR NPs mixture after centrifugation, top image is the same concentration of ATO/CR NPs). (E, F) Flow cytometry analysis and quantifications of MKN45 GC cells (808 nm laser, 1.2 W/cm²) treated with varying concentrations of ATO/CR NPs. (G) Quantitative PI fluorescence at varying concentrations. (H, I) Fluorescence images and quantifications of MKN45 GC cells stained with Calcein AM/PI under different treatments. (scale bar: 100 μm, ATO/CR NPs: 18 μM, Laser: 808 nm 1.2 W/cm²). ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In addition, to quantitatively detect the cellular apoptosis induced by ATO/CR NPs, the flow cytometric assay with Annexin V-FITC (Annexin V-fluorescein isothiocyanate) and PI (Propidium Iodide) staining was executed. As shown in Fig. 3E and 3F, the apoptosis rate also presents a dosage dependence with considerable percentages of 5.4, 16.7, 45.2, and 93.6%, and the quantitative analysis of PI fluorescence for each group is displayed in Fig. 3G, all indicating the excellent synergistic efficiency of ATO/CR NPs to fight cancer. Furthermore, the MKN45 cells were irradiated under an 808 nm laser and incubated with Calcein-AM/PI to assess the live/green and dead/red cells ablated via ATO/CR NPs. As presented in Fig. 3H and 3I, more apoptotic cells in the ATO/CR NPs (+) group are observed than in the control group, revealing the potent synergistic cancer ablation effect of ATO/CR NPs.

2.4. Cellular uptake, anti-angiogenesis, and subcellular co-localization

Fig. 4A shows that it presents brighter cytoplasmic fluorescence when cancer cells are co-incubated with ATO/CR NPs than that in the control group, which is very important for biological imaging and will guide the FLI in vivo. The quantitative flow cytometry fluorescence result further indicates that rapid accumulation of ATO/CR NPs occurs in the cancer cell cytoplasm (Fig. 4B and 4C). In addition, Fig. 4D shows that the angiogenesis in the control group regularly forms a robust vascular network from 0 to 5 h by human umbilical vein endothelial cells (HUVECs), while that is considerably prevented with concentration dependence in the ATO/CR NPs (−) group. Similarly, the live cell fluorescence staining of the vascular network at 5 h dramatically exhibits the decreased tendency in crossover points and vessel length, implying that ATO/CR NPs will block tumor neovascularization owing to the ATO (Fig. 4E–4G). Significantly, the cell viability is almost 100% even at 45 μM of ATO/CR NPs without illumination, showing almost no dark cytotoxicity and superior biocompatibility of ATO/CR NPs to HUVECs (Fig. 4H). Regarding subcellular co-localization, Kessel et al. confirmed that the parallel damage in different subcellular organs can promote cellular photo-ablation [46]. Besides, some self-assembly nanocomposites modified by polyethylene glycol (PEG) can improve the targeting capacity of the therapeutic agents [47,48]. As shown in Fig. 4I, Green Mito-Tracker and Lyso-Tracker were employed to confirm the subcellular colocalization behavior of ATO/CR NPs. The co-localization of the red (ATO/CR NPs) and green (organelle tracker) can be detected in the mitochondria with a Pearson's coefficient of 0.771, which indicates that ATO/CR NPs possess the capability for precise targeting and the potential to induce mitochondrial damage.

Fig. 4.

In vitro evaluation on cellular uptake, anti-angiogenesis, and subcellular organelle distribution. (A) Confocal fluorescence images of MKN45 GC cells treated with ATO/CR NPs (18 μM, 4 h) (scale bar: 50 μm, ATO/CR NPs and DAPI excited at 640 and 405 nm, respectively). (B, C) Quantitative flow cytometry analysis and mean fluorescence intensity (MFI) of ATO/CR NPs in MKN45 GC cells after different time points. (D, E) HUVEC capillary tube formation after ATO/CR NPs treatment at different concentrations: bright field (0, 1, 3, 5 h) and fluorescence (5 h) images. (scale bar: 100 μm, Calcein AM excited at 495 nm). (F, G) Branching points and length for the HUVEC capillary structures induced by ATO/CR NPs from (E). ∗∗P < 0.01. (H) MTT assay of HUVECs based on ATO/CR NPs in different concentrations without laser illumination. (I) Subcellular co-localization of ATO/CR NPs in MKN45 GC cells (scale bar: 20 μm, trackers excited at 488 nm).

2.5. Mitochondrial dysfunction

Fig. 5A illustrates that ATO/CR NPs induce apoptosis in MKN45 cells through light-triggered mitochondrial damage. To further verify the dysfunction of mitochondria, their morphological change after administration was observed using Bio-TEM [49]. As shown in Fig. 5B and 5C, the mitochondria in the control group exhibit an intact structure with uniformly distributed cristae. However, the ATO/CR NPs (+) group displays severe mitochondrial damage in blurred cristae edges, cavitation, degeneration, and a decrease in length, indicating obvious mitochondrial dysfunction. In addition, when the mitochondrial membrane potential (MMP, a classical indicator of mitochondria) is high, JC-1 aggregates in the mitochondrial matrix of healthy cells as red fluorescence, but JC-1 conversely exists as monomers in dysfunctional cells as green fluorescence [50]. As shown in Fig. 5D, the red/green fluorescence ratio in MKN45 GC cells displays a decreasing trend in the control, ATO NPs, ATO/CR NPs (−), and ATO/CR NPs (+) groups, indicating that the different degrees of mitochondrial dysfunction are initiated by ATO NPs or ATO/CR NPs under laser illumination. To confirm the inhibited complex III based on OXPHOS blocking of the ETC after endocytosis, the cellular oxygen consumption rate (OCR) was evaluated by oligomycin (OLI), rotenone and antimycin A (R&A), and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Fig. 5E). Fig. 5F shows a sharp decline in OCR in the CR NPs (+) group, attributed to photoablation induced inactivation of key mitochondrial electron transport chain complexes, leading to proton gradient dissipation, membrane damage, and ion homeostasis disruption. Meanwhile, the reduced OCR in the ATO NPs and ATO/CR NPs (−) groups indicates ATO-mediated suppression of cellular oxygen consumption, which severely damages mitochondria, irreversibly blocks OXPHOS, as well as reduces ATP and HSP levels for enhanced cancer PTT. Therefore, the ATP level is considerably reduced by ATO NPs with dosage dependence, implying the cellular energy is significantly inhibited by activated OXPHOS blocking (Fig. 5G).

Fig. 5.

Mitochondrial dysfunction induced by ATO/CR NPs. (A) Schematic illustration of the apoptosis process with mitochondrial dysfunction induced by ATO/CR NPs with 808 nm laser irradiation. (B) TEM analysis of mitochondria ultrastructure in MKN45 cells with ATO/CR NPs treatment (scale bar: 500 nm). (C) Quantification of the mean length and damaged mitochondria from (B). (D) Observations of mitochondria membrane potential analysis by JC-1 dye staining in MKN45 cells (scale bar: 50 μm). (E) Schematic diagram illustrating the workflow for the OCR measurement using Seahorse XFe 24 analyzer. (F) Kinetic study of mitochondria OCR for cells treated with medium, ATO NPs, ATO/CR NPs (−), and CR NPs (+) after sequential addition of OLI, FCCP, and R&A. (G) Cellular ATP level of MKN45 GC cells with ATO NPs treatment. ∗P < 0.05, and ∗∗P < 0.01.

2.6. Metabolomics and molecular mechanisms triggered by ATO/CR NPs

To investigate the metabolic alterations induced by ATO/CR NPs in tumor ablation, a comprehensive metabolomic analysis was conducted (Fig. 6A). Initially, the principal component analysis (PCA) score plot reveals a clear separation of samples into two distinct clusters: control and ATO/CR NPs (+), which indicates that the metabolomic data are robust and suitable for further analysis (Fig. 6B). In addition, to identify differentially expressed metabolites, a volcano map was generated, demonstrating substantial metabolic heterogeneity in the ATO/CR NPs (+) group. A total of 163 metabolites are upregulated, while 177 metabolites are downregulated, suggesting significant metabolic reprogramming during synergistic cancer treatment (Fig. 6C). Besides, variable importance in projection (VIP) analysis highlights key metabolite classes, including benzene derivatives, lipids, and organic heterocyclic compounds, as critical contributors to the observed metabolic shifts (Fig. 6E). Moreover, the heatmap shows a marked downregulation of hypoxanthine, which is a metabolite essential for ATP production, suggesting reduced ATP generation, and contributing to impaired cellular proliferation in response to ATO/CR NPs (Fig. 6D). Furthermore, to further elucidate the metabolic pathways, a differential abundance (DA) score analysis was conducted, providing a systematic overview of global metabolic alterations (Fig. 6F). These results indicate that the ATO/CR NPs promote glutathione metabolism, which is a key antioxidant involved in mitochondrial redox regulation. Concurrently, the treatment downregulates several critical metabolism pathways, including amino acid biosynthesis and energy metabolism (central carbon metabolism, pentose phosphate, glycolysis, and gluconeogenesis), further supporting the inhibitory effects on cancer cell proliferation. Herein, our unbiased metabolomics analysis reveals the metabolic landscape modulated by ATO/CR NPs, emphasizing their role in the mitochondrial redox process and ATP generation, offering valuable mechanistic insights into the metabolic underpinnings of ATO/CR NPs mediated tumor suppression.

Fig. 6.

Metabolomic analysis of ATO/CR NPs induced therapeutic mechanisms. (A) Schematic representation of metabolomics analysis workflow. (B) Principal component analysis (PCA) illustrating the separation between the control group (red) and the ATO/CR NPs (+) group (blue), indicating distinct metabolic profiles. (C) Volcano plots displaying the differentially expressed metabolites between PBS and ATO/CR NPs (+) treatment. (D) The heatmap illustrates the heterogeneity in metabolite levels after ATO/CR NPs (+) treatment. (E) Fold change and variable importance in projection (VIP) scores for key metabolites. (F) Differential abundance (DA) score analysis comparing the metabolic pathways of the control and ATO/CR NPs (+) groups. Red: upregulation, blue: downregulation in (C-E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

To further investigate the anticancer mechanism of ATO/CR NPs, the expression levels of cell cycle-related proteins: Cyclin D1, CDK4, CDK6, metabolism-related proteins: STAT3, P-STAT3, antiapoptosis-related protein: Bcl-2, and HSP-70 in MKN45 GC cells were analyzed by western blot (Fig. 7B–7J). Wherein, the levels of Cyclin D1, CDK4, CDK6, Bcl-2, and P-STAT3/STAT3 are meaningfully decreased in a dose-dependent manner, which indicates that the synergistic anticancer efficacy of ATO/CR NPs can effectively induce cellular apoptosis. Besides, the HSP-70 expressions in the ATO NPs and ATO/CR NPs (+) groups are downregulated compared to that in the Control and CR NPs (+) groups, respectively, proving the decreased ATP supply in mitochondria for inhibited expression of HSPs. The schematic diagram depicting the proposed molecular mechanism by ATO/CR NPs to induce cancer cell apoptosis is shown in Fig. 7A. The released ATO after 808 nm laser illumination can target STAT3 to decrease anti-apoptotic Bcl-2 level and impede cell cycle-related proteins (Cyclin D1, CDK4, CDK6) for cancer suppression, as well as block OXPHOS by inhibiting the complex III of the ETC to inhibit the ATP production, thus restraining the expression of HSPs to promote PTT for enhanced cancer cell apoptosis.

Fig. 7.

Molecular mechanisms of ATO/CR NPs to fight GC. (A) Schematic illustration of the molecular mechanism of cancer cell suppression induced by ATO/CR NPs. (B-D) Western blotting images of Cyclin D1, CDK4, CDK6, Bcl-2, STAT3, P-STAT3, and HSP-70 in MKN45 GC cells after different treatments (I: Control, II: ATO NPs, III: CR NPs (+), IV: ATO/CR NPs (+), V: ATO/CR NPs (−)). (E-J) The density of each band. Data are shown as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

2.7. Cell migration and invasion inhibited by ATO/CR NPs in vitro

Significantly, as an important organelle with physiological functions, lysosomes can directly affect the fate of cells on migration and invasion capacity, which are important processes of cancer development and metastasis [51,52]. Initially, the lysosomal membrane permeability (LMP) assay was performed to assess the integrity of the lysosomal membrane using acridine orange (AO), which displays red fluorescence when trapped in acidic lysosomes, which will be converted into green fluorescence by the leakage of AO to the cytosol when the lysosomes are destroyed with lysosomal proton gradient disorder [53]. As shown in Fig. 8B, the red fluorescence of the ATO/CR NPs (+) group (18 μM) decreases obviously due to the loss of lysosomal membrane integrity initiated by the enhanced photothermal therapeutic effect, displaying strong lysosomal membrane destabilization and lysosomal dysfunction. Simultaneously, the transwell assays were executed to assess the migration and invasion ability of MKN45 cells treated with different agents without/with extracellular matrix in the transwell insert, respectively (Fig. 8A). Expectedly, the cell migration result manifests that the cell number in the lower chamber is declined in dose-dependence, wherein the group treated with 18 μM ATO/CR NPs exhibited a markedly low migration rate of only 6%, indicating potent inhibition (Fig. 8C and 8D). In addition, the assay of cells co-incubated with different agents containing extracellular matrix by transwell insert was conducted to evaluate the cellular invasive ability. As depicted in Fig. 8E and 8F, the cell number in the lower chamber of the CR NPs (−) group (48 h) is just slightly smaller than that in the control group, while the invasion rates of ATO NPs and ATO/CR NPs (−) groups are only 38.6% and 34.0%, respectively, indicating considerably synergistic anti-cancer effect, which will inspire further investigation into the therapeutic potential of ATO/CR NPs in vivo.

Fig. 8.

Inhibitory effect of ATO/CR NPs on MKN45 cell migration and invasion in vitro. (A) Illustration of the migration and invasion behavior by MKN45 cells after treatment with various nanomedicines via transwell assay. (B) Assessment of lysosomal membrane permeability by acridine orange (AO) staining under various treatment conditions (scale bar: 20 μm). (C, D) Representative images from the transwell migration assay and quantifications of MKN45 gastric cancer cells treated with different concentrations of ATO/CR NPs (−) (scale bar: 100 μm). (E, F) Transwell assay and quantifications of MKN45 gastric cell invasion ability initiated by PBS, ATO NPs, CR NPs (−), and ATO/CR NPs (−). Data are shown as mean ± SD (n = 3). ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.8. Biodistribution and anticancer performance of ATO/CR NPs in vivo

Based on the excellent NIR fluorescence emission of ATO/CR NPs through fluorescence spectrum and cellular uptake in vitro, they would be a superb agent for FLI in animal models, so some pre-experiments were performed before the synergistic cancer ablation in vivo. Initially, the fluorescence signal of ATO/CR NPs in different concentrations was detected, and Fig. S9A–S9B shows that the fluorescence signal linearly increases with dosage dependence. In addition, MKN45 tumor-bearing mice were further selected for FLI of ATO/CR NPs. As presented in Fig. 9B and 9C, the fluorescence intensity at the tumor site gradually reaches the maximum at 2 h due to the EPR target effect after i.v. injection and then decreases owing to the ATO/CR NPs metabolism, guiding tumor therapy in vivo. Wherein, the liver and kidney fluorescence are attributed to their metabolic function, demonstrating in vivo biocompatibility of ATO/CR NPs.

Fig. 9.

Permeability and biodistribution of ATO/CR NPs as well as synergistic GC therapy in vivo. (A) Treatment schedule for the MKN45 tumor-bearing mice to evaluate the anti-tumor efficiency of ATO/CR NPs in vivo. (B, C) Fluorescence images (major organs and tumor) and quantifications (tumor) of ATO/CR NPs. (D, E) Photothermal images and temperature changes of MKN45 tumor-bearing mice with or without ATO/CR NPs injection (120 μM, 200 μL). ∗∗P < 0.01. (F, G) Tumor growth curves after different treatments. ∗∗∗∗P < 0.0001. (H) Body weight changes in different groups for 16 days. (I) H&E, TUNEL, and Ki-67 tumor tissue staining photographs in different groups (scale bar: 50 μm). (J) Hematological, liver, and kidney function indicators. (K-M) VEGF and HSP-70 tumor tissue staining photographs in different groups (scale bar: 50 μm, Ⅰ: Control, Ⅱ: ATO NPs, Ⅲ: CR NPs (+), Ⅳ: ATO/CR NPs (+)). ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

To further confirm the EPR targeting performance of ATO/CR NPs, the PTI in vivo was executed at 2 h after i.v. injection. Fig. 9D and 9E presents that the tumor temperature rises to 55.0 °C (ΔT = 20.1 °C) in 6 min, but only a slight temperature increase (ΔT = 4.3 °C) was tested in the control group, manifesting the excellent targeting and PTI activity, which will further guide the cancer photo-ablation of ATO/CR NPs in vivo. Hence, the synergistic antitumor ability of ATO/CR NPs was performed in vivo by employing the xenograft MKN45 nude mice, which were divided into 6 groups (n = 5): (I) PBS as the control group, (II) ATO NPs, (III) CR NPs (-, without laser irradiation), (IV) ATO/CR NPs (−), (V) CR NPs (+, with laser irradiation), (VI) ATO/CR NPs (+). The treatment procedure is depicted in Fig. 9A. Tumor volume was tracked over time following the administration of different treatments, and the results display that tumors steadily grew rapidly within 16 days in the control group. Besides, ATO NPs, CR NPs (−), and ATO/CR NPs (−) groups only partially restrain tumor growth. Whereas the tumors in the ATO/CR NPs (+) group are eliminated completely at the third treatment and there is no recurrence in the period of treatment, showing the amplified tumor ablation effect of ATO/CR NPs in vivo (Fig. 9F and G). Moreover, as depicted in Fig. 9H, the result of body weight is normal during treatment, further implying no side effects of the therapeutic agents. Simultaneously, the results of normal hematology, liver, and kidney function indexes present that there are no notable differences between the ATO/CR NPs (+) and control groups, demonstrating no infection, inflammation, or systemic side effects during treatment (Fig. 9J). Hematoxylin and eosin (H&E) staining (heart, liver, spleen, lung, and kidney) further confirms no toxicity of these therapeutic agents (Fig. S9C). In addition, tumors from six groups were stained by H&E, TUNEL, and Ki-67 immunohistochemistry to further evaluate the tissue damage. It exhibits morphological features of apoptosis, including nuclear condensation and increased vacuolation, in the ATO NPs, ATO/CR NPs (−), CR NPs (+), and ATO/CR NPs (+) groups when compared with control and CR NPs (−) groups, revealing the enhanced antitumor effects of ATO/CR NPs (Fig. 9I). Finally, the fluorescence of VEGF and HSP-70 in ATO NPs and ATO/CR NPs (+) groups present weaker fluorescence when compared with Control and CR NPs (+) groups, respectively, suggesting the restrained tumor angiogenesis by VEGF downregulation and HSP-70 inhibition for decreased ATP generation in tumor tissue, which will avoid tumor recurrence with excellent synergistic therapeutic effect, showing great potential for clinical application (Fig. 9K–9M).

3. Conclusion

In short, a superior nano-agent (ATO/CR NPs) has been successfully acquired by self-assembling ATO with CR for improved gastric cancer photothermal therapy. Wherein, the ATO not only can meaningfully block OXPHOS to decrease the ATP generation with inhibited expression of HSPs but also can specifically target STAT3 to decrease anti-apoptotic Bcl-2 level, and restrain the angiogenesis activity, as well as impede cell cycle-related proteins (Cyclin D1, CDK4, CDK6), inducing ATO-promoted cancer PTT effect. In vitro and in vivo experiments prove that the ATO/CR NPs present superb biocompatibility, minimal dark toxicity, excellent phototoxicity, and desired tumor ablation properties. Conclusively, this rationally prepared nanoconjugate ATO/CR NPs presents a potential approach for ATO-enhanced gastric cancer photothermal therapy in the clinic.

Ethical approval

All animal experiments were performed in accordance with the regulations on animal experimentation and approved by the ethics committee of Anhui Medical University (Date: March 1, 2021; Approval number: 20,210,901).

CRediT authorship contribution statement

Wenbin Wang: Validation, Writing – review & editing. Lingling Wang: Data curation, Writing – original draft. Gege Zhang: Data curation, Writing – review & editing. Yi Zheng: Formal analysis, Software, Writing – review & editing. Qiong Huang: Data curation. Mi Zou: Data curation. Yanling Zhang: Data curation. Shuang Wu: Data curation. Yajie Sui: Data curation. Jianda Qiu: Data curation. Zhao Zhang: Data curation. Qiaojun Fang: Writing – review & editing. Xianwen Wang: Writing – review & editing. Pingping Liang: Funding acquisition, Supervision, Writing – original draft, 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.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.