Materials
Ruthenium chloride (RuCl3), proanthocyanidins, and GSH were purchased from Macklin Co., Ltd. (China). 3,3’,5,5’-Tetramethylbenzidine (TMB), 1,2-diaminobenzene (OPD), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), and polyvinylpyrrolidone (PVP) were purchased from Aladdin Co., Ltd. (China). Phosphate-buffered saline (PBS), RPMI 1640 medium, and trypsin-EDTA solution were purchased from Biosharp Biologics Co., Ltd. (China). Fetal bovine serum (FBS) was purchased from Gibco Co. (USA). The CCK-8 kits were obtained from SparkJade Co., Ltd. (China). ThiolTracker™ Violet was purchased from Thermo Fisher Scientific, Inc. (USA). Calcein-AM/PI and Annexin/PI kits were purchased from Bestbio Co., Ltd. (China). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), JC-1, DAPI, and GSH kits were purchased from Beyotime Co., Ltd. (China).
Preparation of Ru-PC
The Ru-PC nanocomplex was synthesized through a facile coordination-mediated self-assembly approach [19]. Briefly, 33 mg of polyvinylpyrrolidone (PVP) was completely dissolved in 5 mL of methanol under magnetic stirring. Subsequently, 1 mL of methanolic solution containing 20 mg of RuCl3 was introduced dropwise into the PVP solution, followed by continuous stirring in 120r/min for 5 min. Meanwhile, 10 mg of PC was separately dissolved in methanol and then incorporated into the above mixture. The reaction system was maintained under vigorous stirring for 3 h at ambient temperature, during which the color of the solution changed to dark green. The resulting mixture was subjected to overnight dialysis via membranes with molecular weight cutoffs ranging from 8,000 to 14,000 kDa to remove unreacted precursors. Following dialysis, the product was centrifuged at 10,000 rpm, washed three times with ultrapure water, and lyophilized to obtain grayish-black Ru-PC nanoparticles.
Ru-PC characterization
A projected electron microscope (JEM-1400 Plus, Tokyo, Japan) was used to study the morphology and elemental distribution of the samples, and the chemical composition was determined via X-ray photoelectron spectroscopy (XPS) (Thermo Scientific K-Alpha, Beijing, China). A UVEVIS spectrophotometer (Thermo Scientific™ GENESYS™ 50, USA) was used to measure the ultraviolet (UV) absorption spectrum of the substance.
Ru-PC-like enzyme activity and PEITC bioactivity
The validation experiments of POD-like enzymes were performed with TMB and OPD probes. Briefly, different concentrations of Ru-PC were mixed with H2O2, ROS generation was detected by the addition of 1 µmol/L TMB, and the absorbance curves and the change in absorbance at 652 nm of the supernatant after centrifugation were detected via a UV spectrophotometer. Similarly, the absorbance curve and the change in absorbance at 442 nm were detected by replacing TMB with OPD. Glutathione peroxidase-like enzyme activity was verified via the colorimetric reaction of DTNB with GSH. Different concentrations of Ru-PC were mixed and reacted with GSH. DTNB was added to the reaction system after a period of time, and the supernatant was centrifuged to measure the absorbance at 412 nm via a UV spectrophotometer (Thermo Scientific™ GENESYS™ 50, USA).
Characterization and properties of Ru-PC-PEITC-ALG
The nanotherapeutic platform Ru-PC-PEITC-ALG was prepared by mixing Ru-PC or PEITC with 5 mg/mL of the suggested ALG solution and evaluated for its characterization and properties in terms of gel-forming and loading capacity [31, 32]. High-resolution scanning electron microscopy (SEM) images of Ru-PC-PEITC-ALG were obtained by scanning electron microscopy (Tescan -MIRA LMS, Czech Republic). To measure the fluidic properties of Ru-PC-PEITC-ALG, the rheological properties of the hydrogels were evaluated via a rotational rheometer (MCR 302e, Austria). The manufactured ALG, Ru-PC and Ru-PC-PEITC-ALG were formed into gels and then freeze-dried, and the remaining solids were ground and supplemented with potassium bromide. Fourier transform infrared (FTIR) spectra were obtained via a Fourier transform infrared spectrometer (IRTracer 100, Japan). A simulated tumor microenvironment was prepared by adjusting the Ca2+ concentration to 1.8 mm in PBS, and the amount of Ru ions released from Ru-PC and Ru-PC-PEITC-ALG was measured via inductively coupled plasma‒mass spectrometry (ICP) in the simulated tumor microenvironment (TME) environment for 72 h.
Photothermal performance of Ru-PC-PEITC-ALG
To evaluate the photothermal properties of Ru-PC, the temperature changes in a 200 µg/mL Ru-PC solution under NIR irradiation (808 nm) at various power densities were measured over 4 min via an infrared thermal camera. Similarly, the temperature variations of Ru-PC solutions at different concentrations were monitored for 4 min under 808 nm NIR laser irradiation at 1.5 W/cm². The thermal stability of Ru-PC was validated through multiple NIR irradiation cycles. Additionally, the stability of Ru-PC was assessed by comparing its ultraviolet‒visible (UV‒Vis) absorption spectra before and after light irradiation via UV‒Vis spectrophotometry.
Cell line
Human umbilical vein endothelial cells (HUVECs) and murine breast cancer 4T1 cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). HUVECs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. 4T1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. All the cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
In vitro cytotoxicity of Ru-PC-PEITC-ALG
The cytotoxicity of Ru-PC-PEITC-ALG on HUVECs and 4T1 cells was evaluated via a CCK-8 assay kit. The cells were seeded into 96-well plates and allowed to adhere and reach 80% confluency. The cells were subsequently treated with different concentrations of Ru-PC-PEITC-ALG and coincubated for 6 h. For the groups designated for photothermal treatment, the cells were exposed to 808 nm near-infrared (NIR) irradiation at an intensity of 1.5 W/cm² for 4 min. After irradiation, the supernatant was removed, and the cells were incubated with 10% CCK-8 solution in fresh medium at 37 °C for 2 h to allow color development. The absorbance at 450 nm was measured via a microplate reader (SpectraMax iD3, Molecular Devices, Japan), and the cell viability was calculated on the basis of the optical density values.
The cytotoxic effect of Ru-PC-PEITC-ALG was further evaluated via calcein-AM/PI live/dead staining. 4T1 cells were seeded into confocal dishes and allowed to grow to 80–90% confluency. After treatment with different concentrations of Ru-PC-PEITC-ALG and 6 h of coincubation, the cells designated for PTT were irradiated with an 808 nm NIR laser (1.5 W/cm², 4 min). Following irradiation, the supernatant was removed, and the cells were washed with PBS. The cells were subsequently stained with calcein-AM and PI sequentially at 37 °C for 20 min in the dark to distinguish live (green fluorescence) and dead (red fluorescence) cells. Imaging was performed via a fluorescence confocal microscope (CSIM-130, Sunny Technology, China), and representative images were captured for qualitative analysis of cell viability.
Ru-PC-PEITC-ALG induces apoptosis in 4T1 cells
The apoptotic effect of Ru-PC-PEITC-ALG on 4T1 cells was evaluated via Annexin V-FITC/PI staining combined with flow cytometry. The cells were seeded into 6-well plates, treated with different concentrations of Ru-PC-PEITC-ALG, and coincubated for 6 h. For photothermal treatment, the cells were irradiated with an 808 nm NIR laser (1.5 W/cm², 4 min). After irradiation, both the supernatant and adherent cells were collected, centrifuged, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) at 4 °C in the dark. Apoptotic rates were quantified via a flow cytometer (BD FACSCalibur, USA).
To assess intracellular reactive oxygen species (ROS) levels, DCFH-DA staining was performed. 4T1 cells were seeded into confocal dishes, treated with Ru-PC-PEITC-ALG, and coincubated for 6 h followed by NIR irradiation. The supernatant was discarded, and the cells were incubated with 10 µM DCFH-DA at 37 °C for 30 min in the dark. Nuclei were counterstained with DAPI (1 µg/mL) for 5 min. Fluorescence images were captured via a confocal microscope (CSIM-130, Sunny Technology, China), and semiquantitative analysis of the ROS fluorescence intensity was conducted via ImageJ software.
Exploring the synergies of the PEITC
To explore the synergistic role of PEITC, changes in the mitochondrial membrane potential were analyzed via JC-1 staining. 4T1 cells were seeded into confocal dishes and incubated to the appropriate confluency. After treatment with Ru-PC-PEITC-ALG and incubation for 6 h, the cells were irradiated with an 808 nm NIR laser (1.5 W/cm², 4 min). The supernatant was removed, and the cells were washed thoroughly with PBS, followed by staining with JC-1 dye (5 µg/mL) at 37 °C for 20 min in the dark. The cells were then washed three times with precooled PBS, and the nuclei were counterstained with DAPI (1 µg/mL) for 5 min. All procedures were performed on ice to minimize nonspecific enzymatic activity. Fluorescence images were captured via a confocal microscope (CSIM-130, Sunny Technology, China), with JC-1 aggregates (red fluorescence) and monomers (green fluorescence) visualized to assess mitochondrial damage.
Additionally, the depletion of intracellular GSH by PEITC was evaluated via ThiolTracker™ Violet. After treatment with Ru-PC-PEITC-ALG and NIR irradiation, the 4T1 cells were stained with 20 µmol/L ThiolTracker™ Violet at room temperature for 30 min in the dark. Fluorescence images were acquired via confocal microscopy, and semiquantitative analysis of GSH levels was performed via measurement of fluorescence intensity via ImageJ software. The reduced ThiolTracker™ Violet signal (green fluorescence) indicated GSH consumption, reflecting PEITC-mediated redox modulation.
Transcriptomic and metabolomic profiling of Ru-PC-PEITC-ALG mechanisms
To elucidate the antitumor mechanism of Ru-PC-PEITC-ALG, transcriptomic sequencing and metabolomic analyses were performed on 143b cells. Cells from the control and treatment groups (Ru-PC-PEITC-ALG treatment for 6 h followed by 808 nm NIR irradiation at 1.5 W/cm² for 4 min) were lysed via TRIzol reagent (Invitrogen, USA) for total RNA extraction. mRNA quantification, purification, reverse transcription, and sequencing were conducted by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). Gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using a fold-change threshold of ≥ 2 and a false discovery rate (FDR) < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were performed to annotate functional pathways associated with the DEGs.
In vivo antitumor experiments
Animal experiments were performed according to the guidelines established by the Institutional Animal Care and Use Committee of Anhui Medical University (approved by LLSC20220731). The backs of 4-week-old BALB/c mice were shaved, and 1 × 106 4T1 cells were inoculated subcutaneously. After the tumor volume reached 80–100 cm3 after 7 days, the mice were randomly divided into 7 groups, each consisting of 5 individuals. The treatments used were as follows: (1) control; (2) ALG + laser; (3) Ru-PC-ALG; (4) PEITC-ALG; (5) Ru-PC-PEITC-ALG; (6) Ru-PC- ALG + laser; and (7) Ru-PC-PEITC-ALG + laser. All reagents were used at the following concentrations: ALG, 5 mg/mL; Ru-PC, 200 µg/mL; and PEITC, 20 µmol/L. Twelve hours after the injection, the tumor tissues in groups (2), (6), and (7) were treated with near-infrared (NIR) light at 808 nm NIR, 1.5 W/cm2 for 4 min. Tumor length and width and animal weight were measured every 2 days after treatment, and tumor volume was calculated as (tumor length) × (tumor width)2/2. At the end of the measurements on day 10, the mice were euthanized, and bluntly isolated tumor tissues were removed for imaging and fixation. The collected tumor tissues were subjected to H&E staining, immunohistochemical staining for Ki67 and Caspase 3, and immunofluorescence staining for TUNEL and ROS.
Biosafety assessment
Hemolysis Assay: BALB/c mice were sacrificed, and blood was collected from the eyeballs. Intact erythrocytes are centrifuged for hemolysis. PBS and different concentrations of NPs were added to the erythrocyte suspension, and the mixture was incubated for 6 h. The supernatant was collected after centrifugation, and the absorbance was measured at 577 nm via a UV‒visible spectrophotometer (Thermo Scientific Biomate 160, USA). NP-DSF-ALG solution was injected into the right lower abdomen of BALB/c nude mice (female, 6 weeks old, 18–20 g).
Hematological analysis: Mice were killed before injection and on days 4, 7 and 14 postinjection. Blood and biochemical parameters, including red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin (HGB) level, hematocrit (HCT) level, neutrophil (NEU) count, lymphocyte (LYM) count, platelet (PLT) count, hemoglobin (HGB) level, hematocrit (HCT) level, and platelet (PLT) count, were also measured. The PLT, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CRE) and blood urea nitrogen (BUN) levels were also measured.
Evaluation of major organs: Mice were killed before injection and on days 4, 7 and 14 postinjection. Histological examination of major organs, including the heart, liver, spleen, lung and kidney, was performed via H&E staining.
Data processing
All the experiments were conducted at least twice or in triplicate. The findings presented in this report are representative. The mean and standard deviation (SD) were used to express quantitative data. Differences between groups were assessed via the standardized t test and were considered statistically significant when the p value was less than 0.05. NC: negative control; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
