Materials
Te (CAS: 60-54-8, chemical purity ≥ 98%), copper (II) chloride (CuCl2), TCPP, PVP, GSH, H2O2, ammonium molybdate ((NH4)2MoO4), granulocyte-macrophage colony-stimulating factor (GM-CSF) and methylene blue (MB) were got from Aladdin Biochemistry Technology Co., Ltd. (Shanghai, China). LA, lipopolysaccharide (LPS), fetal bovine serum (FBS), and the bicinchoninic acid (BCA) Protein Assay Kit were obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). Indocyanine green (ICG), hydroxyphenyl fluorescein (HPF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (USA). 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) and Ellman’s Reagent (DTNB) were obtained from Thermo Fisher Scientific (USA). Anti-high mobility group box 1 (HMGB1) mouse monoclonal antibody (PTR2339), anti-calreticulin (CRT) monoclonal antibody, and anti-β-actin mouse polyclonal antibody were purchased from Immunoway Biotechnology (USA). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was obtained from Abcam (Shanghai, China). 2,2,6,6-Tetramethylpiperidine (TEMP) was obtained from Energy Chemical (Shanghai, China). Anti-PDC-E2 (Dihydrolipoamide S-acetyltransferase, DLAT) antibody and recombinant anti-ferredoxin 1 (FDX1) rabbit antibody were purchased from Cohesion Biosciences (Suzhou, China). PE anti-mouse CD206/MMR antibody, FITC anti-mouse CD86 antibody, APC anti-mouse CD80 antibody, FITC anti-mouse CD3 antibody, PerCP anti-mouse CD8 antibody, and APC anti-mouse CD4 antibody were purchased from Elabscience (Wuhan, China). Hoechst 33342, Rhodamine 123, Annexin V-FITC/Propidium iodide (PI) apoptosis detection kit, enhanced chemiluminescence (ECL) substrate kit, and Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Urea, Ethylene Diamine Tetraacetic Acid (EDTA), and Tween-20 were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Yancheng Saibao Biotechnology (Jiangsu, China). Mouse interleukin-10 (IL-10) and mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Solarbio Life Science (Beijing, China). Mouse interleukin-6 (IL-6) and mouse tumor necrosis factor-α (TNF-α) ELISA kits were purchased from MultiSciences (Hangzhou, China). Recombinant interleukin-4 (IL-4) was provided by Novoprotein Scientific Co., Ltd. (Suzhou, China). TUNEL apoptosis detection kit were purchased from YEASEN Biotech (Shanghai, China). Hydroxyphenyl fluorescein (HPF) was obtained from Maokang Biotechnology (Shanghai, China). Hematoxylin and Eosin (H&E) Staining Solution was purchased from Biosharp Life Science (Hefei, China).
Synthesis and characterization of CuTT, P/CuTT, and LP/CuTT
The synthesis of the nanoparticles was initiated by mixing Te (1 mg/mL, 1000 µL) with CuCl2 (1 mM, 1000 µL) and then stirring at 0 °C. Subsequently, TCPP (1 mg/mL, 100 µL) was added dropwise to the above solution under stirring conditions to obtain the unmodified nanoparticles CuTT. To prepare P/CuTT, PVP (1 mg/mL, 1000 µL) was introduced into the CuTT suspension. For LP/CuTT synthesis, PVP (1 mg/mL, 100 µL) and LA (1 mg/mL, 900 µL) were added to the CuTT system. All samples were stabilized overnight at 4 °C. Finally, the samples were placed in a 7 kDa dialysis bag and dialyzed for 12 h. The nanoparticles were stored at 4 °C in the dark. Te and TCPP were measured for content using ultraviolet and visible (UV–vis) spectroscopy by a UV–vis spectrophotometer (Shimadzu, Japan). The particle size and zeta potential measurements were performed using dynamic light scattering (DLS) on a Zetasizer Nano ZS (Malvern Instruments, UK). To assess the stability of nanoparticles, CuTT, P/CuTT, and LP/CuTT were suspended in saline, and their particle sizes during a three-day incubation at 4 °C were measured using DLS. The morphologies of the nanoparticles were determined using transmission electron microscopy (TEM, HT7700, Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) measurements were carried out on a Thermo Fisher Scientific ESCALAB 250 A spectrometer.
To investigate the intermolecular forces governing self-assembly, EDTA (20 mM), Tween-20 (20 mM), urea (20 mM), DMSO (10%), and NaCl (0.9%) were introduced to CuTT, P/CuTT, and LP/CuTT systems, and the nanoparticle size variations were monitored.
Detection of ROS
The degradation of MB was used to determine the generation of ·OH in a H2O2 (1 mM) environment. Cu2+ (50 µM), CuTT, P/CuTT, or LP/CuTT (50 µg/mL) were mixed with MB (2 µg/mL), and then treated with or without a 680 nm laser (1 W/cm2, 2 min). After 10 min of stabilization, the UV–vis spectra were detected by a UV–vis spectrophotometer. Electron spin resonance (ESR) measurement was used to determine the singlet oxygen (1O2) with TEMP as a spin trap to capture the 1O2.
GSH and H2O2 consumption
GSH (1 mM, 200 µL) and Cu2+, CuTT, P/CuTT, or LP/CuTT (50 µg/mL, 200 µL) were added into a 1.5 mL centrifuge tube, respectively. After treatment with a 680 nm laser (1 W/cm2, 2 min), the DTNB (0.5 mM, 200 µL) and NaOH (5 µM, 50 µL) were added for further testing. GSH depletion was calculated by detecting the absorption at 412 nm on a microplate reader.
$${\rm{GSH\,consumption}}\left( {\rm{\% }} \right) = \left( {1 – {{{{\rm{E}}_{{\rm{A}}0}} – {{\rm{E}}_{{\rm{A}}1}}} \over {{{\rm{C}}_{{\rm{A}}0}} – {{\rm{C}}_{{\rm{A}}1}}}}} \right) \times 100{\rm{\% }}$$
Where EA0 and EA1 represent the absorbance changes of the experimental group, and CA0 and CA1 represent the absorbance changes of the control group.
For the detection of H2O2, 200 µL of LA (50 µg/mL), CuTT (50 µg/mL), P/CuTT (50 µg/mL), or LP/CuTT (50 µg/mL) was added into a 1.5 mL centrifuge tube containing 200 µL of H2O2 (5 mM) and incubated at 37 °C for 10 min, then (NH4)2MoO4 (200 µL, 10mM) was added, and the absorbance at 405 nm was measured on a microplate reader.
$${{\rm{H}}_2}{{\rm{O}}_2} {\rm{\,consumption}}\left( {\rm{\% }} \right) = \left( {1 – {{{{\rm{E}}_{{\rm{A}}0}} – {{\rm{E}}_{{\rm{A}}1}}} \over {{{\rm{C}}_{{\rm{A}}0}} – {{\rm{C}}_{{\rm{A}}1}}}}} \right) \times 100{\rm{\% }}$$
Where EA0 and EA1 represent the absorbance changes of the experimental group, and CA0 and CA1 represent the absorbance changes of the control group.
GSH responsiveness
The morphological changes of CuTT, P/CuTT, and LP/CuTT before and after exposure to GSH (1 mM) were analyzed by TEM.
Cellular experiments
Cell lines
B16-F10, NIH/3T3 and RAW 264.7 cell lines were obtained from the Cell Resource Center of Shanghai Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). B16-F10 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium, and NIH/3T3 and RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM). All media were supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Female C57 mice (6–8 weeks) from Wushi Experimental Animals Center (Fuzhou, China) were used for dendritic cells (DCs) isolation and induction. Red blood cells were collected from ICR mice.
Cellular uptake and membrane permeability evaluation
B16-F10 cells were seeded in 12-well plates at a density of 1 × 105 cells/well. After 12 h of adherent growth, the cells were treated with CuTT, P/CuTT, or LP/CuTT at a concentration of 50 µg/mL for 2, 4, and 6 h. Subsequently, the cells were harvested and analyzed using flow cytometry (Beckman CytoFLEX, China).
To evaluate membrane permeability, B16-F10 cells were first treated with either saline or LA (50 µg/mL) for 24 h, followed by the addition of PI dye. The uptake of PI dye was measured by flow cytometry at 2, 4, and 6 h.
MTT assay
MTT assay was performed according to the previous report [22]. In brief, B16-F10 or NIH/3T3 cells were inoculated into 96-well plates at a density of 5 × 103 cells per well and cultured for 12 h. Then, different concentrations of materials were added separately, and an MTT assay was performed 24 h later. If laser irradiation is required, it is performed 6 h after drug administration. Laser parameters (wavelength: 680 nm, power density: 1 W/cm2, irradiation time: 2 min).
Detection of intracellular total ROS and ·OH
B16-F10 cells were seeded in a 12-well plate with cell crawling slices. Subsequently, CuTT, P/CuTT, or LP/CuTT (50 µg/mL) was added, and the cells were incubated for 6 h. After that, the cells were incubated with DCFH-DA (10 µM), followed by 680 nm laser (1 W/cm², 2 min) treatment, and finally stained with Hoechst 33342 (10 µM) for another 20 min. The generation of ROS was monitored using a confocal laser scanning microscope (CLSM, Leica TCS SP8, Germany).
For the detection of ·OH, B16-F10 cells were seeded into 12-well plates. The methodology employed for processing the cells is analogous to that described above, except for using HPF (10 µM) probes for staining purposes. After treatment with different materials and laser, the cells were harvested and subjected to flow cytometric analysis.
Detection of intracellular GSH
B16-F10 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well. The CuTT, P/CuTT, and LP/CuTT (50 µg/ml) groups were then treated with or without a 680 nm laser (1 W/cm², 2 min) after 6 h of cultivation. The cells were then harvested and disrupted, and the intracellular GSH content was quantified as described in the “GSH and H2O2 consumption” section.
Detection of macrophage polarization
RAW264.7 cells were seeded and cultured in a 12-well plate (1 × 105 cells/well). Then, the cells were polarized into the M2 phenotype using IL-4 (20 ng/mL) and to the M1 phenotype using LPS (100 ng/mL) for 24 h [22]. The cells are stained with APC-CD80 and PE-CD206, and flow cytometry analysis is performed to confirm the successful induction of M1 and M2 phenotypes. CuTT, P/CuTT, and LP/CuTT (50 µg/mL) were added to M2 macrophages, followed by a 680 nm laser (1 W/cm2, 2 min) treatment after 6 h. After an additional 6 h of cultivation, the polarization of M1 and M2 macrophages was detected using the aforementioned methods.
Detection of mitochondrial membrane potential (MMP) and cell apoptosis
MMP was measured using Rhodamine 123 dye. B16-F10 cells were inoculated in 12-well plates with cover slips. Cells were treated with CuTT, P/CuTT, and LP/CuTT (50 µg/mL), followed by a 680 nm laser (1 W/cm², 2 min) treatment after 6 h. The cells were then stained with Hoechst 33342 (10 µM) and Rhodamine 123 (10 µM) dyes, respectively, and subsequently imaged using a CLSM. Cell apoptosis was analyzed using flow cytometry with Annexin V-FITC/PI staining kit.
Scratch assay
Cell migration was evaluated using a scratch assay. B16-F10 cells were seeded into 6-well culture plates at a density of 2 × 10⁵ cells per well and cultured until reaching desired confluency. A straight wound was created in the cell monolayer using a sterile 1000 µL pipette tip. After removing the floating cells by washing thrice with PBS, the cells were incubated in medium containing either CuTT, P/CuTT or LP/CuTT. Wound closure was monitored by optical microscopy, and the wound area was quantified using ImageJ software by normalization to the initial gap area. The migration rate was calculated using the following formula:
$${\rm{Migration\,rate}}\left( {\rm{\% }} \right) = {{{{\rm{A}}_0}-{{\rm{A}}_1}} \over {{{\rm{A}}_0}}} \times 100{\rm{\% }}$$
where A0 represents the initial wound area, and A1 represents the remaining area after 24 h.
Western blotting
B16-F10 cells were seeded into 6-well plates (1 × 106 cells/well). After cultivation for 12 h, CuTT, P/CuTT, and LP/CuTT (50 µg/mL) were added. Following another 12 h of cultivation, cells were collected and protein extraction was performed using a whole protein extraction kit. Protein concentrations were quantified using the BCA method. After boiled in loading buffer, protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on pre-cast Bis-Tris 12% gels. Proteins were transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membrane and blocked with 5% BSA in Tris buffered saline tween (TBST) for 1.5 h at room temperature. The membranes were then incubated at 4 °C overnight with primary antibodies including DLAT (1:1000), FDX1 (1:1000), or β-actin (1:2000). After washing three times with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1:10000) for 1 h at room temperature. The protein bands were finally visualized with an ECL substrate kit.
Immunofluorescence assay of CRT and HMGB1
B16-F10 cells were inoculated into a 24-well plate containing slides and treated with CuTT, P/CuTT, and LP/CuTT (50 µg/mL). After 12 h of cultivation, the treated cells were fixed with 4% formaldehyde for 10 min and then sealed with a blocking solution for 30 min. Then, the cells were incubated with CRT antibody for 2 h, followed by incubation with Alexa Fluor® 488 at 37 °C for another 30 min [23]. The same procedure was applied for HMGB1 antibody. Finally, cells were stained with Hoechst 33342 before observation using CLSM.
Bone marrow-derived dendritic cell (BMDC) maturation assay
BMDCs were derived from bone marrow cells isolated from the femurs of female C57 mice (6–8 weeks) [24]. After filtering out tissue fragments and lysing red blood cells, BMDCs were induced using IL-4 (10 ng/mL) and GM-CSF (20 ng/mL). B16-F10 cells were seeded and cultured in 12-well plates, and treated with CuTT, P/CuTT, and LP/CuTT (50 µg/mL), followed by 680 nm laser irradiation. After 12 h of cultivation, the cell culture supernatant was collected and mixed with fresh RPMI-1640 medium containing 10% FBS at a 1:1 ratio [25]. This mixture was then added to pre-seeded BMDCs in 6-well plates and cultured for an additional 24 h. Subsequently, the cells were stained with APC-CD80 and FITC-CD86 antibodies. The maturation of BMDCs was detected using flow cytometry.
Hemolysis assay
Red blood cells were collected from ICR mice and placed in centrifuge tubes containing sodium citrate solution. After centrifugation at 1500 rpm for 10 min, the collected red blood cells were washed 3 times with saline, and then diluted to a 2% suspension with normal saline. A 0.5 mL aliquot of this suspension was mixed with deionized water (ddH2O), saline, CuTT, P/CuTT, and LP/CuTT (0.5 mL) at final concentrations of 200 µg/mL. Red blood cell suspensions treated with saline and ddH2O were served as negative and positive controls, respectively. After 1 h of incubation at room temperature, the mixture was centrifuged at 10,000 rpm, and the absorbance of the supernatant was measured at 540 nm using a microplate reader. The hemolysis ratio was calculated with the following Formula [26]:
$${\rm{Hemolysis}}\left( {\rm{\% }} \right) = {{{{\rm{A}}_{{\rm{sample}}}} – {{\rm{A}}_{{\rm{negative}}}}} \over {{{\rm{A}}_{{\rm{positive}}}} – {{\rm{A}}_{{\rm{negative}}}}}} \times 100{\rm{\% }}$$
Animal experiments
Animals
Female ICR mice (6–8 weeks) were purchased from the Wushi Experimental Animals Center (Fuzhou, China). All related animal experimental procedures were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of Fuzhou University.
Establishment of tumor model
The B16-F10 melanoma model was established by subcutaneous injection of B16-F10 cells (5 × 106 cells, 100 µL) into the right flank of the female ICR mice. The mice were monitored daily for tumor growth and general health status.
In vivo imaging of biodistribution
For in vivo fluorescence imaging studies, nanoparticles were labeled with ICG. Briefly, CuTT, P/CuTT and LP/CuTT were dispersed in an aqueous solution containing ICG (500 µg/mL) and subjected to dialysis for 6 h. The hydrodynamic diameter of ICG-labeled nanoparticles was characterized using DLS.
ICG alone and ICG-labeled nanoparticles (ICG-P/CuTT and ICG-LP/CuTT) were administered to ICR mice via tail vein injection at a dose of 1.5 mg/kg in 100 µL volume. Fluorescence imaging was performed at predetermined time points (1, 2, 4, 6, 8, 10, 12, 24, and 48 h post-injection) using a PerkinElmer Caliper IVIS Lumina XR III imaging system (Waltham, Massachusetts, USA).
In vivo antitumor efficacy
Thirty mice with subcutaneous melanoma cancer model were randomly divided into five groups (n = 6), including a saline control group, P/CuTT, P/CuTT with laser irradiation (P/CuTT + L), LP/CuTT, LP/CuTT with laser irradiation (LP/CuTT + L). Once the tumors reached approximately 100 mm3, the B16-F10 tumor-bearing mice were intravenously injected with saline, P/CuTT (1.5 mg/kg), or LP/CuTT (1.5 mg/kg), every 2 days for a total of three treatments. The tumor volume and body weight of the mice were measured every 2 days for 17 days. The laser treatment groups received laser irradiation (680 nm, 1.0 W/cm2, with an effective exposure of 3 min) 10 h post-injection. The tumor volume was measured using a vernier caliper and calculated with the formula V = (a × b2)/2, where a is the long axis and b is the short axis. The mice were sacrificed on the 17th day.
Detection of ·OH and apoptosis (TUNEL) in tumor
B16-F10 tumor-bearing mice were intravenously injected with saline, P/CuTT, or LP/CuTT (1.5 mg/kg). After 10 h, a laser (680 nm, 1.0 W/cm2, with an effective exposure of 3 min) was applied. The laser was removed for 30 s after each 1 min exposure to prevent skin burns on the mice. Subsequently, tumor tissues were sliced using a frozen sectioning machine. HPF (10 µM) and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) apoptosis detection kit were used to detect ·OH and tumor cell apoptosis respectively. Finally, Hoechst 33342 was used for staining before observation with a CLSM.
Histopathology analysis
Tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) were collected after 17 days of treatment, fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with H&E. The stained sections were then examined under a fluorescence microscope (Zeiss, Germany).
Analysis of immune cells and cytokine production
Tumor tissues and spleens were digested to create single-cell suspensions after 17 days of treatment. The cell suspensions were stained with FITC-CD3/APC-CD4 for CD4+ T cells, FITC-CD3/PerCP-CD8a for CD8+ T cells, and FITC-CD11b/APC-CD80 for M1 macrophages for 30 min at room temperature. Following staining, the cell suspensions were washed three times with PBS and analyzed using flow cytometry. Thirty thousand events were recorded for each sample, and the data were analyzed using FlowJo software. The levels of IL-10, IL-1β, and TNF-α in serum were measured according to the instructions provided with the assay kits.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Experimental data are expressed as mean ± standard deviation (SD) for at least three independent biological replicates. For comparisons between two groups, a two-sided Student’s t-test was applied. For multiple group comparisons, either one-way or two-way analysis of variance (ANOVA) was conducted. Statistical significance was defined as p < 0.05. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns (not significant).