Synthesis of BP quantum Dots (BPQDs)
The BPQDs were synthesized via a straightforward liquid exfoliation method as previously described [19]. In brief, 1 g of NaOH was added to 30 mL of ultra-dry N-methyl-2-pyrrolidone (NMP) and sonicated for 10–20 min. 30 mg of BP crystals was added into the alkalized NMP solution and sonicated for 6–8 h in an ice bath. Subsequently, the mixed solution was centrifuged at 4000 rpm for 10 min, the precipitate was reintroduced into the alkalinized NMP solution and again sonicated for 6–8 h in an ice bath. After sonication, the mixed solution was centrifuged at 4000 rpm for 10 min, then the supernatant was centrifuged at 10,000 rpm for 10 min. Finally, the resulting supernatant was centrifuged at 13,000 rpm for 30 min. The resulting precipitate was washed twice to obtain the black phosphorus quantum dots (BPQDs).
Preparation of liposomes
50 mg of SPC, 10 mg of cholesterol, 10 mg of DSPE-PEG2000 and 10 g of DOTAP were dissolved in 3 mL of chloroform. The solvent was removed by vacuum distillation at 50 oC for 1 h in a 100 mL flask to form a thin film. Then, deionized water was added to the flask. After the above solution was sonicated and treated with liposome extruder (polycarbonate membrane, pore size 100 nm). Finally, deionized water was supplemented and volume was fixed to 5 ml. Freeze-dried by adding lyophilized protective agent.
Preparation of BPQDs-Liposomes
Thin film hydration strategy was used to prepare the liposomes [20]. In brief, 50 mg of SPC, 10 mg of cholesterol, 10 mg of DSPE-PEG2000 and 10 g of DOTAP were dissolved in 3 mL of chloroform. The solvent was removed by vacuum distillation at 50 oC for 1 h in a 100 mL flask to form a thin film. Then, deionized water was added to the flask. After the above solution was sonicated and treated with liposome extruder (polycarbonate membrane, pore size 100 nm), BPQDs were added to the flask, magnetically stirred for 1 h, and dialyzed with a nano-dialysis device (polycarbonate membrane, pore size 30 nm). Finally, deionized water was supplemented and volume was fixed to 5 ml. Freeze-dried by adding lyophilized protective agent.
Synthesis of BPQDs@Lipo-YSA
The YSA-modified liposomes containing BPQDs were prepared by the thin-film hydration method [21]. 50 mg of SPC, 10 mg of cholesterol, 10 mg of DSPE-PEG-YSA and 10 g of DOTAP were dissolved in 3 mL of chloroform. The solvent was removed by vacuum distillation at 50 oC for 1 h in a 100 mL flask to form a thin film. Then, deionized water was added to the flask. After the above solution was sonicated and treated with liposome extruder (polycarbonate membrane, pore size 100 nm), BPQDs were added to the flask, magnetically stirred for 1 h, and dialyzed with a nano-dialysis device (polycarbonate membrane, pore size 30 nm). Finally, deionized water was supplemented and volume was fixed to 5 mL. Freeze-dried by adding lyophilized protective agent.
Characterizations
Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) was conducted on the JEM-3200FS (JEOL, Japan) at an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) was conducted on the Thermo Fisher ESCALAB 250Xi XPS. X-ray diffraction (XRD) was conducted on the SmartLab X-ray diffractometer (Rigaku, Japan). The size distribution and zeta potential were determined by dynamic light scattering (DLS) using the Zetasizer 3000 HAS (Malvern Instruments Ltd., UK). The UV-Vis-NIR absorption spectra were acquired on an ultraviolet-visible spectrophotometer (U-3900, Hitachi, Japan).
Determination of the liposomal encapsulation efficiency
The liposomal encapsulation efficiency was determined by using inductively coupled plasma optical emission spectrometry (ICP) [22]. A total of 200 µL blank liposomes were used as the control group. The sample was digested with nitric acid until it reached an atomic state. The encapsulation efficiency was calculated with the formula EE% = (1-Co/C, ) x 100%.
Cell culture
The NCI-H2009 cell line was maintained in RPMI 1640 medium, enriched with 10% fetal bovine serum, 4 mM L-glutamine, and penicillin/streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. This cell line was acquired from Cellverse Bioscience Technology Co. Routine mycoplasma testing, performed via PCR assays or DAPI staining to inspect for nuclear periphery abnormalities, confirmed the absence of mycoplasma contamination in our cultures.
Preparation of fluorescent BPQDs-Liposome composite
10 µL of Atto532 DOPE was introduced into the lipid formulation to yield red fluorescent liposomes (designated as liposome-Atto532 DOPE). Separately, 10 µL of Cy5.5 dye was mixed into the BPQDs suspension, which was then agitated at 600 rotations per minute overnight in the dark. The following day, the BPQDs-Cy5.5 mixture underwent centrifugation at 13,000 revolutions per minute for an hour and was rinsed thrice with double-distilled water (ddH2O). The purified BPQDs-Cy5.5 and the liposome-Atto532 DOPE were combined and vigorously homogenized 300 times. This blend was passed through a 0.45 μm filter three successive times, collected, and preserved at a temperature of 4 degrees Celsius, with all steps conducted under shielded light conditions.
Cell viability assay
NCI-H2009 cells were seeded into 96-well culture plates at a density of 8,000 cells per well. Post-treatment, 10 µl of Cell Counting Kit-8 (CCK-8) reagent (supplied by Boster, Wuhan, China) was introduced into each well in adherence to the manufacturer’s protocol. Following a 1-hour incubation period at 37 °C, the absorbance at a wavelength of 450 nm was recorded for each well using a microplate reader.
In Vitro Anti-Tumor Efficacy Evaluation
NCI-H2009 cells were seeded onto a 96-well plate at a density of 5 × 104 cells/mL and incubated for 24 h. The medium was subsequently replaced with that containing BPQDs@Lipo-YSA. For the anti-tumor assays, cells were subjected to the following treatments: phosphate-buffered saline (PBS; Control group), BPQDs@Lipo-YSA (Mono-therapy group), and BPQDs@Lipo-YSA plus NIR (Combined Photothermal-Chemotherapy group). Following a 4-h incubation, cells in the photothermal and combined therapy groups were exposed to a 660 nm laser (1.0 W/cm2) for 5 min. Cells in the Control group were also laser-irradiated for 5 min to ascertain the inherent cytotoxicity of NIR. Thereafter, all groups were further incubated for 24 h, and cell viability in each group was quantified using the CCK-8 assay kit. In the live/dead assay, the medium was discarded and replaced with Calcein-AM (5 µg/mL) and propidium iodide (PI; 5 µg/mL). After a 15-min incubation period, cells were rinsed with PBS, and fluorescent images indicative of cellular viability were captured using an IX71 fluorescence microscope (Olympus, Japan).
Flow cytometry analysis
Cells were cultured in media containing phosphate-buffered saline (PBS; Control group), BPQDs@Lipo-YSA (Chemotherapy group), and BPQDs@Lipo-YSA + NIR (Combined Photothermal-Chemotherapy group). For phototherapy, cells were exposed to a near-infrared (NIR) laser at 660 nm with a power density of 1.0 W/cm2 for 5 min. After a 48-h incubation, cells were fixed and stained with propidium iodide for cell cycle analysis. For apoptosis assessment, cells were harvested and resuspended in 200 µL of PBS, followed by staining with both propidium iodide and Annexin V-FITC.
Reactive oxygen species (ROS) assays
In conducting the reactive oxygen species (ROS) assay, cells were initially plated onto 24-well culture plates. Post-treatment, the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), included in the ROS Assay Kit procured from Beyotime (Shanghai, China), was administered to the cells. These cells were next incubated at 37 °C in a humidified cell incubator for 20 min to facilitate probe penetration and intracellular oxidation, marking ROS presence. Afterwards, cells were meticulously rinsed thrice with serum-free medium to discard extracellular, unreacted probe remnants. Ultimately, the accumulation of ROS within the cells was imaged and quantified utilizing laser scanning confocal microscopy (Nikon A1R/A1), affording both visual and numerical data on the intracellular oxidative stress status.
Mitochondrial membrane potential (MMP) assessment
MMP was evaluated using a JC-1 staining kit (Yeasen, Shanghai) following the manufacturer’s protocol. Podocytes were washed thrice in PBS, stained with JC-1 solution for 20 min at 37 °C in darkness, and rinsed three times with PBS. MMP was visualized under a confocal microscope (LSM 800, Zeiss) at 400x magnification, where green fluorescence (Ex/Em ≈ 514/529 nm) signified monomers and red fluorescence (Ex/Em ≈ 585/590 nm) indicated aggregates. Fluorescence intensity was analyzed in five random fields by calculating the aggregates-to-monomers ratio using ImageJ 1.48v software.
Mitochondrial ROS measurement
Accumulation of mitochondrial ROS (mtROS) was quantified using mitoSOX red fluorescent dye (Invitrogen, Carlsbad, CA). Post-treatment with TNF-α or ZWT-supplemented serum, podocytes were washed three times in Hank’s Balanced Salt Solution (HBSS), followed by 10-min incubation at 37 °C with 2.5 µM mitoSOX in light-protected conditions. Post-washing thrice more in HBSS, podocytes were imaged under a confocal microscope (LSM 800, Zeiss, Germany) at 400x magnification. Five random fields were captured, and mitoSOX fluorescence intensity was analyzed via ImageJ 1.48 software.
Detection of mitophagy
Mitophagy in living cells was monitored using a method provided by Dojindo Molecular Technologies. Chemotherapeutic drugs were employed to induce mitochondrial autophagy. The extent of mitochondrial autophagy was quantified based on the Mtphagy dye-positive area within individual cells. Additionally, colocalization between Mtphagy and lysosomal stains was analyzed. All specimens were examined using laser confocal microscopy (Carl Zeiss LSM 710, Oberkochen, Germany).
Indicated deletion mutagenesis
For protein purification, cDNA encoding MYC-tagged full-length AKT1 (1-480 amino acids) and its truncated variants (1-150, 108–480, 408–480 amino acids) were cloned into the GEYB417 plasmid for expression in E. coli. Additionally, cDNA encoding FLAG-tagged full-length PRKN (1-465 amino acids) was cloned into the GEYB657 plasmid for expression in E. coli. All constructed plasmids were validated through DNA sequencing, with detailed sequence information available upon request. Transient transfections were carried out using Lipofectamine 2000 and Opti-MEM medium (Thermo Fisher Scientific 31,985,070), following the manufacturer’s protocol.
Mitochondrial fraction extraction
Prepare the lysis buffer by dissolving 0.42 g of Tris powder (pH 7.2), 0.15 g of NaCl, and 0.05 g of MgCl2 in 1 L of distilled water (ddH2O). For Buffer A, dissolve 10.95 g of sucrose, 0.04 g of EDTA, and 0.12 g of Tris (pH 7.2) in 100 ml of ddH2O. The balance buffer is made by dissolving 1.7 g of Tris (pH 7.2), 0.58 g of NaCl, and 0.19 g of MgCl2 in 40 ml of ddH2O. Muscle tissue or C2C12 cells were processed in a manual homogenizer in the presence of lysis buffer supplemented with PMSF (Beyotime, ST507). Post-homogenization, add the balance buffer, then centrifuge the sample at 15,000 g for 20 min to isolate the mitochondrial and cytosolic fractions. Resuspend the mitochondrial fraction, found in the pellet, in Buffer A. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23,227).
Immunoblotting and Immunoprecipitation
For Western blot analysis, tissues and cells were lysed using Cell Lysis Buffer for Western and IP (Beyotime, P0013) with PMSF. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23,227) and a spectrophotometer (MultiSkan GO, Thermo Fisher Scientific) at 562 nm. Protein samples were separated via SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Bio-Rad 1,620,177). Membranes were blocked in a solution of Tris-buffered saline (BOSTER, AR0031) with 0.1% Tween 20 (Aladdin, 9005-64-5) containing 3% nonfat dry milk or BSA (BioFROXX, 4240GR250), followed by incubation with primary antibodies overnight at 4 °C and horseradish peroxidase-conjugated secondary antibodies (Abbkine, A21020 and A21010) for 1 h at room temperature. Antibodies were diluted in the blocking solution. The primary antibodies utilized for Western blot analysis are detailed in the Reporting Summary. Western blot images were captured using the SINSAGE MiniChemi 610 and SageCapture Pro software.
For immunoprecipitation studies, specific IP antibodies were introduced into the protein lysates and incubated on a rotator at 4 °C overnight. Protein A/G Magnetic Beads (Bimake, B23202) were then added to the samples and incubated on a rotator at 4 °C for 4 h. Following five washes with PBS containing 0.1% Tween 20 (PBS-T), the immunoprecipitates were eluted by boiling in loading buffer for 10 min and analyzed by Western blot as previously described.
To assess protein ubiquitination, cells were treated with MG132 (Selleck 1,211,877-36-9; 20 µM) 2 h prior to harvest. Cells were lysed in RIPA lysis buffer (Beyotime, P0013B) containing PMSF (100 µg/mL) and subsequently analyzed by immunoprecipitation as outlined above.
Antibody
Prkn (abcam, ab77924, 1000), PINK1 (abcam, ab216144, 1:1000), BNIP3 (abcam, ab109362, 1:1000), MAP1LC3A/B (Cell Signaling Technology 12,741; 1:1000), mTOR (phospho S2448)(abcam, ab109268, 1:1000), mTOR (abcam, ab134903, 1:1000), AKT1 (phospho S473) (abcam, ab81283, 1:1000), AKT1 (abcam, ab179463, 1:1000), PI3K (abcam, ab302958, 1:1000), Bax ((abcam, ab32503, 1:1000), Bcl-2 (abcam, ab182858, 1:1000), p53 (abcam, ab26, 1:1000), Cleaved Caspase-3 (abcam, ab2302, 1:1000), ubiquitin (abcam, ab134953; 1:1000), MYC-tag (Bioworld Technology, AP0031M; 1:5000), FLAG-tag (Ray Antibody, RM1002; 1:1000), β-Actin (abcam, ab8226, 1:10000).
Immunofluorescence
For cellular immunofluorescence analysis, cells were fixed using 4% paraformaldehyde, permeabilized with methanol, and blocked with a 3% BSA solution. The cells were then incubated with the corresponding primary antibodies, followed by staining with Cy3-labeled goat anti-rabbit IgG (Beyotime, P0183), FITC-labeled goat anti-mouse IgG (Beyotime, P0196), MitoTracker Green (Beyotime, C1048), and LysoTracker Red (Beyotime, C1046). Imaging was performed using a Zeiss LSM 800 microscope equipped with Airyscan technology and Zeiss Zen software (ZEN 2.3 Blue edition).
In vitro assessment of Immunogenic cell death
Cells were cultivated in media comprising phosphate-buffered saline (PBS; Control group), BPQDs (Chemotherapy group), BPQDs@Lipo + NIR (Combined Photothermal-Chemotherapy group), and BPQDs@Lipo-YSA + NIR (Combined Photothermal-Chemotherapy group). For phototherapy, cells were exposed to a 660 nm laser at a power density of 1.0 W/cm2 for 5 min. Following incubation with anti-calreticulin (CRT) antibody and subsequent labeling with AF597-conjugated secondary antibody, cell analysis was performed using flow cytometry and inverted fluorescence microscopy. Anti-high mobility group box 1 (anti-HMGB1) antibody, along with AF488-conjugated secondary antibody, was utilized for HMGB1 detection. ATP release was quantified using an ATP assay kit.
Animals
Male C57BL/6 mice, aged approximately 6–8 weeks and weighing 18–22 g, were obtained from the Huaxing Experimental Animal Center located in Zhengzhou, China. These animals were housed under standard laboratory conditions, featuring a 12-h light-dark cycle, a controlled temperature of 25 ± 2 °C, and a relative humidity of 50 ± 5%. They were provided with ad libitum access to food and water to ensure their wellbeing.
In vivo immunological study
Following the evaluation of antitumor efficacy, tumors were excised and dissociated into single-cell suspensions of lymphocytes. These cells were then labeled with a panel of mouse antibodies (CD3-APC, CD4-FITC, CD8-PE, CD80-FITC, CD86-PerCP/Cyanine5.5, CD11c-PE, CD25-PE, and Foxp3-Alexa Fluor 488) and analyzed via flow cytometry. Subsequent to the in vivo antitumor study, blood serum was collected, and cytokine levels in the serum, including tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ), interleukin-10 (IL-10), and interleukin-12 (IL-12), were quantified using enzyme-linked immunosorbent assay (ELISA) kits.
Immunohistochemical analysis
Following the in vivo antitumor experiment, tumors and major organs were harvested for histopathological assessments. Sections of tumors and vital organs were stained with Hematoxylin and Eosin (H&E) for general histomorphology. Moreover, to assess apoptotic activity, immunohistochemistry (IHC) staining for Ki67 and Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) were performed on tumor sections.
Targeted delivery of BPQDs to tumors
After intravenous administration of Cy5.5-labeled BPQDs, BPQDs@Lipo + NIR, and BPQDs@Lipo-YSA + NIR (each at a dose of 10 mg/kg), the fluorescence signals of Cy5.5 throughout the body were detected at predefined time points (6, 12, 24, 36, and 48 h) using an IVIS Spectrum system (Caliper IVIS Spectrum, PerkinElmer, USA). Mice were euthanized 24 h post-injection for ex vivo examination of fluorescence distribution in tumors and major organs (heart, liver, spleen, lungs, and kidneys). All images were normalized and analyzed using the Living Image 4.2 software.
Approximately 0.2 g of each tissue sample was weighed, digested overnight in 65% nitric acid, and the concentration of black phosphorus (BP) was subsequently determined using an inductively coupled plasma-optical emission spectrometer (ICP-OES, model 7000DV, PerkinElmer, USA).
Photothermal response at tumor sites
Twenty-four hours following the administration of BPQDs@Lipo-YSA, the mice were anesthetized, and the complete tumor area was exposed to a 660 nm laser at a power density of 1.0 W/cm2 for 5 min. The temperature elevation of tumors during laser irradiation was monitored using an infrared thermal imaging camera (Ti27, Fluke, USA).
Tumor Implantation and Treatment
The procedure for tumor implantation (TI) adhered to previous descriptions (22, 23). In summary, mice in the tumor-bearing group (T) were subcutaneously injected (s.q.) in the right flank with Lewis lung carcinoma cells (LLC cells, 1 × 106 cells, catalog CRL1642, obtained from the American Type Culture Collection, Manassas, VA, USA), while control groups received an equivalent volume of heat-killed LLC cells (HK) at day 7. Once tumors became palpable (approximately 1 cm in diameter, around day 7 post-TI, designated as day 0), distinct groups of tumor-bearing mice were administered one of the following substances via intravenous administration: phosphate-buffered saline (PBS; Control group), BPQDs (Chemotherapy group), BPQDs@Lipo + NIR (Mono-therapy group), or BPQDs@Lipo-YSA + NIR (Combined Photothermal-Chemotherapy group), with treatments continuing for a maximum of 18 days. Food intake, body weight, tumor dimensions, and volume were assessed at baseline prior to TI (day 7) and again at the conclusion of the study. Final measurements were expressed as ratios relative to their respective baseline values.
Statistical analysis
Statistical evaluations in this study were conducted using GraphPad Prism software, version 9.0. All datasets are expressed as the mean ± standard deviation (SD). Comparisons between two groups were performed utilizing the independent samples t-test. For analyses involving three or more groups, a one-way analysis of variance (ANOVA) was employed, complemented by Tukey’s post hoc test for parametric data or the Kruskal-Wallis test for non-parametric data. Statistical significance was inferred at a threshold of p < 0.05.