Materials and reagents
Sodium selenite (Na2SeO3), bismuth(III) nitrate pentahydrate (Bi(NO3)3·5H2O), gelatin (Gel), sodium alginate (SA) and hydrogen peroxide (H₂O₂) were purchased from Aladdin (Shanghai, China). Ethylene glycol (EG), polyvinylpyrrolidone (PVP), 3,3’,5,5’-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), terephthalic acid (PTA), 1,3-diphenylisobenzofuran (DPBF), reduced glutathione (GSH), and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Macklin (Shanghai, China). Phosphate-buffered saline (PBS), trypsin EDTA solution, a Cell Counting Kit-8 (CCK-8), a catalase detection kit, and 2-(4-amidinophenyl)6-indoleaminourea dihydrochloride (DAPI) were purchased from SparkJade (Shandong, China). Gibco DMEM/F12 Dulbecco’s modified Eagle’s medium (DMEM/F12), Gibco fetal bovine serum (FBS), and Gibco Antibacterial Antimycotic (100X) were purchased from Thermo Fisher Scientific (New York, USA). Calcein-AM/PI and the membrane-associated protein V-FITC/PI were purchased from Bestbio (Shanghai, China). 2’,7’-Dichlorofluorescein diacetate (DCFH-DA) and JC-1 were purchased from Biyotime (Shanghai, China). A mouse glutathione peroxidase 4 (GPX4) ELISA kit was purchased from JONLNBIO (Shanghai, China). FerroOrange and Liperfluo were purchased from DOJINDO LABORATORIES (Kyushu, Japan). p53, p62, Caspase3, GPX4, NRF2, SLC7A11, and FTH1 antibodies were purchased from Proteintech (Wuhan, China).
Preparation of Bi2Se3 NSs
In accordance with previously described methods [48], 0.242 g of Na₂SeO₃ was dissolved in 40 mL of EG, and 0.452 g of Bi(NO3)3·5H2O was dissolved in 15 mL of EG. Subsequently, 1.0 g of PVP was dissolved in 50 mL of ethylene glycol (EG) and transferred to a 250 mL round-bottom flask. The dissolved Na₂SeO₃ and Bi(NO3)3·5H2O were added while stirring at 30 °C. The flask was hermetically sealed with nitrogen (N₂) and heated to 180 °C. The solution gradually became white. Subsequently, 2 mL of NH3-H2O was dissolved in 20 mL of EG and introduced into the flask. The reaction mixture was observed to immediately blacken and then cooled to room temperature to allow the reaction to complete. After centrifugation at 10,000 rpm for 10 min, the cells were washed three times with deionized aqueous water and acetone. They were then dried in a vacuum freeze dryer to obtain Bi₂Se₃ NSs.
Characterization of Bi2Se3 NSs
The prepared Bi2Se3 NSs were characterized via transmission electron microscopy (TEM) to obtain micrographs, SAED images, and elemental mapping images. X-ray diffraction (XRD) maps were obtained via an X-ray diffractometer. Zeta particle size and zeta potential analyses were carried out on the Bi2Se3 NSs via a dynamic light scatter meter. Bi2Se3 NSs were analyzed via Fourier transform infrared (FTIR) spectroscopy.
Determination of the Bi2Se3/Gel-SA hydrogel
In accordance with a previously reported method [49], gelatin and sodium alginate were weighed and mixed at 10% and 1% ratios, and deionized water was added. The Gel-SA solution was obtained by stirring at 60 °C in a water bath for 1 h. The injectable solution of Bi2Se3 + Gel-SA was generated by effectively combining Bi2Se3 and Gel-SA. A hydrogel was formed using 3% CaCl2 to mimic the high concentration of Ca2+ in the tumor environment crosslinked with sodium alginate. FTIR spectroscopy was performed via a Fourier transform infrared spectrometer. The frequency, dynamic step strain, and temperature rise properties of the hydrogels were verified via a rotational rheometer. Scanning electron microscopy (SEM) was subsequently used to further evaluate the alignment and obtain elemental mapping images of the Bi2Se3/Gel-SA hydrogel. Comparisons were made by simulating the state of the tumor and the solvent at different temperatures. The morphology of the hydrogel was subsequently observed, captured, and recorded.
Ion release rate
One milliliter of Bi2Se3/Gel-SA hydrogel was added to the dialysis bag and then placed in the dialysate prepared in PBS. The dialysate was sampled periodically, and the ion concentration was determined via inductively coupled plasma emission spectroscopy. The cumulative ion release rate over time was calculated.
Photothermal performance test
First, 1 mL of Bi2Se3 NSs at different concentrations was placed in a colorimeter and irradiated with an 808 nm laser at a power of 2.0 W cm− 2 for 5 min. Pictures were taken via infrared thermography, and temperature changes were recorded. Next, different irradiation powers were used at the same concentration (100 µg mL− 1) for 5 min, and the temperature changes were recorded. Then, three heating/cooling photothermal cycles were monitored for the Bi2Se3 NSs.
Evaluation of free radical production
Four groups were set up: (I) control (PBS), (II) Bi2Se3/Gel-SA without a laser, (III) PBS with 808 nm laser irradiation, and (IV) Bi2Se3/Gel-SA with 808 nm laser irradiation (2.0 W cm− 2, 5 min). The commonly used DPBF probes were added to different groups to evaluate their radical generation properties. Free radical production was detected via electron spin resonance (ESR) spectroscopy.
GSH depletion
GSH depletion generates glutathione peroxidase (GSH-Px), which is involved in this process. DTNB was added to a mixture of GSH, PBS, and Bi2Se3/Gel-SA, and the absorbance of the mixtures was measured at λ = 412 nm at different times to assess the scavenging efficiency (rate).
In vitro antitumor assay
Cell uptake assay
To determine whether the material can work in both tumor and normal tissues, cell uptake assays were carried out in human umbilical vein endothelial cells (HUVECs), NCTCclone929 (L929) and rat nasopharyngeal carcinoma (FAT) cells. These cells were inoculated in 6-well plates, incubated for 24 h, and then replaced with medium containing Bi2Se3 NSs and Cy5.5. The cells were transferred to centrifuge tubes at 0.5, 1, 2, 3, and 6 h, after which the level of cellular uptake was assessed via flow cytometry.
CCK8 assay
To evaluate the in vitro cytotoxicity of Bi2Se3 NSs, HUVECs and L929 cells were inoculated into 96-well cell culture plates. After the safe concentration of Bi2Se3 NSs was determined, FAT cells were inoculated into 96-well cell culture plates. After a series of safe concentrations of Bi2Se3 NSs (0, 20, 40, 60, 80, and 100 µg mL− 1) were added to the cells and incubated for 12 h, the cells were irradiated with an 808 nm laser at a power of 2.0 W cm− 2 for 5 min. The relative cell viability was determined via a CCK-8 assay, and the therapeutic concentration of Bi2Se3 NSs was determined.
Live/dead cell assay
The impact of the Bi2Se3/Gel-SA combination on adipose tissue was evaluated via a calcein-AM/PI live-dead cell assay kit. In particular, the FAT cells were inoculated and incubated in confocal dishes for 24 h, according to the subgroups previously tested for free radical generation, and divided into four groups. The cells were subsequently treated in different ways for 12 h. The cells were then stained with the calcein-AM/PI working solution for 30 min at 37 °C in the dark, after which they were observed under a laser confocal microscope.
ROS detection at the cellular level
DCFH-DA is a probe that is commonly used to detect the production of intracellular ROS. The FAT cells were inoculated and incubated in confocal dishes for 24 h. Subsequently, the cells were treated in different ways for 12 h. The cells were then stained with DCFH-DA working solution in the dark for 30 min. The cells were observed and analyzed via laser confocal microscopy and flow cytometry.
Mitochondrial membrane potential assay
Changes in the mitochondrial membrane potential were identified via JC-1 staining. FAT cells were inoculated and incubated in confocal dishes for 24 h. Thereafter, the cells were subjected to different treatments for 12 h. Staining was then conducted according to the instructions provided in the Mitochondrial Membrane Potential Kit, with the staining process taking place on ice. Fluorescence images were subsequently captured via a laser confocal microscope.
Determination of GPX4 activity
A GPX4 ELISA kit was used to measure intracellular GPX4 activity. FAT cells were inoculated in 6-well plates and incubated for 24 h. Following this, the cells were subjected to different treatments for 12 h. Per the kit instructions, the cells were transferred to a centrifuge tube and disrupted with a cell crusher. The OD values were then measured at λ = 450 nm via an enzyme marker and calculated per the kit instructions.
LPO test
Liperfluo is an analog of Spy-LHP used to detect LPO. FAT cells were inoculated and incubated in confocal dishes for 24 h. Thereafter, the cells were subjected to different treatments for 12 h. Subsequently, the cells were stained with a working solution, prepared per the instructions of the Liperfluo staining kit, for 30 min in the dark. Subsequently, fluorescence images were captured via a laser confocal microscope.
Assessment of intracellular Fe2+ levels
A FerroOrange probe was used to detect intracellular Fe2+ levels. FAT cells were inoculated and incubated in confocal dishes for 24 h. Subsequently, the cells were treated in different ways for 12 h. The working solution was prepared for 30 min in the dark according to the instructions of the FerroOrange staining kit, after which fluorescence images were captured via a laser confocal microscope.
Apoptosis assay
A propidium iodide/annexin V-FITC probe was used to quantify the extent of apoptosis. FAT cells were seeded in 6-well plates and incubated for 24 h. Then, the cells were subjected to different treatment regimens for 12 h. The treated cells were transferred to centrifuge tubes and stained in accordance with the instructions provided with the propidium iodide/annexin V-FITC Staining Kit. The resulting levels of apoptosis were then assessed via flow cytometry.
Western blotting analysis
Western blotting is a widely employed method for detecting protein expression. In the present study, the expression of several proteins, including p53, SLC7A11, p62/SQSTM1, NRF2, FTH1, GPX4, and Caspase 3, was investigated. FAT cells were treated differently for 12 h, harvested by trypsin digestion, and subjected to centrifugation. The total protein was extracted from the cells via a buffer solution with the composition recommended for this purpose. The total protein concentration of each sample was determined via a BCA protein assay kit in accordance with the manufacturer’s instructions. The proteins were subsequently subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with skim milk and then incubated with a diluted primary antibody at 4 °C overnight. Following a brief rinse, the membrane was incubated with a diluted secondary antibody (1:5000) for 30 min at room temperature. The chemiluminescent signal was then detected, and images were captured via a protein imager. The data were subjected to analysis via computer software.
In vivo evaluation of tumor treatment
All the animal experiments were conducted by the Animal Protection and Utilization Committee of Soochow University (No. SUDA20210708A03). The animal model used in this study was BALB/c nude mice (female, 6 weeks old, 18–20 g). A suspension of 1 × 10⁷ cells in 150 µL of PBS was injected subcutaneously into the left dorsum of each mouse to establish the FAT subcutaneous model. After seven days, the tumor size reached approximately 100 mm³. Organ distribution experiments were first performed to observe the metabolic process of Bi2Se3 NSs in mouse tumors/whole body. The mice were sacrificed on days 1, 7 and 14 after Bi2Se3/Gel-SA hydrogel injection to dissect the tumors, important organs and muscles, after which they were crushed with a tissue crusher and immersed in aqua regia overnight. The supernatants were collected and assayed via ICP. The mice were randomly divided into four groups (n = 5 per group). Each group comprised five mice, with an injection volume of 100 µL for in situ injection and irradiation with an 808 nm laser for a 5 min treatment period. The mice were then subjected to thermal imaging via an infrared thermal imager. The groups were as follows: (I) control (PBS), (II) Bi2Se3/Gel-SA without a laser, (III) PBS with 808 nm laser irradiation, and (IV) Bi2Se3/Gel-SA with 808 nm laser irradiation (1.0 W cm− 2, 5 min). Tumor size was measured at 2-day intervals via calipers, and tumor volume was calculated via the following formula: tumor volume = (tumor length) × (tumor width)2/2. The body weight of each mouse was recorded at two-day intervals. Necropsies were performed on the mice that died during the dosing period to obtain tumor data. The remaining mice were necropsied on day 14, dissected, photographed, and subjected to histological staining, including hematoxylin and eosin (H&E), tumor cell proliferation (Ki-67), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) staining. The following staining techniques were employed: UTP nick end labeling (TUNEL), reactive oxygen species (ROS), p53, SLC7A11, p62/SQSTM1, NRF2, FTH1, GPX4, and Caspase 3. The resulting sections were imaged via fluorescence microscopy.
Evaluation of postoperative free radical scavenging
The MB probe was added to different concentrations of Bi2Se3 to evaluate its postoperative free radical scavenging performance. A UV spectrophotometer was subsequently used to quantify the absorbance at λ = 664 nm. A superoxide dismutase (SOD) assay kit was used to assess superoxide radical scavenging directly. The adsorption capacity of the mixtures for H₂O₂ was quantified via a hydrogen peroxide assay kit to evaluate the catalase (CAT) level. The capacity of the mixture to decompose H₂O₂ into O₂ was determined via a dissolved oxygen meter.
In vitro anti-inflammatory and tissue healing assays
Evaluation of inflammatory factor levels in vitro
The administration of Bi2Se3/Gel-SA to HUVECs resulted in a dose-dependent alteration in the extracellular levels of proinflammatory cytokines. HUVECs were incubated in 6-well plates for 24 h. The medium was then replaced with fresh medium mixed with LPS and Bi2Se3/Gel-SA for 24 h. The medium was then collected, and the levels of proinflammatory cytokines, including IL-10, IL-6, IL-1β, and TNF-α, were assessed via an ELISA kit. The collected medium was centrifuged to detect cytokines, and the concentrations were calculated.
Evaluation of postoperative MDA levels
HUVECs were incubated in 6-well plates for 24 h. After 24 h of replacement incubation with fresh medium mixed with LPS and Bi2Se3/Gel-SA, the medium was collected and tested according to the MDA kit instructions to evaluate the cellular MDA levels.
Evaluation of postoperative ATP levels
HUVECs were incubated in 6-well plates for 24 h. The medium was then replaced with fresh medium mixed with LPS and Bi2Se3/Gel-SA for 24 h. The medium was then collected and tested to assess the cellular ATP levels per the instructions provided with the ATP kit.
Postoperative live/dead cell assay
The protective effect of Bi2Se3/Gel-SA on postoperative peritumoral normal cells was assessed via a calcein-AM/PI live-dead cell assay kit. Specifically, HUVECs were inoculated and incubated in confocal dishes for 24 h. Subsequently, the cells were treated in different ways for 12 h. The cells were stained with calcein-AM/PI working solution at 37 °C for 30 min in the dark and then observed under a laser confocal microscope.
ROS detection at the cellular level during the postoperative period
The capacity of Bi2Se3/Gel-SA to neutralize residual ROS during the postoperative period was evaluated via the use of a DCFH-DA probe. HUVECs were inoculated and incubated in confocal dishes for 24 h. Thereafter, the cells were treated in different ways for 12 h. The staining mixture was then replaced with fresh medium containing DCFH-DA for 30 min, after which the cells were observed and imaged via laser confocal microscopy.
Repolarization of the mitochondrial membrane potential after surgery
Inflammatory injury typically disrupts the average polarization of the mitochondrial membrane potential. The efficacy of Bi2Se3/Gel-SA in repolarizing the mitochondrial membrane potential was evaluated via the use of a JC-1 probe. HUVECs were inoculated and incubated in confocal dishes for 24 h. Thereafter, the cells were treated in different ways for 12 h. Staining was then performed in the dark on ice according to the instructions provided with the mitochondrial membrane potential kit. The cells were observed and imaged via a laser confocal microscope.
Cell scratch assay
L929 cells were seeded in a 6-well plate and cultured until they reached 90% confluence. The culture medium was then replaced with serum-free medium, and a sterile needle was used to create uniform scratches on the cell surface. Cell migration was evaluated in the (1) LPS group and (2) LPS + Bi2Se3/Gel-SA group, and images were captured at 0, 12, and 24 h.
Colony formation assay
The L929 cell suspension was serially diluted, inoculated into a 6-well plate, and incubated for 12 h according to the grouping set in the scratch experiment. The culture medium was then replaced with fresh medium for 2 weeks until visible colonies appeared in the culture dish. After washing with PBS, 4% paraformaldehyde was added, and the samples were fixed for 5 min. Then, the fixative was removed, an appropriate amount of crystal violet staining solution was added for 30 min, and the samples were observed after air drying. Colony formation was evaluated in the (I) control, (II) LPS, (III) LPS + Bi2Se3/Gel-SA (5% gel), and (IV) LPS + Bi2Se3/Gel-SA (10% gel) groups.
Animal models of postoperative inflammation and tissue healing
All animal experiments were conducted by the Animal Protection and Utilization Committee of Soochow University (No. SUDA20210708A03). The animal models used in this study were female BALB/c nude mice (female, 6 weeks old, 18–20 g). A solution comprising LPS and/or Bi2Se3/Gel-SA was administered subcutaneously into the left dorsal region, establishing a subcutaneous inflammation model. The mice were randomly assigned to 3 groups, with 5 mice per group. The experimental groups were as follows: (1) control (PBS), (2) LPS, and (3) LPS + Bi2Se3/Gel-SA. The mice were euthanized on day 7, dissected, and photographed, and blood was taken from the eyes and subjected to ELISA to assess the tissue levels of IL-10, IL-6, IL-1β, TNF-α, ATP, and MDA. Furthermore, histological staining, including H&E, Masson, Ki-67, TUNEL, ROS, IL-10, IL-6, IL-1β, TNF-α, CD31 and collagen staining, was conducted. The resulting sections were imaged via fluorescence microscopy.
In vivo safety assessment
BALB/c nude mice (female, 6 weeks old, 18–20 g) were injected intravenously with Bi2Se3/Gel-SA or PBS as controls. The mice were euthanized for H&E staining of significant organs 14 days after injection. Complete hematological and serum biochemical examinations were also performed on mouse blood samples. Among the serum biochemical tests, blood urea nitrogen (BUN) and creatinine (CRE) are two important indicators of renal function, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are essential indicators of liver function. Other relevant hematological parameters are as follows: lymphocyte count (LYM), mean platelet volume (MPV), mean platelet volume (MPV), mean erythrocyte hemoglobin concentration (MCHC), hemoglobin (HGB), platelet distribution width (PDW), mean corpuscular volume (MCV), erythrocyte compression volume (HCT), white blood cell (WBC) count, red blood cell (RBC) count and mean red cell hemoglobin (MCH) count.
Statistical analysis
All the quantitative results were obtained from at least three samples. Graphs were created, and the statistical data were analyzed via Origin 2024. Comparisons between the two groups were made via unpaired t tests. The following levels of statistical significance were employed: *p < 0.05, **p < 0.01 and ***p < 0.001.