3-Bromopyruvate-loaded bismuth sulfide nanospheres enhance most cancers therapy by synergizing radiotherapy with modulation of tumor metabolism | Journal of Nanobiotechnology

Supplies

All reagents used on this work have been of analytical grade. Bismuth nitrate pentahydrate (Bi(NO₃)₃·5H₂O, ≥ 99.99%), nitric acid (HNO₃), sodium hydroxide (NaOH, ≥ 97.0%), polyvinylpyrrolidone (PVP, Mw ≈ 10,000 Da), ethylene glycol (EG, > 99%), thioacetamide (TAA, ≥ 99%), ethanol, acetone have been bought from Aladdin Reagent Co., Ltd. (China). Methoxy polyethylene glycol thiol (mPEG2K-SH, Mw ≈ 2000 Da) was bought from Xi’an ruixi Organic Expertise Co., Ltd (China). Cell Counting Equipment-8 (CCK-8), 3-Methyladenine (3MA) and rapamycin (Rapa) have been obtained from MedChemExpress (USA). 2-Deoxy-D-glucose (2DG) and rotenone have been bought from ApexBio Expertise (USA). Enhanced ATP Assay Equipment, Crystal Violet Staining Resolution, Reactive Oxygen Species Assay Equipment, BCA Protein Assay Equipment, 4,6-Diamidino-2-phenylindole (DAPI) and a couple of,7-dichlorodihydrofluorescein diacetate (DCFH-DA) have been bought from Beyotime Biotechnology (China). Lactic Acid Content material Take a look at Equipment, Glucokinase Exercise Assay Equipment, the monodansylcadaverine (MDC) staining equipment, Triton X-100, bovine serum albumin and anti-fluorescence quenching agent have been obtained from Beijing Solarbio Science & Expertise Co., Ltd (China). 2.5% glutaraldehyde was obtained from Macklin (China). Spurr resin combination was obtained from SPI-CHEM (USA). ROS-ID® Hypoxia/Oxidative stress detection equipment was obtained from Enzo Life Sciences (USA). 3BP was bought from Sigma-Aldrich (USA). Roswell Park Memorial Institute (RPMI)-1640 full medium was bought from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (China). Distilled water was obtained from a Millipore water system. All chemical compounds have been of analytical grade and used with out additional purification.

Synthesis of Bi₂S₃-3BP

To synthesis Bi₂S₃-3BP, 364 mg Bi(NO₃)₃·5H₂O was dissolved in 10 mL HNO₃ resolution (1 mol/L). Then, 108 mg of NaOH, 1.2 g of PVP, and 50 mL of EG have been added into the answer. After sufficiently mixing, the above combination was transferred right into a stainless-steel autoclave with Teflon liner, sealed, and maintained at 150 °C for 3 h. After cooling all the way down to room temperature, the obtained milk-white product was precipitated by centrifugation and washed with distilled water. The Bi₂O₃ have been utilized because the bismuth precursor and template to acquire extremely porous Bi₂S₃ nanospheres.

Subsequent, 100 mg of TAA was dissolved in 30 mL of distilled water, after which 10 mL of Bi₂O₃ suspension (in distilled water) was added. The combination was then transferred right into a stainless-steel autoclave, maintained at 150 °C for 12 h. The obtained black product was centrifuged, after which completely washed with distilled water and absolute ethyl alcohol, respectively. The precipitate was dried in a vacuum oven at -50 °C.

For PEG coating, 10 mg of as-synthesized Bi₂S₃ product was dispersed in 10 mL absolute ethyl alcohol, after which 10 mg mPEG2K-SH was added. The combination was subjected to rotary evaporation for 1 h. Extra mPEG2K-SH have been eliminated by centrifugation and repeated washing utilizing distilled water. The ultimate Bi₂S₃ with PEG modification was resuspended in distilled water for additional use. Subsequent, 3BP was connected to the pores by magnetic stirring. Intimately, 10 mL of Bi₂S₃ with PEG modification (1 mg/mL) and 30 mg 3BP have been nixed and stirred in a single day. Bi₂S₃-3BP was purified by centrifugation and repeated washing utilizing distilled water.

Characterization of Bi₂S₃-3BP

Transmission electron microscopy (TEM, Hitachi H-7600, Japan) was used to characterize the morphology of the pattern. The crystalline type of the merchandise was characterised based mostly on X-ray diffraction (XRD) utilizing an X-ray diffractometer (Panalytical X’ Pert PRO, Shimadzu, Japan) with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was used to characterize the basic composition of intermediate and ultimate merchandise utilizing Thermo Scientific Ok-Alpha (Thermo Scientific, USA). Measurement distribution of the nanospheres was measured utilizing dynamic mild scattering (DLS, Malvern Devices, Malvern, UK). The porosity of Bi₂S₃ nanospheres was analyzed by nitrogen adsorption–desorption isotherms (APSP 2460, Micromeritics, USA).

The infrared spectrum of Bi₂S₃, mPEG2K-SH and Bi₂S₃ with PEG modification have been measured by utilizing an FT-IR spectrometer (Nicolet 6700, Thermo Scientific, USA). The absorption spectra of Bi₂S₃ with PEG modification and Bi₂S₃-3BP (dissolved in deionized water) have been decided with a UV-vis spectrophotometer (US-2550, Shimadzu, Japan). Totally different concentrations of free 3BP have been used to acquire a calibration curve utilizing UV-vis spectrometry, which was used to measure unloaded 3BP within the supernatant. Then the encapsulation effectivity and drug loading effectivity of Bi₂S₃-3BP will be calculated:

Encapsulation effectivity (EE) = (weight of loaded 3BP in Bi₂S₃-3BP / complete weight of 3BP added) × 100%.

Drug loading effectivity (DLE) = (weight of loaded 3BP in Bi₂S₃-3BP / complete weight of 3BP and Bi₂S₃ added) × 100%.

To research the drug launch conduct of 3BP, Bi₂S₃-3BP (loaded 3BP: 6.8 mg) was dispersed in PBS (2.0 mL) after which stirred at 37 ° C. At a predetermined time factors, the supernatant was extracted, then changed with an equal quantity of recent PBS, the supernatant was collected, and the corresponding customary calibration curve was used to investigate the 3BP launch.

Cell tradition and 4T1 tumor-bearing mice mannequin

Murine breast most cancers 4T1 cell traces have been obtained from Procell Life Science & Expertise Co., Ltd (China), and cultured in RPMI-1640 full tradition medium underneath a humidified ambiance with 5% CO₂ at 37 °C. All animal research and procedures have been performed underneath a protocol authorised by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical College (Evaluate Lot No. 2021 − 717). All animal experiments have been performed in accordance with the rules of the Chongqing Administration Strategy of Laboratory Animal. All animals (feminine BALB/c mice with a weight of ≈ 20 g) have been bought from Hunan Slyke Jingda Laboratory Animal Co., Ltd (China). 4T1 tumor-bearing mice have been established by subcutaneous administration of a cell suspension (1 × 10⁶ cells in 100 µL phosphate-buffered resolution (PBS) for every mouse). The tumor dimension was measured utilizing a vernier caliper, and tumor volumes have been calculated as [0.5 × length × (width)²].

Mobile uptake of Bi₂S₃-3BP

The mobile uptake of Bi₂S₃-3BP was investigated by laser scanning confocal microscopy (CLSM, Nikon Eclipse Ti, Tokyo, Japan) and stream cytometry (FACS, CytoFLEX Move Cytometer, Becton Coulter, Brea, CA). Briefly, the 4T1 cells have been seeded at a density of 1 × 10⁵ cells per CLSM dish and incubated for twenty-four h. Cells have been additional cultured in recent RPMI-1640 medium containing FITC-labeled Bi₂S₃-3BP (Bi: 60 µg mL^− 1) and incubated for 0, 1, 2, 3, 4 h. All cells have been mounted with 4% formaldehyde (10 min), washed with PBS, and stained with DAPI (15 min) for CLSM statement. Intracellular uptake of Bi₂S₃-3BP at completely different time factors was additionally quantified by stream cytometry.

Detection of glycolysis inhibition after Bi₂S₃-3BP therapy

Tumor cells cultured in dishes have been randomly divided into 6 teams (n = 3), and incubated with PBS, Bi₂S₃ (Bi: 60 µg mL^− 1), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1), 3BP (10 µg mL^− 1), Rotenone (10 µg mL^− 1), and 2DG (500 µg mL^− 1) for six h, respectively. Western blot (WB) assay was carried out to detect the expression of hexokinase-II (HK-II) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 4T1 cells after completely different remedies. Complete protein was quantified utilizing the BCA protein quantification equipment. The detailed experimental process of WB is proven within the supplementary materials. Tumor cells have been divided into 3 teams (n = 3), and incubated with PBS, Bi₂S₃ (Bi: 60 µg mL^− 1), and Bi₂S₃-3BP ((Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1)) for six h, respectively. The viability of HK-II, intracellular lactate, and intracellular ATP have been measured by the Glucokinase Exercise Assay Equipment, Lactic Acid Content material Take a look at Equipment, and the Enhanced ATP Assay Equipment in keeping with customary protocols, respectively.

Analysis of hypoxia standing in vitro after Bi₂S₃-3BP therapy

Tumor cells after adherence have been incubated with management (untreated), Bi₂S₃ (Bi: 60 µg mL^− 1) or Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) for six h, the tradition vessels have been sealed with a sealant to forestall oxygen alternate. The dissolved oxygen within the medium earlier than and after 6 h of the co-incubation was detected utilizing the dissolved oxygen meter (B949712069, Mettler-Toledo Devices Co., Ltd.). The intracellular hypoxia standing was additional detected with the ROS-ID® Hypoxia/Oxidative stress detection equipment. Sometimes, cells have been seeded in cell-culture dishes at a density of 1 × 10⁵ cells/dish, and cultured in a single day. Then cells have been cultured in serum-free RPMI-1640 medium containing both Bi₂S₃ (Bi: 60 µg mL^− 1) or Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) for an additional 6 h, and the system was sealed or not sealed with sealant. Cells handled with DFO, a chemical hypoxia inducer, have been used as a optimistic management. All dishes have been stained with hypoxia crimson detection reagent for 30 min. After washing with PBS, the dishes have been noticed by CLSM.

In vitro anti-tumor efficacy

To estimate cytotoxicity in vitro, 4T1 cells have been seeded in 96-well plates, and have been cocultured with media containing completely different concentrations of Bi₂S₃ or Bi₂S₃-3BP for six h, and uncovered to X-ray irradiation (A Linear accelerator (VARIAN, 21EX) was used. Cells/mice have been positioned 100 cm from the X-ray supply. Power of radiation was 6 MV.). Cell viability was decided by a CCK-8 technique. Chou-Talalay’s isobolographic technique [34] was calculated for Bi₂S₃-based RT and 3BP on Bi₂S₃-3BP + X-ray utilizing the equation mixture index (CI) = (C_BIS(+3BP)/C_BIS) + (C_3BP(+BIS)/C_3BP) + α[(C_BIS(+3BP))(C_3BP(+BIS))/(C_BIS)(C_3BP)], the place C is the focus of BIS (Bi₂S₃ + X-ray) and 3BP (Bi₂S₃-3BP) alone or together (Bi₂S₃-3BP + X-ray) at a given molar ratio to acquire a given fa. The impact fraction (fa) is the cell loss of life measured by the CCK8 technique. Utilizing the extra conservative reciprocal assumption (α = 0), CI < 1, CI = 1 and CI > 1 point out synergistic, additive and antagonistic results, respectively. For the detection of intracellular ROS era. Tumor cells cultured in dishes have been randomly divided into 5 teams, together with management (untreated), X-ray (solely), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1), Bi₂S₃ (Bi: 60 µg mL^− 1) + X‑ray, and Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) + X‑ray. Intracellular ROS era after completely different remedies have been detected by CLSM after DCFH-DA staining.

Cloning experiments and DNA injury examination have been additional performed underneath anaerobic and cardio situations, respectively. 4T1 cells have been seeded into 6-well plates at a density of 6000 cells per properly and divided into 3 teams together with management (untreated), Bi₂S₃ (Bi: 60 µg mL^− 1), and Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1). All teams acquired X-ray. Subsequent, recent RPMI-1640 medium was used to tradition cells for added 7 days. Lastly, cells have been rinsed with PBS, mounted by 4% paraformaldehyde, and stained with crystal violet about 30 min. Cell colonies have been then counted. As for DNA injury evaluation by γ‑H2AX immunofluorescence staining, 1 h after X‑ray publicity, the cells have been washed with PBS, mounted by 4% formaldehyde, permeabilized with PBS containing 0.5% (v/v) Triton X-100, handled with 5% bovine serum albumin, and incubated with γ‑H2AX (Rabbit monoclonal antibody, 1:250, Abcam) major antibody at 4 °C in a single day. After washing with PBS, cells have been incubated with Rhodamine (TRITC)–Conjugated Goat anti-rabbit IgG secondary antibody (1:100, ZSGB-BIO, Beijing, China) at 37 °C for 1 h, and stained with DAPI for CLSM statement. The optimistic space of DNA injury in each pattern was analyzed by ImageJ software program.

Detection of mobile autophagy

Expression of autophagy-related proteins P62 and LC3 was detected by Western blot assay. Moreover, immunofluorescence staining was used to detect the formation of LC3 dots. Cytotoxicity of autophagy inhibitor/promoter was detected by CCK-8 equipment. The main points have been described within the supplementary supplies. Autophagosomes shaped throughout autophagy have been stained with the MDC staining equipment, 4T1 cells have been seeded in CLSM dishes for 4 teams: Management (untreated), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1), Bi₂S₃ (Bi: 60 µg mL^− 1) + X-ray, and Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) + X-ray, respectively. Then 4T1 cells have been incubated with MDC (Solarbio, G0170) in keeping with the specification for 30 min. After washed by PBS for thrice, the autophagosomes dots have been monitored by CLSM.

Autophagosomes have been noticed extra intuitively by TEM, 4T1 cells have been handled with Management (untreated), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1), Bi₂S₃ (Bi: 60 µg mL^− 1) + X-ray, and Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) + X-ray, respectively. Then 4T1 cells have been mounted with 2.5% glutaraldehyde for greater than 4 h. After washed by PBS for thrice, 4T1 cells have been mounted by osmium tetroxide for two h, dehydrated with graded ethanol and transferred to absolute acetone for 20 min, then embedded in Spurr resin combination to type ultrathin sections. Lastly, the autophagosomes in these cell ultrathin sections have been detected by TEM.

For the detection of cytotoxicity of RT with autophagy inhibitor/promoter, 4T1 cells have been seeded in a 96-well plate and handled as follows: Bi₂S₃ (Bi: 60 µg mL^− 1) + RAPA (3.5 µg mL^− 1), Bi₂S₃ (Bi: 60 µg mL^− 1) + 3-MA (6 µg mL^− 1), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1), Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) + RAPA (3.5 µg mL^− 1), and Bi₂S₃-3BP (Bi: 60 µg mL^− 1, 3BP: 10 µg mL^− 1) + 3-MA (6 µg mL^− 1), respectively. Cells have been incubated with the corresponding nanospheres and drug for six h, after which uncovered to X-ray. After 12 h incubation, CCK-8 equipment have been used to detect the cell viabilities.

Analysis of tumor hypoxia aid and autophagy in vivo

Tumor oxyhemoglobin ranges have been assessed by PA imaging in an oxyhem mode. After post-injection with Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL) and saline, oxyhemoglobin saturation (sO₂ Avr Complete) within the tumor at completely different time factors (0, 2, 4, 6 and 12 h) have been decided. Furthermore, intertumoral hypoxia standing was evaluated by immunofluorescence staining. 4T1 tumor-bearing mice have been assigned into 3 teams (6 mice per group) as follows: (1) Management (200 µL of saline injection), (2) Bi₂S₃ (Bi: 5 mg mL^− 1, 200 µL), and (3) Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL). The mice have been intravenously injected with corresponding nanospheres. After 6 h of the injection, 3 mice in every group have been sacrificed, and the tumor tissues have been harvested for hypoxia-inducible issue 1 (HIF-1α) immunofluorescence staining. The opposite 3 mice in every group have been intravenously injected with pimonidazole hydrochloride (60 mg kg^− 1) (Hypoxyprobe-1 plus equipment, Hypoxyprobe Inc.). After nearly an hour and a half, tumor tissues have been harvested for the detection of Pimonidazole. The fluorescence of HIF-1α and pimonidazole was noticed by CLSM and the typical fluorescence depth was analyzed by ImageJ software program.

In vivo ROS and autophagy ranges of tumor tissue after completely different remedies have been evaluated by immunofluorescence staining. 4T1 tumor-bearing mice have been assigned into 6 teams (6 mice in every group) as follows: (1) Management (200 µL of saline injection), (2) Bi₂S₃ (Bi: 5 mg mL^− 1, 200 µL), (3) Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL), (4) X-ray, (5) Bi₂S₃ (Bi: 5 mg mL^− 1, 200 µL) + X-ray and (6) Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL) + X-ray. The mice have been intravenously injected with corresponding nanospheres. After 6 h, the mice in X-ray, Bi₂S₃ + X-ray and Bi₂S₃-3BP + X-ray teams have been uncovered to X-ray. Then 3 mice in every group have been intravenously injected with DCFH-DA (10 µM, 200 µL). After 60 min, these mice have been sacrificed and tumor tissues have been harvested by frozen tumor slides for detecting ROS. Different 3 mice in every group have been executed and the tumor tissues have been harvested for detecting LC3 (autophagy stage) by immunofluorescence staining.

Anti-tumor efficacy in vivo

The tumor-bearing mice have been randomly divided into six teams (5 mice in every group) as follows: (1) Management (200 µL of saline injection), (2) Bi₂S₃ (Bi: 5 mg mL^− 1, 200 µL), (3) Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL), (4) X-ray, (5) Bi₂S₃ (Bi: 5 mg mL^− 1, 200 µL) + X-ray and (6) Bi₂S₃-3BP (Bi: 5 mg mL^− 1,3BP: 0.8 mg mL^− 1, 200 µL) + X-ray. The mice have been intravenously injected with the corresponding nanospheres on day 0 and day 4, respectively. Mice in X-ray, Bi₂S₃ + X-ray, and Bi₂S₃-3BP + X-ray teams acquired X-ray irradiations at 6 h after the injection. Their physique weight and tumor diameter have been recorded each different day in the course of the 14 days of statement. The tumor volumes have been calculated as [0.5 × length × (width)²]. On day 14, the mice have been sacrificed, and the tumors from every group have been harvested and photographed. Tumor tissues from every group have been mounted in 4% paraformaldehyde for histological evaluation, together with hematoxylin & eosin (H&E), proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick finish labeling (TUNEL) assays. The imply fluorescence depth was analyzed by ImageJ software program.

Statistical evaluation

All information have been expressed as imply ± customary deviation (SD). The GraphPad Prism 8.0 software program was used for statistical evaluation. Two-tailed Pupil’s t-test and one-way ANOVA take a look at have been used to detect statistical variations between two teams and a number of teams, respectively. p-Values are indicated as n.s. = p > 0.05, *p < 0.05; **p < 0.01; ***p < 0.001, respectively.