Skip to main content
Download PDF

3-Bromopyruvate-loaded bismuth sulfide nanospheres improve cancer treatment by synergizing radiotherapy with modulation of tumor metabolism

Journal of Nanobiotechnology volume 21, Article number: 209 (2023) Cite this article

Abstract

Background

Radiotherapy (RT) is one of the most mainstream cancer therapeutic modalities. However, due to the lack of specificity of the radiation adopted, both normal and cancerous cells are destroyed indiscriminately. This highlights the crucial need to improve radiosensitization. This study aims to address this issue by constructing a multifunctional nanospheres that can sensitize multiple aspects of radiotherapy.

Results

Nanospheres containing high atomic element Bi can effectively absorb ionizing radiation and can be used as radiosensitizers. Cell viability after Bi2S3 + X-ray treatment was half that of X-ray treatment alone. On the other hand, exposed 3-bromopyruvate (3BP) could reduce the overactive oxygen (O2) metabolism of tumor cells and alleviate tumor hypoxia, thereby promoting radiation-induced DNA damage. The combination index (CI) of 3BP and Bi2S3-based RT in Bi2S3-3BP + X-ray was determined to be 0.46 with the fraction affected (fa) was 0.5 via Chou-Talalay’s isobolographic method, which indicated synergistic effect of 3BP and Bi2S3-based RT after integration into Bi2S3-3BP + X-ray. Under the combined effect of 3BP and RT, autophagy was over-activated through starvation-induced and redox homeostasis dysregulation pathways, which in turn exhibited pro-death effects. In addition, the prepared nanospheres possess strong X-ray attenuation and high near-infrared (NIR) optical absorption, thus eliminating the need for additional functional components and could serve as bimodal contrast agents for computed tomography/photoacoustic (CT/PA) imaging.

Conclusions

The rational design of multifunctional nanospheres with the unique properties provided a novel strategy to achieving high therapeutic efficacy in RT. This was accomplished through simultaneous activation of multiple sensitization pathways by increasing ionizing radiation, reducing tumor oxygen consumption, inducing pro-death autophagy, and providing multiple-imaging guidance/monitoring.

Introduction

Radiotherapy (RT), particularly external beam RT, is a mainstream cancer therapeutic modalities, with over 50% of cancer patients receiving it at some point during their illness [1, 2]. Generally, ionizing radiation induces cell death via two mechanisms - direct interaction with key cellular components to cause DNA damage or production of cytotoxic reactive oxygen species (ROS) [3, 4]. However, RT has certain limitations, such as unsatisfactory therapeutic efficiency with low X-ray does, and toxicity to the surrounding normal tissues associated with high intensity or multiple frequencies radiation beams [2, 5]. Therefore, there is an urgent need to introduce effective radiosensitization, which could improve curative effect of RT and reduce various side effects. Over the last few decades, nanospheres have emerged as promising tools to enhance the efficacy of RT, and have generated considerable interest. Nanospheres with high atomic number Z (e.g., iodine, gold, bismuth, gadolinium and platinum) have been reported to augment RT against targeted tissues by increasing secondary and oxyelectron production with relative low dose of X ray, thereby reducing the radiation damage to healthy normal tissues [5,6,7]. Bismuth (Bi) based nanomaterials are designed for use in RT due to their excellent X-ray attenuation capabilities [8]. Currently few nanoparticles are available on the market, which may be due to the difficulty of mass production with adaptive characteristics and the high cost of the entire process [6]. However, bismuth-containing composites or drugs have been extensively studied for their lower toxicity risk, good biocompatibility, cost effectiveness and longer blood circulation, making them an attractive candidate for radiosensitization [9]. Notably, porous bismuth sulfide (Bi2S3) nanospheres have attracted much attention for their ability to deliver good drugs [10].

Radioresistance is also associated with hypoxia in tumors, which can impair the efficacy of RT. Available oxygen can stabilize RT-induced DNA damage and produce stable organic peroxides that prevent DNA repair, thereby enhancing the degree of cellular damage [11]. In addition, RT-induced ROS generation capacity is closely related to the oxygen content of tumor tissue [12]. However, the poor vascularization, increased metabolic demand of the tumor cells, and increased interstitial pressure often led to hypoxia within solid tumors, resulting in resistance or failure of RT [13, 14]. Therefore, it is essential to develop effective strategies to alleviate tumor oxygen deprivation to improve the efficacy of RT. Strategies for mitigating the tumor hypoxia mainly include increasing oxygen supply and reducing oxygen consumption. Some studies have supplemented oxygen by using hydrogen peroxide (H2O2)-disintegrating materials or nanomaterials loaded with oxygen [15, 16]. However, nanomaterials used for decomposing H2O2 may suffer from low levels of H2O2 [17] or slow reactions with H2O2 [15]. Oxygen carriers (e.g., perfluorocarbons-based nanomaterials and oxygen-based microbubbles) also process some limitations, such as low oxygen loading [18] and rapid oxygen leakage [19]. These strategies may not prevent the development of tumor hypoxia due to the hyperactive oxygen metabolism prevalent in tumor cells, so it is particularly important to find ways to reduce oxygen consumption and thus alleviate the hypoxic microenvironment at its root [20].

3-bromopyruvate (3BP), an analogue of pyruvate and lactate, is a small molecule alkylating agent that alters tumor metabolism by inhibiting tumor cell glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), which can significantly reduce the rate of intracellular oxygen consumption, effectively relieving tumor hypoxia and enhancing radiosensitization [21, 22].

Moreover, accumulating evidences have indicated that autophagy is activated under conditions of nutritional starvation and oxidative stress through highly regulated pathways associated with energy metabolism [23]. 3BP inhibits respiration, cuts off energy supply, and causes cell starvation, which can induce an increased level of cellular autophagy [24]. Additionally, the generation of ROS caused by ionizing radiation can also induce the occurrence of autophagy [25]. Autophagy is a catabolic process that degrades cytoplasmic proteins or organelles through the formation of autophagic lysosomes to achieve cell homeostasis and organelle renewal [26]. Based on the threshold effect of autophagy regulating cell death/survival, excessive activation of autophagy can promote cell death when it exceeds a certain threshold, and it may be more promising to play a positive role of autophagy in RT [27]. Under the combined effect of 3BP and RT, autophagy is over-activated through starvation-induced and redox homeostasis dysregulation pathways. Meanwhile, inhibition of aerobic glycolysis can directly induce apoptosis by reducing adenosine-triphosphate (ATP) levels in tumor cells [28]. As ATP is essential for supporting DNA replication and maintaining cell proliferation, inhibition of ATP can also enhance the efficacy of RT [28, 29]. However, the small molecular size of 3BP is difficult to retain in tumor tissue due to the enhanced permeability and retention (EPR) effect [30]. The past few decades have witnessed the evolution of nanomedicine from a biologically inert entity to a more intelligent system, aimed at improving in vivo functionality [31]. Therefore, the use of drug delivery vehicles such as mesoporous Bi2S3, to load 3BP is expected to overcome pathophysiological barriers to maximize efficacy.

In this work, we rationally constructed multifunctional nanospheres (Bi2S3-3BP) for multiple RT sensitization by increasing the ionizing radiation effect through high atomic number nanospheres containing Bi, alleviating the tumor hypoxic microenvironment with 3BP, and co-activating cellular pro-death autophagy under nutrient starvation as well as oxidative stress conditions (Scheme 1). These Bi2S3-3BP nanospheres can effectively accumulate at the tumor site through EPR effect. The intertumoral enrichment can be monitored by CT/PA dual-modal imaging to formulate RT plans [32]. After X-ray irradiation, Bi2S3 in nanospheres induces DNA damage by directly absorbing ionizing radiation and generating large amounts of ROS [33]. While 3BP reduces oxygen consumption by inhibiting tumor cell metabolism, the relatively more sufficient oxygen to react with the broken ends of DNA to generate stable organic peroxides, which cannot be easily repaired and greatly enhance the degree of RT-induced cellular damage. Furthermore, 3BP and RT jointly triggered the occurrence of intracellular excessive autophagy, which showed a pro-death effect, further enhancing the efficacy of RT. The efficacy of radiosensitization by Bi2S3-3BP was systematically evaluated both in vitro and in vivo. By sensitizing RT in multiple ways, this work provides new insights to improve the accuracy and sensitivity of RT against tumor.

Scheme 1
scheme 1

Schematic illustration of the synthesized Bi2S3-3BP nanospheres for effective radiosensitization through ionizing radiation enhancement, oxygen consumption reduction, pro-death autophagy activation, and CT/PA dual imaging monitoring/guidance

Full size image

Materials and methods

Materials

All reagents used in this work were 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 were purchased from Aladdin Reagent Co., Ltd. (China). Methoxy polyethylene glycol thiol (mPEG2K-SH, Mw ≈ 2000 Da) was purchased from Xi’an ruixi Biological Technology Co., Ltd (China). Cell Counting Kit-8 (CCK-8), 3-Methyladenine (3MA) and rapamycin (Rapa) were obtained from MedChemExpress (USA). 2-Deoxy-D-glucose (2DG) and rotenone were purchased from ApexBio Technology (USA). Enhanced ATP Assay Kit, Crystal Violet Staining Solution, Reactive Oxygen Species Assay Kit, BCA Protein Assay Kit, 4,6-Diamidino-2-phenylindole (DAPI) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Beyotime Biotechnology (China). Lactic Acid Content Test Kit, Glucokinase Activity Assay Kit, the monodansylcadaverine (MDC) staining kit, Triton X-100, bovine serum albumin and anti-fluorescence quenching agent were obtained from Beijing Solarbio Science & Technology Co., Ltd (China). 2.5% glutaraldehyde was obtained from Macklin (China). Spurr resin mixture was obtained from SPI-CHEM (USA). ROS-ID® Hypoxia/Oxidative stress detection kit was obtained from Enzo Life Sciences (USA). 3BP was purchased from Sigma-Aldrich (USA). Roswell Park Memorial Institute (RPMI)-1640 complete medium was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (China). Distilled water was obtained from a Millipore water system. All chemicals were of analytical grade and used without further purification.

Synthesis of Bi2S3-3BP

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

Next, 100 mg of TAA was dissolved in 30 mL of distilled water, and then 10 mL of Bi2O3 suspension (in distilled water) was added. The mixture was then transferred into a stainless-steel autoclave, maintained at 150 °C for 12 h. The obtained black product was centrifuged, and then thoroughly 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, and then 10 mg mPEG2K-SH was added. The mixture was subjected to rotary evaporation for 1 h. Excess mPEG2K-SH were removed by centrifugation and repeated washing using distilled water. The final Bi2S3 with PEG modification was resuspended in distilled water for further use. Next, 3BP was attached to the pores by magnetic stirring. In detail, 10 mL of Bi2S3 with PEG modification (1 mg/mL) and 30 mg 3BP were nixed and stirred overnight. Bi2S3-3BP was purified by centrifugation and repeated washing using distilled water.

Characterization of Bi2S3-3BP

Transmission electron microscopy (TEM, Hitachi H-7600, Japan) was used to characterize the morphology of the sample. The crystalline form of the products was characterized based on X-ray diffraction (XRD) using an X-ray diffractometer (Panalytical X’ Pert PRO, Shimadzu, Japan) with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was used to characterize the elemental composition of intermediate and final products using Thermo Scientific K-Alpha (Thermo Scientific, USA). Size distribution of the nanospheres was measured using dynamic light scattering (DLS, Malvern Instruments, 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 were measured by using an FT-IR spectrometer (Nicolet 6700, Thermo Scientific, USA). The absorption spectra of Bi2S3 with PEG modification and Bi2S3-3BP (dissolved in deionized water) were determined with a UV-vis spectrophotometer (US-2550, Shimadzu, Japan). Different concentrations of free 3BP were used to obtain a calibration curve using UV-vis spectrometry, which was used to measure unloaded 3BP in the supernatant. Then the encapsulation efficiency and drug loading efficiency of Bi2S3-3BP can be calculated:

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

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

To investigate the drug release behavior of 3BP, Bi2S3-3BP (loaded 3BP: 6.8 mg) was dispersed in PBS (2.0 mL) and then stirred at 37 ° C. At a predetermined time points, the supernatant was extracted, then replaced with an equal volume of fresh PBS, the supernatant was collected, and the corresponding standard calibration curve was used to analyze the 3BP release.

Cell culture and 4T1 tumor-bearing mice model

Murine breast cancer 4T1 cell lines were obtained from Procell Life Science & Technology Co., Ltd (China), and cultured in RPMI-1640 complete culture medium under a humidified atmosphere with 5% CO2 at 37 °C. All animal studies and procedures were conducted under a protocol approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Review Lot No. 2021 − 717). All animal experiments were conducted in accordance with the guidelines of the Chongqing Management Approach of Laboratory Animal. All animals (female BALB/c mice with a weight of ≈ 20 g) were purchased from Hunan Slyke Jingda Laboratory Animal Co., Ltd (China). 4T1 tumor-bearing mice were established by subcutaneous administration of a cell suspension (1 × 106 cells in 100 µL phosphate-buffered solution (PBS) for each mouse). The tumor size was measured using a vernier caliper, and tumor volumes were calculated as [0.5 × length × (width)2].

Cellular uptake of Bi2S3-3BP

The cellular uptake of Bi2S3-3BP was investigated by laser scanning confocal microscopy (CLSM, Nikon Eclipse Ti, Tokyo, Japan) and flow cytometry (FACS, CytoFLEX Flow Cytometer, Becton Coulter, Brea, CA). Briefly, the 4T1 cells were seeded at a density of 1 × 105 cells per CLSM dish and incubated for 24 h. Cells were further cultured in fresh RPMI-1640 medium containing FITC-labeled Bi2S3-3BP (Bi: 60 µg mL− 1) and incubated for 0, 1, 2, 3, 4 h. All cells were fixed with 4% formaldehyde (10 min), washed with PBS, and stained with DAPI (15 min) for CLSM observation. Intracellular uptake of Bi2S3-3BP at different time points was also quantified by flow cytometry.

Detection of glycolysis inhibition after Bi2S3-3BP treatment

Tumor cells cultured in dishes were randomly divided into 6 groups (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 6 h, respectively. Western blot (WB) assay was performed to detect the expression of hexokinase-II (HK-II) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 4T1 cells after different treatments. Total protein was quantified using the BCA protein quantification kit. The detailed experimental procedure of WB is shown in the supplementary material. Tumor cells were divided into 3 groups (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 6 h, respectively. The viability of HK-II, intracellular lactate, and intracellular ATP were measured by the Glucokinase Activity Assay Kit, Lactic Acid Content Test Kit, and the Enhanced ATP Assay Kit according to standard protocols, respectively.

Evaluation of hypoxia status in vitro after Bi2S3-3BP treatment

Tumor cells after adherence were incubated with control (untreated), Bi2S3 (Bi: 60 µg mL− 1) or Bi2S3-3BP (Bi: 60 µg mL− 1, 3BP: 10 µg mL− 1) for 6 h, the culture vessels were sealed with a sealant to prevent oxygen exchange. The dissolved oxygen in the medium before and after 6 h of the co-incubation was detected using the dissolved oxygen meter (B949712069, Mettler-Toledo Instruments Co., Ltd.). The intracellular hypoxia status was further detected with the ROS-ID® Hypoxia/Oxidative stress detection kit. Typically, cells were seeded in cell-culture dishes at a density of 1 × 105 cells/dish, and cultured overnight. Then cells were cultured in serum-free RPMI-1640 medium containing either Bi2S3 (Bi: 60 µg mL− 1) or Bi2S3-3BP (Bi: 60 µg mL− 1, 3BP: 10 µg mL− 1) for another 6 h, and the system was sealed or not sealed with sealant. Cells treated with DFO, a chemical hypoxia inducer, were used as a positive control. All dishes were stained with hypoxia red detection reagent for 30 min. After washing with PBS, the dishes were observed by CLSM.

In vitro anti-tumor efficacy

To estimate cytotoxicity in vitro, 4T1 cells were seeded in 96-well plates, and were cocultured with media containing different concentrations of Bi2S3 or Bi2S3-3BP for 6 h, and exposed to X-ray irradiation (A Linear accelerator (VARIAN, 21EX) was used. Cells/mice were placed 100 cm from the X-ray source. Energy of radiation was 6 MV.). Cell viability was determined by a CCK-8 method. Chou-Talalay’s isobolographic method [34] was calculated for Bi2S3-based RT and 3BP on Bi2S3-3BP + X-ray using the equation combination index (CI) = (CBIS(+3BP)/CBIS) + (C3BP(+BIS)/C3BP) + α[(CBIS(+3BP))(C3BP(+BIS))/(CBIS)(C3BP)], where C is the concentration of BIS (Bi2S3 + X-ray) and 3BP (Bi2S3-3BP) alone or in combination (Bi2S3-3BP + X-ray) at a given molar ratio to obtain a given fa. The effect fraction (fa) is the cell death measured by the CCK8 method. Using the more conservative reciprocal assumption (α = 0), CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic effects, respectively. For the detection of intracellular ROS generation. Tumor cells cultured in dishes were randomly divided into 5 groups, including control (untreated), X-ray (only), 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 generation after different treatments were detected by CLSM after DCFH-DA staining.

Cloning experiments and DNA damage examination were further conducted under anaerobic and aerobic conditions, respectively. 4T1 cells were seeded into 6-well plates at a density of 6000 cells per well and divided into 3 groups including control (untreated), Bi2S3 (Bi: 60 µg mL− 1), and Bi2S3-3BP (Bi: 60 µg mL− 1, 3BP: 10 µg mL− 1). All groups received X-ray. Next, fresh RPMI-1640 medium was used to culture cells for additional 7 days. Finally, cells were rinsed with PBS, fixed by 4% paraformaldehyde, and stained with crystal violet about 30 min. Cell colonies were then counted. As for DNA damage assessment by γ‑H2AX immunofluorescence staining, 1 h after X‑ray exposure, the cells were washed with PBS, fixed by 4% formaldehyde, permeabilized with PBS containing 0.5% (v/v) Triton X-100, treated with 5% bovine serum albumin, and incubated with γ‑H2AX (Rabbit monoclonal antibody, 1:250, Abcam) primary antibody at 4 °C overnight. After washing with PBS, cells were 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 observation. The positive area of DNA damage in every sample was analyzed by ImageJ software.

Detection of cellular autophagy

Expression of autophagy-related proteins P62 and LC3 was detected by Western blot assay. Besides, immunofluorescence staining was used to detect the formation of LC3 dots. Cytotoxicity of autophagy inhibitor/promoter was detected by CCK-8 kit. The details were described in the supplementary materials. Autophagosomes formed during autophagy were stained with the MDC staining kit, 4T1 cells were seeded in CLSM dishes for 4 groups: Control (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 were incubated with MDC (Solarbio, G0170) according to the specification for 30 min. After washed by PBS for three times, the autophagosomes dots were monitored by CLSM.

Autophagosomes were observed more intuitively by TEM, 4T1 cells were treated with Control (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 were fixed with 2.5% glutaraldehyde for more than 4 h. After washed by PBS for three times, 4T1 cells were fixed by osmium tetroxide for 2 h, dehydrated with graded ethanol and transferred to absolute acetone for 20 min, then embedded in Spurr resin mixture to form ultrathin sections. Finally, the autophagosomes in these cell ultrathin sections were detected by TEM.

For the detection of cytotoxicity of RT with autophagy inhibitor/promoter, 4T1 cells were seeded in a 96-well plate and treated 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 were incubated with the corresponding nanospheres and drug for 6 h, and then exposed to X-ray. After 12 h incubation, CCK-8 kit were used to detect the cell viabilities.

Evaluation of tumor hypoxia relief and autophagy in vivo

Tumor oxyhemoglobin levels were 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 Total) in the tumor at different time points (0, 2, 4, 6 and 12 h) were determined. Moreover, intertumoral hypoxia status was evaluated by immunofluorescence staining. 4T1 tumor-bearing mice were assigned into 3 groups (6 mice per group) as follows: (1) Control (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 were intravenously injected with corresponding nanospheres. After 6 h of the injection, 3 mice in each group were sacrificed, and the tumor tissues were harvested for hypoxia-inducible factor 1 (HIF-1α) immunofluorescence staining. The other 3 mice in each group were intravenously injected with pimonidazole hydrochloride (60 mg kg− 1) (Hypoxyprobe-1 plus kit, Hypoxyprobe Inc.). After almost an hour and a half, tumor tissues were harvested for the detection of Pimonidazole. The fluorescence of HIF-1α and pimonidazole was observed by CLSM and the average fluorescence intensity was analyzed by ImageJ software.

In vivo ROS and autophagy levels of tumor tissue after different treatments were evaluated by immunofluorescence staining. 4T1 tumor-bearing mice were assigned into 6 groups (6 mice in each group) as follows: (1) Control (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 were intravenously injected with corresponding nanospheres. After 6 h, the mice in X-ray, Bi2S3 + X-ray and Bi2S3-3BP + X-ray groups were exposed to X-ray. Then 3 mice in each group were intravenously injected with DCFH-DA (10 µM, 200 µL). After 60 min, these mice were sacrificed and tumor tissues were harvested by frozen tumor slides for detecting ROS. Other 3 mice in each group were executed and the tumor tissues were harvested for detecting LC3 (autophagy level) by immunofluorescence staining.

Anti-tumor efficacy in vivo

The tumor-bearing mice were randomly divided into six groups (5 mice in each group) as follows: (1) Control (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 were 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 groups received X-ray irradiations at 6 h after the injection. Their body weight and tumor diameter were recorded every other day during the 14 days of observation. The tumor volumes were calculated as [0.5 × length × (width)2]. On day 14, the mice were sacrificed, and the tumors from each group were harvested and photographed. Tumor tissues from each group were fixed in 4% paraformaldehyde for histological analysis, including hematoxylin & eosin (H&E), proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. The mean fluorescence intensity was analyzed by ImageJ software.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). The GraphPad Prism 8.0 software was used for statistical analysis. Two-tailed Student’s t-test and one-way ANOVA test were used to detect statistical differences between two groups and multiple groups, respectively. p-Values are indicated as n.s. = p > 0.05, *p < 0.05; **p < 0.01; ***p < 0.001, respectively.

Result and discussion

Synthesis and characterization of Bi2S3-3BP

To fabricate 3-BP loaded nanospheres, we first synthesized Bi2S3 nanospheres by a facile and effective hydrothermal method, using Bi2O3 as the bismuth precursor. To improve the dispersion and optimize the colloidal stability of Bi2S3 nanospheres, we stably attached mPEG2K-SH to the surface of Bi2S3 nanospheres by rotary evaporation. Next, 3BP was loaded into the pore by magnetic stirring to obtain Bi2S3-3BP (Scheme 1). As shown in Figure S1-S2, the Bi2O3 nanoparticles, bismuth precursor, revealed a spherical morphology. The obtained Bi2S3 nanospheres showed a distinct star-shaped structure (Fig. 1a and b). The elemental composition of Bi2S3 nanospheres was analyzed by XPS. The full spectrum scanning results showed that Bi2S3 nanospheres primarily contained Bi, S, O and C elements (Fig. 1c). Bi 4f7 and S 2p3 correspond to Bi3+ (158.2 eV) and S2− (160.9 eV), respectively, providing direct evidence for the successful synthesis of Bi2S3 (Fig. 1d). XRD further showed that the representative diffraction peaks of Bi2S3 nanospheres aligned well with the standard pattern of Bi2S3 (JCPDS Card No. 17–0320), indicating the successful preparation of Bi2S2 crystals (Fig. 1e). The porosity of Bi2S3 was studied by Brunauer-Emmet-Teller (BET) analysis of nitrogen adsorption-desorption isotherm. Bi2S3 exhibit type-IV isotherms, whose hysteresis curves correspond to mesoporous/microporous structures (Fig. 1f). The BET specific surface area of Bi2S3 was ~ 30.31 m2g− 1. In addition, the pore size distribution shows that Bi2S3 is dominated by mesoporous pores of 2 to 4 nm (Figure S3). FT-IR was used to confirm the successful surface modification of PEG (Fig. 1g). Comparing the spectra of the three materials, it was found that the characteristic peaks of Bi2S3 with PEG modification samples were from both those of mPEG2K-SH and Bi2S3, indicating the successful conjugation of mPEG2K-SH onto Bi2S3. The obtained Bi2S3 with PEG modification exhibited good stability and dispersion in various physiological conditions (Figure S4-S5). UV-Vis spectra (Fig. 1h) confirmed the successful loading of 3BP, the Bi2S3-3BP showed a broad absorption peak at ~ 212 nm compared with bare Bi2S3, which was corresponding to 3BP. The wavelength at 212 nm is too close to the initial wavelength of UV–vis spectrum measurement of 200 nm, the absorbance peak here showed red-shifted, therefore a calibration curve was obtained at 282 nm (Figure S6). According to the UV-Vis absorbance results, the EE of 3BP in Bi2S3-3BP were calculated to be 22.9 ± 1.6% and and DLE for 3BP was determined as 17.2 ± 1.2%, respectively. To examine drug release properties, Bi2S3-3BP solutions were investigated. The results showed that 58.1% of 3BP was released after 18 h (Figure S7). The average size of Bi2S3-3BP was about 260 nm as measured by DLS (Fig. 1i).

Fig. 1
figure 1

Synthesis and characterization of Bi2S3-3BP. (a) SEM image of Bi2S3. (b) TEM image of Bi2S3. (c) XPS survey spectrum of Bi2S3. (d) XPS spectra of Bi 4f, and S 2p orbits of Bi2S3. (e) XRD patterns of Bi2S3. (f) N2 adsorption-desorption isotherm of Bi2S3 nanospheres. (g) FT-IR spectra of mPEG2K-SH, Bi2S3 with PEG modification, and Bi2S3. (h) Absorbance spectra of free 3BP, Bi2S3 with PEG modification, and Bi2S3-3BP as recorded by a UV–vis spectrophotometer. (i) Size distribution by intensity of Bi2S3 and Bi2S3-3BP as measured by DLS.

Full size image

Cellular uptake of Bi2S3-3BP and inhibition of glycolysis

Efficient cellular uptake is critical for the subsequent therapeutic approaches. The cellular uptake of Bi2S3-3BP nanospheres was investigated by CLSM and flow cytometry analysis. In a prolonged observation, increasing green fluorescence intensity was observed in 4T1 cells after 4 h of co-culture (Fig. 2a), which can be assigned to Bi2S3-3BP (labeled with FITC). The effective uptake is also substantiated by flow cytometry results (Fig. 2b). These results indicate a potentially efficient accumulation of Bi2S3-3BP in tumor cells.

Fig. 2
figure 2

Cellular uptake of Bi2S3-3BP and inhibition of glycolysis. (a-b) Intracellular uptake of Bi2S3-3BP (labeled with FITC) observed by CLSM and quantified by flow cytometry analysis. (c) HK-II and GAPDH expression measured by western blot. (d-e) Quantitative analysis of relative HK-II and GAPDH expression (n = 3). (f) Intracellular lactate levels (n = 3). (g) Intracellular ATP levels (n = 3). (h) Schematic illustration about measuring the O2 consumption. (i) Relative dissolved oxygen (D.O.) changes in the cell media of different groups (n = 3). (j-k) Cells stained with ROS-ID in different groups observed by CLSM and quantified by flow cytometry. n.s.: no significance, *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

Tumor hypoxia severely hampers the therapeutic efficacy of RT. Regulating the metabolism of tumor cells is an emerging approach to alleviate hypoxia [35] In this study, we introduced 3BP to regulate cellular respiratory metabolism. Considering that HK-II and GAPDH are key enzymes involved in glycolysis and OXPHOS [22], a western blot analysis was performed to detect the expression of these two enzymes in 4T1 cells after various treatments. A typical glycolysis inhibitor (2DG) and oxidative phosphorylation inhibitor (Rotenone) were used as controls. Bi2S3 without 3BP loading had no significant effect on the expression levels of HK-II and GAPDH. When 4T1 cells were incubated with Bi2S3-3BP and free 3BP, the expression levels of HK-II decreased by 23.6% and 36.3% (Fig. 2c-d), demonstrating the function of 3BP. Meanwhile, HK-II viability was also significantly reduced by 33.5% after Bi2S3-3BP treatment (Figure S8). The expression levels of GAPDH also decreased by 23.7% and 33.5%, correspondingly (Fig. 2c and e). Therefore, it can be seen that 3BP played a key role in the inhibition of HK-II and GAPDH, and the inhibitory capacity is comparable to that of 2DG, a typical glycolysis inhibitor. When the glycolytic pathway was inhibited, lactate, the end product of glycolysis, decreased. As expected, the intracellular lactate concentration decreased from 7.7 µM to 1.2 µM after Bi2S3-3BP treatment (Fig. 2f). In contrast, Bi2S3 without 3BP did not affect intracellular lactate production. Aerobic glycolysis, also known as the Warburg effect, has been widely documented as the main energy source in most cancer cells [36]. In this study, 3BP disrupted the energy supply by inhibiting glycolysis, which can affect a series of biological events, as ATP is required for numerous biological processes, such as DNA replication and transcription [37]. ATP levels of 4T1 cells after different treatments were measured quantitatively. Bi2S3 barely affected the intracellular ATP level. When 4T1 cells were incubated with Bi2S3-3BP, the intracellular ATP level decreased to 50.1% (Fig. 2g), promoting tumor inhibition.

In addition to inhibiting glycolysis, 3BP also inhibits mitochondrial OXPHOS, resulting in a significant reduction in intracellular oxygen consumption. Therefore, oxygen consumption in the cell culture media was monitored by measuring changes in dissolved oxygen (D.O.) (Fig. 2h-i). After 6 h treatment with PBS or Bi2S3, compared with the untreated group, the D.O. decreased sharply to 53.5% and 54.7%, respectively. The D.O. of Bi2S3-3BP treatment group dropped much less, which was only 26.9%. (Fig. 2j). These results indicated that Bi2S3-3BP could effectively reduce oxygen consumption in cancer cells and potentially counteract hypoxia. To verify this, intracellular hypoxia level was monitored with a hypoxia detection kit. Under the hypoxia condition, both Bi2S3-treated cells and untreated cells showed intense red fluorescence similar to that of DFO-treated cells (positive control) (Fig. 2i), whereas the red fluorescence intensity of Bi2S3-3BP-treated cells was significantly reduced. These results are consistent with the flow cytometry analysis (Fig. 2k) and fluorescence quantification (Figure S9) obtained from ImageJ. These results confirmed that 3BP-containing nanospheres could effectively reduce the consumption of oxygen and alleviate intracellular hypoxia, which would improve the efficacy of RT.

Synergistic effect of Bi2S3-3BP on in vitro RT

The efficacy of RT was positively correlated with oxygen levels, which can prevent repair of DNA damage and thus achieve radiosensitization. After verifying that Bi2S3-3BP can effectively alleviate hypoxia in vitro, we next evaluated the effect of Bi2S3-3BP on radiosensitization at the cellular level. In the absence of X-ray irradiation, Bi2S3 did not exhibit any cytotoxicity even when the concentration reached 80 µg mL− 1. X-ray alone exhibited only slight ionizing radiation toxicity, while Bi2S3 + X-ray showed a gradual increase in toxicity with elevated concentrations of Bi2S3, which should be ascribed to the intrinsically enhanced ionizing radiation of Bi. Meanwhile, Bi2S3-3BP exhibited significant cytotoxicity in the absence of radiation, with a cell viability of 42.4%, which could be attributed to the cell growth inhibition of 3BP. In contrast, Bi2S3-3BP + X-ray could further reduce cell activity, with a cell viability of only 11.2% (Fig. 3a). And the therapeutic effect of the combined treatment (Bi2S3-3BP + X-ray) presented much stronger cell growth inhibition than that of Bi2S3-based RT (Bi2S3 + X-ray), which demonstrated the crucial role of 3BP on RT. To prove the synergistic effect (not only additive effect) of 3BP and Bi2S3-based RT, the CI was calculated. When the influence score (fa) = 0.5, the CI of 3BP and Bi2S3-based RT was 0.46, indicating that 3BP and Bi2S3-based RT had a significant synergistic effect after integrating Bi2S3-3BP + X-ray.

Fig. 3
figure 3

In vitro RT effect of Bi2S3-3BP. (a) Cell viability after different treatments (n = 5). (b) Intracellular ROS level observed by CLSM after different treatments. (c) DNA damage staining after different treatments. Red: γ-H2AX signal (dsDNA damage staining); blue: DAPI (nuclear staining). (d) Statistics of DNA damage staining in (c) (n = 3). (e-f) Images of 4T1 cell clones after different treatments and the corresponding quantification (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

After the reduction of oxygen consumption by 3BP, more intracellular oxygen is available for RT. Intracellular ROS generation during radiotherapy was investigated using DCFH-DA as a fluorescent ROS probe. Cells treated with Bi2S3-3BP + X-ray showed a distinct green fluorescence after X-ray irradiation (Fig. 3b). In contrast, only slight green fluorescence was observed in cells treated with Bi2S3 + X-ray, indicating the contribution of 3BP for ROS generation and providing a solid basis for achieving RT enhancement under hypoxic conditions. The γ-H2AX focus in the nucleus is commonly used as a quantitative marker for DNA double-strand breaks. To demonstrate the level of DNA damage, cells after various treatments were stained with γ-H2AX for CLSM observation. A significant increase in γ-H2AX focus was observed after Bi2S3-3BP + X-ray pretreatment under both normoxic and hypoxia conditions (Fig. 3c-d). Furthermore, the proliferation of tumor cells under X-ray irradiation was assessed by cell colony formation assay (CFA). Tumor cells were incubated with different drugs for 4 h under normoxic and hypoxia conditions, then irradiated with X-ray. The formed cell colonies were counted after 7 days. Under normoxic conditions, Bi2S3 + X-ray group had significantly fewer colonies (284 clones) than the X-ray-only group (836 clones), indicating that Bi2S3 can effectively sensitize RT. In contrast, the number of colonies in the Bi2S3 + X-ray group (436 clones) was higher under hypoxia than in the normoxic group, suggesting that hypoxia affects the efficacy of RT. The number of cell colonies (normoxic: 159 clones, hypoxia: 106 clones) was the lowest in the Bi2S3-3BP + X-ray treated group under both normoxic and hypoxia conditions (Fig. 3e-f). The data consistently demonstrated the important role of these 3BP-containing nanospheres on RT.

Pro-death atophagy induced by Bi2S3-3BP

Indeed, autophagy can be activated under conditions of nutrient starvation as well as oxidative stress through highly regulated pathways associated with energy metabolism [38, 39]. Dysregulation of redox homeostasis, resulting from excessive production of ROS induced by the combined effect of 3BP and RT, can induced the onset of pro-death autophagy, and further effectively improve the therapeutic outcome [40, 41]. In order to obtain insight into the role of autophagy during RT, we investigated cellular autophagy levels following different treatments. The expression level of autophagy-related proteins (LC3 and P62) were evaluated by western blot assay. During autophagy, LC3-I is converted into LC3-II, while the autophagic substrate P62 is degraded. The LC3-II/LC3-I ratio in the combined treatment group (Bi2S3-3BP + X-ray group) was higher than that in the Bi2S3-3BP group and Bi2S3 + X-ray group, while the expression of P62 was reduced to 13.8% (Fig. 4a - c). Furthermore, the level of autophagy was investigated by visualizing and tracking LC3 puncta using immunofluorescence imaging (Fig. 4d). Compared with Bi2S3-3BP group and Bi2S3 + X-ray group, the red fluorescence was notably enhanced in Bi2S3-3BP + X-ray group. Acidic vesicular autophagosomes generated during autophagy could be investigated by MDC staining (Fig. 4f). The Bi2S3-3BP + X-ray group resulted in much stronger green fluorescence of acidic vesicular autophagosomes than that in Bi2S3-3BP group and Bi2S3 + X-ray group. These results indicated that the combination of Bi2S3-3BP and RT significantly increased the level of intracellular autophagy. The accumulation of autophagosomes was directly observed by TEM (Fig. 4e), while there were fewer autophagosomes in the control group. In contrast, multiple autophagosomes were clearly observed in the Bi2S3-3BP + X-ray group, suggesting the evident presence of autophagy.

Fig. 4
figure 4

Pro-death autophagy of Bi2S3-3BP. (a) LC3-II, LC3-I, and P62 expression after different treatments as measured by western blot. (b-c) Quantitative analysis of relative LC3-II/LC3-1 and P62 expression. (d) Representative immunofluorescence images of LC3 punctate dots and MDC in different groups. (e) TEM images showing the formation of autophagosomes after different treatments. Zoomed-in TEM images in typical structures of autophagosomes are indicated with arrows. (f-g) Cell viability of 4T1 cells after being treated with Bi2S3 + X-ray or Bi2S3-3BP + X-ray in the presence of RAPA or 3-MA (n = 5). n.s.: no significance, *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

To investigate whether over-activated autophagy plays a pro-death or pro-survival role during RT, the cytotoxicity of different treatments was examined after the addition of autophagy initiators (Rapa) and autophagy inhibitors (3-MA). Rapa or 3-MA alone showed almost no cytotoxicity (Figure S10). The CCK-8 assay results showed that cytotoxicity was slightly enhanced in the Bi2S3 + X-ray group after Rapa promoted autophagy (Fig. 4g). In contrast, after 3-MA inhibited autophagy, the cytotoxicity of Bi2S3 + X-ray was not significantly affected. However, when Bi2S3-3BP was combined with RT, 3-MA inhibition of autophagy enhanced the survival of 4T1 cells, while the cytotoxicity of Bi2S3-3BP + X-ray was significantly enhanced after Rapa promoted-autophagy (Fig. 4f). Taken together, the integration of 3BP into RT can promote the occurrence of pro-death autophagy in cells, which holds great promise in improving the therapeutic efficacy.

PA and CT imaging

Effective accumulation of Bi2S3-3BP at the tumor site is essential for subsequent in vivo radiation therapy. Therefore, monitoring the biodistribution of Bi2S3-3BP by real-time imaging is necessary. Considering the excellent NIR optical absorption properties of Bi2S3-3BP, it could act as a PA contrast agent [42]. For PA imaging assessments, 715 nm was selected as the optimal wavelength for Bi2S3-3BP (Figure S11). The PA signal strength of Bi2S3-3BP increased linearly from 0.301 to 1.147 with increasing Bi2S3-3BP concentration (Figure S12). Further in vivo PA imaging was performed to detect the accumulation of Bi2S3-3BP in the tumor tissue. A gradual increase in PA signal was observed in the tumor region after intravenously administration, and peaked at 6 h after injection (Figure S13). The quantitative analysis result is also corresponding well with PA imaging (Figure S14).

Given the excellent X-ray attenuation properties and high biocompatibility of elemental bismuth [43], Bi2S3-3BP is also expected to be an ideal contrast agent for CT imaging. Therefore, we systematically investigated the in vitro and in vivo CT contrast imaging of Bi2S3-3BP. Similar to the PA results, the CT enhancement showed a good linear correlation with Bi2S3-3BP concentration (Figure S15). After intravenously administration of Bi2S3-3BP, a significant bright effect was observed in the tumor area (Figure S16), indicating the effective accumulation of Bi2S3-3BP in the tumor site.

In vivo hypoxia relief, ROS generation and autophagy

In vitro assessments demonstrated that Bi2S3-3BP could effectively alleviate cellular hypoxia, and we expect to exhibit a similar effect in vivo. The ongoing changes of tumor hypoxia levels were monitored using oxyhemoglobin signals captured by the PA imaging system. As shown in Fig. 5a, the oxyhemoglobin signal gradually increased and peaked at 6 h after intravenously administration of Bi2S3-3BP. The result also showed a significantly higher signal than that of the control group. Quantitative analysis (Fig. 5b) showed that the oxyhemoglobin signal intensity of Bi2S3-3BP group increased from 0.3% to 11.8% at 6 h post-injection, while the control group (treated with saline) showed almost no change. HIF-1α immunofluorescence staining was also employed to confirm the ability of Bi2S3-3BP to relieve tumor hypoxia in vivo. Extensive red fluorescence was observed in both the saline and Bi2S3 groups, suggesting the intratumoral hypoxia. In contrast, the tumor area exhibited only a faint red fluorescence after intravenous injection of Bi2S3-3BP (Fig. 5c). Semi-quantitative analysis showed that the fluorescence intensity of tumor tissues after Bi2S3-3BP injection was reduced by 21% as compared to the control group (Fig. 5e). Furthermore, pimonidazole was used as the hypoxic probe to detect the hypoxia status, where hypoxic areas tend to exert green fluorescence. Corresponding to HIF-1α fluorescence, the group treated with Bi2S3-3BP exhibited much weaker green fluorescence than the groups treated with saline and Bi2S3 (Fig. 5d and f). These results well verified that 3BP could effectively relieve tumor inner hypoxia status, which is favorable for enhanced RT.

Fig. 5
figure 5

In vivo hypoxia relief, ROS generation and autophagy. (a) PA images of tumor sites in oxy-hemoglobin mode at different time points. (b) Quantification of oxyhemoglobin saturation at tumor sites (n = 3). (c-d) Immunochemical staining of HIF-1α and pimonidazole on tumor sections from 4T1 tumor-bearing mice after various treatments. (e-f) Mean fluorescence intensity semiquantitative analysis of HIF-1α and pimonidazole (n = 5). (g-j) Immunochemical staining of ROS and LC3 on tumor sections from 4T1 tumor-bearing mice, and the corresponding semiquantitative analysis (n = 5). n.s.: no significance, *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

Next, ROS in vivo generation was measured. Mice bearing 4T1 tumors were randomly divided into six groups, including the control group (treated with saline), Bi2S3 only group, Bi2S3-3BP only group, X-ray only group, Bi2S3 + X-ray group, and Bi2S3-3BP + X-ray group. Much stronger green fluorescence of ROS was observed in Bi2S3-3BP + X-ray group as compared with other groups (Fig. 5g). Semi-quantitative analysis (Fig. 5h) showed that the fluorescence intensity of ROS in the Bi2S3-3BP + X-ray group was twice higher than that of Bi2S3 + X-ray group. This result indicated that Bi2S3-3BP + X-ray group could effectively alleviate tumor hypoxia and stimulate a substantial increase in ROS production, thereby amplifying the therapeutic efficacy of RT.

We further used immunofluorescence imaging of LC3 in tumor sections to assess the level of autophagy following various treatments in vivo. Notably, the Bi2S3-3BP + X-ray group showed significantly higher red fluorescence levels than other treatments (Fig. 5i). Semi-quantitative analysis (Fig. 5j) showed that the fluorescence intensity of LC3 in the Bi2S3-3BP + X-ray group was 3.6 times higher than that of Bi2S3 + X-ray group. This finding suggested that the Bi2S3-3BP + X-ray group induced a higher level of autophagy. Overall, these results indicate that 3BP can alleviate hypoxia and enhance the autophagy occurrence in tumor cells during RT, thus maximizing the therapeutic effect.

Fig. 6
figure 6

Anti-tumor efficacy in vivo. (a) Schematic illustration of the Bi2S3-3BP-based RT process. (b) Representative photographs of 4T1 tumor-bearing mice of six groups during the 14 d period. (c) Photographs of tumors dissected from mice of six groups after various treatments. (d) Tumor growth curves of six groups after various treatments. (n = 5). (e) Body-weight curves of six groups during the observation period. (n = 5). (f) Weight of tumors at 14 d post various treatments and tumor growth inhibition rate (n = 5). (g) H&E, TUNEL and PCNA staining of tumors from 4T1 tumor-bearing mice after various treatments. (h) Mean fluorescence intensity semiquantitative analysis of PCNA and TUNEL (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

Anti-tumor efficacy in vivo

Encouraged by the in vitro therapeutic efficacy of Bi2S3-3BP-mediaed RT, as well as the in vivo hypoxia relief, ROS generation, and autophagy results, the anti-tumor efficacy in vivo was investigated next. Tumor-bearing mice were randomly divided into six groups, including the control group (treated with saline), Bi2S3 only group, Bi2S3-3BP only group, X-ray only group, Bi2S3 + X-ray group, and Bi2S3-3BP + X-ray group. Nanospheres were intravenously injection into mice on days 0 and 4, with X-ray irradiation administrated to the tumor site at 6 h after the injection (Fig. 6a). Tumor volumes were monitored after different treatments (Fig. 6b and c), and normalized by relative tumor volume (V/V0) (Fig. 6d).

It was found that the Bi2S3 group did not show significant tumor growth inhibition (Fig. 6f). Conventional RT without any sensibilization showed a slight tumor suppressive effect with a tumor growth inhibition rate of 36%. However, when Bi2S3 as an ionizing radiation sensitizer was introduced, the therapeutic efficacy of RT in the Bi2S3 + X-ray group was significantly enhanced, achieving a tumor growth inhibition rate of 57%. In contrast, tumors treated with Bi2S3-3BP + X-ray group were significantly reduced, with a tumor growth inhibition rate reaching 79.3%. This could be ascribed to ionizing radiation enhancement, oxygen consumption reduction, and pro-death autophagy activation, which sensitized the cells to RT. Consistently, in vitro tumor visualization (Fig. 6c) and weight (Fig. 6f) followed a similar trend with no significant body weight loss during the 14-day observation period (Fig. 6e).

In addition, H&E, TUNEL, and PCNA staining were performed to test the therapeutic efficacy at a pathological level (Fig. 6g). HE staining of tumor sections showed that the Bi2S3-3BP + X-ray group had significantly more deformed cell nuclei (nuclear consolidation, nuclear fragmentation, and nuclear lysis) than tumor sections from other groups, indicating severe necrosis of cancer cells. The level of apoptosis observed with TUNEL (green fluorescence) was consistent with that of H&E staining. TUNEL staining showed that Bi2S3-3BP + X-ray group induced more apoptotic cells than the Bi2S3 + X-ray and X-ray only groups. In contrast, the trend was reversed when examining PCNA staining (red fluorescence), which reflects the level of cell proliferation.

Finally, semi-quantitative analysis of apoptotic and proliferative regions (Fig. 6h) showed that the fluorescence intensity of PCNA in the Bi2S3 + X-ray group was 3-fold higher than that of Bi2S3-3BP+ X-ray group, while the fluorescence intensity of TUNEL for the Bi2S3 + X-ray was only one-third of Bi2S3-3BP + X-ray group. These results suggest that enhanced RT efficiency was achieved in 4T1 breast cancer tumors when incorporating Bi2S3-3BP nanospheres, as compared with the conventional RT without any sensitization method.

Conclusion

In this study, we successfully designed Bi2S3-3BP nanospheres to effectively sensitize the impact of RT by enhancing ionizing radiation, reducing oxygen consumption and activating pro-death autophagy in tumor cells. These nanospheres are enriched in tumor tissues through the EPR effect, as monitored by CT/PA imaging. The Bi element acted as a radiation therapy sensitizer, effectively absorbing ionizing radiation during RT treatment. Meanwhile, 3BP effectively alleviated the hypoxia status of tumor tissues by inhibiting the tumor glycolytic metabolic pathway and mitochondrial OXPHOS, further aggravating the DNA damage during RT. Additionally, the ROS generation induced by the combined effect of RT and 3BP increased the ROS level. The cellular autophagy induced by the overproduction of intracellular ROS and the starvation effect of 3BP exhibited a pro-death effect, further optimized the final outcome of RT. We compared Bi2S3 + X-ray with Bi2S3-3BP + X-ray under the same experimental conditions, and the cell survival rate of Bi2S3-3BP + X-ray was only a quarter of Bi2S3 + X-ray. Although in vitro effects of different metal nanoparticles in radiation therapy have been observed in previous studies, few studies have combined metal nanomaterials with autophagy. Our study achieved desirable anti-tumor effects at both in vitro and in vivo. It may provide a new idea for the study of nanoradiosensitizers and offer a strategy for a precision radiation oncology approach for clinical tumor radiosensitization.

Data Availability

All data used to obtain the present results are available within the paper and the Supporting Information.

References

  1. Meng L, Cheng Y, Tong X, Gan S, Ding Y, Zhang Y, Wang C, Xu L, Zhu Y, Wu J, et al. Tumor Oxygenation and Hypoxia Inducible Factor-1 functional inhibition via a reactive oxygen species responsive nanoplatform for enhancing Radiation Therapy and Abscopal Effects. ACS Nano. 2018;12:8308–22.

    Article  CAS  PubMed  Google Scholar 

  2. Wang K, Tepper JE. Radiation therapy-associated toxicity: etiology, management, and prevention. CA Cancer J Clin. 2021;71:437–54.

    Article  PubMed  Google Scholar 

  3. Srinivas US, Tan BWQ, Vellayappan BA, Jeyasekharan AD. ROS and the DNA damage response in cancer. Redox Biol. 2019;25:101084.

    Article  CAS  PubMed  Google Scholar 

  4. Xiong Y, Xiao C, Li Z, Yang X. Engineering nanomedicine for glutathione depletion-augmented cancer therapy. Chem Soc Rev. 2021;50:6013–41.

    Article  CAS  PubMed  Google Scholar 

  5. Yu H, Yang Y, Jiang T, Zhang X, Zhao Y, Pang G, Feng Y, Zhang S, Wang F, Wang Y, et al. Effective Radiotherapy in Tumor assisted by Ganoderma lucidum Polysaccharide-Conjugated Bismuth Sulfide Nanoparticles through Radiosensitization and dendritic cell activation. ACS Appl Mater Interfaces. 2019;11:27536–47.

    Article  CAS  PubMed  Google Scholar 

  6. Retif P, Pinel S, Toussaint M, Frochot C, Chouikrat R, Bastogne T, Barberi-Heyob M. Nanoparticles for Radiation Therapy Enhancement: the key parameters. Theranostics. 2015;5:1030–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Xie J, Gong L, Zhu S, Yong Y, Gu Z, Zhao Y. Emerging strategies of nanomaterial-mediated Tumor Radiosensitization. Adv Mater. 2019;31:e1802244.

    Article  PubMed  Google Scholar 

  8. Huang H, He L, Zhou W, Qu G, Wang J, Yang N, et al. Stable black phosphorus/Bi(2)O(3) heterostructures for synergistic cancer radiotherapy. Biomaterials. 2018;171:12–22.

    Article  CAS  PubMed  Google Scholar 

  9. Sun L, Hou M, Zhang L, Qian D, Yang Q, Xu Z, Kang Y, Xue P. PEGylated mesoporous Bi2S3 nanostars loaded with chlorin e6 and doxorubicin for fluorescence/CT imaging-guided multimodal therapy of cancer. Nanomedicine. 2019;17:1–12.

    Article  PubMed  Google Scholar 

  10. Liu Z, Wang P, Xie F, Chen J, Cai M, Li Y, et al. Virus-inspired Hollow Mesoporous Gadolinium-Bismuth Nanotheranostics for magnetic resonance imaging-guided synergistic photodynamic-radiotherapy. Adv Healthc Mater. 2022;11:e2102060.

    Article  PubMed  Google Scholar 

  11. Chen Q, Chen J, Yang Z, Xu J, Xu L, Liang C, Han X, Liu Z. Nanoparticle-enhanced radiotherapy to trigger Robust Cancer Immunotherapy. Adv Mater. 2019;31:e1802228.

    Article  PubMed  Google Scholar 

  12. Huang C, Wang FB, Liu L, Jiang W, Liu W, Ma W, Zhao H. Hypoxic tumor radiosensitization using engineered probiotics. Adv Healthc Mater. 2021;10:e2002207.

  13. Zhou X, Meng Z, She J, Zhang Y, Yi X, Zhou H, Zhong J, Dong Z, Han X, Chen M, et al. Near-Infrared light-responsive nitric oxide delivery platform for enhanced Radioimmunotherapy. Nanomicro Lett. 2020;12:100.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Abdel-Wahab AF, Mahmoud W, Al-Harizy RM. Targeting glucose metabolism to suppress cancer progression: prospective of anti-glycolytic cancer therapy. Pharmacol Res. 2019;150:104511.

    Article  CAS  PubMed  Google Scholar 

  15. Wang T, Zhang H, Han Y, Liu H, Ren F, Zeng J, Sun Q, Li Z, Gao M. Light-enhanced O(2)-Evolving nanoparticles boost photodynamic therapy to elicit Antitumor Immunity. ACS Appl Mater Interfaces. 2019;11:16367–79.

    Article  CAS  PubMed  Google Scholar 

  16. Yang Z, Wang J, Ai S, Sun J, Mai X, Guan W. Self-generating oxygen enhanced mitochondrion-targeted photodynamic therapy for tumor treatment with hypoxia scavenging. Theranostics. 2019;9:6809–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ulliman SL, McKay G, Rosario-Ortiz FL, Linden KG. Low levels of iron enhance UV/H(2)O(2) efficiency at neutral pH. Water Res. 2018;130:234–42.

    Article  CAS  PubMed  Google Scholar 

  18. Krafft MP. Alleviating tumor hypoxia with perfluorocarbon-based oxygen carriers. Curr Opin Pharmacol. 2020;53:117–25.

    Article  CAS  PubMed  Google Scholar 

  19. Zhang L, Wang D, Yang K, Sheng D, Tan B, Wang Z, Ran H, Yi H, Zhong Y, Lin H, et al. Mitochondria-Targeted Artificial “Nano-RBCs” for Amplified Synergistic Cancer Phototherapy by a single NIR Irradiation. Adv Sci (Weinh). 2018;5:1800049.

    Article  PubMed  Google Scholar 

  20. Xiang Q, Qiao B, Luo Y, Cao J, Fan K, Hu X, Hao L, Cao Y, Zhang Q, Wang Z. Increased photodynamic therapy sensitization in tumors using a nitric oxide-based nanoplatform with ATP-production blocking capability. Theranostics. 2021;11:1953–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yadav S, Pandey SK, Singh VK, Goel Y, Kumar A, Singh SM. Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: implication in designing of novel anticancer therapeutic strategies. PLoS ONE. 2017;12:e0176403.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Deng Y, Song P, Chen X, Huang Y, Hong L, Jin Q, Ji J. 3-Bromopyruvate-conjugated Nanoplatform-Induced Pro-Death Autophagy for enhanced photodynamic therapy against hypoxic tumor. ACS Nano. 2020;14:9711–27.

    Article  CAS  PubMed  Google Scholar 

  23. Dikic I, Elazar Z. Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol. 2018;19:349–64.

    Article  CAS  PubMed  Google Scholar 

  24. Zou W, Hao J, Wu J, Cai X, Hu B, Wang Z, Zheng Y. Biodegradable reduce expenditure bioreactor for augmented sonodynamic therapy via regulating tumor hypoxia and inducing pro-death autophagy. J Nanobiotechnol. 2021;19:418.

    Article  CAS  Google Scholar 

  25. Li F, Li Z, Jin X, Liu Y, Zhang P, Li P, Shen Z, Wu A, Chen W, Li Q. Ultra-small gadolinium oxide nanocrystal sensitization of non-small-cell lung cancer cells toward X-ray irradiation by promoting cytostatic autophagy. Int J Nanomedicine. 2019;14:2415–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nat Cell Biol. 2013;15:713–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017;17(9):528–42.

    Article  CAS  PubMed  Google Scholar 

  28. Yadav S, Pandey SK, Kumar A, Kujur PK, Singh RP, Singh SM. Antitumor and chemosensitizing action of 3-bromopyruvate: implication of deregulated metabolism. Chem Biol Interact. 2017;270:73–89.

    Article  CAS  PubMed  Google Scholar 

  29. Liu J, Hou M, Yuan T, Yi G, Zhang S, Shao X, Chen J, Jia X, He Z. Enhanced cytotoxic effect of low doses of metformin combined with ionizing radiation on hepatoma cells via ATP deprivation and inhibition of DNA repair. Oncol Rep. 2012;28:1406–12.

    Article  CAS  PubMed  Google Scholar 

  30. Fan T, Sun G, Sun X, Zhao L, Zhong R, Peng Y. Tumor Energy Metabolism and potential of 3-Bromopyruvate as an inhibitor of aerobic glycolysis: implications in Tumor Treatment. Cancers (Basel). 2019;11(3).

  31. Li J, Kataoka K. Chemo-physical strategies to Advance the in vivo functionality of targeted nanomedicine: the Next Generation. J Am Chem Soc. 2021;143:538–59.

    Article  CAS  PubMed  Google Scholar 

  32. Liu J, Zheng X, Yan L, Zhou L, Tian G, Yin W, Wang L, Liu Y, Hu Z, Gu Z, et al. Bismuth sulfide nanorods as a precision nanomedicine for in vivo multimodal imaging-guided photothermal therapy of tumor. ACS Nano. 2015;9:696–707.

    Article  CAS  PubMed  Google Scholar 

  33. Wang S, Li X, Chen Y, Cai X, Yao H, Gao W, et al. A facile one-pot synthesis of a two-dimensional MoS2/Bi2S3 composite theranostic nanosystem for multi-modality tumor imaging and therapy. Adv Mater. 2015;27:2775–2782.

    Article  CAS  PubMed  Google Scholar 

  34. Li J, Li Y, Wang Y, Ke W, Chen W, Wang W, et al. Polymer Prodrug-Based Nanoreactors activated by Tumor Acidity for Orchestrated Oxidation/Chemotherapy. Nano Lett. 2017;17:6983–90.

    Article  CAS  PubMed  Google Scholar 

  35. Petricciuolo M, Davidescu M, Fettucciari K, Gatticchi L, Brancorsini S, Roberti R, Corazzi L, Macchioni L. The efficacy of the anticancer 3-bromopyruvate is potentiated by antimycin and menadione by unbalancing mitochondrial ROS production and disposal in U118 glioblastoma cells. Heliyon. 2020;6:e05741.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hanafy NA, Dini L, Citti C, Cannazza G, Leporatti S. Inihibition of glycolysis by using a Micro/Nano-Lipid bromopyruvic chitosan carrier as a Promising Tool to improve treatment of Hepatocellular Carcinoma. Nanomaterials (Basel). 2018;8(1).

  37. Ko YH, Niedzwiecka K, Casal M, Pedersen PL, Ulaszewski S. 3-Bromopyruvate as a potent anticancer therapy in honor and memory of the late Professor Andre Goffeau. Yeast. 2019;36:211–21.

    Article  CAS  PubMed  Google Scholar 

  38. Ornatowski W, Lu Q, Yegambaram M, Garcia AE, Zemskov EA, Maltepe E, Fineman JR, Wang T, Black SM. Complex interplay between autophagy and oxidative stress in the development of pulmonary disease. Redox Biol. 2020;36:101679.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ferro F, Servais S, Besson P, Roger S, Dumas JF, Brisson L. Autophagy and mitophagy in cancer metabolic remodelling. Semin Cell Dev Biol. 2020;98:129–38.

    Article  CAS  PubMed  Google Scholar 

  40. Zhang Y, Zhang L, Gao J, Wen L. Pro-death or pro-survival: contrasting paradigms on Nanomaterial-Induced Autophagy and Exploitations for Cancer Therapy. Acc Chem Res. 2019;52:3164–76.

    Article  CAS  PubMed  Google Scholar 

  41. Arya JK, Kumar R, Tripathi SS, Rizvi SI. 3-Bromopyruvate elevates ROS and induces hormesis to exert a caloric restriction mimetic effect in young and old rats. Arch Physiol Biochem. 2020:1–8.

  42. Zheng Z, Chen Q, Dai R, Jia Z, Yang C, Peng X, Zhang R. A continuous stimuli-responsive system for NIR-II fluorescence/photoacoustic imaging guided photothermal/gas synergistic therapy. Nanoscale. 2020;12:11562–72.

    Article  CAS  PubMed  Google Scholar 

  43. Luo K, Zhao J, Jia C, Chen Y, Zhang Z, Zhang J, Huang M, Wang S. Integration of Fe3O4 with Bi2S3 for Multi-Modality Tumor Theranostics. ACS Appl Mater Interfaces. 2020;12:22650–60.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82171949). We also appreciated the financial support from the First Affiliated Hospital of Chongqing Medical University (Grant Nos. CYYY-BSYJSCXXM-202230 and CYYY-BSHPYXM-202305).

Author information

Authors and Affiliations

  1. Department of Ultrasound, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, P.R. China

    Yiman He, Huan Yao, Liang Zhang & Dong Wang

  2. Department of Oncology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, P.R. China

    Huawan Chen

  3. Department of Nuclear Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, P.R. China

    Wenbo Li & Lu Xu

  4. Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, P.R. China

    Yang Cao & Zhigang Wang

  5. Department of Radiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, P.R. China

    Di Zhou

Authors
  1. Yiman He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huawan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wenbo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huan Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yang Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhigang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Liang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Di Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YMH, DZ, ZGW, and DW conceived and designed the study. YMH performed the experiments. YMH, LX, YC and WBL performed in CT and PA imaging. YMH and HWC performed in X-ray therapy. HY and DZ provided important technical support during the experiments. YMH wrote the manuscript and LZ revised the final version. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Dong Wang or Di Zhou.

Ethics declarations

Competing interests

The authors declare no competing interests.

Consent for publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Additional file: Fig S1-S2

TEM images of Bi2O3 nanoparticles and Bi2S3 nanospheres. Fig. S3 The porosity of Bi2S3 nanospheres. Fig. S4 Stability of Bi2S3 nanospheres with or without PEG modification in PBS. Fig. S5 The Tyndall effect of Bi2S3 nanospheres in PBS. Fig. S6 (a) UV-vis absorbance spectra of 3BP at various concentrations. (b) The relative absorbance of 3BP at 282 nm against concentration. Fig S7. Cumulative release of 3BP from Bi2S3-3BP. Fig. S8 HK-II viability of 4T1 cells. Fig. S9 Mean Fluorescence intensity quantitative analysis of hypoxia red fluorescence. Fig. S10 Cell viability of 4T1 cells after treat with Rapa and 3-MA. Fig. S11 The recorded PA intensity of Bi2S3-3BP at λ = 700–950 nm. Fig. S12 In vitro PA contrast images and PA values of Bi2S3-3BP at different concentrations. Fig. S13 In vivo PA images of tumors in tumor-bearing mice after intravenously injection of Bi2S3-3BP at different time points. Fig. S14 Changes of PA-signal intensities within tumor regions at corresponding time points. Fig. S15 In vitro CT contrast images and HU values of Bi2S3-3BP at different concentrations. Fig. S16 In vivo CT images of tumor-bearing mice after intravenously injection of Bi2S3-3BP at different time points

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

He, Y., Chen, H., Li, W. et al. 3-Bromopyruvate-loaded bismuth sulfide nanospheres improve cancer treatment by synergizing radiotherapy with modulation of tumor metabolism. J Nanobiotechnol 21, 209 (2023). https://doi.org/10.1186/s12951-023-01970-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12951-023-01970-8

Keywords

Journal of Nanobiotechnology

ISSN: 1477-3155

Contact us