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

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 (f_a) 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.

Keywords: 3-bromopyruvate; Bismuth (Bi) chalcogenides; Hypoxia relief; Pro-death autophagy; Radiotherapy.

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

Fig. 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.

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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