Human serum albumin-based KBiF4@HSA nanoclusters for dual-energy computed tomography and glutathione-scavenging radiotherapy of breast cancer

Abstract

Background: Breast cancer remains the most common malignancy among women worldwide. While traditional computed tomography (CT) scans and image-guided radiotherapy are widely used for breast cancer diagnosis and treatment, their efficacy is often limited.

Methods and results: In this study, we successfully synthesized human serum albumin (HSA)-based KBiF₄ nanoclusters through a simple one-pot biomimetic mineralization strategy. Compared to the clinical contrast agent iohexol, KBiF₄@HSA significantly enhances dual-energy CT (DECT) imaging contrast at high keV levels, offering improved diagnostic accuracy for breast cancer. Furthermore, KBiF₄@HSA exhibits a remarkable ability to scavenge elevated glutathione (GSH) levels and promote reactive oxygen species (ROS) generation. When combined with radiotherapy, KBiF₄@HSA substantially increases X-ray dose deposition at tumor sites, leading to enhanced DNA damage and suppression of breast cancer progression. Importantly, KBiF₄@HSA demonstrates excellent biocompatibility in vivo, with no significant tissue damage or inflammation observed.

Conclusions: This study presents a novel approach for the development of biocompatible DECT contrast agents and radiosensitizers, offering a promising strategy to enhance breast cancer diagnosis and treatment. However, the efficacy of this approach needs to be further validated across diverse breast cancer subtypes to ensure its broad applicability, which emphasizes the necessity for continued research to fully translate this innovative technology into clinical practice.

Keywords: Bismuth; Breast cancer; Contrast agents; Dual-energy CT; Radiotherapy.

Scheme 1

Scheme of the synthesis and degradation of HSA-based KBiF₄ nanoclusters for DECT imaging and GSH-scavenging radiotherapy for breast cancer

Fig. 1

(A) TEM image of KBiF₄@HSA (scale bar: 200 nm). (B) SEM image of KBiF₄@HSA. (C) EDX mapping of KBiF₄@HSA. (scale bar: 0.5 μm). (D) AFM images of KBiF₄@HSA. (E) TEM images and DLS analysis of KBiF₄@HSA at different timepoints during reaction (scale bar: 200 nm)

Fig. 2

(A) High-resolution Bi 4f spectrum of KBiF₄@HSA. (B) High-resolution F 1s spectrum of KBiF₄@HSA. (C) High-resolution K 2p and C 1s spectrum of KBiF₄@HSA. (D) XPS survey spectra of KBiF₄@HSA. (E) UV − vis absorption spectra of KBiF₄@HSA. (F) XRD pattern of KBiF₄@HSA. (G) Spectra of the main elements of KBiF₄@HSA. (H) FT-IR of KBiF₄@HSA. (I) TEM of KBiF₄@HSA incubated with GSH for 0 min, 5 min, 15 min, 30 min, 60 min (scale bar: 200 nm). (J) DLS analysis and photographs of KBiF₄@HSA with different concentrations of GSH

Fig. 3

(A) Cell viability of SHZ-88 cells and NRK cells after incubation with KBiF₄@HSA for 24 h. (B) Hemolysis experiment of KBiF₄@HSA. (C) Cell viability of 4T1 cells after incubation with KBiF₄@HSA for 24 h. (D) Typical images of colony formation of 4T1 cells treated with radiotherapy. (E) Two-photon digital scanned light-sheet microscopy images of 4T1 cells with mito-tracker and lyso-tracker. (F) Fluorescence images of 4T1 cells stained with DCFH-DA were taken to observe intracellular ROS after treatments (scale bar: 200 μm). (G) Changes in GSH levels in 4T1 cells after PBS or KBiF₄@HSA treatments. (H) Fluorescence images of 4T1 cells stained with calcein-AM/PI following diverse treatments. (I) Fluorescence quantitative analysis of calcein-AM/PI

Fig. 4

(A) In vitro CT imaging at different keV and energy spectrum curves of KBiF₄@HSA and iohexol. (B-F) In vitro CT imaging of KBiF₄@HSA and iohexol at (B) 40 keV, (C) 60 keV, (D) 80 keV, (E) 150 keV, (F) 180 keV. The data are shown as the mean ± standard deviation (SD), as calculated using two-way ANOVA followed by post hoc tests with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 5

(A) DECT imaging of a tumor with iohexol at different radiation energies. (B) DECT imaging of a tumor with KBiF₄@HSA at different radiation energies. (C) DECT imaging of the tumor at different anatomical positions with KBiF₄@HSA and iohexol. (D) DECT imaging of the tumor at the effective atomic numbers (Z_eff) with KBiF₄@HSA. (E) DECT values at different keV in the tumor region with KBiF₄@HSA. (The blue circle indicates the tumor location.)

Fig. 6

Radiosensitizing effects of KBiF₄@HSA in vivo. (A) Flow chart of the radiotherapy experiment in vivo. (B) Tumor growth curves of 4T1-bearing tumor mice. (C) Body weights and (D) tumor weights of mice after various treatments. (E) Heatmap diagram of differentially expressed mRNAs screened from PBS and KBiF₄@HSA group (n = 3). (F) Heatmap diagram of differentially expressed mRNAs screened from KBiF₄@HSA group and KBiF₄@HSA + X group (n = 3). (G) KEGG enrichment analysis of the PBS group versus the KBiF₄@HSA group (Top 20 pathways). (H) The results of KEGG pathway enrichment analysis of the PBS group versus the KBiF₄@HSA + X group (Top 20 pathways). (I) H&E, TUNEL, Ki67, γ-H2AX staining images of tumors after treatments (scale bar: 100 μm). The data are shown as the mean ± SD, calculated using three-way ANOVA, followed by one-way ANOVA and post hoc tests with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 7

Liver function indicators: ALT, ALP (A); Ca, K, P, Na, Cl (B); ALB, TP, GLOB, A/G (C) with KBiF₄@HSA pre-injection and post-injection at 1 and 7 days. Kidney function indicators: CREA, UREA, TG (D) with KBiF₄@HSA pre-injection and post-injection at 1 and 7 days. (E) H&E staining of tumor sections from tumor-bearing rats (scale bar: 50 μm)