One-Pot Synthesis of Oxygen Vacancy-Rich Amorphous/Crystalline Heterophase CaWO4 Nanoparticles for Enhanced Radiodynamic-Immunotherapy

Abstract

Radiodynamic therapy that employs X-rays to trigger localized reactive oxygen species (ROS) generation can tackle the tissue penetration issue of phototherapy. Although calcium tungstate (CaWO₄) shows great potential as a radiodynamic agent benefiting from its strong X-ray absorption and the ability to generate electron-hole (e^--h⁺) pairs, slow charge carrier transfer and fast e^--h⁺ recombination greatly limit its ROS-generating performance. Herein, via a one-pot wet-chemical method, oxygen vacancy-rich amorphous/crystalline heterophase CaWO₄ nanoparticles (Ov-a/c-CaWO₄ NPs) with enhanced radiodynamic effect are synthesized for radiodynamic-immunotherapy of cancer. The phase composition and oxygen vacancy content of CaWO₄ can be easily tuned by adjusting the solvothermal temperature. More intriguingly, the amorphous/crystalline interfaces and abundant oxygen vacancies accelerate charge carrier transfer and suppress e^--h⁺ recombination, respectively, enabling synergistically improved ROS production from X-ray-irradiated Ov-a/c-CaWO₄ NPs. In addition to directly inducing oxidative damage of cancer cells, radiodynamic generation of ROS also boosts immunogenic cell death to provoke a systemic antitumor immune response, thereby allowing the inhibition of both primary and distant tumors as well as cancer metastasis. This study establishes a synergistic enhancement strategy involving the integration of phase and defect engineering to improve the ROS generation capacity of radiodynamic-immunotherapeutic anticancer nanoagents.

Keywords: CaWO4 nanoparticles; enhanced radiodynamic effect; heterophase; oxygen vacancies; radiodynamic‐immunotherapy.

Scheme 1

Schematic representation of the one‐pot solvothermal synthesis of Ov‐a/c‐CaWO₄ NPs with enhanced radiodynamic effect for low‐dose X‐ray‐triggered radiodynamic‐immunotherapy of cancer.

Figure 1

Characterization of Ov‐a/c‐CaWO₄ NPs. a) TEM and b) high‐resolution TEM images of Ov‐a/c‐CaWO₄ NPs. b₁) The representative crystalline domain marked by the red square in (b) and b₂) its fast Fourier transform (FFT) pattern. b₃) The representative amorphous domain marked by the green square in (b) and b₄) its FFT pattern. c) SAED image of Ov‐a/c‐CaWO₄ NPs. d) XRD patterns of CaWO₄ NPs prepared at different temperatures including RT, 60 °C, 110 °C, or 160 °C. e) Survey and f) O 1s XPS spectra of CaWO₄ NPs prepared at different temperatures. O_L: lattice oxygen, O_V: oxygen vacancies, O_A: surface‐adsorbed oxygen species. g) The ratios of Ov in O 1s XPS spectra. h) EPR spectra of CaWO₄ NPs prepared at various temperatures.

Figure 2

Radiodynamic ROS generation performance and mechanism of Ov‐a/c‐CaWO₄ NPs. a) Fluorescence spectra of 2′,7′‐dichlorodihydrofluorescein (DCFH) mixed with various CaWO₄ NPs synthesized at different temperatures (including RT, 60, 110, or 160 °C) after X‐ray irradiation (0.5 Gy). The generation of b) •OH, c) O₂ ^•−, and d) ¹O₂ from diverse CaWO₄ NPs under X‐ray irradiation at 0.5 Gy, measured by aminophenyl fluorescein (APF), dihydrorhodamine 123 (DHR 123), and singlet oxygen sensor green (SOSG), respectively. e) The band structures of Ov‐a/c‐CaWO₄ NPs and the energy levels of •OH‐, O₂ ^•−‐, and ¹O₂‐generating processes. f) EIS and g) RL spectra of CaWO₄ NPs prepared at different temperatures. h) Schematic illustration of the synergistic enhancement mechanism of amorphous/crystalline heterophase interfaces and oxygen vacancies on the radiodynamic effect of Ov‐a/c‐CaWO₄ NPs.

Figure 3

In vitro RDT performance of Ov‐a/c‐CaWO₄ NPs. a) Cell viability of 4T1 cells exposed to different concentrations of Ov‐a/c‐CaWO₄ NPs for 12 h and then treated with or without X‐ray irradiation (0.5 Gy). n = 3. b) Cell viability of Ov‐a/c‐CaWO₄ NPs‐incubated 4T1 cells after exposure to X‐ray irradiation at various doses ([Ov‐a/c‐CaWO₄ NPs] = 100 µg mL⁻¹). n = 3. c) Calcein‐AM/PI costaining of 4T1 cells treated with X‐ray irradiation, Ov‐a/c‐CaWO₄ NPs, or Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation. d) Flow cytometry analysis of cancer cell apoptosis after diverse treatments. Fluorescence images of 4T1 cells stained with e) DCFH‐DA or f) C11‐BODIPY^581/591 after treatment with X‐ray irradiation, Ov‐a/c‐CaWO₄ NPs, or Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation (0.5 Gy). Data are presented as mean ± SD. ^*** p <0.001.

Figure 4

Radiodynamically generated ROS‐triggered ICD and immune response by Ov‐a/c‐CaWO₄ NPs in vitro. Immunofluorescence staining of a) HMGB1 release and b) CRT exposure in 4T1 cells treated with X‐ray irradiation, Ov‐a/c‐CaWO₄ NPs, or Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation. c) The levels of released ATP in cell culture medium from different groups. n = 3. d) Schematic illustration of co‐culture system established for evaluating the maturation of DCs after exposure to 4T1 cells pre‐treated with Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation (0.5 Gy). e) Flow cytometry and f) quantitative analysis of CD80⁺CD86⁺ DCs after exposure to 4T1 cells with different treatments (gated on CD11c⁺ cells). n = 3. The secretion of g) IL‐6, h) IL‐12p70, and i) TNF‐α in cell culture medium from various groups. n = 3. Data are presented as mean ± SD. ^** p <0.01, ^*** p <0.001.

Figure 5

In vivo tumor accumulation and X‐ray‐triggered antitumor efficacy of Ov‐a/c‐CaWO₄ NPs. a) Therapeutic schedule for 4T1 tumor‐bearing mice. b) Fluorescence images of 4T1 tumor‐bearing mice and c) the corresponding fluorescence intensity in tumors after i.v. injection of Cy5.5‐labeled Ov‐a/c‐CaWO₄ NPs. n = 3. d) Tumor growth curves of 4T1 tumor‐bearing mice after treatment with PBS, X‐ray irradiation, Ov‐a/c‐CaWO₄ NPs, or Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation. n = 6. e) Body weight curves of different groups of mice. n = 6. f) H&E and g) TUNEL staining of tumor sections harvested from various groups. Data are presented as mean ± SD. ^*** p <0.001.

Figure 6

Tumor metastasis inhibition by Ov‐a/c‐CaWO₄ NPs‐mediated radiodynamic‐immunotherapy. a) Therapeutic schedule for bilateral 4T1 tumor model. Tumor growth curves of b) primary and c) distant tumors in bilateral 4T1 tumor‐bearing mice after treatment with PBS, X‐ray irradiation, Ov‐a/c‐CaWO₄ NPs, or Ov‐a/c‐CaWO₄ NPs plus X‐ray irradiation (0.5 Gy). n = 6. d) Survival curves of mice after different treatments. n = 10. e) Quantitative analysis by flow cytometry of mature DCs (CD80⁺CD86⁺ DCs) in tumor‐draining lymph nodes of mice after various treatments (gated on CD11c⁺ cells). n = 3. Quantitative analysis by flow cytometry of CD4⁺ T cells (CD3⁺CD4⁺) and CD8⁺ T cells (CD3⁺CD8⁺) in f, g) spleens or h, i) distant tumors of different groups of mice (gated on CD3⁺ cells). n = 3. The levels of j) IL‐6, k) IL‐12p70, l) TNF‐α, and m) IFN‐γ in the serum of mice from various groups. n = 3. n) H&E staining of lung tissues harvested from different groups on day 28. Red arrows indicate the metastatic nodules. Data are presented as mean ± SD. ^** p <0.01, ^*** p <0.001.