Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2022 May 24;7(22):18795–18803. doi: 10.1021/acsomega.2c01591

PVP-Modified Multifunctional Bi2WO6 Nanosheets for Enhanced CT Imaging and Cancer Radiotherapy

Yifan Hao †,‡, Bo Peng §, Chao Si †,‡, Bo Wang §, Chengfeng Luo §, Menghao Chen §, Cheng Luo ∥, Baijuan Gong ∥,*, Zhimin Li §,*
PMCID: PMC9178605  PMID: 35694478

Abstract

graphic file with name ao2c01591_0008.jpg

Malignant tumors are one of the main causes of human death. The clinical treatment of malignant tumors is usually surgery, chemotherapy, radiotherapy, and so forth. Radiotherapy, as a traditional and effective treatment method for cancer, is widely used in clinical practice, but the radiation resistance of tumor cells and the toxic side effects to normal cells are still the Achilles heel of radiotherapy. Multifunctional inorganic high-atom nanomaterials are expected to enhance the effect of tumor radiotherapy. Tungsten and bismuth, which contain elements with high atomic coefficients, have strong X-ray energy attenuation capability. We synthesized Bi2WO6 nanosheets (NSs) using a hydrothermal synthesis method and modified polyvinylpyrrolidone (PVP) on their surface to make them more stable. PVP–Bi2WO6 NSs have a variety of effects after absorbing X-rays (such as the photoelectric effect and Compton effect) and release a variety of particles such as photoelectrons, Compton electrons, auger electrons, and so forth, which can react with organic molecules or water in cells, generate a large number of free radicals, and promote cell apoptosis, thereby improving the effect of radiotherapy. We show through γ-H2AX and DCFH-DA probe analysis experiments that PVP–Bi2WO6 NSs can effectively increase cell DNA damage and reactive oxygen species formation under X-ray irradiation. Clone formation analysis showed that PVP–Bi2WO6 NSs can effectively suppress cell colony formation under X-ray irradiation. These versatile functions endow PVP–Bi2WO6 NSs with enhanced radiotherapy efficacy in animal models. In addition, PVP–Bi2WO6 NSs can also be used as contrast agents for X-ray computed tomography (CT) imaging with obvious effects. Therefore, PVP–Bi2WO6 NSs can be used as CT imaging contrast agents and tumor radiotherapy sensitizers and have potential medical applications.

Introduction

Oral squamous cell carcinoma is the most common malignant tumor in the oral and maxillofacial region. Its incidence is gradually increasing,1,2 and it is considered to be the main cause of death from oral diseases in many countries.3 Moreover, the global cancer diagnosis rate and mortality rate have been on the rise in the past decade.4 At present, clinical treatment methods for malignant tumors often include surgery, chemotherapy, radiotherapy, and so forth. Radiotherapy, as a traditional and effective treatment method for cancer, is widely used in clinical practice. Radiotherapy is often used in conjunction with other treatment methods to treat tumors.5,6 Radiotherapy is the use of high-energy ionizing radiation such as X-rays and γ-rays as well as particle radiation, including particles such as α particles or β particles, electrons, protons, or neutron beams for tumor ablation.7,8 The basic principle of radiotherapy is that ionizing radiation reacts with substances in tumor cells during radiotherapy. Ionizing radiation can directly or indirectly react with biological molecules (such as proteins, lipids, and so forth) in the tissue, and DNA is the main target molecule.4 On the one hand, it can directly ionize DNA molecules, causing DNA single-strand or double-strand breaks and base cross-linking and other types of damage, leading to the termination of cell division and cell proliferation; on the other hand, high-energy rays indirectly react with water in the tissue to generate reactive oxygen species (ROS) such as •OH, •H, H2O+, and •O2–. ROS can damage biomolecules through chemical reactions; especially, •OH can bind to DNA, cause DNA to undergo electron transfer and be oxidized, and induce cell DNA damage, leading to cell damage or apoptosis.9−12 However, in the process of clinical treatment, the indirect damage of ionizing radiation to normal tissues, the hypoxic environment inside tumor cells, and the radiation resistance of tumor cells have limited the further development of radiotherapy in the clinic to a certain extent. Moreover, in a low-oxygen environment, DNA double-strand damage can be repaired.13 Therefore, studying how to reduce the radiation resistance of tumor cells, improve the radiation sensitivity of tumor cells, and improve the tumor microenvironment has certain clinical value.11,14,15

The emergence of radiotherapy sensitizers may be expected to solve this problem, which can increase the deposition of radiation in the tumor site, improve the hypoxia in the tumor site, and enhance the production of ROS in tumor cells.16−18 In recent years, studies have found that nanoparticles containing a high atomic number can be expected to be used as radiotherapy sensitizers to enhance the radiosensitivity of tumor cells, thereby improving the effect of radiotherapy and reducing normal tissue damage.19−22 Similar to heterogeneous photocatalysis, the mechanism of radiocatalysis is also under the irradiation of high-energy rays through a series of reactions to generate highly active singlet oxygen, thereby damaging DNA.23 Nanoparticles with a high atomic number have a variety of effects (such as the photoelectric effect and Compton effect) after absorbing rays and release a variety of particles such as photoelectrons, Compton electrons, and auger electrons that react with organic molecules or water in cancer cells to generate a large number of free radicals, which can improve the effect of radiotherapy and have potential medical applications.24 Compton scattering is an inelastic scattering of photons by electrons, and it is one of the most important interactions in radiotherapy. After colliding with the electrons, the energy of the incident photons is reduced, so the radiation dose can be deposited at a local location. The photoelectric effect is that the electrons in the atomic shell absorb the energy of incident rays and are emitted to ionize surrounding biomolecules. High-Z nanomaterials can be enabled to increase the ionizing radiation dose at the tumor site.25 With the development of nanomedicine, multifunctional inorganic high atomic coefficient nanomaterials have attracted widespread attention and have clinical significance in enhancing the effect of cancer radiotherapy.26,27 W (Z = 74) and Bi (Z = 83) containing high atomic coefficient elements have strong X-ray energy attenuation ability. Hossain and Su controlled the concentration of nanoparticles to 350 mg·g–1 under a 50 kVp radiation source, and the radiosensitization effect of nano-bismuth was 1.25 times and 1.29 times stronger than that of nano-gold and nano-platinum, respectively. It is concluded that bismuth nanoparticles have a stronger sensitizing effect than gold and platinum nanoparticles at the same nanometer size, particle concentration, and action site.28 At 100 keV, the X-ray mass attenuation coefficients of Bi and W elements are 5.74 and 4.44 cm2/g, respectively.29,30 By modifying the surface of nanoparticles in different ways, nanoparticles can also be used for active targeting or therapy, and multimodal imaging, which is expected to be used as a contrast agent for X-ray computed tomography (CT) imaging. CT is one of the most widely used imaging methods in clinical practice, which has the advantages of high efficiency, short scanning time, and high resolution. Ordinary CT is suitable for different tissues with large density differences, but it is difficult for the density differences between soft tissues to form a clear contrast on the image. Therefore, the application of contrast agents can selectively enhance the image contrast between the target soft tissue and surrounding tissues and improve CT sensitivity.31 Iodide is the earliest and most widely used CT contrast agent in clinical practice, but the imaging time of this contrast agent is short, and its high osmotic pressure, high viscosity, and electric charge often trigger contrast agent reactions, posing a threat to the life safety of patients.32 Compared with traditional small-molecule contrast agents, nanoparticles can load a large number of contrast elements; have long blood circulation half-life, low renal clearance, and capillary leakage; and can accumulate in the tumor site through the enhanced permeation and retention effect.33 In recent years, Bi2WO6 nanosheets (NSs) have been studied as a powerful multifunctional platform with the ability of photothermal therapy and photodynamic therapy.34 Bi2WO6 NSs can kill tumor cells and achieve local photothermal ablation of tumors under the irradiation of ultraviolet to near-infrared lasers. At the same time, •OH free radicals are generated without consuming oxygen molecules, so that these Bi2WO6 NSs can play a photodynamic killing effect in an oxygen-free manner during cancer treatment.35 In addition, Bi2WO6 NSs have a variety of effects after absorbing X-rays (such as the photoelectric effect and Compton effect) and release a variety of particles such as photoelectrons, Compton electrons, and auger electrons, which have the potential to be used in tumor radiotherapy.36

Therefore, we studied the radiosensitization effect of Bi2WO6 NSs in tumor radiotherapy. In this study, we use a reasonable design to increase the amount of nanomaterials in tumors, enhance the sensitivity of nanomaterials under X-ray, and enhance their ability to produce singlet oxygen and better kill tumor cells. Therefore, we prepared Bi2WO6 NSs. In addition, the surface of the NSs was modified with PVP, which is stable and negatively charged. In the blood circulation, it can effectively avoid being marked and removed by the same negatively charged labeling factors. Therefore, PVP–Bi2WO6 NSs were beneficial to the accumulation in tumors, and the accumulation rate showed a trend of increasing first and then decreasing gradually, reaching the highest at about 24 h. PVP–Bi2WO6 NSs contain both tungsten and bismuth nanomaterials that are radiosensitizing elements, and the sheet structure has a large specific surface area. These factors greatly enhance the radiosensitization capabilities of nanomaterials. Moreover, because of the presence of two metal elements, tungsten and bismuth, PVP–Bi2WO6 NSs can also be used as CT imaging contrast agents to locate tumors. The lamellar structure has a large specific surface area, which can increase the ray absorption cross section, deposit high-energy ray energy, promote the generation of free radicals, and enhance DNA damage, which greatly improves the radiosensitization ability of nanomaterials. In addition, due to the high atomic numbers of the two metal elements, tungsten and bismuth, PVP–Bi2WO6 NSs can also be used as CT imaging contrast agents to show good CT imaging capabilities. In vitro anti-tumor results show that PVP–Bi2WO6 NSs have the function of enhancing the destruction of cancer cells under radiation irradiation. The in vivo anti-tumor results also clearly show that the presence of PVP–Bi2WO6 NSs after radiation exposure can effectively reduce solid tumor tissue. We believe that after continuous exploration and research, PVP–Bi2WO6 NSs will definitely play an active role in future clinical treatments.

Results and Discussion

We synthesized Bi2WO6 NSs by a simple hydrothermal method, and the morphology is shown in Figure 1a, which is a two-dimensional lamellar structure. The X-ray diffraction (XRD) spectrum showed their good crystallinity, and it can be well matched with orthorhombic Bi2WO6 (JCPDS: 73-1126, Figure 1b). Then, we modified PVP on their surface to prepare PVP–Bi2WO6 NSs. The TEM image showed that the obtained PVP–Bi2WO6 NSs basically maintained the same morphology as Bi2WO6 NSs (Figure 1c). PVP modification greatly improved the water-solubility of Bi2WO6 NSs. On the one hand, PVP is negatively charged and can maintain the colloidal stability through electrostatic repulsion. On the other hand, space-occupying PVP ligands can stabilize Bi2WO6 NSs through spatial effects.37 The enhanced water-solubility could enhance the stability of NSs. PVP can also reduce the toxicity of nanoparticles. Many studies have shown that PVP is a kind of ligand with very low toxicity and has certain advantages in improving biocompatibility.38,39

Figure 1.

Figure 1

Open in a new tab

(a) TEM image of Bi2WO6 NSs. (b) Powder XRD patterns of Bi2WO6 NSs. (c) TEM image of PVP–Bi2WO6 NSs.

We tested and analyzed the in vitro cytotoxicity of PVP–Bi2WO6 NSs through CCK-8. As shown in Figure 2a, CAL27 cell viability is still above 90% at concentrations of 100 and 200 μg/mL. At the highest concentration (400 μg/mL), the cytotoxicity is still low, and the cell vitality is still above 85%. This further proves that PVP–Bi2WO6 NSs have lower cytotoxicity and also means that PVP–Bi2WO6 NSs have good biocompatibility in biomedical applications. In addition, we used flow cytometry to detect the effect of PVP–Bi2WO6 NSs on the apoptosis of CAL27 cells. The results are shown in Figure 2b. There is no significant difference in the apoptosis rate of cells compared with the control group at different concentrations, even though at a higher concentration (200 μg/mL), the effect of PVP–Bi2WO6 NSs on cell apoptosis is still very small and can be ignored.

Figure 2.

Figure 2

Open in a new tab

(a) In vitro cytotoxicity of PVP–Bi2WO6 NSs with different concentrations was tested by CCK-8. (b) In vitro cytotoxicity of PVP–Bi2WO6 NSs with different concentrations was tested by flow cytometry.

We analyzed the apoptosis rate of cells under different irradiation doses using flow cytometry. As shown in Figure 3a, compared with the control group, there was no significant difference in the apoptosis rate of the PVP–Bi2WO6 group, and the apoptosis rate of the simple X-ray irradiation group increased; compared with the simple irradiation group, the proportion of live cells in the PVP–Bi2WO6 + X-ray irradiation group is less, and the apoptotic rate is higher (Figure S1). Whether it is a low dose or a high dose, the cell apoptosis rate after co-culturing with the PVP–Bi2WO6 NSs is higher than that of the irradiation alone group (Figure 3b). We did a clone formation test to evaluate the radiosensitization effect of PVP–Bi2WO6 NSs. As shown in Figure 3c, the combination of PVP–Bi2WO6 NSs and X-ray can inhibit the clone formation to a great extent. After co-culturing with PVP–Bi2WO6 NSs alone, the survival rate of CAL27 cells was 97.7%. When only X-ray irradiation (6 Gy) was applied, the survival rate of CAL27 cells decreased to 17.7%. However, after co-cultivation with PVP–Bi2WO6 and X-ray irradiation, the survival rate of the cells dropped to 1.5%, which was significantly lower than that in the X-ray irradiation group (Figure 3d). Under different irradiation doses (2, 4, 6, and 8 Gy), the colony formation rate of the cell group co-cultured with PVP–Bi2WO6 NSs was significantly lower than that of the control group (Figure 3e).

Figure 3.

Figure 3

Open in a new tab

(a) The apoptosis of CAL27 cells under different treatments was conducted by flow cytometry analysis. (b) The apoptosis of CAL27 cells under different treatments and X-ray dose was conducted by flow cytometry analysis. (c) Clonogenic assay of CAL27 cells under different treatments (X-ray dose: 6 Gy). (d) Survival fraction of CAL27 cells under different treatments (X-ray dose: 6 Gy). (e) Survival fraction of CAL27 cells under different treatments and X-ray doses. **P < 0.01 and ***P < 0.001.

In addition, ROS probe DCFH-DA can combine with ROS to emit green fluorescence, and the relative strength of fluorescence can reflect the relative content of ROS in cells. The intensity of green fluorescence in the control group and PVP–Bi2WO6 NS group was extremely low, which proved that ROS generation was very low, and simple co-culture with PVP–Bi2WO6 NSs could not improve the ROS content in CAL27 cells. X-ray irradiation can produce a small amount of ROS in cells, but the content is relatively low. Under X-ray, PVP–Bi2WO6 NSs can generate a large number of ROS, which helps to promote cell apoptosis (Figure 4a). In addition to direct comparison of fluorescence photographs, the fluorescence intensity was also quantitatively analyzed. The results showed that the fluorescence intensity of the PVP–Bi2WO6 NSs + X-ray group was significantly higher than that of other groups, which proves that PVP–Bi2WO6 NSs can effectively increase the level of ROS in CAL27 cells under X-ray irradiation (Figure 4b).

Figure 4.

Figure 4

Open in a new tab

(a) ROS detection in CAL27 cells under different treatments by using DCFH-DA as the probe (scale bar is 100 μm). (b) Average signal intensity of DCFH-DA after different treatments. (c) Average signal intensity of γ-H2AX after different treatments. (d) γ-H2AX staining in CAL27 cells under different treatments (scale bar is 50 μm). **P < 0.01 and ***P < 0.001.

Finally, immunofluorescence staining was used to measure DNA damage in cells. Damaged DNA can be recognized by γ-H2AX and labeled with red fluorescence. Both PVP–Bi2WO6 NSs and X-ray alone have a low effect on DNA damage. However, under X-ray irradiation, PVP–Bi2WO6 NSs can cause large areas of DNA damage (Figure 4d). Quantitative analysis of fluorescence intensity showed that the fluorescence intensity of the PVP–Bi2WO6 NSs + X-ray group was 2.65 times that of the X-ray group, and the effect was very obvious (Figure 4c). These experiments indicate that PVP–Bi2WO6 NSs are expected to be used as radiotherapy sensitizers to enhance the radiotherapy effect on tumors.

In view of the remarkable results of the cell experiment, we further studied the radiotherapy effect of PVP–Bi2WO6 NSs in animals. After subcutaneously inoculating mice with CAL27 cells to establish the tumor models, the mice were randomly divided into four groups: (I) control group, (II) PVP–Bi2WO6 group, (III) X-ray irradiation group, and (IV) PVP–Bi2WO6 + X-ray irradiation group. In the exploration of the optimal time to apply radiation, the experimental results showed that the accumulation rate of PVP–Bi2WO6 NSs in CAL27 tumors showed a trend of gradually increasing at first and then gradually decreasing, and it reached the highest value at 24 h, which was 6.19% (Figure S2). Therefore, 24 h was identified as the point in time to apply X-ray in animal experiments. In subsequent experiments, the tumor volume and the weight of the mice were measured every day. On the 18th day, the tumor volume and weight data could be used to intuitively compare the treatment effects. As shown in Figure 5a–c, the tumor volumes of the mice in the control group and in the PVP–Bi2WO6 group continued to increase. By the end of the treatment, the tumors had grown to about 300 mm3. The fluctuation in the mice’s weight suggested that the body’s metabolism is out of whack, and the tumors are not well treated (Figure 5f). Compared with these two groups, the tumor volumes of the mice in the simple X-ray group were suppressed to a certain extent, and the tumor volumes were reduced. However, the tumor volumes of mice in the PVP–Bi2WO6 + X-ray group were significantly reduced, and the growth rate of the tumors was significantly inhibited (Figure 5a–c). At the same time, the fluctuation of body weight was small, and the mice were basically in a normal growth state, indicating that the tumors were highly treated and the growth of mice was not affected. Besides, this not only proved the low toxicity of nanomaterials, but it also proved the effective protection to the mouse during irradiation (Figure 5f). In Figure 5d,e, tumor weights and H&E-stained tumor sections also confirm the antitumor effects of PVP–Bi2WO6 NSs in vivo. Finally, we performed H&E staining on major organs (including heart, spleen, lung, liver, kidney, and testis) and observed the pathological manifestations of the tissues under an optical microscope. We observed that no obvious pathological manifestations were observed in the tissues of the four groups of mice (Figure S3). At the same time, we performed liver and kidney function analysis on the blood of each group of mice (Figures S4–S6). Compared with the control group, each treatment group showed no obvious toxicity. The above results all proved the good compatibility of PVP–Bi2WO6 NSs in vivo and low biological toxicity.

Figure 5.

Figure 5

Open in a new tab

In vivo synergistic therapy. The mice are divided into four groups: control group, PVP–Bi2WO6 group, X-ray irradiation group, and PVP–Bi2WO6 + X-ray irradiation group. (a) Tumor volume of mice during 18 days. (b) Photographs of tumors at the end of treatment. (c) Photographs of typical mice at the end of treatment. The average tumor volume is 311, 292, 162, and 24 mm3 for each group. (d) H&E staining of tumors at the end of treatment. The scale bar is 100 μm. (e) Average weights of tumors at the end of treatment. (f) Body weight curves of mice during 18 days. ns P > 0.05 (no significant difference) and ***P < 0.001.

The results of in vitro CT imaging experiments showed that the CT imaging ability of PVP–Bi2WO6 NSs and meglumine is similar at a low concentration, but at a high concentration, the CT imaging ability of PVP–Bi2WO6 NSs is stronger (Figure 6a,b). In order to further prove the CT imaging ability of PVP–Bi2WO6 in mice, we injected PVP–Bi2WO6 NSs and diatrizoate meglumine into the mice via tail vein, respectively, and then compared their imaging effects. As shown in Figure 6c, the HU value of the tumor site in the control group is 544, and the HU value of the tumor site in the PVP–Bi2WO6 group is as high as 809, which further proves that PVP–Bi2WO6 NSs can reach the tumor site through the EPR effect and obtain better therapeutic effects.

Figure 6.

Figure 6

Open in a new tab

(a) CT images of diatrizoate meglumine and PVP–Bi2WO6 NSs. (b) HU value of diatrizoate meglumine and PVP–Bi2WO6 NSs in CT imaging at different concentrations. (c) HU value in CT imaging 24 h after injection of diatrizoate meglumine and PVP–Bi2WO6 NSs into the tail vein of mice, respectively.

Conclusions

We successfully prepared PVP-modified Bi2WO6 NSs by hydrothermal synthesis. The sheet-like structure gives it a large surface area and makes it have a strong absorption effect on X-ray. We have proved its good effect of inhibiting cell proliferation through the clone formation experiment, and the ROS experiment proved that it has a better effect of increasing the level of intracellular ROS. The γ-H2AX experiment further proved that PVP–Bi2WO6 NSs can significantly increase the level of DNA fragmentation under X-ray irradiation. In vivo experiments have further proved its powerful radiosensitization effect. Through H&E staining of organs and liver and kidney function tests, we have not observed its toxicity to organs and tissues. The above experiments have effectively proved that PVP–Bi2WO6 NSs can be expected to be used as a radiotherapy sensitizers, thereby improving the therapeutic effect of radiotherapy and reducing its side effects, which has potential medical application value.

Experimental Section

Materials

Bi(NO3)3·5H2O (99.0%) and Na2WO4·2H2O (99.5%) were purchased from Aladdin. HNO3 (AR) and KOH (AR) were purchased from Sinopharm Chemical Reagent Company. Poly(vinylpyrrolidone) (PVP, Mw ∼ 1 300 000) was purchased from Sigma-Aldrich. Cell counting kit-8 (CCK-8), immunostaining permeabilization solution with Triton X-100, ROS assay kit, immunol staining fix solution, blocking buffer for immunol staining, immunol staining wash buffer, crystal violet staining solution, and Hoechst 33342 were all purchased from Beyotime. An annexin V-FITC/PI apoptosis detection kit was purchased from Solarbio. γ-H2AX (phosthoS139) antibody [EP854(2)Y] (AlexaFluor568) (ab206901) was purchased from Abcam.

Synthesis and Modification of PVP–Bi2WO6 NSs

First, 10 mmol Bi(NO3)3·5H2O was dissolved in 4.5 mL of 20% HNO3 aqueous solution under ultrasound, and then it was slowly added dropwise into the Na2WO4·2H2O aqueous solution (5 mmol Na2WO4·2H2O dissolved in 40 mL of deionized water). Next, the mixture was stirred for 1 h. After adjusting the pH to 7.5 by KOH, the mixture was transferred to a Teflon-lined autoclave and treated at 180 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 6000 rpm for 5 min, and then the precipitate was re-dispersed in water. This step needs to be repeated twice.36

In order to modify PVP, the PVP aqueous solution (30 mg of PVP dissolved in 5 mL of deionized water) was slowly added dropwise into the Bi2WO6 aqueous solution (30 mg of Bi2WO6 dissolved in 5 mL of deionized water). After sonicating for 2 h, the mixture was centrifuged at 6000 rpm for 5 min, and then the precipitate was re-dispersed in water. This step needs to be repeated twice. The final sample PVP–Bi2WO6 NSs were dispersed in deionized water.

Cell Experiments In Vitro

CAL-27 cells were selected for the study. CAL-27 cells, also named Centre Antoine Lacassagne-27 cells, were developed by J. Gioanni in 1982, derived from a lesion in the middle of the tongue of a 56-year-old white male. For the in vitro cytotoxicity assay, 100 μL of CAL27 cell suspension (5000 cells/well) was placed in a 96-well plate, and the culture plate was placed in an incubator for 24 h (37 °C, 5% CO2). Then,10 μL of PVP–Bi2WO6 at different concentrations was added to each well (0, 6.25, 12.5, 25, 50, 100, 150, 200, and 400 μg/mL). The 96-well plate was placed in an incubator and was allowed to continue to incubate for 24 h. Then, the original medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS). 10 μL of CCK8 solution was added to each well. The culture plate was incubated in an incubator for 2 h. The absorbance at 450 nm was measured with a microplate reader. In the cell apoptosis assay, 100,000 CAL27 cells were added to each well of a six-well plate, and they were placed in an incubator for 24 h. Different concentrations of PVP–Bi2WO6 (0, 20, 50, 100, 150, and 200 μg/mL) were added to the wells and incubated with the cells overnight. The original culture medium was collected, the cells were washed 3 times with PBS, and then the cells were resuspended in a binding buffer. A annexin V-FITC/PI apoptosis detection kit was used for flow cytometric analysis of apoptosis. In the clonogenic assay, 1,500 of CAL27 cells were added to each well of a six-well plate, the culture plate was placed in an incubator for 24 h (37 °C, 5% CO2), and then, 20 μL of PVP–Bi2WO6 (concentration of 100 μg/mL) was added to each well. After culturing for 24 h, each orifice plate was irradiated with different doses (0, 2, 4, 6, and 8 Gy) of X-ray, and they were put in an incubator to continue culturing. The medium was changed every 2–3 days, and the formation of cell colonies was observed frequently until the formation of visible colonies (50–100). The original medium was discarded in the well plate, the cells were washed with PBS 3 times, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, stained with crystal violet staining solution for 10 min, and the six-well plate was washed with distilled water. Under the microscope, the number of cells proliferating more than 50 was counted, the number of each well was recorded, and photographs were taken of the six-well plate. The average survival score was obtained from three parallel samples. In the apoptosis assay, CAL27 cells (50,000 cells per well) were added to a six-well plate and placed in an incubator for 24 h. After the cells were completely attached, PVP–Bi2WO6 (100 μg/mL) was added to the wells to co-culture these cells overnight. Then, they were irradiated with X-rays, culturing was continued for 24 h, the original culture fluid was collected, the cells and centrifuge were digested, the cells were washed with PBS 3 times, and the cells were resuspended with the binding buffer. An annexin V-FITC/PI apoptosis detection kit was used for flow cytometric analysis of apoptosis. In the ROS experiment in vitro, CAL27 cells were added to a six-well plate (50,000 cells per well), 20 μL of PVP–Bi2WO6 (100 μg/mL) was added after culturing overnight, and co-culturing was continued for 4 h. The cells were washed 3 times with PBS. Serum-free medium was used to dilute the DCFH-DA probe by a thousand times; then, the diluted probe was added to the well and incubated for 20 min, and the cells were carefully washed with serum-free medium 3 times. Finally, 1.5 mL of serum-free culture medium was added to each well. The cells were irradiated with X-ray (6 Gy) and then detected using a fluorescence microscope (excitation wavelength: 488 nm, emission wavelength: 525 nm). In the DNA double-stranded breaks assay, CAL27 cells were cultured in a confocal dish with a density of 50,000 cells per dish and cultured in an incubator for 24 h (37 °C, 5% CO2). Then, the cells were divided into four groups: (1) control group, (2) PVP–Bi2WO6 group, (3) X-ray irradiation group, and (4) PVP–Bi2WO6 + X-ray irradiation group. Then, PVP–Bi2WO6 (100 μg/mL) was added to the cells of groups (2) and (4). It was incubated with the cells for 24 h, and the cells in groups (3) and (4) were irradiated at 6 Gy (1 Gy/min) with X-ray. After culturing for 4 h, immunofluorescence staining of γ-H2AX was performed. The cells were fixed with immunostaining fixative for 10 min and then washed 3 times with immunostaining washing solution. Then, the cells were permeabilized with immunostaining permeate for 15 min, and the cells were washed 3 times. Then, the cells were blocked with immunostaining blocking solution for 2 h. The blocking solution was discarded, diluted γ-H2AX (diluted 1:1000 with the primary antibody dilution solution) was added, and the cells were incubated overnight at 4 °C in the dark. Next, excess antibodies were removed, the cells were washed with a washing solution 3 times, the nuclei were stained with Hoechst 33342 (diluted 1:100 with PBS), and then, a laser confocal microscope was used to detect the fluorescence intensity of each group. (Primary antibody excitation wavelength: 590 nm, emission wavelength: 617 nm; Hoechst33342 excitation wavelength: 346 nm, emission wavelength: 460 nm.)

CT Imaging Assay In Vitro

In the CT imaging assay in vitro, deionized water was used to configure PVP–Bi2WO6 NSs into solutions of different concentrations, with concentration gradients of 0.1, 0.5, 1, 2.5, and 5 mg/mL, respectively. A CT machine was used to test the effect of CT imaging. To more intuitively reflect the effect of CT imaging, the corresponding CT signal values of each sample were read in the reading system, and the signal intensity of each group was compared with a numerical value. (The voltage is 60 kV, the current is 1 mA, and the radiation dose is 0.1 mGy.)

Animal Experiments

Five-week-old BALB/c nude mice were selected, and the mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of College of Basic Medical Sciences, Jilin University. After being adaptively raised in a barrier environment for 1 week, a tumor-bearing mouse model was established. 1,200,000 of CAL27 cells were injected subcutaneously into the middle part of the back of the mouse. After that, the mice were raised normally, the tumor size of the mice was observed and measured, and the tumor volume was estimated according to the formula: V = ab2/2 (where a and b are the length and width of the tumor, respectively). When the tumor volume reached 50–60 mm3, the mice were randomly divided into four groups (n = 3): (I) control group, (II) PVP–Bi2WO6 group, (III) X-ray irradiation group, and (IV) PVP–Bi2WO6 + X-ray irradiation group. Then, mice in groups (I) and (III) were injected with 40 μL of normal saline through the tail vein, and tumor-bearing mice in groups (II) and (IV) were injected with 40 μL of PVP–Bi2WO6 (5 mg/mL) through the tail vein; (III) and (IV) X-ray irradiation was performed 24 h after the tail vein injection in the group, the irradiation dose was 6 Gy, 1 Gy/min. After irradiation, the mice were raised normally, and the body weight and tumor size of the mice were measured and recorded every day to compare the treatment effects of each group. After 18 days of continuous measurement, the mice were sacrificed, and the tumors and major organs (including heart, spleen, lung, liver, kidney, and testis) of the mice were taken out, and these tissues were weighed and stained. The tumors and main organs of the mice were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and frozen sectioned. Finally, the histological section was co-stained with hematoxylin and eosin (H&E) and then imaged using an optical microscope to analyze and compare the pathological manifestations of each tissue. The blood of the mice was centrifuged to obtain the upper serum, and the serum was tested for liver and kidney function.

Characterization

XRD was measured using an Empyrean X-ray diffractometer with Cu K radiation (λ = 1.5418 Å). Transmission electron microscopy (TEM) was conducted using a JEM-2100F transmission electron microscope (200 kV). The absorbance of cells in the plate was detected using a Bio-tek microplate reader at a 490 nm wavelength. The apoptosis was measured using a BD Biosciences FACScan flow cytometer. The ROS fluorescence intensity was monitored using an Olympus U-LH100HG fluorescence microscope. The DNA double-stranded break staining was observed using C2 laser scanning confocal microscopy. The X-ray irradiation was treated with a PXi X-RAD 320 X-ray irradiator (1 Gy/min). The CT imaging capability was assessed with Planmeca Cone beam CT (60 kV, 1 mA). The HU value was measured using Invivo 5.0 software..

Acknowledgments

This work was supported by the Medical Engineering Intersection Joint Funds of the Natural Science Foundation of Liaoning Province, China (2021-YGJC-18) and Science and Technology Project of Jilin Provincial Department of Education (JJKH20201109KJ).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c01591.

  • Additional flow cytometry data, tumor accumulation rates, H&E-stained organ slices, blood liver function test data, blood renal function test data, and blood ion detection data (PDF)

Author Contributions

⊥ Y.F.H. and B.P. contributed equally.

Author Contributions

Y.F.H. and B.P. contributed equally to this work. Z.M.L. and B.J.G. supervised and proposed the project. Y.F.H. and B.P. designed and performed the experiments and co-wrote the paper. C.S., B.W., C.F.L., M.H.C., and C.L. participated in most experiments. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao2c01591_si_001.pdf (514.6KB, pdf)

References

  1. Ng J. H.; Iyer N. G.; Tan M.-H.; Edgren G. Changing epidemiology of oral squamous cell carcinoma of the tongue: A global study. Head Neck 2017, 39, 297–304. 10.1002/hed.24589. [DOI] [PubMed] [Google Scholar]
  2. Du M.; Nair R.; Jamieson L.; Liu Z.; Bi P. Incidence Trends of Lip, Oral Cavity, and Pharyngeal Cancers: Global Burden of Disease 1990-2017. J. Dent. Res. 2020, 99, 143–151. 10.1177/0022034519894963. [DOI] [PubMed] [Google Scholar]
  3. Almangush A.; Mäkitie A. A.; Triantafyllou A.; de Bree R.; Strojan P.; Rinaldo A.; Hernandez-Prera J. C.; Suárez C.; Kowalski L. P.; Ferlito A.; Leivo I. Staging and grading of oral squamous cell carcinoma: An update. Oral Oncol. 2020, 107, 104799. 10.1016/j.oraloncology.2020.104799. [DOI] [PubMed] [Google Scholar]
  4. Huang R.-X.; Zhou P. K. DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduction Targeted Ther 2020, 5, 60. 10.1038/s41392-020-0150-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barton M. B.; Jacob S.; Shafiq J.; Wong K.; Thompson S. R.; Hanna T. P.; Delaney G. P. Estimating the demand for radiotherapy from the evidence: A review of changes from 2003 to 2012. Radiother. Oncol. 2014, 112, 140–144. 10.1016/j.radonc.2014.03.024. [DOI] [PubMed] [Google Scholar]
  6. Wang H.; Mu X.; He H.; Zhang X.-D. Cancer Radiosensitizers. Trends Pharmacol. Sci. 2018, 39, 24–48. 10.1016/j.tips.2017.11.003. [DOI] [PubMed] [Google Scholar]
  7. Wang X.; Guo Z.; Zhang C. Y.; Zhu S.; Li L. L.; Gu Z. J.; Zhao Y. L. Ultrasmall BiOI Quantum Dots with Efficient Renal Clearance for Enhanced Radiotherapy of Cancer. Adv. Sci. 2020, 7, 1902561. 10.1002/advs.201902561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Giulietti A.Laser-driven particle acceleration for radiobiology and radiotherapy: where we are and where we are going. Conference on Medical Applications of Laser-Generated Beams of Particles IV—Review of Progress and Strategies for the Future, Prague, Czech Republic, April 24–25; SPIE-International Society for Optics and Photonics: Prague, Czech Republic, 2017.
  9. Song G. S.; Cheng L.; Chao Y.; Yang K.; Liu Z. Emerging Nanotechnology and Advanced Materials for Cancer Radiation Therapy. Adv. Mater. 2017, 29, 1700996. 10.1002/adma.201700996. [DOI] [PubMed] [Google Scholar]
  10. Jiang W.; Li Q.; Xiao L.; Dou J.; Liu Y.; Yu W.; Ma Y.; Li X.; You Y.-Z.; Tong Z.; Liu H.; Liang H.; Lu L.; Xu X.; Yao Y.; Zhang G.; Wang Y.; Wang J. Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer Radiotherapy via DNA Damage Enhancement and Concomitant DNA Repair Prevention. ACS Nano 2018, 12, 5684–5698. 10.1021/acsnano.8b01508. [DOI] [PubMed] [Google Scholar]
  11. Chen X.; Song J.; Chen X.; Yang H. X-ray-activated nanosystems for theranostic applications. Chem. Soc. Rev. 2019, 48, 3073–3101. 10.1039/c8cs00921j. [DOI] [PubMed] [Google Scholar]
  12. Wang R.; Deng J.; He D.; Yang E.; Yang W.; Shi D.; Jiang Y.; Qiu Z.; Webster T. J.; Shen Y. PEGylated hollow gold nanoparticles for combined X-ray radiation and photothermal therapy in vitro and enhanced CT imaging in vivo. Nanomedicine 2019, 16, 195–205. 10.1016/j.nano.2018.12.005. [DOI] [PubMed] [Google Scholar]
  13. Xia D.; Hang D.; Li Y.; Jiang W.; Zhu J.; Ding Y.; Gu H.; Hu Y. Au-Hemoglobin Loaded Platelet Alleviating Tumor Hypoxia and Enhancing the Radiotherapy Effect with Low-Dose X-ray. ACS Nano 2020, 14, 15654–15668. 10.1021/acsnano.0c06541. [DOI] [PubMed] [Google Scholar]
  14. Jiang W.; Li Q.; Zhu Z.; Wang Q.; Dou J.; Zhao Y.; Lv W.; Zhong F.; Yao Y.; Zhang G.; Liu H.; Wang Y.; Wang J. Cancer Chemoradiotherapy Duo: Nano-Enabled Targeting of DNA Lesion Formation and DNA Damage Response. ACS Appl. Mater. Interfaces 2018, 10, 35734–35744. 10.1021/acsami.8b10901. [DOI] [PubMed] [Google Scholar]
  15. De Ruysscher D.; Niedermann G.; Burnet N. G.; Siva S.; Lee A. W. M.; Hegi-Johnson F. Radiotherapy toxicity. Nat. Rev. Dis. Prim. 2019, 5, 13. 10.1038/s41572-019-0064-5. [DOI] [PubMed] [Google Scholar]
  16. Zhu D.; Lyu M.; Huang Q.; Suo M.; Liu Y.; Jiang W.; Duo Y.; Fan K. Stellate Plasmonic Exosomes for Penetrative Targeting Tumor NIR-II Thermo-Radiotherapy. ACS Appl. Mater. Interfaces 2020, 12, 36928–36937. 10.1021/acsami.0c09969. [DOI] [PubMed] [Google Scholar]
  17. Lyu M.; Zhu D.; Kong X.; Yang Y.; Ding S.; Zhou Y.; Quan H.; Duo Y.; Bao Z. Glutathione-Depleting Nanoenzyme and Glucose Oxidase Combination for Hypoxia Modulation and Radiotherapy Enhancement. Adv. Healthcare Mater. 2020, 9, 1901819. 10.1002/adhm.201901819. [DOI] [PubMed] [Google Scholar]
  18. Liu Y.; Zhen W.; Wang Y.; Song S.; Zhang H. Na2S2O8 Nanoparticles Trigger Antitumor Immunotherapy through Reactive Oxygen Species Storm and Surge of Tumor Osmolarity. J. Am. Chem. Soc. 2020, 142, 21751–21757. 10.1021/jacs.0c09482. [DOI] [PubMed] [Google Scholar]
  19. Dong X.; Cheng R.; Zhu S.; Liu H.; Zhou R.; Zhang C.; Chen K.; Mei L.; Wang C.; Su C.; Liu X.; Gu Z.; Zhao Y. A Heterojunction Structured WO2.9-WSe2 Nanoradiosensitizer Increases Local Tumor Ablation and Checkpoint Blockade Immunotherapy upon Low Radiation Dose. ACS Nano 2020, 14, 5400–5416. 10.1021/acsnano.9b08962. [DOI] [PubMed] [Google Scholar]
  20. Fan Y.; Tu W. Z.; Shen M. W.; Chen X. M.; Ning Y. S.; Li J. J.; Chen T. F.; Wang H.; Yin F. F.; Liu Y.; Shi X. Y. Targeted Tumor Hypoxia Dual-Mode CT/MR Imaging and Enhanced Radiation Therapy Using Dendrimer-Based Nanosensitizers. Adv. Funct. Mater. 2020, 30, 1909285. 10.1002/adfm.202070082. [DOI] [Google Scholar]
  21. Duo Y. H.; Huang Y. Y.; Liang W. Y.; Yuan R. M.; Li Y.; Chen T. F.; Zhang H. Ultraeffective Cancer Therapy with an Antimonene-Based X-Ray Radiosensitizer. Adv. Funct. Mater. 2020, 30, 1906010. 10.1002/adfm.201906010. [DOI] [Google Scholar]
  22. Shen Z.; Liu T.; Yang Z.; Zhou Z. J.; Tang W.; Fan W. P.; Liu Y. J.; Mu J.; Li L.; Bregadze V. I.; Mandal S. K.; Druzina A. A.; Wei Z. N.; Qiu X. Z.; Wu A. G.; Chen X. Y. Small-sized gadolinium oxide based nanoparticles for high-efficiency theranostics of orthotopic glioblastoma. Biomaterials 2020, 235, 119783. 10.1016/j.biomaterials.2020.119783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sahu S. P.; Cates E. L. X-ray Radiocatalytic Activity and Mechanisms of Bismuth Complex Oxides. J. Phys. Chem. C 2017, 121, 10538–10545. 10.1021/acs.jpcc.7b00776. [DOI] [Google Scholar]
  24. Yang S. N.; Yang Y. L.; Yang Y.; Zhao X. Y.; Wang Q.; Li B.; Dong L.; Tian R.; Bao Z. R. Iron-Palladium Decorated Carbon Nanotubes Achieve Radiosensitization via Reactive Oxygen Species Burst. Front. Bioeng. Biotechnol. 2021, 9, 405. 10.3389/fbioe.2021.683363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin R. F.; Feinendegen L. E. The quest to exploit the Auger effect in cancer radiotherapy - a reflective review. Int. J. Radiat. Biol. 2016, 92, 617–632. 10.3109/09553002.2015.1136854. [DOI] [PubMed] [Google Scholar]
  26. Xie J. N.; Gong L. J.; Zhu S.; Yong Y.; Gu Z. J.; Zhao Y. L. Emerging Strategies of Nanomaterial-Mediated Tumor Radiosensitization. Adv. Mater. 2019, 31, 1802244. 10.1002/adma.201802244. [DOI] [PubMed] [Google Scholar]
  27. Chen H.; Gu Z.; An H.; Chen C.; Chen J.; Cui R.; Chen S.; Chen W.; Chen X.; Chen X.; Chen Z.; Ding B.; Dong Q.; Fan Q.; Fu T.; Hou D.; Jiang Q.; Ke H.; Jiang X.; Liu G.; Li S.; Li T.; Liu Z.; Nie G.; Ovais M.; Pang D.; Qiu N.; Shen Y.; Tian H.; Wang C.; Wang H.; Wang Z.; Xu H.; Xu J.-F.; Yang X.; Zhu S.; Zheng X.; Zhang X.; Zhao Y.; Tan W.; Zhang X.; Zhao Y. Precise nanomedicine for intelligent therapy of cancer. Sci. China: Chem. 2018, 61, 1503–1552. 10.1007/s11426-018-9397-5. [DOI] [Google Scholar]
  28. Hossain M.; Su M. Nanoparticle Location and Material-Dependent Dose Enhancement in X-ray Radiation Therapy. J. Phys. Chem. C 2012, 116, 23047–23052. 10.1021/jp306543q. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gong L.; Xie J.; Zhu S.; Gu Z.; Zhao Y. Application of Multifunctional Nanomaterials in Tumor Radiosensitization. Acta Phys.-Chim. Sin. 2018, 34, 140–167. [Google Scholar]
  30. Fan W.; Tang W.; Lau J.; Shen Z. Y.; Xie J.; Shi J. L.; Chen X. Y. Breaking the Depth Dependence by Nanotechnology-Enhanced X-Ray-Excited Deep Cancer Theranostics. Adv. Mater. 2019, 31, 1806381. 10.1002/adma.201806381. [DOI] [PubMed] [Google Scholar]
  31. Wathen C.; Foje N.; Avermaete T.; Miramontes B.; Chapaman S.; Sasser T.; Kannan R.; Gerstler S.; Leevy W. In vivo X-Ray Computed Tomographic Imaging of Soft Tissue with Native, Intravenous, or Oral Contrast. Sensors 2013, 13, 6957–6980. 10.3390/s130606957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singh J.; Daftary A. Iodinated contrast media and their adverse reactions. J. Nucl. Med. Technol. 2008, 36, 69–74. 10.2967/jnmt.107.047621. [DOI] [PubMed] [Google Scholar]
  33. Lusic H.; Grinstaff M. W. X-ray-Computed Tomography Contrast Agents. Chem. Rev. 2013, 113, 1641–1666. 10.1021/cr200358s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhang C.; Ren J.; Hua J.; Xia L.; He J.; Huo D.; Hu Y. Multifunctional Bi2WO6 Nanoparticles for CT-Guided Photothermal and Oxygen-free Photodynamic Therapy. ACS Appl. Mater. Interfaces 2018, 10, 1132–1146. 10.1021/acsami.7b16000. [DOI] [PubMed] [Google Scholar]
  35. Feng L.; Yang D.; Gai S.; He F.; Yang G.; Yang P.; Lin J. Single bismuth tungstate nanosheets for simultaneous chemo-, photothermal, and photodynamic therapies mediated by near-infrared light. Chem. Eng. J. 2018, 351, 1147–1158. 10.1016/j.cej.2018.06.170. [DOI] [Google Scholar]
  36. Zang Y.; Gong L.; Mei L.; Gu Z.; Wang Q. Bi2WO6 Semiconductor Nanoplates for Tumor Radiosensitization through High-Z Effects and Radiocatalysis. ACS Appl. Mater. Interfaces 2019, 11, 18942–18952. 10.1021/acsami.9b03636. [DOI] [PubMed] [Google Scholar]
  37. Heuer-Jungemann A.; Feliu N.; Bakaimi I.; Hamaly M.; Alkilany A.; Chakraborty I.; Masood A.; Casula M. F.; Kostopoulou A.; Oh E.; Susumu K.; Stewart M. H.; Medintz I. L.; Stratakis E.; Parak W. J.; Kanaras A. G. The Role of Ligands in the Chemical Synthesis and Applications of Inorganic Nanoparticles. Chem. Rev. 2019, 119, 4819–4880. 10.1021/acs.chemrev.8b00733. [DOI] [PubMed] [Google Scholar]
  38. Ren X.; Huo M.; Wang M.; Lin H.; Zhang X.; Yin J.; Chen Y.; Chen H. Highly Catalytic Niobium Carbide (MXene) Promotes Hematopoietic Recovery after Radiation by Free Radical Scavenging. ACS Nano 2019, 13, 6438–6454. 10.1021/acsnano.8b09327. [DOI] [PubMed] [Google Scholar]
  39. Liu X.; Xu Y.; Wu Z.; Chen H. Poly(N-vinylpyrrolidone)-Modified Surfaces for Biomedical Applications. Macromol. Biosci. 2013, 13, 147–154. 10.1002/mabi.201200269. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao2c01591_si_001.pdf (514.6KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES