Synergistic Combination of an Intelligent Nanozyme and Radiotherapy for Treating Renal Cancer

Introduction

According to the cancer statistics estimated in 2023, the incidence rate of tumors in the urinary system ranked among the top ten cancers.¹ Among all the estimated cases of urinary cancer, cancers occurring in the kidney and renal pelvis account for one-third of new diagnoses with renal cell carcinoma accounting for 90% of the cases with kidney cancer.² Surgery including partial nephrectomy as well as radical nephrectomy is widely performed during the treatment of renal cell carcinoma patients.^3,4 Targeted therapies utilizing chemotherapeutic drugs and immunotherapy are being utilized as strategies for systemic therapy.⁵ However, it is necessary to preserve as much nephron as possible during the treatment of renal cell carcinoma since the kidney plays an important role in the excretion of waste from the body. Meanwhile, incomplete clearance of cancer cells might lead to tumor recurrence.^6,7 Therefore, to improve the patient’s quality of life as well as inhibit tumor recurrence, researchers are exploring nanocomposites, which can specifically respond to the tumor microenvironment (TME) or can be tailored according to the tumor characteristics.^8–10 The introduction of nanocomposite promotes tumor ablation without impairing kidney function, suggesting its promising potential for treating renal cell carcinoma patients.

Compared to normal tissues, tumor tissues with high metabolism tend to consume more glucose. Due to this, glucose oxidase (GOx) which catalyzes the conversion of glucose and oxygen to gluconolactone and hydrogen peroxide has gained increasing attention in the nanomedicine field.^11–14 The energy depletion caused by these enzymes, and the release of hydrogen peroxide (H₂O₂) cause tumor cell death. In a recent study, tri-metal nanospheres decorated with glucose oxidase termed AuPtAg-GOx were engineered for synergistic tumor therapy via starvation therapy and photothermal therapy to augment immunotherapy efficacy. Under laser irradiation, the glucose depletion efficiency of AuPtAg-GOx was augmented, which enhanced the therapeutic outcome.¹⁵ Zhang et al reported a MnSiO₃@Met@GOx nanocomposite that could be triggered in mild-acidic pH and GSH overexpressing TME. H₂O₂ derived from glucose in the presence of the nanocomposite was converted to toxic hydroxyl radicals (OH) by decomposed Mn²⁺, leading to cell death and tumor ablation.¹⁶ To effectively inhibit glioma tumor recurrence after surgery, Li et al developed a sprayed gel containing GOx@MnCaP nanoparticles (NPs), which improved the therapeutic outcome. GOx decomposes glucose to produce H₂O₂, which is then converted to cytotoxic reactive oxygen species (ROS) by Mn²⁺ to induce tumor cell apoptosis and suppress proliferation.¹⁷

Recently, enzyme mimicking nanocomposites termed nanozymes are extensively being investigated.^18–20 Compared with natural enzyme, these nanozymes possess advantages such as physiological stability, suitability for mass production and high catalytic efficiency.^21,22 Numerous nanozymes have been applied for treating cancers, owing to their catalase, superoxide dismutase, peroxidase (POD) or oxidase mimicking properties.^23–25 Specifically, with high accumulation of H₂O₂ in the TME, nanozymes with peroxidase-mimicking property are capable of turning these H₂O₂ molecules into cytotoxic ROS, killing tumor cells. According to Dong et al, CaF₂ nanozyme was synthesized to enable Ca²⁺ release and ROS generation from ultrasonic (US) amplified POD-catalytic activity, which promoted Ca²⁺ overload and mitochondrial dysfunction. This strategy, based on CaF₂ nanozyme, showed enhanced ability to kill both breast tumor and liver tumor upon US exposure.²⁶ A titanium nitride-based nanoparticle termed TLGp was reported by Liu et al for photo-enhancing POD catalytic activity. GOx in TLGp produced abundant H₂O₂, which was then converted to ROS to achieve tumor therapy while minimizing side effects.²⁷ In addition, Nan et al reported iron phthalocyanine-based Fe (II) Pc-A for cascade reaction, including POD, CAT and OXD activities, where H₂O₂ was decomposed to ROS to enhance oxidative stress in cancer cells and to realize multi-modal cancer therapy.²⁸

Ionizing irradiation is widely used for suppressing tumor growth and elimination of tumors. To completely ablate tumor tissues while protecting normal tissues and augment radiotherapy (RT) efficacy, numerous noble elements have been utilized to design nanoplatforms.^20,29,30 Here, we constructed a novel nanozyme (CeO₂@Au-PEG) by decorating gold nanozyme on CeO₂ nanorods and then modified this nanocomposite with PEG. Under X-ray irradiation, energy deposited on both gold and cerium was enhanced, which further enhanced anti-tumor therapeutic efficacy of RT. Notably, although there is excessive amount of H₂O₂ in the TME compared with normal tissues, the ROS generated with the assistance of POD-mimicking nanozymes may not thoroughly eliminate tumor cells, leaving the possibility for tumor recurrence. Considering that abundant H₂O₂ is produced via the process of glucose oxidization, this nanozyme was designed to possess dual-catalytic properties including glucose-depletion as well as POD-mimicking activity (Scheme 1). In other words, it could produce sufficient H₂O₂ from intracellular glucose, which deprived cancer cells of the nutrients. Next, the generated H₂O₂ was decomposed into toxic ROS, inducing tumor cell death. Both in vitro and in vivo experiments demonstrated that the nanocomposites possessed desired biocompatibility, and that CeO₂@Au-PEG not only converted intracellular glucose into ROS by dual-catalytic properties, but also enhanced the cellular damage caused by ionizing radiation during RT to achieve highly efficient therapeutic outcome against renal cell carcinoma.

Results and Discussion

Preparation and Characterization of CeO₂@Au-PEG

The preparation of CeO₂@Au-PEG consisted of three steps, including the synthesis of porous CeO₂ nanorods with precursor of Ce(NO₃)₃·6H₂O and NaOH, loading of gold nanozyme on CeO₂ nanorods to obtain CeO₂@Au nanorods and decoration of polyethylene glycol (PEG) on nanorods to obtain CeO₂@Au-PEG nanocomposite (Figure 1a). As shown in Figure 1b, CeO₂ displayed the morphology of nanorods with a length of approximately 90 nm. CeO₂@Au was found to be composed of gold nanozyme coated around CeO₂ (Figure 1c). TEM images of Au-PEG were shown in Figure S1. Besides, the zeta potential of CeO₂, CeO₂@Au and CeO₂@Au-PEG were measured to be −11.5 mV, −21.0 mV and −23.0 mV, respectively. The change in zeta potential from −21.0 mV to −23.0 mV suggested the improved colloidal stability after PEGylation. After CeO₂@Au-PEG and Au-PEG were dissolved in phosphate buffered solution (PBS) for 24 h (Figure S2), the solution still remains uniform and clarified, demonstrating satisfied stability of CeO₂@Au-PEG and Au-PEG.

Figure 1 (a) Schematic illustration of the preparation of CeO2@Au-PEG nanocomposite NPs, TEM images showing the morphology of (b) CeO2 and (c) CeO2@Au, (d) Zeta potential of CeO2, CeO2@Au and CeO2@Au-PEG NPs.

Next, X-ray diffraction (XRD) analysis was conducted to further confirm the structure of CeO₂ and CeO₂@Au. Several peaks corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0) and (3 3 1) facets in XRD pattern of CeO₂ were identified as shown in Figure 2a. Diffraction peaks representing gold nanozyme including (1 1 1), (2 0 0), (2 2 0) and (3 1 1) crystal plane were observed in the XRD pattern of CeO₂@Au (Figure 2b). X-ray photoelectron spectroscopy (XPS) wide scan spectra of CeO₂ and CeO₂@Au are displayed in Figures S3 and 2c. It can be seen that cerium and oxygen were present in the obtained CeO₂. And XPS spectra of CeO₂@Au showed typical peaks including Ce 3d, O 1s and Au 4f, confirming the existence of cerium, oxygen and gold. Further investigation of high-resolution spectra of cerium from CeO₂ and CeO₂@Au are shown in Figures S4 and 2d, which possessed similar binding energy peaks. Typically, the Ce 3d spectra of CeO₂@Au showed five peaks labeled u and five peaks labeled which can be attributed to 3d_5/2 and 3d_3/2 respectively. Moreover, the binding energy peaks of gold in the high-resolution spectra at 87.6 eV and 83.8 eV were attributed to 4f_5/2 and 4f_7/2 respectively. The binding energy peaks at 531.3 eV and 529.6 eV were designated to surface oxygen and lattice oxygen (Figure 2e). The above results thus demonstrated the successful fabrication of CeO₂@Au-PEG.

Figure 2 (a and b) XRD patterns of CeO2 and CeO2@Au NPs. (c) XPS analysis of CeO2@Au NPs. (d–f) High-resolution XPS spectra of Ce 3d, Au 4f and O 1s.

Figure 3 (a) Schematic illustration of ROS generation by CeO2@Au-PEG in cells. (b and c) Evaluation of oxidation of glucose by measuring pH values and H2O2 concentration. (d) Evaluation of peroxidase-like activities of various CeO2@Au-PEG concentrations.

Evaluation of the in vitro Catalytic Activity of the Nanocomposite

Endowed with dual-catalytic properties, CeO₂@Au-PEG was internalized by cells, generating sufficient H₂O₂ from glucose, the primary source of cellular energy. Next, H₂O₂ was then converted to ROS by POD-mimicking properties of CeO₂@Au-PEG (Figure 3a). Ionizing irradiation caused significant damage in cells when treated in combination with CeO₂@Au-PEG. To evaluate these two reaction processes, both POD-mimicking catalytical activity and glucose consuming of CeO₂@Au-PEG were measured. The oxidation of glucose by gold nanozyme of CeO₂@Au-PEG can produce H₂O₂ and gluconic acid, resulting in a decrease in the pH value. As shown in Figure 3b, a reduction of pH values was seen as a consequence of gluconic acid generation, indicating the oxidation of glucose in the presence of CeO₂@Au-PEG. Moreover, an increase of H₂O₂ concentration was observed in the group treated with glucose + Au-PEG (Figure 3c). The POD-mimicking activity was also measured using 3, 3’, 5, 5’ - tetramethylbenzidine (TMB), which turned blue in the presence of ·OH. As shown in Figure 3d, typical absorption peaks at wavelength of 450 nm increased as the concentration of CeO₂@Au-PEG increased, which was due to the generation of ·OH by the catalytic activity of CeO₂@Au-PEG. The solution became more blue as concentration of CeO₂@Au-PEG increased. This confirmed that CeO₂@Au-PEG contributed to production of ·OH derived from H₂O₂, which was in turn generated upon the oxidation of glucose.

Evaluation of the Catalytic Performance of CeO₂@Au-PEG in Cancer Cells

Inspired by the above results, we further evaluated the antitumor effects of CeO₂@Au-PEG in vitro. Before the application of CeO₂@Au-PEG to cancer cells, it was necessary to confirm its biocompatibility. Cytotoxicity of CeO₂@Au-PEG was tested at various concentrations on tubular epithelial cells (TECs) and murine renal adenocarcinoma RENCA cells. As shown in Figure 4a, no obvious decrease of cell viability was observed in both cell lines. And over 80% cells survived at a concentration of 200 μg/mL. After PEGylation, Cy 5 labeled-CeO₂@Au-PEG exhibited rapid cellular uptake 1 h after co-incubation (Figure 4b). Moreover, the efficacy of RT was enhanced by CeO₂@Au-PEG in vitro. To investigate the amount of ·OH produced during this process, ROS-staining was conducted using HPF probes. As shown in Figure 4c, no apparent green fluorescence was observed in both the control and glucose free groups while a weak fluorescence was seen in the glucose free + H₂O₂ group, which could be attributed to the POD-mimicking property of CeO₂@Au-PEG. On the contrary, a significantly high level of ROS was detected in the high glucose + H₂O₂ group, which was generated from the cascade catalytical reaction mediated by CeO₂@Au-PEG. Next, to validate the dual-catalytic activities on cells, 8 treatment groups were established, including glucose free, glucose free + H₂O₂, glucose free + RT, glucose free + H₂O₂ + RT, high glucose, high glucose + H₂O₂, high glucose + RT and high glucose + H₂O₂ + RT (Figure 4c). It can be seen that in the absence of glucose, CeO₂@Au-PEG showed a weak toxicity on RENCA cells. After the introduction of H₂O₂, there was a decrease in cell survival due to the POD-catalytic activity of CeO₂@Au-PEG. In addition, cell viability was lower upon exposure to RT. The survival rate of cells in the glucose free + H₂O₂ + RT group decreased to 45.7% due to the therapeutic damage mediated by ·OH generation and ionizing irradiation. Cells treated with high glucose only exhibited suppression due to ROS generation from CeO₂@Au-PEG mediated POD-mimicking process. When adding H₂O₂, a decreasing in cell viability was observed thanks to outstanding POD-like property of CeO₂@Au-PEG. A slightly higher cell death was caused by high glucose + H₂O₂ + RT group than high glucose + RT group, which was due to the fact that the H₂O₂ level in mimicking tumor microenvironment was relatively low. In addition, with the addition of glucose and H₂O₂, CeO₂@Au-PEG suppressed the tumor cells, which was likely due to the ROS involved in cascade reaction of glucose oxidase and H₂O₂ depletion. Over 70% of RENCA cells were not viable in the high glucose + H₂O₂ + RT group. Compared with the glucose free + H₂O₂ + RT group, the presence of glucose greatly elevated the cytotoxicity of the nanocomposite. Furthermore, DNA damage post various treatments was assessed by γ-H₂AX staining (Figure 4e). Consistent with the result of CCK 8 assay, the most apparent red fluorescence was observed in the group treated with high glucose + H₂O₂ + RT, suggesting the most severe DNA damage in this group. Further investigation of the expression of caspase 3 in various group confirm the apparent cell apoptosis in CeO₂@Au-PEG enhanced RT in glucose and H₂O₂ situation (Figures S6). Collectively, the above results indicated that CeO₂@Au-PEG was capable of catalyzing the conversion of intracellular glucose to ROS and decreasing tumor cell survival under RT.

Figure 4 (a) Viability of TEC and RENCA cells pre-treated with CeO2@Au-PEG. (b) Cellular uptake of Cy 5 labeled CeO2@Au or CeO2@Au-PEG by RENCA cells (Scale bar: 50 μm). (c) Confocal fluorescence images showing ·OH generation in RENCA cells treated with CeO2@Au-PEG at various concentrations using HPF probe (green) (Scale bar: 50 μm). (d) Viability of RENCA cells measured by CCK8 assay after various treatments (Scale bar: 50 μm). (e) DNA damage assessment by γ-H2AX staining (Scale bar: 20 μm; group 1: Glucose free, group 2: Glucose free+H2O2, group 3: Glucose free+RT, group 4: Glucose free+H2O2+RT, group 5:High glucose, group 6: High glucose+RT, group 7: High glucose+H2O2, group 8: High glucose+H2O2+RT). *** p < 0.001. Statistical analyses were conducted using t-test.

Figure 5 (a) H&E staining of excised organ tissues post injection of saline, CeO2-PEG or CeO2@Au-PEG NPs (Scale bar: 50 μm). (b-g) Blood routine analysis of mice under different treatment.

Figure 6 (a) The tumor growth curves and (b) body weight change curves during different treatments (n = 5). (c–e) H&E staining and dihydroethidium (DHE) staining and DCFH-DA staining of tumor tissues after various treatments (Scale bar: 50 μm). *** p < 0.001. Statistical analyses were conducted using t-test.

Assessment of in vivo Biocompatibility

To confirm the biosafety of CeO₂-PEG and CeO₂@Au-PEG in vivo, main organs including heart, liver, spleen, lung and kidney of mice injected with saline, CeO₂-PEG or CeO₂@Au-PEG were analyzed by hematoxylin and eosin (H&E) staining (Figure 5a). No apparent lesions were observed in mice administrated with CeO₂-PEG or CeO₂@Au-PEG. Blood routine analysis of parameters including hematocrit (HCT), hemoglobin (HGB), platelet (PLT), red blood cell (RBC), white blood cell (WBC) and mean platelet volume (MPV) in Figure 5c-g demonstrated that blood samples from mice administrated with CeO₂-PEG or CeO₂@Au-PEG were close to the values in the control group. These comprehensive results demonstrated that CeO₂-PEG and CeO₂@Au-PEG had good biocompatibility in vivo.

In vivo Efficacy of CeO₂@Au-PEG in a Mouse Model of Renal Cancer

To verify the anti-renal tumor efficacy of CeO₂@Au-PEG in combination with radiotherapy, subcutaneous tumors were established in BALB/c mice. First of all, biodistribution of CeO₂@Au-PEG in mice post injection via tail vein was analyzed. As shown in Figure S6, most apparent fluorescence intensity was detected in tumor region at 12 h post injection. Four groups were set up including the control, RT, CeO₂@Au-PEG and CeO₂@Au-PEG + RT groups. Tumor volume and body weight in each group were measured every 3 days and mice were executed on day 16 post treatment. As shown in Figure 6a and b, tumor volume in the group treated with PBS showed significant increase with time. Apparent tumor suppression was observed in the group treated with CeO₂@Au-PEG, thus proving the cascade catalytical reaction mediated by CeO₂@Au-PEG. However, therapeutic efficacy of either RT or CeO₂@Au-PEG alone failed to meet the demand for strong tumor inhibition. By striking contrast, the application of CeO₂@Au-PEG + RT dramatically impeded the tumor growth. Besides, by the end of tumor growth monitoring, tumor volume in the control group and RT group was 5 times and 3 times higher than that in the CeO₂@Au-PEG group, respectively. The image of tumors in four groups in Figure S7 further confirmed the results. There was no significant difference in the body weight of mice body among the four groups.

The in vivo effects of different treatments were evaluated by H&E staining and dihydroethidium (DHE) staining of the tumors. As shown in Figure 6c, moderate cell damages were observed in the RT group and CeO₂@Au-PEG group compared with the control group. H&E images demonstrated severe injuries in the CeO₂@Au-PEG + RT group. Consistent with the H&E staining results, the CeO₂@Au-PEG + RT group showed the brightest red fluorescence which was positively correlated to the amount of superoxide anion (O₂•^‾). Meanwhile, green fluorescence representing hydroxyl radicals (•OH) further confirmed the generation of ROS by CeO₂@Au-PEG + RT in vivo.

Conclusion

In conclusion, a novel CeO₂@Au-PEG nanocomposite with dual-catalytic activities was synthesized to enhance the anti-tumor efficacy of RT in a mouse model of renal cancer. On one hand, CeO₂@Au-PEG could convert glucose in tumor cells into gluconic acid and H₂O₂, due to its ability to oxidize glucose, cutting off the main source of energy to the cancer cells. On the other hand, the resultant H₂O₂ generated in this process was then decomposed into cytotoxic ·OH, causing cell damage. Moreover, composed of two noble elements, CeO₂@Au-PEG enhanced the therapeutic efficacy of RT, leading to stronger anti-tumor effects. CeO₂@Au-PEG showed great biocompatibility both in vitro and in vivo. After treating renal tumor bearing mice with CeO₂@Au-PEG, the tumor cells internalized it, leading to a series of chain reactions which generated a large amount of ROS. Meanwhile, CeO₂@Au-PEG amplified the therapeutic effects of RT. The tumor volume shrank remarkably in the group that received the combination of CeO₂@Au-PEG + RT. In summary, our work provides a novel strategy to treat renal tumors via cascade reaction and radiosensitization mediated by CeO₂@Au-PEG nanocomposite.