Enzymatic Activity Tunable Ceria Nanoparticles For The Prevention Of Chemotherapy-induced AKI

Chemotherapy-induced acute kidney injury（AKI）is an adverse event with high morbidity and mortality that severely affects the therapeutic efficiency and survival of cancer patients receiving chemotherapy.Various chemotherapeutic agents excreted by kidney can damage the structure and functional constituents of renal cells through the generation of reactive oxygen species（ROS）,thus inducing AKI.Hence,ROS are one of the critical targets in the prevention and therapy of AKI.Small molecular antioxidants can be utilized to scavenge excessive ROS in clinic,which has shown some success in the protection of kidney from chemotherapeutic agents.Although small molecular antioxidants can alleviate AKI by reducing ROS,they can also reduce ROS level generated by chemotherapy of tumor site due to their lack of specificity,which seriously affects the therapeutic effect of chemotherapeutic drugs,resulting in accelerated tumor progression and metastasis.This limits further application of these small molecular antioxidants in the prevention and treatment of AKI.Alternatively,nanomaterials with ROS-regulating capacities have been studied to broaden the therapeutic strategies for AKI.However,these nanomaterials that indiscriminately reduce oxidative stress in tumour tissues are argued to spur the growth and metastasis of tumors,hindering their potential application for chemotherapy-induced AKI prevention in cancer patients.Therefore,it is of great significance to develop materials that can selectively scavenge ROS at the target site for the prevention and treatment of AKI caused by chemotherapeutic drugs.Previous reports demonstrated that the microenvironment of tumor is slightly acidic compared with the kidney and other organs as well as tissues.Consequently,by exploiting the pathophysiological differences between kidney and tumour,especially the p H discrepancy,we report enzymatic activity tunable ceria nanoparticles that can reconcile AKI prevention and potent chemotherapy.In this paper,we first designed and synthesized oleylamine-coated ceria nanoparticles,then charactered the structure of them by transmission electron microscopy（TEM）,high-reslution transmission electron microscopy（HRTEM）and X-ray photoelectron spectroscopy（XPS）.These results showed that these ceria nanopartilces with a diameter of～3 nm had a near-spherical morphology and excellent crystallinity.The result also confiemed that Ce³⁺and Ce⁴⁺co-exist on the surface of ceria nanoparticles.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy（polyethylene glycol）-2,000](DSPE-PEG_2K)was used to impart the colloidal stability of the CNPs in aqueous solution.The TEM and dynamic light scattering（DLS）were employed to analyze the dispersibility and size of CNPs.The results showed that CNPs had a good monodispersity with a size of～9.7 nm.The enzymatic activity of CNPs was measured using a specific oxygen electrode on Multi-Parameter Analyzer under different p H conditions.The results showed that CNPs exhibited high catalase（CAT）activity at p H 7.4 while showed no CAT activity at p H 6.0.Additionally,the chemical mechanism for the tunable enzymatic activity of CNPs was further investigated by X-ray diffraction（XRD）and Raman spectrum.The results indicated that the redox reaction is initiated between CNPs and surface-absorbed H₂O₂.The decomposition products of H₂O₂ are released to re-expose the surface of CNPs.While under acidic condition,excess H⁺can inhibit the conversion of Ce⁴⁺to Ce³⁺that catalyzes the decomposition of surface-absorbed H₂O₂,which in turn disrupts the re-exposure of active catalytic sites and blocks the antioxidant cycles of CNPs.The cytoprotective effects of CNPs on HK-2 cells and ES-2 cells was analyzed by SRB assay under different p H conditions.The results showed that CNPs exhibited protective effects on HK-2 cells and ES-2 cells at p H 7.4,but can not inhibit the cisplatin（DDP）-induced cytotoxicity at p H6.5,indicating their context-dependent cytoprotective ability.Sigificantly,the cytoprotective activity of CNPs cannot be recovered upon pre-treatment with H₂O₂ at acidic condition.The molecular mechanism underlying the capability of CNPs to prevent chemotherapy-induced AKI was further interrogated by flow cytometry,westren blot,q RT-PCR,and TUNEL staining.The results showed that CNPs significantly inhibited the DDP-induced apoptosis in HK-2 cells.The level of ROS in HK-2 cells was analyzed by flow cytometry;the activity of SOD and level of MDA was detected to assess the cellular redox homeostasis.The in vitro results showed that CNPs effectively scavenge the excessive ROS and restore the cellular homestasis.The advantages of CNPs in the prevention of chemotherapy-induced AKI was confirmed by comparing with N-acetylcysteine（NAC）.The in vivo results demonstrated that both CNPs and NAC treatments showed success in protection of kidney.The anti-tumour efficiency of DDP is found to be suppressed by NAC due to the decrement of tumoricidal ROS.However,it is not affected by CNPs whose ROS scavenging activities are turned off once exposed to the acidic tumour microenvironment.By artificially raising the p H of the tumor microenvironment,the effect of enzymatic activity tunable CNPs on chemotherapeutic efficacy was further verified.The results showed that CNPs weakened the anti-tumour efficiency of DDP when the intratumoral p H is raised.The effect of CNPs on the overall chemotherapeutic outcome was examined by examining the survival rates of mice after different treatments.The results showed that CNPs can significantly improve the survival rate of mice receiving high-dose chemotherapy drugs.The in vivo results showed that CNPs regulated ROS-invovled genes to quench the ROS in kidneys.Additionally,the effects of CNPs on ROS-related pathways were investigated by western blot,q RT-PCR,and gene knockout techniques.The results showed that CNPs participated the upregulation of Nrf2 and activated the Nrf2/Keap1 pathway to regulate ROS-related genes,thus ultimately reducing the intracellular ROS levels.The anti-apoptotic effect of CNPs on Si Nrf2 transfected HK-2 cells was further investigated by western blot and the results showed that CNPs failed to suppress the expression of apoptotic proteins.In summary,this enzymatic activity tunable CNPs can protect renal cells under neutral conditions,and do not affect the cytotoxicity of chemotherapeutic agents to tumor cells under acidic conditions.The CNPs can activate the Nrf2/Keap1 pathway to scavenge the excessive ROS and restore the cellular redox homeostasis,thus inhibiting apoptosis and preventing chemotherapy-induced AKI.Additionally,CNPs exerted no effect on the potency of chemotherapeutic agents in acidic tumour microenvironment.The CNPs could significantly enhance the survival rate of tumour-bearing mice treated with a high dose of DDP.This CNPs incorporated with context-dependent ROS scavenging ability that can open a novel avenue for future design and development of drugs for AKI prevention and treatment.