#5050 ANTIOXIDANT PORPHYRIN-BASED NANOZYME WITH SINGLE RUTHENIUM FOR TREATMENT OF ACUTE KIDNEY INJURY
Authors: Chen Shanshan , LI Qian , Cheng Chong , Su Baihai 
Published: June 2023

Abstract

Background and Aims
Acute kidney injury (AKI) describes a sudden loss of kidney function characterized by elevated serum creatinine (CRE) levels and reduced urinary output. AKI patients have a high mortality rate, but limited treatment options are currently available. During the AKI progression, an excess of reactive oxygen species (ROS) leads to oxidative stress, which often results in cell damage and renal failure. Therefore, ROS scavenging has been considered a promising strategy for AKI treatment. Here we report a porphyrin-based nanozyme with single ruthenium (Por-Ru) as a ROS scavenger to cure AKI.

Method
The Por-Ru nanozyme was synthesised by one-pot synthesis. First, the 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (Por) was added to the solvent containing N, N-dimethylformamide. The RuCl3 was then introduced to form Ru-N coordinated porphyrin networks. We analysed the morphology and chemical structure using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). We also tested its enzyme-mimicking abilities to scavenge ROS. The catalase-like (CAT) property was tested using a peroxide-titanium complex method, and the superoxide dismutase-like (SOD) property was tested using a •O2− scavenging assay. In vitro, the protective effect of Por-Ru nanozyme against ROS damage was examined using the mouse tubular epithelial (TCMK-1) cell line. After biocompatibility evaluation, the therapeutic efficacy of Por-Ru nanozyme was investigated in glycerol-induced AKI mice.

Results
1. Characterizations of Por-Ru nanozyme: The SEM image showed the Por-Ru nanozyme was spherical with an average diameter of 6-8 nm (Figure 1a). Meanwhile, the XPS revealed the presence of Ru and showed the detailed elemental components (Figure 1b). As shown in Figures 1c and d, the Por-Ru nanozyme had excellent CAT-like and SOD-like capabilities. Furthermore, the DPPH• assay confirmed the antioxidant property (Figure 1e).
2. Protective effect of Por-Ru nanozyme in vitro and in vivo: The results of cell counting kit-8 assay showed that Por-Ru nanozyme was not cytotoxic to TCMK-1 cells at concentrations ranging from 10 to 100 µg/mL. We further examined the cytoprotective effects of Por-Ru nanozyme against ROS damage. The flow cytometry results showed that the intracellular ROS level increased after treatment with H2O2, while the ROS level obviously decreased with the addition of Por-Ru nanozyme (Figure 2a, b). The protective effect of Por-Ru nanozyme against H2O2-induced apoptosis was also investigated. As shown in Figures 2c and d, the rate of cell apoptosis was significantly reduced with the addition of Por-Ru nanozyme. Before the in vivo experiments, the haemolysis rate of Por-Ru nanozyme was examined and found to be less than 5% even at a dose of 200 µg/mL. Additionally, the in vivo toxicity assessment showed that Por-Ru nanozyme at test concentrations had no noticeable impacts on blood parameters, liver and kidney functions, and histopathology of major organs in normal mice. Then, we tested the therapeutic effect of Por-Ru nanozyme in AKI mice. As shown in Figures 2e and f, the CRE and blood urea nitrogen (BUN) levels of AKI mice treated with Por-Ru nanozyme were significantly lower than those of AKI mice group. Additionally, the results of haematoxylin and eosin (H&E) staining showed that many casts (marked as triangles) were observed in the kidney tissue of AKI mice, whereas no obvious damage area was found in AKI mice after the treatment of Por-Ru nanozyme.

Conclusion
Our findings demonstrate that the Por-Ru nanozyme has excellent anti-ROS enzyme-mimicking capabilities. In vitro experiments revealed that Por-Ru nanozyme alleviated ROS-induced cell apoptosis. In AKI mice, Por-Ru nanozyme effectively improved renal function and attenuated renal tissue damage. Taken together, Por-Ru nanozyme may become a novel agent for AKI treatment.