Redox‐Mediated Artificial Non‐Enzymatic Antioxidant MXene Nanoplatforms for Acute Kidney Injury Alleviation

Abstract Acute kidney injury (AKI), as a common oxidative stress‐related renal disease, causes high mortality in clinics annually, and many other clinical diseases, including the pandemic COVID‐19, have a high potential to cause AKI, yet only rehydration, renal dialysis, and other supportive therapies are available for AKI in the clinics. Nanotechnology‐mediated antioxidant therapy represents a promising therapeutic strategy for AKI treatment. However, current enzyme‐mimicking nanoantioxidants show poor biocompatibility and biodegradability, as well as non‐specific ROS level regulation, further potentially causing deleterious adverse effects. Herein, the authors report a novel non‐enzymatic antioxidant strategy based on ultrathin Ti3C2‐PVP nanosheets (TPNS) with excellent biocompatibility and great chemical reactivity toward multiple ROS for AKI treatment. These TPNS nanosheets exhibit enzyme/ROS‐triggered biodegradability and broad‐spectrum ROS scavenging ability through the readily occurring redox reaction between Ti3C2 and various ROS, as verified by theoretical calculations. Furthermore, both in vivo and in vitro experiments demonstrate that TPNS can serve as efficient antioxidant platforms to scavenge the overexpressed ROS and subsequently suppress oxidative stress‐induced inflammatory response through inhibition of NF‐κB signal pathway for AKI treatment. This study highlights a new type of therapeutic agent, that is, the redox‐mediated non‐enzymatic antioxidant MXene nanoplatforms in treatment of AKI and other ROS‐associated diseases.

LiF/HCl etching solution, and the mixture reacted for 2 d at 35 C under magnetic stirring. The resultant Ti 3 C 2 T x suspension was repeatedly washed with deionized water and centrifuged to remove the residual LiF. The delamination of Ti 3 C 2 T x was conducted by prolonged sonication treatment under N 2 atmosphere for 1 h, and followed by centrifugation for 30 min at 3500 rpm to obtain a homogeneous supernatant with delaminated Ti 3 C 2 (MXene). The concentration of MXene colloidal solution was determined by filtering a known volume of the solution on a polypropylene filter (Celgard 3501 coated PP) and measuring the weight of the resulting freestanding film after vacuum drying. For the surface modification of Ti 3 C 2 nanosheets, 10 mL Ti 3 C 2 (5 mg mL -1 ) and 200 mg PVP were dissolved into 100 mL of anhydrous ethanol and refluxed at 50 °C for 4 h. The excess PVP was removed by centrifugation at 10 000 rpm for 30 min. Afterward, the resulting Ti 3 C 2 -PVP was washed with ethanol and water for further use.
Characterizations: Scanning electron microscopy (SEM) images were recorded using a JSM-5900LV SEM microscope (JEOL, Japan) at an accelerating voltage of 15 kV. Transmission electron microscopy (TEM) images were obtained on a high-resolution transmission electron microscope (Tecnai G2 F20S-TWIN) equipped with a field emission gun operating at 200 kV. X-ray photoelectron spectrum (XPS) was performed on an XSAM800 X-ray Photoelectron Spectrometer (Kratos Company, UK) with the Al Kα radiation (hv =1486.6 eV). X-ray diffraction (XRD) analysis was carried out on a Japan Rigaku X-ray diffractometer (UItima Ⅳ) from 2θ angle of 5 ° to 45 ° using Cu Kα radiation (λ = 0.154056 nm) at a scanning speed of 10° min −1 . The UV-Vis-NIR absorption was measured in the wavelength range of 300-1000 nm using an ultraviolet-visible near-infrared (UV-vis-NIR) spectrophotometer (UV3600, Shimadzu, Japan). Tapping  Spin-polarization density functional theory (DFT) calculations within the generalized gradient approximation (GGA) using the Perdew-Burke-Ernzerhof (PBE) formulation. We have chosen the projected augmented wave (PAW) potentials to describe the ionic cores and take valence electrons into account using a plane wave basis set with a kinetic energy cut-off of 450 eV. Partial occupancies of the Kohn−Sham orbitals were allowed using the Gaussian smearing method and a width of 0.05 eV. The electronic energy was considered self-consistent when the energy change was smaller than 10 −6 eV. A geometry optimization was considered convergent when the energy change was smaller than 0.05 eV Å −1 . Finally, the adsorption energies (E ads ) were calculated as E ads = E ad/sub -E ad -E sub , where E ad/sub , E ad , and E sub are the total energies of the optimized adsorbate/substrate system, the adsorbate in the gas phase, and the clean substrate, respectively. The free energy was calculated using the equation: G = E + ZPE -TS, where G, E, ZPE and TS are the free energy, total energy from DFT calculations, zero point energy and entropic contributions (T was set to be 300K), respectively.
Cell culture: The immortalized proximal tubule epithelial (HK-2) cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The HK-2 cells were cultured in Keratinocyte-SFM (Gibco, Rockville, MD, USA) supplemented with recombinant epidermal growth factor (0.5 ng mL -1 ) and bovine pituitary extract (25 μg mL -1 ) at 37 °C in an incubator supplied with a humidified atmosphere with 5% CO 2 .
In vitro ROS scavenging using TPNS: HK-2 cells were seeded into a 96-well plate at 5000 cells per well and incubated at 37 °C for 24 h under 5% CO 2 . Then, TPNS with different concentrations (0, 5, 10 μg mL -1 ) were added to culture media, and after 30 min incubation, cells were treated with 250 µM H 2 O 2 . After 24 h incubation under 5% CO 2 at 37 °C, cell viability was determined by cell counting kit-8 (CCK-8, Dojindo, Japan) assay. Republic of China). Informed consent was obtained for any experimentation with human subjects, and all regulations (e.g. IRB) were fulfilled for using human blood. In brief, 2 mL of whole blood was centrifuged for 10 min at 1000 rpm to collect RBCs and gently washed thrice with PBS solution. Then, 10 μL of diluted RBCs suspension was mixed with 990 μL TPNS dispersion at various concentrations (1-100 μg mL -1 ). The mixed dispersions were incubated for 3 h at 37 °C and then centrifuged at 3500 rpm for 5 min before observing and recording the hemolysis phenomenon. The hemolysis ratio was quantified by measuring the absorbance value of supernatant at 540 nm with a microplate reader.
Deionized water and PBS solution were used as the positive and negative control, respectively.
Furthermore, after incubation for 3 h, 50 μL of the mixed dispersions were added to paraformaldehyde fixative. The fixed dispersions were centrifuged for 5 min at 3500 rpm, and then re-suspended with 1 mL deionized water. 50 mL of suspended RBCs dispersion was casted on RBCs smear, and was images using a 3D laser scanning microscope (VK-150K, EYENCE, Japan).
The cytotoxicity of TPNS was determined by the CCK-8 assay in vitro. Briefly, HK-2 cells were seeded into 96-well culture plates at the density of 5000 cells per well and incubated at 37 °C in an incubator with 5% CO 2 for 24 h. Afterwards, the cell culture medium was aspirated and fresh culture media containing various concentrations of TPNS (0-50 μg mL -1 ) were added. After 24 incubation, cells were gently washed twice with sterile PBS and then treated with 100 μL fresh culture medium and 10 μL CCK-8 solution, and further incubated at 37 °C for 2 h. The cell viability was then quantified by measuring the absorbance value at 450 nm by a microplate reader (Bio-Rad, Hercules, CA). To investigate the in vivo degradation and metabolism process, the urine and feces of mice were collected at different time points (6, 12, and 24 h) after intravenous injection of TPNS into normal mice. The Ti contents in urine and feces were quantitatively determined by ICP-MS. were sacrificed to harvest major organs including heart, liver, spleen, lung, and kidney, and the fluorescence images of these organs were obtained by a fluorescence (Caliper Life Sciences, IVIS Spectrum) imaging system.

Treatment of AKI mice:
Two hours after the AKI model induction, different treatments were performed on AKI model mice: group 1 was healthy mice treated with PBS (n = 5); group 2 was healthy mice (n = 5) treated with TPNS (5 μg in 100 μL PBS), group 3 was AKI mice treated with PBS (PBS, n = 5); group 4 was AKI mice treated with TPNS (5 μg in 100 μL PBS, n = 5). The TPNS was administrated once. The survival curve were monitored for 2 weeks after treatment. The body weight variations in each group after treatment were monitored for 24 h.

Kidney function test:
Kidney function tests were performed to evaluate the treatment of AKI. After 24 h post injection, mice were sacrificed to collect blood samples for detecting the BUN and CRE levels using the corresponding detection kits according to the manufacturer's instructions.

Confocal imaging of ROS production in kidneys:
To assess superoxide production histologically, kidneys of mice were collected and stored in optimum cutting temperature (O.C.T.) specimen matrix for cryostat sectioning at -20 °C. The frozen kidneys were further sectioned into 5 μm tissue slices.
Frozen kidney tissue slice were washed with PBS and stained with 1 mM dihydroethidium (DHE) for 30 min to detect ROS formation. Then a cover glass was applied to each slide and confocal imaging was performed using a Nikon A1R confocal microscope.
In vivo toxicity assessment: Healthy mice were randomized into two groups: group 1 was healthy mice intravenously injected with 100 µL PBS as the control group (n = 5), the other group was healthy mice intravenously injected 5 µg of TPNS in 100 µL PBS (n = 5). The mice were euthanized at 24 h post-injection. Subsequently, the major organs (heart, liver, spleen, lung, and kidney) and blood samples were harvested. Histological changes in organs were analyzed by H&E staining, and whole blood samples were used for hematology analysis. The plasma of blood samples was collected after centrifugation at 2000×g for 15 min at 4 °C, and the serum biochemistry test included two important indicators of hepatic function as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and two indicators of kidney function as blood urea nitrogen (BUN) and creatinine (CRE).
Serum TNF-α and IL-6 levels were quantified by the ELISA assay.

RT-PCR array:
For PCR analysis, total RNA was isolated with an RNA extraction Kit (Vazyme, China) according to the manufacturer's protocol. RNA (1 µg) was transcribed into cDNA using reverse transcriptase (Vazyme, China). Each PCR array was a 96-well plate containing gene specific optimized real-time PCR primer sets for 90 genes related to NF-κB signaling pathway. All treatments were performed in triplicate to calculate statistical significance and the results were calculated using the 2-ΔΔCt method. Genes with fold-changes more than 2 or less than 1/2 were considered to have biological significance.

Western blot analysis:
The total proteins from kidney tissue were extracted using ice-cold RIPA lysis buffer containing phosphatase and protease inhibitor cocktail (Beyotime, China). The concentrations of extracted proteins were determined using a BCA protein assay kit (Biosharp, China). An equal amount of protein from each sample was run in 10% SDS-PAGE gel, then transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). PVDF membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies (1:1000, Abcam, UK) for 1 h at room temperature. The intensity of bands was visualized and determined using a ChemiDoc™ XRS detection system (Bio-Rad, USA). Primary antibodies used were: Drp-1 (1/3000, Novus, USA), Opa-1 (1/3000, Novus, USA), NF-κB p65 (1/1000, proteintech), p-NF-κB p65 (Ser536)  Statistical Analysis: Quantitative data were presented as mean ± standard deviation (mean ± SD).
One-way analysis of variance was performed to determine the statistical differences; differences were accepted as significant at a P-value lower than 0.05. Figure S1. a) FTIR spectra of Ti 3 C 2 , PVP, and Ti 3 C 2 -PVP, indicating the successful surface modification of Ti 3 C 2 with PVP. b) TGA curves of Ti 3 C 2 , PVP, and Ti 3 C 2 -PVP.