Clinically translatable gold nanozymes with broad spectrum antioxidant and anti-inflammatory activity for alleviating acute kidney injury

Rationale: Acute kidney injury (AKI) is associated with aberrant generation of oxidative species and inflammation, leading to high mortality of in-hospitalized patients. Although N-acetylcysteine (NAC) showed positive effects in alleviating contrast-induced AKI, the clinical applications are strongly restrained due to the low bioavailability, low renal accumulation, short renal retention time, and high dosage-induced toxicity. Methods: We addressed the clinical dilemma of NAC by developing ultrasmall gold nanoclusters (1-2 nm) capped with NAC (denoted as Au NCs-NAC) as a nanozyme-based antioxidant defense system for AKI alleviation. Rhabdomyolysis-induced AKI mice model was developed, and the same dose of free NAC (as a control) and NAC onto Au NCs (Au NCs-NAC) was used for in vivo investigation of AKI restoration. Results: The as-developed gold nanozyme exhibited high bioavailability and good physicochemical stability as compared to NAC. Meanwhile, Au NCs-NAC showed broad-spectrum antioxidant activity of Au NCs-NAC, offering in vitro renoprotective effects, as well as macrophages by relieving inflammation under hydrogen peroxide or lipopolysaccharide stimulation. Notably, owing to the smaller size than kidney threshold (5.5 nm), Au NCs-NAC displayed preferential renal enrichment (< 2 h) and longer retention (> 24 h) in AKI mice as revealed by fluorescence imaging, thereby largely enhancing the restoration of renal function in AKI mice than free NAC by protecting the kidneys from oxidative injury and inflammation without systemic toxicity, as demonstrated by tissues staining, inflammatory cytokines and biomarkers detection, and mice survival rate. Conclusion: Owing to the synergistic anti-inflammatory/antioxidative effects, and enhanced bioavailability and renal accumulation/retention, Au NCs-NAC displayed far superior therapeutic performance than NAC alone. This work will facilitate the development of high-performance antioxidative nanoplatforms, as well as overcome the clinical limitations of small molecular drugs for AKI treatment and other inflammatory diseases.

The kinetic experiments of Au NCs-NAC for POD-like activity was conducted at room temperature in phosphatic buffer solution (pH 7.4) with Au NCs-NAC (30 μg/mL) in the presence of H 2 O 2 and TMB. The kinetic analysis of Au NCs-NAC with TMB as a substrate was performed by varying the concentrations of TMB at a fixed H 2 O 2 concentration and vice versa. Finally, the catalytic parameters were calculated by fitting the Michaelis-Menten equation.

Hydroxyl radical (·OH) scavenging activity of Au NCs-NAC
The ·OH scavenging activity of Au NCs-NAC was evaluated using a radical antioxidant capacity (HORAC) assay kit (Cell Biolabs, Inc., USA). Experiments with different concentrations of Au NCs-NAC were carried out according to the instructions provided by the manufacturer. Further, 5 mM H 2 O 2 , 50 μM FeCl 2 , and 100 μg/mL of Au NCs-NAC or free NAC were mixed in PBS (pH 7.4), and ESI-MS spectra were recorded.

ABTS radical (ABTS·) scavenging activity of Au NCs-NAC
The ABTS radicals (ABTS·) were first obtained by mixing 7 mM ABTS with 2.45 mM potassium persulfide for 12 h. Next, the absorbance of ABTS· solution with 12.5-100 μg/mL Au NCs-NAC (A S ) and pure ABTS· solution (A A ) was determined at 734 nm. The scavenging efficiency of Au NCs-NAC was calculated by the following formula: [(A A -A S )/A A ] * 100. All measurements were made in triplicate.

Superoxide anion radical (O 2 ·-) scavenging activity of Au NCs-NAC
The O 2 ·scavenging capacity of various concentrations of Au NCs-NAC was assessed by a SOD assay kit (Sigma-Aldrich, USA). The experiment was conducted in full accordance with the instructions provided by the supplier. The ESR signals of sample containing 100 mM DMPO, 100 μg/mL of Au NCs-NAC and 10 mM KO 2 were recorded.

Cytochrome c (Cyt c) electron transfer experiment
Au NCs-NAC (100 μg/mL) were mixed with Cyt c (400 μM) in PBS (pH = 7.4) in dark for 1 h. Then, UV-vis spectrophotometer was used to record the absorption of the resultant solution.
To determine whether Cyt c was oxidized by the dissolved oxygen, N 2 gas was pumped for 1 h in the stock solutions of Cyt c, Au NCs-NAC solution, and PBS, simultaneously.
Subsequently, the experiment was repeated following the above-described process.

Hemolysis assay
For hemolysis assay, the whole blood was drawn from all mice and washed with PBS to obtain a suspension of red blood cells (RBC). Then, RBC suspension (0.2 mL) was mixed with different concentrations (0.2 mg/mL, 0.8 mL) of Au NCs-NAC for 6 h, followed by the centrifugation. Finally, the absorbance of a supernatant was measured at 541 nm. The hemolysis rate of Au NCs-NAC was calculated as follow: Hemolysis (%) = (A s -A PBS )/(A water -A PBS ) × 100%, where A s , A PBS , and A water are the absorbance of the sample groups, the PBS group, and the water group, respectively.