MOF-derived bimetallic nanozyme to catalyze ROS scavenging for protection of myocardial injury

Rationale: Myocardial injury triggers intense oxidative stress, inflammatory response, and cytokine release, which are essential for myocardial repair and remodeling. Excess reactive oxygen species (ROS) scavenging and inflammation elimination have long been considered to reverse myocardial injuries. However, the efficacy of traditional treatments (antioxidant, anti-inflammatory drugs and natural enzymes) is still poor due to their intrinsic defects such as unfavorable pharmacokinetics and bioavailability, low biological stability, and potential side effects. Nanozyme represents a candidate to effectively modulate redox homeostasis for the treatment of ROS related inflammation diseases. Methods: We develop an integrated bimetallic nanozyme derived from metal-organic framework (MOF) to eliminate ROS and alleviate inflammation. The bimetallic nanozyme (Cu-TCPP-Mn) is synthesized by embedding manganese and copper into the porphyrin followed by sonication, which could mimic the cascade activities of superoxide dismutase (SOD) and catalase (CAT) to transform oxygen radicals to hydrogen peroxide, followed by the catalysis of hydrogen peroxide into oxygen and water. Enzyme kinetic analysis and oxygen-production velocities analysis were performed to evaluate the enzymatic activities of Cu-TCPP-Mn. We also established myocardial infarction (MI) and myocardial ischemia-reperfusion (I/R) injury animal models to verify the ROS scavenging and anti-inflammation effect of Cu-TCPP-Mn. Results: As demonstrated by kinetic analysis and oxygen-production velocities analysis, Cu-TCPP-Mn nanozyme possesses good performance in both SOD- and CAT-like activities to achieve synergistic ROS scavenging effect and provide protection for myocardial injury. In both MI and I/R injury animal models, this bimetallic nanozyme represents a promising and reliable technology to protect the heart tissue from oxidative stress and inflammation-induced injury, and enables the myocardial function to recover from otherwise severe damage. Conclusions: This research provides a facile and applicable method to develop a bimetallic MOF nanozyme, which represents a promising alternative to the treatment of myocardial injuries.

Additionally, a dissolved-oxygen portable meter was used to monitor the generation of oxygen directly. Typically, 30, 60, 90, 120,150 and 200 μM H2O2 was mixed with different concentrations of nanozymes (1 mL) in 15 mL of tris-HCl buffer with different pH (0.1 M, pH 5.5, 6.8 and 8.8) at 20 ℃ and 37 ℃. Then, the production of oxygen was detected by using a dissolved-oxygen portable meter in 10 min.

Kinetic analysis
The NBT kinetic assay of SOD-like activities measured at 550 nm using a UV-vis spectrophotometer was recorded and imported in GraphPad Prism 8.0 to analyze enzyme kinetic data. The inhibition rate was calculated by the formula: Inhibition rate% = (AbsorbanceblankA-Absorbancesample)/ (AbsorbanceblankA-AbsorbanceblankA0) × 100%. As for the kinetic analysis of CAT-like activities of Cu-TCPP and Cu-TCPP-Mn, the generation of dissolved O2 under different conditions was measured and recorded at different time points.
The Michaelis-Menten constant Km and Vmax were also automatically calculated by GraphPad Prism 8.0 software.

Cytotoxicity assay
RAW264.7, bEnd.3 or H9C2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM), which contained 1% penicillin-streptomycin (10000 U/mL) and 5% fetal bovine serum. Cells were then seeded with a density of 1 × 10 4 per well in 96-well plates for 24 h at 37 ℃. Different concentrations (5, 10, 20, 50 μg/mL) of Cu-TCPP and Cu-TCPP-Mn nanozymes were added in cells for another 24 h separately. Afterwards, the cells were gently washed by PBS (pH 7.4) for three times. 5 μg/mL Thiazolyl Blue Tetrazolium Bromide (MTT) was added into the 96-well plates for another 4 h in the dark. The cell supernatant was pipetted by micropipettor and 150 μL DMSO was added to the cell supernatant. After horizontal jitter for 10 min to fully dissolved the crystallization of MTT, cell viability was determined by a microplate reader at the absorbance of 490 and 570 nm.

Hemolysis assay
The fresh blood sample (1 mL) was collected from healthy C57BL/6 mouse. The blood sample was centrifugated at 2000 rpm for 5 min to remove the upper serum and obtain red blood cells (RBCs). The RBCs were further washed with 0.9% saline for three times and diluted to 2% RBC suspension. Then, different concentrations of Cu-TCPP-Mn (0.8 mL, 1-H9C2 cells were seeded in confocal dishes and then incubated with different concentrations of enzyme mimics (1, 2.5, 5 μg/mL) for 4 h at 37 ℃. The cells were then incubated at 37 ℃ for 4 h with H2O2 (100 μM), and stained with 500 nM propidium iodide (PI) for 15 min. The treated cells were washed gently by PBS for three times and collected by centrifugation. Then, the fluorescence intensity of all treated cells was assessed by a flow cytometer.

Evaluation of intracellular ROS-scavenging ability by LSCFM and flow cytometry
The intracellular levels of ROS were monitored by using the fluorescent probe of H2DCFH-DA, which reacts with intracellular free radicals and produces a fluorescent product, dichlorofluorescein (DCF). The excitation and emission wavelengths of the fluorescent product were 488 and 525 nm, respectively. RAW264.7 cells were planked in 10 mm confocal petri dishes with a density of 5 × 10 5 cells per well for 24 h at 37 ℃. Then, 1 μg/mL LPS (Rosup) and 5 μg/mL of nanozymes were added to each well for another 24 h. After being gently washed by PBS (pH 7.4) in triplicate, 10 μM H2DCFH-DA was added to RAW264.7 cells followed by 30 min incubation in the dark. After 30 min, cells were gently washed by fresh medium. 1 μg/mL Hoechst 33342 dye were added and incubated for another 5 min.
Then, LSCFM was used to observe the fluorescence of RAW264.7 cells. Similar protocol was applied to flow cytometry experiment for DCF fluorescence detection expect for trypsin digestion of cells followed by DCFH-DA staining.

In vivo biodistribution analysis
Male C57BL/6 (6-8 weeks old, 20-25 g) mice which were subjected to cardiac thoracic surgery or sham surgery were divided into MI and non-MI (sham) groups, respectively.

In vivo assessment in acute MI models
All of our animal studies were approved by the Committee for Experimental Animals Welfare and Ethics of Jinling Hospital, Medical School of Nanjing University. Male C57BL/6 (6-8 weeks old, 20-25 g) mice were subjected to cardiac thoracic surgery as permanent ligation of the left anterior descending (LAD) coronary, or sham surgery (thoracotomy without ligation) [47,48]. Mice were anesthetized initially with 4% isoflurane inhalation followed by 1% isoflurane inhalation for anesthesia maintenance, and ventilated on a rodent respirator via a tracheostomy. A 1.5-cm incision was made between the third and fourth ribs in lower-left sternum, and the pectoral major and pectoral minor muscles were separated to expose the fourth intercostal space. After exposing the heart through thoracic cavity, the LAD artery was

Evaluation of anti-inflammation in MI mice
The evaluation of therapeutic efficacy of Cu-TCPP-Mn nanozyme on MI mice was mainly operated by histological study. Frozen sections of hearts tissue from each group were selected and cut into cross sections for ROS staining (DHE and DCFH-DA, respectively). The ROS fluorescence image of each heart tissue was photographed by CLSFM using the same parameter. Quantitative analysis for the ROS fluorescent intensity was determined by ImageJ software. Three of remaining heart tissues from each group were paraffin-embedded and cut into cross sections for Masson's trichrome staining and immunofluorescent staining for TUNEL, myeloid cell markers (CD45), angiogenesis markers (α-SMA, CD31). Quantitative analysis for the collagen contents and each positive marker of heart tissues was operated by ImageJ software.

Infarct size evaluation
3 days post nanozyme injection, three fresh hearts of each group were excised and frozen in -80 ℃ refrigerator for 20 min. The hearts were dissected into 2 mm slices and stained with 2% triphenyltetrazolium chloride (TTC) for 20 min at 37 ℃ in the dark. After washing with PBS, heart slices were then imaged and weighed. Infarct size was represented by white region whereas viable tissues were represented by red region. The quantitative analysis of the infarct area was calculated by ImageJ software. The treatments were performed once every day for 3 times. After treatments, all rats were performed with cardiac MR imaging on a 9.4 T micro MR scanner, which was combined the ECG trigger with a respiration signal to start data acquisition. T1-weighted MR imaging and cine imaging was performed before (baseline) and at 7 day and 28 day post injection to observe the change of cardiac morphology and function. The T1-weighted cardiac MRI parameters were used as follows: TR 104.169 ms, TE 2.500 ms, FOV 50 × 50 mm 2 , flip angel 40°, slice thickness 1 mm, matrix 256 × 256. Cine FLASH cardiac MRI parameters were used as follows: TR 8.000 ms, TE 1.600 ms, FOV 50 × 50 mm 2 , flip angel 15°, slice thickness 1mm, matrix 192 × 192.

In vivo biosafety and toxicity evaluation
To evaluate the biosafety and toxicity of the nanozyme, healthy and I/R male SD rats were used. Different concentration of nanozyme or equal volume of 0.9% NaCl was administered by lateral cerebral ventricle injection. The rats were dissected, and the main organs (brain, heart, liver, spleen, lung and kidney) were harvested for HE staining.

Statistical Analysis
All data were presented as mean ± S.D. Data were compared by one-way ANOVA with Bonferroni correction for multiple-group comparisons. Significant difference between treatment and control groups: *indicates P ＜ 0.05, **indicates P ＜ 0.01, ***P < 0.001, ****P < 0.0001, ns indicates P ＞ 0.05 with no significance.