A Multifunctional Integrated Metal‐Free MRI Agent for Early Diagnosis of Oxidative Stress in a Mouse Model of Diabetic Cardiomyopathy

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  Scheme 1. Schematic illustration of ROS‐response contrast‐enhanced MRI nanoprobes for the early diagnosis and further therapy of diabetic cardiomyopathy. a) The introduction of multiple hydroxyl groups onto their surface greatly improved the aqueous dispersion of functionalized FCNs, and the coexistence of TEMPO moieties and C—F bonds successfully combined the ROS‐sensitive property and the high resolution and the anatomical information deriving from MRI. b) When exposed to ROS‐rich microenvironment of the injured hearts, RCMNs nanoprobes were actually functionalized by the captured ROS groups and equipped with a stronger interaction with the surrounding microenvironment. This promoted a brief enrichment of the material in the myocardium, caused a variation of the corresponding MRI signal and thus realized the in vivo MRI imaging of ROS. Furthermore, the TEMPO moieties of RCMNs could scavenge ROS to alleviate oxidative stress.

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  Figure 1. Structure characterization and schematic of the formation process for different FCNs and RCMNs. a) Schematic of the formation process for RCMN. Firstly, FCNs were fabricated with graphene oxide (GO) as raw materials via direct gas fluorination. Next, utilizing the chemical reactivity of the introduced C—F bonds, AT and THAM were covalently grafted onto FCNs’ carriers, and eventually RCMN nanoprobes were obtained. FTIR spectra b) and XPS survey spectra c) of GO and different FCNs. d) XPS C1s spectra of FCN‐1. e) DTG curves of FCNs and RCMNs samples at a heating rate of 10 °C min−1 under N2 atmosphere. f) XRD patterns of GO, different FCNs and corresponding RCMNs. g) XPS C1s spectra of RCMN‐1.

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  Figure 2. a) Schematic illustration of the functions of different groups as well as chemical structure of corresponding nanoprobes. SEM image a), statistical analysis of the particle size distribution b), scale bar: 5 µm; and AFM image c) and the corresponding height profiles of RCMNs d), scale bar: 1 µm. e) Changes in zeta potentials of FCNs before and after grafting. f) T2‐weighted images of RCMN‐1, RCMN‐2 and RCMN‐3 in phosphate buffered saline (PBS) medium with the concentration of RCMNs decreasing gradually from 2 mg mL−1 to 0, and corresponding T2 relaxation rates (1/T2) plotted against F concentrations g). h) Schematic diagram for the multifunctional integration strategy and the fabrication of different nanoprobes. i) Electron paramagnetic resonance (EPR) spectra of different RCMNs and the control sample RCMN‐C1. The evaluation of the dispersion stability of RCMNs according to the changes of T2 relaxation rates j), zeta potentials k), and particle sizes l) in 2 weeks.

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  Figure 3. a) Illustration of RCMNs’ capturing and scavenging ROS at the cellular level. b) EPR scanning validation of RCMN‐1 capture ability in cell levels (ROS‐up represents a higher level of ROS in cells). c) Fluorescence profiles of RCMN‐1 before and after being loaded with FITC. d) ROS flow cytometry results of H9C2 cells after different treatment. e) Confocal images of cells after ingestion of probes, scale bar: 20 µm. Blue fluorescence: nuclei stained with DAPI, red fluorescence: cytoskeleton stained with Rhodamine‐Phalloidin, green fluorescence: the nanoprobes RCMNs loaded with FITC. f) The apoptosis of H9C2 cardiomyocytes induced by RCMN‐1 (100 mm based on F) for 24 h was evaluated by using flow cytometry. g) The cytotoxicity of RCMN‐1 nanoprobe tested on H9C2 cells with a CCK‐8 kit.

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  Figure 4. a) Schematic illustration of RCMN‐1 for early diagnosis of DCM. T2 maps b) and T2 values c) before and after probe injection (The “Apex”, “Middle” and “Basal” represent the apical, middle and basal slices of the heart, respectively.). Cardiac MRI black blood images e) and quantitative analysis d) at different time points after intravenous injection of RCMN‐1 into db mice. Cardiac MRI T2 black blood images f) and relative quantitative calculation i), T2 maps g), and T2 values j) of the three groups of mice before and 30 min after injection (Group A: db mice injected with RCMN‐1, group B: db mice injected with the contrast probe RCMN‐1 without ROS‐response properties, and group C: normal mice injected with RCMN‐1). h) Confocal images of heart slices of different groups of mice after administration, scale bar: 100 µm. Blue fluorescence: nuclei stained with DAPI, green fluorescence: the nanoprobes RCMNs loaded with FITC. Data in (c), (i), and (j) represent mean values ± standard deviations (SD), n = 5. Statistical differences were determined by unpaired Student's t‐test. NS means no significant difference. ***p < 0.001.

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  Figure 5. a) Schematic illustration of RCMN for ROS scavenging in vivo. MRI black blood images b) and myocardial signal e), T2 maps c) and values f) before and after injection of different reagents (PBS or RCMN‐1, “native” and “post” represent MRI T2 maps before and after injection of the material, respectively.). ROS d), 8‐OHdG g), and CD31 h) of mice heart sections after administration of different treatment regents (PBS or RCMN‐1), scale bar: 100 µm. i) H&E staining of different organs in mice, scale bar: 100 µm. j) Changes of oxidative stress indicators (lipid peroxidation products: malondialdehyde, MDA; 8‐iso‐prostaglandinF2α , 8‐ios‐PGF2α , and antioxidant enzymes: catalase, CAT; superoxide dismutase, SOD) and cardiac function indicators (vascular endothelial growth factor, VEGF and Creatine Kinase‐MB, CKMB). Data in (e) and (f) represent mean values ± SD, n = 5. Statistical differences were determined by unpaired Student's t‐test. NS means no significant difference. ***p < 0.001.