Fluorescent Nanocomposite for Visualizing Cross-Talk between

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A Novel Fluorescent Nanocomposite for Visualizing CrossTalk between MicroRNA-21 and Hydrogen Peroxide in Ischaemia-Reperfusion Injury in Live Cells and in Vivo Limin Yang, Yanfei Ren, Wei Pan, Zhengze Yu, Lili Tong, Na Li, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03701 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 5, 2016

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Analytical Chemistry

A Novel Fluorescent Nanocomposite for Visualizing Cross-Talk between MicroRNA-21 and Hydrogen Peroxide in IschaemiaReperfusion Injury in Live Cells and in Vivo Limin Yang, Yanfei Ren, Wei Pan, Zhengze Yu, Lili Tong, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China. fax: (86)531 86180017. E-mail: [email protected]; [email protected]. ABSTRACT: MicroRNAs (miRNAs) and reactive oxygen species (ROS) are concurrently implicated in heart ischemiareperfusion (IR) injury. There may exist mutual cross-talk between miRNAs and ROS in cardiac IR injury process. In this study, we developed a novel crown-like silica@polydopamine-DNA-CeO2 nanocomposite by assembly of silica@polydopamine-DNA1 nanoparticles decorated with satellite CeO2-DNA2 nanoparticles for detecting and imaging of microRNA-21 (miR-21) and hydrogen peroxide (H2O2) in simulated IR injury in living cells and in vivo. The miRNA-21 was found to be regulated by H2O2 via PI3K/AKT signaling pathways for the first time in H9C2 cells in simulated ischemiareperfusion injury. H2O2 and miRNA-21 are over produced during mimicked heart ischemia-reperfusion injury, suggesting that they are closely related to reperfusion injury. All these results reveal that there is definite cross-talk between miR-21 and H2O2 in IR injury. The current method can provide a promising strategy to further explore the interplaying roles between ROS and miRNAs in other pathological processes.

MicroRNAs (miRNAs) plays a significant role in cancer and cardiovascular disease.1‒4 Aberrant expression of miRNAs is associated with myocardial ischemiareperfusion (IR) injury, which will lead to heart attack and stroke.5 Reactive oxygen species (ROS) regulates cellular processes including aging and gene expression.6,7 Moreover, abnormal generation or accumulation of ROS is closely correlated with cancer, cardiovascular diseases and IR injury.8‒10 Remarkably, miRNAs and ROS are concurrently existing in cancer, IR injury and cardiovascular diseases. Therefore, it makes us wonder whether there is mutual cross-talk between miRNAs and ROS in these pathologies, especially in heart IR injury process. It has been reported that hydrogen peroxide (H2O2) was over produced during IR injury.11,12 While the expression of microRNA-21 (miR-21) was reported up or down regulation in heart IR injury.13,14 Because the present methods for H2O2 or miR-21 detection in cardiac IR injury are usually based on the historrhexis and cell lysate, which are complicated and time-consuming. Importantly, they can not represent the natural situation after these operations. As a consequence, there is an urgent need for exploring new methods for in situ visualizing miR-21 and H2O2 with distinct signals in living cells and in vivo, which are favorable to further understanding the

mechanism of the occurrence and evolvement of heart attack. Fluorescent imaging analysis was well-suited to detect miR-21 and H2O2 in living cells and in vivo. Currently, many fluorescence probes have been developed for the sensing and imaging of miR-2115‒18 and H2O219‒24. As these fluorescent probes are specific to microRNA (miRNA) or H2O2, a single fluorescence probe which can simultaneously detect miRNA and H2O2 was not reported until now. For miRNA detection, the design strategy of probes is combination of quencher and fluorophore modified nucleic acid. In the presence of targets, fluorescence signal is produced due to the separation between fluorophore and quencher. For H2O2 detection, fluorescence probes are comprised of fluorescence reporter and masking group. The fluorescence signal is changed after H2O2 reacting with masking group. Nevertheless, if a single probe is prepared by combination of miRNA probe and H2O2 probe, the signal of fluorescence reporter of H2O2 was always quenched upon addition of H2O2 due to the presence of quencher of miRNA. Up to now, it is very difficult to achieve simultaneous detection of miRNA and H2O2 in living cells and in vivo using a single fluorescent probe. In this study, we develop a fluorescent nanocomposite for in situ imaging of miR-21 and H2O2 in living cells and

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in vivo during simulated IR injury. Silica@polydopamineDNA1 nanoparticles (SiO2@pDA-DNA1 NPs) as the miR-21 detection unit were constructed by assembling DNA1 on SiO2@pDA NPs surface through π–π interaction.25 CeO2DNA2 as the H2O2 detection unit was prepared via the coordination effects between CeO2 NPs and DNA2.26 Then, the SiO2@pDA-DNA-CeO2 nanocomposite (SPDCN) comprised many satellite CeO2-DNA2 around the SiO2@pDA-DNA1 core NPs was developed by the strong π–π stacking and coordination effects. In the presence of miR-21 and H2O2, DNA1 and DNA2 was released from SPDCN, which could then produce distinct fluorescence signals correlated with the relative amount of the miR-21 targets and H2O2, respectively. As far as we know, the developed SPDCN is firstly reported simultaneously sensing ROS and miRNA. The designed SPDCN is used to visualize the changes of miR-21 and H2O2 in living cells and in vivo for studying their communication. The details of this approach are shown in Scheme 1. Scheme 1. Schematic illustration of the SPDCN for detection of miR-21 and H2O2

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EXPERIMENTAL SECTION Materials and Instruments. 4-(2-hydroxyethyl)-1piperazineethanesulfo nic acid (HEPES), tetraethyl orthosilicate (TEOS), ammonia solution (25-28%), Cerium(III) nitrate (99.5%, Ce(NO3)3·6H2O), hypochlorite (NaOCl), ferrous chloride (FeCl2), dimethyl malonate (DM), hydrogen peroxide (H2O2) sodium hydroxide (NaOH) and 3-morpholinosydnonimine hydrochloride were purchased from China National Pharmaceutical Group Corp. Dopamine, Xanthine (XA), Xanthine Oxidase (XO), LY294002 and 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Hydroxyl radical (•OH) was generated through H2O2/Fe2+ system. Singlet oxygen (1O2) was obtained through the reaction of H2O2 with ClO. Superoxide anion (O2•−) was obtained form XA and XO. Nitric oxide (NO) was prepared from 3morpholinosydnonimine hydrochloride. The rat heart myoblast cells H9C2 were purchased from Procell Life Science Co., Ltd. DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai China). All the chemicals were analytical grade and used without further purification.

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Detailed DNA sequences and modifications were shown in Table S1. High resolution transmission electron microscopys (HRTEM) was carried out on a JEM-2100 electron microscope. Fluorescence spectra measurements were carried out on an FLS-980 Edinburgh Fluorescence Spectrometer. Confocal fluorescence imaging experiments were carried out on a Zeiss 880 confocal laser scanning microscopy. X-ray diffraction (XRD) analysis was carried out on a D/Max 2500 V/PC X-ray diffractometer. Absorption spectra were taken on a pharmaspec UV-1700 UV-visible spectrophotometer (Shimadzu, Japan). RT-PCR experiments were performed on an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA). MTT assay was recorded using a microplate reader (Synergy 2, Biotek, USA). Fluorescence animal imaging was acquired with Caliper IVIS Lumina III imaging system. Preparation of CeO2 Nanoparticles (CeO2 NPs). The CeO2 NPs were prepared by a hydrothermal method.27 0.868 g of Ce(NO3)3·6H2O and 0.016 g of NaOH were dissolved in 5 and 35 mL of doubly distilled water, respectively. Then, these two solutions were mixed and stirred for 30 min at room temperature. Subsequently, the white slurry mixture solution was transferred into an autoclave. The autoclave was subjected to hydrothermal treatment at 180 °C for 24 h. After that, the precipitates were separated by centrifugation, washed with deionized water and ethanol several times and drying at 60 °C overnight. The products after drying were yellow powders. Synthesis of Silica@Polydopamine Core–Shell Nanoparticles (SiO2@pDA NPs). SiO2 was synthesized by a modified Stöber method.28 0.6 mL TEOS was added to 16 mL mixed solvent (ethanol/H2O = 7:1 v/v). Then, 0.3 mL of ammonia solution was added. The mixture were stirred for 2 h. Then, the SiO2 was obtained by centrifuged and washed three times with ethanol. For the synthesis of SiO2@pDA NPs, 300 µL SiO2 solution was dispersed to 10 mL of Tris-HCl (pH=8.5) solution.29 Then, 20 mg dopamine was added to the above solution and stirred for 2 h at room temperature. Subsequently, the SiO2@pDA NPs were obtained by centrifugation washing with deionized water. Fluorescence Quenching Assay. DNA1 (50 nM) was added to various concentrations of SiO2@pDA NPs (5, 10, 15, 20, 25, 40, 60 µg/mL) in buffer solution (10 mM HEPES, 150 mM NaCl, pH 7.4). The fluorescence intensities were recorded with λex = 645 nm, λem = 665 nm. DNA2 (50 nM) was mixed with various concentrations of CeO2 NPs (0, 2, 4, 6, 8, 10, 15, 20 µg/mL) in buffer solution (10 mM HEPES, 150 mM NaCl, pH 7.4). The fluorescence intensities were recorded with λex = 495 nm, λem = 518 nm. Preparation of the Silica@Polydopamine-DNACeO2 Nanocomposite (SPDCN). 50 nM DNA2 was added to a solution of CeO2 NPs (10 µg/mL) and 50 nM DNA1 was added to a solution of SiO2@pDA NPs (25 µg/mL). Then, two solution was mixed and stirred for 0.5 h. The SPDCN was obtained by centrifugation. The


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concentrations of DNA1 and DNA2 were determined according to the standard linear calibration curve. Fluorescence Response to miR-21 and H2O2. For the detection of H2O2, various concentrations of H2O2 were added to the SPDCN solution (35 µg/mL). After incubation for 15 min at 37 °C, the fluorescence of the samples was measured at λex/λem = 495/518 nm. To investigate the selectivity of the SPDCN toward H2O2, reactive oxygen/nitrogen species (OCl-, 1O2, O2•−, ONOO-, NO, •OH) and metal ions (Fe3+, Cu2+, Mg2+, Zn2+) were examined. The concentration is 100 µM for each. The measuring procedure was the same as above. For miR-21 detection, different concentrations of miR-21 targets were added to the SPDCN solution (35 µg/mL). After incubation for 15 min at 37 °C, the fluorescence intensities were recorded with λex/λem = 645/665 nm. To investigate the selectivity of the SPDCN toward miR-21, mismatched targets was examined. The measuring procedure was the same as above. Kinetics. The SPDCN (35 µg/mL) were incubated with miR-21 targets (200 nM), and H2O2 (200 µM), respectively. The fluorescence intensity was determined with increasing time (0, 2, 5, 10, 20, 30, 40, 50, 60 minutes). The fluorescence intensities of DNA2 were recorded with λex/λem = 495/518 nm and the fluorescence intensities of DNA1 were recorded with λex/λem = 645/665 nm. RT-PCR. MicroRNA was isolated from the cell line using the miRcute miRNA Isolation Kit (Tiangen). cDNA synthesis was generated using miRcute miRNA FirstStrand cDNA Synthesis Kit (Tiangen) in accordance with manufacturer’s instructions. RT-PCR of miRNAs was carried out with miRcute miRNA qPCR Detection Kit (Tiangen) on an ABI PRISM 7000 RT-PCR instrument. Relative level of miRNA was calculated from the quantity of miRNA PCR products and the quantity of U6 PCR products. Nuclease Assay. Two groups of SPDCN (35 µg/mL) in buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) were placed in a 96-well fluorescence microplate at 37 °C. Then, 1.3 µL of DNase I (2 U/L) was added to one group. The fluorescence of these samples was monitored for 1 h and was collected at intervals during this time period. Then 200 nM miR-21 targets and 200 µM H2O2 were paralleled added into the two samples with incubation for 1 h at 37 °C, repectively. Cell Culture and MTT Assay. The cells were cultured in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine serum and 100 U/mL of 1% antibiotics penicillin/streptomycin and maintained in a 100% humidified atmosphere containing 5% CO2 at 37 °C. Then, the cells were incubated with the SPDCN (35, 60, 90 μg/mL) for 12 and 24 h, respectively. Subsequently, 150 μL of MTT solution (0.5 mg/mL) was added to each well. The MTT solution was removed after 4 h and 150 μL of DMSO was added to each well. The absorbance was measured at 490 nm with a RT 6000 microplate reader. Confocal Fluorescence Image Assay. The cells were plated on chamber slides for 24 h. The SPDCN (35 μg/mL) was applied all the cell experiments. To investigate the

effect of H2O2 on miR-21 expression, SPDCN treated H9C2 cells were incubated with H2O2 (100 μM) at different times. Cell imaging was carried out after washing the cells with PBS (pH 7.4) for three times. Green channels (DNA2) and red channels (DNA1) were excitated at 488 and 633 nm and the emission emission were collected between 500-580 nm and 650-750 nm, respectively. To simulate ischaemia, SPDCN treated H9C2 cells were cultured in hypoxic recording buffer (156 mM NaCl, 2 mM MgSO4, 1.25 mM K2HPO4, 2 mM CaCl2, 10 mM HEPES, 10 mM sodium lactate, 14.8 mM KCl, pH 6.4) in the argon atmosphere to maintain hypoxia for 0.5 h. To simulate reperfusion, the cells were cultured in DMEM containing 10% FBS with 5% CO2 for different times. In Vivo Imaging Assay. All animal experiments were carried out according to the Principles of Laboratory Animal Care (People's Republic of China) and the Guidelines of the Animal Investigation Committee, Biology Institute of Shandong Academy of Science, China. Mice were housed under normal conditions with 12 h light and dark cycles and given access to food and water ad libitum. For the ischemic-reperfusion injury experiment, the living mice were anesthetized using 4% chloral hydrate (200 μL). Heart ischemia was induced by clamping the left anterior descending coronary artery of the heart. Ten minutes later, the ischemic heart was reperfused by opening the vascular clamp for 2 h followed by injection of SPDCN. The mice were examined by Caliper IVIS Lumina III imaging system with 480 nm excitation for H2O2 detection and 620 nm excitation for miR-21 detection, respectively.

RESULTS AND DISCUSSION Design and Synthesis of SPDCN. First, the CeO2 27 NPs were synthesized via a hydrothermal method. As shown in Figure 1a, the size of CeO2 was about 10 nm. The X-ray diffraction (XRD) pattern revealed that CeO2 was fluorite cubic structure (Figure S1). The uniformed SiO2@pDA NPs were fabricated through the polymerization of dopamine under alkaline conditions 29 in the presence of SiO2. As shown in Figure 1c, the size of SiO2@pDA NPs was about 90 nm with the pDA shell thickness around 10 nm. The SiO2@pDA-DNA1 and CeO2-DNA2 were prepared through the π–π and coordination effects, respectively. The as-prepared SPDCN composed of many satellite CeO2 NPs around the SiO2@pDA core NPs has been seen in Figure 1d, clearly indicating the formation of the crown-like nanocomposite. The preparation of the SPDCN was further verified by the UV-vis absorption spectra. As shown in Figure 1e, the maximum absorption of the SPDCN at 290 nm was attributed to the CeO2 absorbance, suggesting the successful embellishing of CeO2 on the SiO2@pDA surface. Meanwhile, a physical mixture of CeO2 NPs and SiO2@pDA NPs without DNA was prepared for comparison under same conditions. No obvious absorption was observed when CeO2 and SiO2@pDA were directly mixed without DNA. Thus,


Analytical Chemistry b

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Figure 1. TEM images of CeO2 (a), SiO2 (b), SiO2@pDA (c) and SPDCN (d). UV-vis absorption spectra of CeO2, SiO2@pDA, SiO2@pDA + CeO2 and SPDCN (e). the strong π–π stacking and coordination effects between the DNA nucleobases and pDA or CeO2 were considered to contribute for the assembly of CeO2 NPs onto the SiO2@pDA NPs. It reveals DNA is an indispensable part for successfully constructing SPDCN. In Vitro Studies of the SPDCN. To achieve the best sensing performance, the amount of SiO2@pDA and CeO2 were first optimized. The fluorescence intensity of DNA1 and DNA2 are efficiently quenched by 25 μg/mL SiO2@pDA and 10 μg/mL CeO2, respectively a

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Figure 2. Kinetics of the SPDCN in the absence of (black curve) and in the presence of (red curve) miR-21 targets (a) and H2O2 (b), respectively. F0 and F are the fluorescence intensity of SPDCN without and with miR-21 (a) and H2O2 (b), respectively. F/F0 are the value that F0 divided by F. (c) Fluorescence spectra of SPDCN (blue) in the presence of miR-21 (black) and H2O2 (red). (λex = 495 nm). (d) Fluorescence spectra of SPDCN (blue) in the presence of H2O2 (black) and miR-21 (red). (λex = 645 nm).

Figure 3. (a) Fluorescence spectra of SPDCN with various concentrations of H2O2 (λex = 495 nm). (b) F/F0 value against the concentration of H2O2, where F0 and F are the fluorescence intensity of SPDCN without and with H2O2, respectively. (c) Fluorescence spectra of the SPDCN with various concentrations of miR-21 targets (λex = 645 nm). (d) F/F0 value against the concentration of miR-21 targets, where F0 and F are the fluorescence intensity of SPDCN without and with miR-21 targets, respectively. (Figure S2). Thus, the SPDCN was prepared under these conditions. The carrying contents of DNA1 and DNA2 on the SPDCN were determined to be about 1.2 nmol/mg and 1.5 nmol/mg, respectively. Details of the characterization are provided in Figure S3. Kinetic studies showed that the SPDCN responded rapidly to H2O2 and the miR-21 targets within 10 min (Figure 2 a and b). We next evaluated the feasibility of the SPDCN for the simultaneous detection of H2O2 and miR-21. As shown in Figure 2c, the signals with excited at 495 nm did not obviously change in the presence of miR-21 target compared with background fluorescence. In contrast, 9.8-fold increases in fluorescence signal was achieved upon addition of H2O2. Similarly, the fluorescence intensity did not obviously change in the presence of H2O2 compared with background fluorescence while 19-fold increases in fluorescence signal was observed in the presence of miR-21 target with excitation at 645 nm (Figure 2d). These results indicate that SPDCN is capable of simultaneously monitoring H2O2 and miR-21 changes without mutual interference. Under optimized conditions, the fluorescence spectra of SPDCN upon incubation with various concentrations of H2O2 and miR-21 targets were performed. As shown in Figure 3a, the fluorescence intensity increased gradually with the increasing concentrations of H2O2. As a results, a good linearity was obtained from 10 μM to 100 μM of H2O2 (Figure 3b). Furthermore, the response of the SPDCN toward miR21 was further investigated. The fluorescent intensity of


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Figure 5. The fluorescence images of H9C2 cells under reperfusion for different times. The excitation wavelengths of green and red channels are 488 and 633 nm, respectively. expression of miR-21 in H9C2 cells. Although H2O2 and miR-21 are closely with IR injury, the cross-talk between H2O2 and miR-21 during heart IR injury is still unknown until now. Here, we used an 31 oxygen and glucose deprivation reperfusion model to imitate IR conditions aimd to gain insight into the relationship between H2O2 and miR-21 in IR inside H9C2 cells. As shown in Figure 5, the green fluorescence intensity gradually enhanced after reperfusion indicating the H2O2 levels was elevated under these conditions. Whereas, the red fluorescence intensity was weak for the first 1 h after reperfusion,indicating no change of miR-21 expression in the initial reperfusion stage. Then, the red fluorescence intensity increased progressively from 1 h to 6 h. It suggested that the expression of miR-21 gradually increased at this period. Meanwhile, we a

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the SPDCN increased with increasing concentration of the miR-21 targets (Figure 3c), thus indicating that the hybridization of SPDCN and miR-21 targets led to fluorescence recovery. A good linearity was obtained from 10 nM to 100 nM of miR-21 targets (Figure 3d). The excellent response results of the SPDCN made the simultaneous detection of miR-21 and H2O2 in living cells and in vivo possible. Notably, SPDCN exhibited high selectivity for H2O2 and miR-21, respectively (Figure S4). Nuclease stability is a key property of probes for applications in living cells. To evaluate the nuclease stability of SPDCN, the experiments were conducted us30 ing the enzyme deoxyribonuclease I (DNase I). The fluorescence intensity of the SPDCN treated with DNase I was not obviously changed compared the SPDCN without DNase I. When the H2O2 (Figure S5a) or miR-21 target (Figure S5b) were added to the SPDCN-only and SPDCN/DNase I solutions, respectively, the fluorescence intensity in both solutions was enhanced greatly. This suggested that SPDCN showed high resistance to nuclease. It further verified that the fluorescence enhanced was indeed owing to the present of H2O2 and miR-21 target, respectively. MTT assay results further demonstrated that the developed SPDCN was of little toxicity in H9C2 cells (Figure S6). Imaging of miR-21 and H2O2 in Living Cells and in Vivo. In an effort to explore the cross-talk between H2O2 and miR-21, we investigated fluorescence imaging in H9C2 cells treated with H2O2 over time. As shown in Figure 4, the green fluorescence intensity dramatically increased after culturing with H2O2 within 1 h and then it was almost not changed between 1 h and 6 h. It indicated that the concentrations of H2O2 increased at first 1 h in H9C2 cells and remained unchanged from 1 h to 6 h. Meanwhile, we found that the red fluorescence signal was gradually enhanced in H9C2 cells after H2O2 stimulation with increasing time, indicating the rising level of miR-21. RT-PCR further confirmed that the level of miR-21 expression increased after H9C2 cells treated with H2O2 (Figure S7). All these results revealed that H2O2 could up regulate the

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Figure 4. The fluorescence images of H9C2 cells exposed to H2O2 at different times. The excitation wavelengths of green and red channels are 488 and 633 nm, respectively.

Figure 6. The fluorescence images of H9C2 cells under different conditions. (a) and (d) were the control group, (b) and (e) were IR, (c) and (f) were the IR in the presence of LP. (g) Proposed schematic diagram of H2O2 regulate the expression of miR-21 in mimicked IR injury in H9C2 cells. The excitation wavelengths of green and red channels are 488 and 633 nm, respectively.


Analytical Chemistry found a delayed rise in miR-21 levels compared with H2O2 after reperfusion. Inspired by the above study results, we speculate that whether the increased of H2O2 leads to elevate expression of miR-21 during IR injury. Subsequently, dimethyl malonate (DM) was used in the following experiments because it could 31 inhibit the ROS production. As shown in Figure S8, the green and red fluorescence signal was almost not changed compared to the control in IR injury in the presence DM. It indicated that the H2O2 concentrations and miR-21 levels were not changed during IR injury in the presence of DM. The RT-PCR results were consistent with imaging experiments (Figure S9). These results suggested that H2O2 could up regulate the miR-21 levels during IR injury in H9C2 cells. To expand on our discovery, we next investigate the effects of H2O2 on miR-21 expression in the 32 presence of PI3K/AKT inhibitor LY294002 (LY). As shown in Figure 6, the red signal of LY treated H9C2 cells during IR injury was lower than in H9C2 cells only IR treatments but higher than control cells without any treatment. RT-PCR further confirmed this phenomenon (Figure S10). The generation of H2O2 was not influenced by the LP during IR injury. It indicated that the effect of H2O2 on miR-21 expression was weakened by the PI3K/AKT inhibitor LY, revealing that H2O2 could control the expression of miR-21 via PI3K/AKT signal pathway (Figure 6g). In vivo imaging was then successfully realized in mice with heart IR injury. In situ images were acquired from simulated heart surgery. As shown in Figure 7, the injured heart emitted bright fluorescence signals corresponding to H2O2 and miR-21 after IR injury comparing with the control. These results indicated that H2O2 and miR-21 were over generated after simulated heart surgery IR injury. The results reveal that communication between H2O2 and miR-21 exists, and a cooperative effect of these two factors may contributing to heart IR injury. Therefore, we believe that SPDCN is a promising tool for simultaneously monitoring changes in H2O2 and miR-21 levels in vivo.

CONCLUSIONS In summary, we have developed a novel fluorescent nanocomposite for detecting and imaging of miR-21 and H2O2 in living cells and in vivo. In vitro assays revealed that the SPDCN was a sensitive and selective nanoprobe for simultaneous measurement of miR-21 and H2O2. By utilizing SPDCN for simultaneous fluorescence imaging of miR-21 and H2O2, we found that expression of miR-21 was up regulated in H9C2 cells after treatment with H2O2. During mimicked IR injury, the elevated levels of H2O2 resulted in the expression of miR-21 increased via PI3K/AKT signaling pathways. Further experiment results showed that H2O2 and miR-21 were over generated during mimicked heart IR injury in mice. More importantly, applications of SPDCN in living cells and in vivo imaging have demonstrated that the existence of mutual cross-talk between H2O2 and miR-21 in mimicked IR injury. Therefore, SPDCN is believed to be a promising tool for further exploring the relationship between ROS and microRNAs in other diseases.

ASSOCIATED CONTENT Supporting Information DNA sequences, XRD pattern, 0ptimization experiments standard linear calibration curves, specificity, nuclease stability, cell viability, fluorescence images, RT-PCR results. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Fax: (86)531 86180017 *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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This work was supported by 973 Program (2013CB933800) and National Natural Science Foundation of China (21390411, 21535004, 21422505, 21375081, 21505087), and Natural Science Foundation for Distinguished Young Scholars of Shandong Province (JQ201503).

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Figure 7. Representative images (pseudocolor) of mice for H2O2 (a, b) and miR-21 (c, d). (a) and (c) were normal conditions. (b) and (d) were IR conditions. The excitation wavelength in (a) and (b) for H2O2 were 480 nm and the excitation wavelength of (c) and (d) for miR-21 were 620 nm.

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DNA1

CeO2

DNA2

SiO2@pDA

miR-21 H2O2

H2O2 miR-21 H2O2

miR-21

for TOC only


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Fluorescent Nanocomposite for Visualizing Cross-Talk between

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