In Vivo Lighted Fluorescence via Fenton Reaction: Approach for

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In Vivo Lighted Fluorescence via Fenton Reaction: A New Approach for Imaging of Hydrogen Peroxide in Living Systems Changhui Liu, Weiju Chen, Zhihe Qing, Jing Zheng, Yue Xiao, Sheng Yang, Lili Wang, Yinhui Li, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00267 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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Analytical Chemistry

In Vivo Lighted Fluorescence via Fenton Reaction: A New Approach for Imaging of Hydrogen Peroxide in Living Systems

Changhui Liu,†,‡, § Weiju Chen,§ Zhihe Qing, †,* Jing Zheng,§ Yue Xiao,§ Sheng Yang,† Lili Wang,§ Yinhui Li,§ and Ronghua Yang†,§,*

†

School of Chemistry and Biological Engineering, Changsha University of Science and

Technology, Changsha, 410004, P. R. China; ‡

Department of Chemistry and Environmental Engineering, Hunan City University, Yiyang,

413000, P. R. China; §

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha, 410082, P. R. China

*To whom correspondence should be addressed: E-mail: [email protected], [email protected]; Fax: +86-731-8882 2523.

1

ACS Paragon Plus Environment


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ABSTRACT: By virtue of its high sensitivity and rapidity, Fenton reaction has been demonstrated as a powerful tool for in vitro biochemical analysis, however, in vivo applications of Fenton reaction still remain to be exploited. Herein, we report, for the first time, the design, formation and testing of Fenton reaction for in vivo fluorescence imaging of hydrogen peroxide (H2O2). To realize in vivo fluorescence imaging of H2O2 via Fenton reaction, a functional nanosphere, Fc@MSN-FDNA/PTAD, is fabricated from mesoporous silica nanoparticle (MSN), a Fenton reagent of ferrocene (Fc), ROX-labeled DNA (FDNA), and a cationic perylene derivative (PTAD). The ferrocene molecules are locked in the pore entrances of MSN, and exterior of MSN is covalently immobilized with FDNA. As a key part, PTAD acts as not only the gatekeeper of MSN, but also the efficient quencher of ROX. H2O2 can permeate into the nanosphere and react with ferrocene to product hydroxyl radical (⋅OH) via Fenton reaction, which cleaves FDNA to detach ROX from PTAD, thus in turn, lights the ROX fluorescence. Under physiological condition, H2O2 can be determined from 5.0 nM to 1.0 µM with a detection limit of 2.4 nM. Due to the rapid kinetics of Fenton reaction and high specificity for H2O2, the proposed method meets the requirement for real applications. The feasibility of Fc@MSN-FDNA/PTAD for in vivo applications is demonstrated for fluorescence imaging of exogenous and endogenous H2O2 in cells and mice. We expect that this work will not only contribute to the H2O2-releated studies, but also open up a new way to exploit in vivo Fenton reaction for biochemical research.

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INTRODUCTION Hydrogen peroxide (H2O2), as one of representative reactive oxygen species (ROS) in living organism, is an incomplete reduction product of oxygen and endogenously generated in a variety of enzyme-catalyzed reactions, which plays important roles in regulation of many physiological processes, including cell proliferation, differentiation, and migration.1 However, an increasing body of evidence demonstrates that excess H2O2 secretion will lead to oxidative stress, which can subsequently cause cellular damage. The relationships have been discovered between its overabundance and some serious human diseases, including diabetes,2 cancer,3 neurodegenerative disorders,4 and cardiovascular aging.5 Thus, H2O2 has been a potential diagnostic marker for several diseases, and the development of effective strategies for physiological H2O2 monitoring is urgently needed. Recently, by virtue of the convenient read-out and high spatial resolution, various of fluorescent sensing methods have been developed for the detection and imaging of H2O2.6-14 For example, a series of boronate-based probes, which can be activated by reacting with H2O2 for live-system imaging, have been successfully developed.8-11 Alternatively, benzil molecule has been also reported as a recognition unit for H2O2 based on the H2O2-mediated signal transformation from benzil to carboxylic acids.12 In addition, peroxalate nanoparticles have been developed to image in vivo H2O2 by a three-component chemiluminescent reaction.15 Fenton reaction, in which H2O2 is catalyzed by transition metal ion of low oxidation state (e.g., Fe2+) to produce hydroxyl radical (⋅OH),16,17 has been widely applied as a powerful tool for biochemical analysis. For example, based on ⋅OH-induced nucleic acid cleavage, a series of methods have been developed for DNA damage, H2O2 detection and H2O2-mediated biomolecule sensing.18-22 Though the Fenton reaction-based biochemical analysis has shown excellent sensitivity and rapid kinetics, it was limited to the applications in buffer or cell

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lysate. To the best of our knowledge, there is no report on exploiting Fenton reaction as a signal transformation tool for in vivo applications. In this work, we have developed a fluorescent approach for in vivo imaging of H2O2 via Fenton reaction. To realize in vivo fluorescence imaging of H2O2 via Fenton reaction, a functional nanosphere, Fc@MSN-FDNA/PTAD, is fabricated. The design mechanism and the synthetic

route

of the

functional

nanosphere

are

demonstrated

in

Scheme

1.

Fc@MSN-FDNA/PTAD is fabricated from mesoporous silica nanoparticle (MSN), a Fenton reagent of ferrocene (Fc), a 6-carboxylrhodamine (ROX)-labeled single stranded DNA (FDNA), and a cationic perylene derivative (PTAD). The ferrocene molecules are locked in the pore entrances of MSN, the exterior of MSN is covalently immobilized with 6-carboxylrhodamine (ROX)-labeled single-stranded DNA (FDNA). Then, a cationic perylene derivative, perylene tetracarboxylic acid diimide dimmer (PTAD), is aggregated on the MSN surface by noncovalent assembly with the FDNA.23,24 It is worth noting that, as a key part of the approach, the PTAD acts as not only the blocking cap to control closing of MSN pores, but also the efficient quencher to quench ROX fluorescence through fluorescence resonance energy transfer (FRET).25-27 When the functional nanosphere is endocytosed or injected into the living system, Fenton reaction can be triggered by H2O2 to generate ⋅OH,28 which will subsequently induce the oxidative cleavage of the immobilized FDNA to detach ROX from PTAD, thus in turn, lighting the ROX fluorescence. By virtue of the rapid kinetics and excellent specificity of Fenton reaction for H2O2 over other ROS, as well as high cell membrane permeability and good biocompatibility of the silica nanoparticle, the functional nanosphere will provide a new assay platform for sensitive detection and in vivo imaging of H2O2 in cells and tissues.

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EXPERIMENTAL SECTION Chemicals and Apparatus. 3-isocyanatopropyltriethoxysilane (3-ICP) was purchased from Aladdin Industrial, other small-molecule substances were obtained from Alfa Aesar (China) Chemical Ltd., all reagents were of at least analytical grade and used as purchase without any treatment. The oligonucleotide was synthesized by TaKaRa Biotechnology Co. Ltd. (Dalian, China) and purified by HPLC. Human cervical cancer cell (HeLa), human lung carcinoma cell (A549) and human embryonic kidney cell (HEK 293) were obtained from the biomedical engineering center of Hunan University (Changsha, China). Athymic BALB/c nude mice were purchased from SLAC (Changsha, China) Laboratory Animal Co. Ltd.. ROS were prepared following the reported literature.29 Ultrapure water was obtained from a Millipore water purification system (18.2 MΩ cm-1; Millipore Co., Billerica, MA). All apparatus used in this work are detailedly described in Supporting Information. Preparation of the Nanosphere. MCM-41 silica nanoparticles were prepared according to the literature procedure,30 and then functionalized with 3-isocyanatopropyltriethoxysilane (MSN-ICP) according to our previous method.31,32 The purified MSN-ICP (30 mg) was dispersed in 1.0 mL DMSO solution containing 5.0 mM Fc at room temperature. After stirring for 24 h, the Fc-loaded MSN-ICP (denoted as Fc@MSN-ICP) was centrifuged, washed, and dried under vacuum, and the loading capacity of 15.8 mg/g SiO2 was calculated by potentiometric titration. Then, the resulted solid was dispersed in 2.0 mL of deionized water, followed by addition of amino-modified FDNA (200 µM). The mixture was stirred overnight at room temperature to obtain DNA-modified and ferrocene-loaded MSN (Fc@MSN-FDNA). The purified Fc@MSN-FDNA was then shaken with PTAD (8.0 µM), which was synthesized according to the literature procedure (Figure S1, Supporting Information),23,33 for 8 h at room temperature to obtain the functional nanosphere (Fc@MSN-FDNA/PTAD) by centrifuging, washing, and drying under vacuum. 5



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Spectrophotometric Measurements. A stock solution was prepared by addition of Fc@MSN-FDNA/PTAD in Tris-HCl buffer (20 mM, pH 7.2) and the final concentration of the nanosphere was controlled at 0.1 mg/mL. For H2O2 assay, 1.0 mL of the nanosphere solution was first introduced into a quartz cell, followed by addition of different concentrations of H2O2. After incubation at ambient temperature for 5 min, the fluorescence intensity was measured with excitation wavelength at 560 nm. Cell Imaging. Cells were dispersed on 15 mm glass cover slips at 37 oC in 5% CO2 for 24 h. For monitoring endogenous H2O2, cells were pretreated with or without 0.5 µM PMA for 1.0 h or 1.0 mM NAC for 1 h, followed by incubation with the nanosphere (0.1 mg/mL) for 2.0 h. The cells were washed twice and then imaged. The fluorescence signal was recorded with the excitation of 559 nm. In Vivo Imaging. For imaging of exogenous H2O2, 100 µL of Fc@MSN-FDNA/PTAD (1.0 mg/mL in saline) was mixed with various amounts of H2O2 (0, 2.0 and 20 µM), and the mixture were then intramuscularly injected into the legs of anaesthetized female athymic BALB/c nude mice at a depth of ~ 3 mm. The fluorescence images were acquired over 1 h with a 60 s acquisition time using an IVIS Imaging System (Xenogen). Similarly, for imaging of endogenous H2O2, HeLa-tumor xenografts were allowed to grow to a volume of ~100 mm3. 100 µL of β-lapachone (β-Lap, 2.0 µM) was injected into the tumor of mouse models (n = 3) for 12 h, and NAC (1.0 mM) for 1.0 h, followed by 100 µL of the nanosphere (1.0 mg/mL in saline). The fluorescence images were acquired over 1.0 h with a 60 s acquisition time using an IVIS Imaging System (Xenogen).

RESULTS AND DISCUSSION FDNA/PTAD Assembly and Its Fluorescence Restoration via Renton Reaction. To demonstrate the mechanism for Fenton reaction-triggered signal generation, a ROX-labeled 6


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ssDNA (Rox-5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′, FDNA)24 was used for assembly with the quencher PTAD, and H2O2-indueced DNA cleavage with fluorescence restoration in the presence of ferrous ions. As shown in Figure 1A, strong fluorescence emission at ~610 nm is observed from FDNA in Tris-HCl buffer solution (10 mM, pH 7.2) due to fluorophore ROX (curve a, Figure 1A). As a polyanion, FDNA was able to induce noncovalent assembly with cationic PTAD to form DNA-templated ensemble (FDNA/PTAD), then the PTAD aggregation on the DNA strands can effectively quench the fluorescence of the adjacent ROX via FRET (curve b, Figure 1A)25,26. And the fluorescence intensity of FDNA was decreased dramatically with the increase of PTAD concentration, a largest quenching efficiency of up to 94% is achieved when the ratio of PTAD to FDNA reaches 30 : 1 (Figure S2, Supporting Information). Upon addition of H2O2 into the solution containing FDNA/PTAD, the fluorescence intensity was almost constant (curve c, Figure 1A), indicating that H2O2 itself had no influence on the FDNA/PTAD assembly. However, when in the presence of Fe2+ ions in the H2O2-contained solution, a remarkable fluorescence restoration of FDNA/PTAD was observed (curve d, Figure 1A), indicating that Fenton reaction was successfully triggered and its product ⋅OH can efficiently cleave the FDNA into short segments, with the release of ROX molecules. The fluorescence intensity of ROX increased gradually with the increasing of H2O2 concentration (Figure S3, Supporting Information), and a 7.5-fold fluorescence enhancement was obtained when the concentration of H2O2 reached 20 µM. The assembly of FDNA with PTAD and the H2O2-indueced cleavage of FDNA in the presence of Fe2+ were also visually confirmed by gel electrophoresis. As shown in Figure 1B, compared with the FDNA itself (lane 1), there is negligible influence from Fe2+ (lane 2) or H2O2 (lane 3). When the FDNA is assembled with PTAD, the band of FDNA/PTAD moves slower, with weaker fluorescence (lane 4). Expectedly, after treatment with both of H2O2 and Fe2+, one can see that the band of FDNA/PTAD has disappeared (lane 5). In summary, these results successfully 7



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demonstrated that the non-fluorescent FDNA/PTAD can be effectively assembled, and its fluorescence can be lighted by Fenton reaction via cleavage-mediated fluorescence restoration. Construction of the Functional Nanosphere. To realize H2O2-dependent fluorescence restoration in living system, we fabricated a functional nanosphere, Fc@MSN-FDNA/PTAD, from mesoporous silica nanoparticle (MSN), a Fenton reagent of ferrocene (Fc), a 6-carboxylrhodamine (ROX)-labeled single stranded DNA (FDNA), and a cationic perylene derivative (PTAD). The Fc@MSN-FDNA was prepared according to the procedure previously described.32,33 TEM micrographs showed that MSN displays uniform and monodispersed nanosphere in shape and well-ordered hexagonal porous structure with an average diameter of about 90 nm (Figure S4A and S4B, Supporting Information). The powder X-ray diffraction (XRD) and N2 adsorption-desorption isotherms (Figure S5 and Table S1, Supporting Information) further revealed that the MSN is of well-defined MCM-41 type of mesostructure.34 After modification with 3-isocyanatopropyltriethoxysilane (3-ICP) on the exterior of MSN (MSN-ICP), the Fourier transform infrared (FT-IR) spectra of MSN-ICP showed a strong and sharp peak of N=C=O functional group at 2275 cm-1 (Figure S6, Supporting Information). Next, ferrocene molecules were gated into the pores of MSN-ICP with loading capacity of 15.8 mg/g SiO2 (denoted as Fc@MSN-ICP, Figure S7, Supporting Information), and NH2-labeld FDNA was then covalently grafted on the surface along with a marked decrease of zeta potential (Figure S4C, Supporting Information) and a disappeared peak at 2275 cm-1 in FT-IR spectroscopy (Figure S6, Supporting Information). Finally, Fc@MSN-FDNA was wrapped through self-assembly of positively charged PTAD molecules on FDNA to obtain the nanosphere, Fc@MSN-FDNA/PTAD. Compared with the bare MSN, the obtained nanosphere was a little larger in size (Figure S4B, Supporting Information), and its nanopores were capped along with an obvious border on the exterior edge (Figure S4D, 8


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Supporting Information). These direct evidences demonstrated that the functional nanosphere was successfully constructed. As expected, Fc@MSN-FDNA/PTAD displayed negligible fluorescence signal (Figure S8, Supporting Information), this was due to the overlap of the emission spectrum of ROX and the absorption spectrum of PTAD aggregation (Figure S9, Supporting Information). The stability of the nanosphere under various conditions was tested. The results indicated that it was stable at physiological temperature, physiological pH range, high ionic concentration, and even in the complex biological fluid (Figure S10, Supporting Information). In addition, the Fc@MSN-FDNA/PTAD displayed excellent capability to protect its FDNA against enzymatic cleavage (Figure S11, Supporting Information).35 These results demonstrated that the proposed nanosphere holds promising function-stability under physiological conditions and exhibited great potential for application in living system. Fluorescent Sensing of H2O2 with Fc@MSN-FDNA/PTAD. After preparation, the nanosphere was used as a fluorescent probe for H2O2 detection. Figure 2A shows the fluorescence spectra of Fc@MSN-FDNA/PTAD in Tris-HCl buffer containing different concentrations of H2O2. Unnoticeable fluorescence signal is observed in the absence of H2O2, while significant fluorescence enhancements with the increasing in H2O2 concentration. There was a linear relationship between fluorescence enhancement and H2O2 concentration in the range from 5.0 nM to 1.0 µM (Figure 2B), with a detection limit of 2.4 nM (by 3σ/k).36 It is worth noting that, when a very low concentration of H2O2 (5.0 nM) was added to the detection system, a detectable fluorescence enhancement can also be recorded, indicating that the nanosphere was sensitive enough to meet the requirement for sensing endogenous H2O2 in living system.37,38 To verify the efficacy of the loaded ferrocene molecules for H2O2 detection, a control nanosphere without encapsulated ferrocene (MSN-FDNA/PTAD) was prepared and tested. As shown in Figure S12 (Supporting Information), the fluorescence emission of 9



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MSN-FDNA/PTAD at 610 nm did not show any changes after successive addition of H2O2, indicating that the response of the nanosphere toward H2O2 is dependent on the ferrocene-catalyzed Fenton reaction. The kinetics of Fc@MSN-FDNA/PTAD toward H2O2 was investigated by real-time monitoring of the fluorescence intensity as a function of the introduction of H2O2. As shown in Figure 2C, the response of Fc@MSN-FDNA/PTAD toward H2O2 is prompt, and the fluorescence intensity reaches saturation in 3 min, indicating that the nanosphere is a rapid-response tool for H2O2 detection.

To

demonstrate the selectivity of

the

Fc@MSN-FDNA/PTAD for H2O2 detection, the fluorescence responses toward other ROS were examined under the same conditions. As shown in Figure 2D, only H2O2 induces a large fluorescence enhancement, whereas other ROS, including hypochoorite (ClO-), singlet oxygen (1O2), superoxide anion (O2⋅−), peroxynitrite (ONOO−), etc, do not exert obvious interference, suggesting a high selectivity of the nanosphere toward H2O2 over other ROS. Imaging of H2O2 in Living Cells with Fc@MSN-FDNA/PTAD. To conduct Fc@MSN-FDNA/PTAD for monitoring H2O2 in living cells, the cell membrane permeability of the nanosphere was first examined. When Fc@MSN-FDNA/PTAD was incubated with human cervical cancer cell line (HeLa) and normal immortalized human embryonic kidney cell line (HEK 293), TEM micrographs of cells show that most nanospheres were endocytosed (Figure S13, Supporting Information). These results confirmed that Fc@MSN-FDNA/PTAD can be readily taken in by both cancerous and normal cells, and it showed great potential for cellular assay. The cellular toxicity was then evaluated using MTT assay.39 Figure S14 (Supporting Information) shows that the nanosphere has no apparent cytotoxicity to living cells even at high concentrations after 24 h of incubation, suggesting good biocompatibility for biological applications. Next, we investigated whether the nanosphere can efficiently differentiate the native H2O2 10


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level of tumor cells from that of normal cells. Two types of tumor cells, HeLa cell and human lung carcinoma cell (A549), and normal HEK 293 cell were incubated with the nanosphere at 37 oC, then the fluorescence signal within cells were analyzed by confocal laser scanning microscopy (CLSM). As shown in Figure 3, strong fluorescence signal is recorded from tumor cells, while very faint fluorescence signal is detected from the normal HEK 293 cells. It was in accordance with the fact that the concentration of H2O2 in tumor cells was much higher than that in normal cells.40 To further prove that the generation of fluorescence signal was dependent on H2O2, phorbol-12-myristate-13-acetate (PMA) that can induce H2O2 generation,41 and N-acetylcysteine (NAC) that can scavenge free-radicals in cancer cell,42 were used as modulators of intracellular H2O2 level. As shown in Figure 4, the fluorescence of the PMA-treated cells is significantly enhanced compared with the untreated cells, while the fluorescence intensity in the NAC-treated cells is much weaker than that in the cells without treatment. These results demonstrated that the functional nanosphere had good capability for imaging of intracellular H2O2, and sufficient sensitivity for distinguishing the fluctuations of H2O2 level. Imaging of H2O2 in Living Tissues with Fc@MSN-FDNA/PTAD. To further assess the in vivo application of Fc@MSN-FDNA/PTAD, we applied it for monitoring and imaging of H2O2 in mouse models. To demonstrate, 100 µL of nanosphere (1.0 mg/mL) was premixed with H2O2 at different concentrations, then they were injected into the legs at intramuscular depth of ~ 3 mm. As shown in Figure 5A, very faint fluorescence signal is observed under the in vivo imaging system when the mouse is injected with the nanosphere only, while the fluorescence signal is significantly enhanced when the mouse is injected with the nanosphere and H2O2. It suggested that the fluorescence signal of the tissue was indeed caused by H2O2, and the H2O2-activated nanosphere showed good deep-tissue imaging capability. Importantly, when the concentration of H2O2 was increased, higher fluorescence signal was obtained, 11



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indicating that the fluorescence intensity of the tissue was proportional to the amount of the concentration of H2O2. Finally, the ability of the nanosphere for imaging endogenous H2O2 levels in tumor tissue was assessed. Four mice bearing HeLa tumor were used as models, β-lapachone (β-Lap) as a stimulus for H2O2 production,43 and NAC as a scavenger for H2O2.9 To demonstrate, mice were given an intratumoral injection of β-Lap, NAC and the nanosphere. Figure 5B shows that the mice treated with the nanosphere exhibit greater fluorescence intensity than the mice treated with saline, demonstrating that the nanosphere is capable of imaging H2O2 generated in vivo. It is worth noting that the mice pretreated with β-Lap had much higher fluorescence signal than the control mice. However, the mice treated with β-Lap and NAC before imaging showed a ca. 20% decrease in total fluorescence compared to the mice treated with β-Lap only. These observations are in accord with the results of cells imaging, as shown in Figure 4, which demonstrates that the as-constructed nanosphere is sensitive enough to image endogenous H2O2 in living tissue.

CONCLUSIONS In summary, a fluorescent approach for in vivo imaging of H2O2 via Fenton reaction is reported for the first time. Compared to the known H2O2 fluorescent sensing and imaging methods, our proposed approach possesses three remarkable features: (1) by taking the advantage of Fenton reaction, rapid kinetics and good detection capability have displayed for H2O2 sensing. Only 3 min is required for signal saturation, and a detection limit of 2.4 nM is achieved, with negligible interference from other analogues. (2) Due to the high function-stability, good biocompatibility and desirable membrane-permeability of the functional nanosphere, H2O2 in living cells and tissue can be efficiently imaged through in vivo Fenton reaction-lighted fluorescence of the nanosphere. (3) As the first example, the powerful Fenton reaction is successful exploited to mediate signal transformation in living 12


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system, which may be beneficial to a wide range of fields, especially in vivo biochemical sensing and biomedical application.

ASSOCIATED CONTENT Supporting Information More experimental details, results and figures as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Correspondence author: * E-mail: [email protected], [email protected]; Fax: 86-731-88822523. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful for the financial support through the National Natural Science Foundation of China (21135001, 21405038, 21575018) and the Foundation for Innovative Research Groups of NSFC (21521063).

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Figures：：

Scheme 1. Schematic illustrations for in vivo imaging of H2O2 in living system via Fenton reaction. (A) PTAD-aggregated quenching of FDNA fluorescence and H2O2-induced fluorescence restoration via Fenton reaction. (B) The construction of the activable functional nanosphere by integrating the functional DNA and ferrocene with MSN, and its application in living system for H2O2 detection.

17



6

6

Fluorescence,10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 18 of 23

B

a

1

4

2

3

4

5

d 2 bc 0 550

600

650

700

Wavelength/nm

Figure 1. (A) The fluorescence emission spectra of FDNA in Tris-HCl buffer solution containing (a), 0.25 µM FDNA; (b), a) + 7.5 µM PTAD; (c), b) + 20 µM H2O2; and (d), c) + 20 µM Fe2+. (B) Gel electrophoresis shifts for the reaction products of FDNA with H2O2 and Fe2+. From left to right: Lane 1, FDNA; Lane 2, FDNA + Fe2+; Lane 3, FDNA + H2O2; Lane 4, FDNA/PTAD and Lane 5: FDNA/PTAD + Fe2++ H2O2.

18


10 5

20 µM

8

[H2O2]

6

B

10

8

F/F0

4

5 4

6

2 1

2

2

0 0.0 0.2 0.4 0.6 0.8 1.0

0 550

0 600

650

700

[H2O2]/µ M

0

C

8

12

6 4 2

5 10 15 20 25

[H2O2]/µM

F/F0 H 2O 2

D

9 6 3

Time/s

2

2

O

0

O H H 2O

800

tO

600

Bu

400

-

200

1

0

-

0 O 2 C lO O N O O

5

3

4

0

-

Fluorescence, 10

12

A

Wavelength/nm Fluorescene, 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


F/F0

Page 19 of 23

Figure 2. Fluorescence detection of H2O2 in buffer solution with the nanosphere: (A) Fluorescence emission spectra of Fc@MSN-FDNA/PTAD (λex= 560 nm) activated by H2O2 with different concentrations (0, 0.005, 0.05, 0.1, 0.3, 0.5, 1.0, 2.0, 5.0, 10, 20 µM). (B) Fluorescence enhancement (F/F0) of Fc@MSN-FDNA/PTAD toward different concentrations of H2O2. Inset: the linear relationship between F/F0 and H2O2 concentration. (C) Real-time monitoring of the fluorescence as a function of the addition of H2O2 (10.0 µM). (D) Fluorescence responses of Fc@MSN-FDNA/PTAD to H2O2 (10.0 µM), as well as various ROS (50.0 µM). 0.1 mg/mL Fc@MSN-FDNA/PTAD is used for all detections in Tris-HCl buffer (20 mM, pH 7.2) at 25 oC.

19



A549

HEK 293

12 9 6 3 29 3

HE K

A5 49

eL

a

0

Normalized Fluorescence

HeLa

H

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Figure 3. Confocal microscopy images of intracellular H2O2 in HeLa cells, A549 cells and HEK 293 cells with Fc@MSN-FDNA/PTAD. The bar graph is the quantification of relative fluorescence intensity of these cells treated with nanosphere, normalized to the HEK 293 cells. Before imaging, all cells are incubated with Fc@MSN-FDNA/PTAD (0.1 mg/mL) for 2.0 h at 37 °C. Scale bars: 20 µm.

20


A

B

C

D

1.6 1.2 0.8 0.4

AC N

A PM

on

tro

l

0.0 C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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Figure 4. Confocal microscopy images of HeLa cells under different conditions. (A) Cells incubated with the nanosphere (0.1 mg/mL) for 2.0 h; (B) Cells pretreated with PMA (0.5 µM) for 1.0 h and then incubated with Fc@MSN-FDNA/PTAD (0.1 mg/mL) for 2.0 h; (C) Cells pretreated with NAC (1.0 mM) for 1.0 h and then incubated with Fc@MSN-FDNA/PTAD (0.1 mg/mL) for 2.0 h. (D) Graph showing quantification of relative fluorescence intensity of cells treated with Fc@MSN-FDNA/PTAD in the presence of PMA or NAC, normalized to the control incubated with only nanosphere. Scale bars: 20 µm.

21


A

a2

a1

12

a3

a4

a5 9 6 3 0 0

2

20

b1

b2

1.6

b3

b4 b5 1.2 0.8 0.4

−L a

p+

β

N AC

p −L a

tr o

l

0.0


[H2O2]/µM

B

C on

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Page 22 of 23



β

Figure 5. In vivo imaging of exogenous and endogenous H2O2 with the nanosphere. (A) Representative images of the legs of mice treated with Fc@MSN-FDNA/PTAD (1.0 mg/mL) and exogenous H2O2: a1), saline; a2) only Fc@MSN-FDNA/PTAD (control); a3), Fc@MSN-FDNA/PTAD + 2.0 µM H2O2; a4), Fc@MSN-FDNA/PTAD + 20.0 µM H2O2, and a5), total fluorescence intensity integrated for 1.0 h after injection with the mixture of Fc@MSN-FDNA/PTAD and H2O2. (B) Representative images of HeLa-xenograft tumor models

treated

with

β-lapachone

(2.0

µM)

and

NAC

(1.0

mM),

following

Fc@MSN-FDNA/PTAD (1.0 mg/mL): b1), saline; b2) only Fc@MSN-FDNA/PTAD (control); b3),

β-lapachone

+

Fc@MSN-FDNA/PTAD;

b4),β-lapachone

+

NAC

+

Fc@MSN-FDNA/PTAD, and b5), total fluorescence intensity, integrated for 1.0 h after injection with β-lapachone ± NAC and the nanosphere in sequence.

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Graphic for TOC only

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In Vivo Lighted Fluorescence via Fenton Reaction: Approach for

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