MnO2 Platform for Label

Subscriber access provided by AUBURN UNIV AUBURN

Biological and Medical Applications of Materials and Interfaces

DNA-Templated Silver Nanoclusters/Porphyrin/MnO2 Platform for Label-free Intracellular Zn2+ Imaging and Fluorescence/ Magnetic Resonance Imaging Guided Photodynamic Therapy Yao Yao, Na Li, Xing Zhang, Jeremiah Ong’achwa Machuki, Dongzhi Yang, Yanyan Yu, Jingjing Li, Daoquan Tang, Jiangwei Tian, and Fenglei Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01530 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 48 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

ACS Applied Materials & Interfaces

DNA-Templated Silver Nanoclusters/Porphyrin/MnO2 Platform for Label-free Intracellular Zn2+ Imaging and Fluorescence/Magnetic Resonance Imaging Guided Photodynamic Therapy Yao Yaoa, Na Lia, Xing Zhanga, Jeremiah Ong’achwa Machukia, Dongzhi Yanga, Yanyan Yua, Jingjing Lia, Daoquan Tanga,*, Jiangwei Tianb, and Fenglei Gaoa,* a. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy,

Xuzhou

Medical University, 221004, Xuzhou, China b. School of Traditional Chinese Pharmacy, China Pharmaceutical University, 211198,

Nanjing, China

__________________________ *Corresponding

author,

Email

address:

[email protected]

[email protected] (D. Tang).

1

ACS Paragon Plus Environment

(F.

Gao);

ACS Applied Materials & Interfaces 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

Abstract Developing a theranostic platform that integrates diagnosis and treatment in one single nanostructure is necessary for efficient tumor treatment. Here, we presented a novel theranostic nanoprobe for non-labeled fluorescence images of Zn2+ and 635 nm-red light triggered photodynamic therapy (PDT) by a multifunctional DNA-templated silver nanoclusters/porphyrin/MnO2 nanoplatform. MnO2 nanosheets adsorbed hairpin DNA-silver nanoclusters (AgNCs) and porphyrin (P) by facile physisorption, which accelerate the transfection of nanoprobes and P into tumor cells. After entering the cell, the biodegradation of MnO2 nanosheets by glutathione and acidic hydrogen peroxide released AgNCs for label-free Zn2+ fluorescence imaging by the hairpin DNA-fueled dynamic self-assembly of three-way-DNA junction architectures, and the released Mn2+ could act as an effective magnetic resonance imaging (MRI) contrast agent. In addition, MnO2 was decomposed in the acidic H2O2-ample environment and produced O2 to overbear hypoxia-related PDT resistance, highly efficient PDT was obtained by much singlet oxygen (1O2) release of P-AgNCs-MnO2 nanoprobes under light irradiation compared with free P. In vitro and in vivo study confirmed that the P-AgNCs-MnO2 exhibited high fluorescence specificity, MRI, excellent PDT effect and good biocompatibility. This theranostic platform provided a new avenue for the fluorescence and MRI diagnosis of tumor and simultaneously efficient tumor treatment. Keywords: MnO2; Silver nanoclusters; MRI; Fluorescence; Photodynamic therapy.

2


Page 2 of 48

Page 3 of 48 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


1. Introduction The theranostic platform has acquired strong attention in the individualized nanomedicine and clinical application due to its diagnosis and treatment in one single nanostructure1-9. Photodynamic therapy (PDT) as a noninvasive technology was prominently concerned according to the weak side effects, high selectivity, short healing process and potential repeatability10-14. The photosensitizer plays a crucial role to achieve PDT in tumor therapy, however, usual photosensitizers are lacking effective targeting ability15,16. As circulation of photosensitizers in the body, the retention of reagent in the tumor tissues is minimal. In this circumstance, the therapeutic effect of PDT will unavoidably be greatly reduced in the absence of tumor imaging17. To achieve efficient tumor treatment, precise diagnosis is a determinant procedure and essential factor. The imaging probe, as another important component of the theranostic agent, is the diagnostic tool for determining the site and size of tumors, tracking the metabolic distribution of therapeutic agents, and evaluating the therapeutic effects18. Biological processes consist of various chemical reactions that are participated by many metal ions19. Metal ion homeostasis in biological systems play critical roles in their normal functions20. Numerous of studies suggest that the damage of metal ion homeostasis was an important cancer-related signal21. Among these metals, Zn2+ is involved in many biological functions such as gene transcription and signal transmission22,23. In addition, Zn2+ also one of the critical cancer-related biomarkers, including breast cancer, prostate cancer and so on22,23. In the past time, a variety of 3



methods have been established to detect Zn2+. The common methods are atomic absorption spectrometry24, electrochemical25, colorimetric26, Raman27 methods and so on28,29. However, the above methods all need complex chemical treatments of samples and are impossible to detect the concentration and distribution of Zn2+ in living cells. Fluorescence imaging is a powerful method for in-situ detection of zinc ions activity in cells due to its stability and simplicity. Li et al30 have developed a fluorescence nanoprobe based on AuNPs-DNAzyme to trace intracellular Zn2+. Yang et al31 have also developed a two-photon AuNPs-DNAzyme nanoprobe to obtain the imaging of intracellular Zn2+. Although the above-mentioned fluorescence detection methods have obtained prospective results in targets detection, the dye-labeled fluorescent probe was expensive and time-consuming, had poor anti-bleaching effect, or needed to construct a donor-acceptor pair, which limit its applications in practice. Therefore, it is required to develop a novel type of non-labeled fluorescent nanoprobe for imaging of Zn2+. Recently, fluorescent metal nanoclusters have obtained extensive attention because of its excellent photostability, sub-nanometer size and low toxicity for the development of molecular signals and sensors32-35. Especially, silver nanoclusters (AgNCs) employing DNA as a template have been used to biolabels and biosensors36,37. Its fluorescence intensity and wavelength could be changed by modulating the sequence and length of oligonucleotides38. In recent times, Werner et al39 confirmed that the fluorescence intensity of AgNCs would be extremely increased when AgNCs were approached to a G-rich overhang mediated by hybridization. In 4


Page 4 of 48

Page 5 of 48 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


view of this new property of AgNCs, “turn-on” fluorescent strategy has been designed to detect DNA and protein

40-44,

individually. It is hopefully that the engineering

DNA-based specific, no conjugation, and signal-on probe for Zn2+ detection will be developed based on the “turn-on” phenomenon of AgNCs. In addition, a weaker fluorescence or no fluorescence was observed in the fluorescence methods for endogenous Zn2+ imaging due to the ultralow concentrations of free Zn2+ in cells30-32. In order to obtain strong fluorescence, the above-mentioned methods all need to add additional Zn2+ into cells before imaging, which was lacking in fluorescence sensitivity according to the one-to-one binding30,31. In this regard, it is imperative to improve the sensitivity of the fluorescence methods. To get a more sensitive detection of tumor-related Zn2+, amplification strategies were designed for RNA-cleaving DNAzyme probes. Several signal amplification strategies have been reported in intracellular fluorescence imaging, such as cascade hybridization reaction, hybridization chain reaction, DNAzyme and catalytic hairpin assembly (CHA)45-53. The catalytic self-assembly amplification to form three-way-DNA junction architectures is a new development pattern of CHA. In comparison with the traditional CHA, the three-way-DNA junction architectures can provide a recyclable, faster and stable platform to achieve signal amplification strategy54-56. Therefore, a theranostic platform combining three-way-DNA junction architectures and biodegradable MnO2 nanosheet is imperative to develop for sensitive detection of Zn2+ and PDT in tumors. MnO2 nanosheets was widely applied in live-cell imaging because of its excellent physicochemical properties57-62, such as adsorbing DNA probes to enter cells through 5



endocytosis. It has been reported that MnO2 nanosheets could be decomposed by overexpressed hydrogen peroxide (H2O2) in tumor cells to generate O2, thereby overcoming the tumor hypoxic environment63-66. In addition, MnO2 nanosheets could consume intracellular glutathione (GSH) to shelter the singlet oxygen (1O2) produced by the photosensitizer and then enhance the PDT efficacy. In view of excellent physicochemical properties of MnO2 nanosheets, we selected MnO2 to carry hairpin DNA probes. During the three-way-DNA junction architectures, we introduced the G-rich sequence to induce the fluorescence enhancement of AgNCs. As illustrated in Scheme 1, in the existence of Zn2+, the substrate strand was cleaved by the DNAzyme (H0) to produce initiator DNA (T), which could trigger a cascade of hairpin-silver nanoclusters (H1、H2、H3) assembly reaction to produce three-way-DNA junction architectures, leading to an enhanced fluorescence readout. Meanwhile, the released Mn2+ offered strong magnetic resonance imaging contrast agent. In addition, highly efficient PDT was obtained by much 1O2 release of P-MnO2 under light irradiation. As executing detailed experiments, the function of the platform confirmed its the application prospect in tumor.

2. Experimental 2.1. Synthesis of MnO2 Nanosheets. MnO2 nanosheets was prepared in accordance with the recent published literature step with minor change51. Before the synthesis reaction, 0.6 M tetramethylammonium hydroxide (TMA·OH) in 3wt % H2O2 and 0.3 M MnCl2·4H2O aqueous solution were first prepared. In short, 40 mL mixing solution of TMA·OH and 20 mL aqueous solution of MnCl2·4H2O were 6


Page 6 of 48

Page 7 of 48 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


mixed in 15 s. Then the mixing solution changed dark brown and was stirred powerfully 12 h in the bare air at 25 ℃. Afterwards, centrifuging was used to gather the pre-product of MnO2 nanosheets which was washed many times and further was dried at 55 ℃ for 24 h. To get MnO2 nanosheet with small size, 20 mg dried MnO2 nanosheet was dissolved in 40 mL water and ultrasonicated for 5 h by ultrasonic cleaning machine and ultrasonic cell crasher for 0.5 h. 2.2. Synthesis of Fluorescent AgNCs. DNA stabilized AgNCs were prepared based on the reported literature with some modifications40. In brief, the 15 μM hairpin DNA probes (H1, H2, H3) of C-rich sequence were dissolved with 10 mM sodium phosphate buffer (PB, pH 7.4). In order to gain the stable stem-loop hairpin structures, each hairpin DNA probe was hold at 95 °C for 5 min and programmed to cool down to 25 °C before use. A 10 mM solution of AgNO3 was added into solution of each hairpin probe by vigorous shaking for 2 min and then chilled on ice for 0.5 h. A 2 mM solution of NaBH4 was added into previous solution by strong shake for 1 min and then maintained at 4 ºC overnight before use. The final concentration ratio among each hairpin DNA probe, AgNO3 and NaBH4 was 1:21:21. In the process of preparation of AgNCs, dark room condition should be provided to guarantee the fluorescence characteristic of silver nanoclusters. 2.3. Preparation of the P-AgNCs-MnO2. The preparation of hairpin DNA probes and P were mixed with MnO2 nanosheets for 20 min at 25 ºC to adsorb hairpin DNA probes and P on MnO2 nanosheets by the interaction of physisorption. Then, the PB (10 mM, pH 7.4) was added, and the mixing solution was kept for another 40 min at 7



25 ºC. 2.4. Electrophoresis Experiment. Similar to previous experiments, H0, H1, H2 and H3 was hold at 95 °C for 5 min and programmed to cool down to 25 °C before use. For the reaction of DNA assembly, the concentration of H0 was 100 nM and other hairpin DNA probes were 500 nM and then kept for 4 h at 37 °C. 8 μL of each sample and mixed with loading buffer were added into the 2.5% agarose gel electrophoresis. The agarose gel was stained with gentle nucleic acid dye before preparing it and imaged with Tanon-1600R gel imaging system (Shanghai, China) under UV irradiation. 2.5. Detection of Zn2+ in Vitro. The P-AgNCs-MnO2 was prepared by previous method. For the fluorescence detection, 1 mM GSH was pour into the P-AgNCs-MnO2 in PB (10 mM, pH 7.4) to reduce MnO2 nanosheets for 5 min. Zn2+ of various concentrations were added into the mixture of solution and then incubated at 37 ºC for 3 h. Finally, the fluorescence emission was recorded by fluorescence spectrophotometer (Shimadzu FL4600). 2.6. In Situ Imaging of Zn2+ with the P-AgNCs-MnO2. Before incubating the nanoprobes, the prepared adherent MCF-7 cells were treated with PBS, TPEN (150 μM), DTDP (150 μM) and Zn2+ (5 μM) for 30 min and then the cells were cleaned with fresh RPMI-1640. The preparation of P-AgNCs-MnO2 in fresh RPMI-1640 was added into cells at 37 °C for 4 h and then the cells were washed 3 times with PBS and fixed with a cell fixing solution. Finally, the cells were observed under a fluorescent imaging system. 8


Page 8 of 48

Page 9 of 48 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


2.7. Detection of 1O2. Singlet oxygen sensor green (SOSG) which was extremely sensitive to 1O2, was used to evaluate the level of 1O2 during the detection process. Free P and P-AgNCs-MnO2 with or without MCF-7 cell lysis solution were mixed with SOSG in PB (10 mM, pH 7.4) and then irradiated under 635 nm for 10 min. After that, the generated 1O2 was determined immediately by detecting restored SOSG ﬂuorescence. 2.8. Cytotoxicity Assay. In one group, the prepared adherent MCF-7 cells treated with numerous concentrations of MnO2 nanosheets for 24 h. In another group, PBS, AgNCs-MnO2, P and P-AgNCs-MnO2 were introduced to the cell medium for 4 h incubation and then substituted with fresh RMPI-1640. The irradiated group was exposure to white light for 1 h, and the other group did the dark treatment. After 24 h incubation, cells culture medium substituted with 10 µL of CCK-8 and 90 µL of fresh culture medium and incubated for about 40 min. Then, the absorbance of each well was detected by using a microplate reader (Thermo Scientific™ Varioskan™ LUX). 2.9. Live/Dead Cells Staining. The prepared adherent MCF-7 cells were incubated with PBS, AgNCs-MnO2, P and P-AgNCs-MnO2 in fresh RMPI-1640 at 37 °C for 4 h and then substituted with fresh RMPI-1640. The irradiated group was exposure to white light for 1 h and the other group was kept in the dark. After 12 h incubation, the cells were cleaned 2 times with mild PBS and then 500 μL of Calcein AM/PI solution was mixed and reacted for 40 min at 25 °C without light. After that,

the calcein

AM/PI solution was substituted with 100 μL PBS. Finally, The MCF-7 cells were subjected to flourescence imaging using the inverted fluorescence microscope 9



(Olympus IX73). 2.10. In Vivo Image and PDT Efficacy. For vivo image, 100 μL PBS and 100 μL P-AgNCs-MnO2 nanoprobe (10 μM P, 10 μM AgNCs and 180 μg mL-1 MnO2) were intratumorally injected into the tumor-bearing mice, respectively. Imaging was carried out using an imaging system at 6 h after injection. For PDT, 100 μL PBS, 100 μL AgNCs-MnO2 (10 μM AgNCs and 180 μg mL-1 MnO2), 100 μL of 10 μM P and 100 μL of P-AgNCs-MnO2 nanoprobe (10 μM P, 10 μM AgNCs and 180 μg mL-1 MnO2) were intratumorally injected into the tumor-bearing mice, respectively. After 6 h, MCF-7 tumor position of nude mice was illuminated by 635 nm laser (1 W cm-2) for 5 min. The entire tumor was evenly illuminated by moving the light. Tumor diameter was measured by vernier caliper and the volume of tumor was obtained from the formula: V =0.5×ab2, where a and b were the width and height of the tumor. After 16 days, MCF-7 tumor tissue was removed from the mouse for weighing and to examine the histopathology by H&E staining.

3. Results and Discussion 3.1. Theory of Signal Amplification Strategy. The theory of the label-free and cyclic amplification nanoplatform for Zn2+ detection and 635 nm-red light triggered PDT was depicted in Scheme 1. In this nanoplatform, four specific DNA probes were used: the process of three-way-DNA junction architectures consisted of four hairpin structures (H0, H1, H2, H3), which were painstakingly designed to prevent spontaneous self-assembly without the substrate Zn2+. In order to realize fluorescence enhancement, the C-rich sequence (12-nt sequences long) at the 3’ end of each hairpin 10


Page 10 of 48

Page 11 of 48 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


DNA-silver nanoclusters (H1, H2, H3) was partially complementary to the G-rich sequence (18-nt sequences long) at the 5’ end of each neighboring DNA. The van der waals force powerfully mediated the basal plane of MnO2 nanosheets adsorption of all hairpin DNA. Due to their excellent biocompatibility, MnO2 nanosheets could transfer the four hairpin DNA probes into cells by endocytosis, thereby preventing them from enzymatic degradation. After endocytosis, the MnO2 nanosheets promptly converted to Mn2+ dissolved in cytoplasm by the reduction of GSH and acidic H2O2 in cells, and then the adsorbed hairpins were free. In the absence of target Zn2+, no fluorescence could be found, because all hairpin DNA formation were closed, and the AgNCs and the G-rich sequence labeled on the one probe were spatially isolated by additional nucleotide sequences, because the fluorescent intensity was enhanced due to the AgNCs close-enough proximity to G-DNA. In the existence of Zn2+, the substrate strands were cut off by the DNAzyme (H0) to produce initiator DNA (T) which could trigger a cascade of hairpin (H1 、 H2 、 H3) assembly reaction to produce three-arm-DNA junction architectures. In this state, an enhanced fluorescence was observed when the AgNCs on a cytosine-rich probe was sufficiently close to the guanine-rich DNA probe. The designed sensing system could amplify detection of Zn2+ through the hairpin DNA-fueled dynamic assembly signal amplification mechanism. Furthermore, the MRI contrast agent properties of Mn2+ make it efficient MRI. Meanwhile, once endocytosed, P-AgNCs-MnO2 nanoprobe was freed in the acidic and H2O2-ample environment of tumor cells to initiate in situ production of O2 and overbear hypoxia-related PDT resistance and then generate much 1O2 to kill 11



Page 12 of 48

tumor cells. As a result, the designed theranostic platform of label-free P-AgNCs-MnO2 nanoprobes could effectively detect Zn2+ and realize therapeutics of cancer. 3.2.

Characterization

of

the

P-AgNCs-MnO2

nanoprobe.

Multiple

characterization methods were employed to demonstrate the obtained AgNCs. As displayed in Figure 1A, the transmission electron microscope (TEM) image indicated that the AgNCs with an average diameter of ~2 nm was uniformly distributed, because the ssDNA possessed pre-formed templates and prevents the growth of nanoclusters once an expected size was received. In addition, AgNCs had an excellent monodispersity in aqueous solution (inset of Figure 1A). The high-resolution TEM image (Figure S1A) displayed the distinct lattice fringes of the prepared AgNCs with an interplanar distance of 0.23 nm, equaling to (111) plane of an Ag crystal. Atomic force microscope (AFM) was also applied to display the AgNCs for studying the size of AgNCs. The AgNCs were distributed evenly in Figure 1B. An AFM image (inset of Figure 1B) shows the topographic heights of AgNCs range from 1.7 nm to 2.2 nm (average height of approximately 2 ± 0.2 nm), which was aligned with the TEM image. As displayed in Figure 1C, two distinct peaks were found at 366.8 and 373.8 eV, which should be corresponded to the 3d Ag (0) excited photoelectrons, stating that Ag (0) NCs had been formed and existed. What’s more, the fluorescence spectra of the AgNCs was displayed in Figure 1D (Ex-1, Em-1), the maximum emission was found at 545 nm when excited at 462 nm. We also found that the fluorescence enhancement was extremely obvious and the 12


Page 13 of 48 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


maximum emission was found at 677 nm after the G-probe was added (Figure 1D, Ex-2, Em-2). The results showed that such hybridization could make the dark AgNCs extremely close to the G-rich sequence and greatly increase its fluorescence in the buffer. The excellent property of MnO2 nanosheets acts a crucial role in our designed strategy. As displayed in Figure 1E and 1F, the MnO2 nanosheets presented an obvious two-dimensional single-layer sheet structure with ~200 nm in width and a size of height profile ~1.5 nm. In addition, the dynamic light scattering (DLS) results displayed that the mean size of MnO2 nanosheets was approximately 200 nm (Figure S2A). The zeta potential value of MnO2 nanosheet was measured to be about -26.7 mV (Figure 1H), showing that the surface was negatively charged. This charge was due to the formation of Mn-vacancies on the MnO2 nanosheet and performed excellent water dispersibility (Inset of Figure 1E). In addition, the UV-Vis spectra of the MnO2 nanosheets showed a maximum absorption peak at ~370 nm. The XPS of MnO2 nanosheets exhibited the characteristic peaks equaling to Mn 2p1/2 (642.0 eV), Mn 2p3/2 (653.9 eV), and O 1s (Figure S2B; Figure 1K, curve a). The phase and crystallographic structure of the MnO2 nanosheets were examined by X-ray diffraction (XRD). As shown in Figure 1L, significant XRD peaks were observed at 2θ = 12.16, 36.49, and 65.81 and could be attributed to the (001), (100), and (110) planes of birnessite type MnO2, separately. The prepared MnO2 nanosheets proved to be successful by above-mentioned experimental results. Similar to the situation of those previously reported MnO2 nanosheets, we speculated the absorption of hairpins DNA onto MnO2 nanosheets results from physisorption. TEM, AFM, XPS, UV-vis 13



and XRD were applied to examine the binding of the AgNCs to MnO2 nanosheets. TEM and AFM observation clearly showed that the dots were well interspersed on the face of MnO2 nanosheets (Figure 1I and Figure 1J). By the way, the TEM images of AgNCs-MnO2 and P-AgNCs-MnO2 were almost indistinguishable because the photosensitizer P was an amorphous organic compound (Figure S1B). In addition, the UV-vis spectrum of AgNCs-MnO2 nanosheets presented a strong absorption peak at about 260 nm (Figure 1G, curve b) compared with pure MnO2, which could be well-assigned to the adsorption of the DNA strand, showing that the AgNCs successfully bound to the surface of MnO2 nanosheet. In addition, XPS and XRD were used to validate the fabrication process. From the XPS image of AgNCs-MnO2 composites, the characteristic peaks at 464 and 458.4 eV were clearly obtained, which are belonged to Ag 2p1/2 and Ag 2p3/2. It was important to note that for AgNCs-MnO2 composite, at 2θ = 37.9° gained strong characteristic peak, which was almost consistent with the peak of an Ag (111) lattice in position, suggesting that the AgNCs on the surface of MnO2. Furthermore, the significant new three peaks at 400.2 eV were observed in the XPS image of P (Figure 1K, curve c), which was corresponding to N 1s of P. The XRD pattern of P-AgNCs-MnO2 displayed reduced peak intensities in comparison with the two other samples (Figure 1L, curve c), which confirmed the immobilization of P on the face of MnO2. The above data indicated that P-AgNCs-MnO2 was successfully synthesized. 3.3. Feasibility of the Strategy for Zn2+ Detection. The catalytic self-assembly amplification to form three-way-DNA junction architectures was first confirmed by 14


Page 14 of 48

Page 15 of 48 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


electrophoretogram. As shown in Figure 2A, hairpin DNA probes (H0) exhibited individual clear band, because H0 had no RNA-cleaving reaction without Zn2+ (lane a). The Zn2+-specific DNAzyme of hairpin DNA probes (H0) could release initiator DNA (T) (below band) and substrate strands (upper band) in the presence of Zn2+, which produced two new band compare lane a. The three hairpins (H1, H2 and H3) mixture shows only one band, because of the same number bases and good metastability respectively of the three hairpins (lane c). In the presence of Zn2+, the migration of the mixture of H0, H1 (lane d) was slower than lane c, which attributed to the hybridization of H1 and T (the fragment of H0). And the T/H1/H2 (Lane e) generated by the reaction solution of H0, H1 and H2 had a low mobility with respect to the site of the band in Lane d. Furthermore, the mixture of Zn2+, H0, H1, H2 and H3 in Lane f showed a brighter band with lower mobility compared with Lane e, and could release initiator DNA (T) (below band), indicating that the three-arm-DNA junction architectures were successfully formed by the hairpin DNA-fueled dynamic self-assembly. The three-way DNA junction architectures was also characterized by AFM. As shown in Figure 2B, the emergence of DNA networks was observed in the three-arm DNA branched shape marking with the ring. The three-arm DNA branched junctions had strong rigidity and excellent dispersibility with an average length of 20 nm, which was consistent with the theoretical length of the DNA structure. Therefore, the AFM visualization of DNA networks on the surface validates the ability of the three-way DNA junction architectures. 15



The feasibility of the designed nanoprobes for Zn2+ detection was further verified by the fluorescence measurement on the different mixtures. As shown in Figure 2C, the mixed solution of H0, H1, H2, and H3 had the smallest fluorescent signal (curve a) because the interaction of the three hairpin DNA was weak in absence of the target. In addition, the mixed solution of H1, H2, H3 and Zn2+ had also the smallest fluorescent intensity (curve b). However, the fluorescence intensity of H0-H1 did not increase compared to curve a and b (curve c), further demonstrating that the fluorescence signal was due to the close-enough proximity of AgNCs to G-DNA. When Zn2+ was put into the mixed solution of H0, H1, and H2, the fluorescence intensity was obviously enhanced (curve d). This enhancement was mostly related to the reason that the cleaved DNA strand of H0 in presence of target Zn2+, triggered one-step hybridization between H1 and H2 and AgNCs brought into close-enough proximity to G-DNA on the adjacent DNA strand, leading to fluorescent intensity enhancement. Although there was a significant increase, the incubation of Zn2+ ions mixed with H0, H1, H2, H3 enhances more significantly in fluorescent response (curve e), showing that the three-way DNA junction nanostructures were successfully assembled. Thus, these results showed that the intense signal amplification efficiency of the designed three-arm DNA junction architectures could be used to detect Zn2+. In addition, the fluorescence property of the prepared P-AgNCs-MnO2 nanoprobes was evaluated with or without 1 mM GSH (Figure S3A). To obtain the optimal experimental conditions of best sensing performance, the incubating time and the concentration of MnO2 nanocarrier were studied (Figure S3B, S3C). Under 16


Page 16 of 48

Page 17 of 48 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


the best conditions, the fluorescence signals of the nanoprobe and various concentrations of Zn2+ were measured. In a typical experiment, P-AgNCs-MnO2 (50 μg mL-1) were treated with Zn2+ for 3 h. Figure 2D indicated that the fluorescence intensity of the nanocomplex enhanced with Zn2+ of various concentrations (0, 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 200, 300 nM). Through the correlation of fluorescence changes at 639 nm, a calibration curve was acquired for continuous concentration of the Zn2+ from 0 to 300 nM (Figure 2E). The result showed that the fluorescence spectra was positively correlated with the concentration of Zn2+, and the limit of detection of the probe was 0.023 nM (3σ/slope). The detection scope of the nanoprobe satisfies the sensing needs of Zn2+, since the concentration of Zn2+ in living cells is usually 0-300 nM23. The high sensitivity of this strategy was closely related to the three-way-DNA junction architectures, confirming that the method could be effectively applied to detect Zn2+. To validate the feasibility of the P-AgNCs-MnO2 nanoprobe to detect Zn2+ in intricate biological environment, this strategy was exploited to the detection of various biologically relevant metal ions and intracellular biomolecules for control experiments, including Mn2+ (25 μM), Mg2+ (25 μM), Cu2+ (1 mM), Ca2+ (1 mM), Na+ (1 mM), K+ (1 mM), Fe3+ (25 μM), lysozyme (20 μM), mRNA (20 μM), telomerase (20 μM), glucose (5 mM), ascorbic acid (5 mM), uric acid (20 μM) and dopamine hydrochloride (20 μM) separately. As shown in Figure 2F, the fluorescence signal of Zn2+ (25 nM) was obviously higher than other metal ions and intracellular biomolecules even when they had higher concentration. These 17



experimental data showed that such a P-AgNCs-MnO2 nanoprobe could be applied for detecting Zn2+ in complex biological system, which proved that the DNAzyme sequence was very specificity to Zn2+. 3.4. Fluorescence of P-AgNCs-MnO2 in Living Cells. To investigate the practicality of this strategy in complex intracellular environments, we selected MCF-7 cells for cellular uptake and Zn2+ imaging. MnO2 nanosheets as nanocarriers can be degraded by GSH and acidic H2O2 of cellular species in cancer cells, enabling P and hairpin DNA probes delivery and releases for living cells. After endocytosis of the P-AgNCs-MnO2 nanocomplex, with the transfection of the hairpin probes H0, H1, H2 and H3, the cells showed a more pronounced red fluorescence image corresponding to endogenous Zn2+ (Figure 3E). In addition, we transfected into cells employing only three DNA probes H0, H1 and H2. As expected, the fewer and weaker fluorescence image was acquired in the cell (Figure 3D). It was worth noting that the cleaved DNA strand of H0 with the target Zn2+, triggered one-step hybridization between H1 and H2, and DNA-templated AgNCs were close enough proximity to G-DNA on the adjacent hairpin DNA, resulting in a slight increase in fluorescent intensity. Therefore, this result showed that the fluorescence signal was really lower without three-way DNA junction amplification. Control experiments using cells treated with the H0-H1-MnO2 and H1-H2-H3-MnO2 nanoprobes showed that there was no fluorescence (Figure 3B and Figure 3C). In addition to studies of adherent cells, flow cytometry was also used to examine the feasibility of this strategy on post-digestion suspension cells. As displayed in Figure 3F, flow cytometry results of the three 18


Page 18 of 48

Page 19 of 48 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


groups also indicated that three-way DNA junction amplification exhibited much higher fluorescence signals than one-step hybridization between H1 and H2. These findings clearly proved that three-way DNA junction amplification was expected to provide higher amplification efficiency for sensitive detection of Zn2+ in living cell imaging. To further verify that the fluorescence signal of P-AgNCs-MnO2 was not related to P, P-MnO2 was treated with MCF-7 cells for 4 h and no fluorescence image was observed (Figure 3A). The results indicated that P-AgNCs-MnO2 was successful for detection Zn2+ in the MCF-7 cells. The feasibility of using the strategy for in situ imaging of Zn2+ in cells was also tested by dynamic observation of AgNCs fluorescence after the transfection of the probes (Figure S4). In the first hour, no significant fluorescence imaging was found. After 1 h, a slight fluorescence was found in the cytoplasm, and the intensity enhanced with the prolonged incubation time until it attained a maximum at 4 h. The increase in fluorescence intensity was closely associated to the formation of many three-way DNA junction architectures, indicating the presence of Zn2+ in the cytoplasm. To further confirm the fluorescence signal generated by endogenous Zn2+ of the cells, the designed method was applied to measure the changes of intracellular Zn2+ after different stimulation. Before incubating with the P-AgNCs-MnO2 probes, the cells were stimulated with N, N, N0, N0-tetrakis (2-pyridyl) ethylenediamine (TPEN), 2,20-dithiodipyridine (DTDP) for 30 min, and the PBS group was used as a control. In these drug stimulation treatments, TPEN could increase the concentration of intracellular Zn2+, and B could reduce 19



Page 20 of 48

the concentration of intracellular Zn2+. The TPEN-treated MCF-7 cells had lower fluorescence intensity (Figure 4B), whereas DTDP pre-incubated cells had higher fluorescence intensity (Figure 4C), comparing to the PBS group (Figure

4A).

Moreover,

obvious

increase

of

fluorescent

intensity

of

P-AgNCs-MnO2 was found when the cells were added gradually with Zn2+ (Figure 4D) provided from outside the cells. All the results showed that the fluorescence signal was produced by the endogenous Zn2+. Subsequently, to further verify the applicability distinguishing cancer cells from normal cells of P-AgNCs-MnO2 nanoprobes, we chose five different cell lines for research. Different cancer cell lines were observed various fluorescence intensity, indicating that Zn2+ levels were different in each of these cells. Brighter fluorescence images were found for cancer cells (MCF-7 cells (Figure 5A), HeLa cells (Figure 5B), HepG2 cells (Figure 5C)), indicating that the selectivity of the P-AgNCs-MnO2 nanoprobe was excellent and the Zn2+ levels of cancer cells were higher than normal cells (LO2 cells (Figure 5D) and L929 cells (Figure 5E)). MCF-7 cells had stronger fluorescence intensity than other cells, because Zn2+ was a tumor-associated biomarkers of breast cancer cells. Overall, these results confirmed that the P-AgNCs-MnO2 nanoprobe could effectively detect Zn2+ of various concentration in cancer cells by cell fluorescence image, providing an efficient diagnostic tool for identifying cancer cells from normal cells. 3.5. Fluorescence and MRI Imaging. Accurate diagnosis can effectively guide tumor treatment. To validate the effectiveness of our strategy for theranostics, we 20


Page 21 of 48 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


performed a fluorescence imaging study of MCF-7 tumor-bearing mice for the diagnosis. As shown in Figure S8A and S8B, significant fluorescence was noticed at the tumor region of the P-AgNCs-MnO2 group at 6 h after injection compared with PBS control. These results indicated that our P-AgNCs-MnO2 nanoprobe could be employed in the sensitive detection of Zn2+ in vivo. To further investigate the feasibility of P-AgNCs-MnO2 in MR imaging, the MRI signals of P-AgNCs-MnO2 solutions were first detected in prosthesis. With the increase of the P-AgNCs-MnO2 concentrations, the longitudinal relaxation rates (1/T1) increases (r1=8.23 mM-1·S-1), and the MR imaging was brighter, indicating that P-AgNCs-MnO2 could be employed as a contrast agent for MRI (Figure S9A and S9B). The MR imaging of P-AgNCs-MnO2 was then found in vivo, and the comparable MR images were obtained by data reconstruction and multiple analysis of acquired intensity data. After injection with P-AgNCs-MnO2 solutions at 6 h, the MRI signals were markedly enhanced in the tumor site compared with PBS control (Figure S9C and S9D), verifying that P-AgNCs-MnO2 could accumulate in the tumor position. In summary, MR images in vitro and in vivo confirmed that P-AgNCs-MnO2 was the outstanding and prospective contrast agent for MR images of tumors. 3.6. P-AgNCs-MnO2 Triggered 1O2 Generation. As is well known, MnO2 has an ability to catalyze H2O2 to produce O2 in neutral solutions. For another, malignant cancer cells generate excessive amounts of H2O2 compared with normal cells, resulting in a substantial increase in H2O2 levels inside and outside the tumor cells. Therefore, there are an acidic and H2O2-rich microenvironment in solid tumors. In an 21



acid environment, although MnO2 is a catalyst, it can also oxidize H2O2 to produce O2 with the release of Mn2+ and then the enhancement of O2 contributes to the improvement of PDT because of the photocatalytic effect of O2 to harmful 1O2 by 635 nm laser irradiated. As displayed in Figure S5, when the concentration of H2O2 attained 100 μM in pH = 5.5, MnO2 nanosheets was completely decomposed to produce a large amount of oxygen. An oxygen quenched indicator ([Ru(dpp)3]Cl2) was involving in further confirming the production of oxygen. To verify the versatility of P-AgNCs-MnO2 self-tracking oxygen supply, fluorescence cell imaging analysis was performed using the [Ru(dpp)3]Cl2 channel. As shown in Figure 6, the fluorescence gradually weakened with the incubation time prolonged and attained a minimum at 8 h, which was caused by the production of O2 with the decomposition of MnO2. In view of this results, we believe that the MnO2 increased oxygenation within tumor cells. We also used SOSG as a fluorescent indicator to verify that P-AgNCs-MnO2 and P generates 1O2 under the 635 nm-red light triggered activation. The fluorescent SOSG endoperoxide was produced by 1O2 oxidation of SOSG, which provided another indirect method for reporting 1O2 levels. Figure S6A showed that the P-AgNCs-MnO2 generated a large of 1O2 under irradiation by SOSG compared with pure P in the existence of MCF-7 cell lysates. In addition, the P-AgNCs-MnO2 in the existence of MCF-7 cell lysates displayed an enhanced 1O2 generation than that of P-AgNCs-MnO2 in the absence of MCF-7 cell lysates. The results displayed that the effect of the MnO2 on the photosensitizers was obvious due to the energy resonance transfer, which would have an influence on the PDT performance. In addition, the 22


Page 22 of 48

Page 23 of 48 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


production of intracellular ROS was verified employing a DCFH-DA probe. DCFH-DA could be oxidized to DCF by 1O2 to show green fluorescence. After entering the cell, DCFH-DA deacetylated to 2’,7’-dichlorofluorescin (DCFH) under the catalysis of intracellular esterases, and then converted to the fluorescent DCF by the oxidation of 1O2. As prospective, the P-AgNCs-MnO2 group produces stronger green fluorescence than the P group under light irradiation (Figure 7). As displayed in Figure S6B, the flow cytometry results of three groups of cells also indicated that the fluorescence signal of P-AgNCs-MnO2 was remarkably stronger than that of P, which was aligned with the result of enhanced SOSG intensity after 1O2 production. The light-sensitive P group also showed weak green fluorescence under light irradiation. In contrast, no fluorescence was found in the untreated group and only the MnO2 group. This phenomenon was owing to the oxygen produced by the decomposition of H2O2 catalyzing by the MnO2 nanozyme and the concentration of GSH drops, which protected the 1O2 formed by the reaction of GSH with MnO2. The formation of 1O2 in these cells may offered an excellent PDT platform to achieve cancer cells therapy. 3.7. Photodynamic Therapy. The ability of the P-AgNCs-MnO2 to generate 1O2 in extracellular environment has been confirmed, and then the PDT efficiency of P-AgNCs-MnO2 should also be investigated in cancer cells. The live cell-staining assay and cell-viability assay were involved in investigating the therapeutic efficiency. To further verify the PDT efficacy of P-AgNCs-MnO2, free P as comparison was also treated with cells. As displayed in Figure 8B, there are a large quantity of dead cells in cells incubated with P-AgNCs-MnO2 under light irradiation, the group of free P 23



also had some dead cells, but less than P-AgNCs-MnO2 group. Figure 8A showed no obvious cytotoxicity in cells incubated with PBS, AgNCs-MnO2, free P, or P-AgNCs-MnO2 without light irradiation and no clear differences was observed. What’s more, similar results were achieved by the CCK-8 cell-viability assay compared with the fluorescence live/dead cell staining. Figure S7A showed that the cell viability of P-AgNCs-MnO2 nanocomplex (21.6%) was obvious lower than free P (59.8%) under light irradiation, and other groups displayed higher cell viability. With the concentration of P enhanced, the cytotoxicity of P-AgNCs-MnO2 nanocomplex was significantly increased and free P also increased under light irradiation (Figure S7B). The cell viability of P-AgNCs-MnO2 nanocomplex reaches 19.6 % and free P reaches 51.6 % when the concentration of P increases to 3.5 μM. However, the cell viability of P-AgNCs-MnO2 was slightly changed when the concentration of P increased from 0 to 3.5 uM without light irradiation. Next, Annexin V-FITC/PI double staining by flow cytometry was involved in verifying the cell death, apoptosis and necrosis assay. As depicted in Figure 8C, MCF-7 cells treated with P-AgNCs-MnO2 nanocomplex exhibited higher apoptosis (about 75.2% of apoptotic cells). This result was attributed to the correlation between cell toxicity and apoptosis or necrosis. Those above results showed that the PDT efficacy of P could be enhanced by MnO2 nanocarriers in tumor cells and then the remarkable treatment effect of P-AgNCs-MnO2 nanocomplex was achieved by apoptosis or necrosis. With the vitro experiments achieving gratifying results, P-AgNCs-MnO2 was used as a multifunctional reagent to evaluate PDT in vivo. Mice bearing MCF-7 tumor were 24


Page 24 of 48

Page 25 of 48 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


applied for experiments. After intratumoral injection with PBS, AgNCs-MnO2, P, or P-AgNCs-MnO2, MCF-7 tumor bearing mice were exposed to 635 nm-red light for 5 min (1 W cm-2). The size of the tumor was gauged by calipers every 2 days after starting treatment. The results of relative tumor volumes growth curves suggested that the tumor growth of the group P-AgNCs-MnO2 was largely suppressed after PDT with light (Figure 9B). The difference in weight of tumors further indicated that the therapeutic efficiency of experimental group was significantly higher than the control groups (Figure 9C). The tumor photograph in each group were displayed in Figure 9A. These results indicated that the PDT of P-AgNCs-MnO2 could achieve tumor inhibited and without regrowth during our experiment period. The hematoxylin and eosin staining of tumor tissue indicated that cancer cells of the group of P-AgNCs-MnO2 generated significant morphological changes and the other control groups remained normal. Taken together, P-AgNCs-MnO2 possess great potentials as excellent agents for tumor therapy in vivo by PDT. In addition, the main organs containing heart, lung, kidney, liver, and spleen of different experimental groups were executed the histological analysis after experiment period of 16 days. The results shown in Figure S10, indicating that the typical organs treated with P-AgNCs-MnO2 with 635 nm-red light displayed no pathological changes, and hepatic and renal sections showed no abnormalities in hepatocytes and glomerular structures, and pulmonary fibrosis was not seen in the lung specimens.

4. Conclusions To sum up, we have designed a novel theranostic platform that could integrate the 25



detection of intracellular Zn2+ levels and PDT in one single nanostructure based on DNA-templated silver nanoclusters/porphyrin/MnO2 multifunctional nanoplatform. The as-fabricated P-AgNCs-MnO2 displayed sensitivity detection of endogenous or exogenous Zn2+ by hairpin DNA-fueled dynamic self-assembly of three-way-DNA junction architectures. Cellular fluorescence image of Zn2+ verified that our platform could sensitively distinguish tumor cells from non-tumor cells. In addition, the released Mn2+ could be employed as contrast agents for MRI. Moreover, the target Zn2+ fluorescence imaging and MRI together guide an effective tumor therapy. Highly efficient PDT was obtained by much 1O2 release of P-AgNCs-MnO2 under light irradiation compared with free P and the tumor growth was efficiently inhibited. It is undoubtedly that this uncomplicated fluorescence/MR dual-mode imaging strategy with high superiority will have promising applications in tumor imaging and simultaneously efficient tumor treatment.

Associated Content Supporting Information Materials and methods, Cytotoxicity of the nanoprobe, HRTEM image of an individual AgNCs (Figure S1), Characterization of MnO2 nanosheets (Figure S2), Condition optimization (Figure S3), Time optimization (Figure S4), UV-Vis of MnO2 nanosheets treated with H2O2 (Figure S5), Fluorescence analysis of 1O2 (Figure S6), Optimization of the nanoprobe cytotoxicity (Figure S7), In vivo fluorescence images (Figure S8), In vitro and in vivo MR images (Figure S9) and H&E stained tissue sections (Figure S10). This information can be obtained free of charge via the Internet at http://pubs.acs.org/. 26


Page 26 of 48

Page 27 of 48 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


Author Information Corresponding Author *Email address: [email protected] (F. Gao); [email protected] (D. Tang).

Acknowledgment This work was funded by the Natural Science Foundation of Jiangsu Province (BK20171174), China Postdoctoral Science Foundation (2017M610355), and Jiangsu Postdoctoral Science Foundation (1701045C).

References (1) Baek, S.; Singh, R. K.; Kim, T. H.; Seo, J. W.; Shin, U. S.; Chrzanowski, W.; Kim, H. W. Triple Hit with Drug Carriers: pH- and Temperature-responsive Theranostics for Multimodal Chemo- and Photothermal Therapy and Diagnostic Applications. ACS Appl. Mater. Interfaces 2016, 8, 8967-8979. (2) Chakraborty, A.; Boer, J. C.; Selomulya, C.; Plebanski, M. Amino Acid Functionalized Inorganic Nanoparticles as Cutting-Edge Therapeutic and Diagnostic Agents. Bioconjugate Chem. 2018, 29, 657-671. (3) Cao, Y.; Dong, H. F.; Yang, Z.; Zhong, X. M.; Chen, Y.; Dai, W. H.; Zhang, X. J. Aptamer-conjugated Graphene Quantum Dots/Porphyrin Derivative Theranostic Agent

for

Intracellular

Cancer-Related

MicroRNA

Detection

and

Fluorescence-guided Photothermal/Photodynamic Synergetic Therapy. ACS Appl. Mater. Interfaces 2017, 9, 159-166. (4) Mieszawska, A. J.; Mulder, W. J. M.; Fayad, Z. A.; Cormode, D. P. Multifunctional Gold Nanoparticles for Diagnosis and Therapy of Disease. Mol. 27



Page 28 of 48

Pharmaceutics 2013, 10, 831-847. (5) Zhu, H.; Wang, Y.; Chen, C.; Ma, M. R.; Zeng, J. F.; Li, S. Z.; Xia, Y. S.; Gao, M. Y. Monodisperse Dual Plasmonic Au@Cu2–xE (E= S, Se) Core@Shell Supraparticles: Aqueous Fabrication, Multimodal Imaging, and Tumor Therapy at in Vivo Level. ACS Nano 2017, 11, 8273-8281. (6) Wang, X.; Ma, Y. C.; Sheng, X.; Wang, Y. C.; Xu, H. X. Ultrathin Polypyrrole Nanosheets Via Space-Confined Synthesis for Efficient Photothermal Therapy in the Second Near-infrared Window. Nano Lett. 2018, 18, 2217-2225. (7) Chen, D. Q.; Dougherty, C. A.; Zhu, K. C.; Hong, H. Theranostic Applications of Carbon Nanomaterials in Cancer: Focus on Imaging and Cargo Delivery. J. Control. Rel. 2015, 210, 230-245. (8) Kong, F. P.; Liang, Z. Y.; Luan, D. R.; Liu, X. J.; Xu, K. H.; Tang, B. A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging. Anal. Chem. 2016, 88, 6450-6456. (9) Liu, J. T.; Du, P.; Mao, H.; Zhang, L.; Ju, H. X.; Lei, J. P. Dual-Triggered Oxygen Self-Supply Black Phosphorus Nanosystem for Enhanced Photodynamic Therapy. Biomaterials 2018, 172, 83-91. (10) Fan, W. P.; Lu, N.; Xu, C.; Liu, Y. J.; Lin, J.; Wang, S.; Shen, Z. Y.; Yang, Z.; Qu, J. L.; Wang, T. F.; Chen, S. P.; Huang, P.; Chen, X. Y. Enhanced Afterglow Performance

of

Persistent

Luminescence

Implants

for

Efficient

Repeatable Photodynamic Therapy. ACS Nano 2017, 11, 5864-5872. (11) Zhang, Y.; Wang, F. M.; Liu, C. Q.; Wang, Z. Z.; Kang, L. H.; Huang, Y. Y.; 28


Page 29 of 48 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


Dong, K.; Ren, J. S.; Qu, X. G. Nanozyme Decorated Metal–Organic Frameworks for Enhanced Photodynamic Therapy. ACS Nano 2018, 12, 651-661. (12) Choi, Y.; Kim, S.; Choi, M. H.; Ryoo, S. R.; Park, J.; Min, D. H.; Kim, B. S. Highly Biocompatible Carbon Nanodots for Simultaneous Bioimaging and Targeted Photodynamic Therapy In Vitro and In Vivo. Adv. Funct. Mater. 2014, 24, 5781-5789. (13) Li, X.; Kolemen, S.; Yoon, J.; Akkaya, E. U. Activatable Photosensitizers: Agents for Selective Photodynamic Therapy. Adv. Funct. Mater. 2017, 27, 1604053-1604063. (14) Lismont, M.; Dreesen, L.; Wuttke, S. Metal-Organic Framework Nanoparticles in Photodynamic Therapy: Current Status and Perspectives. Adv. Funct. Mater. 2017, 27, 1606314-1606329. (15) Yu, Z. Z.; Ge, Y. G.; Sun, Q. Q.; Pan, W.; Wan, X. Y.; Li, N.; Tang, B. A Pre-Protective Strategy for Precise Tumor Targeting and Efficient Photodynamic Therapy with a Switchable DNA/Upconversion Nanocomposite. Chem. Sci. 2018, 9, 3563-3569. (16)Cao, Y.; Meng, X. D.; Wang, D. D.; Zhang, K.; Dai, W. H.; Dong; H. F.; Zhang, X. J. Intelligent MnO2/Cu2-xS for Multimode Imaging Diagnostic and Advanced Single-Laser Irradiated Photothermal/Photodynamic Therapy. ACS Appl. Mater. Interfaces 2018, 10, 17732-17741. (17)Hong, H.; Zhang, Y.; Sun, J. T.; Cai, W. B. Molecular Imaging and Therapy of Cancer with Radiolabeled Nanoparticles. Nano Today 2009, 4, 399-413. 29



(18)Yan, L.; Amirshaghaghi, A.; Huang, D.; Miller, J.; Stein, J. M.; Busch, T. M.; Cheng, Z.; Tsourkas, A. Protoporphyrin IX (PpIX)-Coated Superparamagnetic Iron Oxide Nanoparticle (SPION) Nanoclusters for Magnetic Resonance Imaging and Photodynamic Therapy. Adv. Funct. Mater. 2018, 28, 1707030-1707038. (19) Uzzo, R. G.; Crispen, P. L.; Golovine, K. Diverse Effects of Zinc on NF-κB and AP-1 Transcription Factors: Implications for Prostate Cancer Progression. Carcinogenesis 2006, 27, 1980-1990. (20) Masanta, G.; Chang S. L.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. A Mitochondrial-Targeted Two-photon Probe for Zinc Ion. J. Am. Chem. Soc. 2011, 133, 5698-5700. (21)Franklin, R. B.; Costello, L. C. Zinc as an Anti-tumor Agent in Prostate Cancer and in Other Cancers. Arch. Biochem. Biophys. 2007, 463, 211-217. (22) Taylor, K. M.; Hiscox, S.; Nicholson, R. I.; Hogstrand, C.; Kille, P. Protein Kinase CK2 Triggers Cytosolic Zinc Signaling Pathways by Phosphorylation of Zinc Channel ZIP7. Sci. Signal 2012, 210, 1-10. (23) Hu, P.; Wang, R., Zhou, L.; Chen, L.; Wu, Q. S.; Han, M. Y.; El-Toni, A. M.; Zhao, D. Y.; Zhang, F. Near Infrared-activated Upconversion Nanoprobes for Sensitive Endogenous Zn2+ Detection and Selective On-Demand Photodynamic Therapy. Anal. Chem. 2017, 89, 3492-3500. (24) Gonzales, A. P. S.; Firmino, M. A.; Nomura, C. S.; Rocha, F. R. P.; Oliveira, P. V.; Gaubeur, I. Peat as a Natural Solid-phase for Copper Preconcentration and Determination in a Multicommuted Flow System Coupled to Flame Atomic 30


Page 30 of 48

Page 31 of 48 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


Absorption Spectrometry. Anal. Chim. Acta 2009, 636, 198-204. (25) Zhuang, H. J.; Wang, Z. Z.; Zhang, X. C.; Hutchison, James A.; Zhu, W. F.; Yao, Z. Y.; Zhao Y. L.; Li, M. A Highly Sensitive SERS-Based Platform for Zn (II) Detection in Cellular Media. Chem. Commun. 2017, 53, 1797-1800. (26) Li, L.; Zhang, Y.; Zhang, L. N.; Ge, S. G.; Yan, M.; Yu, J. H. Steric Paper Based Ratio-type Electrochemical Biosensor with Hollow-Channel for Sensitive Detection of Zn2+. Sci. Bulletin 2017, 62, 1114-1121. (27) Lee, S.; Nam, Y. S.; Lee, H. J.; Lee, Y.; Lee, K. B. Highly Selective Colorimetric Detection of Zn (II) Ions Using Label-Free Silver Nanoparticles. Sens. Actuators, B 2016, 237, 643-651. (28) Karami, H.; Mousavi, M. F.; Yamini, Y.; Shamsipur, M. On-Line Preconcentration and Simultaneous Determination of Heavy Metal Ions by Inductively Coupled Plasma-Atomic Emission Spectrometry. Anal. Chim. Acta 2004, 509, 89-94. (29) Becker, J. S.; Zoriy, M. V.; Pickhardt, C.; Palomero-Gallagher, N.; Zilles, K. Imaging of Copper, Zinc, and Other Elements in Thin Section of Human Brain Samples (hippocampus) by Laser Ablation Inductively Coupled Plasma Mass Spectrometry. Anal. Chem. 2005, 77, 3208-3216. (30) Li, L.; Feng, J.; Fan, Y. Y.; Tang, B. Simultaneous Imaging of Zn2+ and Cu2+ in Living Cells Based on DNAzyme Modified Gold Nanoparticle. Anal. Chem. 2015, 87, 4829-4835. (31) Yang, C.; Yin, X.; Huan, S. Y.; Chen, L. L.; Hu, X. X.; Xiong, M. Y.; Chen, K.; 31



Zhang, X. B. Two-Photon DNAzyme–Gold Nanoparticle Probe for Imaging Intracellular Metal Ions. Anal. Chem. 2018, 90, 3118-3123. (32) Si, H. B.; Sheng, R. J.; Li, Q. L.; Feng, J.; Li, L.; Tang, B. Highly Sensitive Fluorescence Imaging of Zn2+ and Cu2+ in Living Cells with Signal Amplification Based on Functional DNA Self-assembly. Anal. Chem. 2018, 90, 8785-8792. (33) Ritchie, C. M.; Johnsen, K. R.; Kiser, J. R.; Antoku, Y.; Dickson, R. M.; Petty, J. T. Ag Nanocluster Formation Using a Cytosine Oligonucleotide Template. J. Phys. Chem. C 2007, 111, 175-181. (34) Shamsipur, M.; Molaabasi, F.; Hosseinkhani, S.; Rahmati, F. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes. Anal. Chem. 2016, 88, 2188-2197. (35) Obliosca, J. M.; Babin, M. C.; Liu, C.; Liu, Y. L.; Chen, Y. A.; Batson, Robert A. Ganguly, M. P.; Jeffrey T.; Yeh, H. C. A Complementary Palette of Nanocluster Beacons. ACS Nano 2014, 8, 10150-10160. (36) Chen, Y.; Phipps, M. L.; Werner, J. H.; Chakraborty, S.; Martinez, J. S. DNA Templated Metal Nanoclusters: From Emergent Properties to Unique Applications. Acc. Chem. Res. 2018, 51, 2756–2763. (37) Chen, J. Y.; Ji, X. H.; Tinnefeld, P.; He, Z. K. Multifunctional Dumbbell-shaped DNA-templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules. ACS Appl. Mater. Interfaces 2016, 8, 1786-1794. 32


Page 32 of 48

Page 33 of 48 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


(38) Dong, F. Y.; Feng, E. D.; Zheng, T. T.; Tian, Y. In Situ Synthesized Silver Nanoclusters for Tracking the Role of Telomerase Activity in the Differentiation of Mesenchymal

Stem

Cells

to

Neural

Stem

Cells.

ACS

Appl.

Mater.

Interfaces 2018, 10, 2051-2057. (39) Yin, J. J.; He, X. X.; Wang, K. M.; Xu, F. Z.; Shangguan, J. F.; He, D. G.; Shi, H.

Label-Free and Turn-on Aptamer Strategy for Cancer Cells Detection Based on a

DNA–silver Nanocluster Fluorescence upon Recognition-Induced Hybridization. Anal. Chem. 2013, 85, 12011-12019. (40) Li, J. J.; Zhong, X. Q.; Cheng, F. F.; Zhang, J. R.; Jiang, L. P.; Zhu, J. J. One-Pot Synthesis of Aptamer-Functionalized Silver Nanoclusters for Cell-Type-Specific Imaging. Anal. Chem. 2012, 84, 4140-4146. (41) Yeh, H. C.; Sharma, J.; Han, J. J.; Martinez, S. J.; Werner, H. J. A DNA−Silver Nanocluster Probe that Fluoresces Upon Hybridization. Nano Lett. 2010, 10, 3106-3110. (42) Li, J. J.; Zhong, X. Q.; Zhang, H Q.; Le, C. X.; Zhu, J. J. Binding-Induced Fluorescence Turn-on Assay Using Aptamer-Functionalized Silver Nanocluster DNA Probes. Anal. Chem. 2012, 84, 5170-5174. (43) Yeh, H. C.; Sharma, J.; Shih, I. M.; Vu, M. D.; Martinez, S. J.; Werner, H. J. A Fluorescence Light-up Ag Nanocluster Probe that Discriminates Single-nucleotide Variants by Emission Color. J. Am. Chem. Soc. 2012, 134, 11550-11558. (44) Wang, W. J.; Huang, S.; Li, J. J.; Rui, K.; Bi, S.; Zhang J. R.; Zhu, J. J. Evaluation of Intracellular Telomerase Activity Through Cascade DNA Logic Gates. Chem. Sci. 2017, 33



8, 174-180. (45) Cheglakov, Z.; Cronin, T. M.; He, C.; Weizmann, Y. Live Cell MicroRNA Imaging Using Cascade Hybridization Reaction. J. Am. Chem. Soc. 2015, 137, 6116-6119. (46) Wu, C. C.; Cansiz, S.; Zhang, L. Q.; Teng, I. T.; Qiu, L. P.; Li, J.; Liu, Y.; Zhou, C. S.; Hu, R.; Zhang, T.; Cui, C.; Cui, L.; Tan, W. H. A Nonenzymatic Hairpin DNA Cascade Reaction Provides High Signal Gain of mRNA Imaging Inside Live Cells. J. Am. Chem. Soc. 2015, 137, 4900-4903. (47) Wang, F.; Elbaz, J.; Orbach, R.; Magen, N.; Willner, I. Amplified analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires. J. Am. Chem. Soc. 2011, 133, 17149-17151. (48) Wu, Z.; Liu, G.-Q.; Yang, X.-L.; Jiang, J.-H. Electrostatic Nucleic Acid Nanoassembly Enables Hybridization Chain Reaction in Living Cells for Ultrasensitive mRNA Imaging. J. Am. Chem. Soc. 2015, 137, 6829-6836. (49) Zhu, G.; Zhang, S.; Song, E.; Zheng, J.; Hu, R.; Fang, X.; Tan, W. Building Fluorescent DNA Nanodevices on Target Living Cell Surfaces. Angew. Chem., Int. Ed. 2013, 52, 5600-5606. (50) Choi, H. M. T.; Chang, J. Y.; Trinh, L. A.; Padilla, J. E.; Fraser, S. E.; Pierce, N. A. Programmable In Situ Amplification for Multiplexed Imaging of mRNA Expression. Nat. Biotechnol. 2010, 28, 1208-1212. (51) Ge, Z. L.; Lin, M. H.; Wang, P.; Pei, H.; Yan, J.; Shi, J. Y.; Huang, Q.; He, D. N.; Fan, C. H.; Zuo, X. L. Hybridization Chain Reaction Amplification of MicroRNA 34


Page 34 of 48

Page 35 of 48 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


Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor. Anal. Chem. 2014, 86, 2124-2130. (52) Tang, Y.; Zhang, X.; Tang, L.; Yu, R.; Jiang, J. In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (ligation-bHCR). Anal. Chem. 2017, 89, 3445-3451. (53) Liu, L.; Liu, J. W.; Wu, H.; Wang, X. N.; Yu, R. R.; Jiang, J. H. Branched Hybridization Chain Reaction (bHCR) Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells. Anal. Chem. 2018, 90, 1502-1505. (54) Ravan, H.; Amandadi, M.; Esmaeili-Mahani, S. DNA Domino-Based Nanoscale Logic Circuit: A Versatile Strategy for Ultrasensitive Multiplexed Analysis of Nucleic Acids. Anal. Chem. 2017, 89, 6021-6028. (55) Li, X.; Xie, J. Q.; Jiang, B. Y.; Yuan, R.; Xiang, Y. Metallo-Toehold-Activated Catalytic Hairpin Assembly Formation of Three-Way DNAzyme Junctions for Amplified Fluorescent Detection of Hg2+. ACS Appl. Mater. Interfaces 2017, 9, 5733-5738. (56) Qing, Z.; He, X.; Huang, J.; Wang, K.; Zou, Z.; Qing, T.; He, D. Target-Catalyzed Dynamic Assembly-Based Pyrene Excimer Switching for Enzyme-Free Nucleic Acid Amplified Detection. Anal. Chem. 2014, 86, 4934-4939. (57) Choi, C. A.; Lee, J. E.; Mazrad, Z. A. I.; In, I.; Jeong, J. H.; Park, S. Y. Redoxand pH-Responsive Fluorescent Carbon Nanoparticles-MnO2-Based FRET System for Tumor-targeted Drug Delivery in Vivo and in Vitro. J. Ind. Eng. Chem. 2018, 63, 208-219. 35



(58) Garg, D.; Mehta, A.; Mishra, A.; Basu, S. A Sensitive Turn on Fluorescent Probe for Detection of Biothiols Using MnO2@Carbon Dots Nanocomposites. Spectrochim. Acta A Mol. Biomol Spectrosc. 2018, 192, 411-419. (59) Li, J.; Li, D. X.; Yuan, R.; Xiang, Y. Biodegradable MnO2 Nanosheet-Mediated Signal Amplification in Living Cells Enables Sensitive Detection of Down-Regulated Intracellular MicroRNA. ACS Appl. Mater. Interfaces 2017, 9, 5717-5724. (60) Zhao, Z. L.; Fan, H. H.; Zhou, G. F.; Bai, H. R.; Liang, H.; Wang, R. W.; Zhang, X. B.; Tan, W. H. Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging Via MnO2 Nanosheet−aptamer Nanoprobe. J. Am. Chem. Soc. 2014, 136, 11220-11223. (61) Fan, H. H.; Zhao, Z. L.; Yan, G. B.; Zhang, X. B.; Yang, C.; Meng, H. M.; Chen, Z.; Liu, H.; Tan, W. H. A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing. Angew. Chem. 2015, 127, 4883-4887. (62) Ou, M.; Huang, J.; Yang, X. H.; Quan, K.; Yang, Y. J.; Xie, N. L. Wang, K. M. MnO2 Nanosheets Mediated “DD-A” FRET Binary Probes Forsensitive Detection of Intracellular mRNA. Chem. Sci. 2017, 8, 668-673. (63) Liu, J. T.; Du, P.; Liu, T. R.; Bernardino J. Córdova Wong, Wang, W. P.; Ju, H. X.; Lei, J. P. A Black Phosphorus/Manganese Dioxide Nanoplatform: Oxygen Self-Supply Monitoring, Photodynamic Therapy Enhancement and Feedback. Biomaterials 2019, 192, 179-188. (64) Fan, W.; Bu, W.; Shen, B.; He, Q.; Cui, Z.; Liu, Y.; Zheng, X.; Zhao, K.; Shi, J. Intelligent MnO2 Nanosheets Anchored with Upconversion Nanoprobes for 36


Page 36 of 48

Page 37 of 48 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


Concurrent pH-/H2O2-Responsive UCL Imaging and Oxygen-Elevated Synergetic Therapy. Adv. Mater. 2015, 27, 4155-4161. (65) Zhang, C.; Chen, W. H.; Liu, L. H.; Qiu, W. X.; Yu, W. Y.; Zhang, X. Z. An O2 Self-Supplementing

and

Reactive-Oxygen-Species-Circulating

Amplified

Nanoplatform via H2O/H2O2 Splitting for Tumor Imaging and Photodynamic Therapy. Adv. Funct. Mater. 2017, 27, 1700626-1700639. (66) Zhu, P.; Chen Y.; Shi, J. Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic Tumor Oxygenation. ACS Nano. 2018, 12, 3780–3795.

37



Scheme

1.

Label-free

for

intracellular

tumor-related

Page 38 of 48

Zn2+ imaging and

fluorescence/MRI bimodal platform guided photodynamic therapy based on DNA-templated silver nanoclusters/porphyrin/MnO2.

38


Page 39 of 48 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


Figure 1. (A) TEM, (B) AFM, (C) XPS of AgNCs; (D) Fluorescence spectra of AgNCs without or with G-DNA; (E) TEM, (F) AFM of MnO2 nanosheets; (G) UV-vis of (a) MnO2 nanosheets and (b) AgNCs-MnO2; (H) Zeta potential of MnO2 nanosheets; (I) TEM, (J) AFM of AgNCs-MnO2; (K) XPS of (a) MnO2, (b) AgNCs-MnO2, (c) P-AgNCs-MnO2; (L) XRD of (a) MnO2, (b) AgNCs-MnO2, (c) P-AgNCs-MnO2. Inset of Figure 1A, 1E, 1I: photos of AgNCs, MnO2, AgNCs-MnO2.

39



Figure 2. (A) Agarose gel electrophoresis image of (a) H0 without Zn2+, (b) H0 with Zn2+, (c) H1-H2-H3 with Zn2+, (d) H0-H1 with Zn2+, (e) H0-H1-H2 with Zn2+, (f) H0-H1-H2-H3 with Zn2+; (B) AFM image of the three-way DNA junction nanostructures; (C) Fluorescence spectra of H0-H3 without Zn2+ (a), H1-H3 with Zn2+ (b), H0-H1 with Zn2+ (c), H0-H2 with Zn2+ (d), H0-H3 with Zn2+ (e); (D) Fluorescence spectra of the P-AgNCs-MnO2 probe in the existence of different concentrations of Zn2+ (a to m; 0, 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 200, 300 nM); (E) Plot of fluorescence intensity vs various Zn2+ concentrations; (F) Specificity of the P-AgNCs-MnO2 nanoprobe over (a) Zn2+, (b) Mn2+, (c) Mg2+, (d) Cu2+, (e) Ca2+, (f) Na+, (g) K+, (h) Fe3+, (i) lysozyme, (j) mRNA, (k) telomerase, (l) glucose, (m) ascorbic acid, (n) uric acid, and (o) dopamine hydrochloride.

40


Page 40 of 48

Page 41 of 48 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


Figure 3. Confocal fluorescence image of MCF-7 cells treated with (A) P-MnO2 nanoprobe, (B) P-H0-H1-MnO2 nanoprobe, (C) P-H1-H2-H3-MnO2 nanoprobe, (D) P-H0-H1-H2-MnO2 nanoprobe, (E) P-AgNCs-MnO2 nanoprobe after 4 h, (F) Flow cytometry assay of MCF-7 cells treated with (a) PBS, (b) P-H0-H1-H2-MnO2 nanoprobe, (c) P-AgNCs-MnO2 nanoprobe (Scale bar = 20 μm).

41



Figure 4. Confocal microscopy images of MCF-7 cells treated with (A) PBS, (B) TPEN, (C) DTDP and (D) 5 μM Zn2+ after treated with 50 μg ml-1 P-AgNCs-MnO2 nanoprobe for 4 h (Scale bar = 20 μm).

42


Page 42 of 48

Page 43 of 48 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


Figure 5. Confocal microscopy images of (A) MCF-7 cells, (B) HeLa cells, (C) HepG2 cells, (D) LO2 cells, and (E) L929 cells treated with 50 μg ml-1 P-AgNCs-MnO2 nanoprobe after 4 h (Scale bar = 20 μm).

43



Figure 6. Confocal microscopy images of MCF-7 cells after treatment with 5 µM [Ru(dpp)3]Cl2 for 2 h, followed by incubation with P-AgNCs-MnO2 nanoprobe for 0-8 h (A-E) (Scale bar = 20 μm).

44


Page 44 of 48

Page 45 of 48 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


Figure 7. Confocal microscopy images of MCF-7 cells under (A) PBS, (B) MnO2, (C) P, (D) P-AgNCs-MnO2 nanoprobe after incubation with DCFH-DA (Scale bar = 100 μm).

45



Figure 8. Fluorescence images of Calcein AM/PI-costained cells with no light (A) or with light (B) and flow cytometry assay of apoptosis cells (C) after incubated with different treatments (Scale bar = 200 μm).

46


Page 46 of 48

Page 47 of 48 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


Figure 9. (A) Photographs of representative mice experienced different treatments (16th d after the first treatment), excised MCF-7 tumors form tumor-bearing mice and its histological analyses; (B) Relative tumor volumes growth curves; (C) Weight of tumors.

47



For TOC only

48


Page 48 of 48

MnO2 Platform for Label

Recommend Documents