DNA-Stabilized Silver Nanoclusters for Label-Free Fluorescence

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Article Cite This: Anal. Chem. 2018, 90, 14368−14375

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DNA-Stabilized Silver Nanoclusters for Label-Free Fluorescence Imaging of Cell Surface Glycans and Fluorescence Guided Photothermal Therapy Jing Wu, Na Li, Yao Yao, Daoquan Tang,* Dongzhi Yang, Jeremiah Ong’achwa Machuki, Jingjing Li, Yanyan Yu, and Fenglei Gao* Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 221004 Xuzhou, China

Anal. Chem. 2018.90:14368-14375. Downloaded from pubs.acs.org by UNIV OF KANSAS on 01/20/19. For personal use only.

S Supporting Information *

ABSTRACT: A multifunctional nanoplatform that enables the integration of biological detection, imaging diagnosis, and synergistic therapy into a single nanostructure holds great promise for nanoscience and nanomedicine. Herein, a novel theranostic platform was presented for label-free imaging of cell surface glycans based on DNA/silver nanoclusters (AgNCs) via hybridization chain reaction (HCR) and fluorescence guided photothermal therapy (PTT). In this strategy, a dibenzocyclooctyne (DBCO)-functionalized DNA and two hairpin structures of DNA/AgNCs probes were involved. Following metabolic glycan labeling, the binding of DBCO-functionalized DNA to cell surface initiated HCR, and then cell surface glycans were specifically labeled by DNA/AgNCs fluorescent probes. Furthermore, this signal amplification strategy was adopted in quantitative analysis, and the detection limit could be achieved as low as 20 cells in 200 μL of binding buffer. Moreover, the remarkable photothermal properties of DNA/AgNCs via HCR led to efficient killing of cancer cells and inhibited the tumor growth under imaging guide. In this strategy, DNA/AgNCs were utilized to detect the cellular glycans, which aided in overcoming the high cost and instability of fluorescent dyes. Simultaneously, the HCR process avoided the introduction of excessive azido-sugars under the precondition of ensuring apparent fluorescence. These results indicated that the developed nanoplatform has great potential for specific cell surface glycans imaging and fluorescence guided PTT.

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including cell growth and differentiation, intercellular signaling, immune responses, pathogen interactions, and intracellular signaling responses.7,8 Consequently, it is necessary to analyze multiple glycans on the cell surface, which visually reflects the correlation between the particular glycan patterns and their roles in cancers status and development.9,10 To date, a series of methods, such as mass spectrometry, electrochemical biosensors, and fluorescence imaging, have been developed for glycan analysis. Mass spectrometric analysis is a powerful tool for the detection of glycomics because of its broad detection range, high mass resolution, and capability for multicomponent analysis.11 Electrochemistry is an important field for detection because of its advantages of high sensitivity, fast response, and low cost.12,13 However, the above-mentioned methods are unsuitable for in situ imaging, and certain glycans were underestimated during the cumbersome cell lysis, enzymatic cleavage, as well as derivatization processes.14 With the development of confocal laser scanning microscopy, fluo-

heranostic nanoplatforms integrating diagnostic capability and therapeutic functions into one system have attracted intense attention as promising alternatives to traditional diagnosis and synergistic therapy, and they act as next-generation nanocarrier systems in the biomedical field.1,2 Photothermal therapy (PTT) as a new type of a potentially hyperthermia-driven therapeutic approach in the field of oncology has attracted increasing attention due to its deep tissue penetration ability and fewer side effects.3 The therapeutic effect of PTT without tumor targeting will inevitably be greatly reduced.4 Accurate diagnosis is a pivotal step and crucial prerequisite for efficient treatment of tumor.3 As the other necessary part of the theranostic progress, the imaging diagnosis is an important tool to deeply understand the location and size of tumor, track the biodistribution of theranostic agents, and assess the treatment efficiency.5 Protein glycosylation is a universal post-translational modification process, and it is related to many properties of proteins.6 Glycans are modified on the cell surface through glycosylation, and they are classified into O-linked glycans and N-linked glycans according to their mode of linkage with proteins. Aberrant alterations in O- and N-linked glycans are involved in many types of cancers for their biological roles, © 2018 American Chemical Society

Received: August 23, 2018 Accepted: November 28, 2018 Published: November 28, 2018 14368

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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glycochemistry (Scheme 1). Unnatural sugars with azide groups were tagged to the cell surface via the metabolic glycan

rescence-based detection method has been developed that enables real-time in situ imaging of cell surface glycans.15 Using fluorescence-labeled glycan recognition probes that specifically recognize and bind to cell surface glycans, fluorescence imaging of cells can be performed directly, which can help to monitor the dynamic changes of glycosylation on the cell surface and provide a potentially working channel for diagnostic and therapeutic usage.16,17 In fact, dye-labeled fluorescent probes are relatively expensive and time-consuming, therefore, development of novel label-free fluorescent probes for imaging of glycans on cell surfaces is highly desirable. Recently, fluorescent metal nanoclusters (NCs) have become emerging luminescent nanomaterials in biosensing and bionanotechnology due to their ease of synthesis, excellent photostability, low toxicity, and controllable size. Therefore, they can act as promising alternatives to organic dyes and semiconductor quantum dots. In particular, DNA as a ligand for controlling the passivation of NCs, is well suited for the task of producing fluorescent NCs.18,19 Silver nanoclusters (AgNCs) in situ synthesized using cytosine-rich loop DNA templates have attracted special attention owing to their subnanometer size, water solubility, as well as good photostability and biocompatibility.20,21 Furthermore, the fluorescent AgNCs highly depend on the DNA template sequence. Different DNA sequences can lead to the synthesis of AgNCs with different excitation, emission positions, and fluorescence intensities, providing an easy way for the effective design of fluorescent probes.22 Therefore, DNA-stabilized AgNCs instead of traditional organic dyes, as a new type of label-free fluorescent material, are widely used in bioanalytical research.23,24 Nevertheless, the application of DNA/AgNCs probes in in situ imaging of cell surface glycans has rarely been investigated. Inspired by excellent characteristics and wide applicability of DNA/AgNCs, a label-free fluorescence strategy for in situ imaging of cell surface glycans based on DNA/AgNCs probes was presented in this study. However, the fluorescence intensity was restricted owing to the limited number of AgNCs fluorophores grown on the DNA skeleton. Moreover, corresponding carbohydrates on the cell surface could be indiscriminately changed to unnatural sugars by carbohydrate metabolism. As a result, the addition of high concentrations of unnatural sugar might interfere with the normal physiological function of cells. Therefore, using as few azido-sugars as possible for the metabolic labeling of glycans on the cell surface would be a more meaningful approach to study the glycosylation process. In recent years, the use of nucleic acid hybridization as a source of energy to amplify signals or to drive molecular motors has been a relatively new concept.25−27 Hybridization chain reaction (HCR) was a characteristic isothermal and enzyme-free signal amplification strategy.28 Introduction of a primer strand DNA can trigger a selfassembly of two metastable hairpin probes with partial complementarities to alternately form a nicked doublestranded polymer.29 Recently, HCR was applied to highly sensitive in situ fluorescence imaging of intracellular biomacromolecules.30,31 Herein, a label-free and enzyme-free signal amplification strategy was reported for the in situ imaging of cell surface glycans by skillful combination of DNA/AgNCs and HCR method. DNA/AgNCs as fluorescent probe were prepared, and nucleic acid amplification technology was applied to

Scheme 1. Schematic Illustration of DNA/AgNCs and HCRBased Theranostic Nanoplatform for Label-Free Fluorescence Imaging of Cell Surface Glycans and Fluorescence Guided PTT

labeling technique.32,33 Dibenzocyclooctyne (DBCO)-functionalized DNA reacted with an azide group through copperfree click chemistry.34,35 The DNA assembled on the cell surface served as the chain initiator and initiated two hairpin structures of DNA/AgNCs to absorb more NCs for signal amplification. Glycans on the cell surface could be imaged using a confocal microscope. Precious metals nanoparticles (NPs) have been extensively studied owing to their good photothermal conversion effect, and AgNPs exhibit great application prospects in photothermal conversion for its unique localized surface plasmon resonance and cost effectiveness than gold NPs.36,37 To the best of our knowledge, DNA/AgNCs absorbing light and then efficiently converting optical energy into local heat, producing an excellent PTT efficacy has never been reported. In this study, we first reported the use of remarkable photothermal properties of DNA/ AgNCs, which made it possible to efficiently kill cancer cells and inhibited the tumor growth under imaging guide. Using the designed strategy, a label-free fluorescent probe was successfully prepared for in situ imaging of cell surface glycans, which also acted as a promising novel candidate for theranostic agent for imaging guided PTT.

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EXPERIMENTAL SECTION Synthesis and Characterization of Silver Nanoclusters. For the preparation of fluorescent AgNCs, hairpin structure DNA (H1, H2) was diluted with phosphate buffer (20 mM, pH = 7.4, containing 1 mM Mg2+) and contents were heated at 95 °C for 5 min. Further the solution was cooled down to room temperature, and AgNO3 (10 mM) was added into the DNA solution. The contents were then placed in the ice bath for 15 min, and NaBH4 (2 mM) was quickly added to reduce the resultant solution. Subsequently, the mixture solution was shaken vigorously for 1 min and kept in the dark at 4 °C for approximately 5 h prior to use. The final concentrations of reaction mixture were as follows: hairpin DNA structure DNA (0.5 μM), AgNO3 (10 μM), and NaBH4 (10 μM). The fluorescence measurements were carried out in the range from 600 to 800 nm with the excitation wavelength at 585 nm. Fluorescence emission spectra were collected using a bandwidth of 5 nm. All these measurements were recorded at room temperature. Ultraviolet−visible (UV−vis) absorption 14369

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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on the target protein. In conjunction with bio-orthogonal reactions, labeling and imaging of glycans could be achieved. The azide group of azido-sugars labeled on the cell was covalently coupled with DBCO-functionalized DNA (H3) through copper-free click chemistry, which facilitated the introduction of DNA (H3) on the cell surface. The assembled DNA (H3) could initiate HCR reaction in the presence of two hairpin structures of DNA/AgNCs (H1 and H2), leading to dynamic grafting of DNA/AgNCs onto the long DNA nanowire at the cell surface glycans, which afforded HCR assembly with high fluorescence responses and provided the in situ imaging of cell surface glycans. Furthermore, the remarkable photothermal properties of DNA/AgNCs resulted in efficient killing of cancer cell under guided imaging. Therefore, the proposed label-free DNA/AgNCs probes theranostic platform could be efficient in detecting surface glycans and therapeutics of cancer cell. Characterization of the DNA/AgNCs Probe. The fluorescence of DNA/AgNCs was strongly affected by the surrounding medium, stabilizer, aggregation state, and particles size. Therefore, the UV−vis spectra and AFM were used to characterize the obtained DNA/AgNCs. The UV−vis spectrum of the Ag clusters shows two absorption bands at 440 and 567 nm (Figure 1A), which is consistent with the results for reported DNA/AgNCs.38 To study the size of DNA/AgNCs, AFM was used to characterize the DNA/AgNCs (Figure 1B). Clearly, the DNA/AgNCs are distributed evenly. An AFM image gives the topographic heights of AgNCs, which are in the range between 1.7 and 2.1 nm (average height is about 1.9 ± 0.2 nm), which is consistent with the definition of NCs (2 nm) because the ssDNA provides preformed templates and stops the NCs from growing once a desired size is reached. Further, the XPS was adopted to inspect the valence of Ag atoms in DNA/AgNCs. A P 2p peak occurred at 133.1 eV, which corresponded to the phosphate group, indicating the presence of DNA (Supporting Information (SI), Figure S1). Two peaks are clearly observed at 366.8 and 373.8 eV, which correspond to the photoelectrons excited from 3d Ag(0), indicating the formation and presence of Ag(0) NCs (Figures 1C and SI, Figure S1). Moreover, the excitation and emission spectra are shown in Figure 1D, and the maximum emission of the DNA/AgNCs is shown at 677 nm when excited at 585 nm. Furthermore, the fluorescence signal of DNA/AgNCs is stable over the time as presented in SI, Figure S2B. Then, the feasibility of HCR was verified based on DNA/AgNCs. First, the morphology of the long HCR products was characterized through AFM. Figure 1E demonstrated that with the progress in HCR, longer and curved DNA nanostructures with the length more than 2 μm were obtained owing to the formation of the HCR product involving thousands of repeated sequences. Furthermore, the AgNCs were separated well over a longer portion of the DNA and only could be observed on the DNA skeletons, confirming the feasibility of HCR. The associated height profiles showed that the size of AgNCs was about 2 nm and the height of the DNA nanostructures was about 2 nm, which was found to be consistent with the AFM result of DNA/AgNCs and matched with the diameter of DNA. Moreover, fluorescence analysis was carried out to further confirm the deposition of more AgNCs on the DNA via HCR. Figure 1F exhibited a weak fluorescence signal in the presence of only AgNCs probe H1 or AgNCs probe H2. After adding the probe H3 into the AgNCs probe H1, the fluorescence was still not changed. In contrast, when probe

spectra of DNA/AgNCs were measured on a UV spectrophotometer (MAPADA UV-6100). Fluorescence spectra of DNA/ AgNCs and the HCR products were obtained using a fluorescence spectrophotometer. The size of DNA/AgNCs was characterized by atomic force microscopy (AFM, Agilent 5500 AFM/SPM), and the valence of Ag atoms in DNA/ AgNCs was inspected by X-ray photoelectron spectroscopy (XPS, ESCALAB 250 spectrometer). Photothermal temperature was recorded using an infrared (IR) thermal camera (FOTRIC 228S). Cell Culture and Imaging. MCF-7, HeLa, HepG2, LO2, L929, and A549 cells used in this study were cultured in DMEM supplemented with 10% FBS, penicillin (100 μg mL−1), and streptomycin (100 μg mL−1) at 37 °C under a humidified atmosphere containing 5% CO2. For the imaging of cell surface glycans, MCF-7 cells were seeded in 35 mm confocal dishes (glass bottom dish) at 5 × 103 cells per dish and grown for 24 h at 37 °C. After discarding the old medium, the adherent cells were incubated in culture medium containing Ac4ManNAz at 50 μM for 48 h. For the click reaction, the metabolically labeled cells were first fixed using paraformaldehyde (4%) for 20 min followed by incubation with 2 μM DBCO-functionalized DNA (H3) for 30 min. For the HCR reaction, the DBCO−DNA modified cells were treated with the AgNCs probe H1 and the AgNCs probe H2 in phosphate buffer at 25 °C for 2 h. The cells were washed three times with phosphate buffer after each step and imaged using the laser confocal microscope. Evaluation of in Vitro Cell Killing. First, live/dead cell staining assay was conducted to verify the photothermal effect of DNA/AgNCs. MCF-7 cells were seeded into a 6-well plate at a density of 2 × 104 cells per well and cultured for 12 h. After cells were adherent, the medium was discarded and replaced by fresh medium containing 50 μM Ac4ManNAz and cultivated for 48 h. Then the cells were washed twice with PBS and incubated with 8 μM DBCO-functionalized DNA (H3) at 37 °C for 30 min. This was followed by the treatment of DNA/AgNCs probe H1 (8 μM) and DNA/AgNCs probe H2 (8 μM) at 37 °C for another 30 min. For PTT, cells were exposed to 808 nm near-IR (NIR) laser (1 W cm−2) for 10 min. After the light irradiation treatment, ac alcein-AM/ propidium iodide (PI) double stain kit was used to inspect the viability of released cells. Briefly, MCF-7 cells were stained with 2 μM calcein-AM and 8 μM PI at 37 °C for 30 min and observed under a fluorescence microscope. Next, evaluation of apoptosis after PTT was carried out by flow cytometry. After MCF-7 cells labeled with DNA/AgNCs probe were exposed to 808 nm near-IR (NIR) laser (1 W cm−2) for 10 min, the supernatant was discarded and substituted with fresh cell culture medium followed by further cultivation for 12 h. Further, cells were harvested for apoptosis detection using an Annexin V-FITC/PI cell apoptosis kit via flow cytometry.

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RESULTS AND DISCUSSION Principle of Signal Amplification Strategy. Scheme 1 demonstrated that the process of HCR consist of two hairpin structures (H1, H2) and target sequence (H3). Studies had reported that by using metabolic glycan labeling technique, cell surface glycosylation could be studied at the living level. Utilizing the tolerance of the substrate by each glycosyltransferase in the glycosylation pathway, an analogue of a precursor monosaccharide (unnatural sugar) was introduced that can be recognized by a glycosyltransferase to be modified 14370

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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final temperature were proportional to the DNA/AgNCs concentration. The high temperature could generate significant local heat for effective ablation of cancer cells. This indicated that DNA/AgNCs would be able tofunction as PTT agents in cancer therapeutics. In Situ Monitoring of Cell Surface Glycans. To further investigate the application of this signal amplification strategy for detection of cell surface glycans after certificating hybridization of DNA/AgNCs probes in buffer, laser-scanning confocal microscopy (LSCM) was used for obtaining the image of metabolically glycan-labeled cells (Figure 2A). After

Figure 2. (A) Confocal laser scanning microscopy images of Ac4ManNAz-labeled MCF-7 cells after being incubated without DNA/AgNCs probe or with different probes: DNA/Ag NCs probe H1 + DNA/AgNCs probe H2, H3 + DNA/AgNCs probe H1, and H3 + DNA/Ag NCs probe H1 + DNA/AgNCs probe H2. (B) Flow cytometric histogram of Ac4ManNAz-labeled MCF-7 cells incubated without AgNCs probes (red line) or with two different probes: H3 + DNA/AgNCs probe H1 (blue line) and H3 + DNA/Ag NCs probe H1 + DNA/AgNCs probe H2 (orange line).

MCF-7 cells were labeled with azido-sugar, no fluorescence signal was observed in the nanoprobe-untreated cells. Moreover, cells undergoing incubation with AgNCs probe H1 and AgNCs probe H2 failed to display the fluorescence signal. When cells were incubated with AgNCs probe H1 and probe H3 without the HCR process, weak fluorescence was emanated from the cell surface. However, the treatment with the mixture of AgNCs probe H1, AgNCs probe H2 and probe H3 allowed the observation of strong fluorescence signal through the HCR reaction, indicating that the probe H3 was not only an important trigger of HCR reaction but also a bridge between the nanoprobe and cells by click reaction. Apart from the research on fixed cells, flow cytometric assays were carried out to verify the feasibility of this method on living cells (Figure 2B). These results confirmed that the long DNA was engineered on the cell surface and signal amplification strategy could be a feasible method for the in situ imaging of metabolically glycan-labeled cells. Specificity of Glycans Recognition. To prove that sialic acid on the cell surface labeled with Ac4ManNAz is the specific site for the recognition and binding of DNA/AgNCs probe, tunicamycin was used to inhibit the biosynthesis of N-linked glycans in cells. Tunicamycin has been reported as a reagent that can block the formation of protein N-glycosidic linkages by inhibiting the transfer of N-acetylglucosamine 1-phosphate to dolichol monophosphate.39 For Ac4ManNAz-labeled cells, after the treatment with tunicamycin, the fluorescence intensity significantly decreased compared to untreated cells, which could be attributed to the inhibition expression of sialic acid on the cell surface. In contrast, Ac4GalNAz-labeled cells did not show obvious change, supporting sialic acid as the specific site (Figure 3). These results indicated the good recognition

Figure 1. (A) UV−vis spectra of DNA/AgNCs. (B) AFM image of DNA/AgNCs probe. (C) XPS characterization of DNA/AgNCs. (D) Fluorescence excitation and emission spectra of DNA/AgNCs probe. (E) AFM image of the HCR product of DNA/AgNCs probe H1, DNA/AgNCs probe H2 and H3. (F) Fluorescence spectra of the system under different conditions: H3 + DNA/AgNCs probe H1, H3 + DNA/AgNCs probe H2, and H3 + DNA/AgNCs probe H1 + DNA/AgNCs probe H2. (G) Temperature change curves of the aqueous solutions containing 8 μM DNA/AgNCs and PBS irradiated with 808 nm laser for 10 min at 1 W cm−2. (H) Thermal images of DNA/AgNCs at different concentrations ranging from (a) 0.5, (b) 1, (c) 2, (d) 4, and (e) 8 μM.

H3 was added to the mixture of AgNCs probe H1 and AgNCs probe H2, an increased fluorescence was obviously observed (curve c), indicating that HCR caused significant enhancement in fluorescence. These results undoubtedly demonstrated that the HCR reaction based on DNA/AgNCs could be a feasible detection method for the purpose of signal amplification. Furthermore, the photothermal conversion ability of DNA/ AgNCs was also systematically investigated by irradiating with 808 nm laser at 1 W cm−2 for 10 min. Figure 1G showed that the temperature of the DNA/AgNCs solution (8 μM) reached 53.6 °C and the temperature increase speed was 2.86 °C min−1, while the temperature of PBS remained 27.5 °C. Moreover, the temperature increased monotonically with the increase in the concentration of DNA/AgNCs in the range of 0.5−8 μM (Figure 1H). The rate of temperature rise and the 14371

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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signal on the surface of LO2 and L929 cells was very weak, indicating low expression of sialic acid on normal cell surface. These results demonstrated the phenomenon that sialic acid was less distributed on the surface of normal cells and overexpressed on the surface of tumor cells. Therefore, our platform for the imaging of cell surface glycans is widely applicable and is a reliable way to achieve dynamic monitoring of cell surface glycans. Quantification of Cell Surface Glycans. Under the optimized conditions, quantitative assay of MCF-7 cells was implemented to investigate the sensitivity of this signal amplification strategy based on DNA/AgNCs and HCR. MCF-7 cells with the number ranging from 0 to 200000 cells in 200 μL of binding buffer were prepared by gradient dilution. To carry out target cell quantification, the labeled events appearing in the upright window were counted. Figure 5

Figure 3. Confocal images of Ac4ManNAz-labeled MCF-7 cells incubated with DNA/AgNCs probes after being treated without or with tunicamycin.

specificity of metabolic glycan labeling technique through labeling unnatural sugars with reactive groups to the end of corresponding glycan chains on the cell surface. Universality Study of Our Platform. According to the way of linking with proteins or lipids, glycans are divided into the following three major categories: N-linked glycans (glycans linked to proteins via nitrogen atoms), O-linked glycans (glycans linked to proteins via oxygen atoms), and lipid-linked glycans (glycans linked to lipids). Different types of azidosugars are used to metabolically incorporate a sugar analogue into corresponding cellular glycans through underlying biosynthetic machinery. To verify the applicability of the imaging of cell metabolically labeled glycans based on the HCR, two types of azido-sugars, including Ac4ManNAz (for labeling cell surface N-linked glycans) and Ac4GalNAz (for labeling cell surface O-linked glycans) were selected to label cells. The confocal results displayed that the MCF-7 cells labeled with different azido-sugars showed different fluorescence imaging, which indicated that the contents of different glycans on the same cell surface were different (Figure 4A). To further verify the applicability of this method to all types of cells, we selected two normal cells and three tumor cells as experimental cell lines, which were labeled with 50 μM Ac4ManNAz. Figure 4B shows a bright fluorescence signal from the surface of tumor cells. Conversely, the fluorescence

Figure 5. Flow cytometry assays of Ac4ManNAz-labeled MCF-7 cells with decreasing cell number from 0 to 200000 in 200 μL of binding buffer.

exhibited that the DNA/AgNCs labeled MCF-7 cells locating in the upper right region decrease gradually with the reduction of cell number. Background signal was tested without any cells in the 200 μL of binding buffer. Counts obtained by subtracting the background count plus three times the standard deviation could be considered valid. The calibration curve presented a good linear relationship with a regression equation of log Y = 1.0324logX−0.1578, where X and Y represent cell numbers measured using a hemocytometer and by this strategy, respectively (SI, Figure S5). The lowest cell number actually detected was 20 cells in 200 μL of binding buffer, which reflected broad detection range and relatively low detection limit. The detection limit and linear range of the new proposed method was comparable to other reported amplified sensors, and the comparison results are summarized in SI, Table S1. The sensitivity of this method was higher than other methods. The result indicated that signal amplification strategy based on DNA/AgNCs and HCR reaction exhibited the ability for the sensitive detection of low amounts of target cells. Evaluation of in Vitro Cell Killing. To investigate the PTT efficacy of DNA/AgNCs in in vitro, comparative studies on the viability of MCF-7 cells were conducted upon incubation with 8 μM DNA/AgNCs probe under NIR irradiation (808 nm, 1 W cm−2, 10 min). Fluorescent live/ dead cell costaining with calcein AM/PI was applied to directly

Figure 4. (A) Confocal images of MCF-7 cells incubated with DNA/ AgNCs probes after being metabolically labeled with Ac4GalNAz or Ac4ManNAz at 50 μM. (B) Confocal images of different cells incubated with DNA/AgNCs probes after being metabolically labeled with 50 μM Ac4ManNAz. 14372

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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in vivo investigation for detection of cell surface glycans in the nude mice bearing MCF-7 tumors. According to literature, glycans on the cell surface of tumor cells were overexpressed and would be metabolically labeled with most azido-sugars, which could lead to the accumulation of DBCO-H3 at the tumor site through copper-free click chemistry. This led to sitespecific generation of H3 groups on tumor cells. Furthermore, tumor tissue could rapidly uptake DNA/AgNCs (H1, H2) due to the enhanced permeability and retention effect40 and sitespecific DNA hybridization engineering.41 SI, Figure S7 shows that no visible fluorescence signal could be detected in the control group. In contrast, the weak fluorescence signal could be observed after injection of Ac4ManNAz and DNA/AgNCs probe without HCR and the signal intensity enhanced with the HCR process. These results powerfully verified that this signal amplification strategy could be a feasible method for in vivo detection of cell surface glycans. In Vivo Photothermal Imaging. To explore the in vivo potential photothermal conversion of DNA/AgNCs, temperature change in tumor was analyzed in a mouse model bearing MCF-7 tumor and recorded in real time using an IR thermal camera. Figure 7A manifests that after laser irradiation at 808 nm at a power density of 1 W cm−2, the tumor temperature in mice injected with Ac4ManNAz and DNA/AgNCs coupled with HCR increased rapidly up to 54.3 °C at 10 min postirradiation, which was able to effectively ablate malignant tumors. However, tumor temperature of the group treated with Ac4ManNAz and DNA/AgNCs increased only up to 41.8 °C,

observe the result of PTT. Calcein AM could be used for the staining of living cells and showed green fluorescence, and PI could be used for the staining of dead cells and showed red fluorescence. Figure 6A demonstrated that the cells treated

Figure 6. (A) Confocal fluorescence images of MCF-7 cells which were treated with PBS, DNA/AgNCs and DNA/AgNCs + HCR with or without laser irradiation. The cells were all costained with CalceinAM (live, green) and PI (dead, red). (B) Flow cytometry analysis of apoptosis of MCF-7 cells with blank control treatment, DNA/AgNCs, and DNA/AgNCs or DNA/AgNCs based on HCR with NIR light irradiation.

with DNA/AgNCs probe without NIR irradiation show green fluorescence without red fluorescence, indicating very little effect of DNA/AgNCs on cell viability. In contrast, the green fluorescence decreased and red fluorescence increased when MCF-7 cells were incubated with DNA/AgNCs probe under NIR irradiation, indicating that some MCF-7 cells were killed by the good efficacy of PTT of DNA/AgNCs. The amount of DNA/AgNCs absorbed on the cell surface affects the efficacy of photothermal therapy. Figure 6A showed that all the treated cells were almost dead after HCR reaction, while the death ratio of the group without HCR was relatively low, thus confirming the outstanding photothermal killing capacity of DNA/AgNCs in tumor cells. Similarly, the cytotoxicity test demonstrated a higher killing rate in the group treated with DNA/AgNCs based on HCR than other two groups, respectively, treated with PBS and DNA/AgNCs without HCR after the irradiation laser of 808 nm at a power density of 1 W cm−2 for 10 min (SI, Figure S2F). Moreover, significant cell killing was also assessed by flow cytometry apoptosis analysis. It was found that more than 66.34% MCF-7 cells treated with DNA/AgNCs probe based on HCR were in apoptosis state after the irradiation for 10 min, while less cells treated with either DNA/AgNCs probe and irradiation without HCR or only DNA/AgNCs probe were in apoptosis/necrosis status (Figure 6B). These results indicated that the DNA/ AgNCs based on HCR signal amplification and PTT are excellent for therapeutic efficiency. Detection of in Vivo Cell Suface Glycans. The in vitro evaluation displayed that our label-free and enzyme-free platform based on DNA/AgNCs with HCR exhibited specific and effective detection of glycans on cell surface. To further assess the tumor-marking effect, this method was applied to the

Figure 7. (A) In vivo photothermal images of mice after intravenous injection of PBS, Ac4ManNAz + DNA/AgNCs, and Ac4ManNAz + DNA/AgNCs based on HCR. (B) Relative tumor volume after different treatments. (C) Average tumor weight of mice on the 18th day after different treatments. (D) Photographs of tumor tissues in different groups after PTT. 14373

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guide. Our strategy is label-free, cost-effective, isothermal, and involves enzyme-free fluorescence signal amplification using DNA/AgNCs as the source of fluorescence with HCR. Therefore, the proposed label-free DNA/AgNCs probes theranostic platform combines efficient imaging of cancer cell surface glycans and cancer therapeutics, which holds great potential in biomedical application.

and no apparent temperature increase was detected in mice injected with PBS. These observation sevidently declared the predominant in vivo photothermal effect of DNA/AgNCs combined with HCR. In Vivo Tumor Therapy. Inspired by the advantageous therapeutic efficiency in in vitro and the acceptable photothermal conversion in in vivo, the in vivo antitumor effect of DNA/AgNCs was further investigated. When the initial tumor volume reached about 100 mm3, the mice were classified into three groups at random, where two groups were selected to receive DNA/AgNCs injection and Ac4ManNAz pretreatment and the third group treated with PBS served as a control. Figure 7B demonstrated that under the laser irradiation at 808 nm at a power density of 1 W cm−2 for 10 min, tumors in mice injected with Ac4ManNAz and DNA/AgNCs increased rapidly during the therapy, which was similar to the phenomenon in the PBS-treated group, failing to inhibit tumor growth through corresponding treatments. In significant contrast, tumors in the group treated with Ac4ManNAz and DNA/AgNCs coupled with HCR showed efficient tumor regression, and the relative tumor volume was 0.75 on the 18th day. Furthermore, the corresponding tumor graphs also verified the optimal tumor suppression of DNA/AgNCs under NIR irradiation (Figure 7D). Moreover, hematoxylin and eosin (H&E) staining was performed after PTT (SI, Figure S8A). Upon the treatment of DNA/AgNCs without HCR, highly polymorphic nucleus and mitosis in most malignant cells could be observed from tissue sections owing to the limited destruction from the laser therapy, which would result in the revival of tumor growth. However, tumor tissues in the group combined with DNA/ AgNCs and HCR displayed obvious tumor cell damage, including severe cell shrinkage and nuclear condensation. Overall, these results certificated that our multifunctional platform exhibited superior PTT effect resulting from large number of DNA/AgNCs probes absorbed on the cell surface. In Vivo Biosafety Evaluation. To further evaluatethe side effects of DNA/AgNCs, the body weight was monitored (Figure 7C). In the measurement, neither noticeable weight loss nor obvious abnormalities was observed, manifesting the negligible biotoxicity of our theranostic nanoagent. Furthermore, H&E images of major organs including liver, spleen, kidney, heart, and lung demonstrated no apparent histopathological abnormalities or lesions in the two groups treated with DNA/AgNCs compared to the control group (SI, Figure S8B). Collectively, these results indicated the outstanding biocompatibility of DNA/AgNCs without noticeable toxicity and supported its potential phototherapy application in nanoscience and nanomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03837.

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Experimental procedures: reagents and materials, instruments, preparation of fluorescent silver nanoclusters, apoptosis experiment, inhibition of N-glycosylation by tunicamycin treatment, quantification of cell surface glycans, and cytotoxicity assay; cytotoxicity of the DNA/ AgNCs probe. Full XPS of DNA/AgNCs, condition optimization, time optimization, cytotoxicity of the DNA/AgNCs probe, the calibration curve, in vivo fluorescence imaging, and histological examination of tumor (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone/Fax, +86-516-83262138; E-mail, jsxzgfl@sina.com (F.G.). *E-mail: [email protected] (D.T.). ORCID

Fenglei Gao: 0000-0002-9367-8368 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21565002), Natural Science Foundation of Jiangsu Province (BK20171174), China Postdoctoral Science Foundation (2017M610355), Jiangsu Postdoctoral Science Foundation (1701045C), and Qing Lan Project.

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CONCLUSIONS A novel theranostic platform was developed for label-free and enzyme-free imaging of cell surface glycans based on DNA/ AgNCs with HCR and fluorescence-guided PTT. On the basis of metabolic glycan labeling, reactive organic functional groups could be built on the cell surface for the click reaction. Through HCR, DNA length could be increased to absorb more AgNCs for signal amplification. The in situ imaging of glycans on cell surface could be achieved using a confocal microscope. Importantly, this strategy was successfully applied to quantitative analysis through flow cytometry. Furthermore, the excellent photothermal characteristics of DNA/AgNCs with HCR resulted in efficient killing of cancer cells and inhibited the tumor growth under the surface glycans imaging 14374

DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375

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DOI: 10.1021/acs.analchem.8b03837 Anal. Chem. 2018, 90, 14368−14375