Instantaneous pH-Boosted Functionalization of Stellate Gold

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Instantaneous pH-Boosted Functionalization of Stellate Gold Nanoparticles for Intracellular Imaging of miRNA Jisun Ki,† Eunji Jang,† Seungmin Han,† Moo-Kwang Shin,† Byunghoon Kang,† Yong-Min Huh,*,‡ and Seungjoo Haam*,† †

Department of Chemical and Biomolecular Engineering, Yonsei University, Yonsei-ro 50, Seoul 120-749, South Korea Department of Radiology, College of Medicine, Yonsei University, Seoul 120-752, South Korea

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S Supporting Information *

ABSTRACT: Various types of nanoprobes have recently been utilized to monitor living organisms by detecting and imaging intracellular biomarkers, such as microRNAs (miRs). We here present a simple one-pot method to prepare stellate gold nanoparticles functionalized with miR-detecting molecular beacons (SGNP-MBs); low pH conditions permitted the rapid-high loading of MBs on the surface of SGNPs. Compared to the conventional gold nanoparticle-based MBs, SGNPs carried a 4.5-fold higher load of MBs and exhibited a 6.4-fold higher cellular uptake. We demonstrated that SGNP-MBs were successfully internalized in human gastric cancer cell lines and could be used to accurately detect and image intracellular miRs in an miR-specific manner. Furthermore, the relative levels of intracellular miRs in three different cell lines expressing miR-10b (high, moderate, and low levels) could be monitored using SGNP-MBs. Consequently, these results indicated that SGNP-MBs could have applications as highly potent, efficient nanoprobes to assess intracellular miR levels in living cells. KEYWORDS: branched gold nanoparticles, molecular beacon, miRNA sensing, cancer imaging, nanobiosensor

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INTRODUCTION Real-time sensing of various biomarkers inside living cells provides insights into our understanding of the pathological status of cell and biomolecular interactions.1−3 Detection and imaging systems based on nanomaterials have facilitated spatiotemporal observation of intracellular target molecules.4−7 For example, gold nanoparticles (GNPs) have been exploited to deliver fluorescent molecular beacons (MBs),8,9 which target mRNA (mRNA) or microRNAs (miRs) with specific sequences, into target cells due to the capacity of GNPs to act as carriers and effective quenchers.10−13 Compared with conventional transfection agents used to transfect MBs into cells, GNPs exhibit excellent cytocompatibility, thereby increasing the capacity of MB delivery, and can protect against unnecessary interactions with biomolecules, including nucleases.14,15 In particular, anisotropic GNPs, such as nanostars, nanorods, and bipyramids, increase cellular uptake efficiency13,16,17 and permit the delivery of large amounts of MBs owing to their high surface areas.18,19 Owing to these strengths, extensive studies have been performed to improve GNP-based sensing systems and to validate their performance. These efforts have involved modulation of the tuning sizes and shapes of GNPs as well as the functionalization of GNPs with probe molecules such as MBs.19,20 For instance, Zhang et al. reported that low-pH citrate buffer facilitates adsorption of nucleotides on the surfaces of GNPs by reducing the time © XXXX American Chemical Society

required for the MB-functionalizing step as compared with traditional salt aging methods.21−24 Here, we report a timesaving one-pot synthesis method for stellate GNPs (SGNPs) functionalized with MBs (SGNP-MBs) for real-time sensing of intracellular miRs in living cells. In our method, large amounts of MBs rapidly adsorbed onto SGNPs spontaneously under low pH conditions, resulting in anisotropic growth of GNPs to SGNPs with increased surface area. SGNP-MBs could be prepared within 1 h and showed enhanced cellular uptake efficiency without cytotoxicity.25,26 Additionally, SGNP-MBs could specifically sense cytoplasmic miRs. miRs have been shown to act as reliable biomarkers, and their abnormal expression is closely related to cancer progression, pathological grade, malignancy, and apoptosis.27−29 As a model system to validate the performance of SGNP-MBs, we chose miRNA-10b, a known onco-miR exhibiting high expression in metastatic gastric cancer.30,31 In three different cell lines expressing miR10b (Hs746T, AGS, and YCC-16), SGNP-MBs successfully distinguished different levels of intracellular miR-10b. Received: December 21, 2016 Accepted: May 5, 2017

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DOI: 10.1021/acsami.6b16452 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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RESULTS Synthesis and Characterization of SGNP-MBs. SGNPMBs were synthesized in a simple and time-saving method involving simultaneous seed-mediated synthesis of a SGNPMBs at low pH (pH 3) and surface modification of SGNPs with MBs (Scheme 1a). Reduction of Au ions occurred

Scheme 1. (a) Schematics of Molecular Beacons (MBs) Loading on Stellate Gold Nanoparticles (SGNPs) Using Low pH Assisted Method and (b) on Gold Nanoparticles (GNPs) Using Salt Aging Method

Figure 1. Characterization of SGNP-MBs. Transmission electron microscopy (TEM) images of (a) SGNP-MBs and (b) GNP-MBs (scale bar: 20 nm). (c) Surface charge as determined by zeta potential and hydrodynamic diameters. (c) Number of particles in MKN-45 cells after treatment for 2 h. (d) Molecular beacon loading values on GNP-MBs and SGNP-MBs.

and the target miR.33−35 The fluorescence intensities of SGNPMBs modified with various MBs having different reporter probe lengths were determined for varying miR-10b target concentrations for 1 h. The difference in fluorescence signal was clear when the 15mer reporter probe was used (Supporting Information, Figure S4). We selected miR-10b, an miR that is highly expressed in gastric cancer and promotes migration and invasion. In addition to the high loading capacity, enhanced stability of SGNP-MBs is necessary to protect MBs from degradation in the physiological environment. Accordingly, we monitored the fluorescence intensities of SGNP-MBs incubated with nuclease. As shown in Figure 2a, the fluorescence of SGNP-MBs was stably quenched (88%) in the presence of 0−1 mU DNase I, whereas the fluorescence signal of SGNP-free MBs increased up to 73% depending on the concentration of DNase I, indicating that SGNP-MBs exhibited nuclease resistance compared with SGNP-free MBs. To evaluate the stability of SGNP-MBs, we monitored the fluorescence intensity of SGNP-

instantly using ascorbic acid in the presence of Ag ions, resulting in the growth of SGNPs with a stellate shape and average size of 95 ± 7.1 nm (Figure 1a). Silver ions assisted the anisotropic growth of Au branches on specific crystallographic facets.32 In Figure 1b, the size of GNP-MBs used as control group was 13.8 ± 0.4 nm. Furthermore, the size control of the SGNPs was possible by using a different ratio of HAuCl4 to gold seeds concentration (Supporting Information, Figure S1). Stirring with MBs for 1 h was sufficient to stabilize the SGNPs. After purification, the SGNP-MBs exhibited a negative surface charge (−23 ± 2.4 mV; Figure 1c) and were well dispersed in distilled water and several buffers (Supporting Information, Figure S2). The MBs were conjugated to SGNPs via terminal thiol-functional groups and annealed to a reporter probe that was conjugated with an organic fluorophore (Supporting Information, Table S1). The number of MBs attached to a single SGNP was calculated by fluorescence measurement. SGNP-MBs had 389 ± 37 MBs on average (Figure 1d), which was 4.5 times higher than that on single seed GNPs due to the anisotropic growth of SGNPs increasing the surface area of SGNPs. These results indicated that SGNPMBs were effectively and efficiently prepared. To confirmed morphology effect of nanoparticles, we examined the loading capacity of MBs of sphere gold nanoparticles of 90 nm in diameter (90GNPs; Supporting Information, Figure S3). 90GNPs functionalized with miR-detecting molecular beacons (90GNP-MBs) had 232 ± 21 MBs on average, which was 60% of loading efficiency on SGNP-MBs. To prepare an efficient miR sensor, the reporter probe length was adjusted from a 13 mer to a 15 mer because we assumed that the sensitivity was related to the free energy between MBs

Figure 2. (a) Quenching efficiency of SGNP-MBs against DNase I compared with control MBs for 12 h. (b) Stability of SGNP-MBs against of endolysomal pH. B

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those two single-mismatched molecules was significantly reduced compared with that of the perfectly matched target molecule. In particular, detection performance with a mutation in the distal region was decreased by 74%, whereas a mutation in the proximal region decreased by 62% as compared with that of the perfectly matched molecule. Second, to estimate the target sensitivity of SGNP-MBs, the limit of detection (LOD) of SGNP-MBs was determined by 0.1-fold serial dilutions of the target concentration (Figure 3d). At target molecule concentrations ranging from 10 pM to 10 nM, the fluorescence signal of SGNP-MBs gradually decreased. Owing to the excellent functionality of SGNPs in quenching of the fluorescence signal, the LOD of SGNP-MBs was determined to be as low as 10 pM. In Vitro Delivery Profile of SGNP-MBs. To determine whether SGNP-MBs could be utilized as an in vitro sensing platform, we investigated in vitro cytotoxicity using the human gastric cancer cell lines Hs746T and YCC 1 (Supporting Information, Figure S6; Scheme 2). The cells were treated with

MBs incubated at various pH similar to that of the harsh endosomal environment in the presence of nuclease (Figure 2b). No significant differences in fluorescence preference and intensity of SGNP-MBs were observed after incubation at pH 6.5 and 5 compared with that at pH 7.2. The quenching efficiency and nuclease resistance character of 90GNP-MBs represented similar preference to SGNP-MBs (Supporting Information, Figure S5). Target Sensitivity and Selectivity of SGNP-MBs. To evaluate the sensing potential of SGNP-MBs, we prepared two SGNP-MBs designed to detect miR-10b (SGNP-10b) and miR200b (SGNP-200b), respectively. miR-200b was chosen as a second target owing to its distinct role in the regulation of epithelial-mesenchymal transition (EMT).36,37 For both SGNP10b and SGNP-200b, the fluorescence signal of 1 nM SGNPMBs increased as the target concentration increased in a range from 0 to 140 nM (Figure 3a). In particular, the sensing

Scheme 2. Schematic Illustration of the SGNP-MBs to Sense the miR-10b Expression Dependent Fluorescence Intensity Change

Figure 3. (a) Fluorescence spectra of SGNP-10b over a target miRNA concentration profile for 1 h. The kind of target miRNA is miR-10b and miR-200b. (b) Fluorescence images indicated target specific binding. (c) Variation of the fluorescence intensity of SGNMBs in the presence of perfectly matched target and two types of mutants with single nucleotide mismatch, proximal (5 terminal) and distal (3 terminal). (d) The florescence graph of the limit of detection.

SGNP-MBs at various concentrations (0.1−10 nM; the concentration of MBs ranged from 38.9 to 389 nM) for 4 h at 37 °C. Treatment with SGNP-MBs did not induce significant cytotoxicity, indicating that SGNP-MBs remained highly biocompatible without inhibitory effects on cell proliferation. To assess in vitro delivery of SGNP-MBs as a detection platform, the cellular internalization of SGNP-MBs was observed using transmission electron microscopy (TEM) and inductively coupled plasma-atomic emission spectrometry (ICP-OES) analysis. TEM images (Figure 4a) showed that the morphology of cells was intact, and SGNP-MBs were observed in the cytoplasm after incubation of Hs746T cells with 1 nM SGNP-MBs for 2 h. Moreover, enlargement of the red box region shows some monodispersed SGNP-MBs sustained their original morphologies and size after uptake in the cells. To quantitatively estimate the endocytosis of SGNP-

performance of SGNP-10b to detect miR-10b was not attenuated in the presence of miR-200b at an equivalent concentration. In addition, fluorescence images (Figure 3b) revealed that, at the target concentration of 40 nM, SGNP-10b and SGNP-200b exhibited distinct and specific hybridization with their complementary targets in a sequence-specific manner, even in the presence of nontarget molecules. In the absence of a perfect target, the fluorescence intensity of SGNPMBs showed no notable changes. To further assess the sensing ability of SGNP-MBs, we analyzed the selectivity and sensitivity of SGNP-MBs (Figure 3c). First, to estimate the target selectivity of SGNP-MBs, we recruited two different singlemismatched molecules for which the position of the mutation was placed at either the distal or the proximal end from the SGNP surface. The detection performance of SGNP-MBs with C

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Figure 4. (a) TEM images of (i) SGNP-10b uptake for 2 h (scale bar: 0.5 μm). (ii) Magnified images of the SGNP-10b in the cytoplasm. (b) Number of particles in MKN-45 cells after treatment for 2 h. (c) Fluorescence image of uptake of SGNP-10b at 40× magnification. The red color is the cy3 fluorescence intensity of flare probes. (d) Flow cytometric analysis of SGNP-10b after incubation for 0, 0.5, 1, and 2 h.

MBs in Hs746T cells, the number of SGNP-MBs that accumulated in Hs746T cells was calculated using ICP-OES. The results showed that 2662 ± 63 particles were internalized in each Hs746T cell (Figure 4b). In addition, we determined whether the stellate shape of SGNPs affected the delivery of MBs to the target cells in vitro by comparing their cellular uptake with that of GNP-MBs. After incubation with 1 nM GNP-MBs for 2 h, 410 ± 22 GNP-MBs were internalized into each Hs746T cell, indicating that SGNPs delivered 6.4-fold more MBs into the target cells than did GNPs. In Vitro Detection and Imaging of Intracellular miRs Using SGNP-MBs. To validate the ability of SGNP-MBs to detect intracellular miRs in living cells, we treated Hs746T cells, which show high expression of miR-10b, with SGNP-10b and then assessed changes in fluorescence over time. As shown in Figure 4c, the fluorescence intensity increased significantly after incubation of Hs746t cells with 1 nM SGNP-10b for 2 h. To quantitatively assess the detection capacity of SGNP-MBs, we performed quantified signal enhancement in cells treated with SGNP-10b for 0, 0.5, 1, and 2 h by flow cytometry. As shown in Figure 4d, the signal for miR-10b in Hs746t cells gradually increased until 2 h after SGNP-MB treatment. Specifically, 8.92%, 51.42%, and 57.24% of cells were positive for miR-10b at 30 min, 1 h, and 2 h, respectively, indicating that a 1-h incubation with SGNP-10b was sufficient for real-time detection of intracellular miR-10b in Hs746T cells. We performed a control experiment with two negative control conditions for evaluating the possibility of off-target effects. To determine the specificity for detection of miR-10b in Hs746T cells by SGNP-10b, we treated cells with an inhibitor of miR10b (anti-miR-10b) prior to incubation with SGNP-10b for 2 h. Shown in Figure 5, the fluorescence signal of miR-10b in the cytoplasm of Hs746T cells was decreased from 56% to 0.44%, as determined by both fluorescence laser confocal microscopy and flow-cytometric analysis. As negative controls for intracellular miRs, Hs746T cells were treated with SGNP-200b to detect miR-200b, which was expressed at much lower (90-fold lower) levels than miR-10b in Hs746T cells. No significant fluorescence signal was observed by fluorescence laser confocal microscopy or flow-cytometric analysis (Figure 5b). As expected, preincubation of Hs746T cells with anti-miR-200b prior to treatment with miR-200b had no marked effects on the fluorescence signal.

Figure 5. In vitro imaging of intracellular miR-10b in Hs746T cells. (a) Confocal microscopic images and (b) flow cytometric analysis of Hst46T cells incubated for 1 h with SGNP-10b, SGNP-200b, and SGNP-10b after preincubation for 3 h with miR-10b inhibitor, and SGNP-200b after preincubation for 3 h with miR-200b inhibitor.

Real-Time Detection of miR-10b Using SGNP-MBs in Various Living Cells. Finally, to test whether SGNP-MBs are classified cell types based on endogenous miR-10b expression, three gastric cancer cell lines (Hs746t, AGS, and YCC-1) were treated with SGNP-10b and the relative expression levels of miR-10b were estimated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR; Supporting Information, Figure S7). The cells were treated with varying concentrations of SGNP-10b and incubated for 1 h at 37 °C. Fluorescence images of Hs746T cells treated with SGNP-10b were obtained using fluorescence laser confocal microscopy (Figure 6a). After treatment of Hs746T cells with SGNP-10b, a significantly greater increase in Cy3 intensity was observed compared with that in AGS and YCC-16 cells. Similarly, in flow cytometric analysis, 2- and 4-fold higher miR-10b expression was observed in Hs746T cells than in AGS and YCC16 cells, respectively (Figure 6b).

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DISCUSSION Oligonucleotide-functionalized GNPs have been developed to detect various biomolecules with high sensitivity and biocompatibility. Accordingly, many studies have focused on three fields, i.e., anisotropicity, surface modification methods, and adjusting sequences, to develop advanced monitoring systems. First, the anisotropic spiky shell of SGNPs is advantageous for the preparation of SGNP-MBs because the increased surface area after anisotropic growth permits higher amounts of MBs to attach to SGNPs than on common GNPs.38 Increasing the MB loading density on SGNPs might improve uptake as well as transfection efficacy. Moreover, because the endocytosis of nonspherical nanoparticles is generally more efficient owing to the larger contact area, the stellate D

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SGNP-MBs via a facile method with few limitations. Instead of salt aging, we used low-pH conditions, increasing the loading amount of oligonucleotides through protonation of cytosine and adenine bases and reducing the electrostatic repulsion between negatively charged GNP surfaces. Thus, adsorption of oligonucleotides to the surface of GNPs was favorable under low pH conditions, which was also required for the growth of SGNPs. Consequently, the synthesis of SGNP-MBs, growth of stellate shells on GNPs, and adsorption of MBs simultaneously could be achieved in a one-pot synthesis method. Third, to enhance the sensitivity of the SGNP-MB system, we adjusted the sequences of the MBs. First, a spacer of 10 nucleotides was added to the end of the recognition portion of the capture probe to increase sensitivity by avoiding steric hindrance induced by the gold surface. Second, the reporter probe was extended from a 13mer to a 15mer, leading to efficient detachment of the reporter probe when the capture probe was bound with the target miR. Specifically, by extending the reporter probe to a 15mer, the overlap of the domain between the target miR and reporter probe was increased. Because the longer overlapping domain of the reporter probe induced more conformational constraints, the binding constant between the reporter probe and capture probe was decreased, promoting efficient detachment of the reporter probe with higher sensitivity. In the selectivity test, the reason for the reduced change in the fluorescence signal was that a single nucleotide sequence position (between the target and capture probe) affected the hybridization thermodynamic. It is worth noting that the proposed assay may have potential applications in single nucleotide polymorphism analysis, providing insights into the optimal design for MBs to match the seed region. To evaluate potential in vitro applications, several gastric cancer cell lines were treated with SGNP-MBs, and efficient cellular uptake and targeting ability to specific miRs were confirmed. The results of this study clearly demonstrated that only 1 h was sufficient to detect intracellular miRs in living cells. In offtargeting tests, SGNP-MBs exhibited high selectivity in living cells and had identical effects when miR-10b MBs were changed to the miR-200b complementary sequence. Furthermore, the possibility of multiplex detection was proven by simultaneous delivery of SGNP-MBs functionalized with two types of MBs (Supporting Information, Figure S8). SGNP-MBs permitted the successful visualization of endogenous miRs, thus offering the potential for clinical diagnosis.

Figure 6. In vitro quantitative detection of miR-10b in three gastric cancer cell linesHs746T, AGS, and YCC-16. (a) Confocal microscopic images of three cells incubated for 1 h with SGNP-10b. (b) miR-10b expression in three cells was performed using flow cytometric analysis with the cells treated with SGNP-10b. Hs746T showed higher expression of miR-10b than AGS and YCC-16.

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CONCLUSION We developed a simple and robust method to formulate SGNPMBs targeting sense intracellular miRs in living cells. With their high capacity for MB loading and high cellular uptake efficiency, SGNP-MBs showed excellent potential for detection and imaging of the relative expression levels of miRs in various cell lines. Therefore, miRNA monitoring in human gastric cancer using SGNP-MBs represents a promising diagnostic imaging tool and extends the scope of therapies involving knockdown of specific genes related to disease pathways.

morphology improves the efficiency of SGNP-MB endocytosis compared with that of spherical particles.39 Consequently, the highly loaded SGNP-MBs could improve uptake as well as in vitro monitoring at short times. Second, the salt-aging method is a common modification technique in which salt is gradually added over 1−2 days. Salt diminishes the electrostatic repulsion between oligonucleotides and GNPs, resulting in faster adsorption kinetics for increased loading of oligonucleotides. Although this method is effective for modification, the saltaging process has several limitations, such as the requirement for time-consuming steps and the aggregation of GNPs under high-salt conditions. Moreover, because large (>50 nm) anisotropic-shaped particles tended to aggregate, additional surfactants, such polyethylene glycol or sodium dodecyl sulfate, are needed to prevent particle aggregation.40 In this study, we described an advanced intracellular miR sensing system, using

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EXPERIMENTAL SECTION

Materials. Hydrogen tetrachloroaurate hydrate (HAuCl4·3H2O), sodium citrate, silver nitrate, l-ascorbic acid, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and potassium cyanide (KCN) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphatebuffered saline (PBS, 10 mM, pH 7.4), Dulbecco’s modified Eagle’s medium (DMEM), and minimum essential medium (MEM) were

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ACS Applied Materials & Interfaces purchased from Gibco (Invitrogen, Grand Island, NY, USA). Functionalized DNA oligonucleotides and AccuTarget Human miRNA inhibitors were purchased from Bioneer Inc. (Daejeon, Korea). The sequences of the DNA oligonucleotides are summarized in Table S1. Ultrapure deionized water was used for all the syntheses. All of the other chemicals and reagents were of analytical grade. Preparation of SGNP-MBs. MBs were prepared by annealing a mixture of two DNA oligonucleotides, a capture probe, and a flare probe at a molar ratio of 1:1 at 95 °C for 4 min and then at 70 °C for 10 min, followed by slow cooling to 25 °C. MBs were activated by TCEP solution (0.1 M) prior to use. SGNP-MBs were synthesized by a seed-mediated growth method. The seed solution (15 nm in diameter) was prepared by adding 15 mL of 1% citrate solution to 100 mL of 1 mM HAuCl4·3H2O solution at 95 °C under vigorous stirring. After 15 min, the solution was cooled and kept at 4 °C for long-term storage. To synthesize SGNP-MBs, a growth solution was prepared by adding 100 μL of the seed solution to 10 mL of 0.25 mM HAuCl4· 3H2O solution (pH 3) at 25 °C under moderate stirring (700 rpm). Next, 100 μL of 1 mM AgNO3 solution and 50 μL of 100 mM ascorbic acid solution were added simultaneously, followed by quick injection of 2 nmol MBs. Within 30 s, a greenish-black SGNP-MB solution was obtained. SGNP-MBs were washed twice by centrifugation at 900 rpm for 5 min. Characterization of SGNP-MBs. The morphology of SGNP-MBs was observed using high-resolution TEM (HR-TEM; JEM-2100F; JEOL, Japan) at an accelerating voltage of 200 kV. The average hydrodynamic diameters and zeta potentials of SGNP-MBs were measured using dynamic laser scattering (ELS-Z; Otsuka Electronics, Osaka, Japan). The optical absorbance of SGNP-MBs was measured using an Optizen UV−vis spectrometer (2120UV; Mecasys, Korea). The MB loading capacity of SGNP-MBs was analyzed by measuring the fluorescence intensity of SGNP-MBs (excitation/emission: 540/ 570 nm) after SGNPs were etched with KCN solution (0.1 M). A hybrid multimode microplate reader (Synergy 2 multimode reader; VT, USA) was used to measure the fluorescence intensity. To assess the detection ability of SGNP-MBs against a target molecule, we used a synthetic target oligonucleotide and mismatched analogs with several single nucleotide polymorphisms. SGNP-MBs were incubated with the target or nontarget oligonucleotides at various concentrations for 1 h at 37 °C, followed by measurement of the fluorescence intensity using the hybrid multimode microplate reader. Fluorescence optical images of SGNP-MBs were obtained using an IVIS Spectrum in vivo imaging system (PerkinElmer, MA, USA). To ensure stable performance of SGNP-MBs in the physiological environment, we observed the interaction of SGNP-MBs with target molecules under various pH conditions by measuring changes in fluorescence intensity. The resistance of SGNP-MBs against nucleotidase-induced degradation was validated by comparing the quenching efficiency of SGNP-MBs with control MBs (cMBs) with black hole quencher-2 (BHQ-2) instead of the nanostructured quencher (SGNPs). SGNP-MBs and cMBs (40 nM) were incubated with DNase I (0.1−1 mU) in DNase reaction buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) for 12 h at 37 °C. Cell Lines and Culture Conditions. Human gastric cancer cell lines (Hs746T, AGS, and YCC16) were used to evaluate the in vitro sensing performance of SGNP-MBs. Hs746T and AGS cells were purchased from the Korean Cell Line Bank (Seoul, Korea), and YCC16 cells were purchased from the Yonsei Cancer Research Institute (Seoul, Korea). Hs746T cells were cultured in DMEM, and AGS and YCC16 cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics. All cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Biocompatibility Test for SGNP-MBs. The biocompatibility of the SGNP-MBs was quantified using a colorimetric assay based on the cellular reduction of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). The cells were seeded in 96-well plates at a density of 2 × 104 cells/well, incubated overnight, and treated with 100 μL DMEM (10% FBS) containing SGNP-MBs at various concentrations for 4 h at 37 °C. Then, the cells were washed with PBS and treated with yellow MTT solution. Formazan crystals formed

from MTT by viable cells with active metabolism were solubilized with 10% sodium dodecyl sulfate in 0.01 M HCl. The absorbance of the resulting purple solution was measured at 575 and 650 nm as reference using a hybrid multimode microplate reader. Finally, cell viability was determined from the intensity ratio of the treated to untreated cells and is represented as the average ± standard deviation (n = 3). Cellular Uptake of SGNP-MBs in Vitro. To analyze the cellular uptake of SGNP-MBs, Hs746T cells were seeded at a density of 5.0 × 104 cells/well in 6-well plates and incubated at 37 °C overnight. The cells were treated with 1 nM SGNP-MBs for 0.5, 1, and 2 h and then harvested. Intracellular accumulation of SGNP-MBs over the incubation time was observed by fluorescence microscopy (Observer/z1; Zeiss, Barcelona, Spain) and flow-cytometric analysis (FACSCalibur; Becton Dickinson and Co., Franklin Lakes, NJ, USA). To compare the intracellular uptake of SGNP-MBs with GNP-MBs, we measured Au ions in Hs746T cells using ICP-OES (IRIS Intrepid II XSP; Thermo Fisher Scientific, Boston, MA, USA). The cells were treated with 5 nM SGNP-MBs and GNP-MBs for 2 h at 37 °C. The cells were washed and then lysed in aqua regia for 12 h at 120 °C. The obtained concentration of Au ions in Hs746T cells was converted to the concentration of SGNP-MBs or GNP-MBs in the cells. To observe the intracellular localization of SGNP-MBs in Hs746T cells, the cells were treated with 5 nM SGNP-MBs, harvested, and postfixed with 1% OsO4. The prepared specimens were observed by TEM (JEM-1011; JEOL) at an acceleration voltage of 80 kV. Quantification of Intracellular miRs by Real-Time PCR. To measure miR expression levels in Hs746T, AGS, and YCC16 gastric cancer cells, RT-qPCR was performed with internal standards. Total RNA from each cell type was extracted using a MasterPure Complete DNA and RNA Purification Kit (Epicenter, Madison, WI, USA). cDNA was synthesized with 1.5 μg of total RNA using a miScript cDNA synthesis kit (Qiagen, Valencia, CA, USA). RT-qPCR was performed in triplicate with a miScript SYBR Green PCR Kit and miScript Primer Assay (Qiagen) on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Relative miR expression was normalized to endogenous reference genes (snRNA-68 and snRNA-72) and was calculated by the ΔCt method from triplicate experiments. Intracellular miR Detection in Live Cells. To evaluate the performance of SGNP-MBs to detect intracellular miR expression, we used Hs746T cells with high expression of miR-10b and low expression of miR-200b. Hs746T cells were seeded into 6-well plates (3 × 105 cells/well) and incubated at 37 °C overnight. The cells were treated with 1 nM SGNP-10b or SGNP-200b in DMEM (with 10% FBS) at 37 °C for 2 h. As negative control groups, cells were pretransfected with anti-miR-10b and anti-miR-200b (AccuTarget Human miRNA inhibitors) using Lipofectamine RNAiMAX (Invitrogen, Life Technologies, USA) according to the manufacturer’s protocols. Expression of miR-10b and miR-200b was observed using a laser scanning confocal microscope (LSM700; Carl Zeiss, Jena, Germany) with a 63× oil immersion objective at an excitation wavelength of 540 nm for Cy3 and quantified using flow cytometry (FACSCalibur). To detect intracellular miR expression, we used three gastric cancer cell lines: Hs746T cells, which exhibited high expression of miR-10b; AGS cells, which exhibited moderate expression of miR10b; and YCC16 cells, which exhibited low expression of miR-10b (see the Supporting Information). Each cell line was seeded in 35 mm μDishes (Ibidi, Madison, WI, USA) at a density of 4 × 105 cells/dish and incubated at 37 °C overnight. After washing, the cells were incubated with 1 nM SGNP-10b in DMEM (with 10% FBS) at 37 °C for 2 h. The cell nuclei were stained with Hoechst 33342 (Invitrogen; 1 μL/mL) for 30 min at 37 °C, and cells were rinsed twice with PBS (10 mM, pH 7.4). The relative expression of intracellular miR-10b was observed using a laser scanning confocal microscope (LSM700) with a 63× oil immersion objective at excitation wavelengths of 405 and 540 nm for Hoechst 33342 and Cy3, respectively. Differential expression of miR-10b among the three cell lines was determined quantitatively using flow cytometry (FACSCalibur). F

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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/acsami.6b16452. Transmission electron microscopy (TEM) images and absorbance spectra of SGNP-MBs with various size and morphology. Stability of SGNP-MBs in different solutions presented by size distribution and absorbance spectra. Sequence information on the molecular beacon consisting of a capture probe and a reporter probe. Fluorescence spectrum of SGNP-MBs using various MBs having different lengths of the reporter probe. Cell viability of Hs746T cells and YCC-1 cells treated with SGNP-MBs. miR-10b and miR-200b expression levels of Hs746T, AGS, and YCC-16 cells using qRT-PCR. Fluorescence spectrum using SGNP-MBs to detect both miR-10b and miR-200b. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Fax: +82-(2)-312-6401. E-mail: [email protected]. *Fax: +82-(2)-312-6401. E-mail: [email protected]. ORCID

Seungjoo Haam: 0000-0003-1533-8357 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by BioNano Health-Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as “Global Frontier Project” (Grant No. H-GUARD_2013M3A6B2078946).

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DOI: 10.1021/acsami.6b16452 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX