Target-Cell-Specific Fluorescence Silica Nanoprobes for Imaging and

Feb 27, 2014 - Institute of Life Sciences, Joint Center for Nanobody R&D between SEU and Egens Bio, Southeast University, Sipailou District,. Nanjing ...
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Target-Cell-Specific Fluorescence Silica Nanoprobes for Imaging and Theranostics of Cancer Cells Henan Li,† Yawen Mu,‡ Jusheng Lu,† Wei Wei,† Yakun Wan,‡ and Songqin Liu*,† †

School of Chemistry and Chemical Engineering, Southeast University, Jiangning District, Nanjing 211189, P.R. China Institute of Life Sciences, Joint Center for Nanobody R&D between SEU and Egens Bio, Southeast University, Sipailou District, Nanjing 210000, P.R. China

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S Supporting Information *

ABSTRACT: MicroRNAs (miRNAs) has been identified as diagnostic and prognostic biomarkers and predictors of drug response for many diseases, including a broad range of cancers, heart disease, and neurological diseases. The noninvasive theranostics system for miRNAs is very important for diagnosis and therapy of the cellular disease. Herein, a target-cell-specific theranostics nanoprobe for target-cell-specific delivery, cancer cells and intracellular miRNA-21 imaging, and cancer cell growth inhibition was proposed. The nanoprobe (FS-AS/MB) was prepared by simultaneously coupling of the AS1411 aptamer and miRNA-21 molecular beacon (miR-21MB) onto the surface of Ru(bpy)2+ 3 -encapsulated silica (FS) nanoparticles. The FS nanoparticles synthesized by a facile reverse microemulsion method showed nearly monodisperse spherical shape with a smooth surface, good colloidal stability, a fluorescence quantum yield of ∼21%, and low cytotoxicity. The antibiofouling polymer PEG grafted onto a silica shell reduced nonspecific uptake of cells. The ability of FS-AS/MB for target-specific cells delivery, simultaneous cancer cells, intracellular miRNA-21 imaging, and inhibition of miRNA-21 function and suppression of cell growth in vitro, were also demonstrated. The results of the present study suggested that the proposed nanoprobes would be a promising theranostics for different cancers by imaging and inhibiting other intracellular genes.

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an oncogene in tumors by blocking expression of key apoptosisenabling genes. Thus, the important functions of miRNA-21 make it a promising candidate for cancer diagnostics and therapy. Recent significant progress in optical imaging techniques has offered the opportunities of noninvasive and repeated real-time analysis of miRNA in living cells.17 Several noninvasive miRNA imaging methods, based on stem loop- or linear-structured DNA molecular beacon (MB) containing perfectly complementary oligonucleotides, have been developed to image endogenous miRNAs.18−23 Additionally, to knock down overexpressed miRNA, chemically modified oligonucleotide analogues (anti-miR) have been used as miRNA inhibitors, including locked nucleic acids and 2′-O-methyl modified oligonucleotides, which inhibits the function of miRNA by hybridization with its target miRNA forming a complex.24,25 Thus, the combination of MB and anti-miR can be a promising diagnostic and therapeutic biomarker in cancers by simultaneously imaging miRNA biogenesis and disrupting miRNAdependent regulatory circuits. However, with the miRNA-21 as a biomarker in theranostics, a stable and targeted delivery without potential side effects is a significant challenge.

icroRNAs (miRNAs) are small, highly conserved noncoding RNAs that play key roles in many biological processes by regulate gene expression at a post-transcriptional level.1,2 Abnormal expression of specific miRNAs was implicated in a number of diseases, including a broad range of human cancers, heart disease, and neurological diseases.3−5 Consequently, miRNAs are identified as novel types of tumor suppressors or oncogens for diagnostic and prognostic biomarkers and predictors of therapy response.3,6 Recent studies demonstrate great potentials for the application of miRNA-based therapy in cancer treatment.7−13 Among these miRNAs, microRNA-21 (miRNA-21) is one of the most prominent endogenous miRNAs involved in the genesis and progression of human cancers, which functions as an oncogene and is commonly overexpressed in many human cancers.14 At the molecular level, miRNA-21 is commonly and evidently upregulated in human glioblastoma and that inhibiting the miRNA-21 expression triggers activation of caspase and leads to increased apoptotic cell death.15 Furthermore, miRNA-21 is highly overexpressed in breast cancer cells compared to the matched normal breast cells. Knockdown of miRNA-21 in cultured breast cancer cells causes cells growth inhibition associated with increased apoptosis and decreased cell proliferation, which could be in part owing to the downregulation of the antiapoptotic Bcl-2 in cancer cells.16 These results suggest that overexpressed miRNA-21 may function as © 2014 American Chemical Society

Received: January 15, 2014 Accepted: February 27, 2014 Published: February 27, 2014 3602

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Scheme 1. Schematic of the Synthesis of FS-AS/MB and Strategy of Cancer-Targeting Theranostics using FS-AS/MB

biocompatibility and allow subsequent bioconjugation, respectively. Cell-specific delivery is achieved by functionalizing FS nanoparticles with AS1411 aptamer, which leads to the delivery of MB only inside targeted cells with nucleolin protein. The simultaneous cell and intracellular miRNA-21 imaging can be accomplished under the same excitation wavelength without any cross-talk. Most importantly, the released miR-21-MB from the nanoprobe can hybridize with miRNA-21 and inhibit cell growth in vitro.

Recently, various nanostructured materials have been used for the fabrication of the MB delivery system.21,26 The polyethylenimine-grafted graphene nanoribbon has been used for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA.26 Multifunctional SnO2 nanoparticles were proposed by using folic acid for cell-specific delivery and MB conjugated to fluorescence SnO2 nanoparticles with a disulfide linkage for imaging intracellular targets miRNAs-21.21 Dye-doped silica nanoparticles exhibit several advantages such as high photostability and good biocompatibility.27 The functional moiety on nanoparticles allows for the incorporation of various cell-specific targeting, imaging, and therapeutic functions into a single dye-doped silica nanoparticle, which is designed for theranostics without losing the individual properties of each component. The combination of fluorescent silica nanoparticles and aptamers to distinguish cancer cells and display their potential in tumor diagnosis was achieved.28,29 Multicolor fluorescence bioimaging is the most effective way to understand the functions of biomarkers in pathological and physiological fields.30−32 Especially, multicolor fluorescent bioimaging by single-wavelength excitation has been proved to be a powerful tool for simultaneous monitoring of multiple targets in cells.33,34 Using a single-wavelength excitation could considerably simplify the instrumentation requirements, and there is no need for multiple laser lights to excite fluorescent probes separately in the assay.35 In this study, we design a multifunctional fluorescence SiO2 nanoprobe, which contains a cell-target moiety as well as a conjugated 2′-O-methyl-modified miRNA-21 molecular beacon (miR-21-MB) to specifically recognize the miRNA-21 in breast cancer cells (MCF-7). The tris(2′,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)2+ 3 ) doped SiO2 (FS) nanoparticles were synthesized by a one-pot two-step reverse microemulsion method. Polyethylene glycol (PEG) and functionalized amine groups were conjugated to these FS nanoparticles, which provide better

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EXPERIMENTAL SECTION Materials and Reagents. Tetraethoxysilane (TEOS), 3chloropropyltriethoxysilane, 4′,6-diamidino-2-phenylindole (DAPI), formaldehyde, tris(2′,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)32+), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-Hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich Trading Company, Ltd. (Shanghai). Triton X-100 and (3aminopropy)triethoxysilane (ATPS) were received from Acros Organic (Geel, Belgium). 2-[Methoxypoly(ethyleneoxy)propyl]trimethoxysilane (MPETS) was obtained from Gelest Inc.. 3-(4,5-Dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT) was purchased from KeyGen Biotech. Company Ltd. (Nanjing, China). 9-Fluorenylmenthyl choroformate (Fmoc-Cl) and hexahydropyridine were gained from Sinopharm Chemical Reagent Company, Ltd. (Shanghai, China). Deoxyribonuclease I (DNase I) was purchased from Sangon Biological Engineering Technology & Company Ltd. (Shanghai, China). Antifade mounting medium was gained from Beyotime (Beijing, China). All other reagents were of analytical grade. The 0.1 M phosphate buffer solution (PBS) was prepared by mixing the stock solution of Na2HPO4 and NaH2PO4. Distilled water was used throughout the study. The oligonucleotides were purchased from Sangon Biological Engineering Technology & Company Ltd. Their sequences were AS1411 apatmer: 5′-HOOC-TTGGTGGTGGTGGTT3603

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The amine moieties on the surface of FS nanoparticles and FS-AS/MB were determined by using the standard Fmoc quantification protocol.38 Mixed together with continuous stirring overnight under N2 at room temperature were 3 mL of 20 mg mL−1 FS nanoparticles or FS-AS/MB anhydrous DMF solution and 5 mL of 30 mg mL−1 Fmoc-Cl anhydrous DMF solution. After centrifugation of the solution mixture, precipitated FS nanoparticles or FS-AS/MB with complete Fmoc protection on the amine moieties were collected, washed thoroughly with methanol, and dried under vacuum overnight. After this precipitation was weighed, 2 mg of Fmoc protected FS nanoparticles or FS-AS/MB was resuspended with 0.8 mL of DMF by sonication for 30 min. Then, 0.2 mL of hexahydropyridine was added to the mixture and sonicated for 20 min. The supernatant was collected by centrifugation at 13200 rpm for 30 min. Amine quantification was calculated by Lambert−Beer law. The extinction coefficient of 7800 mol−1 dm3 cm−1 at 300 nm was used in this work. UV absorption should lie between about 0.3 and 1.2 absorbance units in each test. Cell Culture and Cytotoxicity Assay. The MCF-7 human breast cancer cell line was cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Wisent) supplemented with 10% fetal calf serum (FCS, Sigma), 100 μg mL−1 penicillin, and 100 μg mL−1 streptomycin at 37 °C in a humidified 5% CO2containing atmosphere. Cell numbers were monitored by using a Petroff-Hausser cell counter. MCF-10A human normal mammary epithelial cells were cultured similarly, except that DMEM was replaced by DMEM:nutrient mixture F-12 (DMEM/F12). For the cytotoxicity assay, 2.5 × 103 MCF-7 cells were cultivated in 100 μL media in each well of a 96-well plate for 12 h. After discarding the previous media, 100 μL of fresh medium alone or medium containing different amounts of FS nanoparticles, FS-AS, or FS-AS/MB was added to each well and cultivated for various times. Then 20 μL of 5 mg mL−1 MTT was added to each well. The mixture was allowed to incubate for 4 h, and then the medium was removed and 150 μL sodium dodecyl sulfate (DMSO) was added to each well to solubilize the formazan dye. After vortexing for 15 min, the absorbance of each well was measured by using a model 550 microplate reader Bio-Rad 550 at 570 nm (Bio-Rad). Confocal Laser Microscopy Assay. Cancer and normal cells were seeded on 25 mm diameter cover-glass slips at the bottom of each well by adding 1 × 104 cells to each well to culture at 37 °C for 12 h. After washing with 10 mM pH 7.4 PBS three times, 500 μL of fresh DMEM containing 100 μg mL−1 FS-AS/MB was added to each well and incubated at 37 °C for 3 h. The redundant nanoparticles were removed by thoroughly washing the cover-glass slips with PBS. Then 200 μL of 4% paraformaldehyde solution was added and gently shaken for 20 min to fix the cells on the surface of cover-glass slips. After additional washing with PBS, the cover-glass slips were treated with 500 μL of 1 μg mL−1 DAPI in PBS for 5 min to stain the nucleus of the fixed cells. Finally, the cover-glass slip was coated by a glass slide with 7 μL of mounting medium in the place of both glass slips. The resulting glass slide aggregation was inverted and placed above a 40× oil-immersion objective lens on the confocal laser scanning microscopy (Leica TCS SP5 II, Germany). Excitation of DAPI was carried out with a Diode laser at λ = 405 nm, and emissions were collected in the blue channel. For excitation of FAM and FS, an Ar laser

GTGGTGGTGGTGG-3′; miRNA-21 target: 5′-TAGCTTATCAGACTGATGTTGA-3′; miRNA-16 target: 5′-TAGCAGCACGTAAATATTGGCG-3′; miRNA-26a target: 5′TTCAAGTAATCCAGGATAGGCT-3′; single-base mismatch stand of miRNA-21 target: 5′-TAGCTTATCAGTCTGATGTTGA-3′; and three-based mismatch stand of miRNA-21 target: 5′-TAGCTTTTCAGTCTGAAGTTGA-3′. MiR-21-MB was designed to form a partially double-stranded oligonucleotide using a pair of oligonucleotides (Scheme 1). The long oligonucleotide (MBL) contained a miRNA-21 binding sequence, which is perfectly complementary to mature miRNA-21, with a carboxyl moiety and a disulfide linkage at the 5′ end and carboxyfluorescein (FAM, excitation/emission =488/518 nm) at the 3′ end. The short oligonucleotide (MBS) has a quencher (DABCYL). The MBL was 5′-HOOCAA-S-S-gtcaacatcagtctgataagctaTGTCGCTT-carboxyfluorescein (FAM)-3′ (lowercase: 2′-O-methyl base, uppercase: DNA) and MBS was 5′-DABCYL-GCGACAtagct-3′ (lowercase: 2′-Omethyl base, uppercase: DNA). Instruments. The morphologies of nanoparticles were analyzed by using transmission electron microscopy (TEM, JEOL JEM-2010, Japan) and scanning electron microscopy (SEM, JEOL JSM-7001F, Japan) at an accelerating voltage of 200 and 10 kV. X-ray photoelectron spectroscopy (XPS) was performed by using ESCALAB 250 (Thermo). Fluorescence spectra were recorded on a FluoroMax-4 spectrofluorometer with xenon discharge lamp excitation (Horiba). Zeta potentials and sizes of the nanoparticles were determined at room temperature by a Zeta Plus Potential Analyzer (Brookhaven Instruments Corporation) and dynamic light-scattering measurement (DLS, 90 Plus/BI-MAS Brookhaven). In these experiments, the nanoparticles were dispersed in ultrapure water (≥18 MΩ cm−1, Milli-Q, Millipore), and the ionic strength of the suspensions was kept constant by adjusting with different NaCl concentrations. Preparation of Multifunctional Cancer Cell Theragnostic Nanoprobes. FS nanoparticles were prepared with modification, according to our previous work.36,37 Briefly, 1.8 mL of Triton X-100, 7.5 mL of cyclohexane, 1.6 mL of 1hexanol, and 480 μL of 3 mg mL−1 Ru(bpy)2+ 3 aqueous solution were mixed together with continuous stirring. When 100 μL of TEOS was added to the mixture, the hydrolyzation was initiated by adding 60 μL of NH3·H2O (28%). The reaction was allowed to continue for 24 h at room temperature. Then 15 μL of TEOS, 10 μL of MPETS, and 5 μL of ATPS were added to the reaction mixture and allowed to continue the reaction for another 12 h with stirring. After centrifugation of the solution mixture, the precipitate (FS nanoparticles) was collected, washed thoroughly with ethanol and water, and dried under vacuum. For construction of the target-cell-specific nanoprobes, 2 nmol of AS1411 and 2 nmol of miR-21-MB were mixed with EDC and NHS (molar ratio of DNA:EDC:NHS = 1:100:250) in aqueous solution under gentle stirring for 30 min at room temperature. Then 2 mg of FS nanoparticles were added to the mixture. The suspension was gently stirred at 4 °C for 12 h. Finally, the AS1411 and miR-21-MB modified FS (FS-AS/MB) was collected by centrifugation, washed three times with PBS, redispersed in 2 mL of pH 7.4 PBS, and stored at 4 °C for later experiments. Conjugation of only AS1411 aptamer or miR-21MB with FS nanoparticles (FS-AS, FS-MB) was prepared by similar procedures. 3604

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Figure 1. (A) TEM image of FS nanoparticles. Inset: TEM image of FS-AS/MB. (B) Absorbance and fluorescence spectra (λex = 480 nm) of FS nanoparticles (black) and Ru(bpy)2+ 3 (red) in water. Inset: (left) photograph of the Tyndall effect of FS solution and (right) FS solution under 365 nm irradiation. (C) DLS characterization. (D) Zeta potential and agarose-gel electrophoresis of FS nanoparticles and FS-AS/MB, respectively. The gel electrophoresis was performed by 0.5% agarose gel in PBS, and the resulting migration pattern was revealed under UV irradiation.

at λ = 488 nm was used and emissions were recorded in green and red channels, respectively.

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RESULTS AND DISCUSSION Characterization of the AS1411 and miR-21-MBModified FS Nanoparticles. FS nanoparticles were prepared by the two-step reverse microemulsion method as shown in Scheme 1. First, Ru(bpy)2+ 3 -encapsulated silica nanoparticle was generated by doping Ru(bpy)2+ inside the silica network 3 though a water-in-oil reverse microemulsion method. Then, MPETS and ATPS were added to drive the postcoating of −NH2 groups and PEG on FS nanoparticles. The TEM image revealed that the resulting FS nanoparticles have an average size of ∼50 nm and nearly monodisperse spherical shape with a smooth surface (Figure 1A). The SEM image showed that the as-prepared FS nanoparticles display an ordered monolayer with equal spacing arrayed on the substrate, indicating that the resulting FS nanoparticles have uniform and monodispersed sizes (Figure S1 of the Supporting Information). The postcoating of −NH2 groups and PEG onto FS nanoparticles by hydrolysis of MPETS and ATPS was confirmed by XPS spectra. FS nanoparticles showed the binding energy of the core electrons for the Si 2p line at 101.75 eV, generated from the Si−O groups and the Ru 3d line at 284.89 eV (Figure 2). A small peak of N 1s located at 409 eV was observed, which can be assigned to the NH2 group, while a strong O 1s peak located at 539.5 eV corresponded to both the PEG moiety and the SiO2 intrinsic matrix.39 The prepared FS nanoparticles could be homogeneously dispersed in an aqueous solution, and no aggregation could be observed. Moreover, the FS suspension showed a good colloidal stability, where even the salt concentration changed to 1 M and pH values varied from 1 to 13 (Figure S2 of the Supporting Information). The colloidal stability was supported

Figure 2. The XPS survey spectrum of FS nanoparticles from 0 to 1200 eV. Inset: narrow-scan XPS of the N 1s regions.

by the well-defined Tyndall effect of FS suspension (Inset in Figure 1B). The good stability of FS suspension was due to the high negative-charged surface of FS that resulted from the coating of the PEG moiety on FS nanoparticles, which prevented the nanoparticles from coagulation and nonspecific uptake by the strong electrostatic repulsion effect between the nanoparticles.40 The FS nanoparticles displayed the same color as Ru(bpy)2+ 3 itself and a strong absorption peak at around of 450 nm, corresponding to the Ru(bpy)2+ 3 moiety. The FS nanoparticles emitted orange luminescence excited by the 365 nm ultraviolet lamp (Inset in Figure 1B), with a fluorescence quantum yield of ∼21% (Figure S3 of the Supporting Information). Comparing with the free Ru(bpy)2+ 3 in water, fluorescence spectrum of FS nanoparticles was shifted about 12 nm to blue (Figure 1B). This was due to that Ru(bpy)2+ 3 embedded inside the silica shell 3605

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Figure 3. (A) Emission fluorescence spectra of FS-AS/MB (100 μg mL−1) in the absence and presence of target DNA (1 nM). (B) Plot of the fluorescence intensity vs the concentration of the target. Inset: linear plot for target determination.

Figure 4. (A) Fluorescence spectra of complementary, single-base mismatch, three-base mismatch, miRNA-16, miRNA-26a, and blank. (B) Histograms for the spectra in part A.

cytotoxicity may ascribed to the negative charged surface of FS nanoparticles and the presence of PEG on the surface of FS nanoparticles, which could enhance the biocompatibility in vivo and in vitro.43 Fluorescence Properties of FS-AS/MB in the Presence of Target. When AS1411 and miR-21-MB were coupled to FS nanoparticles, the fluorescence spectra of FS-AS/MB displayed a smart peak corresponding to Ru(bpy)2+ 3 moiety at 596 nm (Figure 3A). Negligible fluorescent signal of FAM in FS-AS/ MB could be observed, confirming the high fluorescent quenching efficient by Dabcyl quencher. After addition of miRNA-21 target, the fluorescence intensity at 596 nm remained constant as FS nanoparticles, while the fluorescence emission at 518 nm enhanced significantly (Figure 3A). The hybridization between miRNA-21 target and miR-21-MB allowed the Dabcyl quencher to release from miR-21-MB, which recovered the fluorescence of FAM and resulted in the increasing of the fluorescence intensity at 518 nm. The recovery of the fluorescence of FAM in the presence of the perfectly complementary miRNA-21 target could be used for miRNA-21 target detection. It was found that the fluorescence intensity at 518 nm increased with the increasing concentration of the miRNA-21 target. The linear range for miRNA-21 target detection was from 0.5 to 40 nM with a detection limit of 0.18 nM (Figure 3B). In addition, no fluorescence resonance energy transfer (FRET) between FAM and Ru(bpy)2+ 3 embedded inside the silica shell was observed. Previous studies demonstrated that the distance between chromophore and quencher must be less than 10 nm for the generation of a FRET signal.44,45 In our case, two chromophoric groups of Ru(bpy)2+ 3 and FAM were separated by the 33-mer oligonucleotide. With the assumption that the distance between DNA bases was 0.34 nm, the distance

experience a less polar and quite different environment compared to its freedom in water.41 The amine moieties presented on the surface of FS nanoparticles were used for coupling of AS1411 aptamer and miR-21-MB, both containing a 5′-end of the −COOH group through aqueous carbodiimide coupling chemistry. The amine moieties on the surface of FS nanoparticles were determined to be ∼4.5 nmol mg−1. After coupling with AS1411 aptamer and miR-21-MB, the remaining amine groups on FS were quantified to be 2.3 nmol mg−1, resulting in a conjugation efficiency of 48.9% for AS1411 and miR-21-MB. After coupling with AS1411 and miR-21-MB, the morphology of FS-AS/MB was the same as FS nanoparticles and displayed a uniform array, good colloidal stability, and no aggregation (inset in Figure 1A). The DLS analysis showed that the hydrodynamic size increased from 52 to 62 nm for FS nanoparticles and FS-AS/ MB (Figure 1C), respectively. Meanwhile, the zeta potential changed from −35 to −46 mV for FS nanoparticles and FS-AS/ MB (Figure 1D, left), respectively. The gel electrophoresis showed that FS-AS/MB had slightly more migration toward the positive electrode than the FS nanoparticles (Figure 1D, right). All these results suggested the successful conjugation of AS1411 aptamer and miR-21-MB to FS nanoparticles. Moreover, the relatively small size and negative charges of FS nanoparticles may aid in the reduction of nonspecific cellular uptake.42 To evaluate the cytotoxicity of FS nanoparticles, a MTT assay was employed to assess the cell viability. MCF-7 cells were treated with FS nanoparticles with various dosages for 3 days. The cell viability was maintained at 85%, corroborating the fact that FS did not show any acute cytotoxicity to the cell even at the nanoparticle levels up to 400 μg mL−1 over 72 h (Figure S4 of the Supporting Information). The low 3606

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respectively, indicating that the nonspecific uptake of nanoparticles was negligible (Figure S6, panels A and B, of the Supporting Information). In addition, receptor-deficient MCF10A cells were treated with FS-AS under similar conditions to the MCF-7 cells. No red fluorescence was observed, further verifying the target-cell-specific delivery (Figure 5B). The results of confocal microscopy analysis clearly showed that FSAS/MB bind specifically to the targeted cells. Moreover, if the miR-21-MB are digested by DNase I endonuclease, the fluorescence intensity will increase because the donor dye molecule will separate from the quenchers. After incubation of DNase I endonuclease and FS-AS/MB or free miR-21-MB, no obvious fluorescence could be observed, indicating that 2′-Omethyl oligonucleotide possesses high resistance to the enzymatic cleavage (Figure S7 of the Supporting Information) Theranostics of FS-AS/MB. The property of target-cellspecific delivery of FS-AS/MB could be used for cancer cells imaging under single wavelength excitation of 488 nm. As shown in Figure 6, two kinds of fluorescence spots with red for FS nanoparticles and green for FAM were clearly observed in the cytoplasm of the MCF-7 cells at the red and green fields, respectively. The green fluorescence came from the recovery of the fluorescence of FAM in the presence of intracellular miRNA-21, which hybridized with miR-21-MB and released the Dabcyl quencher from miR-21-MB. It should be noted that the red and green spots were almost around the cell nucleus (blue spots) but lack colocalization, indicating the release of miR-21MB from FS-AS/MB. The release of miR-21-MB occurred through the cleavage of the disulfide linkage between the miR21-MB and FS nanoparticles in the intracellular reductive and acidic environment.48 The designed 2′-O-methyl-modified miR-21-MB can hybridize with intracellular miRNA-21 of MCF-7 cells and inhibits the cell growth, resulting in a therapeutic function. When MCF-7 cells were transfected with miR-21-MB, the amount of intracellular miRNA-21 in the cells determined by reversetranscriptase quantitative PCR (RT-PCR) was half of that without miR-21-MB transfection (Figure S8 of the Supporting Information). On the other hand, the cell viability was also measured using the MTT assay after MCF-7 cells were transfected with miR-21-MB at various concentrations. A significant reduction of cell viability (75.6%) was obtained after the cells were treated with 50 nM of miR-21-MB (Figure S9 of the Supporting Information). This is in good agreement with the previous report,15 indicating that the designed miR-21MB can provide an efficient and straightforward way to block the intracellular miRNA-21 function. When MCF-7 cells were treated with FS-AS/MB, the MTT assay showed an obvious reduction in MCF-7 cell viability. The cell viability was decreased by increasing the treatment time and reached a minimum value of 78% for 72 h of treatment (Figure 7). In contrast, only 3% reduction in MCF-7 cell viability was observed after treatment with FS nanoparticles, FS-AS, and FSMB in the same conditions. This demonstrated that the proposed FS-AS/MB have great potential for cancer theranostics.

between two chromophoric groups is more than 10 nm. Therefore, fluorescence signal from Ru(bpy)32+ was not regulated by coupling of miR-21-MB to FS nanoparticles and no FRET of Ru(bpy)2+ 3 could be observed in FS-AS/MB. Furthermore, the cross interference of the fluorescence by FAM and Ru(bpy)2+3 inside the silica shell was investigated. Upon single wavelength excitation (488 nm), the mixture of MBL and FS nanoparticles showed two distinct emissions at 518 and 596 nm for FAM and FS nanoparticles, respectively (Figure S5A of the Supporting Information). Moreover, their fluorescence intensity simultaneously increased with the increasing concentration of MBL and FS nanoparticles, while the locations of the fluorescence characteristic peaks had little influence. When the amount of FS nanoparticles kept constant, the fluorescence emission at 518 nm increased proportionally with MBL concentration and the emission of FS remained unchanged (Figure S5B of the Supporting Information). Therefore, no cross-talk between FAM and FS nanoparticles was observed, making the simultaneous imaging under single wavelength excitation possible and feasible. The sequence specificity of miR-21-MB was also studied. For comparison, the miRNA-21 target with a single-base mismatch, three-base mismatch, miRNA-16 target, and miRNA-26a target were used under the same conditions. The perfectly complementary miRNA-21 target showed a fluorescence intensity of 2 and 2.9 times larger than that for single-base and three-base mismatch sequence, respectively. The response to miRNA-16 and miRNA-26a did not obviously change in comparison to the background fluorescence (Figure 4). These results suggested that the proposed miR-21-MB was of high sequence specificity. Target-Cell-Specific Delivery. Target-cell-specific delivery is very important for the development of theranostics because it can limit side effects from nonspecific delivery and reduce the quantity of the gene probe needed for treatment.46 AS1411 aptamer, targeting nucleolin protein, which is highly expressed in the membrane of cancer cells, was conjugated to the FS nanoparticles as a target-specific moiety.47 To confirm the specific cell-targeting ability of the theranostics nanoprobe, confocal microscopy analysis by using breast-cancer cells (MCF-7) that express nucleolin protein and human normal mammary epithelial cells (MCF-10A) deficient in nucleolin protein was evaluated. With incubation of FS-AS with MCF-7, a strong red fluorescence is observed for MCF-7 cells (Figure 5A). In contrast, negligible fluorescence was observed after MCF-7 cells were treated with FS nanoparticles and FS-MB,

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CONCLUSION In summary, this work proposed a target-cell-specific fluorescence silica nanoprobe for imaging and theranostics of cancer cells. The FS nanoparticles synthesized by a facile reverse microemulsion method showed nearly monodisperse spherical shape with a smooth surface, good colloidal stability, a

Figure 5. Confocal images of (A) MCF-7 cells and (B) MCF-10A treated with FS-AS (A, 100 μg mL−1) at 37 °C for 3 h (blue field = nucleus staining and red field = FS nanoparticles fluorescence). 3607

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Figure 6. Confocal images of MCF-7 cells treated with FS-AS/MB (100 μg mL−1, 50 nm linked MB) at 37 °C for 3 h (blue field = nucleus staining, green field = FAM-labeled MB, and red field = FS nanoparticles fluorescence).

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ACKNOWLEDGMENTS We thank Professor Xiangdong Liu (Institute of Life Sciences, Southeast University) for his helpful advice. This work was supported by the National Basic Research Program of China (Grant 2010CB732400), the Key Program (Grant 21035002) from the National Natural Science Foundation of China and National Natural Science Foundation of China (Grants 21175021 and 21375014), and the Fundamental Research Funds for the Central Universities.

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Figure 7. Cancer-targeting therapy by FS-AS/MB. MCF-7 cells treated with FS nanoparticles (100 μg mL−1), FS-AS1411 (100 μg mL−1), FSMB (100 μg mL−1), and FS-AS/MB (100 μg mL−1, 50 nm linked MB), respectively.

fluorescence quantum yield of ∼21%, and low cytotoxicity. The antibiofouling polymer PEG grafted onto silica shell reduced the nonspecific uptake of cells. The target cell-specific delivery of the resulting nanoprobes was illustrated by the special imaging ability of cancer cells. AS1411 aptamer and conjugated nanoparticles should be useful for the specific targeting of cancer cells. The simultaneous cell imaging and intracellular miRNA detection can be accomplished under the same excitation wavelength without any cross-talk, simplifying the requirements of the excitation source to a single-wavelength laser. Most importantly, the miR-21-MB can be released from FS-AS/MB in the intracellular reductive and acidic environment of cancer cells and which hybridizes with miRNA-21 and inhibits cell growth in vitro with minimized invasiveness and side effects. The multifunctional MB-based nanoprobe in this report can be used as a target-specific theranostics probe for many different cancers by detecting and inhibiting other genes in other cancers.

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S Supporting Information *

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*E-mail: [email protected]. Tel: 86-25-52090613. Fax: 86-2552090618. Notes

The authors declare no competing financial interest. 3608

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