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A Binary System for MicroRNA-Targeted Imaging in Single Cells and Photothermal Cancer Therapy Ruo-Can Qian, Yue Cao, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01804 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 3, 2016
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Analytical Chemistry
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A Binary System for MicroRNA-Targeted Imaging in Single Cells and Photothermal Cancer Therapy 10
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Ruo-Can Qian, Yue Cao, Yi-Tao Long* 12
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Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China.
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Fax: +86 021-64250032; E-mail:
[email protected];
[email protected] 15 17
16 ABSTRACT: Abnormal expression of microRNAs (miRNAs) is often associated with tumorigenesis, metastasis and progression. Among them, miRNA-21 is found to be overexpressed in most of the cancer cells. Here, a binary system is designed for miRNA-21 targeted imaging and photothermal treatment in single cells. The binary system is composed by a pair of probes (probe-1 and probe2), which are encapsulated in liposomes for cell delivery. Both of the two probes adopt gold nanoparticles (AuNPs) as the core material, and the AuNPs are functionalized with Cy5-marked molecular beacon (MB-1/MB-2 for probe-1/probe-2 respectively). The loop part of MBs are designed to be complementary with miRNA-21. Therefore, after the binary system enters into the cytoplasm, MBs can be opened upon miRNA-21 triggered hybridization, which turns “on” the fluorescence of Cy5 for the localization of miRNA-21. At the same time, a crosslinking between the probes occurs since the far ends of MB-1 and MB-2 are designed to be complementary with each other. The miRNA-induced aggregation shifts the absorption of AuNPs to near-infrared, which can be observed under dark-field microscopy (DFM) and used for the following photothermal therapy. Under near-infrared (NIR) irradiation, MCF-7 breast cancer cells are successfully killed. The proposed system can be further applied in tumor-bearing mice, and shows significant therapeutic effect. This work provides a new tool for intracellular miRNA analysis and targeted treatment against cancer.
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30 INTRODUCTION MicroRNAs (miRNAs), typically 18-25 nucleotides, are a class of small non-coding RNA molecules widely exist in eukaryotic cells.1-3 MiRNAs play key roles in a series of post-transcriptional regulatory events, including cell proliferation, differentiation, metabolism and apoptosis.4-7 Over the past decade, the significance of miRNAs in the regulation of cell bioprocesses and functions has been widely known and attracted more and more attention. Recent studies show that the dysregulated expression of miRNAs is closely associated with the genesis of various cancers.8-10 For example, miRNA-21 is found to be overexpressed in most of the tumor cells.11 Therefore, miRNAs have been considered as one of the most promising diagnostic markers for cancer cells.12-14 Owing to the significance of miRNAs in cancer diagnosis and therapy, a variety of techniques have been developed for miRNA analysis, including real-time quantitative PCR,15,16 northern blotting,17,18 microarray analysis,19 and electronic detection.20 Despite of their good performance, most of these methods have limited their applications in routine miRNA detection. The further exploration of the intracellular imaging of miRNAs and miRNA-targeted cancer therapy is still a challenge. Recently, noble metal nanoparticles, especially gold nanoparticles (AuNPs), have attracted considerable interest due
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to their favorable biocompatibility and unique optical properties.21-23 As an attractive nanomaterial, AuNPs owe the capability to resonantly absorb and scatter the incident light, inducing strong localized surface plasmon resonance (LSPR).24-27 Notably, AuNPs can be used for photothermal treatment due to their strong absorption and high heat conversion efficiency.26 Although several AuNP-based cancer phototherapies has been reported,28-30 these photosensitizers lack the ability to selectively target or track cancer marker miRNAs. In this respect, it is of great significance to design innovative AuNP-based photothermal agents for highly efficient and selective cancer therapy. In addition, considering the clinical application, the desired photothermal AuNPs should be designed to absorb long wavelengths of light in near-infrared (NIR) region, in order to enable deeper tissue penetration.30 To achieve this goal, AuNPs of different shapes and sizes have been prepare for NIR illumination and achieved therapeutic effect.31-34 Unfortunately, most of these particles are larger than 50 nm, thus the endocytosis efficiency is not very perfect.35-37 This motivates us to design a smart system, which can efficiently deliver small-sized AuNPs (less than 20 nm in diameter) into the cytoplasm and then assemble in cancer cells to shift the absorption light to NIR region for the following photothermal therapy. Herein, a binary system based on AuNPs of 15 nm was developed for in situ imaging of intracellular miRNAs and the
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Analytical Chemistry following miRNA-targeted NIR cancer therapy (Scheme 1). The binary system was composed by a pair of molecular beacon (MB)-functionalized AuNP probes (MB-1/MB-2 for probe-1/probe-2 respectively). The designed MBs were conjugated on the surface through their thiol-labeled 5’ end, and formed a hairpin structure with the Cy5-tagged 3’ end. After modification, the fluorescence of Cy5 was quenched by AuNPs via fluorescence resonance energy transfer (FRET).38 The loop part of MBs was designed to be complementary with miRNA-21. In the presence of miRNA-21, the hairpin could be opened due to the hybridization, turning on the fluorescence of Cy5 due to the departure of Cy5 from the surface of AuNPs. Thus, the location of miRNA could be observed by fluorescence imaging. Then a crosslinking occurred between probe-1 and probe-2, because the far ends of the opened hairpin were designed to be complementary with each other. The miRNA-triggered AuNP aggregation shifted the absorption of AuNPs to NIR region for the following photothermal therapy (Scheme 1a). Using MCF-7 breast cancer cells over expressing miRNA-21 as a model, probe-1 and probe-2 were encapsulated in liposomes for cell delivery. After the binary system was taken in by cells, the miRNA-21-triggered fluorescence could be observed. At the same time, the miRNA-induced aggregation intensity of AuNPs could be monitored with dark-field microscopy (DFM) and the plasmon resonance Rayleigh scattering (PRRS) spectroscopy (Scheme 1b).39-41 Upon endocytosis and NIR irradiation, obvious cell apoptosis was achieved. The proposed system could also destroy cancer cells selectively in tumor-bearing mice, thus provided a facile and high efficient photosensitizer for miRNA-targeted NIR cancer therapy with tracing ability.
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47 Scheme 1. Schematic illustration of the binary system for miRNA tracking in single cells and targeted NIR photothermal therapy. (a) MiRNA-21 induced AuNP aggregation and fluorescence “off-on”. (b) In situ imaging of miRNA-21 in MCF-7 cells and the miRNA-targeted NIR therapy.
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EXPERIMENTAL SECTION Materials and Reagents. Chloroauric acid (HAuCl4•4H2O) was obtained from Sigma-Aldrich Inc. (USA). Hydrogen peroxide (H2O2), sulfuric acid (H2SO4) were purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Annexin V-FITC/propidium iodide (PI) apoptosis detection
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kit, DAPI detection kit, 3-(4,5-dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT), and MCF-7 cells were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). Lipofectamine-2000 (Lipo-2000) and LysoTracker Green was obtained from Invitrogen China Ltd. Phosphate buffer saline (PBS, pH 7.4) contained 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, and 1.41 mM KH2PO4. All other reagents were of analytical grade. All aqueous solutions were prepared using ultrapure water ( 18 MΩ, Milli-Q, Millipore). The RNA sequences were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) with the following sequences: miRNA-21: 5’-UAG CUU AUC AGA CUG AUG UUG A3’; anti-miRNA-21: 5’-UCA ACA UCA GUC UGA UAA GCU A-3’; mismatched miRNA-21: UAG CUU AUC AGA CAG AUG UUC A. The molecular beacon (MB) sequences were purchased from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China) with the following sequences: MB-1: 5’-HS-(CH2)6-CCGTTCTA TCA ACA TCA GTC TGA TAA GCT A TAGAACGG-Cy5-3’; MB-2: 5’-HS-(CH2)6-TAGAACGG TCA ACA TCA GTC TGA TAA GCT A CCGTTCTA-Cy5-3’. Characterization. The transmission electron microscopic (TEM) images were performed on a JEM-2010 high-resolution transmission electron microscope (JEOL Ltd., Japan). The dark-field images were observed on an inverted microscope (eclipse Ti-U, Nikon, Japan) equipped with a darkfield condenser (0.8 < NA < 0.95) and a 40× objective lens (NA = 0.8). The plasmon resonance scattering light was excited by a white light source (100 W halogen lamp). The scattering light was split by a monochromator (Acton SP2300i, PI) equipped with a grating (grating density 300 lines/mm; blazed wavelength 500 nm), and the scattering spectra were recorded by a spectrometer CCD (CASCADE 512B, Roper Scientific, PI). The fluorescence images were observed on the same microscope excited by a mercury lamp (100 W Epi illuminator). MTT assay were performed on a microplate reader (Synergy 5, Biotech, USA). The NIR irradiation was performed by a 680 nm laser (Laserwave Optoelectronics Technology Co., Ltd., China). Preparation of 13 nm AuNPs. Gold Nanoparticles (AuNPs) were prepared using a classical one-pot method.42 Briefly, 200 mL of HAuCl4 (0.01%) was added to a roundbottom flask with vigorous stirring and heated to 100 °C. Afterward, 5 mL of trisodium citrate solution (1%) was quickly added to the boiling solution under continuous stirring for 1 h, until the color of the solution turned dark red. The obtained solution was stored at 4 °C. Preparation of MB-Functionalized AuNPs (probe-1 and probe-2). For the preparation of probe 1, a volume of 10 μL Cy5-tagged MB-1 (100 μM) was mixed with 1 mL AuNP solution and then stirred overnight at room temperature. After that, 0.1 mL PBS buffer (containing 2 M NaCl) was added to the mixture dropwise to stabilize the obtained MB-
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Analytical Chemistry
1 functionalized probe-1. The above solution was centrifuged and washed with PBS twice, then resuspended in 1 mL PBS. The supernatant containing excess MB was collected for fluorescence detection. MB-2 modified probe-2 was prepared using the same method. Preparation of Probe-1 and Probe-2 Co-Encapsulated Liposome (the Binary System). Liposomal nanovesicles were applied to deliver the binary system into the cytoplasm. Briefly speaking, 10 μL of probe-1 solution and 10 μL of probe-2 solution were mixed with 20 μL of serum-free medium. Then, 1.0 μL of Lipo-2000 was dissolved in 20 μL of serum-free medium and incubated at room temperature for 10 min. After the above two solutions were mixed and incubated for 20 min at room temperature, the probe-1 and probe-2 coencapsulated liposome was formed as the binary system. The probe-1 or probe-2 only encapsulated liposome was also prepared for control experiments with the same method. Cell Culture. MCF-7 cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), streptomycin (100 μg mL-1), and penicillin (100 μg mL-1) in a humid atmosphere with 5% CO2 at 37 °C. Cell number was calculated by a Petroff-Hausser cell counter. Sample Preparation for DFM Detection and Scattering Spectroscopy. For DFM analysis, the AuNPs were immobilized on the bottom surface of culture dishes. The dishes were first ultrasonic treated in ethanol for 1 h. Then the dishes were sonicated in pure water for 1 h and dried for use. The AuNP solution was dropped on the bottom surface of a clean culture dish for DFM imaging. To image the AuNPsingested cells, 1 mL of the cell suspension (1×106 mL-1) was seeded in a clean culture dish overnight. Then, 60 μL of the AuNPs encapsulated liposome was added into the dish and incubated with the cells at 37 °C for a certain time. Then, the cell-attached culture dish was washed and immersed in PBS to perform the DFM imaging. The scattering light of AuNPs was split by a monochromator and recorded by a CCD spectrometer to obtain the scattering spectra. Demonstration of MiRNA-21 Triggered Aggregation of AuNPs. The miRNA-21 induced aggregation of the binary system was tested in solution. The mixture of 0.5 mL probe1 and 0.5 mL probe-2 was incubated with different concentrations of miRNA-21 (10 μL) for 15 min at 37 °C, and the corresponding UV-vis spectra, fluorescence spectra and DFM images were observed. In Situ Tracing and Quantification of Intracellular MiRNA-21 with the Binary System. MCF-7 cells (1 mL, 1×106 mL-1) were cultured in each culture dish for 24 h. After the cells were incubated with the binary system (60 μL) at 37 °C for different times. Afterwards, the cells were sent for fluorescence and DFM imaging. To quantitatively detect the amount of intracellular miRNA-21, MCF-7 cells were transfected with different amounts of anti-miRNA-21 as an inhibitor of miRNA-21, thus provided a set of cell samples with different miRNA-21 expression levels to obtain the calibration curve. These anti-miRNA-21 treated MCF-7 cells were divided into two groups. The first group was centrifuged to prepare cell extracts containing various amounts of miRNA-21 to obtain the Cy5 fluorescence spectra. From the
peak intensity and Cy5 standard curve, the amount of miRNA-21 in a single cell (N) was calculated. The second group was incubated with the binary system (60 μL) and observed under a fluorescence microscope (red fluorescence of Cy5 excited under a green filter). From the corresponding cell images, the average red channel brightness intensity (RI) of the cell area was read by Adobe Photoshop software. Therefore, the plot of RI vs. N was obtained for the quantification of intracellular miRNA-21. MTT Assay. The cytotoxicity of the binary system was analyzed by MTT kit. MCF-7 cells (100 μL, 1×106 mL-1) were seeded in a 96-well plate for 4 h. Then, the binary system (60 μL) was added to each well and incubated for different times at 37 °C (cells incubated in culture medium were tested as control). Afterwards, the medium was discarded and the cells in each well were treated with MTT (50 μL, 1 mg mL-1) for 4 h. After washing with PBS, 100 μL dimethyl sulfoxide (DMSO) was added, and the plate was vibrated for 15 min. The absorbance (A) at 450 nm was observed to calculate the cell viability (Atest/Acontrol×100%). Cell Apoptosis Assay. The apoptosis experiments were performed with Annexin V-FITC/PI staining. MCF-7 cells (1 mL, 1×106 mL-1) were seeded in culture dishes overnight, the cells were treated with the binary system. Then the cells were irradiated with an 680 nm laser for 30 min at a power of 0.5 W cm−2. Afterwards, the cells were stained with Annexin V-FITC/PI for 10 min following the manufacturer’s instruction, washed with PBS and the cell death observed with fluorescence microscope. Targeted Photothermal Therapy on MCF-7 Cells. After MCF-7 cells (1 mL, 1×106 mL-1) were cultured and incubated with the binary system, the cells were irradiated with the 680 nm laser for different times at 0.5 W cm−2 at 37 °C. The apoptosis process was visualized under a DFM/fluorescence microscope. Monitoring Photothermal Therapy in Living Mice. Female BALB/c nude mice (pathogen-free, 5-6 weeks) were obtained from Bioray Laboratories Co., Ltd. (Shanghai, China). All animal experiments followed the regulations of institutional animal use and care. MCF-7 tumor model was established by subcutaneous injection of MCF-7 cells (in logarithmic growth phase) on the flank of the nude mice under anesthesia with isoflurane. The volume of the tumor was measured with a vernier caliper and calculated using 0.5 × length × width2.43 The tumor-bearing mice were then intravenously injected with the binary system. 24 h after injection, the tumor region was irradiated with a 680 nm laser for 30 min at a power of 0.5 W cm−2 to perform the photothermal therapy. The therapeutic effect was evaluated by monitoring the tumor volume after the NIR treatment. After the NIR treatment, the mice were euthanized and the tumor tissues and other organs were harvested for histological study by hematoxylin-eosin (H&E) staining. The mice without NIR treatment groups were used as control. RESULTS AND DISCUSSION Characterization of AuNP Probes and MiRNA-21 Induced Aggregation and Fluorescence Recovery of the Binary System. Bare AuNPs were synthesized following a conventional one-pot method.17 The TEM image of the AuNPs showed a homogeneous distribution with an average
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Analytical Chemistry size about 13 nm (Figure 1a). The UV-Vis spectra of MB-1 functionalized probe-1 and MB-2 modified probe-2 showed the characteristic peaks of DNA at 260 nm and AuNPs at 523 nm, as the latter showed a tiny red shift (~4 nm) compared with the peak at 519 nm of bare AuNPs (Figure 1b). In addition, the probe solutions were treated with mercaptoethanol, and the supernatant showed obvious fluorescence from the released MB (Figure S1), indicating the successful conjugation of MB. From the fluorescence intensity of the supernatants containing excess cy5-tagged MB (Figure S2), the amount of MB on each AuNP was estimated to be around ~102.44,45 After MB modification, the AuNP probes showed good stability under high salt and acidic conditions in a pH 5.5 PBS solution containing 0.1 M NaCl (Figure S3).
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49 Figure 1. (a) TEM image of the prepared AuNPs. (b) UVvis spectra of bare AuNPs, probe-1 and probe-2. (c) Photograph of probe-1 (0.5 mL) and probe-2 (0.5 mL) mixed solution incubated with miRNA-21 for 15 min with different concentrations (final miRNA-21 concentration: control, 25, 65, 100, 125 and 150 nM from A to F). Corresponding (d) UV-vis spectra, (e) FL intensity, (f) Fluorescence spectra (Inset: Plot of FL intensity vs. Cy5 concentration), and (g) DFM images of samples A to F (dots in circles amplified in the boxes in the lower left). Cy5 excitation: 648 nm; emission: 688 nm.
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After that, the miRNA-21 triggered aggregation of the binary system composed by probe-1 and probe-2 was first detected in PBS solution. The mixed solution of probe-1 and probe-2 was incubated with different concentrations of miRNA-21 (Figure 1c), and their UV-vis absorptions were tested at 37 °C (Figure 1d). The photograph of these solutions showed that the color changed gradually from red to purple with the increasing concentration of miRNA-21. The corresponding absorption peak of AuNPs showed a continuous red shift from 520 nm to 680 nm in UV-vis spectra with the red side tail covering 750 nm, demonstrating the miRNA-21 induced aggregation. Control experiment showed no aggregation in the presence of miRNA-21 using probe-1 or probe-2 solution alone, indicating the specific conjugation between probe-1 and probe-2 induced by miRNA-21 (Figure S4). To evaluate the simultaneous Cy5fluorescence recovery in the presence of miRNA-21, the fluorescence intensity (FL) of the mixed probe solution was observed (Figure 1e). The FL value increased with the growing concentration of miRNA-21 and reached a plateau at 125 nM, demonstrating the miRNA-21 responsible fluorescence switch. The corresponding fluorescence spectra were shown in Figure 1f. From the standard curve of cy5 (Figure 1f, inset), the amount of miRNA-hybridized MB molecules was calculated to be 0, 1.54×1013, 3.91×1013, 6.05×1013, 7.20 ×1013, 7.20×1013 from A to F. Kinetic studies showed that the mixed probe solution responded rapidly to the hybridized target miRNA within 10 min, and the situation in single probe-1 or probe-2 solution was almost the same (Figure S5), thus indicating that the crosslinking between probe-1 and probe-2 did not affect the miRNA-targeted Cy5 fluorescence recovery. To verify the specific recognition between probes and miRNA-21, a mismatched miRNA-21 control sequence was used to incubate with the mixed probe solution and negligible fluorescence was detected, confirming the specific binding of miRNA-21 to the probes (Figure S6). The miRNA-21 induced aggregation of probe-1 and probe2 was further observed by DFM images (Figure 1g). As the concentration of miRNA-21 increased, the AuNP aggregates emerged and their color changed from green to orange and red, with larger size and stronger scattering light intensity. The corresponding TEM images further confirmed the miRNA-21 induced AuNP aggregation (Figure S7). Therefore, the proposed binary system could successfully form AuNP aggregates for the following NIR irradiation and realize the localization by fluorescence at the same time. In Situ Imaging and Quantification of Intracellular MiRNA-21. Before intracellular experiments, the cytotoxicity of the binary system was first detected by MTT assay. After 4 h incubation, the viability of the cells still maintained at 90% (Figure S8), confirming the low cytotoxicity of the proposed system for the intracellular experiments. For the following intracellular detection, MCF-7 cells were first seeded in a culture dish. Afterwards, 60 μL of the probe-1 and probe-2 entrapped liposomes (the binary system) were added into the culture dish, which was observed under microscope over time to test the feasibility of the binary system for in situ tracking of intracellular miRNA-21. At the same time, the cells were stained by a lysosomal tracker (LysoTracker Green) to monitor the fate of AuNP probes
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26 Figure 2. (a) Time course microscopic images of MCF-7 cells incubated with the binary system (A1-A6: Cy5 fluorescence images; B1-B6: DFM images; Ⅰ-Ⅲ: the detail view of aggregated AuNP probes in B4-B6; i1-i3: corresponding scattering spectra of AuNP probes in circles 1-3 of Ⅰ-Ⅲ; C1-C6: overlapped area of Cy5 fluorescence and AuNP DFM dots; D1-D6: LysoTracker Green stained lysosomes; E1-E6: overlapped area of AuNP aggregates entrapped in lysosomes). (b) Microscopic images of MCF-7 cells treated with different amounts of anti-miRNA-21 (final concentration: 200, 100, 50, and 0 nM from A to D) and then incubated with the binary system for 1.5 h. (c) Histogram of the average RI value in cell areas corresponding to A-D in figure b. Inset: plot of RI vs. N (amount of miRNA-21 per cell).
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after cell uptake. As shown in Figure 2a, within the first 30 min, there was no obvious fluorescence signal. After 50 min, the red fluorescence of Cy5 emerged in the cytoplasm, and its intensity kept growing with the incubation time until reaching a plateau after 1.5 h (Figure 2a, A1-A6). The recovering of the Cy5 fluorescence indicated the opening of the hairpin structure (MB) on the surface of AuNP probes, and thus demonstrated the existence of the miRNA-21. To study the miRNA-21 triggered aggregation of AuNP probes, the corresponding DFM images at different incubation times were captured. As shown in Figure 2a, B1-B6, efficient accumulation of AuNP probes was observed in MCF-7 cells after 70 min. The aggregate accumulation increased at a longer incubation time (Figure 2a, B4-B6, Ⅰ-Ⅲ), appearing as strong orange scatters. The scattering peak of the AuNP aggregates was at approximately 670~690 nm, with the peak wavelength and the scattering intensity increased with the incubation time (Figure 2a, i1–i3). In addition, the DFM images showing the location of AuNP aggregates were overlapped with the fluorescence images of Cy5. As shown in Figure 2a, C1–C6, the AuNP dots and the Cy5 fluorescence regions almost overlapped with each other completely. Therefore, the miRNA-21 induced AuNP aggregation was
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demonstrated for the binary system in living cells, and the aggregates inside of cells could absorb light in NIR and farred region for photothermal therapies. At the same time, the fluorescence images showing the location of lysosomes were observed with LysoTracker Green (Figure 2a, D1–D6). Thus the subcellular distribution of AuNPs was analyzed by overlapping the DFM images and the fluorescence images of LysoTracker Green. As shown in Figure 2a, E1–E6, only a fraction of AuNP aggregates was entrapped in the lysosomes, while most of the AuNP aggregates escaped from the lysosomes into the cytoplasm. By dividing the overlapped area of AuNP aggregates in lysosomes (Figure 2a, E6) by the area of all the AuNP aggregates (Figure 2a, B6), the percentage of AuNPs entrapped in lysosomes after 2 h incubation was estimated to be 20%. Thus, the corresponding percentage of released AuNPs was 80%, showing a quick escape speed of the AuNP probes from the lysosomes possibly due to the lipid-membrane fusion between the membrane of lysosomes and the liposomal wall of the binary system. In this case, the proposed binary system could efficiently deliver the AuNP probes into the cytoplasm, which was favorable for miRNA-21 targeted imaging and therapy. In a control experiment, MCF-7 cells were treated with only probe-
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Analytical Chemistry 1 or probe-2 and observed under microscope (Figure S9). No aggregation of AuNPs was observed, indicating that the miRNA-21 induced AuNP aggregation only occurred under the treatment of the binary system containing both probe-1 and probe-2. For the quantification of intracellular miRNA21, the anti-chain of miRNA-21 (anti-miRNA-21) was used as a model miRNA-21 inhibiting agent to change the level of miRNA-21 in MCF-7 cells. After transfecting MCF-7 cells with different amounts of anti-miRNA-21, a series of cell samples with different miRNA-21 levels were obtained and these cells were divided into two groups. One group was sent for fluorescence and DFM imaging (Figure 2b). With the increasing amount of anti-miRNA-21, the fluorescence of Cy5 decreased significantly, indicating the inhibition effect of anti-miRNA-21 to miRNA-21 level. The corresponding DFM images also showed a reduced extent of AuNP aggregation due to the decreased expression of miRNA-21. From the Cy5 fluorescence images of MCF-7 cells, the average red channel intensity (RI) in cell area was read using Adobe Photoshop software (Figure 2c). At the same time, the other group of anti-miRNA-21 treated MCF-7 cells was centrifuged, and the cell extracts was added into the mixed solution of probe-1 (0.5 mL) and probe-2 (0.5 mL). After 15 min incubation, the fluorescence intensity of Cy5 was detected, and the amount of miRNA-21 per cell (N) could be calculated from the Cy5 standard curve (Figure 1f, inset). Therefore, a calibration curve was obtained, showing a linear relationship between the RI value and the amount of miRNA in each cell (Figure 2c, inset). With the calibration curve, the amount of miRNA-21 in a single MCF-7 cell was estimated to be around ~104 copies per cell, which was comparable with other literature.46,47 Evaluation of Photothermal Therapeutic Efficiency in Living Cells. Since the above experiments have successfully demonstrated that the proposed binary system could efficiently deliver the AuNP probes into living cells and form aggregates in the presence of miRNA-21, it was further researched if the AuNP aggregates could be used for photothermal therapies to kill tumor cells under NIR irradiation. MCF-7 cells were used to evaluate the therapeutic effect. After incubation with the binary system for 1.5 h, the cells were irradiated with a 680 nm laser at 0.5 W cm−2 at 37 °C. After different times of illumination, the cells were observed under microscope (Figure 3a). Before NIR treatment, the binary system transfected MCF-7 cells grew normally with a regular cell shape. After NIR irradiation, cell apoptosis gradually appeared. After 30 min of illumination, the apoptotic MCF7 cells ruptured, indicating the therapeutic ability of the binary system to kill tumor cells under NIR illumination. In addition, the NIR treated MCF-7 cells were stained by Annexin V-FITC/PI to visualize the cell death under fluorescent microscope. As shown in Figure 3b, the NIR treated cells displayed significant fluorescence of apoptotic features, indicating the cells were at the late-stage of apoptosis. In a control experiment, after the MCF-7 cells were incubated with the binary system for 2 h or treated with NIR irradiation
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Figure 3. (a) Microscopic images of MCF-7 cells transfected with the binary system for 2 h and then treated with NIR irradiation at 0.5 W cm-2 for different times. (b) Microscopic images of Annexin V-FITC/PI stained MCF-7 cells after NIR irradiation (from left to right: bright-field (BF), Annexin V-FITC/PI staining fluorescence, DFM, and overlapped area of BF and AuNP DFM dots).
for 30 min without transfection at 37 °C, both cell groups maintained a good shape and no apoptotic fluorescence was observed (Figure S10), indicating that both NIR irradiation and the binary system transfection were necessary to induce the cell apoptosis. Coupled plasma atomic emission spectroscopy (ICP-AES) was used to analyze the cellular uptake of the AuNP probes. The binary system treated MCF-7 cells showed increasing uptake amounts of probes with the incubation time (Figure S11) owning 6×103 probes per cell after 2 h incubation, verifying the trend observed by microscopic images in Figure 2a. In Vivo NIR Treatment in MCF-7 Tumor-Bearing Living Mice. Based on the cell NIR experiments, the designed binary system was further applied to monitor the NIR treatment effect in living mice. Female BALB/c nude mice (pathogen-free, 5-6 weeks) were used to establish the tumor model. MCF-7 tumor was implanted on the flank of the nude mic by subcutaneous injection of MCF-7 cells in logarithmic growth phase under anesthesia with isoflurane. Afterwards, the tumor bearing mice were intravenously injected with the solution of the binary system. The mice were sent for in vivo fluorescence imaging to assess the effect of the proposed binary system. As shown in Figure 4, the AuNP probes could efficiently reach the tumor region after 8 h post injection and retain in the tumor tissues even after 48 h (Figure 4a, tumor region indicated with arrows, and the enlarged images provided in Figure S12), indicating the favorable sustainability of the binary system in tumor. The distribution of AuNP probes in main organs and tumor after 8 h post injection also indicated the accumulation of probes in tumor, and a negligible amount of the probes in heart, lung, kidney, spleen and colon (Figure S13). The MCF-7 tumor bearing mice injected with the binary system were
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then irradiated with a NIR laser for 30 min at a power of 0.5 W cm−2 to perform the therapy and the mice without NIR treatment were used as control. The tumor status was monitored after the treatment, and the major organs and tumor tissues were collected after 7 days post NIR treatment for histological study. Negligible organ damage or tumor metastasis was observed from the H&E stained tissue slices. (Figure 4b), demonstrating the low side-effect of the designed therapy. Notably, prominent necrosis was observed on the tumor tissue slice, while the control sample showed no noticeable cell destruction (Figure 4c), indicating the significant therapeutic effect of the NIR therapy based on the
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proposed system. In addition, the tumor volumes of tumorbearing mice without or with NIR treatment were tracked and measured for 2 weeks to evaluate the therapeutic effect. As shown in Figure 4d, the tumor growth was significantly inhibited after the NIR therapy compared with the untreated sample, indicating the potential of the designed therapy for clinical application. CONCLUSIONS In summary, we have designed a binary system composed by a pair of functionalized AuNP probes to achieve specific recognition and imaging of intracellular miRNA-21 with the therapeutic effect for inducing cell apoptosis. The miRNAinduced fluorescence recovery and aggregation of AuNP probes can be used for not only in situ imaging and quantification of intracellular miRNA-21, but also for the following photothermal therapy under NIR irradiation. With the proposed NIR therapy, an efficient destruction of MCF-7 breast cancer cells has been successfully achieved, which provides an in vivo monitoring method for cancer therapy. In addition, the proposed system has been further applied in tumor-bearing mice, which exhibits significant therapeutic effect without observable side-effect, which has confirmed the practicality of the system. These impressive results pave the way for further studying the biological roles of miRNAs in cancer cells, and provide new insight into the design of smart system for intracellular miRNA imaging and targeted treatment against cancer with clinical potentials.
ASSOCIATED CONTENT 29
Supporting Information Supplementary methods; evaluation of MB assembled on AuNP probes; enhanced stability of MB-functionalized AuNP probes; specific aggregation of the binary system; kinetics of miRNA-21 triggered fluorescence recovery; demonstration of the miRNA-21 specific recognition of the binary system; cytotoxicity of the binary system; intracellular behavior of probe1/probe-2; cell apoptosis situation under single treatment of the binary system or NIR irradiation. This material is available free
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of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION 41 42
Corresponding Author
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Fax +86 021-64250032. E-mail:
[email protected].
4 Notes
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The authors declare no competing financial interests.
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ACKNOWLEDGMENT 48 49 51
50 Figure 4. (a) Real-time fluorescence images of MCF-7 tumor-bearing mouse after intravenous injection of the binary system for different times (Left: real photo of the tumor-bearing mouse). (b) H&E stained tissue sections from different organs of mice and (c) tumor tissue sections without and with NIR treatment after 7 days. (d) Change of tumor volume without and with NIR treatment for two weeks.
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This research was supported by the National Natural Science Foundation of China (21327807), the Science Fund for Creative Research Groups (21421004), the Programme of Introducing Talents of Discipline to Universities (B16017), the China Postdoctoral Science Foundation (2015M570335) and the State Key Laboratory of Analytical Chemistry for Life Science Open Foundation (SKLACLS1512).
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