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Optimized ultrasound conditions for enhanced sensitivity of molecular beacons in the detection of MDR1 mRNA in living cells Qiumei Zhou, Yi Ma, Zhaohui Wang, Ke Wang, Ruonan Liu, Zhihao Han, Min Zhang, Siwen Li, and Yue-Qing Gu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04503 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 9, 2016
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Analytical Chemistry
Optimized ultrasound conditions for enhanced sensitivity of molecular beacons in the detection of MDR1 mRNA in living cells Qiumei Zhou‡, Yi Ma‡, Zhaohui Wang, Ke Wang, Ruonan Liu, Zhihao Han, Min Zhang, Siwen Li, and Yueqing Gu* State Key Laboratory of Natural Medicines, Department of Biomedical Engineering, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China ABSTRACT: P-glycoprotein (P-gp), aprognostic indicator for chemotherapy failure, is encoded by multidrug resistance gene (MDR1). MDR1 mRNA expression could serve as a guidance for the personalized medicine. However, the traditional PCR process for mRNA measurement is complicated and cannot realize the real-time detection of mRNA in living single cells. In this work, optimized gold nanoparticle-based molecular beacons were employed to determine MDR1 mRNA levels in living cancer cells. To improve detection sensitivity, ultrasound (US) irradiation was applied to facilitate and enhance cellular uptake of hairpin DNAcoated gold nanoparticle (hDAuNP). The US conditions including irradiation power, exposure time, duty cycle and incubation time were optimized. The slight difference in MDR1 expression manipulated by siRNA silence could be recognized by US assisted hDAuNP beacons, a 10-fold increase of detection sensitivity was achieved compared with the non-ultrasound assistance. Meanwhile, the detection cycle could be shortened from 12 hours to 2 hours. Furthermore, this hDAuNP beacon can serve as an antisense agent to down-regulate P-gp expression and to reverse drug resistance of MCF-7/Adr cells to doxorubicin. Our results demonstrated that MDR1 hDAuNP beacon assisted by US irradiation had great potential to predict chemotherapy sensitivity and to overcome multidrug resistance in cancer cells, and was thus a promising tool for individualized medicine.
Cancer is a leading cause of death throughout the world, with 8.2 million deaths from cancer in 2012 alone. The World Health Organization (WHO) projects that the annual number of new cancer cases will increase, on average, by 8 million within the next two decades.1 Despite the great advances in cancer treatment in recent years, development of resistance to anticancer drugs results in failure many types of chemotherapy. The efficacy of chemotherapy is substantially limited by either intrinsic or acquired gene mutations or over-expression of specific proteins. In the majority of these cases, the tumorspecific mutations or abnormally expressed proteins are potent biomarkers of cellular responses to targeted chemotherapy. Therefore, gaining insight into gene expression is critical for accurate predictions of drug susceptibility in patients. The Garnettet and Barretina groups reported a systematic approach to identify predictive biomarkers of tumor response by highthroughput profiling with detailed genetic annotation of targeted agents against hundreds of human cancer cell lines.2, 3 MDR1 has previously been identified as a reliable biomarker for therapeutic efficacy prediction of the widely used anticancer drug doxorubicin (DOX) and other drugs, the substrates of p-glycoprotein (P-gp).4 P-gp, encoded by the MDR class of genes, was the first human ABC transporter to be identified as reported by Juliano et al.5 P-gp confers drug resistance by pumping a variety of drugs out of tumor cells at the expense of ATP hydrolysis.6 Failure of chemotherapy in clinic is often associated with P-gp overexpression in various hematological malignancies as well as in solid tumors.7 Levels of MDR1 mRNA expression are crucial for predicting patient response to chemotherapy drugs and for
further guiding personalized anti-cancer therapies.8 Currently, the most commonly used techniques for mRNA detection include in situ hybridization (ISH),9 polymerase chain reaction (PCR) 10 and northern blot (NB) analysis.11 However, these single-point and end-point techniques have several limitations, including the requirement for cell lysis, elimination of contaminating nucleases and inability to capture the dynamic expression of mRNA in real time with high precision. Duo to the degradation of target gene mRNA by cell lysis and RNA purification protocols, there has been growing interest in the development of probes for real-time imaging of mRNA expression in situ in living cells. Molecular beacons, comprised of antisense oligonucleotide labeled with a ‘reporter’ fluorophore at one end and a quencher at the other end, 12 are being increasingly adopted as promising tools for the detection and analysis of molecular targets in live cells. Over the past decade, a variety of molecular beacons have been developed, including semiconductor quantum dots,13 magneto fluorescent nanoparticles,14 and gold nanoparticles.15, 16 Among these beacons, gold nanoparticles (AuNPs), which are bio-inert, nontoxic, and have a high quenching efficiency, have garnered the most attention in molecular beacon applications. Based on fluorescence resonance energy transfer energy (FRET) principles, gold nanoparticles coupled with hairpin DNA sequences (hDAuNP beacons) possess numerous attractive properties, including enhanced nucleic acid binding, resistance to degradation, and the ability to enter cells without the need for transfection agents. Our previous reports also demonstrated the ability of hDAuNP beacons for qualitative detection of mRNA expression in live cells.16 Moreover, sev-
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eral groups have recently attempted to define the gene silencing ability of molecular beacons, which may be used as antisense oligonucleotide to down-regulate mRNA expression of target genes.18, 19, 20 Collectively, hDAuNP beacons are ideal ‘theranoutic’ agents, combining intracellular mRNA regulation with real-time detection. The nanoparticulate hDAuNP beacon is typically endocytosed by living cells and does not require the use of transfection agents. The beacon then accumulates in cytoplasm and emits fluorescence upon hybridization with target mRNAs.16 However, our previous study indicated that limited beacons entered cells, resulting in relatively low detection sensitivity. Further, it takes approximately 12 hours for the hDAuNP beacon to enter the cytoplasm, which necessitates longer detection cycles.15, 17 To circumvent these drawbacks, a nondestructive, rapid and high-efficient delivery method of hDAuNP beacons to living cells is urgently needed to detect mRNA with high sensitivity. Several physical methods, including ultrasound (US), 21, 22 electroporation, 23 and gene gun24 have been reported to facilitate the uptake of different nanopartilces into cancer cells. Among these physical transfection methods, US is the easiest way for facilitating cellular uptake of nanoparticles without expensive instruments. Meanwhile, it is comparatively less invasive and is not damaging to cells. However, no study to date has systemically investigated the effects of US on the uptake of DNA conjugated gold beacons into the cells and its detection sensitivity. In this study, we synthesized and optimized hDAuNP beacons for detection of MDR1 mRNA and explored its regulation in cancer cells. US was applied to enhance cellular uptake of hDAuNP beacons. Optimized US conditions including irradiation intensity, exposure time, duty cycles and incubation time were determined, and the detection sensitivity of hDAuNP beacons for MDR1 mRNA was investigated. MDR1 mRNA levels in different cancer cell lines were examined by using the optimized hDAuNP beacon method and its reliability was evaluated by qRT-PCR. Subsequently, the gene regulation by hDAuNP beacons was further investigated. Experimental Section Materials and Reagents. Chloroauric acid (HAuCl4) was obtained from the Shanghai Chemical Reagent Corporation (Shanghai, China). Dithiothreitol (DTT), thiol-polyethylene glycol 2000 (thiol-PEG2000) were purchased from SigmaAldrich (Shanghai, China). MDR1 siRNAs were obtained from Biomic Biotechnologies Co., Ltd. (Nantong, China). The beacon and DNA sequences were purchased from Sangon Biotech (Shanghai) Co., Ltd. All chemicals and solvents were of analytical grade purity. All solutions were prepared using deionized water (Milli-Q grade, Millipore) with a resistivity of 18.2 MΩ cm. The human cell lines L-02 (liver cell line), U87MG (glioblastoma), HepG2 (liver cancer), MCF-7 (breast cancer) and HCT-116 (colon carcinoma) were all purchased from ATCC. DOX-resistant MCF-7/Adr cells and K562/Adr cells were obtained from the Institute of Hematology and Blood Diseases Hospital (Tianjin, China), and their resistance to DOX was confirmed. Synthesis and optimization of hDAuNP beacons. AuNPs with an average diameter of 15 nm were prepared as described by our previous reports.17 AuNPs were first functionalized with thiol-PEG. The MDR1 mRNA sequences were then con-
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jugated to the PEG-modified AuNPs by thiol. The hairpin DNA loaded on the surface of AuNP was quantified as previously reported.25 The following oligonucleotide sequences were used: Beacon sequences: 5’-(FITC)-CTCGCACCTATAGCCCCTTTAACTTG GCGAGAAAAA AAAAA-SH-3’
Target DNA sequences: 5’-CAAGTTAAAGGGGCTATAGGT-3’ Mismatched DNA sequences: 5’-CgAtTTAAcGGaGGcATAGcT-3’ hDAuNP beacon characterization. The size distribution and morphology of AuNPs were analyzed measured using a Mastersizer 2000 Laser Particle Size Analyzer (LPSA) and a transmission electron microscope (200kV, JEM-2100, JEOL, Japan), respectively. To determine the optimized number of DNA sequence coated on AuNPs, different AuNPs-DNA ratios of 1:20, 1:40, 1:60 and 1:80 were prepared and incubated with MCF-7/Adr cancer cells, and the respective fluorescence signals were compared. Subsequently, extracellular hybridization with increasing concentrations of hDAuNP beacon (0.25, 0.50, 0.75, 1.25 and 1.5 nM) was performed. The fluorescence of the target DNA solution (60 nM) was measured using a fluorescence spectrophotometer. The stability of hDAuNP beacons was investigated. DNase I (0.38 mg/L), reduced GSH (10 mM), and DTT (0.35 M) were added to hDAuNP beacon (1 nM) solution. The fluorescence intensity of samples was monitored for 60 min. US Exposure. US irradiation intensity, exposure time and duty cycles were optimized using a modified Sonifier cell disrupter (650-92, BioSafer, Nanjing) equipped with a US generator, transducers, and a glass water tank. The water tank was filled with degassed water and maintained at 37°C. MCF7/Adr cells were seeded at 3 ×105/cm2 in confocal Petri dishes and subsequently maintained overnight at 37°C/5% CO2. The hDAuNP beacon (1 nM) was added to each well and US irradiation was carried out at intensities of 0, 100, 200, 300, 400 and 500 W. MCF-7/Adr cells were then incubated for an additional 2 hours in a 5% CO2 incubator. The effect of US intensity was investigated using Laser Confocal Fuorescence Microscopy (FV1000, Olympus, Japan) and quantified using flow cytometry on a BD FACSCanto. Optimal US irradiation intensity was determined based on differences in intracellular fluorescence signals. In addition, viability of MCF-7/Adr cells 24 hours after irradiation with different US powers was determined by MTT assay. Follow the same procedures as described above, optimal US irradiation duty cycles (DC) and exposure time were investigated. The ripple effect on the surface of the cell culture media was not observed during the US treatment. Each experiment was performed in triplicate. To determine the optimal incubation time, hDauNP beacons (1 nM) were added to cells, which were subjected to US irradiation at 300W and 40% DC for 4 min. Cells were then incubated with hDAuNP beacons for 0, 0.5, 1, 2, 4, 6 or 12 hours in a humidified 5% CO2 incubator. MCF-7/Adr cells were washed three times to remove unbound beacons, and fixed with 4% formalin for 15 min at 4°C. Finally, cover slips were immobilized onto slides for hyperspectral microscopy (CytoViva, Inc., Alabama) analysis. Dark-field optical images of hDAuNP beacons were obtained using a CCD camera (Olympus Microsystems, BX-53, Japan). Further, intracellular fluorescence signals analysis with time lapse was performed using confocal laser scanning microscopy (CLSM).
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Detection of MDR1 mRNA in living cells. To modulate MDR1 expression levels, MDR1 siRNA was transfected into MCF-7/Adr cells with a final siRNA concentration of 0, 20, 30, 50, or 100 nM using Lipofectamine 2000 (Invitrogen, Shanghai). Cells were further incubated for 24 hours, treated with 1 nM hDAuNP beacon, and irradiated with US. Intracellular fluorescent signals were monitored by flow cytometric analysis. Real-time quantitative PCR (qRT-PCR) was used to quantitate the relative expression levels of MDR1 mRNA. Equivalent numbers of HepG2, U87MG, HCT-116, MCF-7, K562 /Adr and MCF-7/Adr cells were seeded into confocal Petri dishes and incubated overnight at 37°C. Cells were then treated with hDAuNP beacons and US. After incubation for additional 2 hours, the intracellular fluorescence was visualized using CLSM and quantitated with flow cytometric analysis. Further, qRT-PCR was used to quantitate the expression levels of MDR1 mRNA, and MTT assay was used to determine the IC50. All assays were performed in triplicate. Down-regulation of P-gp enhances chemosensitivity of MCF-7/Adr cells. To investigate the gene silencing potential of the MDR1 hDAuNP beacon (1nM). qRT-PCR and Western blot analysis were performed on MCF-7/Adr cells transfected with MDR1 siRNA (60 nM) or treated with hDAuNP beacons and US. To investigate the DOX accumulation in MCF-7/Adr cells, the hDAuNP beacons and US were used. After a 48-hour culture, cells were treated with DOX (50 µM), for an additional 2 hours. CLSM and flow cytometry were used to monitor the fluorescent signal of DOX. To investigate the chemo-sensitivity of MCF-7/Adr cells to DOX, MDR1 siRNA and siRNA-NC transfaction were performed using Lipofectamine 2000, and the MDR1-targeted beacon (1 nM) and control beacon (STAT5B hDAuNP beacon, 1 nM) were added and transfected via US treatment. After 48 hours in culture, DOX (0.125 µM to 800 µM) was added and incubated for an additional 48 hours. The response of MCF-7/Adr cells to DOX was evaluated using the MTT assay. Statistical analysis. All values are reported as the mean ± standard deviation. Statistical analysis was performed using student's t-test. Statistical significance was assigned for Pvalues
