Multifunctional Molecular Beacons Modified Gold Nanoparticle as

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Multifunctional Molecular Beacons Modified Gold Nanoparticle as Nanocarrier for Synergistic Inhibition and in Situ Imaging of Drug Resistant Related mRNAs in Living Cells Xiaoting Liu, Xiaowen Xu, Yi Zhou, Nan Zhang, and Wei Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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ACS Applied Materials & Interfaces

Multifunctional Molecular Beacons Modified Gold Nanoparticle as Nanocarrier for Synergistic Inhibition and in Situ Imaging of Drug Resistant Related mRNAs in Living Cells

Xiaoting Liu,† Xiaowen Xu, ‡ Yi Zhou,† Nan Zhang,*,§ and Wei Jiang*,†,‡

†Key

Laboratory of Chemical Biology (Ministry of Education), School of

Pharmaceutical Science, Shandong University, Ji’nan 250012, Shandong, P. R. China. ‡School

of Chemistry and Chemical Engineering, Shandong University, Ji’nan 250100,

Shandong, P. R. China. §Department

of Oncology, Jinan Central Hospital Affiliated to Shandong University,

250012 Ji’nan, Shandong, P. R. China

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ABSTRACT The overexpression of ATP-binding cassette (ABC) transporters is one of primary causes of drug resistance in cancer. Downregulating expression of these transporters by inhibiting the mRNA translation process is an effective approach to cope with this situation. Herein, multifunctional molecular beacons (MBs) modified gold nanoparticle (AuNP) as nanocarrier (MBs-AuNP) is developed for synergistic inhibition and in situ imaging of drug resistant related mRNAs in living cells. MBs-AuNP is composed of: (i) triple specially designed molecular beacons modified on the surface of AuNP, for binding drug resistant related mRNAs, loading doxorubicin (Dox) and reporting fluorescence signal; (ii) AuNP, for loading MBs, introducing them into cells and quenching their fluorescence. After uptake by cells, MBs-AuNP will hybridize with three different drug resistant related mRNAs (MDR1 mRNA, MRP1 mRNA, BCRP mRNA), respectively, which could inhibit their translation to decrease efflux protein expression and lead to AuNP-quenched fluorescence recovery for in situ imaging. qRTPCR and western blot results showed that drug resistant related mRNAs and efflux protein expression were both decreasing. Dox loaded MBs-AuNP performed higher suppression efficacy compared with free Dox against HepG2/ADR (0.35 μM vs 1.06 μM of IC50) and MCF-7/ADR (2.78 μM vs > 5 μM of IC50). Direct observation of intracellular hybridization events and distinguishment of drug resistant cancer cells or non-drug resistant cancer cells could be accomplished through fluorescent imaging analysis. This nanocarrier is capable of downregulating the expression of multiple efflux proteins by gene silencing and allows in situ monitoring of silencing events, and 2


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thus provides a powerful strategy to cope with drug resistance at the gene level.

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KEYWORDS: Nanocarrier; drug resistance; synergistic inhibition; antisense oligonucleotides; in situ imaging

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INTRODUCTION Chemotherapy is the most generally applied treatment of cancer.1 However, the emergence of resistance to chemotherapeutic drugs results in the failure of chemotherapy.2-3 Up-regulation of ATP-binding cassette (ABC) transporters is one of primary reasons of drug resistance. Three key efflux transporters engaged in drug resistance are multidrug resistance protein 1 (MDR1, permeability glycoprotein, P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) as well as multidrug resistance-associated protein 1 (MRP1 or ABCC1).4-6 Overexpressed efflux transporters pump out intracellular foreign drugs, thereby decreasing the concentration of chemotherapeutics in drug resistant tumor cells and eventually leading to a poor chemotherapeutic effect.7 It has been proved that efflux transporters-associated drug resistance could be overcome in some degree via raising the retention time of therapeutic agents in cells and circumventing the drugs excretion by kinds of drugloaded nanocarriers.8-9 Since the released drugs are remaining in cytosol and would be gradually pumped out of cells by active efflux transporters, the efficacy of drug-loaded nanocarriers is still unsatisfied.1, 7 In contrast, directly inhibiting expression of efflux transporters is a more fundamental strategy to reduce intracellular drug excretion and consequently improve sensitivity of chemotherapeutic agents in drug resistant cancer cells.10 Gene therapeutics, mainly including antisense oligonucleotides, microRNA as well as small interfering RNA (siRNAs), can be applied in reducing the expression level of drug resistant relevant transporters by gene silencing to reverse transporters-mediated 5



drug

resistance.11-14

Compared

with

microRNAs

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and

siRNAs,

antisense

oligonucleotides can directly exert effect to suppress the complementary singlestranded mRNA translation process without the formation of RNA-induce silencing complex (RISC).15-18 There are several attempts to utilize the antisense technique to inhibit the expression of single transporters for overcoming drug resistance.10,

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Nevertheless, resistance of cancer cells to chemotherapeutic agents is complicated and induced by various transporters. Once single transporter was suppressed and/or downregulated, another transporter may be upregulated.20-22 Therefore, a multitransporters simultaneously downregulating strategy may be more effective in combating drug resistance and enhancing chemotherapeutic efficacy. Several nanocarriers were developed to deliver antisense oligonucleotides for silencing intracellular drug resistance related mRNA, which could improve their transfection efficiency and intracellular stability.23 It is still difficult to determine their localization in living cells due to lack of appropriate detectable signal from these strategies. “Always on” fluorescence nanocarriers possessed detectable fluorescence signal to monitor bio-distribution of antisense oligonucleotides after delivered into cells.24-25 A major deficiency of “always on” strategies are that the interaction of antisense oligonucleotides with target mRNAs could not be directly observed. Visualization of silencing events is important because it provide a tool to evaluate the silencing efficacy. It is preferred to engineer activated fluorescence nanocarrier,26 which referred that fluorescence was turned on only when antisense oligonucleotides hybridized with target mRNAs, to achieve in situ observation of silencing events. In 6


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turn, fluorescence signal also reflects the expression level of target mRNAs and accordingly can be used to distinguish drug resistant or non-drug resistant cancer cells. Therefore, an all-in-one nanocarrier combined silencing of multiple drug resistant related mRNA with in situ monitoring of silencing events is highly desired for coping with drug resistance. Herein, multifunctional molecular beacons (MBs) modified gold nanoparticle (AuNP) as nanocarrier (MBs-AuNP) is developed for synergistic inhibition and in situ imaging of drug resistant related mRNAs in living cells. MBs-AuNP is consisted of a gold nanoparticle core and triple specially designed molecular beacons modified on AuNP with gold-thiol bond. After uptake by drug resistant cancer cells, MBs-AuNP will hybridize with three different drug resistant related mRNAs (MDR1 mRNA, MRP1 mRNA, BCRP mRNA), respectively, which could inhibit their translation process to decrease efflux protein expression and lead to AuNP-quenched fluorescence recovery for in situ imaging. qRT-PCR and western blot results showed that drug resistant related mRNAs and efflux protein expression were both decreasing. To further investigate resistance overcoming and sensitivity restoring activities of Dox-MBs-AuNP, proliferation and viability of drug resistant cancer cells were evaluated after different treatments. Dox-MBs-AuNP showed preferable cytotoxic efficacy compared with free Dox against HepG2/ADR (0.35 μM vs 1.06 μM of IC50) and MCF-7/ADR (2.78 μM vs > 5 μM of IC50). Direct observation of intracellular hybridization events and distinguishment of drug resistant cancer cells or sensitive cancer cells may be accomplished via fluorescent imaging analysis. This study provided a promising 7



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approach to synergistically reverse drug resistance via silencing multiple drug resistant related mRNAs as well as simultaneously achieve in situ imaging of silencing events, which emerged great values for biomedical applications.

Scheme 1. Schematic illustration of multifunctional molecular beacons modified gold nanoparticles as nanocarrier for synergistic inhibition and in situ imaging of drug resistant related mRNA in living cells. EXPERIMENTAL SECTION Chemicals and materials. MTT (Ultra-Pure Grade), trisodium citrate dihydrate (≥ 99.5%), DMSO (≥ 99.9%), TCEP (tris(2-carboxyethyl)phosphine, ≥ 98.0%) as well as HAuCl4 (≥ 99.9%) were bought from Sigma-Aldrich (Missouri, USA). Reduced glutathione (GSH, ≥ 98.0%) was acquired from Aladdin Industrial Corporation (Shanghai, China). 1,4-(D,L)-dithiothreitol (DTT, ≥ 97%) was provided by BBI Life Sciences Corporation (Shanghai, China). RPMI-1640 cell medium, phosphate buffer 8


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saline as well as fetal bovine serum (FBS) were brought from Biological Industries (Israel). Doxorubicin hydrochloride (Adriamycin, ≥ 99.0%) was purchased from Beijing Huafeng (Beijing, China). DNase I was bought from NEB Inc. (Massachusetts, USA). Ultrapure water to prepare all aqueous solutions, whose electronic conductivity was higher than 18.25 MΩcm-1. Chemical agents not mentioned were analytical reagents and commercially available. DNA sequences in the study were summarized in Table S1 and synthesized by Sangon Biological (Shanghai, China). Apparatus. UV-Vis absorption spectrum was measured by Hitachi U-2910 spectrometer (Japan). Hitachi F7000 fluorospectrophotometer (Japan) were applied in all fluorescent emission spectrum. Zetasizer Nano ZS90 (Malvern, UK) were used to record dynamic light scattering (DLS) results. HT7700 transmission electron microscope (Hitachi, Japan) was implemented for imaging of transmission electron microscope (TEM). Leica (63×) confocal laser scanning microscope (Germany) was employed to record confocal fluorescence images. Thermo Varioskan Plate Reader (USA) was used to record MTT results. K30 metal bath was purchased from Allsheng Instruments (China). Electrophoresis analysis. Native-PAGE electrophoresis was conducted to validate the assembly of molecular beacon (MDR1 MB, MRP1 MB and BCRP MB) and feasibility after incubated with accordingly mRNA targets (MDR1 mRNA target, MRP1 mRNA target and BCRP mRNA target). The electrophoresis was run on 10% native-PAGE in TBE buffer. The ethidium bromide was used to stained the gel for 5 min after electrophoresis and was photographed for the next step with Alpha Innotech 9



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digital imaging system (California, USA). Preparation of MBs-AuNP. 13 nm-AuNP was synthesized through citrate reduction method according to the reference.27 After 2 min stirring of 5 mL HAuCl4 (10 mM) solution and boiling ultrapure water (45 mL), 5 mL trisodium citrate dihydrate aqueous solution (38.8 mM) was poured into boiling solution immediately. Along with the step, faint yellow solution has transformed to colorless as well as ultimately to wine red color. After 10 min boiling, the colloid was removed from heating source and kept stirring to 25 ºC. The ready-made AuNP was filtered with 0.22 µm millipore and then preserved at 4 ºC. The final concentration of AuNPs was measured through UV-Vis method and calculated by absorption values (2.7 ×108 Lmol−1cm−1 of molar absorptivity at 519 nm). Size of ready-made AuNP was about 13 nm, which was determined by transmission electron microscope (TEM). To form the structure of molecular beacon, oligonucleotides sequences of MDR1 MB, MRP1 MB, BCRP MB were heated up to 95 ºC and kept for 5 min, then annealed to 25 ºC slowly. The mixture of AuNPs and thiolated MDR1 MB, MRP1 MB, BCRP MB (TCEP pretreated, in a 1:60:60:60) was stirred for 16 h. After that, 0.2 M phosphate buffer (pH = 7.4) was put into the mixture to reach the final concentration of 10 mM. Injection of NaCl solution (2 M) to the mixture was slowly accomplished in 8 h to meet the ultimate concentration of 0.15 M. After stirring 24 h, poly dA (TCEP pretreated) was added and then stirred over another 12 h. After centrifuged (25 min, 12000 rpm) of the ultimate solution, the supernatant was suck up carefully. To purify functionalized AuNPs for follow-up experiments, precipitate was redispersed in PBS (pH=7.4). Then 10


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this procedure was repeating for three times to remove all free molecular beacons and poly dA. Quantification of molecular beacons on each AuNP. Calculation of the number of each AuNP loaded molecular beacons was determined according to previous researches.28-29 In brief, 1M DTT solution was added into MBs-AuNP (6 nM) to meet 0.35 M of final concentration. The mixture was shaken at 25 ºC for 12 h to release MB completely. Quantification of the released MB was determined by scanning fluorescence spectrum of supernatant after centrifugation of the solution. As for Cy5 labeled MDR1 MB, excitation of fluorescence was set to 635 nm and maximum emission was recorded at 668 nm. Fluorescent intensity at 668 nm of different concentrations of Cy5 labeled MDR1 MB and their concentrations were plotted to a standard curve. Then molar concentrations of Cy5 labeled MDR1 MB was calculated by inserting the recorded fluorescent intensity at 668 nm to the standard curve. As for Cy3 labeled MRP1 MB, excitation of fluorescence was set to 525 nm and maximum emission was recorded at 563 nm. As for FAM labeled BCRP MB, maximum emission of fluorescence was recorded at 518 nm with 488 nm excitation. Concentrations of these two kinds of MBs were determined in the same way to Cy5 labeled MDR1 MB. Hybridization Experiment. For detection of multiplexed analytes, the three complementary targets were added into MBs-AuNP (5 nM). Fluorescence emission spectra of Cy5, Cy3 and FAM were scanned with fluorescence spectrometer after incubation at 37 ºC for 12 h. Stability of MBs-AuNP. DTT (0.35 M), DNase I (3.5 U/L), PBS or GSH (15 mM) 11



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was incubated with 5 nM MBs-AuNP, respectively. The fluorescent intensities of Cy5, Cy3 and FAM were measured every ten minutes in an hour after starting incubation. Cell lines and cell culture. MCF-7 (human breast adenocarcinoma) cells as well as HepG2 (human liver hepatocellular carcinoma) cells were acquired from Cell Bank of Chinese Academy of Sciences. Dox-resistant HepG2 cells (HepG2/ADR) were obtained from Shanghai Aiyan Biology. Dox-resistant MCF-7 cell line (MCF-7/ADR) was generously supplied by Prof. Xiuzhen Han from Shandong University. Cells culture were performed in a 37 °C atmosphere of 5% carbon dioxide. RPMI-1640 containing 10% FBS was selected for cell culture medium. Intracellular fluorescence imaging of MBs-AuNP. HepG2 or HepG2/ADR as well as MCF-7 or MCF-7/ADR were seeded in confocal dishes, respectively. MBs-AuNP (3 nM) were added after 24 h culturing, and incubated for 3 h. Cell imaging by confocal laser scanning microscope (CLSM) was then carried out. Analysis of relative expression level of MDR1 mRNA, MRP1 mRNA as well as BCRP mRNA. Total RNAs of HepG2 or HepG2/ADR as well as MCF-7 or MCF7/ADR cells were extracted by trizol reagent. cDNA was prepared by RevertAid Premium Reverse Transcriptase (Thermo Scientific™ EP0733). Primers of qRT-PCR analysis of mRNA were listed in Table S2 and housekeeping gene GAPDH was selected as normalized control. The qRT-PCR experiments were conducted on ABI Stepone plus. The cycling program was composed of heating to 95 °C and keeping for 3 min, then 45 cycles (95 °C for 3 s followed 60 °C for 30 s). Relative quantities of mRNAs were determined according to the literature.30 12


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Western blot. Cells were lysed by modified RIPA buffer after 48 h incubation with different concentration (1 nM, 2 nM, 3 nM) of MBs-AuNP. Western blot assay was conducted according to manufacturer protocols. The antibodies against GAPDH and Pgp were obtained from Beyotime Biotechnology (Nantong, China). The antibody against MRP1 was obtained from WanleiBio. The antibody against BCRP were purchased from Boster Biological Technology co. Ltd. Quantification of doxorubicin (Dox) loaded in each MBs-AuNP. The mixture of Dox (5 μM) with MBs-AuNP (6 nM), MDR1 MBs-AuNP (6 nM), MRP1 MBs-AuNP (6 nM), or BCRP MBs-AuNP (6 nM) was incubated for 2 h and then centrifugated at 4 °C (12000 rpm, 25 min). 505-650 nm emission spectrum of supernatants was measured by fluorescence spectrometer with exciting at 473 nm. The concentration of Dox inserted into particles could be calculated according to the calibration curve, which was plotting with fluorescent intensities and molar concentrations. Amount of each particle loaded Dox equaled quotient of the concentration of Dox inserted into particles and the particles concentration. Cell viability. HepG2 (3000 cells/well) or HepG2/ADR (8000 cells/well) as well as MCF-7 (3000 cells/well) or MCF-7/ADR (5000 cells/well) were seeded into 96-well transparent cell culture plates. After 24 h culture, different concentrations of Dox were added into the cells for 48 h. HepG2/ADR (8000 cells/well) cells were planted into 96well transparent cell culture plates for 24 h culture before treatments. Different concentrations of free Dox, MBs-AuNP and Dox-MBs-AuNP (all concentrations transferred to Dox) were added in the cells for 48 h, respectively. MCF-7/ADR (5000 13



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cells/well) were planted and cultured in 96-well transparent cell culture plates for 24 h, then treated with different concentrations of free Dox, MBs-AuNP and Dox-MBsAuNP (all concentrations transferred to Dox) for 48 h. Besides, HepG2/ADR (8000 cells/well) cells were treated with Dox, Dox-MBs-AuNP, Dox-MDR1 MBs-AuNP, Dox-MRP1 MBs-AuNP and Dox-BCRP MBs-AuNP (the loaded Dox was 0.5 μM for all particles) respectively for 48 h. MCF-7/ADR (5000 cells/well) cells were treated with Dox, Dox-MBs-AuNP, Dox-MDR1 MBs-AuNP, Dox-MRP1 MBs-AuNP and Dox-BCRP MBs-AuNP (the loaded Dox was 5 μM for all particles) respectively for 48 h. For the next step, media was abandoned and each well was washed by PBS and added 100 μL MTT solution for 4 h incubation. Discarded MTT solution after incubation, 100 μL DMSO was injected to dissolve purple formazan. Absorption at 570 nm were recorded by plate reader after shaking for 4 min. Statistical analysis. Values in this study were displayed as the format of mean ± standard deviation (SD) of three times of independent experiments. When P value was higher than 0.05, it was considered as no statistically difference. It was defined to “very significant difference” or “significant difference” and labelled as “**” or “*” respectively, when P value of student T-test was lower than 0.01 or 0.05. RESULTS AND DISCUSSION Electrophoresis characterization. Native-PAGE electrophoresis was performed to verify self-assembly of molecule beacons (MBs) and hybridization of MBs with specific mRNA targets. Figure S1 illustrated the native-PAGE analysis results, MBs (lane 1, lane 3 and lane 5) moved more quickly than the combinations of MBs and 14


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accordingly mRNA targets (lane 2, lane 4 and lane 6). In addition, the lanes were clear. The results indicated that MBs were successfully assembled and can realize the recognition of mRNA targets. Preparation and characterization of MBs-AuNP. Preparation and characterization of the MBs-AuNP nanocarrier for MDR1 mRNA, MRP1 mRNA, BCRP mRNA binding, imaging and drug delivery was illustrated in Figure 1. The average diameter and morphology of AuNPs were determined through TEM images (Figure 1A). After modified with MBs, MBs-AuNP performed superior dispersibility (Figure 1B), which could more effectively prevent the aggregation of AuNP. The hydrodynamic diameter was growing when AuNP was modified with MBs, from naked AuNP (21.01±1.29 nm) to MBs-AuNP (35.95±2.31 nm) based on dynamic light scattering (DLS) data (Figure 1C). According to UV-Vis spectra, the maximum absorption was red shifted from 519 nm to 523 nm after modified with MBs, which further proved that the surface of AuNPs has successfully modified MBs (Figure 1D).

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Figure 1 TEM images for (A) AuNPs and (B) MBs-AuNP. (C) Dynamic light scattering measurements for MBs-AuNP and AuNPs. (D) UV-Vis spectra for MBs-AuNP and AuNPs. Quantification of triple MBs on each AuNP. To estimate amounts of triple molecular beacons linked to the surface of AuNP, standard calibration curves of Cy5labeled MDR1 MBs (Figure S2A), Cy3-labeled MRP1 MBs (Figure S2C) and FAMlabeled BCRP MBs (Figure S2E) were plotted. MBs-AuNP (Figure S2B, S2D, S2F, black line) was treated with DTT (Figure S2B, S2D, S2F, blue line), which was identified as a strong reductant, for departing MBs completely from the surface of AuNP. The molar concentrations of MBs were then calculated according to the fluorescence intensity of the DTT treated MBs-AuNP and the standard calibration curves. Approximate 21 Cy5-labeled MDR1 MBs were modified on one AuNP. Approximate 19 Cy3-labeled MRP1 MBs were modified on one AuNP. Approximate 17 FAM-labeled BCRP MBs were modified on one AuNP. In vitro study of MBs-AuNP. To evaluate the properties of MBs-AuNP for the simultaneous detection of three mRNA targets, hybridization experiments were conducted. As shown in Figure S3, it was confirmed that fluorescence signal was produced when each kind of MB was specifically hybridized with its own mRNA target. Since nonspecific degradation or destroy of MBs-AuNP in living cells also generating false-positive signals, it was vital to verify the tolerance of intracellular enzymes and reducing agents. DNase I and glutathione (GSH) were put into MBs-AuNP separately, and low fluorescence was obtained (Figure S4, blue and grey line). The data manifested 16


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that MBs-AuNP had high tolerance to enzymes and endogenous reducing agents. But it should be noted that the MBs modified on AuNPs could be released by exogenous strong reducing agent DTT, producing strong fluorescence signal (Figure S4, orange line). Intracellular imaging. MBs-AuNP was then used to simultaneously image triple mRNAs in a couple of liver cancer cell lines, HepG2 as well as doxorubicin (Dox) resistant HepG2/ADR. When treated HepG2/ADR cells with MBs-AuNP, a strong red fluorescent signal of MDR1 mRNA, a strong yellow fluorescent signal of MRP1 mRNA, and a strong green fluorescent signal of BCRP mRNA (Figure 2) were recorded with CLSM. Nevertheless, three fluorescence signals were all very weak after HepG2 cells treated with MBs-AuNP, suggesting that the MBs-AuNP can be employed to distinguish drug resistant cancer cells and sensitive cancer cells. qRT-PCR results indicated that the relative expression levels of the triple mRNAs of HepG2/ADR were all higher, when compared with those of HepG2 cells. The results were coincident with the fluorescence imaging results by CLSM and further revealed that the fluorescence signals of MBs-AuNP had a good correlation with the expression levels of mRNA (Figure 2B, 2C, 2D). These drug resistant related mRNAs were co-overexpressed in HepG2/ADR cells. Correlation between the drug resistant related mRNA expression level and the cellular response to doxorubicin (Dox) was further investigated by MTT assay, and IC50 were calculated. IC50 values of HepG2/ADR cells (1.06 μM) was higher than that of HepG2 cells (0.30 μM), further confirming that there emerged Dox resistance in HepG2/ADR (Figure S5). These results were in accordance with 17



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fluorescence confocal imaging and qRT-PCR results. Another couple of cells, MCF-7 and Dox resistant MCF-7/ADR, CLSM imaging was also applied to estimate MBsAuNP for simultaneously imaging triple mRNAs. After MCF-7/ADR being treated with MBs-AuNP, strong yellow and red fluorescent signals of MRP1 mRNA as well as MDR1 mRNA were obtained under CLSM (Figure 2A). The results suggested that MRP1 mRNA as well as MDR1 mRNA were overexpressed in MCF-7/ADR. While the green fluorescence signal for BCRP mRNA was very faint, showing that the expression level of BCRP mRNA was low. When MCF-7 were treated with the MBsAuNP, all triple fluorescence signals were very dim under the same conditions. The qRT-PCR data further testified that the relative levels of MRP1 mRNA as well as MDR1 mRNA of MCF-7/ADR were both higher, when compared with those in MCF7, while levels of BCRP mRNA were both low in MCF-7/ADR and MCF-7 (Figure 2B, 2C, 2D). IC50 of MCF-7/ADR (10.52 μM) was higher than that of MCF-7 (0.55 μM), confirming that MCF-7/ADR were resistant to Dox (Figure S6). MBs-AuNP could direct observe the interaction of MBs-AuNP and related mRNAs and utilize intracellular mRNA imaging to distinguish drug resistant cancer cells and sensitive cancer cells.

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Figure 2 (A) Intracellular imaging of MDR1 mRNA, MRP1 mRNA and BCRP mRNA in HepG2/ADR, HepG2, MCF-7 and MCF-7/ADR cells under CLSM. Red fluorescence was arisen from Cy5. Yellow fluorescence was arisen from Cy3. Green fluorescence was arisen from FAM. The relative level of (B) MDR1 mRNA, (C) MRP1 mRNA and (D) BCRP mRNA by qRT-PCR. Suppression of drug resistant related mRNA and efflux protein in MBs-AuNPtreated drug resistance cancer cells. Numerous experiments manifested that antisense oligonucleotides can specifically hybridize with related mRNA and then efficiently 19



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inhibit their translation process when properly delivered. Hence, we explored the intracellular silence efficacy of MBs-AuNP in HepG2/ADR as well as MCF-7/ADR via qRT-PCR. According to Figure 3A, levels of three kinds of mRNA of HepG2/ADR cells were all declined, when incubated with MBs-AuNP. As shown in Figure 3B, levels of three kinds of mRNA of MCF-7/ADR were all lowered, when incubated with MBsAuNP. The expression of efflux protein was further investigated by western blotting analysis, and it was shown in Figure 3C and 3D that three kinds of efflux protein were all decreased in HepG2/ADR as well as MCF-7/ADR. All results undoubtedly verified that MBs-AuNP could specifically and efficiently induce downregulation of intracellular drug resistant related mRNA and then decrease levels of efflux transporters in drug resistant cancer cells consequently. Therefore, MBs-AuNP is valuable for the intracellular drug resistant related mRNA binding and imaging-guided mRNA regulation in living cells.

Figure 3 The relative level of MDR1 mRNA, MRP1 mRNA and BCRP mRNA in (A) 20


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HepG2/ADR and (B) MCF-7/ADR cells treated with (grey) and without (black) MBsAuNP by qRT-PCR (*p < 0.05, **p < 0.01, n = 3); Western blotting analyses of (C) HepG2/ADR and (D) MCF-7/ADR cells, GAPDH was internal control. Dox-loading capacity of MBs-AuNP, MDR1 MBs-AuNP, MRP1 MBs-AuNP and BCRP MBs-AuNP. By taking MBs-AuNP, MDR1 MBs-AuNP, MRP1 MBs-AuNP and BCRP MBs-AuNP as nanocarrier, Dox could intercalate into base pairs of double strand DNA of MBs-AuNP (Dox-MBs-AuNP), MDR1 MBs-AuNP (Dox-MDR1 MBsAuNP), MRP1 MBs-AuNP (Dox-MRP1 MBs-AuNP) and BCRP MBs-AuNP (DoxBCRP MBs-AuNP). When Dox intercalate into the stem of molecular beacons, its fluorescence will be quenched. The standard curve of Dox was established (Figure S7) to determine the absolute amount of Dox intercalated into one MBs-AuNP, MDR1 MBs-AuNP, MRP1 MBs-AuNP or BCRP MBs-AuNP. According to decline of fluorescence intensity after incubation with different nanocarriers and standard calibration curve (Figure S8), amounts of Dox loaded in one MBs-AuNP, MDR1 MBsAuNP, MRP1 MBs-AuNP or BCRP MBs-AuNP can be calculated. The results performed that there was about 330 Dox molecules inserted into one MBs-AuNP, MDR1 MBs-AuNP, MRP1 MBs-AuNP or BCRP MBs-AuNP. Cell viability. To further verify that Dox-MBs-AuNP can reverse drug resistance to enhance chemotherapeutic effect as we expected, HepG2/ADR and MCF-7/ADR were utilized for estimating the efficacy of Dox-MBs-AuNP on cell viability. As demonstrated in Figure 4, we compared the cell viabilities of free Dox-treated, DoxMBs-AuNP-treated and MBs-AuNP-treated drug resistant cancer cells utilizing MTT 21



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assay. MBs-AuNP had comparatively low cytotoxicity against HepG2/ADR as well as MCF-7/ADR. As Figure 4A illustrated, proliferation of HepG2/ADR was suppressed by free Dox and Dox-MBs-AuNP in a dose-dependent manner. Compared with free Dox (IC50 value of 1.06 μM), Dox-MBs-AuNP showed better antiproliferative activities (IC50 value of 0.35 μM). Free Dox performed weak antiproliferative effect against HepG2/ADR. It might owe to up-regulated efflux proteins on cell membrane, which was able to immediately pump Dox out of cells. But Dox-MBs-AuNP showed higher inhibitory effect (P < 0.05) than free Dox. Dox-MBs-AuNP could specifically recognize MDR1 mRNA, MRP1 mRNA and BCRP mRNA. The translation process of mRNA can be inhibited, and then the expression of efflux protein would be downregulation. Above mentioned results verified that this MBs-AuNP could greatly increase the chemotherapeutic efficiency on HepG2/ADR cells. As Figure 4B illustrated, Dox-MBs-AuNP exhibited better performance against for MCF-7/ADR than free Dox (2.78 μM vs > 5.00 μM of IC50 values, P < 0.05).

Figure 4 In vitro cytotoxicity of MBs-AuNP, Dox-MBs-AuNP and Dox toward (A) HepG2/ADR and (B) MCF-7/ADR cells (*p < 0.05, **p < 0.01, n = 3). The synergistic effect of the effective suppression of three kinds of efflux proteins by 22


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Dox-MBs-AuNP and the capacity of this nanocarrier to bypass drug efflux restores sensitivity of drug-resistant cancer cells to the released Dox. Single kind of molecular beacons modified AuNPs loaded Dox (Dox-MDR1 MBs-AuNP, Dox-MRP1 MBsAuNP and Dox-BCRP MBs-AuNP), which could only specific bind with single kind of mRNA and downregulate single kind of efflux protein, were constructed as controls. As shown in Figure 5, Dox-MBs-AuNP performed superior suppression efficacy than Dox-MDR1 MBs-AuNP, Dox-MRP1 MBs-AuNP and Dox-BCRP MBs-AuNP (P < 0.05) against HepG2/ADR as well as MCF-7/ADR cells, which referred a high intracellular concentration of Dox as well as a remarkable anticancer performance. Above mentioned results suggested this nanocarrier (MBs-AuNP) had the capacities of synergistical effect in drug-resistant cancer cells.

Figure 5 In vitro cytotoxicity of Dox-MBs-AuNP, Dox-MDR1 MBs-AuNP, DoxMRP1 MBs-AuNP, Dox-BCRP MBs-AuNP and Dox toward (A) HepG2/ADR and (B) MCF-7/ADR cells (*p < 0.05, n = 3). CONCLUSION In this paper, multifunctional molecular beacons modified AuNP as nanocarrier for in 23



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situ imaging and synergistic inhibition of drug resistant related mRNA in living cells. MBs-AuNP facilitates intracellular drug resistant related mRNA imaging to distinguish drug resistant cancer cells and sensitive cancer cells. MBs-AuNP, as antisense agent, was able to effectively downregulate drug resistant related mRNAs and efflux proteins expression and to reverse drug resistance in HepG2/ADR and MCF-7/ADR cells. DoxMBs-AuNP displayed a remarkably enhanced cytotoxicity and performed efficient synergistic efficiency in drug resistant cancer cells, verifying the potent of this method to combat drug resistance and increase therapeutic efficacy. This nanocarrier combined downregulating the expression of multiple efflux proteins by gene silencing with in situ monitoring of silencing events, and was thus an effective strategy to cope with drug resistance at the gene level. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Native-PAGE analysis; quantification of triple MBs on one AuNP; specific recognition of MBs-AuNP to triple mRNA targets; stability of MBs-AuNP after treated with DNase Ⅰ, GSH, PBS and DTT; cell survival rate of HepG2 and HepG2/ADR after incubated with doxorubicin (Dox); cell survival rate of MCF-7 and MCF-7/ADR after incubated with Dox; standard calibration curves of Dox; drug loading experiments of MBs-AuNP, MDR1 MBs-AuNP, MRP1 MBs-AuNP and BCRP MBs-AuNP; DNA sequences; qRT-PCR primer sequences (PDF) Author Information

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Corresponding Author * E-mail: wjiang-sdu.edu.cn. Fax: +86-531-88564464. Tel: +86-531-88363888. * E-mail: [email protected]. Notes The authors declare no conflict of interest. Acknowledgements This work was funded by the National Natural Science Foundation of China (Nos. 21675100, 21675101, 21705094, and 81502525), Major Project of Science and Technology of Shandong Province (2017G006037). References (1) Pan, L.; Liu, J.; He, Q.; Wang, L.; Shi, J. Overcoming Multidrug Resistance of Cancer Cells by Direct Intranuclear Drug Delivery Using TAT-Conjugated Mesoporous Silica Nanoparticles. Biomaterials 2013, 34 (11), 2719-2730. (2) Tu, Z.; Qiao, H.; Yan, Y.; Guday, G.; Chen, W.; Adeli, M.; Haag, R. Directed Graphene-Based Nanoplatforms for Hyperthermia: Overcoming Multiple Drug Resistance. Angew. Chem., Int. Ed. 2018, 57 (35), 11198-11202. (3) Schinkel, A. H.; Jonker, J. W. Mammalian Drug Efflux Transporters of the ATP Binding Cassette (ABC) Family: an Overview. Adv. Drug Deliver. Rev. 2012, 64, 138153. (4) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. 25



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Table of Contents Graphic

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Multifunctional Molecular Beacons Modified Gold Nanoparticle as

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