Endogenous Stimuli-Responsive Nucleus-Targeted Nanocarrier for

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Biological and Medical Applications of Materials and Interfaces

Endogenous Stimuli-Responsive Nucleus-Targeted Nanocarrier for Intracellular mRNA Imaging and Drug Delivery Xiaoting Liu, Lei Wang, Xiaowen Xu, Haiyan Zhao, and Wei Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16345 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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ACS Applied Materials & Interfaces

Endogenous Stimuli-Responsive Nucleus-Targeted Nanocarrier for Intracellular mRNA Imaging and Drug Delivery Xiaoting Liu,† Lei Wang,† Xiaowen Xu,‡ Haiyan Zhao,‡ Wei Jiang*,†,‡

†Key

Laboratory of Chemical Biology, Ministry of Education, School of Pharmacy,

Shandong University, Jinan 250012, P. R. China. ‡Key

Laboratory of Colloid and Interface Chemistry, Ministry of Education, School of

Chemistry and Chemical Engineering, Shandong University, Jinan 250100, P. R. China.

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ABSTRACT Drug resistance arisen from overexpressed efflux transporters increases the efflux of drugs and accordingly restricts the efficacy of chemotherapy. Advances in nanocarriers have provided potential strategies to cope with drug resistance. Herein, endogenous stimuli-responsive nucleus-targeted nanocarrier is developed for intracellular multidrug resistance protein 1 (MRP1) mRNA imaging and drug delivery. This nanocarrier (AuNPmRS-DSs) is composed of three parts: (i) gold nanoparticle (AuNP), for loading DNA and quenching fluorescence; (ii) mRNA recognition sequence (mRS) modified on the surface of gold nanoparticle by gold-thiol bond, for the specific recognition of MRP1 mRNA; (iii) detachable subunit (DS), hybridized with Cy5 labeled DNA linker and nucleolin recognition motif, and grafted onto mRS via the DNA linker, for loading doxorubicin (Dox), binding to nucleolin and reporting signal. Firstly, nucleolin recognition motif of this nanocarrier targets nucleolin, which is overexpressed on cancer cells surface, and after that the whole nanocarrier enters the cell via nucleolin-mediated internalization. Subsequently, mRS will specifically recognize overexpressed MRP1 mRNA, leading to the release of trapped DS and followed by AuNP-quenched Cy5 fluorescence recovery. Finally, by translocation of nucleolin from cytoplasm to nucleus, DS targets nucleus to delivery Dox. By intracellular fluorescence imaging, the differentiation of drug-resistant and non-drug2


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resistant cells could be achieved. Compared with free Dox (IC50 > 8.00 μM), Dox-loaded AuNP-mRS-DSs (IC50 = 2.20 μM) performed superior suppression efficacy towards drugresistant cancer cells. Such a nanocarrier provides an effective strategy to synergistically sense and circumvent drug resistance, which may be exploited as a candidate for personalized medicine.

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KEYWORDS: Nanocarrier, stimuli responsive, nucleus-targeted, MRP1 mRNA imaging, drug delivery

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INTRODUCTION Chemotherapy is one of the most typical means for cancer therapy,1 but its efficacy is usually significantly restricted by emergence of drug resistance.2,3 Drug resistance is often arisen from active efflux of drugs by overexpression of efflux transporters, decreasing drug concentration in cells to levels under the lethal threshold and producing limited therapeutic potency.4 Therefore, it turns into a considerable strategy to bypass the efflux transporters-mediated drug efflux for overcoming drug resistance. Studies have indicated that nanoparticles might escape from the efflux transporters pathway and these nanoparticles could serve as drug carriers to counteract the efflux effects by delivering a high drug concentration to drug-resistant cells.5,6 Nevertheless, drugs delivered by these nanocarriers remaining in cytosol would re-expose to active efflux transporters and be pumped out of cells, leading to unsatisfied chemotherapy efficacy.7 5



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Acting as cellular control center, nucleus is the ultimate targeting site of plentiful firstline chemotherapeutic agents, such as doxorubicin (Dox).8 Hence, a nuclear-uptake nanocarrier that can release chemotherapeutic agents inside nucleus would exceedingly circumvent the efflux transporters-mediated drug resistance and strengthen therapeutic efficacy.9-11 Successfully delivering nanocarriers to nucleus is quite challenged by biobarriers.12,13 Owning to nucleopore restriction (< 9 nm), the nanocarriers have to be small enough to pass through nucleopore into nucleus.14,15 Small size gold nanoparticles (2 nm and 6 nm) were able to get into nucleus, in contrast, large size gold nanoparticles (10 nm and 16 nm) were found merely in cytoplasm.16 After internalizing into cancer cell cytoplasm, the passive nucleus transport of nanocarriers remains unsatisfied, whereas conjugation of nuclear targeting aptamer or other ligands, which facilitates active transport of nanocarriers to deliver drug intranuclearly, performed more effective accumulation.17 Such small nanocarriers, which guaranteed nuclear penetration, would be excreted rapidly by the kidney system from blood stream, resulting in limited tumor accumulation.18,19 On the contrary, large size nanocarriers (50-200 nm) are capable of concentrating at leaky regions of tumor vasculature spontaneously by passive targeting, but are unable to internalize into nucleus.20 Hence, a size-tunable nanocarrier with nucleus-targeted properties are required to break down these bio-barriers and ensure effective intranuclear drug delivery. To obtain size-tunable function, one large nanocarrier should be designed to release 6


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smaller subunits after internalized into tumor cells. Several nanocarriers have been developed to release their small size subunits after stimulated by an exogenous stimulus, such as near-infrared, but they were restricted to apply only in local treatment.21 In systemic treatment, it is critical to treat the metastases, which lead the major cause of cancer mortality.22 Furthermore, it is incapable to ensure that subunits of large nanocarriers would be released inside cells under exogenous stimuli. To guarantee both systemic treatment effects and intracellular release of subunits, the size change triggered by applying an endogenous stimulus from tumor tissue itself is preferred. Tumor markers, such as aberrantly expressed nucleic acids and proteins, possess high cancer cell or tissue specificity and act as an indicator of cancer initiation, development and metastasis.23-25 As an efflux transporter, multidrug resistance protein 1 (MRP1), is overexpressed in many chemotherapeutic drug resistant cell lines and translated by MRP1 mRNA, is often related to resistance of many first-line chemotherapeutic drugs.26 Level of MRP1 mRNA is closely correlated with the progression of drug resistance in prostatic carcinoma, nonsmall-cell lung cancer (NSCLC), breast cancer and so on.27 Antisense oligonucleotide28 (anti-MRP1 mRNA) is not only a potential agent for recognizing target MRP1 mRNA, but also a gatekeeper to control the release of small size subunits. Herein, endogenous stimuli-responsive nucleus-targeted nanocarrier (AuNP-mRS-DSs) is developed for intracellular MRP1 mRNA imaging and drug delivery. The nanocarrier is composed of three parts: (i) gold nanoparticle (AuNP), for loading DNA and fluorescence 7



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quenching; (ii) mRNA recognition sequence (mRS) modified on the surface of gold nanoparticle by gold-thiol bond, for the specific recognition of MRP1 mRNA; (iii) detachable subunit (DS), hybridized with Cy5 labeled DNA linker and nucleolin recognition motif (containing AS1411 aptamer29) and grafted onto mRS via the DNA linker, for loading Dox, binding to nucleolin and reporting signal. Firstly, nucleolin recognition motif of this nanocarrier targets nucleolin, which is overexpressed on cancer cells surface, and after that the whole nanocarrier enters the cell via nucleolin-mediated internalization. Subsequently, mRS will specifically recognize overexpressed MRP1 mRNA, leading to the release of trapped DS and followed by AuNP-quenched Cy5 fluorescence recovery. Finally, by translocation of nucleolin from cytoplasm to nucleus,17,30 DS targets nucleus to delivery Dox. Intracellular fluorescence imaging could achieve the differentiation of drug-resistant and non-drug-resistant cells. Besides, the distribution of released detachable subunits in drug-resistant cells were able to be visualized via fluorescence imaging. IC50 of Dox-loaded AuNP-mRS-DSs against MCF7/ADR cells was 2.20 μM, much lower than free Dox (IC50 > 8.00 μM), and it signified that the nanocarrier performed superior suppression efficacy towards drug-resistant breast cancer cells. This nanocarrier had great capacities of chemotherapy sensitivity prediction and drug resistance circumvention in cancer treatment, and it could be developed as a potent candidate for individualized medicine.

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Scheme 1. Schematic illustration of endogenous stimuli-responsive nucleus-targeted nanocarrier for intracellular mRNA imaging and drug delivery. EXPERIMENTAL SECTION Reagents and materials. DNA oligonucleotides in this study were provided by Sangon Biological (Shanghai, China). The sequences were listed in Table S1. Chloroauric acid (HAuCl4, ≥ 99.9%), DMSO (≥ 99.9%), sodium citrate tribasic dihydrate (≥ 99.5%), TCEP (≥ 98.0%) as well as MTT (Ultra-Pure Grade) were purchased from Sigma-Aldrich Company (St. Louis, Mo, USA). DL-dithiothreitol (DTT, molecular biology grade) was acquired from BBI Life Sciences (Shanghai, China). Glutathione (GSH, biotech grade) was obtained from Aladdin (Shanghai, China). DNase I was purchased from New England Biolabs (Ipswich, MA, USA). Cell medium RPMI 1640 and phosphate buffer saline (PBS) was provided by BI (Israel). Doxorubicin (> 99.0%) was provided by Huafeng Co., Ltd (Beijing, China). M-MLV reverse transcriptase kit was purchased from the Invitrogen by Life Technologies (USA). Hoechst 33342 was provided by Beyotime Biotechnology 9



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(Nantong, China). FastStart Universal SYBR Green Master (ROX) was acquired from Roche Applied Science (USA). Fetal bovine serum (FBS) as well as trypsin were purchased from Solarbio Life Science (Beijing, China). All other aqueous solutions were produced with ultrapure water (> 18.25 MΩcm-1), which was manufactured by Millipore Milli-Q water purification systems. All other reagents (analytical pure) were provided by standard reagent suppliers. Apparatus. All fluorescent emission spectra were scanned with Hitachi F-7000 fluorescence spectrometer (Japan). The UV-vis absorption spectra were scanned with Hitachi U-2910 spectrometer (Japan). Dynamic light scattering (DLS) results were obtained from Zetasizer Nano ZS (Malvern, UK). Transmission electron microscope (TEM) images were recorded by JEM-1011 transmission electron microscope (JEOL, Japan). The confocal fluorescence images were recorded with Olympus IX-81 (60×) confocal laser scanning microscope (Tokyo, Japan). K30 metal bath was provided by Allsheng Instruments (China). The fluorescence images were recorded with Axio Observer microscope (Carl Zeiss, Germany). Total gold content was analyzed by ICP-AES (Thermo, IRIS Advantage). MTT assay was measured by ThermoFisher Scientific Multiskan GO (USA). Electrophoresis analysis. To verify the assembly of mRS-DS and feasibility after treated with MRP1 mRNA target and nonsense sequence, a native-PAGE (nondenaturing polyacrylamide gel electrophoresis) experiment was performed. The electrophoresis was 10


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run on 10% native-PAGE with constant 30 mA in TAE-Mg buffer. After electrophoresis, ethidium bromide was applied to stain this gel with for 5 min, and next, photographed under Alpha Innotech Alpha Imager (San Leandro, USA). Synthesis of AuNP-mRS-DSs. Preparation of 20 nm in diameter gold nanoparticles (AuNPs) were performed by citrate reduction method.31 1.25 mL HAuCl4 (10 mM) solution was poured rapidly to a boiling ultrapure water (48.75 mL) with stirring. After 2 min, 0.875 mL sodium citrate tribasic dihydrate (1%) was put in the boiling solution immediately. Then this solution transformed from faint yellow to colorless and eventually to burgundy. The colloid was keeping stirred to room temperature after 10 min boiling and removing the heating source. The solution was stored at 4 ºC after filtered by a 0.22 µm millipore filter. The concentrations of AuNPs were calculated by their OD values at 450 nm (ε = 5.41 ×108 Lmol−1cm−1).32 Transmission electron microscope (TEM) images exhibited that size of the particles was 20.57±1.54 nm (40 particles sampled). AuNPs was mixed with thiolated mRNA recognition sequence (mRS, TCEP pretreatment) in a 1:1000 molar ratio, then shaken for 16 h. Subsequently, phosphate buffer (pH = 7.4; 0.2 M) was put into the mixture to a final concentration of 0.01 M. Sodium chloride solution (2 M) was injected gradually over eight hours to a final concentration of 0.15 M. After that, the solution was shaken over a period of 24 h. Finally, the resulting solution containing functionalized particles was centrifuged at 12,000 rpm for 25 min. And then precipitate were redisposed in phosphate buffered saline (pH=7.4) to produce the purified 11



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mRS functionalized AuNPs for subsequent experiments. mRS functionalized AuNPs were mixed with DNA linker and nucleolin recognition motif in a 1:500:500 molar ratio. Then this solution was heated to 85 ºC, kept for 10 min and cooled to room temperature gradually. Shake at room temperature for additional 24 h to make hybridization completely. To purify the AuNP-mRS-DSs and remove unhybridized sequences, the AuNP-mRS-DSs mixtures were centrifuged at 12,000 rpm for 25 min, and without disturbing the kermesinus pellet of gold nanoparticles, the supernatant was suck up. Then AuNP-mRS-DSs were resuspended in PBS. Repeating this process for three times resulted in the removal of all unhybridized complementary sequences. Quantification of mRS-DS on one AuNP. The number of mRS-DS, which loaded on single AuNP were calculated by the previous protocol.23 Briefly speaking, DTT solution (1 M) was added to AuNP-mRS-DSs solution (1 nM) to reach a final concentration of 0.35 M. The mRS-DS was released completely after 12 h incubation with stirring at room temperature. The released mRS-DS was separated from AuNP via centrifugation, and then the fluorescence spectra of supernatant were scanned with fluorescence spectrometer. The fluorescence of Cy5 labeled mRS-DS was excited at 635 nm and recorded with 655-850 nm. The maximum fluorescence intensity was at 668 nm and transferred to molar concentrations of mRS-DS by insertion from a standard linear calibration curve, which was calculated by fluorescence intensity and different concentrations of Cy5 labeled DNA linker. 12


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Specificity of fluorescent recovery. DNase I (3.5 U/L), DTT (0.35 M), GSH (15 mM) MRP1 mRNA (500 nM) or PBS was added to AuNP-mRS-DSs (1 nM) and then mixed fully. The fluorescence emission spectra of Cy5 were measured after 12 hours. Stability of AuNP-mRS-DSs. Freshly prepared AuNP-mRS-DSs (1 nM) was added to an equal volume of PBS, medium RPMI 1640 (containing 10% FBS). The fluorescence emission spectra of Cy5 were recorded with an excitation wavelength of 635 nm after incubation for 24 h. Hybridization experiment. For analyte detection, the AuNP-mRS-DSs (1 nM) was incubated with the TK1 mRNA target (200 nM), c-myc mRNA target (200 nM), MRP1 mRNA target (200 nM) and nonsense sequence target (200 nM). The fluorescence intensity of Cy5 was measured by fluorescence spectrometer (λex = 635 nm, λem = 668 nm) after incubation for 2 h at 37 °C. Cell lines and cell culture. HL-7702 (Normal human liver cells), HepG2 (hepatocellular cancer cells), MCF-7 and MDA-MB-231 (human breast cancer cells) were obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences. MCF-7/ADR (Dox-resistant MCR-7 cells) was generously provided by Prof. Yunxue Zhao from Shandong University School of Basic Medical Sciences. All cells were cultured in cell medium RPMI 1640 containing 10% FBS and maintained in a 5% CO2 atmosphere environment at 37 °C. Intracellular imaging of AuNP-mRS-DSs. MCF-7/ADR, MCF-7, HepG2 and MDA13



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MB-231 cells were planted into confocal dishes and cultured for 24 h. 100 μL AuNP-mRSDSs (1.5 nM) were cultured in confocal dishes for 2 h and then prepared for confocal laser scanning microscope (CLSM) imaging. MCF-7/ADR cells were seeded into confocal dishes for 24 h. 100 μL AuNP-mRS-DSs (2 nM) or AuNP-mRS-cDSs (2 nM) were added into confocal dishes, respectively. After 8 h incubation, the samples were subsequently stained by Hoechst 33342 for 20 min. After washed by PBS for three times, they were prepared for fluorescent imaging. Detection of MRP1 mRNA expression by quantitative real-time PCR (qRT-PCR). Total RNAs were extracted from MCF-7/ADR cells, MCF-7 cells, HepG2 cells as well as MDA-MB-231 cells with trizol reagent (Invitrogen), respectively. RNA concentration and quality were determined by spectrometric measurement of the A260/A280 ratio. cDNA was synthesized by the M-MLV reverse transcriptase kit (Invitrogen). qRT-PCR analyses of MRP1 were performed using the primer in Table S2 and normalized to housekeeping gene GAPDH. In particular, the qRT-PCR was conducted on qTOWER 2.0 (Jena, Germany). The cycling program consisted of 55 cycles (10 min at 95 °C; 55 cycles at 95 °C for 15 s and 60 °C for 60 s). The relative level of MRP1 mRNA was calculated by 2-ΔΔCt method.33 Primers of qRT-PCR were as shown in Table S2. Cellular Uptake. MCF-7/ADR cells and HL-7702 cells were planted with density of 150000 cells/well in RPMI 1640 with 10% FBS. After 24 h, AuNP-mRS-DSs (1 nM) were incubated with two types of cells for 7 hours. And AuNP-mRS-cDSs (1 nM) were added 14


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to incubate with MCF-7/ADR cells. After incubation, the cells were gently washed by PBS buffer for three times and digested to remove cells. Then the cells were counted by a Qiujing cell counter (Shanghai, China) and treated with 200 μL of aqua regia (3:1 hydrochloric acid/nitric acid). After overnight incubation, every sample was diluted with ultrapure water. The total gold content of samples was analyzed by ICP-AES (Thermo, IRIS advantage). According to the previous protocols,34,35 the number of nanocarriers within single cell was quantified by ICP-AES. Quantification of doxorubicin (Dox) loaded on one AuNP-mRS-DSs. Dox (10 μM) incubated with AuNP-mRS-DSs solution (2 nM) or AuNP-mRS-cDSs solution (2 nM) for 3 h, the mixture was separated via centrifugation (12000 rpm, 25 min, 4 °C). Fluorescence spectra of supernatants were recorded with fluorescence spectrometer (λex = 480 nm, λem = 505-700 nm). A standard linear calibration curve of fluorescence intensities and concentrations of Dox was generated under the identical condition. The molar concentration of Dox was converted from the fluorescence intensity and divided by the AuNP-mRS-DSs concentration. Measurement of cell viability. HL-7702 (~4000 cells/well) and MCF-7 (~4000 cells/well) cells were plated in 96-well plates and cultured for 24 h. The cells were incubated with Dox, AuNP-mRS-DSs, Dox@AuNP-mRS-DSs and Dox@AuNP-mRScDSs (the loaded Dox were both 4 μM) for 24 h after removed the original medium. MCF7/ADR (~4000 cells/well) cells were plated in 96-well plates and cultured for 24 h, and 15



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then the cells were incubated with various concentrations of Dox, AuNP-mRS-DSs, Dox@AuNP-mRS-DSs and Dox@AuNP-mRS-cDSs (the volume of loaded Dox were both 0.1 μM, 1 μM, 2 μM, 4 μM, 8 μM) for 24 h. Next, the cells were washed gently by PBS for three times. 100 μL of MTT solution (0.5 mg/mL in RPMI 1640) was injected to each well. Solutions of each well were discarded after 4 h incubation and 150 μL/well DMSO was added to resolve purple substance. OD values were recorded at 570 nm after four-minute shaking. Statistical analysis. All data expressed as mean ± standard error was results of three independent experiments. When P value was under 0.01 or 0.05, it was regarded as significant statistical difference and symbolled with ** or *, separately. By the time P value was higher than 0.05, it could be regarded as no statistically different. RESULTS AND DISCUSSION Electrophoresis characterization the assembly of mRS-DS. mRS-DS DNA nanostructure was composed of three DNA sequences. To validate the self-assembly of mRS-DS and the feasibility of design, before linking to AuNPs, the product was verified by polyacrylamide gel electrophoresis (PAGE). As shown in Fig. S1, three DNA sequences combinations (lane 3) moved more slowly than two DNA sequences combinations (lane 1 and lane 2) lacking one DNA sequence. Furthermore, clearness of the lanes confirmed the successful assembly of mRS-DS. Lane 4 and 5 indicated that mRS-DS could recognize MRP1 mRNA target specifically rather than that nonsense sequence. 16


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Synthesis and characterization of AuNP-mRS-DSs. The AuNP-mRS-DSs nanocarriers for MRP1 mRNA detection and targeted drug delivery were successful prepared and characterized (Fig. 1). TEM images exhibited that average diameters of gold nanoparticles were approximately 20.57±1.54 nm (Fig. 1A). After modified with DNA structure, AuNPmRS-DSs nanocarriers showed higher dispersibility, which could prevent the aggregation of gold nanoparticle effectively (Fig. 1B). The hydrodynamic diameter of AuNP-mRS-DSs was larger than naked AuNP according to dynamic light scattering (DLS) results, indicating that diameter of AuNPs increased when surface modified with DNA structure (Fig. 1C). Unlike 524 nm absorption peak of AuNPs spectrum, the UV-vis maximum absorption of the AuNP-mRS-DSs red-shifted to 528 nm (Fig. 1D), which further confirmed the successful modification of the AuNPs by DNA structure.

Fig. 1 The characterization of AuNPs and AuNP-mRS-DSs. (A) TEM image of AuNPs. (B) TEM image of AuNP-mRS-DSs. (C) DLS characterization of AuNPs and AuNP-mRS17



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DSs. (D) UV-vis spectra of AuNPs and AuNP-mRS-DSs. Quantification of mRS-DS on one AuNP. A standard linear calibration curve of Cy5labeled DNA linker (Fig. 2A) was established to evaluate the number of mRS-DS modified on the AuNP surface. AuNP-mRS-DSs (Fig. 2B, purple line) was treated with the strong reductant DTT (Fig. 2B, orange line) for the complete release of mRS-DSs from the AuNPs. Then the molar concentrations were calculated from fluorescence of mRS-DS by the standard linear calibration curve derived from various concentrations of Cy5 labeled DNA linker. Approximate 63 mRS-DSs were conjugated to one AuNP. The nonspecific degradation and interactions in living cells could generate false-positive signals from the AuNP-mRS-DSs. To investigate the stability of AuNP-mRS-DSs resisting intracellular reductants and enzymes, GSH and DNase I were added to AuNP-mRS-DSs respectively, and almost no fluorescent signal was observed (Fig. 2B, black line and gray line). These results indicated that this nanocarrier possessed high resistance to nuclease and reductants. Furthermore, AuNP-mRS-DSs could specifically recognize MRP1 mRNA target (Fig. 2B, green line), producing fluorescence signal.

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Fig. 2 (A) Standard linear calibration curves of Cy5-labeled DNA linker. (B) Fluorescence spectra of AuNP-mRS-DSs (1 nM) after incubation with PBS, GSH, DTT, DNase I and MRP1 mRNA target, respectively. Stability and specificity of AuNP-mRS-DSs. The stabilities of AuNP-mRS-DSs were also investigated in different media, such as PBS, RPMI 1640 with 10% FBS (Fig. S2). Collectively, these results indicated structural stability of AuNP-mRS-DSs. This is a fundamental prerequisite for cellular applications. One of the significant advantages of 19



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AuNP-mRS-DSs is that they have high selectivity to DNA targets. In this study, MRP1 mRNA target, cmyc mRNA target, TK1 mRNA target and nonsense sequence were used to investigate the selectivity of AuNP-mRS-DSs. As shown in Fig. S3, for MRP1 mRNA target, obvious fluorescence signal was detected. However, for the other three targets, almost no fluorescence signal was produced. Thus, AuNP-mRS-DSs showed high sequence selectivity. Intracellular imaging of AuNP-mRS-DSs. The ability of the AuNP-mRS-DSs to independently detect MRP1 mRNA was then studied in living cells. To investigate the specificity of this nanocarrier in living cells, we detected MRP1 mRNA in MCF-7/ADR cells, MCF-7 cells, HepG2 cells and MDA-MB-231 cells, respectively. As shown in Fig. 3A, in MCF-7/ADR cells, which overexpressed MRP1 mRNA in the cytoplasm, the brightest fluorescence was imaged. Nevertheless, dim fluorescence signal was observed in MRP1 mRNA low expressed MCF-7 cells, HepG2 cells as well as MDA-MB-231 cells. The results of qRT-PCR revealed the MRP1 mRNA relative level of MCF-7/ADR cells was much higher than other three cells (Fig. 3B). The confocal imaging results were consistent with the MRP1 mRNA expression level in these cells measured by qRT-PCR. These results indicated that AuNP-mRS-DSs was capable of detecting MRP1 mRNA expression in different cells and facilitated intracellular MRP1 mRNA imaging to differentiate drug-resistant and non-drug-resistant cells.

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Fig. 3 (A) CLSM images of MRP1 mRNA in MCF-7/ADR, MCF-7, HepG2 and MDAMB-231 cells. The red fluorescence was produced by Cy5. The scale bar represents 20 μm. (B) Normalized histogram of the relative abundance of MRP1 mRNA by qRT-PCR. To achieve active tumor targeting, AS1411, an aptamer that can specifically bind to nucleolin overexpressed on tumor cell membrane, was used as the targeting ligand of AuNP-mRS-DSs and the sequence of AS1411 aptamer was contained in nucleolin recognition motif. Besides, nucleolin is capable of translocating between the cell surface and nucleus for its bidirectional nucleus localization sequence. To design control nanocarrier AuNP-mRS-cDSs, AS1411 aptamer sequence was replaced by MUC1 aptamer 21



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sequence.36 MUC1 aptamer could specifically recognize MUC1 protein overexpressed on the cytomembrane of most adenocarcinomas, such as breast cancer cells.37 Compared with AuNP-mRS-DSs, AuNP-mRS-cDSs could only target cytomembrane rather than nucleus. After internalized into drug-resistant cells, AuNP-mRS-DSs or AuNP-mRS-cDSs could specifically recognize MRP1 mRNA, then release small size detachable subunit (DS) or control detachable subunit (cDS). Tracing activated Cy5 fluorescence (red) displayed following path of DSs or cDSs. The intracellular localization of DSs or cDSs were visualized by fluorescence imaging (Fig. 4). It was found that the accumulation of DSs in the nucleus was higher than cDSs, indicating that the conjugation of AS1411 aptamer could facilitate DS into the nuclei to delivery doxorubicin (Dox).

Fig. 4 Fluorescence images of MCF-7/ADR cells incubated with AuNP-mRS-DSs and AuNP-mRS-cDSs. The red fluorescence was produced by Cy5. The scale bar represents 20 μm. Cellular Uptake. To evaluate targeting ability of AuNP-mRS-DSs in living cells, HL7702 cells and MCF-7/ADR cells were used. Nucleolin was overexpressed on the membrane of MCF-7/ADR cells rather than normal hepatocyte line (HL-7702). The 22


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intracellular uptake quantities of AuNP-mRS-DSs in the two cells were determined by an approach based on ICP-AES. As Fig. S4 showed, AuNP-mRS-DSs were able to be uptaken by both these two cells, and the quantity of AuNP-mRS-DSs uptake in a single MCF7/ADR cell was much larger than that in a single HL-7702 cell, which is consistent with overexpression of nucleolin on membrane of MCF-7/ADR cells rather than HL-7702 cells. Furthermore, nucleolin and MUC1 protein were all overexpressed on the membrane of MCF-7/ADR cells. To study the uptake capacity of MCF-7/ADR cells to AuNP-mRS-DSs and AuNP-mRS-cDSs, intracellular uptake quantities of these two nanocarriers in single MCF-7/ADR cell were determined by ICP-AES. The results indicated that there was almost no difference between the uptake amounts of these two nanocarriers by MCF7/ADR cells (Fig. S5). Dox-loading capacities of AuNP-mRS-DSs and AuNP-mRS-cDSs. By taking AuNPmRS-DSs and AuNP-mRS-cDSs as nanocarriers, Dox was able to insert into the DNA base pairs of AuNP-mRS-DSs (Dox@AuNP-mRS-DSs) and AuNP-mRS-cDSs (Dox@AuNPmRS-cDSs). After intercalating into DNA base pairs, the fluorescence of Dox would be quenched.23 To measure the quantity of Dox loaded in single AuNP-mRS-DSs or AuNPmRS-cDSs, we established the standard linear calibration curve of free Dox (Fig. S6). According to fluorescence intensity of Dox after incubation with different concentrations of nanocarriers (0 nM, 2 nM) and standard linear calibration (Fig. S7A and Fig. S7B), the number of Dox loaded in single AuNP-mRS-DSs or AuNP-mRS-cDSs can be calculated. 23



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The results showed that there were approximate 1092 Dox molecules intercalated into single AuNP-mRS-DSs nanocarrier and approximate 1134 Dox molecules intercalated into single AuNP-mRS-cDSs. Cell viability. To examine the tumor inhibition efficacy of Dox@AuNP-mRS-DSs, the MTT assay was performed on MCF-7/ADR cells, HL-7702 cells and MCF-7 cells in vitro. As demonstrated in Fig. 5, about 90% cells were alive when AuNP-mRS-DSs were incubated with the HL-7702 cells. Moreover, compared with free Dox (4 μM) group, Dox@AuNP-mRS-DSs (P < 0.01) and Dox@AuNP-mRS-cDSs (P < 0.01), of which loaded Dox concentrations were both 4 μM, possessed lower cytotoxicity to normal liver HL-7702 cells. This indicated that these two nanocarriers met the requirements of reducing unexpected side effect of free Dox to normal cells. This may be due to the low expression of nucleolin and MUC1 protein on the HL-7702 cell membrane and low expression of MRP1 mRNA in HL-7702 cell. Furthermore, the cell viability of MCF-7 cells incubated with Dox@AuNP-mRS-DSs, Dox@AuNP-mRS-cDSs and free Dox was similar, decreasing to about 42.0%, 44.4% and 33.8%, with even higher efficacy in the sample with free Dox. This may be due to the low expression of MRP1 mRNA in cytoplasm that results in limited release of DS to target nucleus to play antitumor role. As demonstrated in Fig. 6, the growth of MCF-7/ADR cells was inhibited by Dox@AuNP-mRS-DSs and Dox@AuNP-mRS-cDSs in a dose-dependent manner. Dox@AuNP-mRS-DSs had an IC50 of 2.20 μM, which was lower than Dox@AuNP-mRS-cDSs (6.02 μM) and free Dox (> 24


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8.00 μM). Free Dox showed poor tumor suppression efficacy for MCF-7/ADR cells. This may be due to overexpressed drug efflux transporters on the cytomembrane, which can directly expel the free drug from the cells. AuNP-mRS-DSs or AuNP-mRS-cDSs could specifically recognize MRP1 mRNA, then release DS or cDS. Dox@AuNP-mRS-cDSs performed superior suppression efficacy than free Dox (P < 0.05). AuNP-mRS-cDSs could avoid efflux effect to some degree. Dox@AuNP-mRS-DSs performed superior inhibitory efficacy compared with Dox@AuNP-mRS-cDSs (P < 0.01). Owning to that cDS accumulated mainly in the cytoplasm, drugs released from drug delivery system would be re-exposed to efflux transporters. DS could specifically bind nucleolin to effectively target nucleus owning to the conjugated AS1411 aptamer, then release Dox into final target location, nucleus, to inhibit the proliferation of tumor cells. These results demonstrated that this AuNP-mRS-DSs could greatly enhance the therapeutic efficacy on Dox-resistant MCF-7 cells.

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Fig. 5 MTT assay. Cell viability of HL-7702 and MCF-7 cells with different treatments. (n = 3, **p < 0.01).

Fig. 6 MTT assay. Cell viability of MCF-7/ADR cells with different treatments. (n = 3, **p < 0.01, 0.01 ≤ *p < 0.05). CONCLUSION In this paper, an endogenous stimuli-responsive nucleus-targeted nanocarrier loading doxorubicin (Dox) was developed for intracellular mRNA imaging and drug delivery. AuNP-mRS-DSs facilitates intracellular MRP1 mRNA imaging to differentiate drugresistant and non-drug-resistant cells. Besides, the distribution of released detachable subunits in drug-resistant cells could be visualized by fluorescence imaging. Dox loading experiments manifested that each AuNP-mRS-DSs could be inserted by about 1092 Dox molecules. IC50 of Dox-loaded AuNP-mRS-DSs against MCF-7/ADR cells was 2.20 μM, 26


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much lower than free Dox (>8.00 μM), and the cytotoxicity assay confirmed that Doxloaded AuNP-mRS-DSs performed superior suppression efficacy towards drug-resistant cells. This nanocarrier had great latent capacities to sense and circumvent drug resistance in a synergistic way, and it can be exploited as a potent candidate for personalized medicine.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Oligonucleotides sequences (Table S1); PCR primer sequences (Table S2); gel electrophoresis characterization the assembly of mRS-DS (Figure S1); stability of AuNP-mRS-DSs in PBS and 1640 medium (10% FBS) (Figure S2); specificity of AuNP-mRS-DSs to different mRNA target (Figure S3); quantification of internalized AuNP-mRS-DSs in per MCF-7/ADR or HL-7702 cell by ICP-AES (Figure S4); quantification of two nanocarriers in per MCF-7/ADR cell by ICP-AES (Figure S5); standard linear calibration curves of doxorubicin (Figure S6); Dox loading capacities of AuNP-mRS-DSs (Figure S7A) and AuNP-mRS-cDSs (Figure S7B).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: +86-531-88363888. Fax: +86-531-88564464.

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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21475077, 21675100, 21675101, and 21705094). REFERENCES (1) Pan, W.; Yang, H.; Zhang, T.; Li, Y.; Li, N.; Tang, B. Dual-Targeted Nanocarrier Based on Cell Surface Receptor and Intracellular mRNA: an Effective Strategy for Cancer Cell Imaging and Therapy. Anal. Chem. 2013, 85, 6930-6935. (2) Li, Z.; Wang, H.; Chen, Y.; Wang, Y.; Li, H.; Han, H.; Chen, T.; Jin, Q.; Ji, J. pH- and NIR Light-Responsive Polymeric Prodrug Micelles for Hyperthermia-Assisted SiteSpecific Chemotherapy to Reverse Drug Resistance in Cancer Treatment. Small 2016, 12, 2731-2740. (3) Li, W.; Zhang, H.; Assaraf, Y. G.; Zhao, K.; Xu, X.; Xie, J.; Yang, D. H.; Chen Z. S. Overcoming ABC Transporter-Mediated Multidrug Resistance: Molecular Mechanisms and Novel Therapeutic Drug Strategies. Drug Resist. Update. 2016, 27, 14-29. (4) Zhang, R.; Gao, S.; Wang, Z.; Han, D.; Liu, L.; Ma, Q.; Tan, W.; Tian, J.; Chen, X. Multifunctional Molecular Beacon Micelles for Intracellular mRNA Imaging and Synergistic Therapy in Multidrug-Resistant Cancer Cells. Adv. Funct. Mater. 2017, 27, 28


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Endogenous Stimuli-Responsive Nucleus-Targeted Nanocarrier for

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