DNA-Based Nanomedicine with Targeting and Enhancement of

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Biological and Medical Applications of Materials and Interfaces

A DNA-based Nanomedicine with Targeting and Enhance Therapeutic Efficacy of Breast Cancer Cells Yuxi Zhan, Wenjuan Ma, Yuxin Zhang, Chenchen Mao, Xiao-Ru Shao, Xueping Xie, Fei Wang, Xiaoguo Liu, Qian Li, and Yunfeng Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03449 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Applied Materials & Interfaces

A DNA-based Nanomedicine with Targeting and Enhance Therapeutic Efficacy of Breast Cancer Cells Yuxi Zhan1, Wenjuan Ma1, Yuxin Zhang1, Chenchen Mao1, Xiaoru Shao1, Xueping Xie1, Fei Wang2, Xiaoguo Liu2, Qian Li2, Yunfeng Lin1, * 1. State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, P. R. China 2. School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China

*Corresponding Author: Yunfeng Lin

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, P. R. China

Tel: 86-28-85503487; Fax: 86-28-85503487

Email address: [email protected]

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ABSTRACT

Recently, a DNA tetrahedron has been reported to be a novel nanomedicine and a promising drug vector   because of its compactness, biocompatibility, biosafety, and editability. Here, we modified the DNA tetrahedron with a DNA aptamer (AS1411) as a DNA-based delivery system, which could bind to nucleolin for its cancer cell selectivity. Nucleolin is a specific biomarker protein overexpressed on membranes of malignant cancer cells and its deregulation is implicated in cell proliferation. The antimetabolite drug 5-fluorouracil (5-FU) is an extensively used anticancer agent, however, its major limitation is the lack of target specificity. Cyanine 5 (Cy5), a fluorescent probe, can be used to label DNA tetrahedron and enhance photostability with minimal effects on its basic functions. In this study, we additionally attached 5-FU to the DNA-based delivery system as a new tumor-targeting nanomedicine (AS1411-T-5-FU) to enhance the therapeutic efficacy and targeting of breast cancer. We examined the difference of cellular uptake of AS1411-T5-FU between breast cancer cells and normal breast cells and concluded that AS1411T-5-FU had a better targeting ability to kill breast cancer cells than 5-FU. We further


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evaluated the expressions of cell apoptosis-related proteins and genes, which are associated with the mitochondrial apoptotic pathway. Ultimately, our results suggest the potential of DNA tetrahedron in cancer therapies and we develop a novel approach to endow 5-FU with targeting property.

KEYWORDS: 5-FU, AS1411, DNA tetrahedron, cell apoptosis, breast cancer.


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INTRODUCTION Breast cancer is a severe health issue globally, accounting for one-fourth of cancer cases with over 1.65 million newly diagnosed patients and approximately 522,000 related deaths per year.1,

2

Owing to the poor therapeutic effects of traditional medicine

formulations, majority of patients suffering from breast cancer are conventionally treated with surgery, which is also supported by radiotherapy, chemotherapy, or other medical treatments.3-5 Although progressive treatments have been used clinically, studies are still exploring more efficient alternatives to increase the cure rate of this malignant tumor.6, 7 5-Fluorouracil (5-FU) is a pyrimidine analogue and a traditional anticancer drug widely used to treat various cancers, especially colorectal and breast cancers.3,

8-10

Since it

shares the same transport pathway as uracil for entering into cells, 5-FU exerts its cytotoxicity through the erroneous incorporation of fluoronucleotides into DNA and RNA, and downregulation of thymidylate synthase, leading to fatal DNA and RNA damage.11, 12 It has been proved that chemotherapy with 5-FU improves the survival rate of patients with stage III cancer.13 However, a more extensive application is challenging owing to its short half-life in vivo,10 uncertain drug resistance,12,

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and most importantly, lack of


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targeting.3, 15 5-FU acts on not only tumor cells, but also normal cells, which is responsible for its side effects. Besides, efficacious tumor elimination requires a high dosage of 5FU,14,

16

which aggravates the damage on healthy tissues.9 In recent years, several

approaches have been developed to address these issues, including combining other chemotherapies (such as, leucovorin and irinotecan)8, 16, 17 and loaded with drug vehicles (nano-biomaterials).3, 9, 13-15 However, with little measurable success on these strategies, more efficient alternative strategies are still necessary. Recently, DNA tetrahedron, an emerging nanophase biomaterial, has been confirmed to be a multifunctional medicine owing to its anti-inflammatory,18, 19 antioxidative,19 and neuroprotective20,

21

delivery medium,22,

properties. Furthermore, it can be applied as an efficient drug 23

with advantages of biocompatibility, biodegradation, and

editability.24-28 Therefore, various materials such as aptamers and anticancer drugs have been designed to modify or load onto the DNA tetrahedron.27, 29-32 AS1411 is a G-rich DNA oligonucleotide, which is generally used as an aptamer with targeting and anticancer effects.33,

34

Its G-quadruplex structure can bind with nucleolin protein that is

especially present on the surface of tumor cells.34-36 Moreover, it has been detected to be


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a therapeutical agent for several human diseases, such as liver and breast cancer.34, 37 Based on these factors, we modified AS1411 to the DNA tetrahedron (T-AS1411) as a novel drug vehicle,38 which could strengthen the delivery efficiency of drug monomers with targeting.33, 39 Furthermore, Cyanine 5 (Cy5) is a fluorescent probe, which can be attached to nucleotide sequences,18, 24, 40 and be commonly used to track the intracellular location of biomaterials because of its stable fluorescence intensity.33, 41 In this study, we constructed and characterized DNA-based nanomedicine modified with 5-FU and AS1411 (AS1411-T-5-FU), determined its tumor cell killing effects, and then attached with Cy5 to analyze the cellular uptake using flow cytometry and immunofluorescence staining. The results revealed that AS1411-T-5-FU possessed structural stability, biocompatibility, potent toxicity, and preferential killing ability toward cancer cells, converging the multiple benefits of 5-FU, AS1411, and DNA tetrahedron. Moreover, AS1411-T-5-FU also showed enhanced therapeutic efficacy against breast cancer cells (MCF7 cell line) mediated by improved nuclear absorbability than 5-FU. More importantly, the DNA-based nanomedicine did not have side effects on breast normal cells (MCF10A cell line), implying the amelioration of 5-FU.


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RESULTS AND DISCUSSION Here, we present and explain in detail the results of the western blotting, quantitative real-time

polymerase

chain

reaction

(qPCR),

immunofluorescence

staining,

polyacrylamide gel electrophoresis (PAGE), flow cytometry, dynamic light scattering (DLS), cell counting kit-8 (CCK-8) and transmission electron microscopy (TEM) experiments performed. Identification of AS1411-T-5-FU Based on the structure of the DNA tetrahedron, AS1411-T-5-FU theoretically possessed a tetrahedral structure with modifications at two vertices (Figure 1A). To confirm each single strand DNA (ssDNA) and the successful formation of AS1411-T-5FU, 8% PAGE was carried out. The ordinary ssDNAs were shown in Figure 1B: S1, S2, S3, and S4 were of normal lengths as reported,21, 42, 43 and the 5-FU-conjugated S3 (S35-FU) and AS1411-modified S4 (S4-AS1411) were obviously extended compared with S3 and S4. AS1411-T-5-FU exhibited retarded mobility compared with that of the DNA tetrahedron and other intermediate products (Figure 1C).29 To further verify the formation


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of AS1411-T-5-FU, TEM was performed. AS1411-T-5-FU showed a recognizable tetrahedral structure, with a diameter of approximately 20 nm (Figure 1D, red circle).42, 44 Furthermore, to measure the particle size and size distribution, the sample was examined using DLS. The mean size of AS1411-T-5-FU was about 19.00 nm (Figure 1E), which was consistent with the TEM result. In comparison with the mean DNA tetrahedron size reported previously (16.14 nm),29, 45 there was a logical increase caused by the inclusion of AS1411 and 5-FU. Expression of nucleolin Nucleolin, a phosphoprotein, which is implicated in tumor invasion is reported at a constitutively high level in varieties of tumor cells. It is also a crucial target of AS1411 aptamer, the structure of which can be transformed after combining with AS1411 and its normal functions associated with cell proliferation can be constitutively blocked.35,

46

Remarkably, this aptamer-mediated process involved not only cell growth inhibition, the targeting effects on tumor cells make more sense. From Figure 2A, immunofluorescence revealed the overexpression of nucleolin in MCF7 cells and the low expression in MCF10A cells, which were the prerequisite of the targeting ability of AS1411-T-5-FU.


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Effects of AS1411-T-5-FU on cell viability The cytotoxicity of AS1411-T-5-FU was evaluated using the CCK-8 (Figure 2B and C), with a concentration gradient of 125 nM and 250 nM. After pretreatment with AS1411-T5-FU for 24 h, the viability of MCF7 cells was significantly decreased compared with that after pretreatment with free 5-FU in the same concentration, and the difference was especially obvious at 250 nM. Therefore, 250 nM was selected as the optimal concentration and was used in the subsequent experiments. In contrast, AS1411-T-5-FU exhibited no inhibition on growth of MCF10A cells and free 5-FU exhibited a marginal effect irrespective of the concentration. In summary, 5-FU inhibited the growth of both MCF10A and MCF7 cells, but AS1411-T-5-FU exhibited a significant effect on the growth of only MCF7 cells. The cytotoxicity of AS1411-T-5-FU was notably stronger than that of 5-FU on MCF7 cells. This might be attributed to the coexistence of AS1411 and 5-FU on the single strands of DNA tetrahedron, which not only strengthened the killing effect of the free 5-FU, but also enabled AS1411-T-5-FU to kill the cancer cells selectively. Cellular uptake of AS1411-T-5-FU


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To trace the intracellular uptake of AS1411-T-5-FU, S1 was loaded with Cy5.24, 41 As shown in Figure 3A, the fluorescence intensity (Cy5) was conspicuous in MCF7 cells pretreated with AS1411-T-5-FU, whereas it was hardly detectable in cells pretreated with T-5-FU. This result suggested that without the modification of AS1411, T-5-FU could not be considerably delivered into the cell nucleus. Conversely, there was no difference in the delivered amounts between MCF10A cells treated with AS1411-T-5-FU and T-5-FU (Figure 4A). A small amount of these two nanomaterials penetrated the nucleus after crossing the nuclear membrane, because of the low expression of nucleolin proteins in MCF10A cells.37 This limited uptake of AS1411-T-5-FU by MCF10A cells indicated that it might largely avoid side effects on normal breast cells. Correspondingly, flow cytometry (Figure 3B and C, 4B and C) showed that the uptake ratio of AS1411-T-5-FU was two times higher than that of T-5-FU in MCF7 cells, whereas, that of the two groups in MCF10A cells was negligible and showed no difference. This sharp contrast might be due to the targeting property of AS1411, which effectively weakened the cytotoxicity of AS1411-T-5-FU on the normal breast cells. Effects of AS1411-T-5-FU on cell cycle and apoptosis


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The effects of 250 nM AS1411-T-5-FU on cell cycle and cell apoptosis were additionally explored by flow cytometry. The G0, G1, S, G2, and M phases are an integral part of the cell cycle. DNA duplication, which plays a crucial part in cell growth, occurs during the S phase. Therefore, the proportion of cells in S phase can indirectly reveal the potential of cell proliferation.47 The number of MCF7 cells in S phase apparently reduced after 24-h post treatment with AS1411-T-5-FU compared with that following treatment with free 5FU (Figure 5A and C). This implies that less cells were actively proliferating. In line with the results of the CCK-8, MCF10A cells at S phase differed between control and 5-FU groups, whereas almost no difference was detected between the control and AS1411-T5-FU groups (Figure 5E and G). Previous studies have shown that decrease in cellular mitochondrial membrane potential (MMP) and exposure to phosphatidylserine (PS) result in apoptosis by

activating the mitochondrial intrinsic pathway48. Annexin V particularly binds to PS, which is expressed on the outer membrane of undamaged cells. Further, propidium iodide (PI) is a signal of dead cells as it can penetrate damaged membranes.49 Thus, the Annexin


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V/PI assay was performed to calculate the proportion of healthy, necroptotic, and early and late apoptotic cells after treatment with AS1411-T-5-FU and 5-FU. A typical distinction was observed between AS1411-T-5-FU and free 5-FU groups in MCF7 cells (Figure 5B and D). And as expected, almost no difference was observed in MCF10A cells among the treatment groups (Figure 5F and H). These findings further demonstrated that AS1411-T-5-FU was more potent against MCF7 cells than free 5-FU, while its effect on MCF10A cells was negligible. Effects of AS1411-T-5-FU on the mitochondrial apoptotic pathway Cell apoptosis is initiated by signals conveyed from the plasma membranes of dying cells. Two families of molecules are the main factors known to induce the apoptotic process. One is the caspase family including the subgroups of caspase-1, -4 and -5; caspase-2, -8, -9 and -10; and caspase-3, -6 and -7, which play different roles in apoptosis.50, 51 Another is a series of protein members in Bcl-2 family, including Bax, Bcl-2 antagonist/killer (Bak), BH3 interacting domain death agonist (Bid), Noxa, myeloid cell leukemia 1 (Mcl-1), Bcl-2, and Bcl-2 like 11 (Bim). The trigger of the Bcl-2 family forms an apoptosome activating the downstream caspase-3, -6, and -7, which stimulate other


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factors that directly lead to cell death. Both the caspase and Bcl-2 families are associated with the mitochondrial apoptosis pathway,52 in which the downregulation of Bcl-2 and the overexpression of Bax and caspase-3 occur during apoptosis.21, 44, 50, 51 To further validate the enhanced killing effect of AS1411-T-5-FU compared with that of free 5-FU, the expression levels of gene (Bax, Bcl-2, and caspase-3) were measured by qPCR (Figures 6D, 7D, and 8D). In the group treated with AS1411-T-5-FU (250 nM), Bax and caspase-3 were considerably upregulated, with a mean value of 6.8- and 4.5-fold, respectively. Moreover, the expression of Bcl-2 was obviously decreased in the AS1411T-5-FU group by 0.22-fold. In contrast, marginal variation was investigated in the 5-FU group, with 1.3-, 2.3-, and 0.85-fold for Bax, caspases-3, and Bcl-2, respectively. These significant trends in apoptotic related genes reflected the activation of cell apoptosis and the acceleration of apoptotic process in AS1411-T-5-FU group. Immunofluorescence staining revealed the expression levels of the three relevant proteins (Bax, caspase-3, and Bcl-2). The red luminescence represents the expression level of Bax (Figure 6A), which was the strongest in the AS1411-T-5-FU group and weaker in the free 5-FU group, illustrating the effective cell apoptosis of AS1411-T-5-FU.


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A similar tendency was shown in Figure 8A, where red represents caspase-3. This finding was further confirmed by the results presented in Figure 7A, which showed that the fluorescence intensity was apparently weakened after exposure to AS1411-T-5-FU. The corresponding consequences of the western blot analysis were shown in Figures 6B, 7B, and 8B. The relative intensity of Bax and caspase-3 was typically enhanced with a concomitant decline in Bcl-2. Data analysis were exhibited in Figure 6C, 7C, and 8C, Bax, caspase-3, and Bcl-2 levels were approximately 2.5, 1.78, and 0.49 times higher in the AS1411-T-5-FU group than in 5-FU alone group, respectively. The same tendencies were found in both genes and proteins, which further proved the correlation of mitochondrial pathway and cell apoptosis and the high-efficiency of AS1411-T-5-FU. For this investigation, we upgraded DNA tetrahedron by conjugation with Cy5, 5-FU, and AS1411 at thiol groups of S1, S3 and S4, respectively. The DNA tetrahedron was then adopted as a drug carrier to deliver 5-FU to the nucleus of cells, and AS1411 served as an aptamer to target cancer cells. The results showed that the therapeutic efficacy and targeting performance of AS1411-T-5-FU were profoundly improved. Theoretically, as an uracil analogue, 5-FU can be attached to all types of nucleic acid biomaterials in the


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similar way, which may largely improve its inadequacies, including drug resistance, short half-life and low efficiency. And the particular target ability of AS1411 sequence is extensively studied in the long-term researches. It has great potential to be developed into an antineoplastic agent which is applicable to a broad spectrum of cancers. Besides, this study focused on the structural upgrading of DNA tetrahedron, and mainly confirmed the increased inhibitory actions of AS1411-T-5-FU in vitro. Nevertheless, there are still many pivotal problems require to be addressed in the further studies, such as the stability in serum and the practical effects in vivo, the in-depth understanding of signaling pathways, and more relevant test groups (such as DNA tetrahedron or AS1411-T groups) should be exerted to complete this topic. Further experiments would be implemented to perfect this fascinating finding.

CONCLUSIONS In conclusion, we successfully developed AS1411-T-5-FU, a novel anticancer drug with powerful pharmacodynamic actions and selective lethal effects on breast cancer cells. We compared AS1411-T-5-FU with T-5-FU to elucidate the importance of AS1411 and


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through the evaluation of gene expressions of Bax, Bcl-2, and caspase-3, which are the intracellualr signals in the mitochondrial apoptosis pathway, we affirmed the predominant anticancer activity of AS1411-T-5-FU. Besides, the effective concentration of AS1411-T5-FU in this study was 250 nM, which is not harmful to normal breast cells and is consistent with our previous studies.19, 20, 43 These superior characteristics of AS1411-T5-FU suggest that it has the potential to be developed into a novel treatment strategy for malignant tumors. The prospective of this biomaterial would be promising.

EXPERIMENTAL SECTION Materials As represented in Table 1, the single strand DNA (ssDNA) modified with 5-FU and AS1411 was compounded by TAKARA (Dalian, China). 5-FU was purchased from J&K (Beijing, China), and was diluted with dimethyl sulfoxide (DMSO) in a concentration below 0.5%, which would not affect the final consequence.9. Trypsin-ethylenediaminetetraacetic acid (EDTA, 0.25% (w/v)) was bought from Corning (NY, USA). Fetal bovine serum (FBS)


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and penicillin-streptomycin solution were purchased from Gibco (Grand Island, NY, USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM/High Glucose), DMEM/F-12 (1:1) medium, and phosphate-buffered saline (PBS) were bought from HyClone (Logan, UT, USA). Confocal dishes and culture flasks were acquired from Corning (NY, USA). Paraformaldehyde solution (4 w/v %) was purchased from Boster (Wuhan, China). Trishydrochloride (HCl) and magnesium chloride (MgCl2) were obtained from Bio-Rad (Hercules,

CA,

USA).

4′6-Diamidino-2-phenylindole

(DAPI)

and

fluorescein

isothiocyanate (FITC)-labeled phalloidin were purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 assay kit was obtained from Dojindo Technology Chemical Corporation (Shanghai, China). Genomic DNA eliminator and RNeasy Plus mini kit were purchased from Qiagen (Hiden, Germany). Whole cell lysis assay, Annexin V-FITC apoptosis detection and DNA content quantitation assay (cell cycle) kits were acquired from KeyGEN Institute of Biotechnology (Jiangsu, China). PrimeScript reverse transcription-PCR kit, Polyvinylidene fluoride (PVDF) membranes, and cDNA synthesis kit were acquired from Takara (Dalian, China). Glyceraldehyde 3-phosphate


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dehydrogenase (GAPDH) and all apoptosis relevant antibodies (Bax, Bcl-2, caspase-3) were acquired from Abcam (Cambridge, UK). Preparation of Cy5-AS1411-T-5-FU Cy5, 5-FU and AS1411 were conjugated at thiol groups of S1, S3 and S4, respectively.24, 25, 53 Cy5-AS1411-T-5-FU was assembled with four ssDNAs: S1-Cy5, S2, S3-5-FU, and S4-AS1411 using a method reported previously.20,

23, 29, 42

Briefly, four

ssDNAs with different sequences at the same concentration were blended into a buffer solution (containing MgCl2 and Tris, pH 8.0), and mixture system was then heated to 95 °C for 10 min and cooled down to 4 °C for 30 min.20, 23, 29, 42 Characterization of AS1411-T-5-FU PAGE (8%) was carried out to prove the successful synthesis of AS1411-T-5-FU. The samples were separated using a polyacrylamide gel with 1× Tris-acetate-EDTA (TAE) electrophoresis buffer at a voltage of 80 V for 80 min.20, 54 Subsequently, the gel was stained with gel-red (against light) and shaken for 15 min.21 Finally, the gel was captured by an enhanced chemiluminescence (ECL) detection system (Bio- Rad, Hercules, CA, USA).


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The morphology and dimension of the AS1411-T-5-FU particles were examined using TEM. A drop of AS1411-T-5-FU solution was dried on cleaved mica exposed to infrared rays for 5−10 min, and then the TEM images were captured under an accelerating voltage of 120 kV with a Tecnai G2 F20 S-TWIN TEM (FEI Co., OR, USA).42, 44 In addition, the particle size and size distribution of AS1411-T-5-FU were examined using DLS with the Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK).29, 43 Cell culture The human breast cancer cell line (MCF-7) and breast epithelial cell line (MCF10A) were purchased from the American Type Culture Collection (ATCC). MCF-7 cells were incubated using a medium containing DMEM/High Glucose, 1% (v/v) penicillinstreptomycin solution and 10% (v/v) FBS. MCF10A cells were incubated in DMEM/F12, mixed with 1% (v/v) penicillin-streptomycin solution, 5% (v/v) horse serum, 1% (v/v) nonessential amino acids, 10 μg/mL insulin, 0.5 μg/Ml hydrocortisone, and 20 ng/mL epidermal growth factor (EGF). All cells were placed in an incubator under a stable living environment of 5% CO2 at 37 °C. The medium was renewed 1 or 2 times a week. Cell count kit-8


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The effects of AS1411-T-5-FU on cells (MCF10A and MCF7) were evaluated by the cell viability assay. MCF7 and MCF10A cells were incubated for 24 h in 96-well plates (5 x 103 cells per well) under standard conditions. Afterwards, AS1411-T-5-FU and free 5-FU were used at test concentrations of 125 nM and 250 nM and cultivated for 24 h. After washing with PBS three times, CCK-8 solution was added to assess the viability of MCF10A and MCF-7 cells. The optical density (OD), which reflects the inhibition of cell growth, was recorded at a wavelength of 450 nm.29 Cellular uptake of AS1411-T-5-FU Cy5 was attached to S1 to synthesize Cy5-AS1411-T-5-FU and Cy5-T-5-FU.24, 53MCF7 and MCF10A cells were migrated from the culture flasks to the bottom of cell culture dishes. The cells were treated with AS1411-T-5-FU and T-5-FU (250 nM) at 37 °C for 12 h. Culture medium was rinsed by PBS and cells were then fixed with 4% cold paraformaldehyde for 15 min. After rinsing with PBS three additional times, phalloidin and DAPI were applied to tint the cytoskeleton and nucleus for 30 and 10 min, respectively.20, 21, 55

To observe cellular uptake and obtain images, the Nikon N-SIM confocal laser

microscope (Japan) was used.


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In addition, the assimilation state was detected by flow cytometry. The cells were grown in six-well plates for 24 h, and then treated with Cy5-T-5-FU and Cy5-AS1411-T-5-FU (250 nM) for 12 h (as mentioned above). After digested by trypsin, the cells were collected into centrifuge tubes, washed with PBS for three times, and then transferred to polystyrene round bottom tubes and resuspended in PBS. The cells were subsequently counted using a flow cytometer (FC500 Beckman, IL, USA). Changes in cell cycle The variation in the cell cycle was examined by flow cytometry. Briefly, 5-FU and AS1411-T-5-FU were added into the culture plates, followed with incubation for 24 h. Subsequently, the processed and digested cells were collected into centrifuge tubes. The cells were fixed with cold ethanol after washing with PBS three times. The next day, RNase and PI staining solution were added sequentially and incubated for 30 min and 1 h, respectively.21 The changes were observed using a flow cytometer and resolved by the WinMDI2.9 and Win-Cycle 32 software. Quantification of necroptotic and early and late apoptotic cell populations


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The two types of cells were harvested into centrifuge tubes after 24-h-treatment with 5FU and AS1411-T-5-FU and washed with PBS three times. Then, 200 μL staining buffer was used to resuspend the cells, 5 μL each of Annexin-V-FLUOS and PI for 15 min in succession. The samples were protected from light. Besides, single staining with PI and Annexin-V-FLUOS were also performed.21 To analyze the quantity of apoptotic state, a flow cytometer and the software WinMDI2.9 and Win-Cycle 32 were performed. Western blotting The cells were incubated in 6-well plates for 24 h, and then processed with 5-FU and AS1411-T-5-FU for 24 h (as described above). Samples were extracted using a whole cell lysis assay, and then added 5× loading buffer and boiled for 5 min. The proteins were dissociated via 15% sodium dodecyl sulfate (SDS)-PAGE. The gel was transferred on to PVDF membranes and blocked by 5% bovine serum albumin (BSA) for 1 h. Subsequently, apoptosis-related primary antibodies against Bax (1:500; ab53154), Bcl-2 (1:1000; ab182858), and caspase-3 (1:1000; ab13847) were employed at 4 °C overnight. The following day, membranes were incubated with appropriate secondary antibodies (1:3000; Beyotime, Shanghai, China) for another 1 h. All membranes were rinsed with


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Tris-buffered saline with Tween (TBST) for 15 min three times. Finally, the target bands were exposed and visualized by the ECL detection system (Bio-Rad, Hercules, USA). To quantify the intensity of each sample, ImageJ software was used. Besides, internal reference was set by GAPDH. qPCR Briefly, the cells were processed with 5-FU and AS1411-T-5-FU for 24 h. The total RNA was extracted and refined by the RNeasy Plus Mini kit and genomic DNA eliminator, and the corresponding cDNA was reversely transcribed using a cDNA synthesis kit. The quantification of apoptosis relevant genes Bax, Bcl-2, and caspase-3 (the sequence of the primers is listed in Table 2) was analyzed using qPCR with the PrimeScript RT-PCR kit. The amplification procedures were conducted as follows: denaturation at 94 °C for 5 min, 40 amplification cycles comprising 94 °C for 5 s, and 60 °C for 34 s in succession. To investigate the constitution of the mistaken priming and the primer dimer, a melting curve was established for every process. GAPDH was used as an internal reference.21, 44 Immunofluorescence


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MCF-7 and MCF10A cells were transferred into cell culture dishes and treated with AS1411-T-5-FU and 5-FU for 24 h. Subsequently, cells were fixed with 4% cold paraformaldehyde for 15 min, followed by treatment with 0.5% TritonX-100 for 10 min to permeabilize the cell membranes, and incubation with 5% sheep serum for 1 h to block the cells. PBS was used for 15 min between each step. The cells were cultivated with nucleolin (1:200) and antibodies against the apoptosis relevant proteins Bax (1:100), Bcl2 (1:150), and caspase-3 (1:80) at 4 °C overnight, and then secondary antibodies (1:500, Invitrogen, CA, USA) at 37 °C for 1 h the next day.21, 44 DAPI and phalloidin were used to stain the cell nucleus and cytoskeleton, respectively. Confocal laser microscope (Nikon N-SIM) was applied to capture the images of the interest proteins. Statistical analyses All experiments were carried out no less than three times. Data are presented as the means ± standard deviation (SD). The statistical disparity among different groups was analyzed by t-test or one-way analysis of variance (ANOVA). Results with a P-value < 0.05 were considered significant.


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ACKNOWLEDGEMENTS We thank the National Natural Science Foundation of China (81671031) for the financial support. We are grateful to Dr. Chenghui Li (Analytical and Testing Center, Sichuan University) for assisting with the particle size analysis and capturing the laser scanning confocal images.

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Figure 1. Successful synthesis and characterization of AS1411-T-5-FU. (A) Diagrammatic sketch of AS1411-T-5-FU. (B) Certification of each ssDNA (1: S1; 2: S2; 3: S3; 4: S3-5FU; 5: S4; 6: S4-AS1411). (C) Proof of the successful synthesis of AS1411-T-5-FU by PAGE (1: S1; 2: S1+S2; 3: S1+S2+S3; 4: S1+S2+S3-5-FU; 5: TDNs; 6: T-5-FU; 7: T-


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AS1411; 8: AS1411-T-5-FU). (D) Morphological images of AS1411-T-5-FU by TEM (AS1411-T-5-FU: red circle). (E) Size distribution of AS1411-T-5-FU by DLS.

Figure 2. (A) the expression of nucleolin on the nuclear membrane of MCF7 and MCF10A cells. (nucleolin: red, nucleus: blue, cytoskeleton: green). Scale bars are 50 μm. (B) Cell counting kit-8 to evaluate the cytotoxicity of AS1411-T-5-FU and 5-FU on different concentrations (125 nM and 250 nM). MCF7 cells were treated for 24h. Data are


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presented as mean ± SD (n = 4). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001. (C) Cell counting kit-8 to evaluate the cytotoxicity of AS1411-T-5-FU and 5-FU on different concentrations (125 nM and 250 nM). MCF10A cells were treated for 24h. Data are presented as mean ± SD (n = 4). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.


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Figure 3. MCF7 cellular uptake of AS1411-T-5-FU and T-5-FU after treated for 12 h (AST-5 stands for AS1411-T-5-FU). (A) MCF7 treated with Cy5-T-5-FU and AS1411- (Cy5-) T -5-FU in immunofluorescence images (Cy5: red, nucleus: blue, cytoskeleton: green).


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Scale bars are 25 μm. (B) Cellular uptake of AS1411-T-5-FU and T-5-FU by flow cytometry. (C) Quantitative analysis of flow cytometry. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001.


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Figure 4. MCF10A cellular uptake of AS1411-T-5-FU and T-5-FU after treated for 12 h (AST-5 stands for AS1411-T-5-FU). (A) MCF10A treated with Cy5-T-5-FU and AS1411- (Cy5) T -5-FU in immunofluorescence images (Cy5: red, nucleus: blue, cytoskeleton: green). Scale bars are 25 μm. (B) Cellular uptake of AS1411-T-5-FU and T-5-FU by flow cytometry. (C) Quantitative analysis of flow cytometry. Data are presented as mean ± SD (n = 4).

Figure 5. The inhibition on cell proliferation in MCF7 and MCF10A cells. Cells are treated for 24 h. (A) Quantification of cell cycle of MCF7 tested by flow cytometry. Data are presented as mean ± SD (n = 4). Statistical analysis: *p < 0.05. (B) Data analysis of cell apoptosis of MCF7 by flow cytometry. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001. (C) The changes of cell cycle reflected by flow cytometry (MCF7 cells). (D)


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The conditions of cell apoptosis tested by flow cytometry (MCF7 cells). (E) Quantification of cell cycle of MCF10A tested by flow cytometry. Data are presented as mean ± SD (n = 4). Statistical analysis: *p < 0.05. (F) Data analysis of cell apoptosis of MCF10A by flow cytometry. Data are presented as mean ± SD (n = 4). (G) The changes of cell cycle reflected by flow cytometry (MCF10A cells). (H) The conditions of cell apoptosis tested by flow cytometry (MCF10A cells).


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Figure 6. The expression of apoptosis related protein-Bax. (A) After exposure with 5-FU (250 nM) and AS1411-T-5-FU (250 nM) for 24 h, Immunofluorescent images of MCF7 cells (Bax: red, nucleus: blue, cytoskeleton: green). Scale bars are 25 μm. (B) Expression of Bax determined by western blotting. (C) Quantitative analysis of western blotting. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001. (D) Expression of


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Bax detected by quantitative real-time PCR. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001.

Figure 7. The expression of apoptosis related protein-Bcl-2. (A) After exposure with 5-FU (250 nM) and AS1411-T-5-FU (250 nM) for 24 h, Immunofluorescent images of MCF7


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cells (Bcl-2: red, nucleus: blue, cytoskeleton: green). Scale bars are 25 μm. (B) Expression of Bcl-2 determined by western blotting. (C) Quantitative analysis of western blotting. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001. (D) Expression of Bcl-2 detected by quantitative real-time PCR. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p < 0.001.


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Figure 8. The expression of apoptosis related protein-caspase-3. (A) After exposure with 5-FU (250 nM) and AS1411-T-5-FU (250 nM) for 24 h, Immunofluorescent images of MCF7 cells (caspase-3: red, nucleus: blue, cytoskeleton: green). Scale bars are 25 μm. (B) Expression of caspase-3 determined by western blotting. (C) Quantitative analysis of western blotting. Data are presented as mean ± SD (n = 4). Statistical analysis: ***p

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