Aptamer-Modified Tetrahedral DNA Nanostructure for Tumor-Targeted

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Aptamer-modified tetrahedral DNA nanostructure for tumor-targeted drug delivery Qianshun Li, Dan Zhao, Xiaoru Shao, Shiyu Lin, Xueping Xie, Mengting Liu, Wenjuan Ma, Sirong Shi, and Yunfeng Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13328 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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ACS Applied Materials & Interfaces

Aptamer-modified tetrahedral DNA nanostructure for tumor-targeted drug delivery

Qianshun Li, Dan Zhao, Xiaoru Shao, Shiyu Lin, Xueping Xie, Mengting Liu, Wenjuan Ma, Sirong Shi, 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.

*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: +86-28-85582167; E-mail address: [email protected]

Keywords Tetrahedral DNA nanostructures, AS1411, tumor-targeted, drug delivery, anticancer

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Abstract Tetrahedral DNA nanostructures (TDNs) are considered promising drug delivery carriers because they are permeable to cellular membrane and biocompatible and biodegradable. Furthermore, they can be modified by functional groups. To improve the drug-delivering ability of TDNs, we chose anticancer aptamer AS1411 to modify TDNs for tumor-targeted drug delivery. AS1411 can specifically bind to nucleolin, which is overexpressed on the cell membrane of tumor cells. Furthermore, AS1411 can inhibit NF-κB signaling and reduce the expression of bcl-2. In this study, we compared the intracellular localization of AS1411-modified TDNs (Apt-TDNs) with that of TDNs in different cells under hypoxic condition. Furthermore, we compared the effects of Apt-TDNs and TDNs on cell growth and cell cycle under hypoxic condition. A substantial amount of Apt-TDNs entered and accumulated in the nucleus of MCF-7 cells; however, the amount of Apt-TDNs that entered L929 cells was comparatively less. TDNs entered in much less quantity in MCF-7 cells than Apt-TDNs. Moreover, there was little difference in the amount of TDNs that entered L929 cells and MCF-7 cells. Apt-TDNs can inhibit MCF-7 cell growth and promote L929 cell growth, while TDNs can promote both MCF-7 and L929 cell growth. Thus, the results indicate that Apt-TDNs are more effective tumor-targeted drug delivery vehicles than TDNs, with the ability to specifically inhibit tumor cell growth.


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1. INTRODUCTION

DNA nanotechnology has been widely investigated in different biomedical fields such as disease diagnosis, therapeutics, and 3D bioprinting 1 - 8 . Tetrahedral DNA nanostructures (TDNs) have attracted a great deal of attention in biological and biomedical fields because of the biological nature of DNA9 and convenient synthesis. Successfully synthesized TDNs comprise four single stranded DNA (ssDNA) designed by complementary base pairing; each ssDNA forms a face of the DNA tetrahedron. TDNs are permeable to the cellular membrane mainly through caveolin-mediated endocytosis, whereas ssDNA or double stranded DNA (dsDNA) shows limited cell permeability9-12. Aptamers are relatively short ssDNA, RNA oligonucleotides, or peptides that bind to target molecules with high specificity and affinity13-15. G-rich oligonucleotide AS1411 is a 26-mer DNA aptamer, which specifically binds to nucleolin. Previous research has shown that nucleolin is a protein overexpressed on the cell membrane of tumor cells16-20, and nucleolin can transport anticancer ligands from the cell surface to the nucleus21. Many anticancer drugs, such as doxorubicin (DOX), destroy tumor cells by inserting nuclear DNA22. The nucleus-targeting anticancer drugs vehicles have been widely investigated to increase the efficiency of anticancer drugs. As an anticancer aptamer, AS1411 can inhibit NF-κB signaling and reduce the expression of bcl-2. Currently, it is being tested as an anticancer agent in a phase II clinical trial23-26. TDNs are considered a promising drug delivery system because they are permeable to cellular membrane. Furthermore, they are biocompatible and biodegradable and can



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be modified by functional groups27,28. We used anticancer aptamer AS1411 to modify TDNs and improve their drug-delivering ability. Compared with free aptamers, AS1411-modified TDNs carry significantly more drug molecules28. We inferred that AS1411-modified TDNs doubles the antitumor effect owing to combined anticancer effect of AS1411 and the drug molecule. Furthermore, AS1411-modified TDNs form an efficient tumor-target drug delivery system by specifically binding with nucleolin overexpressed on the cell membrane of tumor cells. Normal cell cultures are mostly maintained in vitro at an oxygen concentration of approximately 20%, while cells in physical tissue are maintained at a lower oxygen concentration of approximate 3%29,30. Hypoxia occurs when a tumor develops31. We placed cells under hypoxic condition to simulate the natural microenvironment. In this study, we synthesized AS1411-modified TDNs, determined their characteristics, and explored their effects on tumor cells and normal cells under hypoxic condition.

2. MATERIALS AND METHODS

2.1 Materials

Specific sequence of single-stranded DNA (ssDNA) was synthesized by TaKaRa (Dalian, China), and the specific sequences are displayed in Table 1. Fetal bovine serum (FBS) was acquired from Corning (New York), DAPI and FITC-labeled phalloidin were obtained from Sigma-Aldrich (Shanghai). The cell counting kit-8 (CCK-8) was obtained from Dojindo. (Shanghai), and KeyGEN DNA content Quantitation Assay (Cell Cycle) was purchased from KeyGEN Institute of


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Biotechnology (Jiangsu, China).

2.2 Cell Culture

Mouse fibroblast cell line L929 cells were cultured in RPMI-1640 culture medium with FBS and penicillin-streptomycin solution at the concentration of 10% (v/v) and 1% (v/v) separately. Human breast cancer cell line MCF-7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin solution. The cells were placed under hypoxic condition (oxygen: 1%)29.

2.3 Synthesis of Tetrahedral DNA Nanostructures and Aptamer-modified Tetrahedral DNA Nanostructures

TDNs were synthesized as previously reported10,11,32,33: S1, S2, S3, and S4 were mixed in TM buffer (10 mM Tris-HCl, 50 mM MgCl2·6H2O, pH 8.0) at same concentrations. The mixture solution was quickly heated to 95°C for 10 min, then cooled down to 4°C for 20 min. The aptamer-modified TDNs (Apt-TDNs) were prepared under the same conditions using S1, S2, S3, and S5.

2.4 Characterization of Tetrahedral DNA Nanostructures and Aptamer-modified Tetrahedral DNA Nanostructures

Polyacrylamide gel electrophoresis (PAGE, 8%) was used to verify the synthesis of TDNs and Apt-TDNs 34 . Atomic force microscopy (AFM; SPM-9700 instrument, Shimadzu, Kyoto, Japan) was used to investigate the morphology of Apt-TDNs by



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adding drops of Apt-TDNs solution onto a cleaved mica and air-drying the sample for 15 min35.

2.5 Cellular Uptake of Tetrahedral DNA Nanostructures and Aptamer-modified Tetrahedral DNA Nanostructures

To observe the intracellular localization of Apt-TDNs and TDNs, a confocal laser microscope (TCS SP8; Leica, Wetzlar, Germany) was used to capture cell images. TDNs and TDNs modified with cy5 were added to MCF-7 and L929 cells. MCF-7 cells and L929 cells were seeded at an initial density of 1×104 cells/cm2 under hypoxic condition with the regular medium for 24h, after 24h we changed the regular medium to medium without fetal bovine serum, then added 250nM TDNs and 250nM Apt-TDNs, respectively, for 6h. The cells were washed with PBS thrice and fixed in cold 4% paraformaldehyde solution for 15 min. The cells were re-washed with PBS thrice, and DAPI and phalloidin were used to stain the nucleus and cytoskeleton, respectively36-38.

2.6 Cell Counting Kit-8

L929 and MCF-7 cells were seeded in a 96-well plate (5000 per well) and incubated with the regular medium under hypoxic condition for 24 h and then with TDNs (250 nM) and Apt-TDNs (250 nM), respectively, for 6 h. CCK-8 was used to detect the changes in proliferation of L929 and MCF-7 cells39-41.


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2.7 Cell Cycle Assay

MCF-7 cells and L929 cells were incubated in regular medium for 24h, then treated with Apt-TDNs, TDNs, or serum-free medium for 6 h. The cells were harvested, rinsed with PBS thrice, and fixed with 70% cold ethanol at −20°C for the night. Then, the cells were mixed with 100 µL of RNase for 30min at 37°C and stained with 400 µL of propidium iodide (PI) solution for 30 min at 4°C away from light42. DNA content was measured using a flow cytometer (FC500 Beckman, IL, USA), and the results were analyzed using the software WinMDI2.9 and WinCycle 32.

2.8 Statistical Analysis

All experiments were performed in triplicate and reproduced at least thrice. Statistical analysis of data was performed with SPSS 19.0 (IBM, Silicon Valley, CA, USA) by one-way ANOVA. If the two-tailed p value was

Aptamer-Modified Tetrahedral DNA Nanostructure for Tumor-Targeted

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