Understanding the Biomedical Effects of the Self-Assembled

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Understanding of the Biomedical Effects of the Selfassembled Tetrahedral DNA Nanostructure on Living Cells Qiang Peng, Xiao-Ru Shao, Jing Xie, Si-Rong Shi, Xueqin Wei, Tao Zhang, Xiaoxiao Cai, and Yunfeng Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03786 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 8, 2016

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ACS Applied Materials & Interfaces

Understanding of the Biomedical Effects of the Self-assembled Tetrahedral DNA Nanostructure on Living Cells

Qiang Peng†, Xiao-Ru Shao†, Jing Xie, Si-Rong Shi, Xue-Qin Wei, Tao Zhang, Xiao-xiao Cai， Yun-Feng Lin*

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.

†

Qiang Peng and Xiao-Ru Shao contributed equally.

*Correspondence: Y.F. Lin, Tel: 86 28 85503487 Fax: 86 28 85503487 Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Recently, high attentions have been paid to DNA again due to the successful synthesis of DNA-based nanostructures that can enter cells via endocytosis and thus have great potentials in biomedical fields. However, the impacts of DNA nanostructures on life activities of living cell are unknown. Herein, the promotion effect of tetrahedral DNA nanostructure (TDN) on cell growth and the underlying molecular mechanisms are reported. Upon exposure to TDN, the cell proliferation is significantly enhanced, accompanied with up-regulation of cyclin-dependent kinase like-1 gene, changes in cell cycle distribution and up-regulation of the Wnt/β-catenin signaling-related proteins (β-catenin, Lef 1 and cyclin D). In contrast, single stranded DNA (ssDNA) shows no such functions. Furthermore, TDN is able to reverse the inhibition effect of DKK1, a specific inhibitor for Wnt/β-catenin pathway. Hence, Wnt/β-catenin pathway is the target for TDN to promote cell proliferation. The findings allow TDN to be a novel functional nanomaterial that has great potentials in tissue repair and regeneration medicine.

KEYWORDS: nanomaterials, self-assembly, cell cycle, microarray, flow cytometry, western blot


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INTRODUCTION DNA, as a kind of functional gene, has been used for many years in biomedical fields.1-6 Nowadays, the DNA-based nanostructures and their potential biomedical applications have attracted high attentions.7-13 Compared to native DNA that cannot be transported into cells without carriers, DNA nanostructures can be taken up by living cells via the similar pathways as other nanomaterials, such as clathrin-mediated endocytosis and caveolin-mediated endocytosis.14-15 This property, combined with their excellent biocompatibility, low toxicity and high biostability, confer DNA nanostructures a great potential for advanced drug delivery.16-18 Tetrahedral DNA nanostructure (TDN), a novel type of three-dimension DNA nanostructure, has been assembled by the highly specific and programmable base paring among four single-stranded DNA.19-21 It has been demonstrated that TDNs can be rapidly taken up by cells mainly via caveolin-mediated endocytosis and are able to escape from lysosomes after properly functionalization with nuclear localization signals.15 The ability of lysosomal escape is a key point for successful gene delivery. In fact, TDNs have been successfully used for targeted in vivo delivery of siRNA.22 In addition, the biomedical applications of TDNs have been well-documented elsewhere.8, 18, 23-25 As we know, all of these applications are tightly relevant to TDN-cell interaction which is the first step and very important to comprehensively understand the subsequent biological processes. However, the reports on the interaction between TDN and living cells are heavily lacking. In this present work, we aim to investigate the impacts of TDNs on normal cell proliferation and find out the relevant protein, gene and signal pathway using various molecular biological technologies. The disclosure of how TDNs affect cellular growth will help us better understand the cell responses to TDNs and thus exploit TDNs in a more reasonable manner.



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EXPERIMENTAL SECTION Materials. Single-stranded DNA (ssDNA) with specified sequence was provided by Takara (Dalian, China). KeyGEN DNA content Quantitation Assay (Cell Cycle) was obtained from KeyGEN Institute of Biotechnology (Nanjing, China). Cell Counting Kit-8(cck-8) was purchased from Shanghai Dojindo Technology Chemical Corporation (Shanghai, China). Fetal bovine serum (FBS) was obtained from HyClone(London, USA). All antibodies were purchased from Abcam(Cambridge, UK). All other chemical reagents used in this study were of analytical grade or better. Synthesis of tetrahedral DNA nanostructure (TDN). TDN was synthesized according to a previous method with minor modifications.19 Briefly, four specifically designed DNA single strands (s1, s2, s3, s4) with equal concentration were mixed in a buffer solution (10 mM Tris and 5 mM MgCl2). The mixture solution was heated to 95 oC for 10 min and cooled down to 4 oC for 20 min. The successful synthesis of TDN was confirmed by 8% polyacrylamide gel electrophoresis (PAGE). Effects of TDN on cell proliferation. Mouse L929 fibroblasts were cultured as previously described and the cell proliferation was examined by both RTCA (real-time cell analysis system) and cck-8 assay.26-28 Briefly, L929cells were seeded on the specially made 16-well plates (Eplate 16) at a density of 3000 cells per well for RTCA or on the normal 96-well plates at the same density for cck-8 and other examinations. The cells were cultured at 37 °C/5% CO2 for 24 h in RPMI-1640 culture medium containing 10% fetal bovine serum (FBS). Subsequently, the culture medium was replaced by TDN solutions with concentrations of 62.5, 125, 250 and 500 nM or by fresh medium without TDN (as control). After adding TDN, the cell proliferation was monitored


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with RTCA for 24 h and examined by cck-8 assay at 6 and 24 h, respectively. In cck-8 assay, the mixture of single-stranded DNA (ssDNA) that had the same concentration with their counterpart TDN was also used to treat cells in the same manner as described above. Microarray analysis. The total RNA prepared from L929 cells using an RNeasy MiniKit (Qiagen) was quantified by the NanoDrop ND-2000 (Thermo Scientific) and the RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).29 The sample labeling, microarray hybridization and washing were performed according to the manufacturer’s standard protocols. Briefly, the total RNA were transcribed to double strand cDNA and synthesized into cRNA, followed by labeling with Cyanine-3-CTP and hybridization onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies). The Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze the array images and extract the raw data. Subsequently, the Genespring software was employed for further analysis of the raw data. After normalization of raw data using quantile algorithm, the probes in which at least 75% of the values in any one group have flags in "Detected" were chosen for further data analysis. The differentially expressed genes were screened by fold change and p value calculated from t-test. The threshold set for up- and down-regulated genes was 2-fold change as well as p ≤ 0.05. Afterwards, GO and KEGG analysis was applied to determine the roles of these differentially expressed mRNAs. Finally, Hierarchical Clustering was performed to display the distinguishable genes' expression pattern. Flow cytometry. The cell cycle change upon exposure to TDN was examined by flow cytometry. Briefly, L929 cells were cultured under standard conditions as above for 24, followed by removal of old culture medium, washing, adding TDN (250 nM) or blank serum free culture



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medium, and further culture. At predetermined time intervals, cells were digested and examined using a cell cycle detection kit (KeyGen BioTECH, Nanjing, China) according to its instructions. A flow cytometer (FC500 Beckman, IL USA) was employed and software WinMDI2.9 and WinCycle 32 were used for further analysis. Western blot. The expression level of essential proteins (β-catenin, Lef 1 and cyclin D) involved in canonical Wnt/β-catenin signaling pathway was analyzed by western blot. Briefly, L929 cells were cultured for 24 h and then treated with TDN (250 nM). At fixed time intervals, the culture medium was discarded and the cells were washed with PBS. Afterwards, the total proteins were extracted using the whole cell lysis assay (KeyGen BioTECH, Nanjing, China) according to the manufacturer’s instructions. The collected proteins were analyzed by western blot using primary rabbit anti-mouse monoclonal antibodies (Abcam, Cambridge, UK) and secondary goat anti-rabbit IgG (ZSGB-Bio, Beijing, China). GAPDH was used as internal control. For DKK1 inhibition experiment, DKK1 alone (100 ng/ml) or the mixture of DKK1 and TDN (DKK1 100 ng/ml, TDN 250 nM) was used to treat cells for 24 h. The other procedures were the same as above. Confocal laser microscopy. The expression level of β-catenin was examined by confocal laser microscopy. Briefly, L929 cells were cultured for 24 h and then treated with TDN (250 nM) or DKK1 (100 ng/ml) or both. The treated cells were washed and fixed in 4% paraformaldehyde at 4oC for 15 min, followed by incubation with 0.5% triton-100 at 37 oC (15 min), incubation with goat serum at room temperature (1 h), and immunofluorescent staining using primary rabbit anti-mouse monoclonal antibody (Abcam, Cambridge, UK) and secondary Alexa Fluor 594 donkey anti-rabbit IgG for β-catenin (red), DAPI for nucleus (blue) and phalloidin for


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cytoskeleton (green). The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems, Solms, Germany). RESULTS AND DISCUSSION Self-assembly of TDN and its impact on cell proliferation. The self-assembled TDN is composed of four equimolar ssDNA (s1, s2, s3 and s4, Figure S1A) and each ssDNA forms a triangle that shares its three sides with the other three ssDNA (Figure 1A). The detailed description of each surface and base paring of TDN is shown in Figure S1B. The successful synthesis of TDN was confirmed by polyacrylamide gel electrophoresis (PAGE) according to the previous reports.15, 19 As shown in Figure 1B, the band in Lane 1 at the lower position is the monomer of s1, corresponding to ~30 bp of marker, the bands of s2, s3 and s4 correspond to ~40 bp, and the circled band in Lane T corresponds to ~150 bp which is well matched with the theoretical value of TDN (30 + 40 * 3 = 150). This result indicates the successful assembly of TDN. The band at the upper position in Lane 1 might be resulted from the self-assembly of s1 into a multimer. Similarly, the randomized assembly of ssDNA (s1, s2, s3 and s4) into multimers during the synthesis of TDN could also be observed from Lane T in Figure 1B. As we know, the native PAGE is affected by various factors, including the composition, sequence and possible conformation of ssDNA. Hence, the difference in band position between s1 monomer and the other ssDNA (s2, s3, s4) may be resulted from the internal fold of s1 sequence.



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Figure 1. A) Schematic of self-assembly of TDN from four specific designed ssDNA. B) Confirmation of the successfully assembled TDN by native PAGE (M: marker with 20 bp DNA ladder; 1~4: ssDNA s1~s4 (63 nt); Circled band in Lane T: TDN).

As a novel nanomaterial, TDN has shown great potentials for biomedical use. But its practical use will not be realized until its cellular biological effects are clearly disclosed. Herein, we investigated the effects of TDN on the growth of L929 cells. The real-time cell analysis (RTCA) showed that cell proliferation was significantly improved by TDN in a concentration-dependent manner in the range of 62.5~250 nM (Figure 2A). It is notable that the TDN with the highest exposure concentration (500 nM) strongly promoted cell proliferation within the first 6 h but lost the function thereafter. It was assumed that this might be resulted from the saturated growth space in the used cell culture plate. As we know, RTCA is a novel tool to monitor living cells, which is much more convenient than conventional assays and can provide real-time data throughout the


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experiment. The monitoring mechanism of RTCA system is based on the area occupied by cells (i.e. area-dependent impedance) but not the real cell number.30 Therefore, this technology may have a saturation phenomenon. This mechanism can also explain the reason for the obvious drop in the cell index profile at the time point of adding TDN. The process of adding TDN was at room temperature rather than 37 oC, leading to some extent of shrinkage in cell size that was presented as lower cell index values (Figure 2A). In order to confirm that the cell number increased indeed upon exposure to TDN, we used cck-8 assay to examine the living cells. As a result, the TDN concentration-dependent cell proliferation was quite obvious when the cells were treated by TDN for 6 h (Figure 2B). The living cell number significantly increased with increasing the TDN concentration. For TDN with concentration of 500 nM, the cell proliferation reached maximum (158%) at 6 h but decreased to 113% at 24 h. In contrast, the cell proliferation ratio was 128% for TDN (250 nM) at 6 h but increased to maximum (162%) at 24 h. This result obtained using cck-8 assay (Figure 2B) is perfectly matched with the result obtained using RTCA (Figure 2A), indicating that TDN is an effective stimulus to improve the cell proliferation and that the decrease in cell proliferation at 500 nM TDN (Figure 2A) was not due to the saturated space of RTCA. A more visualized increase in cell number upon exposure to TDN can be found from the micrographs (Figure S2). Interestingly, the single stranded DNA (ssDNA) showed no effects on cell proliferation at any concentration (Figure 2B). This means ssDNA does not induce any bio-signal transduction regarding cell proliferation but TDN does. Undoubtedly, this phenomenon is tightly related to the special structure of TDN. As novel nanomaterials, TDN has shown the ability for cellular uptake through endocytosis, the main pathway for nanomaterials to be taken up by cells.31-33 Although



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caveolin-mediated endocytosis has been demonstrated to be the main pathway for TDN to enter cells, 15 the other two types of endocytosis pathways, namely clathrin-mediated endocytosis and macropynocytosis may also be involved in transporting TDN into cells. But how and why TDN promotes cell proliferation is still unknown.

Figure 2. Cell proliferation of L929 cells upon exposure to TDN with varied concentrations. A) Cell proliferation detected by real-time cell analysis system (RTCA). B) The different effects of ssDNA and TDN on cell proliferation detected by cck-8 assay. Data are presented as mean ± sd (n = 4). Statistical analysis: *p

Understanding the Biomedical Effects of the Self-Assembled

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