Systematic Study in Mammalian Cells Showing No Adverse Response

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Biological and Medical Applications of Materials and Interfaces

A Systematic Study in Mammalian Cells Shows No Adverse Response to Tetrahedral DNA Nanostructure Kai Xia, Huating Kong, Yunzhi Cui, Ning Ren, Qingnuan Li, Jifei Ma, Rongrong Cui, Yu Zhang, Jiye Shi, Qian Li, Min Lv, Yanhong Sun, Lihua Wang, Jiang Li, and Ying Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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ACS Applied Materials & Interfaces

A Systematic Study in Mammalian Cells Shows No Adverse Response to Tetrahedral DNA Nanostructure

Kai Xia,† § # Huating Kong,† # Yunzhi Cui,† § # Ning Ren,† § Qingnuan Li,‡ Jifei Ma,‡ Rongrong Cui,‡ Yu Zhang,† Jiye Shi, ⊥ Qian Li,† Min Lv,† Yanhong Sun,† Lihua Wang,† Jiang Li,†* Ying Zhu†*

†

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron

Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China. ‡

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai

201800, China. ⊥

§

UCB Pharma, Slough, SL1 14EN Berkshire, UK.

University of Chinese Academy of Sciences, Beijing 100049, China.

1

ACS Paragon Plus Environment

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ABSTRACT：The advent of DNA technology has demonstrated great potential in a wide range of applications, especially in the field of biology and biomedicine. However, current understanding of the toxicological effects and cellular responses of DNA nanostructures remains to be improved. Here we chose tetrahedral DNA nanostructures (TDNs), a type of nanocarriers for delivering molecular drugs, as a model for systematic live-cell analysis of the biocompatibility of TDNs to normal bronchial epithelial cells, carcinoma cells and macrophage. We found the interaction behaviors of TDNs in different cell lines were very different; whereas after internalization, most of TDNs in diverse cell lines positioned to lysosomes. By a systematic assessment of cell responses after TDN exposure to various cells, we demonstrate that internalized TDNs have good innate biocompatibility. Interestingly, we found TDN-bearing cells would not affect the cell cycle progression and accompany cell division, TDNs were separated equally into two daughter cells. This study improves our understanding of the interaction of DNA nanostructures with living systems and their biocompatibility, which will be helpful for further designing DNA nanostructures for biomedical applications.

KEYWORDS: Tetrahedral

DNA nanostructures

(TDNs),

Cell,

Interaction,

Biocompatibility, Cell cycle

INTRODUCTION DNA is not only the genetic material for coding, storing and transferring biological information, but also a versatile material for the “bottom-up” construction of exquisite nanostructures.1-4 Since the pioneering work of Seeman et al. in the 2


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1980s,5 there has been great progress in assembling a variety of complicated two- and three- dimensional DNA nanoarchitectures.6-9 Today, these DNA nanoarchitectures with excellent mechanical rigidity and structural stability have demonstrated great potential for a wide range of applications, which include molecular sensoring,10-15 computation,16-17 nanomachines18-19 as well as diagnostics and therapeutics.20-24 Among various biological and biomedical applications, the interaction of DNA nanostructures with living systems and their biocompatibility are important issues. Several reports have indicated that self-assembled DNA nanostructures were permeable to the cellular membrane and accumulated in cells.25-31 Especially, Kim et al. indicated that the entry mechanism of tetrahedral DNA nanostructures (TDNs) into cells was mainly caveolin-mediated endocytosis pathways.31 Following study further confirmed this entry mechanism of TDNs, and revealed that after endocytosis, they mainly located to lysosomes.32 Moreover, most of cell culture studies preliminary evaluated

the

cytotoxicity

of

DNA

nanostructures

by

the

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and indicated that they showed no obvious cytotoxicity to any of cell lines after incubation.21, 22, 24-26, 33

Thus, current understanding of the toxicological effect of DNA nanostructures is

still limited. Further systematic biocompatibility assessment of DNA nanostructure to various cell lines is essential. In this work, we choose tetrahedral DNA nanostructures (TDNs), one of the most practical DNA nanoconstructs, self-assembled from four DNA strands and prepared with a high yield,28, 34 as an representative of DNA nanostructures. Normal bronchial epithelial cells (BEAS-2B), carcinoma cells (HeLa), and macrophage (RAW264.7), which are widely used in biomedical research, were chosen as representative mammalian cell models. We observed different uptake dynamics of TDNs with those 3



cell lines, demonstrated excellent innate biocompatibility of TDNs through cytotoxicity determination, protein expression assay, inflammation and apoptosis monitoring as well as cell organelle function detection. Significantly, we found that TDNs did not alter the cell cycle progression and cell division. All of the findings will deepen our understanding of the interaction of DNA nanostructures with mammalian cells, which will be useful for better designing DNA nanostructure-based bioprobes and biovectors for further applications.

RESULTS AND DISCUSSION Visualization of TDNs - cell interaction We assembled TDNs from four, 63-base ssDNA strands with rationally designed, partially complementary sequences28, 34 (Figure 1a and Table S1). By polyacrylamide gel electrophoresis (PAGE), we confirmed the successful formation of the TDNs (~90% yield) (Figure 1b). Dynamic light scattering (DLS) analysis indicated the hydrodynamic size of TDNs prepared in this work was about 9.5 nm (Figure 1c). Each vertex of the TDNs was labeled with a red-colored fluorophore Cy3 (Cy3-TDNs) to facilitate visualization of TDNs - cell interaction by fluorescence microscopy (Table S1). PAGE and DLS analysis confirmed the successful formation of similar-sized Cy3-TDNs (Figure S1). First, we incubated various cell lines (BEAS-2B, HeLa, and RAW264.7 cells) with Cy3-TDNs and continuously observed the internalization process by confocal microscopy and flow cytometry. Both methods revealed that at the initial 6-h incubation, the fluorescence intensity (FI) inside RAW264.7 cells was much higher than that in HeLa and BEAS-2B cells. From 6 - 24 h after incubation, the FI in RAW264.7 cells slowly increased until 12 h and then gradually decreased, whereas the FI in other two cell lines increased rapidly with time until the cytoplasm was saturated by fluorescence at 24 h 4


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(Figure 2a, b and Figure S2, S3). This indicate cell uptake process of TDNs in different cell lines was very different. At early exposure time (0 - 6 h), compared to other two cell lines, RAW264.7 cells can quickly internalize a large number of TDNs due to their own strong phagocytosis.35 However, with the elapsed time (6 - 24 h), the uptake of TDNs in BEAS-2B and HeLa cells gradually increased and that in RAW264.7 cells decreased gradually after 12 h. This probably due to the shorter average cell doubling time of RAW264.7 cells (11 - 15 h) compared to that of other two cell lines (24 h).35 Next, we tracked the internalization of Cy3-TDNs in diverse cell lines by using real-time live-cell imaging with a DV Elite microscope. We observed that, for

BEAS-2B/HeLa cells, red-colored particle slowly moved along the cell membrane for 60/80 sec and suddenly crossed the membrane and rapidly came into the cytoplasm. For RAW264.7 cells, we observed a completely different process. Red-colored particle approached the cell membrane, moved along and crossed the membrane, and came into

the cytoplasm rapidly. In particular, the retention time on membrane (Phase II) was only 8 sec, which is much shorter than that for other two cell lines. Moreover, its movement speed along the membrane was faster than that for other two cells (Figure 2c, Figure S4 and Video S1-S4). This attributed to the more powerful phagocytosis

ability of RAW264.7 cells than that of other two cells.35

Examination of innate biocompatibility of TDNs Having observed the different interaction process of TDNs with diverse cell lines, we performed a series of innate biocompatibility tests to evaluate the biosafety of TDNs. MTT analysis showed that TDNs showed no obvious toxicity to these cells after 24-h incubation, even at the highest incubation concentration of 100 µg/mL. 5



Although we observed a slight increase in RAW264.7 cells at some TDN concentrations, no statistically significant difference in group means was seen in TDNs–treated cells compared to the control cells. Therefore, this was not considered as treatment-related effects on cell viability (Figure 3a). Fluorescence-based LIVE assays also confirmed this results (Figure S5). SDS-PAGE and two-dimensional (2D) gel electrophoresis indicated TDNs treatment didn’t markedly altered the protein expression profile in diverse cell lines (Figure 3a and Figure S6). The immunogenicity of nanomaterials is one of the important indicators to evaluate its biocompatibility, especially macrophage cell response is the first line in adaptive immunity.36 Thus we measured the secreted cytokine levels in RAW264.7 cells with ELISA, and found that TDN treatment didn’t induce any production of interleukin-6 (IL-6) or interleukin-12 (IL-12), two representative indicators of inflammation, in RAW264.7 cells (Figure 3c). Apoptosis-inducing nanoparticles usually signal through varying the expression of Bcl2 family proteins including pro-apoptotic proteins such as Bax as well as anti-apoptotic protein such as Bcl-2, and the increased ratio of Bax/Bcl-2 was suggested as an indicator for decreased cellular protection against apoptosis as well as potential cytotoxicity.37 Here by western blot analysis, compared to control samples, we didn’t observe any alteration of this ratio in various cell lines after TDN treatment (Figure 3d). All these data indicate that the TDNs are biocompatible at the protein expression level in vitro. Our previous study indicated that for HeLa cells, most TDNs via endocytotic internalization were eventually delivered to lysosomes after long-time incubation (6 – 6


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12 h),32 we thus monitored the subcellular distribution of TDNs in different cell lines and assessed their effects on organelle function after 12 h incubation. Cells were stained with lysotracker, a green fluorophore specifically binding to lysosomes.32 For BEAS-2B, HeLa and RAW264.7, the yellow areas in the confocal images, which were overlapping of red-colored Cy3-TDNs and green-colored labeled lysosomes, were 89%, 84% and 78%, respectively (10-15 cells per group) (Figure 3e). This suggest that localization TDNs in different cell lines is lysosomes. It is well-known that the acidic pH (~ 4.5) of lysosomes is critical for its physiological functions.38 To test whether TDN localization affect the pH in lysosomes, cells were labeled with LysoSensor Green DND-189, a dye accumulating in intracellular acidic organelles with a pH-dependent decrease in FI in response to organelle pH alkalization,39 for comparison of lysosome acidity. Confocal microscopy showed that compared to control groups, there were no significant change in FI of various cells after TDN treatment, while positive control chloroquine (CQ), a lysosomal inhibitor increasing the lysosome pH,40 remarkably decreased FI of various cells (Figure 3f and Figure S7). These data suggest treatment with TDNs will not cause alkalinization of lysosomes in various cell lines. That’s to say, subcellular localization of TDNs in lysosomes will not affect its function. Taken together, we conclude that TDNs have innate potential for further biocompatible nanoimaging and therapeutic delivery.

Assessments of the effects of TDNs on cell cycle The cell cycle leads the cell to its normal division and duplication. Cell cycle 7



stages is regulated by various protein families, among which diverse cyclin proteins and associated cyclin-dependent kinases (CDK) play a crucial role.41 To analyze the effects of TDN exposure on cell cycle progression (Figure 4a), we first examined the expression level of cell cycle-regulated proteins cyclin B1, cyclin D1, phospho-CDC2 and CDC2 (CDK1) by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression level was used as an internal control. Compared to their respective control cells, there were no significant changes in the expression levels of cyclin D1, cyclin B1, CDC2 and phospho-CDC2 of each cell line (Figure 4b). Additionally, cell cycle distribution analysis showed that the TDN exposure did not alter the distribution of cell cycle stages after each kind of cell was incubated with TDNs for 24 h, respectively (Figure S8). Subsequently, accompany cell division, TDNs were separated into two daughter cells. We further quantified the FI of Cy3-TDNs in each cell line. The ratios of TDNs were separated in two daughter cells by 50.3% /49.7%, 48.2% /51.8% and 54.7% /45.3% for BEAS-2B, HeLa and RAW264.7 cells, respectively (Figure 4c). This suggest that TDNs were separated equally into two daughter cells during cell division. In addition, we labeled the cell nuclei and actin with Hoechst 33258 and Actin Green™ 488 Ready Probe, respectively. We found that red-colored Cy3-TDNs were distributed in cytoplasm but not nuclei in various cell lines. Compared to their respective control cells, TDNs spread in cytoplasm didn’t cause obvious morphological changes in the cell nucleus and actin during mitosis (Figure 4c and Figure S9). All these data indicate that these TDN-bearing cells keep the cell 8


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physiological functions on cell cycle progression, which reconfirms good biocompatibility of TDNs to various cell lines.

CONCLUSIONS In this work, we systematically studied the biocompatibility of DNA nanostructure to various cell lines by using TDNs as a representative. Although the interaction behaviors of TDNs with different cell lines are very different evidenced by fluorescence microcopy and flow cytometry analysis, they have good innate biocompatibility to various cells demonstrated by a comprehensive toxicology assessment. Moreover, endocytic TDNs do not disturb the cell physiological functions on cell cycle progression. Our work encourages further studies of DNA nanostructures as promising nanomaterials for further biocompatible nanoimaging and biomolecule delivery researches.

EXPERIMENTAL SECTION TDN preparation. TDNs were prepared according to literature.42 Briefly, four strands (S1 - S4)28, 34 and four Cy3-labeled strands (Cy3-S1 – Cy3-S4) (Table S1) synthesized by TaKaRa Biotechnology (Dalian) Co., Ltd. were dissolved in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA) with concentration of 100 µM, and then diluted in Tris-EDTA buffer (20 mM Tris and 50 mM MgCl2) with a final concentration of 1 µM. The solution was heated to 95°C 10 min, and then cooled to 4°C for 20 min. The prepared TDNs and Cy3-TDNs were characterized using 8% non-denaturing polyacrylamide 9



electrophoresis (PAGE) and diameter analysis with a Delsa NanoC particles Analyzer (BECKMAN COULTER, USA).

Cell lines and treatment. BEAS-2B and HeLa cells were grown in the RPMI-1640 cell culture medium with 10% fetal bovine serum (FBS) and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin). RAW264.7 macrophage-like cells were grown in the Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and antibiotics. All cells are kept at 37°C with 5% CO2-containing atmosphere. Cells were added in 24-well plates with a density of 8×104 cells/well (for BEAS-2B and HeLa) or 105 cells/well (for RAW264.7) and incubated 8 – 12 h to allow for adherence. Following phosphate buffered saline (PBS) washing, each kind of cells was treated with TDNs or Cy3-TDNs at required experimental concentrations and incubation time. The evaluation system contains the following: (1) TDNs-cell interaction observation, (2) biocompatibility assessment of TDNs and (3) cell cycle analysis after TDNs treatment.

ASSOCIATED CONTENT Supporting Information.

One table, five figures and four videos are provided in the supporting information. Figures S1−S5 with accompanying figure legends 10


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Video S1 (AVI) Video S2 (AVI) Video S3 (AVI) Video S4 (AVI)

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program (2016YFA0400902), Key Research Program of Frontier Sciences, CAS (Grant No. QYZDJ-SSW-SLH031), the Open Large Infrastructure Research of Chinese Academy of Sciences, the National Natural Science Foundation of China (11675251, 21390414, U1532119, U1432116, 21675167, 11575278, 21505148), Instrument Developing Project of the Chinese Academy of Sciences (Develop the microscope system for Single Particle Tracking within Living Cells) and the Youth Innovation Promotion 11



Association of CAS (Grant No. 2016236).

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Figure 1. Characterization of TDNs. (a) Schematic showing of preparation of TDNs. (b) Native PAGE of TDNs. Lane M: DNA Marker (20 bp). (c) DLS measurement statistics of TDNs.

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Figure 2. Visualization of TDNs - cell interaction. Cells were treated with 100 nM TDNs for 24 h. (a, b) Confocal images of cells (a) and MFI inside the cells (b) at various timepoints after incubation (Data in b are represented as means ± SD). (c) Cellular uptake of TDNs as visualized in real time with a DV Elite microscope. Upper: diverse stages (Green: Approaching the membrane; Yellow: Moving along the membrane; Blue: crossing the membrane and coming into the cytoplasm). Lower: Speed analysis of the representative trajectory (representative trajectories and videos of internalization of TDNs in each cell line see Figure S4 and VideoS1-S4 in Supporting information). 19



Figure 3. Examination of innate biocompatibility of TDNs. Cells were treated with 0 - 100 nM (a) or 100 nM (b-f) TDNs. (a) Cell viability after incubation with TDNs at various concentration for 24 h. (b) RAW264.7 cells were treated with 100 nM TDNs or 20 nM S-CpG (positive control) for 24 h. The concentrations of IL-6 and IL-12 in culture media were measured after incubation. (c-f) Cells were treated with 100 nM TDNs for 12 h. (c) Protein expression profile in cells after incubation (M: Marker; C: Control; T: TDNs). (d) Representative immunoblots for apoptosis-related proteins Bax, Bcl2 (Upper); Bax/Bcl2 ratio in TDNs treated cells (Lower). Cells treated with 20


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0.5 mM H2O2 for 24 h were used as positive controls for apoptosis induction. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Normalized band densities were shown below each band. No TDNs treatment was defined as 1. (e) The colocalization between lysosomes (green) and TDNs (red) was examined by confocal microscopy (Left) and line profiling of fluorescence intensities (Right). (f) Confocal images of cells after incubation and subsequent staining with LysoSensor Green DND-189. Cells treated with 100 µM CQ for 6 h were used as positive controls for lysosome alkalinize.

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Figure 4. Assessments of the effects of TDNs on cell cycle. (a) General experimental design. (b) Cells were treated with 100 nM TDNs for 24 h. Representative immunoblots for cell cycle related proteins cyclin B1, cyclin D1, CDC2 and p-CDC2 in TDNs treated cells. GAPDH was used as the loading control. Normalized band densities were shown below each band. No TDNs treatment was defined as 1. (c) Cells were treated with 100 nM TDNs for 6 h. Confocal observation of TDNs separation in cells during cytokinesis. The ratios of the red fluorescence intensities of TDNs in separating daughter cells were quantified in each cell line. 22


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TOC

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Systematic Study in Mammalian Cells Showing No Adverse Response

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