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TiO2 Nanorods Arrays with Mesoscopic Micro-nano Interfaces for In Situ Regulation of Cell Morphology and Nucleus Deformation Hongni Liu, Rongxiang He, Meilin Ruan, Jingrong Xiao, Zhengtao Zhang, Chaohui Chen, Weiying Zhang, YiPing Cao, Yumin Liu, and Yong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11257 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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ACS Applied Materials & Interfaces

TiO2 Nanorods Arrays with Mesoscopic Micro-nano Interfaces for In Situ Regulation of Cell Morphology and Nucleus Deformation Hongni Liu,

†,⊥

†,⊥

Meilin Ruan,

Jingrong Xiao,

†,§,⊥

†

†

Zhengtao Zhang, Chaohui Chen, Weiying

Zhang,† Yiping Cao,† Rongxiang He,*,† Yumin Liu*,† and Yong Chen*,†,‡ †

Institute for Interdisciplinary Research & Key Laboratory of Optoelectronic Chemical Materials

and Devices of Ministry of Education, Jianghan University, Wuhan 430056, China. ‡

Département de Chimie, Ecole Normale Supérieure, 24 Rue Lhomond, F-75231 Paris Cedex05,

France. KEYWORDS: nucleus deformation, mesoscopic micro/nano interface, TiO2 nanorods arrays, cancer cells, cell morphology ABSTRACT: Cell morphology and nucleus deformation are important when circultating tumor cells break away from the primary tumor and migrate to a distant organ. Cells are sensitive to the microenvironment and respond to the cell-material interfaces. We fabricated TiO2 nanorods arrays with mesoscopic micro-nano interfaces through a two-step hydrothermal reaction method to induce severe changes in cell morphology and nucleus deformation. The average size of the microscale voids was increased from 5.1 µm to 10.5 µm when the hydrothermal etching time was

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increased from 3 h to 10 h, whereas the average distances between voids were decreased from 0.88 µm to 0.40 µm. The nucleus of the MCF-7 cells on the TiO2 nanorods substrate that was etched for 10 h exhibited a significant deformation, because of the large size of the voids and small distance between voids. The process of nucleus deformation was reversible during the cells cultured on the mesoscopic micro-nano interface in order to proliferate. This reversible process was regulated by the combined role of the uniform pressure applied by the actin cap and the localized pressure applied by the actin underneath the nucleus. Cell morphology and nucleus shape interacted with each other to adapt to the microenvironment. This mesoscopic micro-nano interface provided a new insight into the cell-biomaterials interface to investigate cell behaviors. 1. INTRODUCTION Generally, tumor metastasis can be divided into two major phases: cancer cells physically metastasize from the primary tumor to a distant organ through hematogenous circulation and the colonization of the translocated cells.1 During the process of translation, cancer cells should invade into the matrix and blood vessels, which surrounded the cancer cells . Circulating tumor cells (CTCs) in the bloodstream can exit the circulation and then invade the microenvironment of foreign tissue. Meanwhile, dynamic changes in epithelial and mesenchymal composition have been exhibited on the circulating tumor cells during the translation.2 These changes may be influenced by microenvironments where the cancer cells migrate. The number of the CTCs can be utilized as an indicator of clinical progression.3 Because of its critical importance, many methods based on cell or nucleus sizes, nanosized surface structure, and special antigen have been developed to capture CTCs.4-16 However, few studies about the deformation of the CTCs have been reported.


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Cells can sense and respond to the microenvironments. Virtual cell model had been used to predict cell response to substrate topography.17 Cytoplasmic actin filaments played an important role in the modulation of the shape and function of the cell and nucleus.18 A series of micropatterned rectangular shapes are used to investigate the nuclear orientation and deformation, which are regulated by the lateral compressive forces applied by tension in the central actomyosin fibers.19-21 Researchers have utilized microstructured or nanostructured substrates to investigate cell cytoethology. Micropatterned hydrogel substrates and anisotropically stiff 3D micropillar niches are fabricated to investigate the orientation and shape of human mesenchymal stem cells.22, 23 Cells round up on the flat hydrogel surfaces and enlongate and align parallel to the microplates. Silicon micropillar arrays with various shapes, sizes, and configurations were fabricated to investigate the collective and single glioma cell behaviors at the microsized interfaces.24 When the diameters are greater than 2 µm, tumor-like aggregation and branching of the glioma cells occur. Micropillars induced local changes in actin-rich filopodia protrusions when cells were conformed to the spatial between micropillars. Neuronals cultured on micropatterned surfaces can survive over extanded periods of time, and this is important in the time-dependent analysis of cellular processes.25 When the sizes of the micropillars and the distances between micropillars are smaller than that of the nucleus and the height of the micropillars is higher than 1 µm, nucleus deformation occurs.26,27 The shape of the nucleus is regulated by the size and structure of the micropillars.28-30 When the sizes of the pillars are smaller than 1 µm, nucleus maintains its shape and cell morphology changes to adapt the nanoscale interface. However, the sizes and the distances of the pillars are limited to micrometers, which are utilized by the microscaled characterization of the cells. On the other hand, when cells


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were cultured on nanostructured substrates, such as nanoparticles, nanopillars or nanofibers, cells morphology was mainly affected and the nucleus deformation was not obviously.31-34 Due to the dual characterization of the nanoscale surface morphology and microscale size of the cells, mesoscopic micro-nano interfaces were utilized to increase the cell capture efficiency, such as fractal Au particles14, multifunctional smart particles13, rose petals replicas35, and Rhipsalis structures.36 TiO2 nanorods or nanofibers are biocompatible and selective substrates for capturing and releasing CTCs.37, 38 In the present work ,we fabricated TiO2 nanorod arrays (NRAs) with mesoscopic micro-nano interfaces using two-step hydrothermal reaction to investigate the cell morphology and nucleus deformation. A normal cancer cell line (MCF-7) instead of CTCs was used as a cell model in our experiments. Nanoscale TiO2 NRAs were firstly hydrothermal growth on substrate and microscale voids were fabricated on the TiO2 NRAs through HCl hydrothermal etching. Sizes and the distances between the voids were controlled by etching time, which was coupled with the double characterization of the cells. The cells morphologies on the mesoscopic micro-nano interface substrates were characterized using scanning electronic microscope. Laser confocal fluorescence microscope was utilized to characterize the distribution of the F-actin and vinculin and the nucleus shape. We also discussed the impact of this mesoscopic micro-nano interface on the cell viability and mitosis. This study will provide a promising strategy to investigate the cells behabiors. 2. MATERIALS AND METHODS 2.1 Materials Fluorine-doped SnO2 conductive glasses (FTO, 2.8 cm × 5.0 cm, sheet resistance 10 –15 Ωsq-1) were obtained from Asahi Glass, Japan. Ethanol, acetone, TiCl4, and HCl were purchased from Sinopharm Chemical Reagent Co., Ltd, China. Deionized (DI) water was generated by MILLI-Q


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system (Millipore, MA, USA). Paraformaldehyde (PFA, 36% in water), 4,6-Diamidino-2phenylindole dihydrochloride (DAPI), Triton X-100, Bovine serum albumin (BSA), normal goat serum, and anti-vinculin-FITC antibody were purchased form Sigma-Aldrich. Fetal bovine serum (FBS), 0.25 % Trypsin-EDTA (Gibco, 1x), and Alexa Fluotm 568 Phalloidin were purchased from Invitrogen. To prepared the 0.2 M TiCl4 pretreatment aqueous solution, 3 mL of TiCl4 was slowly added into a mixture solution under magnetic stirring condition for 30 min, which contains 95 mL of DI water and 2 mL of concentrated HCl. To prepared the TiO2 growth solution, 3 mL of TiCl4 was dropwise added into another mixture solution under magnetic stirring condition for 3 h, which contains 30 mL of DI water and 30 mL of concentrated HCl. All reagents in this work were used without additional treatment. 2.2 Micro-nano interfaces fabrication and characterization Two-step hydrothermal technology was used to fabricate the nano-micro highly oriented rutile TiO2 interface according to previous studies39-41. First, FTO glasses were sequentially ultrasonically cleaned in acetone, ethanol, and DI water, followed by drying using compressed nitrogen. Second, in order to deposit TiO2 germ crystals on the FTO glasses surface, the substrates were immersed to a 0.2 M TiCl4 aqueous solution at 70 °C for 40 min. After cleaning three times with DI water, TiCl4 treated FTO substrates were placed in a muffle furnace and annealed in air at 550 °C for 1 h and then ultrasonically cleaned three times with DI water and then dried under compressed nitrogen. After immobilized on polytef-lined stainless hydrothermal reactors, the TiCl4-treated FTO glasses were immersed by 63 mL of TiCl4 growth aqueous. After the hydrothermal reaction in a forced-air convection drying oven for 12 h at 150 °C, TiO2 nanorods arrays (NRAs) were hydrothermal growth on the TiCl4-treated FTO glasses. The asprepared substrates were cleaned three times with DI water and dried under compressed nitrogen.


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To fabricate the micro-nano mesoscopic interface, the as-prepared highly ordered rutile TiO2 NRAs were hydrothermally etched in a mixture solution at 150 °C at different time intervals, which contains 40 mL concentrated HCl and 20 mL DI water. Then, the etched substrates were cleaned three times with DI water and then dried, as reported in a previous study42. The mesoscopic micro-nano interface utilized in this two-step hydrothermal reaction can be fabricated. Surfaces of these nano-micro interfaces were characterized using ultrahigh-resolution cold field scanning electron microscopy (SEM) (SU8010, HITACHI, Japan). 2.3 Cell culture and immunofluorescence staining Cancer cell line MCF-7 was cultured in DMEM, which contained 5% FBS and 1% penicilinstreptomycin, in a cell incubator (37 °C, 5 % CO2, Thermo Forma Series II, Thermo Scientific). After the cancer cells were treated by Tyrisin, the cells were diluted to a concentration of 104 ml1

. The mesoscopic nano-micro TiO2 substrates were washed by phosphate buffered saline (PBS)

at least 3 times. Then, 1 mL cancer cells sample were added on each substrate in a 12 well plate and cultured for 24 h. After being carefully washed with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100. Samples were blocked with 3% BSA for 30 min before incubation with 10 µg ml-1 anti-vinculin-FITC antibody for 2 h at room temperature and 10 µg ml-1 Alexa Fluotm 568 Phalloidin for 2 h at room temperature to label vinculin and F-actin, respectively. After being washed with PBS, 1 µg ml-1 DAPI was used to label the cell nuclei for 10 min.43-45 After being washed with DI water, laser scanning confocal microscope (Leica SP8, Germany) was used to characterize the fluorescence stain. 2.4 Cell morphology characterized by SEM


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After being cultured on the micro-nano mesoscopic TiO2 interface substrates for 24 h, cells were washed with PBS and then incubated with 4% glutaraldehyde overnight in 4 °C refrigeration to fix the cell skeleton. After being washed with DI water three times, the cells were successively dehydrated in ethyl alcohol with concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and absolute ethyl alcohol, respectively.46, 47 Then, the samples were freeze dried for 4 h. After gold sputtering on the samples, SEM was used to characterize cell morphology. 3. RESULTS AND DISCUSSION Two-step hydrothermal reaction was used to fabricate mesoscopic micro-nano TiO2 NRAs interface. The fabrication process is similar to our previous research39, where the micro-nano mesoscopic interface is transferred to poly(dimethylsiloxane) surfaces to increase surface hydrophobicity. First, the TiO2 NRAs nano interface was fabricated. As shown in Figure 1a, highly ordered aligned rutile TiO2 NRAs were fabricated on the TiCl4-treated FTO glass through hydrothermal reaction. The height of TiO2 NRAs was controlled by the hydrothermal reaction time. After reaction for 12 h, the height of the TiO2 film reached approximately 24 µm. The surface morphology of the as-prepared TiO2 NRAs is shown in Figure 2a. The second step was to fabricate the micro-sized voids on the nano-sized TiO2 NRA surfaces by HCl hydrothermal etching, as shown in Figure 1a. The process may be caused by the rapid hydrolysis of the Ti4+ ions over the precursors42 that caused the TiO2 nanorods to bend and assemble together, which was indicated by the white dotted line in Figure 2h. In this way, micro sized voids were generated on the nano sized TiO2 NRA surface.


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Figure 1. (a) Schematic of the TiO2 nanorods arrays with mesoscopic micro-nano interfaces using a two-step hydrothermal reaction. (b) Schematic of the cell morphology and nucleus deformation in response to flat, nanoscale, and mesoscopic micro-nanoscale substrates. The sizes of the voids were influenced by the HCl hydrothermal etching time. The HCI etching times in Figure 2a - 2f were 0, 2, 3, 4, 7, and 10 h. The dimensions of the as-prepared TiO2 nanorods arrays were about 180 nm. The dimension of the peak of the TiO2 nanorods was about 20 nm and the morphologies of the nanorods on the top were turbination with an angle of about30°. TiO2 nanorod arrays with diameter from 160 - 300 nm were used as substrates to capture circulating tumor cells after being modified with BSA-aptamer; the results showed that substrate with diameter of 230 nm had the highest cell capture efficiency.38 Therefore, the size of voids of the as-prepared TiO2 NRAs was coupled with the size of the nanoscale microvilli, filopodia, and extracelluar matrix scaffolds on cancer cell surfaces.5, 7 As shown in Figure 2i,


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after HCl hydrothermal etching for 3 h, the X size of the voids was increased to about 5.1 ± 1.1 µm. The X size was increased to about 10.5 ± 3.5 µm when the etching time increased to 10 h. The sizes of these voids on 10 h etched TiO2 NRAs substrates were similar to the sizes of the cancer cell nucleus. In the previous research, the influence of Si nanopillar length on the cell capture yields was investigated; the results indicated that substrates with Si nanopillars longer than 6 µm achieved a maximum cell capture yield.5 Therefore, this mesoscopic micro-nano interface on the TiO2 NRAs after HCl hydrothermal etching can be well coupled with cells because of the size effect. On the other hand, the ratio of the size (X) and height (Z) of the voids and the distance between voids were characterized, as shown in Figure 2j. The ratio of X and Z increased with increased etching time whereas the average distance between voids decreased from 0.88 µm to 0.40 µm when the hydrothermal etching time increased from 3 h to 10 h. Therefore, this kind of substrate with nanoscale TiO2 nanorods and microscale voids is suitable for investigating the morphology of the cells and the deformation of nucleus.


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Figure 2. SEM images of the TiO2 nanorods arrays hydrothermally etched by HCl for (a) 0 h, (b) 2 h, (c) 3 h, (d) 4 h, (e) 7 h, and (f) 10 h, respectively. Scale bars in (a) - (f) are 10 µm and in the insets in (a) and (b) are 500 nm. (g) and (h) are the SEM images of the cross section of the asprepared TiO2 NRAs and 10 h etched TiO2 NRAs. The white dotted line in (h) indicated the bended TiO2 nanorods after hydrothermal etched by HCl. Scale bars in (g) and (h) are 5 µm. (i) The sizes of the voids were related to the HCl etching time. (j) The distance between voids and the size-height ratio of voids were modulated by the hydrothermal etching time. Nanorough surfaces without any antibodies modified can effectively capture cancer cells because of their strong adhesion between cell extracellular matrix and substrates.48 On the other hand, nanorough surfaces modified with antibodies can specifically increase the capture efficiency of circulating tumor cells.33 During the detaching process from the primary tumors and invading into blood circulation, the epithelial-mesenchymal transition of adherent epithelial cells to migratory mesenchymal state occurs in circulating tumor cells. The nanostructure characterization of the cancer cells surface can contribute to cell migration and entry. Cell morphologies were affected by the microenvironment. The SEM images in Figure 3 show the MCF-7 cells morphology after being cultured on the substrates for 24 h. The results indicated that cells morphology had a noticeable change. Large amounts of microvilli flocked together on the top and the center surface of cells that were cultured on the flat glass surface, as shown in Figure 3a. Surface roughness of flat glass was about 1 µm. Microvillus or pseudopodia on the bottom surface was stretched outside to catch the substrate surface, and the cell membrane was in close contact with the substrate surface. From the zoomed-in SEM image in Figure 3b, the diameter of the microvillus or pseudopodia was changed from 42 nm to 133 nm, which was in agreement with previous reports.49 Length of the expanded pseudopodia can increase to 7.8 µm.


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Figure 3. SEM images of the MCF-7 cells cultured on (a) the flat glass substrate and the TiO2 NRAs substrates etched by HCl for (b) 2 h, (c) 3 h, (h) 4 h, (i) 7 h, and (g) 10 h, respectively. (d) - (f), (k) - (m) are the zoomed-in SEM images corresponding to (a) - (c) and (h) - (j), respectively. Scale bars are all 2 µm. SEM images in Figure 3b, 3c, 3h - 3j show the MCF-7 cell morphology after being cultured on the substrates that have been etched by HCl for 2, 3, 4, 7 and 10 h, respectively. When the HCl hydrothermal etching time increased from 2 h to 10 h, the size of the voids on the surface increased. In this condition, cell morphologies changed dramatically in these micro-nano mesoscopic bio-interfaces microenvironments. The microvillus or pseudopodia morphologies and distribution on cell surfaces also changed. Compared with the cell on the flat substrate in Figure 3a, the amount of the microvillus or pseudopodia on the top and the center of the cell surface decreased, as shown in Figure 3b, 3c, 3h - 3j. Moreover, microvillus or pseudopodia moved extended outward the cell membrane, which resulted to smoother the cell surfaces. The Zoom-in SEM images in Figure 3e, 3f, 3k - 3m indicated that the cell membranes invaded the voids and the cells membrane surfaces became smooth when the void size increased. Previous


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research had indicated that the nucleus is stiffer than the cytoplasm.50 Fluidity of the cell membrane is better than that of the cell nucleus. Therefore, cell membrane was spread widely when the cell height was decreased because the cell nucleus invaded the voids on the micro-nano interface. As illustrated in Figure 1b, the cell nucleus maintained its spherical or ellipsoidal shape on the flat glass substrate but changed to a deformed shape on the mesoscopic micro-nano interface, especially on the TiO2 NRAs substrate etched for 10 h.

Figure 4. (a) The longitudinal section of the nucleus on glass and TiO2 NRAs substrates. (b, d) 3D fluorescence images of the nucleus on 10 h etched TiO2 NRAs substrate and glass substrate, respectively. (c, e) Z slices images of the nucleus corresponding to (b) and (d), respectively. (f,


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g) The relative fluorescence intensity of the cross section of straight-cut nucleus corresponding to (b) and (d), respectively. Scale bars are all 10 µm. As shown in Figure 4a, longitudinal section fluorescence images of the nucleus on glass and TiO2 NRAs substrates indicated that the nucleus invaded the voids when the etching times were increased. When the cells were cultured on the glass substrate and the TiO2 NRAs substrate, the bottom of nucleus was smooth because the surface roughness of substrates was smaller than 1 µm. When the hydrothermal etching times were increased from 3 h to 10 h, the void sizes were larger than 1 µm and the nucleus deformed dramatically from 20% to 90% in the Z-direction. 3D fluorescence image of the nucleus on glass substrate was shown in Figure 4d and the corresponding Z slices images were shown in Figure 4e. The results indicated that the nucleus morphology was hemispheric and the distribution of chromatin is uniform. 3D fluorescence image of the nucleus on 10 h etched TiO2 NRAs substrate was shown in Figure 4b and the corresponding Z slices images were shown in Figure 4c. The results indicated that the nucleus was divided into parts in the voids. Compared to the relative fluorescence intensity of the cross section of straight-cut nucleus on the glass substrate in Figure 4g, the relative fluorescence intensity of that on the 10 h etched TiO2 NRAs substrate in Figure 4f further indicated the nucleus was deformed. On the other hand, the height of the nucleus on the different substrates was related to the height of cell. Results in Figure 3 and Figure 4a indicated that the height of cell was decreased when the void size increased. Cell morphology regulated by the substrates may be explained as contact guidance, that is, cells can sense the surface structures or surrounding topography.51 Cells can sense the surface as continuous or discontinuous and can react accordingly. When the distance between discontinuity is smaller than the size of the microvillus or pseudopodia, cells can distinguish that the substrate


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is flat, such as flat glass. When the distance is larger than the size of the microvillus or pseudopodia but smaller than 1 µm, cells may decide that the substrate is discontinuous and the nucleus can keep its shape.26 Similar results can be demonstrated when the cells are cultured on vertical high density silicon nanocolumn arrays.52 FIB-SEM images of HL-1 cells on nanopillar substrates showed that the nuclear envelope was bended within the range of 1 µm.53 When the distance is larger than 1 µm but smaller than the size of the cell, nucleus can begin its deformation. In the present study, nucleus morphology was regulated by the size of the voids. As shown in Figure 4c, nucleus kept its spherical morphology when cells were cultured on the flat glass substrate. When the HCl hydrothermal etching time increased from 3 h to 10 h, the average size of the voids on TiO2 NRAs substrates increased from about 3.9 µm to about 8.0 µm and the height increased from about 7.5 µm to 10.7 µm. Under this micrometer sized microenvironment, internal cellular structure was reorganized and the nucleus was deformed in response to the surface topography of the substrate.28, 29 Thus, nuclear deformation depends on the void size. As shown in Figure 4a, the nucleus showed severe deformation when the cells were cultured on the TiO2 NRAs etched for 10 h. The voids were parted off from one another by TiO2 NRAs, and the voids were V-shaped. When the hydrothermal etching time was increased from 3 h to 10 h, the average distances between voids were decreased from 0.88 µm to 0.40 µm, as shown in Figure 2j. Nanopillar-induced nuclear deformation was investigated and the deformation was affected by the nanopillar radius, height, and pitch as reported in the previous study.54 Nuclear deformation was determined by nuclear stiffness and the opposing effects from actin and intermediate filaments. Because of the nuclear stiffness, the voids were not filled by nuclear.


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Figure 5. (a) - (f) are the immunofluorescence images of the cells cultured on (a) the flat glass substrate and the TiO2 NRAs substrates etched by HCl for (b) 2 h, (c) 3 h, (d) 4 h, (e) 7 h, and (f) 10 h, respectively. Scale bars are all 20 µm. (g) Cell area on the different substrates. The nucleus shape is tightly regulated by the substrates geometry, and this regulation occurs through a dome-like actin cap which covers the top of nucleus.55 Actin filament bundles in the actin cap are terminated by vinculin-containing focal adhesions, which connect the nucleus and cell basal.55 The integrity of the perinuclear actin cap depends on the F-actin assembly and plays a critical role in nuclear shape.55 These actin cap above the nucleus can apply a uniform pressure on nucleus that pushes the nucleus towards the substrate, as illustrated in Figure 6g. On the other hand, the actin underneath the nucleus applied a localized pressure to pull down the nucleus, as illustrated in Figure 6g. When the cells were cultured on the flat substrate, there are no organized actins or fewer and thinner actin filament bundles. When the cells were cultured on the


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mesoscopic micro-nano interface substrate, a large amount of actin accumulated around the nanorods. The force applied by the localized pressure was independent of the pitch, whereas the force applied by the uniform pressure increased with the square of the pitch.54 In the present work, the force caused by the uniform pressure increased with the increased size of voids and the distance between voids. Therefore, the nucleus was deformed severely when the cells were cultured on the substrate with large voids, as shown in Figure 4a - 4c. These conclusions can also be found from the 3D immunofluorescence stain of the cells, as shown in Figure 5a - 5f. On the flat glass substrate, Alexa Fluotm 568-labled phalloidin stained F-actin fibers distribute parallelly at the edge of the cell whereas the FITC-labeled vinculin distributed homogeneously around the nucleus. F-actin was discretely distributed on the cell membrane, which connected vinculin to maintain the cell shape. When the cells were cultured on the TiO2 NRAs etched by HCl for 10 h, a large amount of F-actin accumulated around the TiO2 nanorods and on the top of the cells, as shown in Figure 5f. The influences of this micro-nano mesoscopic bio-interface on cell morphologies can be found through the cell area. In order to calculate the cell area, 60 ~ 200 cells on the each kind of substrate were counted to characterize the influence of micro-nano biointerface on cell area. As shown in Figure 5g, cell area was increased when the cells were cultured on glass substrate, as-prepared and 2 h etched TiO2 NRAs substrates. When the etched time increased from 2 h to 10 h, the cell area was decreased. Because part of the cell membrane invaded the voids.


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Figure 6. (a) Immunofluorescence image of the cells cultured on the TiO2 NRAs substrate etched by HCl for 10 h. The arrows indicated the cells that are in the process of mitosis. Scale bar is 50 µm. (b, d) 3D fluorescence images of a mitotic cell cultured on the 10 h etched TiO2 NRAs substrate: (b) F-actin and (d) nucleus. (c, e) Fluorescence images of the longitudinal and cross section images of the cell in (b). (f) The relative fluorescence intensity of F-actin and nucleus at the different cross section of straight-cut of the mitotic cell. Scale bars in (b) - (f) are 10 µm. (g) Schematic of the reversible process of cell morphology and nucleus deformation when the cells were cultured on the mesoscopic micro-nano bio-interface. Cell morphology and nucleus shape were regulated by the combination of the uniform pressure and localized pressure, which were applied on the top and underneath of the nucleus, respectively.


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Our results indicated that mesoscopic micro-nano interfaces can induce nucleus deformation and this deformation affected the behavior of the entire cell. Cells grown on this micro-nano interface showed the same level of viability as cells grown on the flat glass substrate. During the mitosis process, sufficient force generated by actomyosin contraction and hydrostatic pressure to round up against substrates to divide.56 As shown in Figure 6a, after cultured on the 10 h-etched TiO2 NRAs substrate for 24 h, a large proportion of the cells had accomplished their mitotic process and reattached on the substrate. The white arrows indicated that mitosis was in progress. The red arrows indicated that the cell recovered its spherical morphology and starts its mitosis. 3D, the longitudinal and cross section fluorescence images of a mitotic cell cultured on the 10 h etched TiO2 NRAs substrate were shown in Figure 6b - 6d. The relative fluorescence intensity of F-actin and nucleus at the different cross section of straight-cut of the mitotic cell was shown in Figure 6e. These results indicated that the nucleus was parallel during the mitosis. As illustrated in Figure 6g, the micro-nano heterostructure can induce nucleus deformation under the combination roles of the uniform and localized pressure. Once the cells entered the process of mitosis, nucleus will recover its spherical shape. When the mitosis process ended, the cells will adapt to the microenvironment. This attachment and detachment process of the cell and nucleus morphology will continue during the cell culture. CONCLUSION Inspired by the cell characterization, micro-nano mesoscopic interface substrates have been fabricated through two-step hydrothermal reaction. The size of the micro scaled voids and the distance between voids were controlled by the hydrothermal etching time. When the etching time increased from 3 h to 10 h, the sizes of the voids increased from 5.1 µm to 10.5 µm whereas the distances between voids decreased 0.88 µm to 0.40 µm. Nucleus invaded into the voids when the


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height of voids increased. Large voids can accelerate the nucleus deformation. Nucleus deformation induced by the micro-nano mesoscopic interface showed no effect on its mitosis during the cell culture. Nucleus deformation was reversible under the combination role of the uniform pressure and localized pressure. Cell morphology and nucleus shape interacted with each other to adapt to the microenvironment. This mesoscopic micro-nano interface in the present work provided a new insight into the cell-biomaterials interface to investigate cell behaviors. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (R. H.). *E-mail: [email protected] (Y. L.) *E-mail: [email protected] (Y. C.). Present Addresses §

Present Address: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology,

Chinese Academy of Sciences, Wuhan 430072, China. Author Contributions ⊥

(H. L. M. R. and J. X.) Liu H.N., Ruan M.L. and Xiao J.R. contributed equally. All authors

contributed to the manuscript preparation. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financial supported by the National Natural Science Foundation of China (Grant No.81402466, 61404060,31600801) and the Wuhan Basic Research for Application Project (2015071704011602, 2015011701011595). We acknowledge the PhD research foundation of Jianghan University. REFERENCES (1) Chaffer, C. L.; Weinberg, R. A. A Perspective on Cancer Cell Metastasis. Science 2011, 331, 1559-1564. (2) Yu, M.; Bardia, A.; Wittner, B. S.; Stott, S. L.; Smas, M. E.; Ting, D. T.; Isakoff, S. J.; Ciciliano, J. C.; Wells, M. N.; Shah, A. M.; Concannon, K. F.; Donaldson, M. C.; Sequist, L. V.; Brachtel, E.; Sgroi, D.; Baselga, J.; Ramaswamy, S.; Toner, M.; Haber, D. A.; Maheswaran, S. Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition. Science 2013, 339, 580-584. (3) Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W. M. M.; Hayes, D. F. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. N. Engl. J. Med. 2004, 351, 781791. (4) Wang, S. T.; Liu, K.; Liu, J. A.; Yu, Z. T. F.; Xu, X. W.; Zhao, L. B.; Lee, T.; Lee, E. K.; Reiss, J.; Lee, Y. K.; Chung, L. W. K.; Huang, J. T.; Rettig, M.; Seligson, D.; Duraiswamy, K. N.; Shen, C. K. F.; Tseng, H. R. Highly Efficient Capture of Circulating Tumor Cells by Using


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