Temperature-gating titania nanotubes regulate migration of

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Functional Nanostructured Materials (including low-D carbon)

Temperature-gating titania nanotubes regulate migration of endothelial cells Sai Wu, Deteng Zhang, Jun Bai, Wang Du, Yiyuan Duan, Yixiao Liu, Xiaohui Zou, HongWei Ouyang, and Changyou Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17530 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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ACS Applied Materials & Interfaces

Temperature-gating titania nanotubes regulate migration of endothelial cells Sai Wua, Deteng Zhanga, Jun Baia, Wang Dua, Yiyuan Duana, Yixiao Liub, Xiaohui Zoub, Hongwei Ouyangb,c, Changyou Gaoa,c* a

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer

Science and Engineering, Zhejiang University, Hangzhou 310027, China b

Centre for Stem-cell and Tissue Engineering, School of Medicine, Zhejiang University, Hangzhou

310027, China. c

Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang

University, Hangzhou 310058, China E-mail address: [email protected]

1

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Abstract External stimuli-responsive biomaterials represent a type of promising candidates for addressing the complexity of biological systems. In this study, a platform based on the combination of temperature-sensitive polymers and a nanotube array was developed for loading of sphingosine 1-phosphate (S1P) and regulating migration of endothelial cells (ECs) at desired conditions. The localized release dosage of effectors could be controlled by the change of environmental temperature. At a culture temperature above the lower critical solution temperature (LCST), the polymers “gatekeeper” with a collapsed conformation allowed the release of S1P, which in turn enhanced migration of ECs. The migration rate of single cell was significantly enhanced up to 58.5%, and the collective migration distance was also promoted to 25.1% at 24 h and 33.2% at 48 h. The cell morphology, focal adhesion, organization of cytoskeleton, and expression of genes and proteins related to migration were studied to unveil the intrinsic mechanisms. The cell mobility was regulated by the released S1P, which would bind with S1PR1 receptor on cell membrane and trigger Rho GTPases pathway. Key words: nanotubes; titanium; cell migration; sphingosine 1-phosphate; temperature-gating

2


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Introduction Titanium and its alloys have experienced wide clinical use nowadays, especially in dental and orthopedic surgery because of their excellent mechanical strength, outstanding corrosion and wear-resistance, and relatively low density.1 They have been made into implants for load-bearing regions such as titanium hips and titanium knees, and applied in cardiovascular implants, orthopedic prostheses and dental implants.2 Nonetheless, the instructive disadvantages within unmodified titanium including the poor integration with hosts’ surrounding tissues, as well as the susceptibility to bacterial infections in some cases have limits its further applications.3 One of the promising ways to improve the success of long-term implants is the acceleration of revascularization, which plays a critical role in improving the integration of implants and decreasing the morbidity of infections.4 So far different approaches of surface modification have been attempted to enhance the revascularization by endowing the surface with micro- and nano-architecture, and desired chemistry and energy states, which can significantly alter the adhesion, migration, proliferation and gene expressions of cells.5 For example, the titania nanotubes on titanium substrate can significantly improve cell compatibility and alter cell behaviors

6-7

due to their similar dimensional micro- and

nano-scale with extracellular matrix architecture. The TiO2 nanostructure on Ti surface has proven to be beneficial in promoting adhesion, growth and migration of endothelial cells, one of the major types of cells for angiogenesis. Mohan et al. found that uniform TiO2 nanoscale modification of different nanostructures on metallic Ti substrate enhances the ECs proliferation, whereas inhibits the proliferation of smooth muscle cells and adhesion of platelets.8 The nanotubular titania surfaces can significantly enhance the proliferation of endothelial cells and help SMC maintain their non-proliferative phenotype, which are essential for resistance of blunt thrombosis and 3



anti-restenosis.9 Meanwhile, because of the well-controlled size, distribution and geometry, surface chemistry, and large specific surface area, the titania nanotube array also constitutes an excellent delivery platform for antibiotics, growth factors and even inorganic bioactive elements to promote the adhesion, proliferation and differentiation of ECs and osteoblasts. 10-12 Cell migration is the primary and important process in regenerative medicine, which is directly related to success of the artificial implants. Therefore, modulation of cell behaviors is important for designing and creating novel biomaterials in tissue repair and regeneration. Smart interface materials are a good choice, which are capable of undergoing rapid changes of conformation and physicochemical properties once triggered. The smart polymer, poly(N-isopropylacrylamide) (PNIPAAm), has the ability to reversibly change its conformation from extended coil to a collapsed globule-like structure below and above the lower critical solution temperature (LCST), respectively.13 Hou et al. fabricated a smart artificial single nanochannel system, which allows control over temperature-tunable ionic transport property through modification inside the nanochannel.14 Our previous work has demonstrated that doxorubicin-loaded PNIPAAm micro-gel particles cause stronger cytotoxicity of lung adenocarcinoma (A549) cells due to their intracellular volume increase and thereby drug release below LCST.15 Cell migration is not only governed by physicochemical properties of a substrate, but also regulated simultaneously by bioactive molecules in their microenvironment.16-18 The potential for exogenous accelerators to enhance cell migration havs been tested in only a few studies. Biologically active sphingosine 1-phosphate (S1P) is a kind of lipids released by activated platelets. It is involved in a range of physiological and pathological processes, including angiogenesis, wound healing, cardiovascular diseases, cancer and autoimmune disorders. It can interact with many types of cells to 4


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enhance cell proliferation, migration, and survival through a family of G protein-coupled receptors.19 Wacker and his coworkers showed that after stimulated with S1P, the mobility of endothelial cells (EC) is enhanced on mPEG hydrogels tethered with RGD peptides.20 Herein we develop a platform of a temperature-gating titania nanotube array for localized and controlled release of S1P. Temperature-driven gatekeeper PNIPAAm molecules are grafted onto the inner walls of nanotubes, which can switch the open and close of nanotubes in response to temperature stimuli. When the platform is cultured at room temperature, the gatekeeper PNIPAAm will block the pore entrance and stably retain the payloads inside the tubes, which is apparently beneficial for the storage and long shelf-life. Once the temperature is raised above the LCST in applications (for example 37°C for cell culture), the gatekeeper undergoes a conformation transition to release the cargos. The mobility of ECs in a single and collective migration way is then studied for the first time in response to the gating (Scheme 1). The mechanisms of activation of S1P are further analyzed and discussed according to the results of cell morphology, focal adhesion, actin organization and expression of relative genes and proteins. 2. Experimental Section Materials Pure titanium sheets (99.5%, 0.25 mm thick, Baoji Sheng Tai Metal Material Co. Ltd., China) were used as substrates for the anodization of TiO2 nanotube arrays. N-Isopropylacrylamide (NIPAAm, Wako Chemical. Co., Japan) was dissolved in hexane and recrystallized for purification, dried in a vacuum oven at 25 ºC, and stored at -20 ºC before use. 4,4’-Azobis(4-cyanovaleric acid) (≥75%), dopamine

(DA),

polyethyleneimine

(PEI),

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. 5



Fluorescein sodium salt (FL) and sphingosine 1-phosphate (S1P) were provided from Abcam. All other reagents were of analytical grade. Milli-Q water and ultrapure nitrogen were used throughout the experiments. Preparation of titania nanotubes arrays The highly ordered TiO2 nanotube arrays were fabricated according to previous literatures using an electrochemical anodization method.21-22 Pure titanium (7 mm×7 mm×0.25 mm) was used as substrate for anodization. After mechanical polishing, the titanium plate was cleaned by sonication in methylbenzene, acetone, ethanol and water for 10 min, respectively, and was dried with a nitrogen gas flow. Next, a two-step anodization of titanium was performed in an electrochemical cell using the titanium plate as an anode and the platinum plate as a cathode. The distance between the anode and cathode was kept as 20 mm. In the first anodization step, a constant DC voltage of 50 V was applied for 12 h in H2O/glycerol (1:1, v/v) electrolyte containing 0.27 M NH4F at room temperature. After removing the resultant TiO2 layer by sonication, the remnant titanium surface with nano-textures was applied for the second anodization, using the same electrolyte at 10 V for 30 min. Synthesis and characterization of PNIPAAm-COOH PNIPAAm-COOH was synthesized according to a method reported previously.23 5 g monomer NIPAAm and 50 mg initiator 4,4′-azobis(4-cyanovaleric acid) were added into a flask and dissolved by 50 mL methanol. The solution was degassed by bubbling with nitrogen and reacted at 65 ºC for 3 h. Then the product PNIPAAm-COOH was precipitated from hot water. After washing twice in hot water, the polymers were dissolved and lyophilized. Mn (130 kDa) and polydispersity index (PDI, 3.2) of PNIPAAm-COOH were determined using gel permeation chromatography (GPC 220, Polymer Laboratories Inc., MA). Tetrahydrofuran (THF) was used as the solvent, and narrow dispersive 6


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polystyrenes

were

used

as

calibration

standards.

Temperature-responsive

property

of

PNIPAAm-COOH was determined by a turbidity method. PNIPAAm-COOH was dissolved in water and phosphate buffered saline (PBS, pH 7.4) respectively, with a final concentration of 1 mg/mL. The transmittance value of the polymer solution was measured as a function of temperature at λ=500 nm, by using a UV-vis spectrophotometer. Immobilization of PNIPAAm-COOH onto TiO2 nanotubes arrays Modification of TiO2 nanotubes (TiNT) by PDA/PEI was accomplished via the copolymerization of dopamine (DA) with PEI according to a previous method.24 DA and PEI were dissolved in 10 mM Tris buffer at pH=8.5 to obtain 1 mg/mL solution, into which TiO2 nanotubes were immediately incubated for a certain period of time under vacuum condition. After rinsed with water for three times, the TiNT was dried by a nitrogen gas flow to obtain the PDA/PEI coated TiNT, which is designated as TiNT-NH2. To graft the polymer PNIPAAm-COOH onto the nanotube walls, 3% (w/v) PNIPAAm-COOH aqueous solution was incubated in EDC and NHS (PNIPAAm: EDC: NHS= 1: 1.2: 1.2 mol) for 1 h at room temperature. Then the TiNT-NH2 was incubated with the activated PNIPAAm solution at a final pH value of 5.5 for 24 h to obtain the PNIPAAm-coated TiNT, which is designated as TiNT-P. Loading and release of cargoes into the nanotubes The nanotubes in the array of TiNT-P were employed as nanoreservoirs to load S1P and fluorescein sodium salt, a model substance for monitoring the loading and release. The nanotubes were loaded with cargos in the “on-state” of nanotubes via a simplified lyophilization method. In brief, 200 L 1 mM solution of S1P (NaOH buffer, 1 mM, pH 7.5) was pipetted onto the TiNT-P (1 cm × 1 cm) to allow even coverage on the surface. The solution was then allowed to dry on the TiNT-P under 7



vacuum at 37 ºC for 2 h. 1 mol S1P were loaded into the nanotubes after several repeating procedures. 1 mL water was immediately added on the surface to eliminate the redundant S1P following the final drying at 28 ºC. The solution was collected after rinsing for further detection. The TiNT-P loaded with S1P is marked as TiNT-P/S1P. The concentrations of the original and the rinse solutions were measured by a S1P ELISA kit. The loading efficiency was thus obtained by 100%*(Co-Cr)/Co, where Co and Cr are the original concentration and the concentration in the rinse solution, respectively. To investigate the release of loaded S1P, the nanotubes were soaked in 500 µL of PBS in a microfuge tube at 37 ºC or 28 ºC with orbital shaking at 70 rpm. At the predetermined time intervals, 200 µL solution was withdrawn to determine the release kinetics, and fresh PBS was replaced to maintain a constant volume. The released S1P was quantified by using a S1P ELISA kit (Echelon, USA), and its concentration was compensated for dilutions because of the replacement with fresh PBS. The gating release of S1P was demonstrated by cyclic exposure to 37 ºC and 28 ºC water bath at the time intervals of 5 min, and the released S1P was measured at each transition point. Surface characterizations The morphologies of the nanotube arrays were investigated by using a field emission scanning electron microscopy (FESEM, Hitachi S-4800, Japan) at an acceleration voltage of 3 kV. The water contact angles on samples were measured by dispensing 5 μL of water onto the surfaces with a DSA 100 contact angle measuring system (Krüss, Germany). In order to measure the thermal sensitivity of TiNT-P, a chamber with circulating water was mounted to the apparatus for the control of temperature. The samples were initially placed into a chamber, and the needle was vertically placed. After the samples were equilibrated to the set temperature for 30 min, a sessile drop of water at the 8


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same temperature was dropped vertically onto the surface, and the water contact angles were measured by using the same method. X-ray photoelectron spectroscopy (XPS, Escalab 250Xi, ThermoFisher, UK) was used to investigate the chemical components on the substrates with a correction of C1s peak at 284.6 eV. The XPS spectra at different depth of the nanotubes were obtained by etching with Ar+ for different time. Cell culture Endothelial cells (ECs) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). ECs were incubated in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Gibco, USA), which was added with 10% fetal bovine serum (FBS, Sijiqing Inc., China), 100 U/mL penicillin and 100 μg/mL streptomycin, in a 37 ºC, 5% CO2 incubator. Cell adhesion and proliferation TiNT-P and TiNT-P/S1P samples were sterilized and placed in a 24-well plate, followed by seeding ECs at a density of 104 cells/cm2. Cells were incubated in 5 μg/mL fluorescein diacetate (FDA) for 5 min and inspected by a fluorescence microscope (IX81, Olympus, Japan) after cultured at 37 ºC and 28 ºC for 4 h and 24 h respectively. The number and spreading area of ECs were calculated according to the fluorescent images by the Image Pro Plus software. For cell proliferation assay, ECs were seeded at a density of 4  104 cells/cm2 and cultured at 37 °C and 28 °C for 1 d and 3 d, respectively. Cells cultured on tissue culture polystyrene (TCPS) at 37 ºC and 28 ºC were used as control groups, respectively. After 1 and 3 days, the quantitative analysis of cell proliferation was performed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.25 At 1 d and 3 d, the samples were taken out and rinsed three times with PBS, 9



and were transferred to a new plate, into which the MTT solution was added. After the cells were further incubated at 37 °C for 4 h, DMSO was added to dissolve the formed formazen. The absorbance value of the solution was detected by a microplate reader (Tecan M200 PRO, Tecan, Switzerland) at 490 nm. Cell proliferation was determined according to the ratio of experimental groups to the control ones, respectively. Cell migration In the wound healing assay experiments, a 2-well silicone insert with a defined narrow gap was used to improve the reproducibility and minimize the surface damage. The ECs were seeded at a density of 5  105/cm2 on each sample, which was partially covered by the silicone insert in the middle position in prior. After the cells were cultured for 12 h to reach confluence on the samples, the 2 well culture-insert was removed, forming a narrow cell-free stripe. Cells were labeled by live cell tracker CM-DiO and incubated for another 2 days in RPMI-1640 with a lower concentration of FBS (0.4%) to suppress cell proliferation. Cells were cultured at 37 ºC and 28 ºC and observed under the fluorescence microscope, respectively. The single cell migration was assayed on TiNT-P and TiNT-P/S1P surfaces at above (37 ºC) and below (28 ºC) LCST of PNIPAAm, respectively. The CM-DiO labeled ECs were seeded at a lower density (104 cells/cm2) in order to avoid cell-cell interaction, and cultured in RPMI-1640 with 10% FBS at 28 ºC to ensure the adhesion of ECs in the first 12 h. Then the cells were cultured in RPMI-1640 with 0.4% FBS, and placed in an incubation chamber, which was mounted onto a fluorescence microscope to in situ monitor the migration process. The images were taken at every 15 min intervals during the whole observation time. Based on the manual tracking and reconstruction of these images, the cell movement trajectories were obtained using NIH Image J software. The 10


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migration distance and rate were calculated by the Chemotaxis software (Ibidi, Germany). More than 50 cells were selected randomly and triplicate experiments were conducted for each sample. Study of interactions between cells and substrate Cell morphology and detailed cell-biomaterial interactions were studied by FESEM. After 12 h incubation of ECs in RPMI-1640 with 10% FBS at 28 ºC for 12 h, the culture medium was replaced with low FBS, and cultured at 37 ºC or 28 ºC, respectively. At the pre-determined time, the samples with attached cells were gently rinsed with PBS, fixed with 4% paraformaldehyde and dehydrated using graded ethanol. Finally, the samples were dried by using a critical point dryer (K850, EMITHCH, UK) to maintain the original feature, and observed by FESEM. Cell nucleus, focal adhesion, and actin fibers were stained by fluorescent dyes respectively, to reveal the cell morphology, focal adhesion complexes and skeleton organization. In Brief, after 12 h incubation in RPMI-1640 with 10% FBS at 28 ºC, and 4 h incubation at 37 ºC or 28 ºC, the cells were gently rinsed with FBS and fixed with 4% paraformaldehyde. After three washes in PBS, the cells were treated with 0.5% Triton X-100/PBS for the increase of permeability. Bovine serum albumin (BSA)/PBS solution (5%, w/v) was used to block the non-specific protein adsorption on the sample. After that, the cells were incubated with monoclonal antibody targeting vinculin, and rinsed with BSA/PBS solution. Next, the samples were treated with the FITC-labeled goat-anti-mouse second antibody, rhodamine phalloidin, and 4’,6-diamidino-2-phenylindole (DAPI) respectively. Then images of cells were taken using a confocal laser scanning microscope (CLSM, LSM 780, Zeiss, Germany). Three independent experiments were carried out. Characterization of EC functions To detect the secretion of PGI-2 and tPA by ECs, the culture medium was collected at 24 h and 11



measured by human PGI-2 kit (Rapid Bio, China) and human TPA ELISA kit (Boster Bio-engineering, China) respectively. The gene expression of CD31, CD144, eNOS, biglycan and THBD was detected by real-time PCR assay. First, total RNA was isolated by a TRIzol® Plus RNA Purification Kit (Invitrogen, USA). The cDNA templates were converted from the RNA population by SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen, USA). Following the generation of a cDNA template, the PCR reaction was optimized, performed and analyzed. ECs-related gene primers were designed and listed in Table S1. As a comparison, the group with 0.5 mM S1P in bulk medium was set up as control, and marked as TiNT-P+S1P. Single-cell gene expression analysis After 12 h incubation of ECs in RPMI-1640 with 10% FBS at 28 ºC, and the subsequent 4 h in low FBS medium at 37 ºC or 28 ºC, trypsin/EDTA solution (0.025% trypsin and 0.01% EDTA in PBS) was used to digest and obtain the cell suspension. Single cells were manually picked up one by one under a phase-contrast microscope. cDNA reverse transcription and pre-amplification of selected genes were performed by using the Single Cell Sequence Specific Amplification Kit (Vazyme). Generated cDNA was diluted with nuclease-free water. The quantitative PCR analysis was performed on the BioMark HD platform (Fluidigm) using 48.48 Dynamic Array integrated fluidics chips (M48, Fluidigm), which allows the simultaneous detection and quantification of 48 genes in each of 48 samples. The primers used in single-cell gene analysis are listed in Table S2. The data of single-cell gene expression was initially analyzed with Fluidigm Data Collection software. The Heatmap and cluster analysis of genes and samples were analyzed in R software by using the Pheatmap package and Vegan package. Principal component analysis was visualized in R using ggbiplot function. Western blotting 12


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After being cultured at 37 ºC or 28 ºC, the ECs were washed twice with PBS and added with radio immunoprecipitation assay buffer (RIPA) for cell lysis. Protease inhibitors was added into RIPA lysis to prevent proteolysis and maintain phosphorylation. After high speed centrifugation of the lysate, the resultant supernatant was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then the proteins were electrotransferred to a PVDF membrane and immunostained. The detection of proteins was performed by the immunological method using an enhanced chemiluminescence system (ECL Western Blotting Substrate, Pierce, USA). The quantitative data of integral optical density (IOD) was determined by Image J software. Rho GTPase activity, which reveals endogenous levels of GTP-bound (active) Rac1 and Cdc42, was assayed by using Active Rac1 Detection Kit and Active Cdc42 Detection kit. Cells were lysed homogenously and centrifuged at a high speed. Rac1 and Cdc42 pull-down assays were conducted by using GST-Pak1 protein-binding domain. After electrophoresis and transfer of the pull-down supernatant, the membrane was blocked and treated with first antibody. Finally, western blot detection with a goat anti-mouse IgG second antibody was performed using SuperSignal® West Dura Extended Duration Substrate. The integrated optical density was analyzed by Image J software. Statistical tests The data are displayed as mean ± standard deviation. Statistical analysis was conducted by one-way ANOVA via the Turkey post hoc method. and the significance was set as p < 0.05. Results and Discussion Preparation and characterization of TiNT-P/S1P As shown in Scheme 1, the temperature-gating controlled-release system was constructed on a titania nanotube array grafted with the thermo-sensitive PNIPAAm polymers. The titania nanotube array was 13



fabricated by a two-step electrochemical anodization approach, which was served as an ideal localized drug-delivery system by loading payloads into the nanotubes. The grafted PNIPAAm can undergo conformation change in response to small variation in temperature. When the local environmental temperature is below the lower critical solution temperature (LCST), the polymer chains are extended to block the tubes and retain the payloads. Once the local environmental temperature exceeds the LCST of PNIPAAm, the polymer chains are collapsed, leading to the open of the nanotubes and thereby releasing the pre-loaded S1P functional molecules. The titania nanotube array was prepared on the outermost layer of a titanium (Ti) substrate by using a previously described anodization method, which is most convenient to generate the nanotube arrays with diameters ranging from 15 nm to 250 nm and lengths up to 1 mm.26 The parameters of anodization including voltage, current, anodization time, electrolyte, etc. determine the structure of nanotube arrays cooperatively. In this work, the nanotube array was generated by electrochemical anodization of Ti substrate at 10 V for 30 min in the glycerol/H2O medium containing 0.27 M NH4F. Figure 1 shows that the obtained nanotubes were vertically ordered array. The average inner diameter of the tubules was 23.6±3.8 nm, and the corresponding density of nanotubes was calculated to be 2.08×1010 cm−2. The wall thickness was about 10 nm. Figure 1d displays the cross section of the nanotube array with a length of ≈ 1 μm, which was primarily determined by the reaction time. As shown in Figure 1a, the small interspaces between nanotubes were resulted from the high density of nanotubes. The dense distribution and small diameter are thought to be linked to the weak chemical dissolution of TiO2 by fluoride ions under low concentration of hydrogen ions.27 Dopamine can form a thin and amino-capping polydopamine (PDA) layer by self-polymerization. However, this layer is not dense and compact enough due to the short chains of PDA oligomers and 14


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the non-covalent connections in PDA. To gain the improvement of PDA layer and subsequent immobilization of PNIPAAm, a denser and more compact layer with more reactive amino groups was prepared by grafting polyethyleneimine (PEI) with long chains on the PDA layer during the self-polymerization. The PDA/PEI macromolecular layer was formed by impregnating the TiNT array into an alkaline DA/PEI solution to allow self-polymerization of dopamine and coupling reaction between PDA and PEI.24 There were no obvious aggregates on the TiNT surfaces (Figure S1), suggesting that the resulted PDA/PEI layer was very homogenous. This PDA/PEI layer on the TiNT-PDA/PEI

was then

served

as the stable intermediate layer to

immobilize the

temperature-sensitive switch, PNIPAAm, via the reaction between amino groups on surface and carboxyl groups of PNIPAAm polymers. The

PNIPAAm-COOH

polymers

were

synthesized

by

using

the

carboxyl

containing

4,4’-azobis(4-cyanovaleric acid) initiators via radical polymerization (Figure S2), whose structure was confirmed by the 1HNMR spectrum (Figure S3). The LCSTs of PNIPAAm-COOH were measured in H2O and PBS with a concentration of 1 mg/mL via UV-vis spectroscopy (Figure S4), respectively. In water the PNIPAAm-COOH solution was transparent below 31.2 ºC, which is consistent with literature.23 However, the LCST in PBS was 2 ºC lower, which is known as a result of salt-out effect.28 The thermo-responsive transition of PNIPAAm is known as the effects of the intermolecular and intramolecular hydrogen interactions between chemical groups and water.29 The PNIPAAm-COOH polymers were easily immobilized onto the tubular walls of TiNT-PDA/PEI by using an EDC/NHS coupling reaction as illustrated in Scheme 1d. The NHS esters were formed by exposing the TiNT-PDA/PEI surface to an aqueous solution of EDC and NHS for 1 h. The NHS ester layer was reacted with an aqueous solution of 3% PNIPAAm-COOH for 24 h (pH 5.5), following by 15



rinsing three times with water to obtain the PNIPAAm grafted TiNT-PDA/PEI (TiNT-P). To verify the success of PNIPAAm grafting and its depth in the nanotubes, the surface chemical compositions of TiNT-P and TiNT-PDA/PEI at different depth (0, 25, 50 nm below the surface) were characterized by XPS (Figure 2 and Figure S5). On the TiNT-PDA/PEI surface (0 nm depth), the percentages of C1s and N1s were 39.3% and 5.3%, respectively, and the detected C/N ratio was 7.4. Given that the C/N ratios in PDA and PEI were 16 and 1.7, respectively, the molar ratio of PDA/PEI in the PDA/PEI layer was calculated as 0.66. On the TiNT-P surface, the relative contents of C1s and N1s were 48.0% and 9.5%, respectively. The calculated C/N ratio of TiNT-P (5.08) is much larger than that of TiNT-PDA/PEI, but is identical to the ratio of 5.14 in PNIPAAm polymers whose C and N contents are 63.72% and 12.39%, respectively. These results demonstrate the successful immobilization of PNIPAAm onto the nanotubes. At a depth of 25 nm below the surface, the intensity of C1s and N1s was weakened obviously. At the distance up to 50 nm away from the surface, the C1s and N1s were below the detection limit, suggesting that no PNIPAAm can be immobilized at this region. The thermo-responsive property of the immobilized PNIPAAm was characterized by measuring the static water contact angle (Figure 3). The as-prepared TiNT surface was very hydrophilic with a water contact angle of 10°. After PDA/PEI modification (TiNT-PDA/PEI), it increased slightly to 15.5±1.6° (Figure 3a, top), revealing that the hydrophilic nature was not changed obviously. The PNIPAAm-grafted TiNT-P surface became less hydrophilic with a contact angle of 41.1±7.9° at 28 ºC, which substantially rose to 62.9±2° at 37 ºC above the LCST, demonstrating that the typical temperature-responsive wettability was maintained (Figure 3a, bottom). Figure 3b shows the graph of the water contact angle on the TiNT-P surface as a function of temperature. The variation of the 16


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contact angles matched a sigmoidal curve model. The maximum change in the profile occurred between 32-35 ºC, according to which the LCST was estimated as about 32 ºC, demonstrating that the surface immobilization does not change the LCST. Release of S1P One of the key abilities of this smart system is to perform gating release on demand by switching the On and Off states. First, the loading efficiency was measured to be 56.7±6.7%, suggesting that the drug can be efficiently loaded and stably retained in the nanotubes against routine washing. Figure 4a shows that S1P was maintained inside the nanotubes with slower release at 28 ºC (off state) in the “gate-keeper” modified TiNT-P system. The amount of released S1P did not change obviously after the rapid release within 60 min. At 37 ºC (on state), the release of S1P was sharply enhanced, and the released amount reached up to 3 times as that at 28 ºC. On the other hand, on the unmodified TiNT surfaces the release profiles were similar at both 28 ºC and 37 ºC as shown in Figure 4b, revealing there is no switch effect without the “gate-keeper” polymers. The ability of gating release was demonstrated by cyclic change between the On and Off states at different temperatures (Figure 4c). This experiment was conducted by cyclic exposure of TiNT-P/S1P at 37 ºC and 28 ºC environment, and the release of S1P was monitored at each transition point. When the TiNT-P/S1P system was exposed to 28 ºC, the S1P released more slowly. Once exposed to 37 ºC, the release rate was enhanced sharply due to the change in nanotube size, which resulted from the conformational change of polymers. The S1P gating release demonstrates that the smart nanotube array is able to switch between the On and Off states, and perform an on-demand release in a controlled way. We have also demonstrated the switched release of fluorescein sodium. Fluorescein sodium has a similar molecular weight and the same negative charge as that of S1P (Figure S6a, b). Figure S6c 17



show that an obvious increase in released amount occurred at 37 ºC for the TiNT-P system. In contrast, the release was kept the same at 37 ºC and 28 ºC for the TiNT system (Figure S6d). These results are in consistence with that of S1P, proving the thermo-controlled gating release of TiNT-P system. Adhesion and proliferation of ECs Prior to cell migration on the biomaterials, the initial interaction of cells to substrates is cell adhesion. Hence, evaluation of cell adhesion was carried out by FDA fluorescent staining at different point of time (Figure 5a). A fewer cells with a round shape were observed at 4 h culture, showing no significant difference regardless of the loading of S1P and culture temperature. The number of adhered ECs increased markedly when the culture time increased to 24 h. At this period of time, the difference in cell numbers became significant between the TiNT-P/S1P at 37 ºC and all the other samples. Many cells were found on the TiNT-P/S1P at 37 ºC, whereas a smaller number of cells with round shape adhered on the other three samples (Figure 5b). The EC cells had a smaller spreading area at 4 h culture time, which was improved obviously 24 h later (Figure 5c). Again, the cell spreading area on the TiNT-P/S1P sample at 37 ºC was significantly larger compared with that of all other samples. A faster cell proliferation is beneficial to the rapid endothelialization on artificial vascular grafts. To compare quantitatively the effect of released S1P in a gating way on ECs proliferation, the cytoviability of all samples was normalized to that of ECs cultured on TCPS at 37 ºC and 28 ºC, respectively. Figure 5d shows that cell viability exhibited no significant difference among the four samples at day 1. At day 3, the cell viability on the TiNT-P/S1P at 37 ºC was statistically higher than that on TiNT-P at 28 ºC, but was in the same level to that of the TiNT-P/S1P at 28 ºC and TiNT-P at 18


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37 ºC. These results demonstrate that the released S1P has a less effective effect on improving ECs proliferation. Moreover, the lower culture temperature does not deter the cell proliferation on the TiNT-P surface when S1P was loaded. Migration of ECs In the repair and generation of various tissues such as bone, blood vessel, and wound, the proliferation and migration of endothelial cells are highly crucial for the formation of new blood vessels to supply nutrients to other types of cells.30 The ability of ECs to reestablish blood vessel integrity and function is critical in repairing and generating tissues. Therefore, it is of paramount significance in developing new biomaterials that can regulate the behaviors of ECs migration in a controlled way. Furthermore, one factor that contributes to such impaired tissue healing is the migration rate. It is believed that the cell migration rate can be improved by physical and chemical cues on substrates or in surrounding microenvironment. S1P has been proven to be related with many crucial physiological processes, including cell growth, survival, migration and so on.31 The wound healing assay is a traditional method to study cell migration, which can mimic the collective migration of cells in wound healing in vivo to a great extent. Endothelium damage in blood vessels will cause the migration of endothelial cells heading to the injured sites eventually. Different from the fibroblasts that migrate as a loosely connected fashion, the endothelial cells will form a dense cell sheet and migrate into wound.32 Because ECs do not proliferate prominently in the first 3 days (Figure 5d), the movement of the cells would be mainly attributed by the collective migration of ECs cell sheet. Figure 6 shows that the migration distance of cells on TiNT-P/S1P at 37 ºC was significantly longer than the other three groups, especially at 48 h. The significant improvement of ECs migration on the TiNT-P/S1P at 37 ºC is reasonably attributed to the released S1P at this 19



condition, which has the function of promoting ECs mobility. Detail behaviors of cell migration were analyzed by in situ tracking the trajectories of single cells on the TiNT-P substrates with and without S1P loading at 28 ºC and 37 ºC, respectively. The movement trajectories were reconstructed into a two-dimensional graphic by setting the starting position as the origin of coordinates (0, 0) (Figure 7). Figure 7 and Figure S7 show that the cells migrated randomly on the sample in all directions. The end-points of trajectories were shown as a rose diagram and analyzed by Rayleigh test, demonstrating a random movement with the p value close to 1 (Figure S7). The ECs cultured on the S1P-released TiNT-P surface travelled a longer distance compared to cells under other conditions. The statistic migration rates of ECs on TCPS, TiNT, TiNT-P and TiNT-P/S1P at 28 ºC and 37 ºC are shown in Figure 7e. The cells migrated on the TiNT-P/S1P at 37 ºC significantly faster than those on the TiNT-P/S1P at 28 ºC or the TiNT-P at 37 ºC, revealing the positive functions of released S1P triggered by the higher temperature. The cell migration rate at 37 ºC was always higher than that at 28 ºC no matter where they were cultured, suggesting that the normal culture temperature does advance cell mobility to some extent. A control experiment shows that the migration of NIH 3T3 cells was independent on the S1P loading and culture temperature (Figure S8), demonstrating that the gating effect of TiNT-P/S1P is specific to ECs. In order to investigate the functions of the released S1P in the medium, we detected the single cell migration after washing out the culture medium and refreshing with the new one. At this condition, the migration rate was 11.0 m/h at 37C (Figure S9), which was slower than that on the TiNT-P/S1P 37C without refreshing (13.0 m/h, Figure 7e), while the rate at 28C (8.2 m/h, Figure S9) remained the same as that on the TiNT-P/S1P 28C (Figure 7e). These results prove that the released S1P in the medium is required to some extent. Nonetheless, the migration of ECs can be still enhanced upon the S1P 20


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peptides are released into the medium. The mobility of ECs depends intrinsically on the cell adhesion and cell-material interactions. In accordance with the time of cell migration, adhesion of ECs at 5 h post cell seeding on each sample was observed by SEM (Figure 8) and CLSM (Figure 9). The ECs cultured on the TiNT-P at 28 ºC maintained a round morphology, whose filopodia were not particularly pronounced. By contrast, those ECs cultured on the TiNT-P/S1P at 37 ºC showed much more obvious filopodia protrusions with elongated tentacles, indicating a more intimate contact between filopodia and the nanotubes. When Cells were cultured on the TiNT-P/S1P at 28 ºC and TiNT-P at 37 ºC, they could also contact closely with the nanotubes, but had a fewer filopodia. The focal adhesion and cytoskeleton organization of ECs under different conditions were further studied through CLSM imaging. Focal adhesion serves as a link between cytoskeleton and substrate, and regulates various cellular behaviors, including cell adhesion, growth, and differentiation. Microfilaments, composed mainly of actin, make up cell skeleton, maintaining the normal structure and functions of cells. Therefore, the focal adhesion complexes and cell skeleton were investigated by by staining actin and vinculin. On the TiNT-P/S1P surfaces, the vinculin was well spreading, and the actin fibers were distributed throughout the whole cell, indicating the strong interaction between the cells and surfaces (Figure 9a, b). By contrast, on the surfaces without loading S1P, cells possessed a rounder shape, with fewer focal adhesion plaques and actin filaments (Figure 9c, d), conveying the weaker interactions with the nanotubes. Functions of ECs In order to benefit for the vascular homeostasis, vascular endothelium releases various antithrombotic and anticoagulant cytokines.33 The gene expression levels of several endothelium function-related 21



factors, including platelet endothelial cell adhesion molecule-1 (CD31), vascular endothelial cadherin (CD144), endothelial nitric oxide synthase (eNOS), biglycan and thrombomodulin (THBD), were investigated. The gene expressions (Figure 10a) showed no obvious difference between the TiNT-P/S1P and TiNT-P+S1P, although some of the genes showed a lower expression in the TiNT-P+S1P conditions, for example, eNOS, biglycan and THBD. To unveil the biological functions of ECs, the following two cytokines, prostacyclin (PGI-2) and tissue-type plasminogen activator (tPA) were detected. PGI-2 can obviously inhibit the aggregation of blood platelet, and tPA can accelerate thrombolysis by activating plasminogen. As shown in Figure 10a, b, the secretions of PGI-2 and tPA on the TiNT-P/S1P 37 ºC were 75.5% and 62.5% higher than on the TiNT-P samples, respectively. Compared to the cells with S1P in bulk medium, ECs on TiNT-P/S1P 37 ºC promoted higher secretion of PGI-2 and tPA. These results may be caused by the higher local concentration of S1P due to the confined area of nanostructure. Single cell gene analysis In order to unveil the intrinsic mechanism regulating the migration of ECs under the stimulation of released S1P, single-cell gene analysis was performed. The tested genes (Table S2) involved actin nucleating and polymerization proteins (VASP, WAVE, and WASP etc.), focal adhesion components (integrins, talin, vinculin, tensin, paxillin, and FAK etc.), and proteins controlling myosin II activation (MLCK, PAK, MLCP, and ROCK etc.). Individual cells were isolated from each sample (TiNT-P/S1P 37 ºC, TiNT-P/S1P 28 ºC, TiNT-P 37 ºC, and TiNT-P 28 ºC), which were marked as S1-12, S13-24, S25-36, and S37-48 (Figure 11a), respectively. The clustering analysis of genes and specimen and the following principal component analysis were performed in all cell samples collected from four culture conditions, in order to establish the S1P-stimulated pattern of gene expression that may aid the 22


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determination of S1P functions. The clustering of cell samples shows that the mRNA levels for these 41 migration-related transcription factors could be separated roughly into two clusters. By setting each cell sample in correspondence with the original culture condition where they were collected from, the percentages of S1P-stimulated cells and non-stimulated cells in each cluster were calculated as 66.7% and 81.8%, which means 66.7% S1P-stimulated cells were separated to one cluster and 81.8% non-stimulated cells were separated to the other. Two clusters of cell samples were separated from each other, in line with the evidence of difference in single cell migration. The data of principal component analysis was shown in Figure 11b where every point stands for one single cell, and different colors represent where it was collected from. Although the two individual clusters can be distinguished clearly, there is also some obvious overlap, indicating that cells cultured on these four nanotube arrays have some similar activity states. This result is caused by the same cell type and their similar expressions in main genes. The expressions of the actin polymerization and focal contact modulators are likely influenced by the extracellular action of released S1P, because the receptors of S1P are required during recognition and binding. There are three S1P receptors, S1PR1, S1PR2 and S1PR3, which have been proven to be related with cell migration and growth. In order to investigate the pathways being activated by S1P to stimulate ECs migration, the gene expressions of S1P receptors, i.e. S1PR1, S1PR2 and S1PR3, were calculated and compared (Figure 11c). On the TiNT-P/S1P at 37 ºC the S1PR1 was expressed with a significantly higher value than that on other samples, whereas the expressions of S1PR2 and S1PR3 were similar on all four samples. These results demonstrate that S1P mainly binds to receptor S1PR1 on cell membrane, but not S1PR2 or S1PR3. We detected the gene expressions of S1PR1 as shown in Figure S10. In the presence of S1P in the bulk medium, the expression of S1PR1 was improved and 23



kept the same with that on the TiNT-P/S1P 37 ºC. These data show that the activation of S1P was maintained no matter it was added in the medium or released from the nanotubes. Western blotting S1P receptors couple to different subsets of heterotrimeric G proteins, thus having different roles on cell migration. In order to investigate the downstream signaling pathways of S1P and activation of the Rac and Rho family of small G proteins, western blotting analysis was carried out to verify this assumption. Cdc42 and Rac1 participate in the regulation of actin polymerization and pseudopodia formation. They act like molecular switches via a switching process binding between guanosine triphosphate (GTP) and guanosine diphosphate (GDP). When GTP is bound, the activated Cdc42 and Rac1 will interact with effector molecules to induce formation of actin fibers, lamellipodia, and filopodia. Therefore, the activation state of GTP-bound Cdc42 and Rac1 at four different culture conditions was quantitatively analyzed (Figure 12a-c). The active Cdc42 and Rac1 were normalized to the total lysate protein amount. The released S1P at TiNT-P/S1P 37 ºC condition significantly upregulated Cdc42 and Rac activity. Rac1 signaling can induce the phosphorylation of myosin and thus regulate cytoskeletal contractility to mediate cell migration. In this work, we have developed a platform based on titania nanotube arrays modified with thermo-responsive polymers as “gatekeeper”, which can be used as a controlled-release system. The pre-loaded effectors were released upon the stimuli was triggered, which then medicated the behaviors and functions of ECs cultured on the platform. PNIPAAm, a kind of temperature-responsive polymers, has drastic conformational change upon subtle environmental stimuli. The essence for the design of PNIPAAm-based temperature-gating system is the conformational changes of temperature-responsive polymers. The reversible conformational changes of PNIPAAm at 24


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different temperature lead to the changes in pore size of nanotubes, translating into the gating effect of nanotubes. The nanochannels grafted with PNIPAAm polymers display an advantage which can adjust the transportation of ion triggered by temperature simultaneously.14 Bojko and his co-workers have fabricated PNIPAAm-grafted nanoporous silicon nitride membranes. The modified nanoporous membranes have a clear ON-OFF temperature-responsive state in diffusion of vitamin B12 and FITC-dextran.34 In our study, the nanotube array acts as nanocarriers, while the 130 kDa PNIPAAm molecules grafted on the inner walls of tubes play a role of “gate-keeper”. The grafting of PNIPAAm was confirmed by several methods such as water contact angle and XPS. The pre-loaded effectors were well maintained inside the nanotubes during storage below LCST, while were released rapidly at the temperature for cell culture. The temperature-responsive release from the TiNT-P can be explained by the change of the effective tube size: increasing temperature induces folded polymer chains and thus larger channel benefiting S1P diffusion. The released effectors, S1P, are potently chemoattractants for endothelial cells. When the responsive nanotube arrays were cultured at 37 ºC for more than 30 min, the concentration of S1P reached its maximum and kept unchanged for the following culture time. The effect of released S1P on ECs was tested by adhesion and migration at various culture conditions in low serum medium, to avoid being muted by binding to lipoprotein in serum. Once the released S1P binding to endothelial cells, it can activate G-coupled receptors on cell membrane, which subsequently release G-protein dimers or trimers to initiate signal transduction and lead to cell migration (Scheme 2).35

Conclusions We have successfully demonstrated a platform based on titania nanotubes arrays covered with 25



thermo-responsive polymers for temperature-gating controlled release and regulating cell migration. The PNIPAAm polymers were used as a temperature “gatekeeper”, which retained the payloads at room temperature, and opened the pores rapidly to allow the release when triggered with heating. The thermo-triggered release was demonstrated by detecting amounts of the model molecules at temperatures below and above the LCST as a function of time. When ECs were cultured on the S1P-loaded TiNT-P, single cell mobility and collective cell migration were enhanced at the state of released S1P (at 37 ºC) compared to that at the retained state (at 28 ºC). Single-cell gene expression analysis and western blotting analysis were used to investigate the related genes and proteins, thus providing a deeper insight into the regulation of S1P on cell migration and its mechanism of Rho GTPase signaling. The combination of smart polymers-modified nanotube array and stimulus source plays a primary role on controlled release and thereby regulated cell migration, which is triggered by S1P and the following activities of Rho GTPases pathways. We believe that the development of such a biocompatible system with smart polymer-gatekeepers in the bio-inert nanotube array provides a versatile method for drug delivery and opens wide range of research opportunities for regenerative biomaterials. Supporting Information: Primer sequences and PCR product length for genes related with EC functions (Table S1) and migration (Table S2). SEM images of the PDA/PEI modified titania nanotube array (Figure S1). Synthesis of –COOH end-capped PNIPAAm (Figure S2) and its characterization with 1H NMR spectrum (Figure S3) and UV-Vis spectroscopy (Figure S4). XPS survey spectra of TiNT-PDA/PEI (Figure S5). Molecular structures of fluorescein sodium and S1P, and release curves of fluorescein sodium (Figure S6). Rose diagram of single cell migration trajectories (Figure S7). Migration traces and statistical migration rate of NIH 3T3 cells (Figure S8) 26


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and ECs (Figure S9). Related gene expression of S1P receptor S1PR1 (Figure S10). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Acknowledgements: This work is financially supported by the National Key Research and Development Program of China (2016YFC1100403), the Natural Science Foundation of China (21434006), the 111 Project of China (B16042), and the Fundamental Research Funds for the Central Universities (2017XZZX001-03B, 2017XZZX008-05).

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of

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Figure captions: Scheme 1. Schematic illustration of the temperature-gating controlled release system. (a) The mobility of endothelial cells is promoted once the “gate-keeper” is triggered above the LCST of PNIPAAm to release S1P payloads. (b) A side view and (c) a top view of one nanotube illustrating how the system works. Temperature-responsive PNIPAAm polymers are immobilized onto the inner wall of nanotubes, which turn the gat Off and On at the extended and collapsed conformation switched by temperature, and in turn regulate the release of pre-loaded effector S1P molecules, respectively. The molecular structure of S1P is shown in the top of (c). (d) Grafting of PNIPAAm onto TiNT surface. After the TiNT surface is treated with an aqueous solution of DA and PEI in vacuum for 2 h, PNIPAAm molecules are immobilized onto the wall of nanotubes by using a standard EDC/NHS coupling reaction. Figure 1. (a-c) Top view and (d) side view SEM images of the original titania nanotubes array at different magnifications. Figure 2. XPS survey spectra of TiNT immobilized with PNIPAAm measured on top surface, and 25 nm and 50 nm below surfaces, respectively. The insets show the C1s and N1s spectra of the TiNT-P at a distance of 25 nm and 50 nm away from the surface. Under Table summarizes the relative contents of N and C elements. Figure 3. (a) Photo images of water drops and static water contact angles of TiNT-PDA/PEI and TiNT-P at 28 ºC and 37 ºC, respectively. (b) Water contact angle measured on TiNT-P as a function of temperature. Figure 4. Release curves of S1P from (a) TiNT-P and (b) TiNT samples at 37 ºC and 28 ºC, respectively. (c) Release of S1P from TiNT-P sample after alternative exposure to 37 ºC and 28 ºC. 31



Figure 5. (a) Fluorescent images of ECs (stained with FDA) being adhered on TiNT-P/S1P and TiNT-P surfaces after being cultured for 4 h and 24 h at 37 ºC and 28 ºC, respectively. Scale bar=100 m. Statistic results of (b) cell number and (c) cell spreading area on TiNT-P/S1P and TiNT-P surfaces after being cultured for 4 h and 24 h at 37 ºC and 28 ºC, respectively. (d) Relative viability of ECs after being cultured on the TiNT-P/S1P and TiNT-P surfaces at 37 ºC and 28 ºC for 1 d and 3 d, respectively. Cells cultured on TCPS at 37 ºC and 28 ºC were used as controls, respectively. Asterisk (*) indicates statistical significant difference at a p < 0.05 level. Figure 6. (a) Representative fluorescent photos of ECs sheets being cultured on TiNT-P/S1P and TiNT-P surfaces at 37 ºC and 28 ºC for 24 h (upper panel) and 48 h (lower panel), respectively. The green dashed lines outline the original boundaries of the cell sheets. Scale bar=200 μm. (b) The statistic results of the migration distance of cell sheets; n=5. Asterisk (*) indicates statistical significant difference at a p < 0.05 level. Figure 7. Migration traces of single ECs on (a, b) TiNT-P/S1P and (c, d) TiNT-P surfaces at (a, c) 37 ºC and (b, d) 28 ºC, respectively. The cells were continuously tracked every 15 min for 8 h. The total numbers of the trajectories were 48 for each sample. Scale bar=50 m. (e) The statistical migration rate of ECs on TiNT-P/S1P, TiNT-P, TiNT and TCPS at 28 ºC and 37 ºC. Asterisk (*) indicates statistical significant difference at a p < 0.05 level. Figure 8. SEM images of ECs being cultured on (a-d) TiNT-P/S1P and (e-h) TiNT-P at (a, b, e, f) 37 ºC and (c, d, g, h) 28 ºC, respectively. (b), (d), (f) and (h) are higher magnification images of (a), (c), (e) and (g), respectively. Cells are endowed with pseudo purple color by the Photoshop software for better discrimination from the substrates. 32


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Page 33 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. CLSM images show the ECs morphology being cultured on (a, b) TiNT-P/S1P and (c, d) TiNT-P at (a, c) 37 ºC and (b, d) 28 ºC, respectively. Rows 1-4 show the nucleus (blue), vinculin (green), and actin, (red), and the merged fluorescence images, respectively. Scale bar = 5 μm. Figure 10. (a) Expression of genes related to ECs on TiNT-P, TiNT-P/S1P and TiNT-P+S1P at 37 ºC and 28 ºC, respectively. Each type of gene expression was detected by RT-PCR and normalized to that on TiNT-P 37 ºC. Secretion levels of (b) PGI-2 and (c) tPA by ECs cultured on TiNT-P, TiNT-P/S1P and TiNT-P+S1P at 37 ºC and 28 ºC, respectively. The data were representative of three independent experiments, mean ± SD, *p < 0.05. Figure 11. (a) A heat map of expression levels for migration-related genes of individual cells being collected from four groups with hierarchical clustering of cells and genes. Red and green colors represent higher and lower expressions, respectively. (b) Principal component analysis plot of the single cells, which are colored according to their origins where the samples have been collected. (c) Gene expression levels of three receptors of S1P (S1PR1, S1PR2 and S1PR3) by single-cell gene expression. Asterisk (*) indicates statistical significant difference at a p < 0.05 level. Figure 12. (a) Western blot analysis of migration-related proteins (Cdc42 and Rac1) expressed by ECs being cultured on TiNT-P/S1P and TiNT-P surfaces at 37 ºC and 28 ºC for 24 h, respectively. Active Cdc42 and Rac1 ratios to total (b) Cdc42 and (c) Rac1 protein abundance, respectively. Scheme 2. Schematic illustration of cellular signaling pathways of migration-related proteins that mediate the cell migration.

33


ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

a

Page 34 of 48

Fast

Slow

37 ºC

NH2

b

c OH

Na O O P O Na O

37 ºC 37 °C

28 ºC

28 °C

Off-state

On-state

d NH2 NH2 NH2 NH2

Scheme 1

O O O O (1) EDC+NHS NH NH NH NH (2)PNIPAM-COOH (3)28°C 34


S1P

Page 35 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


a

b

100 nm

c

d

Figure 1 35



O 1s Surface

Itensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 36 of 48

F 1s Ti 2p C 1s N 1s

25 nm below

50 nm below

1200

1000

800

600

400

200

0

Binding Energy (eV) surface

25 nm below surface

50 nm below surface

N content

9.45

1.57

N.A

C content

47.97

4.05

N.A

Figure 2 36


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


a

b

TiNT-PDA/PEI 15.5±1.6°

Contact angle (°)

Page 37 of 48

TiNT-PNIPAM 41.1±7.9° 62.9±2.0° 37 C

60 50 40 20

28 C

25

30

35

40

Temperature (°C)

Figure 3 37


45


a

b

c

37ºC 28ºC 37ºC 28ºC 37ºC 28ºC 37ºC 28ºC

Figure 4 38


Page 38 of 48

Page 39 of 48

a

TiNT-P/S1P 37ºC

TiNT-P 37ºC

TiNT-P/S1P 28ºC

TiNT-P 28ºC

4h

100

Cell number (/field)

b

80

TiNT-P/S1P 37°C TiNT-P/S1P 28°C TiNT-P 37°C TiNT-P 28°C

c

*

60 40 20 0

4

Spreading area (mm2)

24 h

24

1.5

*

1.0

800 600


*

400 200 0

4

24

Culture time (h)

Culture time (h)

d

Relative cell viability (fold increase/control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46



0.5 0.0

1

3

Figure 5

Culture time (d) 39



a

TiNT-P/S1P 37ºC

TiNT-P 37ºC

TiNT-P/S1P 28ºC

TiNT-P 28ºC

24 h

48 h

b

Migration distance (µm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 40 of 48

TiNT-P/S1P 37°C

150

* 100

TiNT-P 37°C TiNT-P/S1P 28°C TiNT-P 28°C

*

50 0

24

48

Culture time (h)

Figure 6 40


Page 41 of 48

a

TiNT-P/S1P 37ºC

b

TiNT-P/S1P 28ºC

e

c

TiNT-P 37ºC

d

Cell mobility (µm/h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


TiNT-P 28ºC

20

37°C 28°C

*

15

*

10

*

5 0

1P S / T-P N i T

Figure 7 41


T-P N i T

T TiN

PS TC


TiNT-P/S1P 37ºC

TiNT-P/S1P 28ºC

Page 42 of 48

TiNT-P 37ºC

TiNT-P 28ºC

a

c

e

g

b

d

f

h

Figure 8 42


Page 43 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


TiNT-P/S1P 37ºC TiNT-P/S1P 28ºC TiNT-P 37ºC

c1

TiNT-P 28ºC

d1

a1

b1

a2

b2

c2

d2

a3

b3

c3

d3

a4

b4

c4

d4

Figure 9 43


TiNT-P 37°C

2.0

TiNT-P 28°C TiNT-P/S1P 37°C

1.5

TiNT-P/S1P 28°C TiNT-P+S1P 37°C

1.0

TiNT-P+S1P 28°C

0.5 0.0

n D ca 44 31 y OS 1 l D N D HB g C i e T C B

b

c

PGI2

400

*

*

300 200 100 0

Page 44 of 48

2.5

Concentration (pg/ml)

a

Concentration (pg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Relative gene expression


TiNT-P

tPA

*

300

*

37°C 28°C

200 100 0

TiNT-P/S1P TiNT-P +S1P

TiNT-P

Figure 10 44


TiNT-P/S1P

TiNT-P +S1P

a

b

-S1P

+S1P

4

0 -4 -10

-5 5 0 PC1 (40.6% explained var.)

10

c Gene expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


PC2 (17.1% explained var.)

Page 45 of 48

S1PR3

10

TTiN

45


S1PR1 S1PR2

20

0

Figure 11

*

30

1P P/S

°C

°C °C 8° C 37 28 P2 P P 1 T T /S TiN TiN T -P N i T

37

a

1

2

3

4

b

Activated Cdc42

Total Cdc42

2.5

Page 46 of 48

* TiNT-P/S1P 37°C TiNT-P/S1P 28°C

2.0

TiNT-P 37°C

1.5

TiNT-P 28°C

1.0 0.5 0.0

Activated Rac1

c

Total Rac1

GAPDH

1: TiNT-P/S1P 37ºC

3: TiNT-P 37ºC

2: TiNT-P/S1P 28ºC

4: TiNT-P 28ºC

Active Rac1/Total Rac1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Active Cdc42/Total Cdc42


2.0 1.5 1.0 0.5 0.0

Figure 12 46


* TiNT-P/S1P 37°C TiNT-P/S1P 28°C TiNT-P 37°C TiNT-P 28°C

Page 47 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


GTP Rho GTP p65PAK

GTP

Rac

Scar/WAVE

MLCP

Scheme 2 47


ROCK


Page 48 of 48

For TOC use only

Fast

Slow 37 ºC

48


Temperature-gating titania nanotubes regulate migration of

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