Functionalized Carbon Nanowalls as Pro-Angiogenic Scaffolds for

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Functionalized Carbon Nanowalls as ProAngiogenic Scaffolds for Endothelial Cell Activation Vimal Kumar, Sheikh Mohamed Mohamed, Srivani Veeranarayanan, Toru Maekawa, and Sakthikumar Dasappan Nair ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00724 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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ACS Applied Bio Materials

Functionalized Carbon Nanowalls as Pro-Angiogenic Scaffolds for Endothelial Cell Activation Vimal Kumar, M. Sheikh Mohamed, Srivani Veeranarayanan, Toru Maekawa, D. Sakthi Kumar* Bio-Nano Electronics Research Centre, Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Saitama, 350-8585, Japan

* Corresponding author Prof. D. Sakthi Kumar, Ph: 81-492-39-1636 Fax: 81-492-34-2502 E-mail: [email protected]

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Abstract: Substrates that can be utilized to assist in expression of the inherent characteristics of cells under in vitro conditions are necessary to mimic the in vivo scenario to the maximum extent possible. We demonstrate the application of functionalized Carbon Nanowalls (CNW) in facilitating human endothelial cell attachment and proliferation. The CNW films were grown on silicon substrates by Surface-Wave Microwave Plasma-Enhanced Chemical Vapor Deposition (SWMWPECVD) technique. These CNW were then functionalized using Nitrogen (N2) gas plasma to form functionalized N2 doped CNW (CNW-N2). Characterization of CNWs revealed a uniform petal-like morphology with the individual CNW width measured to be 1~5 nm and the film thickness in the range of 10~12 μm. The N2 functionalized CNWs proved to be highly efficient in providing cellanchorage to the endothelial cells. Profiles of major cytoskeletal proteins revealed a higher degree of expression in the functionalized CNWs depicting the maintenance of structural integrity of cells. Interestingly, the CNW-N2 substrate was found to promote proangiogenic factors in the cells, which is observed here for the first time, that could pave way for the utilization of this substrate for detailed studies of angiogenesis processes in vitro and further biomedical applications.

Key Words: Carbon nanowalls, Angiogenesis, Cell adhesion, Cell scaffold, Carbon nanomaterials, Chemical vapor deposition


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1.

Introduction:

Carbon Nanomaterials (CNMs) are a family of nanomaterials with carbon as the basic structural unit. Graphene, Carbon Nanotubes (CNTs), Fullerenes, Nanodiamonds (NDs) etc.1 are a few prominent examples. Their unique physiochemical properties (mechanical, electrical and chemical), aided by the well-established functionalization strategies of carbon chemistry make CNMs highly desirable in numerous applications such as sensors and energy storage, with Graphene2 and CNT3 dominating the field. Carbon nanowalls (CNWs) are nanographitic forms of carbon growing 90° vertical to the substrate surface and characterized by high surface to volume ratio, high aspect ratio, high electrical conductivity and non-reactive electrochemical properties4. Also, unlike CNTs, CNWs are grown without the use of metallic nanoparticles, and thus do not require further purification steps before final utilization and application. Owing to these properties, CNWs have found applications in a variety of fields such as battery electrodes5,6, fuel cell supports7,8 and surface coatings9,10. The as-grown CNWs exhibited super-hydrophobic wettability resulting from the surface roughness. Therefore, common aqueous solvents such as water, buffer solutions and ethanol cannot form a layer on pristine CNW due to the high contact angle between the liquid and the surface of the CNW layer

11,

thus

hindering their biological applications. In order to fabricate a more responsive biomaterial, it is desirable to control the hydrophobicity by incorporating nitrogen, oxygen and other interstitial atoms onto the structure of the nanographitic walls12. Previous reports suggest that many researchers have established the ability of CNWs in the biomedical field as theragnostics agents, scaffolding, sensors, etc. 13–17. It is therefore imperative that CNWs would be a good candidate for use as biomaterial interface material. Designing a biomaterial involves deciphering the underlying surface properties of the material, protein adsorption, and material-associated cellular response for bioapplications and tissue engineering. Numerous studies exist portraying the varied cellular behavior/characteristics which are affected by the surface chemistry of different materials. The relationship between material surface properties (essentially the wettability) and cellular response is mainly influenced by the charge, type, quantity, and conformation of adsorbed protein layer that governs cellular attachment/proliferation. Due to their vertical arrangement and surface roughness, CNWs show potential as substrates or scaffolds for cell attachment and proliferation, as alternatives to standard tissue culture ware, by greatly



enhancing the cellular anchorage. Presently, a few reports of functionalized CNWs exist on the attachment of cellular entities on CNWs surface16,18. Though these studies highlighted the ability of CNWs to serve as biomaterial substrates, they mainly focused on the adhesion and growth of cells, not dwelling into the effect on functional properties of the cells, such as the metabolic profiling. Herein, we try to understand a few major cellular parameters using endothelial cells (ECs) as models on pristine and N2 functionalized CNWs (CNW-N2). The choice of ECs, especially vascular ECs can be attributed to their prominence in tissue engineering and regenerative medicine therapies for various vascular pathologies and since they are omnipresent in all types of tissue engineering cell grafts. A few examples of ECs in practical application include the prevention of thrombosis by endothelialization of vascular grafts, improving the bioavailability of tissue engineered organs by assisting in their vascularization, or augmenting vessel proliferation in ischemic injury19. We analyze three different substrates, namely cell culture compatible glass cover slips (CS), unmodified CNWs and CNW-N2 to study the cell attachment and proliferation of ECs. Our observations showed that CNW-N2 substrate greatly influences the functional behavior of ECs leading to angiogenic protein expression. To our knowledge, this is the first of a kind study establishing the mechanoresponse mediated functional activation of ECs by a substrate, CNW-N2 in this case, without any external chemical cues/mediators. Most of the studies undertaken to study the interaction between CNMs and biological cells have been conducted using graphene20–24 and carbon nanotubes25–28. This work shows that CNWs can be used to achieve superior cell attachment, thus paving way for incorporation of CNWs in future works involving tissue engineering, Lab-on-Chip, Microchannel-on-Chip and biosensors. 2.

Materials and Methods:

2.1. Materials: Zetasizer 100μm tracer polystyrene microsphere particles were purchased from Malvern. Dulbecco’s modified Eagle’s medium (DMEM), Penicillin/Streptomycin, Fetal bovine serum (FBS) and Phosphate buffered saline (PBS) pH 7.1 were purchased from Gibco (Thermo Fisher Scientific). Human brain microvascular endothelial cells (HBMECs) were cultured in Complete classic medium kit (Cell Systems). NucBlue, Phalloidin Rhodamine, Goat anti-mouse IgG (H+L) secondary antibody, DyLightTM 488, Goat anti-


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rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568 and PrestoBlue cell viability reagent were purchased from Invitrogen (Thermo Fisher Scientific). AntiCD31 antibody and Anti-Vimentin antibody were purchased from abcam. Proteome profiler Human Angiogenesis Array and Human Ki-67/MKI67 DuoSet ELISA (R&D systems) was purchased from Funakoshi Ltd. Live/dead cell double staining kit and 2’,7’Dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich. Any additional chemicals or reagents were of analytical grade procured from either Sigma (Merck) or Wako chemicals, Japan. 2.2 Methods 2.2.1 Synthesis of CNWs: CNWs were developed on Silicon (Si) substrates of 20 X 20 mm (P-type, mirrored polished Si Wafers ) using Surface Wave Microwave Plasma Enhanced Chemical Vapor Deposition (SWMPECVD) system (ULVAC, Inc., CN-CVD-200) as shown in Scheme 1. Firstly, H2 gas was introduced at a constant flow rate at 30 Standard Cubic Centimeters per Minute (SCCM) and the chamber pressure was kept at 25 Pascal. This pressure was maintained throughout the whole reaction process. The microwave was tuned to output 350W and the hydrogen plasma was ignited. While igniting the plasma, the substrate was heated to 800 °C at a rate of 100 °C/second. Once the substrate was heated, CH4 gas was introduced at a flow rate of 15 SCCM and a reverse bias of 10 V was applied, these conditions were maintained for 15 minutes after which the substrate was allowed to cool under vacuum at a cooling rate of 12 °C per second. 2.2.2 Functionalization of CNWs: In order to functionalize the CNWs, a low-pressure plasma functionalization technique was implemented using the same apparatus used for CNWs synthesis. First the substrate was kept at 2 Pa in the vacuum chamber. Then, Nitrogen gas was introduced at 40 SCCM in the sample chamber; the plasma was ignited at 0.5 kPa at a microwave power of 350 W for 5 minutes. The temperature was kept at 25 °C in a nitrogen purged atmosphere inside the chamber. Finally, the substrates were taken out of the chamber and used for further characterization and applications.



2.2.3. Characterization of CNWs: Surface morphology characterization of CNWs was carried out using a Scanning Electron Microscope (SEM) (Hitachi SU6600) operated at a voltage of 5.0 kV and beam current of 14.9 µA for all the samples. The transmission electron microscope (TEM) characterization was conducted using a JEOL Ltd. JEM 2200FS equipped with an omega filter; the electron beam voltage was 200 kV and beam current of 156 µA. Surface topography analysis was conducted with an Atomic Force Microscope (Asylum Research MFP3-3D), in AC mode (Tapping mode) with an Olympus OMCL-AC240TS silicon cantilever having a resonance frequency of 70 kHz and spring constant of 2 N/m. The scan size was set to 50.00 μm with a scan rate of 0.25 Hz/s. The physiochemical characterization was performed by Hard X-ray Photoelectron Spectroscopy (HXPS) using ULVAC-PHI, Inc. PHI Quantes instrument. Aluminium (Al) was used for X-ray generation, the monochromatic Al Kα was hν =1486.6 eV, the pass energies were 55 eV and 280 eV for the high resolution and wide scan spectra respectively. Auger Electron Spectroscopy was performed as complimentary technique to XPS. A JEOL JAMP-9510F Field Emission Auger Microprobe, with a dark space resolution of 3 nm, ion gun voltage of 10 keV and current of 1.09×10-7A, with tilt angle of 30.0° was used. Raman Microscope Spectroscopy was performed using LabRAM Horiba HR-800UV equipped with an Ar ion laser, the laser wavelength used for all the experiments was 514 nm and the magnification was kept at 100 X. The contact angles of the samples were measured using sessile contact angle measurement using Kyowa Interface Sciences Drop Master DM 301, using the half angle method, with the droplet size kept at 1.0±0.1 μl. The surface of Si, SiO and Poly (methyl methacrylate) (PMMA) slide were measured as controls to better characterize the hydrophobicity of CNW. Finally, the surface zeta potential for the glass cover slip (CS), Tissue Culture Plastic (TCP), CNW and CNW-N2 were measured using Malvern Zetasizer Nano ZS. Measurements were conducted using water as the dispersant and polyethylene tracer particles of 1 μm diameter, with all experiments conducted at a pH of 7.0. All Contact angle and Surface Zeta Potential experiments were carried using Millipore MilliQ Deionized Distilled water with a resistivity of 18.2 MΩ·cm at 25°C.

2.2.4. Cell culture:


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HBMECs were cultured in T25 flasks (TPP) in Complete classic medium and maintained in a humidified incubator at 37 °C under a 5 % CO2 flow. The sample substrates (placed individually in a 6-well culture plate) were surface sterilized under UV light for 30 min, post which approximately 25,000 cells (dispersed in 500 μl medium) were deposited directly on the substrates. The substrates were transferred to an incubator (37 °C with 5 % CO2) for 30 min to allow the cells suspended in media to settle down. After the short incubation time, 2 ml of media, enough to submerge the substrates was added to the wells and returned to the incubator. All cell-based assays were performed post 72 h incubation of the cells, to provide sufficient time for the cells to interact with the respective substrates. Post-incubation, the contents of the plates were aspirated, replaced by 2 ml of fresh medium and 200 μl of PrestoBlue reagent in each well and incubated at 37 °C with 5 % CO2 for 1h. The emission was recorded at 590 nm with 520 nm excitation using a Microplate Spectrophotometer (Powerscan HT, Dainippon Sumitomo Pharma). Relative cell viability values were expressed as the percentage of the emission from the respective wells. CS was treated as positive control and its viability was set to 100 % and the values observed for CNW and CNW-N2 correlated accordingly. Intracellular reactive oxygen species (ROS) was profiled using the DCFH-DA dye and proliferation marker analysis using Ki67, according to manufacturer’s instructions and read in a microplate spectrophotometer at 450 nm after wavelength correction. The cells were also stained with Calcein/PI live/dead staining kit, actin cytoskeletal, nuclear, vimentin and CD31 staining (according to product instructions) and observed under an upright microscope (Nikon ECLIPSE 80i) at an excitation wavelength of 405 nm (NucBlue), 488 nm (CD31/Calcein) and at 561 nm (Phalloidin Rhodamine/Propidium iodide). The angiogenic profiling was done according to the manufacturer’s instructions and the proteins expressed were analyzed by running them in stringdB database.

2.2.5. Cell preparation for SEM analysis: Cell preparation for SEM evaluation was performed according to previous report29 with modifications. Cells (on respective substrates) were prefixed in 0.1 % glutaraldehyde in culture media for 5 minutes. The medium was then decanted and replaced with 2.0 % glutaraldehyde in PBS at room temperature and fixed for 30 min. Specimens were dehydrated: 5 minutes each with 50 %, 70 %, 90 % and 100 % ethanol, air dried and viewed in a SEM at 5 kV.



3. Results and Discussion 3.1

Morphological Characterization:

The top view and side view of CNW and CNW-N2 are shown in Fig. 1a and 1b, respectively. The width of CNW individual flakes was measured to be 1~5 nm depending on the nanographitic wall’s thickness, while the CNW film height was in the range of 10 ~ 12 μm. The vertical nanographtic layers were in the form of petal-like morphology. Hydrogen etching has been proposed as the reason for the interconnected graphitic petal formations30. There were no considerable topology/morphological differences observed between the pristine CNW and CNW-N2. Morphology of the CNWs suggests that the nitrogen plasma does not cause nanoscale etching on the edge planes of CNW as seen in the width of individual nanographtic walls as shown in Fig. 1a). It was observed that the CNW layer adhesion on Si substrate was significantly improved by applying a bias voltage of 10 V to the substrate. The CNW micro petal grains are also dependent on the substrate material used31, suggesting increased diffusion of carbon radical species in the substrate and directly affecting the synthesis of CNWs thus contributing to the interconnected petal like morphology. The TEM images that are seen in Fig. 1c and d, show that the CNW structure is composed of a few nm layers of graphene that overlap to form the individual walls, the inter lattice fringes of CNWs can be seen in the magnified insets in Fig. 1c and d, showing minimal changes in atomic spacing and lattice dislocation. This confirms the presence of a homogenous micrographitic layer and minimal etching due to nitrogen plasma. The TEM images also highlight the excellent surface to volume ratio and very high aspect ratio of CNWs, thus allowing them to have a very large surface area to promote attachment of cell. The lack of any morphologically identifiable changes between the pristine and N2 functionalized CNWs was again evident from the AFM analysis (Fig. 2a-e, f-j respectively). The lack of cracks in the CNW structure before and after plasma functionalization signifies good mechanical integrity of the nanographtic walls and overall resistance to micro etching. The height for both of the CNW films was found to be 200 nm indicating minimal morphological damage caused by ion etchings during the process. Therefore the nitrogen plasma etching only affects the edge planes of CNW32. The roughness of CNWs as measured with AFM revealed that in both CNW and CNWN2 there were no detectable changes.


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3.2 Physiochemical Characterization: To assess the composition and chemical state of CNWs, HXPS analysis was performed. In the case of C1s bonding as shown in Fig. 3a, the 284.6 eV peak is assigned to C-C bonding33, the 285.7eV peak refers to C-O bonding and the peak at 290 eV signifies the π- π* stacking. In Fig. 3b the N1s peak at 398.3 eV can be seen, clearly indicating the presence of interstitial N2 atoms on the surface layers of the functionalized CNWs. The pristine CNW does not show a prominent N1s peak as can be seen in Fig. 3c. The presence of Oxygen O1s peak at 532 eV (Fig. 3c)34, can be theorized to the existence of high energy nitrogen free radicals in the plasma, lowering the binding energy of the edge plane C-C bond and allowing for the formation of carboxylate and C-O bonds and thus allowing for Oxygen atoms to bind to the surface of the CNW and increasing the Oxygen moiety in the CNW structure. Formation of C5H5N and C4H5N species can also be theorized arising from the graphitic walls and basal edge planes after nitrogen functionalization35. The elemental percentage of Carbon, Oxygen and Nitrogen is presented in Table 1. Auger analysis showed that for CNW-N2, the C1s peak is at 280 eV while the N1s peak is at 400 eV (Fig. 3d). This reconfirms the successful Nitrogen doping on the surface of CNW. Raman spectroscopy revealed a 488 nm Raman shift for non-functionalized CNWs with D and G peaks at 1168 cm-1 and 1373 cm-1 respectively, while the 488 nm Raman shift for CNW-N2 are identified by the D and G peaks located at 1173 cm-1 and 1373 cm-1, respectively (Fig. 4). On the other hand, the D and G peaks while using 514 nm laser, on the samples without plasma treatment were 1373.8 cm-1 and 1562 cm-1, which is similar to that of CNW with nitrogen functionalization. The 2D and G+D peaks at 2073.25 cm-1 and 2929.3 cm-1 signify the sp2 hybridization34( Fig. 4.) This confirms that the nitrogen plasma etching causes increase in defects in the CNW edge planes and is responsible for doping of material as can be seen in the more prominent G peak in Fig. 4. Also, from Table 2, we can infer that the ID/IG ratio for CNW-N2 is 1.11 and for CNW is 0.98. This difference in the ratios is caused due to the increase in localized defects in the graphitic edge basal plane36. The surface zeta potential of CNW and CNW-N2 as shown in Table 3 (Fig. S1) shows the higher negative value for CNW as compared to CNW-N2. This implies that there is a difference in surface charges because of the increase in pyridine formation37,38 due to the incorporation of nitrogen in the graphitic walls thus increasing the surface zeta potential values. The high negative value is also an indicator for good stability of the film.



3.3 Surface Wettability Characterization: The sessile contact angle has been used to indirectly infer the surface energy of the CNW samples as can be seen from Table 4 (Fig. S2). The contact angle is dependent on the surface energy and surface tension, which are in turn dependent on the thin film nanostructure. The hydrophobicity of CNW can be shown using the Cassie-Baxter model39. In the case of hydrophobic CNWs, the contact angle results suggest a CassieBaxter wetting. According to this model the CNW film can be considered a homogenous film and thus the surface tension on the water droplet is high. However, after the introduction of Nitrogen atoms, the surface tension of the CNW-N2 film is seen to drastically reduce. Since the morphology of CNW and CNW-N2 are the same, the contact angle of the samples is not dependent on the functionalized CNW’s surface roughness, but rather the Nitrogen and Oxygen interstitial atoms in the structure of the graphitic walls. 3.4 SEM analysis of cell adhesion and proliferation: In order to evaluate the feasibility of the substrates as cell anchoring platforms, we plated HBMECs, on CS, CNW and CNW-N2 substrates. CS was used as the cell-culture compatible reference, which is an already well-established culture substrate especially for SEM and fluorescent microscopic investigations. SEM observation to understand the morphological characteristics of cultured cells on substrates were performed. The qualitative analysis of cellular architecture after 3-day culture indicated that the cells were well-formed, adhered to and spread on both pristine (Fig. 5c, d) and N2 functionalized CNW (Fig. 5e-i) substrate’s surface area. In contrast, cells on CS though showed wellformed cells (Fig. 5a, b), they were not as spread as those seen on CNW substrates. Furthermore, the cells grown on CNW-N2 substrates exhibited greater cell adhesion (Fig. 5f inset) with the surface due to lower surface energy when compared with CNW or CS. Also, it is to be noted that CNW-N2 substrates exhibited similar zeta potential as that of CS and TCP (common culture substrates) (Tab. 3, Fig. S1), thereby rendering the surface more hydrophilic by means of increasing the concentration of Oxygen-containing molecular groups. It is by this hydrophilic property that anchorage-dependent cells are able to attach to the surface and become confluent. Hydrophilic surfaces are characterized with water contact angles less than 40°

40,

suggesting that the moderate wettability of

CNW-N2 plays a crucial role in promoting higher cell attachment. The cellular preference for hydrophilic surfaces over hydrophobic surfaces is known16 with characteristics as cell spreading, shape, and cytoskeletal organization differing with surface wettability and


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charge. Although cellular morphologies were relatively similar on all three surfaces, the rounded, well spread morphology were predominantly present on CNW-N2 while the bipolar morphology was pronounced on CS. These observations were made even though zeta potentials of CS and CNW-N2 exhibited similar values (Tab. 3, Fig. S1). This can be attributed to the fact that carbon surfaces bind with serum proteins in culture media via electrostatic interactions due to the presence of oxygenated groups and consequently enhance the cell attachment due to a higher population of surface-bound molecules41, compared to the CS surface. Additionally, long cytoplasmic extensions were observed on CNW-N2 substrates which seem to promote cell adhesion and spreading characterized by numerous elongated and sturdy filopodia with surface ruffles (indicated by yellow lines in Fig. 5g-i as well in inset in Fig. 5f). This observation was crucial in determining the preferential cellular adhesion onto CNW-N2 substrates, indicative of the ability of these cells to efficiently adapt and migrate on the surface. Also, we believe that the substrate’s local topography offers anchoring sites for the filopodia that helps with cell crawling, thus guiding cell migration. Such a phenomenon holds high significance when studying the effects of nanoscale topologies on formation of filopodial structures. Compared with cells on CS and CNW, those in contact with CNW-N2 were seen to form intercellular connections with multiple cells (Fig. g-i). Such observations are reflective of the enhanced cell activation index42 which is an indicator for the ‘active metabolic state’ of the cells. Activated cells are usually larger with prominent plasma membrane ruffling, exhibiting enhanced adherence and spreading, along with increased pseudopod formation. These observations were predominantly made in the CNW-N2 samples and though could be seen in CS and CNW as well, however were less pronounced compared to the former. It is believed that differences in surface chemistry and wettability between CNW and CNWN2 influence the protein conformation and adsorption on the respective surfaces which in turn determine the cellular morphology and subsequently their function. 3.5 Cytocompatibility and proliferation kinetics: Once the practicability of the substrates to support cell growth was established, analysis of toxicity, if any, induced by the substrates was evaluated. Live/dead cell staining was performed to visually assess the cytocompatibility of tested substrates. Fig. 6a-f shows representative images of live/dead staining with viable cells (calcein stained in green) and dead cells (PI stained in red). After 72 h of culture, most of the cells on all three substrates were viable and healthy (stained green) (Fig. 6a, c, e). These images clearly showed that



all tested substrates exhibited good cell compatibility. One could also notice the confluency and complete cell coverage in all three substrates. Further, to quantify the metabolic activity of the cells PrestoBlue cell viability reagent was employed. Cells grown on CS were designated as controls (fluorescence value normalized to 100 %). As shown in Fig. 6g, the viability of cells grown on CNWs showed a deficit of 18 % as compared with CS. Though the metabolic activity of the cells was slightly compromised, the cell count remained on par with most cells remaining viable. The microscopic observation (Fig. 6a,c,e) made us to hypothesize that the decrease in metabolic activity was not due to cytotoxic behavior or decreased cell count but due to metabolism differences between the cells grown on substrates. This statement is supported by the fact that cells grown on CNW-N2 substrates exhibited 1.6 times higher metabolic activity when compared to CS, indicating augmented metabolism. The higher metabolic activity in cells cultured on CNW-N2 is directly correlative of EC activation which could be attributed to the higher wettable surface area of CNW-N2 that facilitates adsorption of cell activating proteins. Next, we profiled, the ROS production in similar experimental condition using DCFH dye. Again, the ROS production in both coverslip and CNW grown cells remained similar with quantitatively insignificant variation (Fig. 6h). However, cells grown on CNW-N2 showed 1.3 times higher ROS production that could be attributed either to cell stress or higher metabolic activity. It has to be reckoned that the constant aerobic respiration in the mitochondria is responsible for majority of the cellular ROS43. Also, we examined the ki67 profile in all the samples by ELISA. Ki-67 is a 350-370 kDa nuclear protein associated with the mitotic chromosome-associated proteins, expressed in all cells except those not in the G0 phase of the cell cycle, making it a perfect marker for cell proliferation determination. When analyzed, as expected CNW-N2 grown cells exhibited near to twice expression of Ki-67 when compared to CS and CNW group; whereas the Ki-67 expression in CS and CNW remained similar with insignificant difference (Fig. 6i). This higher expression of Ki-67 re-confirms the ability and role of CNW-N2 substrate in EC activation. 3.6 Cytoskeletal marker analysis: Cytoskeletal alterations, especially actin reorganization greatly influence the cellular morphology and physical characteristics44. Therefore, we analyzed the actin arrangement in the respective test samples. Actin forms a group of globular multi-functional proteins responsible for major cellular activities such as governing the cell dynamics during cell division via a network of microfilaments. A clear difference in actin expression between


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CNW-N2 and the other groups was recorded (Fig. 7c,g,k). Cells grown on CNW-N2 were well spread with distinctly-defined actin stress fibers (Fig. 7k) and actin-rich core at the cell-substratum contact zones that would positively elevate the adhesion kinetics with the extracellular matrix. On the contrary, CS and CNW exhibited a bipolar morphology with comparatively smaller cell area and actin filaments concentrated in the cell periphery (Fig. 7c, g). The higher expression of actin by cells on CNW-N2, is correlative of the SEM observations where the cell-cell and cell-substrata contact was well pronounced (Fig. 5e-i) when compared to CS or CNW. Further, analysis of another major cytoskeletal intermediate filament, vimentin, responsible for maintaining the mechanical integrity and structural support to cells, was assayed. Vimentin is fundamental to the endothelial phenotype, essaying a crucial role involving the physiological EC mechanoresponse. Decreased vimentin expression imparts severe irregularities in cell attachment, migration, signaling, neurite extension, and vascularization45. The expression of vimentin was found to be qualitatively higher in CNW-N2 grown cells throughout the cell body (Fig. 7q), whereas in CS (Fig. 7m) and CNW (Fig. 7o) samples the expression was comparatively lower. Vimentin is known to positively influence EC activation facilitating cell adhesion, migration and angiogenesis. As with actin, the expression profile for vimentin was also highly supportive of the SEM observations. To further understand the EC activation and cell-substrate adhesion phenomenon, we investigated the expression of CD31, a member of the immunoglobulin superfamily present on the intercellular junctions of ECs. CD31 is known to be a major player in the angiogenesis (new blood vessel formation) process and determines the adhesion cascade between endothelia and inflammatory cells46. Confirming the speculation based on previous actin/vimentin expression profiles, CNW-N2 group exhibited higher CD31 expression at cell surface (Fig. 7w) than the cells grown on other two substrates (Fig. 7s, u). The higher expression of vimentin along with CD31 in the cells grown on CNW-N2 indicates the mechanoinduction of EC activation by this substrate. With the absence of any extra chemical cues, we believe that the surface topography of CNW-N2 with suitable wettability and surface charge leads to these observations. As ECs line the blood vessels in the in vivo scenario, they are constantly exposed to blood flow-induced shear stress. Therefore, maintaining the physiologic endothelial phenotype characterized here by the actin, vimentin and CD31 profiling is essential to survive such mechanical cues.



It has to be understood that the flexibility and adaptability of cytoskeletal proteins is a major part of a robust endothelial mechanoresponse. This could be explained by the fact that during shear stress, as with the case of blood flow, EC actin stress fibers realign with the flow direction and micro/nanometer displacements are witnessed with vimentin, so as to minimize shear damage16. So, we believe that cells grown on CNW-N2 substrates undergo such robust cytoskeletal alignment due to substrate adherence mediated mechanoresponse leading to active and functional ECs. 3.7 Angiogenic profiling: As mentioned earlier, ECs play a crucial role in the blood vessel formation and development (angiogenesis), both in the normal and tumorigenic conditions. Therefore, profiling of angiogenesis related proteins expressed by the cells grown on different substratum were investigated. Out of the many angiogenic analytes, 8 were expressed in all three test groups (Fig. 8, Table 5). However, in addition to these, CNW-N2 showed expression of 5 more analytes (MMP8, MMP9, IGFBP3, PDGF-AB/BB and uPA) that were not expressed in CS or CNW. The expression of most of these proteins are positively correlated with activation of ECs and angiogenesis induction (Table 6) with discrete role in the angiogenesis pathway (Fig. S4). The proteins expressed by cells grown on CNWN2 that are positive regulators of angiogenesis, were run in stringdB database to map the protein-protein interaction between the protein of interest and their functional partners. Few proteins of interest were found to be co-occurring functional partners between themselves, underscoring their close interaction in angiogenesis, which are tabulated in Table 7. and the interaction maps of each protein of interest are shown in Figure 8. The co-expressing angiogenic proteins with high co-expression score for the proteins of interest were determined and few of those involved are tabulated in Table 7. These vascularization-related protein groups might be the actual reason of EC activation that leads to increased cell metabolism and other immunochemical observations. It is to be noted that most of the proteins that are predominantly expressed by cells grown on CNWN2 groups were indeed proteins that have a positive role in extracellular matrix remodeling, pericytes proliferation, EC survival/proliferation, migration (as indicated in angiogenesis pathway map in Fig. S3) indicating that mechanostimulus induced by the CNW-N2 substrate can in fact lead to functional activation of ECs modulating cellular proteome expression rather than just being a mechanical support.


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4. Conclusions: Human Endothelial cells were successfully proliferated on N2 functionalized Carbon Nanowall substrates to create biocompatible scaffolds for the cells due to the tunable hydrophobicity of CNWs. The similar morphology of CNW and CNW-N2 is distinguished in their physiochemical characteristics, thus suggesting the formation of –C-O-, -C=O-, C5N5N and C4H5N in the edge planes and the graphitic walls. Increased Oxygen content in CNW-N2 is attributed to the lowering of energy levels in the structure as a result of structural rearrangement due to the Nitrogen radical incorporation in the CNW structures. This surface modification also reflects changes in the contact angle and zeta potential. Most research on alternative substrates based on CNMs for cell growth in vitro is focused on the material’s ability to provide efficient anchorage to the cells in consideration and seldom discuss on the maintenance/enhancement of the inherent characteristics of cells. In our observations, we found that the CNW-N2, not only proved to be a highly compatible and able support for cell anchorage, but was able to retain the basic instincts of the endothelial cells by allowing for the cytoskeletal network rearrangement according to the anchorage platform, mimicking to an extent their similar properties under blood flow conditions in vivo. Also, certain major angiogenic markers were found to be additionally expressed on the CNW-N2 substrates. This distinctive property of the CNW-N2 substrate to act as a pro-angiogenic promoter holds great advantage when considering applications in generating vascularized grafts for ophthalmology/tissue reconstruction/3D organ printing. This work will surely contribute to a better understanding of CNW functionalization and application in the biomedical field, and can be extended to develop materials in tissue engineering, regenerative medicine, lab-on-chip devices and biosensors.

Supporting Information: Additional experimental data as surface zeta potential characterization, contact angle, angiogenesis pathway is available as supplementary information.

Acknowledgement: Vimal Kumar would like to thank the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Government of Japan for financial support in the form of Monbukagakusho scholarship since April 2015.



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FIGURES


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Scheme 1



Figure 1. Electron microscopic characterization: (a), (b) SEM low magnification image of CNW/CNW-N2 respectively. Respective upper right insets present the lateral view and lower right shows the magnified view of the graphitic nanostructures with the distinctive petal-like morphology. (c), (d) shows the TEM images of CNW and CNW-N2 respectively; the insets represent the magnified version, the atomic arrangement of the nanographitic structure is clearly visible in both cases.


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Figure 2. Surface topographic analysis: (a), (b), (c) shows the surface morphology of CNWs from lateral, diagonal and top views respectively while (d) and (e) show the cross section of CNW profile and corresponding height profiles respectively. Similarly figures (f), (g), (h) show the surface morphology of CNW-N2 from the lateral, diagonal and top views respectively while (i) and (j) shows the cross section of CNW-N2 profile and corresponding height profiles respectively. The height profile of the CNW-N2 gives indication of minimal etching in CNW-N2 with similar topological characteristics as the non-functionalized counterpart.



Figure 3. Elemental characterization: (a) shows the XPS C1s peaks of CNW and CNWN2 at 284.8 eV signifying C-C bond (b) The XPS N1s peak position at 398.2 eV, which is characteristic of pyridinic (C5H5N) bonding confirming the presence of N interstitial atoms (c) shows the characteristic O1s peaks from 531 eV to 532.2 eV signifying C-O, C=O and –O-C=O- bonding. (d) Show the AES wide spectrum of CNW and CNW-N2, here C1s is assigned at 285 eV and N1s peak is assigned at 398 eV thus confirming the Nitrogen doping of CNW.


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Figure 4. Raman Spectroscopy: (a) shows the characteristic Raman spectra of graphitic carbon nanostructures. The D, G, D’ and D+G are identified at 1168 cm-1, 1373 cm-1 while the 2D and G+D peaks are at 2073.25 cm-1 and 2929.3 cm-1. The ID/IG ratio is significantly higher in the case of CNW-N2. (b) magnified D and G peak.



Figure 5. Cell adhesion and proliferation on substrates. (a-b, c-d, e-f) represents the cells grown over CS, CNW and CNW-N2, respectively. Prolific cell adhesion and surface ruffling was observed in the case of CNW-N2 substrates. ‘Gripping’ (f, yellow arrows) of the CNWs was clearly observed with nearly all the cells in the functionalized substrate. Also, highly aligned, extended and interconnected cellular morphology was witnessed in CNW-N2 (g-I, yellow lines).


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Figure 6. Cytocompatibility analysis. (a-f) The live and dead population analysis did not show any significant variation among all the test substrates, nullifying their toxicity aspects (scale bars represent 200 µm). (g) cellular metabolic viability investigations revealed a comparatively higher ‘active’ state of the cells in the CNW-N2 group, whereas there was no significant variation observed with CS or CNW. (h). Analysis of ROS depicted a higher percentage of generation in the functionalized substrate, which based on the cell viability and live/dead investigations, was determined to be related to the enhanced metabolism of the mitochondria, releasing the ROS as byproducts. (i). The cellular proliferation marker, Ki67, was evidently higher in expression in cells grown on CNW-N2 than the other substrates. It has to be noted that the values observed with viability, ROS and Ki67 in CS and CNW were not less, but were comparatively lower when related to CNW-N2.



Figure 7. Cytoskeletal organization analysis. The dynamics of actin microfilaments, comprising polymerization and cytoskeletal arrangement on respective substrates was imaged with rhodamine phalloidin. Actin was present in its intact and defined structural form in CNW-N2 cells (i, k), whereas in CS (a, c) and CNW (e, g) the actin network was more pronounced in the peripheral regions. Insets (b,f,j,d,h,l) represent composite images of actin and nuclear region. Double immunocytochemical detection of the cytoskeletal component, vimentin revealed a noticeable difference in expression patterns of CNW-N2 (q) and CS/CNW (m/o). The discrete signals of vimentin throughout the cellular body in CNW-N2 was not as pronounced in the other samples. Insets (n,p,r) represent composite images of vimentin and nuclear region. Similarly CD31 expression was also relatively higher in CNW-N2 (w) as compared with other substrates (s,u). Insets (t,v,x) represent composite images of CD31 and nuclear region. Scale bars for (a,b,e,f,i,j) represent 200 µm and for rest of the images represent 20 µm.


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Figure 8. Angiogenic protein expression and bioinformatic mapping. The relative levels of human angiogenesis related proteins expressed by cells cultured on different substrates shows expression of 5 proteins that are only expressed in CNW-N2 substrates (indicated by blue arrow) along with three other proteins that are expressed by cells grown on all three substrates. These eight angiogenic proteins were bioinformatically analyzed for coexpression protein profiles that are shown (a-h). (a-h) represents bioinformatics coexpression maps of close-knit proteins of MMP8, MMP9, IGFBP, PDGFB, PLAU, FGF2, EGFR, VEGF respectively.



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TABLES Table 1. XPS Atomic concentration in % C1s

N1s

O1s

CNW

99.54

0.16

0.3

CNW-N2

98.58

0.85

0.56

Table 2. Raman Spectra ID/IG ratio CNW

CNW-N2

Wavelength ID

IG

ID/IG

ID

IG

ID/IG

514nm

2.162

0.98

4.01

3.61

1.11

2.125

Table 3. Surface Zeta Potential of materials MATERIAL SURFACE ZETA POTENTIAL (mV)

CS -71

TCP -52

SILICON -31

CNW -127

CNW-N2 -71.6

Table 4. Average contact angles for different samples Material

Θ1

Θ2

Θ3

Θ4

Θ5

Θavg

PMMA

74.1

78.2

77.3

72.4

75.3

75.46

CNW

105.8

114.6

110.2

112.4

104.3

109.46

CNW-N2

12.8

11.7

7.3

4.9

5.8

8.5

Table 5: Parallel determination of relative levels of angiogenesis-related proteins. GDNF EGF Angiogenin PD-ECGF FGF-b Activin A IL-8 VEGF MMP8

CS + + + + + + + + -

CNW + + + + + + + + -


CNW-N2 + + + + + + + + +

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MMP9 IGFBP3 PDGFAB/BB uPA

-

-

+ + +

-

-

+

Table 6: Significantly expressed angiogenic proteins and their functions in terms of endothelial expression. Proteins FUNCTION IN TERMS of Angiogenesis MMP8 ECM Matrix Remodeling MMP9 ECM Matrix Remodeling IGFBP3 Helps IGF to bind IGFR; Activates endothelial migration PDGF-AB/BB Binds PDGFR; Activates pericyte proliferation uPA ECM Matrix Remodeling FGF-b Binds FGFR; Activates endothelial migration EGF Binds EGFR; Activates endothelial proliferation VEGF Binds VEGFR2; Activates endothelial survival Table 7: Expressed angiogenic proteins and their functional co-expressing proteins with co-expression score. PROTEINS FUNCTIONAL PARTNER CO-EXPRESSION SCORE MMP8 MMP9 VEGFA 0.987 PLAU 0.981 IGFBP3 IGF1 0.998 IGF2 0.997 MMP2 0.958 MMP1 0.954 PDGF uPA MMP9 0.981 MAPK1 0.962 FGF-b FGFR 0.999 MAPK3 0.982 EGF EGFR 0.999 SRC 0.994 VEGFA 0.993 IGF1 0.993 INS 0.990 VEGF HIF1A 0.996 EGF 0.993 IGF1 0.991 PIK3CA 0.991



TOC Graphic


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Functionalized Carbon Nanowalls as Pro-Angiogenic Scaffolds for

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