Effect of Polyelectrolyte Multilayers Assembled on Ordered

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Effect of polyelectrolyte multilayers assembled on ordered nanostructures on adhesion of human fibroblasts Marcus S. Niepel, Joao F Mano, and Thomas Groth ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09349 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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ACS Applied Materials & Interfaces

Effect of polyelectrolyte multilayers assembled on ordered nanostructures on adhesion of human fibroblasts Marcus S. Niepela,b, João F. Manoc, Thomas Grotha,b* AUTHOR ADDRESS a

Institute of Pharmacy, Biomedical Materials Group, Martin Luther University Halle-Wittenberg

b

Interdisciplinary Center of Materials Science, Martin Luther University Halle-Wittenberg

D-06099 Halle (Saale), Germany Email: [email protected] [email protected] c

Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro,

3810-193 Aveiro, Portugal Email: [email protected]

KEYWORDS nanolithography, patterning, polyelectrolyte, poly (ethylene imine), heparin, self-assembly, fibroblast

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ABSTRACT

Nanosphere lithography (NSL) and layer-by-layer (LbL) technique are combined here for the first time to design a flexible system to achieve nanotopographical control of cell adhesion. NSL is used to generate regular patterns of tetrahedral gold nanodots of different size and distance. Beside the change in topography, LbL is used to generate a polyelectrolyte multilayer (PEM) system consisting of heparin (HEP) and poly (ethylene imine) (PEI) on top of the gold dots. The localized formation of PEM on gold dots is achieved by prior passivation of the surrounding silicon or glass surface. Properties of PEM are changed by adjusting the pH value of HEP solution to either acidic or alkaline values. Studies with human dermal fibroblasts (HDF) reveal that cells spread to a higher extent on PEM formed at pH 5.0 in dependence on the structure dimension. Further, filopodia formation is highly increased in cells on nanostructures exhibiting HEP as terminal layer. The new system offers a great potential to guide stem cell differentiation in the future owing to its high degree of chemical and topographical heterogeneity.

INTRODUCTION Mammalian cells in tissues are adhesion-dependent, i.e. survival and growth are dependent on cell-matrix as well as cell-cell interactions, which holds for cells in connective, muscle, and nerve tissues as well as epithelia.1 A three-dimensional network of fibrillar proteins like collagens, proteoglycans and glycoproteins, the extracellular matrix (ECM), provides structural stability and presents chemical and mechanical cues to cells.2 Cell surface receptors like integrins regulate the adhesion of cells to this network by linking them to various matrix proteins such as collagen, fibronectin (FN), laminin and others.3 Subsequently to integrin ligation, assembly of focal adhesions and signal transduction is initiated that regulates cellular processes like


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migration, growth, differentiation and survival.4 In addition to the presence of these chemical cues also their spacing5 and mechanical properties of ECM have a strong impact on cell behavior as shown by several studies.6-8 Spatz et al. demonstrated that ligand spacing at the nanometer scale is a determinant of focal adhesions assembly of integrins and subsequent signal transduction in cells.9 Several top-down and bottom-up approaches can be used to modify the topography of materials at the nanoscale for fundamental studies and biomedical applications.10-11 Methods such as electron beam lithography (EBL)11, photolithography12, nanoimprint lithography (NIL)13, microcontact printing (µCP)14-15, or laser interference lithography (LIL)16 are used to machine features top-down into the material surface. In contrast, bottom-up methods use self-assembly of smaller and simpler building blocks (atoms, molecules, nanoparticles, etc.) to assemble larger, more complex structures. Non-templated self-assembly is attractive due to its simplicity and potential efficiency to achieve an ordered structure only by mixing of components. Here, selfassembled monolayers (SAM) or nanostructures from block-copolymers are widely applied.17-19 Further, the layer-by-layer (LbL) technique, based on electrostatic interaction of oppositely charged molecules or other building block (e.g. nanoparticles), can also be used to design nanostructures in lateral as well as z-direction.20 Beside these classical top-down and bottom-up approaches, there are also hybrid techniques that combine the advantages of both. For example, in colloidal lithography the self-organization into hexagonal close-packed (hcp) masks is characteristic for bottom-up, while patterned layers obtained after metal vapor deposition or possible etching can be assigned to top-down approaches.21-22 Nanosphere lithography (NSL) as an example of colloidal lithography is based on the spreading of nanometer-sized polystyrene beads on planar surfaces to be used as mask for


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subsequent gold vapor deposition. After dissolution of polystyrene particles tetrahedral gold structures remain on the surface.22 Due to the different chemistry of background and gold dots, diverse modification steps have to be applied. The former is often silicon dioxide or glass which can be passivated by silanes terminated with oligo ethylene glycol, while gold can be modified with thiols.21 Thereby, size and distance of the nanostructures can be varied using particles of different diameter.23 In a recent work we could demonstrate that decreasing the distance of gold dots was related to increased spreading and growth of fibroblasts.21 The LbL technique on the other hand, as classical bottom-up technique, leads to formation of multilayers on charged material surfaces. Multilayer properties like thickness, viscoelasticity, surface charge, and wettability can be controlled by selection of polyelectrolytes as well as process parameters like pH value, ionic strength, or temperature during multilayer assembly.20, 24-25 Particularly surface charge and wettability of biomaterials are important for adsorption of proteins and adhesion of cells.26-28 Previous studies with LbL have shown that multilayers terminated by heparin as polyanion adsorb low quantities of proteins like FN and are cytophobic, when multilayer formation is carried out at acidic pH and terminal layers are highly hydrophilic. By contrast, protein adsorption and cell adhesion increased on heparin-terminated multilayers prepared at basic pH value, when multilayers became less hydrophilic.29-30 Since NSL allows only a stepwise change of distance of gold dots, a more continuous method to tailor surface properties to control cell spreading would be desirable to program cell differentiation. Hence, adjustment of pH value during multilayer formation is another way to affect cell adhesion and spreading. We try here - to our knowledge for the first time - to combine NSL and LbL to study the effect of nanostructure dimension and pH value during final stages of multilayer assembly on adhesion and spreading of human fibroblasts. Results are reported herein.


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EXPERIMENTAL SECTION Surface cleaning Silicon wafers (Si-Mat, Germany) and glass coverslips (Menzel, Germany) as model surfaces were cleaned before use with ‘RCA clean 1’.31 Briefly, ultrapure water and ammonia (Roth, Germany) were mixed and heated to 75-80°C, and hydrogen peroxide (Roth) was added to achieve a ratio of 5:1:1 (v/v/v) for the removal of organic residues. After 10 min, all samples were excessively rinsed with ultrapure water (6 cycles of 5 min), dried with a stream of nitrogen, and used immediately for further modification. Colloidal lithography Nanostructures were designed by NSL as described elsewhere.21 Briefly, polystyrene nanoparticles (PS-NP, Microparticles, Germany) of different diameters (476, 756, and 1390 nm) were deposited either by spin coating (Süss MicroTec, Germany, Ø 1 µm) on planar substrates to obtain monodisperse monolayers. The assembled, hcp PS-NP templates were used to generate tetrahedral gold nanostructures by electron beam physical vapor deposition (EBPVD).21 Here, the masks were coated with 10 nm chromium and 75 nm gold and distinct nanostructures were generated after removal of the PS-NP using organic solvents. Self-assembled monolayers SAM were used to passivate and activate the nanostructured samples with varying surface chemistry. First, the free substrate surface, either silicon or glass, was blocked by incubating the samples in a 1% solution of 2-[methoxy-(polyethyleneoxy)propyl] trimethoxysilane (OEG-


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silane, ABCR, Germany) in ethanol p.a. (Roth) at room temperature overnight. Thereafter, the samples were rinsed with the solvent and ultrapure water, dried with a stream of nitrogen, and stored in a desiccator until further use. Second, the gold nanostructures were modified by incubating all samples in 2 mM mercaptoundecanoic acid (MUDA, Sigma-Aldrich, Germany) in ethanol p.a. at room temperature overnight to introduce terminal carboxyl groups and, thus, a negative charge similar to silicon or glass, permitting subsequent multilayer formation or specific interaction of cells. Again, all samples were rinsed with the solvent and ultrapure water, dried with a stream of nitrogen, and stored in a desiccator until further use. Polyelectrolyte multilayer formation PEI (750 kDa, Sigma-Aldrich) and HEP (8-25 kDa, Applichem, Germany) were dissolved at 2 mg mL-1 in phosphate buffered saline (PBS, 5.1 mM NaH2PO4, 11.7 mM Na2HPO4, 140 mM NaCl, pH 7.4). The intrinsic pH value of the PEI solution was pH 10.3 ± 0.1 and not controlled during layer formation. However, the intrinsic pH value of the HEP solution was pH 7.4 ± 0.05 and adjusted to either pH 5.0 or pH 9.0 for deposition of the terminal HEP layer only, which represented the 8th overall layer. For visualization of the layer formation, the carboxylic acid group of HEP was labeled with 6-aminofluorescein (Fluka, Germany). Details of the process can be found in the supporting information. Both PEL were allowed to adsorb at room temperature onto the nanostructured samples for 30 min, starting with PEI as first layer. Each adsorption was terminated by rinsing with ultrapure water twice for 5 min. Finally, multilayer formation was halted after eight or nine single layers, abbreviated as (PH)4 for the terminal HEP layer and (PH)4P for the terminal PEI layer.


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Surface morphology The distribution and morphology of pristine or PEM-modified nanostructures was investigated with scanning electron microscopy (SEM, ESEM XL 30 FEG, Philips, The Netherlands), confocal laser scanning microscopy (CLSM, LSM710, Zeiss, Germany), and atomic force microscopy (AFM, Nano-R, Pacific Nanotechnology, USA). Here, SEM was used in highvacuum mode (p = 10−6 mbar) to determine nanostructure dimensions. Further, CLSM was used to characterize PEM formation on top of the nanostructures. Finally, AFM used in close-contact mode at environmental atmosphere with air as ambience revealed nanostructure height and PEM distribution. AFM images were analyzed using an evaluation copy of SPIP (version 6.0.13, Image Metrology, Denmark). Surface potential The surface zeta potential was determined with the SurPASS Electrokinetic Analyzer (Anton Paar, Austria). Special manufactured cover slips ((10x20) mm²) were modified with nanostructures and PEM and mounted with double-sided tape to the adjustable gap cell. A flow rate of 100-150 mL min-1 at a maximum pressure of 300 mbar was adjusted and 1 mM potassium chloride (Roth) was used as model electrolyte. Hydrochloric acid (0.1 N, Roth) was used for pH titration from pH 10.0 to pH 3.0 and the zeta potential was determined using the streaming current. Surface wettability The wetting properties of nanostructured surfaces were characterized with an OCA 15+ (Dataphysics, Germany). Dynamic water contact angles (WCA) were recorded dispensing 5 µL


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ultrapure water with a velocity of 0.2 µL s−1 to the surface and aspirating it with the same velocity. At least two droplets per sample were measured. Overall, three samples of each condition of two independent experiments were dimensioned and mean and standard deviation were calculated. WCA

Effect of Polyelectrolyte Multilayers Assembled on Ordered

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