Nanopattern Gradients for Cell Studies Fabricated Using Hole-Mask

Jun 1, 2016 - (23) These surfaces have been used to study the influence of adhesive patch size on cellular behavior, and the criteria for focal adhesi...
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Nanopattern Gradients for Cell Studies Fabricated Using Hole-Mask Colloidal Lithography Thea Bøggild, Kasper Runager, and Duncan S. Sutherland* Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark S Supporting Information *

ABSTRACT: Culturing cells on gradient nanopatterns provides a useful tool to explore cellular adhesion to mimics of the extracellular matrix or screen for cellular responses to nanopatterns. A method is presented to fabricate complex gradient protein patterns based on hole-mask colloidal lithography, which can generate nanopatterns in multiple materials and of multiple shapes. Gradients of gold structures were functionalized to form gradients of protein nanopatterns of different shapes (bars, dot pairs, and rings), where a key parameter was systematically varied in each gradient. Cells were grown on vitronectin nanopatterns, showing differential adhesion (spread area/focal adhesion size) along the gradients. KEYWORDS: protein nanopattern, hole-mask colloidal lithography, gradient, cell adhesion, C2C12

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Studies of cell behavior at surfaces often require large area samples for statistically relevant data and not all fabrication approaches are suitable.18 Fast, parallel techniques capable of patterning large areas are preferable. Electron-beam lithography (EBL) can be employed for studies requiring more complex patterns.19 Replication of EBL defined patterns with, for example, nanoimprint lithography still require resource costly master fabrication. A number of self-assembly-based approaches have been developed based on block copolymer micelles7,9,20 or colloidal nanoparticles.21 Block copolymer micelles have been applied to pattern peptides at the nanoscale in a number of studies. In one elegant extension a gradient was fabricated into a peptide-functionalized gold nanodot pattern via block copolymer micelles with a gradient of peptide spacings (albeit over a limited range), which was shown to direct cell migration.8 Another often used self-assembly approach is sparse colloidal lithography,3,22 which is fast and capable of large area patterning and has previously been used to fabricate nanopatterned surfaces for cell studies.6,21 With appropriate chemical modifications nanopatterns of circular domains of extracellular matrix proteins can be formed, although more complex patterns have also been applied.23 These surfaces have been used to study the influence of adhesive patch size on cellular behavior, and the criteria for focal adhesion (FA’s) bridging between extracellular matrix patches. The closely related technique of hole-mask colloidal lithography is capable of creating significantly more complex structures.24,25 Briefly summarized, the approach creates suspended hole-masks by applying colloidal lithography on top of a thin polymer layer.

he microenvironment around a cell in vivo provides multiple extrinsic signals that together with intrinsic signals determine cellular decisions and fate.1 In vitro surface adhering cells have been shown to be remarkably sensitive to a wide range of behavioral cues from the underlying surface. Attempts to identify the underlying mechanisms and general trends in the cellular response to varying surface parameters have led to a number of experiments culturing cells on artificial substrates with various characteristics in order to characterize the response. In this way, cells have been shown to respond to substrate rigidity,2−4 chemistry and micro- and nanoscale features, such as topography, 5 and the identity 6 and distribution7 of adhesive ligands. Such substrates have been found to influence proliferation,5 morphology,8 migration,9 apoptosis,8 and differentiation,10 depending on the cell type in question.11 Efforts both to understand the complexity and synergy between different surface parameters on cellular response and to aid in the identification of appropriate parameters for optimized in vitro cell culture systems have led to the development of screening approaches of individual parameters12 or combinations of parameters.13 In vivo ligand density or type often gives information on location within tissue or state (e.g., Selectin presentation at inflamed endothelia14), whereas gradients of ligands or chemokines are considered to provide direction signals and steer cell motility.15 In vitro nanoscale organization of ligands in gradients can be used as mimics for the in vivo situation to study cellular responses,16 studying for example the role of ligand density or organization in directing cell motility. Although gradient-like patterns have been formed in microfluidic devices, the general fabrication of gradient structures is not simple in particular for studying cell populations, and further developments are required.17 © XXXX American Chemical Society

Received: September 4, 2015 Accepted: June 1, 2016

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DOI: 10.1021/acsami.5b08315 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces This allows for the creation of relatively complex features by physical vapor deposition of material through the holes in the hole mask, as multiple depositions can be employed in the fabrication of the desired surface features without the need for realigning the mask. Here, we report the novel extension of hole-mask colloidal lithography for fabricating gradients or screening arrays presenting systematically varied nanostructured features, by modulating the polymer resist thickness across the sample prior to creation of the hole-mask. A gradient in polymer film thickness can be translated into a gradient of nanostructures with varying feature shapes or spacings by using angled physical vapor deposition. We demonstrate here gradients of three different feature types, bars, rings, and pairs of dots. In proof of principle experiments, we show the applicability of the patterns in cell studies, where three different types of continuous gradient patterns and one array were created and functionalized with PEG and protein, and the cellular responses were evaluated and correlated to the gradient properties. A schematic overview of the final steps in the fabrication process is given in Figure 1. Full details are given in the Supporting Information. In brief, thin polymer films are spuncoated on the substrates before applying a spatially asymmetric oxygen plasma to etch differentially across the surface of the polymer-coated substrate creating a polymer thickness gradient across the material. We directed the etching with a dielectric

cover suspended above and partially covering the sample, which restricted the access of the activated plasma species leading to the creation of the gradient. A dispersed colloidal monolayer mask was assembled by electrostatic self-assembly.22 A layer of Ti was deposited and the particles removed to create the hole mask, and the exposed polymer etched away with oxygen plasma down to the sample surface. The diameter of the hole mask was reduced via deposition at a highly oblique angle,25 which allows for the fabrication of smaller features with higher aspect ratios. The hole-mask can be used to generate nanostructures in multiple process steps by angled evaporations onto the substrate, with the shape of the structure controlled by the height of the surrounding polymer layer and control of angle of deposition and rotation. Lift off of the polymer reveals the nanostructure gradient. Here the patterns were functionalized with protein for use in a cell study by a method previously reported.21 In short, the silicon oxide parts of the surface were functionalized with PLLg-PEG, which is widely used as a protein antifouling coating for oxide surfaces,26 to prevent protein binding between the gold nanostructures. Subsequently, vitronectin was deposited onto the nanostructures to provide gradient patterns of cell binding ligands. Nanopatterns of vitronectin have previously been shown to support cell attachment (C2C12 cell line) in a patch size-dependent manner4 and are used here to demonstrate the use of gradients to explore multiple shapes of nanostructures. Cell attachment (judged by cell size, shape, and formation of focal adhesions) was evaluated by fluorescence microscopy for C2C12 cells on vitronectin gradients of different types after 20 h of incubation. Ring, bar, and dot pairs gradients were used, as ring-shaped nanoscale organization of proteins is present in a number of adhesion structures (e.g., podosomes and other adhesions in antigen-presenting immune cells) and bar/dot pair structures can be used to study the role of extracellular ligand distribution on the development of focal adhesions from focal complexes. In the current study, three different geometries were created, (bars, rings, and pairs of dots) where bar length, ring diameter and dot pair separation systematically varied along the gradients. The three geometries and their development from dots, transforming into the specific structures with varied size/ aspect ratio/spacing along the gradient can be seen in Figure 2. Bar fabrication used programmed deposition angle continuously varying between −20° and 20° from normal, without rotation. Pairs of dots were made by sequential depositions at −20° and 20°, whereas rotating the sample at 5 rpm with an angle of 20° gave ring structures. Here, the same material was deposited in each of the dots but the self-aligned hole-mask allows multiple materials to be sequentially codeposited.24 The volume of metal deposited to form each of the structures is constant across the gradient so that as the lateral dimensions of the structures changes along the gradient there is a concomitant and predictable change in thickness. Further discussion of the thickness and the resolution of the technique are present in the Supporting Information. Using asymmetric plasma etching to create the gradient allows for easy adjustment of the gradient steepness. In our approach, simply adjusting the height of the dielectric cover above the sample resulted in a change in the steepness of the gradient. In Figure 3; gradients of dot pairs are shown for three different dielectric cover-sample separation distances (approximately 2, 4, and 6 mm). Some debris from the patterning process is visible in Figure 3C at 250 μm into the gradient, a

Figure 1. Schematic illustrating the final steps of the fabrication process when making a gradient of dot pairs. B

DOI: 10.1021/acsami.5b08315 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

immobilization and C2C12 cell culture is described in the Supporting Information. The cells were fixed, immunostained and examined with fluorescence microscopy; examples are shown in Figure 4 with further images in the Figure S3. The start of the gradient is preceded by a homogeneous vitronectincoated gold region which could be used both as a control and to mark the start of the gradient. Cell behavior was drastically altered on the gradient compared to the homogeneous control region with a larger cell number, increased spreading and more pronounced FA’s on the homogeneous vitronectin surface related to the restriction of the available adhesions area to nanoscale dimensions6 on the gradient. Within the gradients the effect of the nanoscale distribution of vitronectin could be studied. The ring gradient showed a trend of increasing cell numbers along the early part of the gradient (Figure 4, left panel) indicating that the cell adhesion to the surface was influenced by the nanoscale distribution of the vitronectin. The lack of cell attachment to the initial parts of the gradient show that there is a threshold of vitronectin pattern size to which these cells can adhere. The results for the rings show onset of cell attachment corresponding to ∼200 nm diameter rings which fits with previous observations utilizing multiple samples with discrete sizes where the onset of cell attachment lay at 200 nm diameter structures.6 The spread area of the cells was reduced on the gradients compared to at the homogeneously available vitronectin, but clearly increased for cells at longer distances up the gradients (see Figure 4) for both ring and bar gradients (increased at 1.5 mm compared to 0.5 mm into the gradient for bar and ring patterns) showing a stronger interaction with the patterns further along the gradient (see Figure 4 and Tables S2 and S3 and significances in Tables S5 and S6). At ring patterns the increase of cell size was accompanied by concomitant increases in the cell perimeter and Feret (see Figure 4 and Tables S3 and S6). The change of patterns over these ranges can be seen in the right two images of Figure 2A, B and Table S1. At vitronectin bar patterns, adherent cells were seen at similar length structures (∼200 nm) of pattern to the rings but which were significantly smaller in total area (∼40% reduced) suggesting that the critical parameter is not the area of a ligand patch but the length. The length of the focal adhesions formed by cells at the bar patterns also increased along the gradient with no significant increase in the width (see Table S4 and significances in Table S7). The overall size of these adhesions (length from ∼1.5 to ∼4 μm) shows that these must bridge over several nanoscale patches of protein (up to 10 or more); however, the form of these ligand nanopatches determines the development of the focal adhesions and the outcome of the overall cell adhesion. The method presented here has significant flexibility, with several opportunities for varying different parameters of the final gradients. The steepness of the gradient can be varied, as can the shape, size and chemical composition of the features displayed. The height of the polymer layer, the size of the particles, the shrinking of the holes, the etching of the polymer as well as the depositions chosen to create the structures may all be varied to create gradients. Shorter plasma treatment of the PMMA layer after particle removal will lead to a smaller available area for each feature, restraining the size of the final features, as seen in the end of the steepest gradient in Figure 3. Employing smaller polystyrene particles results in smaller features, while changing the shrinking step will affect the range of aspect ratios possible in the final structures. As demonstrated, the lateral height variation in the polymer can

Figure 2. SEM images of three different pattern gradients, showing the change in the structures at different distances from the start of the gradient. (A) Ring structure, (B) bar structure, and (C) pair of dots structures. The scale bars are 0.67 μm.

Figure 3. SEM images of three gradients of different steepness showing the change in the structures at different distances from the gradient start. Gradient A and B utilized 160 nm particles, gradient C utilized 200 nm particles. Scale bars are 500 nm.

discussion on this can be found in the Supporting Information (see also Figure S2). Alternate methods such as drop casting or nanoimprint lithography to generate the thickness difference across the polymer film also exist. Depending on the method employed, the gradient could potentially be made steep enough to allow single cells to explore multiple surface patterns, or gentle enough that each cell would essentially experience a single structure type. The gradients created with the plasma etching are not linear, being steepest at the edge of the polymer. As an alternative multiple etching steps with a mask which is moved between etches can be used to generate step gradients/arrays of homogeneous regions with systematically varied structures suitable for cell array studies. The potential to study cell adhesion with gradients of vitronectin nanopatterns proteins was demonstrated. C2C12 cells have previously been shown to be sensitive to nanopatterns of vitronectin in a size and protein dependent manner (with BSA nanopatterns showing no cell adhesion).6 Protein C

DOI: 10.1021/acsami.5b08315 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Far left panel, quantified data of cell size/shape parameters on ring gradients. Left panel ring gradient; stitched 10× images taken on a ring gradient. Cells are stained red for actin with scale bar of 400 μm. The lower third of the image, below the white dividing line, show the cells on the gold-coated part of the surface, the image above show the first ∼2 mm of the gradient. (A−D) Bar gradient; 63×pictures showing cells stained green for vinculin on different areas of the bar gradient. A was taken approximately 1.5 mm in from the gradient beginning, B ∼1 mm in and C at ∼500 μm in, whereas D shows a cell growing on homogeneous protein. Far right panel quantified cell area and focal adhesion length at bar gradients. Scale bars for bar gradient are 30 μm. * indicates significance at p < 0.05.

be controlled to define the steepness of the gradient, or arrays with step changes in features. Other methods of creating height changes in the polymer could be employed to fabricate different gradient formats. As the placement of the hole mask is fixed relative to the sample, it is possible to use multiple depositions to create the pattern, allowing for the use of different materials, and the possibility to create more complex structures presenting multiple chemistries. Here we have demonstrated a homopattern of a single protein. Multiple chemistries have previously been applied to co-deposit multiple proteins,27 which could be applied to gradients of, for example, codeposited dots with two chemistries in each pair. Creating multiprotein patterns based on adsorption of proteins is an ongoing challenge.28 Hole-mask colloidal lithography has been successfully modified to create three different, gradually changing patterns of variable steepness. The resulting gradients were found suitable for use in proof of principle cell studies, with appropriate functionalization, and C2C12 cells were found to respond to the gradients. On gradients of ring structures, cell spreading was found to increase as the rings grew larger, whereas cells on bar gradients displayed more prominent focal adhesions which elongated as the patterns elongated, despite apparently bridging between patches. The correlation between the cell adhesion and the nanostructure size and shape could be

measured giving insight into for example the development of cell adhesion complexes. In addition to studying biological processes such as cell adhesion, the nanopatterning approach presented here could also be applied to study a range of other physical nanoscale phenomena, for example, plasmonic optical properties and coupling of metallic nanoparticles.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08315. Materials and Methods, pictures of array sample (Figure S1) and pictures of the debris from the patterning process (Figure S2) along with a discussion on this, images of cells on bar gradient (Figure S3), quantified nanostructure data (Table S1) and cell data (Tables S2− S7) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. D

DOI: 10.1021/acsami.5b08315 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been funded by the Danish Research council (FNU Grant 12-126120) and the EU FP7 programme (DIREKT Grant 602699) .

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ABBREVIATIONS EBL, electron-beam lithography FAs, focal adhesions PEG, poly(ethylene glycol) PMMA, poly(methyl methacrylate) BSA, bovine serum albumin

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DOI: 10.1021/acsami.5b08315 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX