Size-Tunable Nanoneedle Arrays for Influencing Stem Cell Morphology, Gene Expression, and Nuclear Membrane Curvature
[component]
unpublished
High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols was used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used the nanoneedle tip
more »
... ameter to control the morphology, Peer reviewed version of the manuscript published in final form at ACS Nano, 2020, DOI: 10.1021/acsnano.9b08689 2 nuclear size and F-actin alignment of interfaced hMSCs, and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays and the degree of nuclear membrane impingement, with the latter clearly visualized using focused-ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring. Keywords: Nanofabrication, high-aspect ratio, deep reactive ion etching (DRIE), nanoneedles, cell-material interactions, biointerface Many aspects of the microenvironment directly influence cell function and differentiation. Bioengineered systems often employ biomimetic physical cues arising from intrinsic properties of the substrate (e.g. chemical identity, mechanical properties, topography) 1-4 or externallyapplied forces (e.g. shear, compression, tension, electric, acoustic, magnetic). [5][6][7][8] In particular, micro-and nanoscale topographies are potent regulators of stem cell adhesion, morphology, migration, proliferation and differentiation. 9-11 Nanoscale structures (e.g. dots, pits, grooves, roughened surfaces) can be created using a variety of fabrication strategies, including electronbeam lithography, 12 photolithography, 13 interference lithography, 14 nanoimprinting, 15 particle self-assembly 16 and electrospinning. 17, 18 Of these structures, vertically-aligned nanowires, nanotubes, and nanostraws have shown great utility for recording intracellular electrical signals, 19 studying mechanosensing pathways 20 and facilitating biomolecule delivery. 21-23 We have previously reported the fabrication of high-aspect-ratio porous silicon nanoneedle arrays for intracellular biochemical sensing 24, 25 and in vivo transfection of plasmid DNA encoding Peer reviewed version of the manuscript published in final form at ACS Nano, 2020, DOI: 10.1021/acsnano.9b08689 3 for vascular endothelial growth factor. 26 Key design features of this platform include the material mesoporosity and the sharp tips of the nanoneedles (50 nm in diameter), which have been shown to enable cargo loading and promote endocytosis. 27 These mesoporous silicon nanoneedles were biodegradable in aqueous environments within 48 hours. This collection of properties was ideal for delivery applications requiring a temporary cell-material interface; however, for long-term cell culture, nanoneedle arrays must remain stable for days to weeks. Here, we describe the fabrication of nondegradable silicon nanoneedles that can provide a continuous topographical interface to human mesenchymal stem cells (hMSCs) for at least five weeks in culture. We used a combination of different reactive ion etching protocols to create solid silicon nanoneedles with tips that could be tuned from 20 to 700 nm in diameter. We used the tunable Tunable diameter of the nanoneedle tips to leverage control over impacted the morphology, polarization, gene expression, Yes-associated protein (YAP) localization, and nuclear deformation of hMSCs cultured on nanoneedle arrays. These results are applicable to the design of biomedical devices, bioelectrodes, and platforms controlling cell behavior using topographical cues, and should provide insight into basic biology and cell-nanomaterial interactions. RESULTS & DISCUSSION Fabrication and Characterization of Nanoneedle Arrays with Different Tip Diameters. We fabricated arrays of nanoneedles with different tip diameters from silicon wafers using a top-down fabrication approach (Figure 1a) . On a nitride-coated wafer, we first patterned a two-dimensional dot array using negative photoresist, then used reactive ion etching (RIE) to transfer this pattern into a hard silicon nitride etch mask. 26 We used deep reactive ion etching (DRIE), 28, 29 with alternating etch and passivation steps, to anisotropically etch vertical silicon pillars. We then sharpened the pillars into nanoneedles using RIE, which isotropically etched Peer reviewed version of the manuscript published in final form at ACS Nano, 2020, DOI: 10.1021/acsnano.9b08689 4 the silicon nitride cap and the top of the pillar. The tip diameter (D tip ), defined as the diameter observed from the top view of the resulting structure, correlated linearly with the RIE process time, allowing us to finely tune Dtip from 20 to 700 nm (Figure 1b) . The other geometric properties were consistent across the surface of a 100 mm diameter silicon wafer, with 5 µm high nanoneedles arranged in a uniform square array with 2 µm spacing. We tested the longterm stability of our nanoneedle arrays against degradation in cell culture medium (Minimum Essential Medium Alpha-Modification) with 10% v/v fetal bovine serum (FBS) under standard cell culture conditions (37°C, 5% CO2). Analysis by scanning electron microscopy (SEM) revealed no visible degradation over 4 weeks (Figure S1a), in contrast to the rapid degradation observed for porous silicon nanoneedles. 26, 30 This aqueous stability enabled us to use the nanoneedle arrays as a continuous topographical interface to influence the long-term culture of hMSCs (Figure S1b). The hMSCs readily adhered to the sharp nanoneedles, nanopillars and flat controls without the need for any additional substrate coating. We assessed the proliferation of hMSCs on the sharp nanoneedles, nanopillars, and flat controls by measuring the number of Ki-67 positive nuclei after 72 h, and the relative gene expression of MKI67 after 24 h of cell culture ( Figure S2 ). We observed a slight reduction in Ki-67 positive nuclei on the nanoneedles and nanopillars compared to the flat controls, and significant reduction in gene-level expression of MKI67 between the blunt and sharp nanostructures after 6 h. The survival with reduced proliferation of hMSCs cultured on nanoneedles was also evidenced by LIVE/DEAD ® staining performed after 35 d, which showed that all substrates supported long-term hMSCs viability ( Figure S2c) . SEM analysis revealed that the hMSCs on the nanopillar array (Dtip = 718 ± 32 nm) had large, flattened cell bodies and relatively few protruding filopodia (Figure 1c) , whereas cells on the sharp nanoneedles (Dtip = 47 ± 7 nm) were highly polarized with extended filopodia (Figure 1d) . In the latter case, we observed that nanoneedles in contact with hMSCs were clearly deformed. Previous studies have shown that silicon-based nanostructures can be Peer reviewed version of the manuscript published in final form at ACS Nano, 2020, DOI: 10.1021/acsnano.9b08689 5 thinned in order to reduce the effective material stiffness and increase the mechanical flexibility. 31, 32 To understand the change in effective substrate stiffness as a function of Dtip, we modeled the flexural stiffness of the nanoneedles using Euler-Bernoulli beam theory. 33, 34 This model was used to calculate the deflection profile when an external force of 300 nN (the maximum traction force reported for a single cell 35 was applied orthogonally to the nanoneedles (Figure 1e) . This analysis showed an increased deflection with increased tip sharpness, and a calculated value of effective stiffness that was linearly dependent upon tip diameter (Figure 1f ). This model suggests that the nanopillars are approximately 15 times stiffer than the sharp nanoneedles. Using this geometric relationship to tune the effective stiffness of the nanoneedle arrays it may be possible to control the morphological and phenotypic changes of interfaced cells through topography-driven mechanotransduction. Tip Diameter of Nanoneedle Arrays Affects Cell Morphology. To investigate the changes in cell morphology in more detail, we cultured hMSCs on flat silicon (as a control), nanopillars (Dtip = 718 ± 32 nm), two different blunt nanoneedles (Dtip = 316 ± 20 nm and 172 ± 6 nm), and one set of sharp nanoneedles (Dtip = 47 ± 7 nm). We fixed the cells for immunostaining at four time-points (6, 12, 24 and 72 h after seeding). We then used image-based cell profiling to analyze 5,372 immunofluorescent microscopy images and extract single-cell morphological and protein localization features for over 100,000 cells (Figure 2a) . From this high-content image analysis, we were able to quantify pronounced, systematic morphological changes as a function of nanoneedle sharpness (Figure 2b) . In particular, decreasing the Dtip reduced the spread area of both cells and nuclei, promoted cell body elongation, and decreased the cell protrusion ratio, which represents the number of protrusions extending from the cell (exemplar raw data for a selection of measurements shown in Figure S3 ) is the area of cell protrusions divided by the total cell area ( Figure S3 ). In addition, nuclear solidity (a measure of nuclear perimeter tortuosity) visualized as a slight scalloping in the in-plane nuclear membrane around Peer reviewed version of the manuscript published in final form at ACS Nano, 2020, DOI: 10.1021 6 the nanoneedles, decreased with increasing nanoneedle sharpness ( Figure S4 ). Backgroundcorrected and batch-normalized intensities of cytoskeletal proteins (F-actin, α-tubulin) were also influenced by changing Dtip. Local cell density, determined by Voronoï tessellation, was also greater on nanoneedles than flat surfaces or nanopillars. Linear Discriminant Analysis Uncovers Complex Morphological Phenotypes that are Impacted by Tip Diameter of Nanoneedle Arrays. While image analysis allowed us to intuitively explore features of interest, we also used linear discriminant analysis (LDA) to uncover less obvious features that were influenced by the nanoneedle tip diameter. While all measured features could be used to train this model, it was important to avoid multicollinearity between samples (inter-dependence between related measurements), since this negatively impacts the stability and robustness of LDA models. 36 Accordingly, we pre-selected 31 features of interest (Table S1) , and then used an automated step-wise procedure to remove stronglycorrelated features ( > 0.75, see Supplementary Information for details). 36 This filtering produced an LDA model with 18 features, which could each be described in terms of the composite potency index, a relative measure that enables interpretation of a model comprising four discriminant functions (Figure 2c) . 37 This model revealed that α-tubulin intensity had the largest impact in separating cells from different substrates. Cell mean radius and cytoplasmic ratio of F-actin and α-tubulin were also observed to strongly influence the cell classification. Separation of the three different target classes (flat substrates, nanopillars, and sharp nanoneedles) was clearly visible when clustered using the two primary discriminant functions (LD1 and LD2) (Figure 2d ). Sharper Nanoneedles Guide Cell Alignment and Polarization by Activated Actomyosin Contractility. High-content image analysis also revealed a strong dependence in cell orientation as a function of nanoneedle tip sharpness ( Figure S5) . Alignment was clearly evident from fluorescence microscopy images, which showed hMSCs bidirectionally polarized Peer reviewed version of the manuscript published in final form at ACS Nano, 2020,
doi:10.1021/acsnano.9b08689.s003
fatcat:zalxdnfyhndizocjdn6wkcnvkq