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Bio-inspired hierarchically structured surfaces for efficient capture and release of circulating tumor cells Xiao-Qiu Dou, Ping Li, Siyu Jiang, Haider Bayat, and Holger Schönherr ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16202 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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ACS Applied Materials & Interfaces

Bio-Inspired Hierarchically Structured Surfaces for Efficient Capture and Release of Circulating Tumor Cells Xiaoqiu Dou*, Ping Li, Siyu Jiang, Haider Bayat, and Holger Schönherr* AUTHOR ADDRESS: Physical Chemistry I and Research Center of Micro and Nanochemistry and Engineering (Cµ), Department of Chemistry and Biology, University of Siegen, AdolfReichwein-Str. 2, 57076, Siegen, Germany

KEYWORDS: Hierarchical Structures, Cancer Cell, Cell Capture, Cell Release, Anti-EpCAM

ABSTRACT: The development of novel bio-inspired surfaces with hierarchical micro- and nanoscale topographic structures for efficient capture and release of circulating tumor cells (CTCs) is reported. The capture of CTCs, facilitated by surface-immobilized epithelial cell adhesion molecule antibodies (anti-EpCAM), was shown to be significantly enhanced in novel three-dimensional hierarchically structured surfaces that were fabricated by replicating the natural micro- and nanostructures of rose petals. Under static conditions, these hierarchical capture substrates exhibited up to 6 times higher cell capture ability at concentrations of 100 cells mL-1 in contrast to flat anti-EpCAM-functionalized polydimethylsiloxane (PDMS) surfaces.

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As indicated by scanning electron microscopy (SEM) and immunofluorescent images, this enhancement can be in large parts attributed to the topographical interaction between nanoscale cell surface components and nanostructures on the substrate. Similarly, the increased surface area affords a higher nominal coverage of anti-EpCAM, which increases the number of available binding sites for cell capture. By treating the substrates with the biocompatible reductant glutathione (GSH), up to 85% of the captured cells were released, which displayed over 98% cell viability after culturing on tissue culture polystyrene (TCP) for 24h. Therefore, these bio-inspired hierarchically structured and functionalized substrates can be successfully applied to capture CTCs, as well as release CTCs for subsequent analysis. These findings provide new prospects for designing cell-material interfaces for advanced cell-based biomedical studies in the future. 1. Introduction During the progression of metastasis, cancer cells that detach from solid primary tumors and enter the bloodstream have been regarded as potentially accessible source for diagnosis and monitoring of cancer progression and treatment. These cancer cells in the peripheral blood are called circulating tumor cells (CTCs). 1 Normally, CTCs are extremely rare in the blood of patients (a few to hundreds per milliliter), which poses considerable challenges for CTCs detection.

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Many of the recently established technologies for CTC detection exploit

nanostructures, including nanodots, 3 nanotubes, 4 ,5 nanopillars, 6 , 7 nanofibers, 8 , 9 nanowires, 10 ,11 nanorods, 12 and some other irregular nanostructures. 13 , 14 These three-dimensional nanobiointerfaces are motivated in parts by the nanostructures found in biological systems, such as in the immune-elimination interface of T lymphocytes and cancer cells, signal-conduction interfaces of neurons, and the materials-exchange interfaces of intestinal villi. At these nanostructured biointerfaces, cell activities, such as recognition, adhesion, nutrient uptake, and


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impulse conduction take place along with molecular interactions.15,16 These facts indicate the importance of combining nanostructures with surface chemistry to design functional cell biointerfaces that achieve effective cell-materials interactions.17 In general, nanostructures have been found to allow for enhanced local topographic interactions between the substrate and nanoscale components of the cellular surface (e.g. filopodia and microvilli) and result in vastly improved cell-capture affinity compared to unstructured flat substrates. 18 Recently, microstructures have been nanotextured to fabricate micro-/nano- hierarchical substrates to achieve enhanced cell capture. 19,20 Compared to plain surfaces, microstructured topographies provide a larger surface area for the immobilization of antibodies (e.g. the epithelial cell adhesion molecule anti-body (anti-EpCAM)), thereby increasing the binding odds between membrane receptors and antibodies and thus enhancing the CTC-capture efficiency. Moreover, the dimensions of CTCs themselves are on the micron length scale, hence rough substrates possessing a microscale topographic structures could afford better contact with the targeted cells and facilitate cell capture.19 Therefore, intricately hierarchically structured substrates exhibiting both microstructures that accommodate cells (via maximized contact between the cells and the microstructure capture surrounding) and nanostructures fitting the cellular pseudopods hold great promise to further enhance the capture of CTCs. Numerous tools and processes are available to fabricate hierarchical surfaces, such as chemical etching,21 chemical vapor deposition,22,23 silicification calcination,24 electrospinning,25 hot embossing,26 lithography,27 etc., but specialized setups and optimized conditions are required for most of these techniques. Noteworthy, multi-level structural hierarchy can often be observed in natural biological systems, such as butterfly wings, gecko feet, shells, and rose petals, etc..28 These natural hierarchical structures can be directly utilized as templates to replicate the micro-


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/nanostructures onto a suitable substrate, which represents a low-cost strategy that is devoid of specialized setups.29 In our work, red rose petals were chosen as a template because the size of their microstructures (typical diameters of the hemispherical micropapillae are 20 - 30 µm), which is similar to that of a CTC, may facilitate cell attachment (Figure 1). In addition, there are many nanoscale wrinkles superimposed on these micro-structures, being conducive for cellular pseudopods to grasp on the surface.30 For replication polydimethylsiloxane (PDMS) was chosen because it has been reported to replicate even individual carbon nanotubes and the stiffness of soft polymeric material is close to extracellular matrix.31 This may lead to enhanced cell adhesion compared to stiff inorganic materials.32 The preparation of hierarchical rose petal-derived PDMS surface was successfully achieved by a facile imprint pattern transfer process. The hierarchical micro-/nanostructures were replicated on the PDMS surface for CTC capture with diameters and depths/heights of micro-concave or convex structures of about 20 - 30 µm and widths of the nanofolds of about 500 - 600 nm. In order to obtain cell-targeting and stimulus-responsive cell release, anti-EpCAM was conjugated to the surface of PDMS via a disulfide bond-containing linker. EpCAM is frequently over-expressed in most carcinomas and absent in hematologic cells, hence it represents a prime capture target.33 The anti-EpCAM antibody on the PDMS surface can direct specific recognition of the antigens present on the surface of EpCAM-positive cells. Because the antibody was conjugated on the surface of PDMS through disulfide bonds, the addition of reductants (e.g. glutathione) affords the on-demand detachment of anti-EpCAM from substrate surface and the concomitant detachment of the captured cells (Figure 1).


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This report shows that rose petal-derived micro-/nano- hierarchical surfaces functionalized with anti-EpCAM as specific recognition molecules exhibit efficient capture of EpCAM-positive cells by exploiting the synergy of molecular recognition and physical topographic interactions. The capture surfaces replicated from rose petals benefit the specific recognition of CTCs in static cell-recognition. Overall, this study may provide promising guidance to develop novel bioinspired hierarchical biointerfaces for improved CTCs capture and beyond.

Figure 1. Schematic of anti-EpCAM modified hierarchical structures for CTC capture. The enhanced capture of EpCAM-positive CTCs was achieved through chemical antibody-antigen recognition and local topographic interactions between cells and hierarchical structures on PDMS. Cell release can be achieved through disulfide bond cleavage by addition of glutathione (GSH) as a reductant.


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2. Experimental Section 2.1 Materials and Reagents. PDMS elastomer and its curing agent, Sylgard 184 were purchased from Dow Corning (Wiesbaden, Germany). (3-Aminopropyl)triethoxysilane (APTES), biotin disulfide N-hydroxysuccinimide ester (NHS-(S-S)-Biotin), streptavidin, glutathione (GSH), poly(vinyl alcohol) (PVA, MW = 22,000 g/mol) and tablets of phosphate buffered saline (PBS) were purchased from Sigma-Aldrich. Anti-EpCAM antibody was purchased from R&D systems. Paraformaldehyde and Triton X-100 were purchased from VWR. Anti-paxillin, anti-mouse IgG Alexa 488 and Phalloidin-Rhodamin were purchased from Invitrongen, Life Technologies. Hoechst 33258 and Fluorescein diacetate (FDA) were purchased from Sigma-Aldrich, and propidium iodide (PI) was purchased from Carl Roth. 24-Well cell culture plates were purchased from Sarstedt AG & Co. (Nürnbrecht, Germany). Throughout the whole study, Milli-Q water was drawn from a Millipore Direct Q8 system (Millipore Advantage A10 system, Schwalbach, with Millimark Express 40 filter, Merck, Germany) with a resistivity of 18.2 MΩ cm. 2.2 Preparation of Hierarchically Structured Surfaces. For fabrication of inverse rose petal structures with concave curvature, the PDMS precursor mixed with the curing agent (10:1) was poured on the untreated fresh rose petals and cured at 70ºC for 2 h. After complete drying of the rose petals embedded into PDMS, they became crisp and could be removed from the PDMS surface by slightly bending the PDMS. To replicate the rose petals (convexly curved microstructures), aqueous PVA solution (10 wt%) was poured onto the fresh rose petals and was exposed to ambient air at 25°C. When the water was evaporated, the PVA film was peeled off affording inverse rose petal structures. Then, the PVA mold was replicated with PDMS following the procedure as shown above (Figure 2a).


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2.3 Surface Modification with anti-EpCAM. Prior to the surface modification, the PDMS substrates were treated with O2 plasma (Plasma Prep2, Gala, Gabler Labor Instrumente GmbH, Germany) for 3 min.34 Then the substrates were modified with 6% (v/v) APTES in absolute ethanol at 25ºC for 24 h. The obtained aminated PDMS surfaces were washed with ethanol and kept at 130ºC for 1 h. Next, the substrates were treated with NHS-(S-S)-Biotin (0.1 mg mL-1) in DMSO (containing 1.2 eq triethylamine) for 24 h, introducing biotin onto the substrates. Afterwards, the substrates were treated with 0.02 mg mL-1 streptavidin in PBS solution at 4ºC for 24 h, then washed with Milli-Q water to remove the excess streptavidin. Finally, 200 µL antiEpCAM (5 µg mL-1 in PBS buffer) was applied to the substrates, followed by incubation at 4ºC for 24 h and subsequently washed with Milli-Q water. The anti-EpCAM modified substrates were sealed and stored at 4ºC before using. 2.4 X-ray Photoelectron Spectroscopy. The different steps of the surface modification were analyzed on cured PDMS thin films obtained by spin-coating on Si wafers (SIEGERT WAFER GmbH, Germany, type/dopant: P/B, resistivity: 1 - 10 Ω cm). The samples were analyzed after each step of modification by XPS (S-Probe ESCA SSX-100s, Surface Science Instruments, USA) using Al Kα radiation of 200 W. The spectra were analyzed using Casa XPS processing software. 2.5 Surface Area Ratio and Roughness. The surface area ratio and roughness of the PDMS substrates were determined with a LEXT OLS4000 Industrial Laser Confocal Microscope (Olympus, Japan). Before the measurements, the samples were sputter coated with gold (8 - 10 nm). The mean value of surface area ratio Sdr and roughness Sa were calculated from five images taken in different regions of the samples. The images were analyzed by Software SPIPTTM 6.6.2 (Image Metrology A/S, Denmark). Sdr expresses the percentage of additional surface area


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contributed by the texture (taking the z height into account) compared with a flat x,y plane of the nominal size of the measurement region: =

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Bioinspired Hierarchically Structured Surfaces for ... - ACS Publications

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