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Fibrinogen Motif Discriminates Platelet and Cell Capture in PeptideModified Gold Micropore Arrays Kellie Adamson,† Elaine Spain,† Una Prendergast,† Niamh Moran,‡ Robert J. Forster,† and Tia E. Keyes*,† †
School of Chemical Sciences, Dublin City University, Dublin 9, Ireland Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland
‡
S Supporting Information *
ABSTRACT: Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin αIIbβ3), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, αIIbβ3. Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.
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controversial.11−13 Whereas the AGD peptide interacts specifically with the αIIbβ3 receptor and is required for platelet aggregation, the role of RGD in platelet aggregation, and whether both motifs bind to discrete or overlapping binding sites within the integrin remains unclear.14−17 Crystallographic data have provided the strongest evidence that the two peptides bind to a common site, suggesting that the HHLGGAKQAGDV motif binds to an extended site at a receptor that includes the RGD-binding site.18 More recently, Sánchez-Cortés and Mrksich, using RGD and AGD monolayer substrates, have found that in agreement with crystal structure studies, αIIbβ3 transfected CHO K1 cells attached, spread, and assembled a cytoskeleton on both ligand surfaces, and inhibition experiments revealed that the two ligands bind competitively to the same receptor.17 In the case of platelets, results varied regarding the extent to which the RGD peptide can block Fg binding. 19−21 Competitive binding studies of radiolabelled Fg suggest that because platelets have other integrin receptors that bind RGD, for example, αvβ3 and α5β3, these integrins mediate Fg binding despite the presence of platelet receptor antagonists.19−21 By contrast, AGD peptides can block Fg binding specifically to integrin αIIbβ3, suggesting the AGD motif is exclusive to platelet integrin αIIbβ3.19,21 Given this specificity, we were
INTRODUCTION The capacity to selectively capture cell types based on surface chemistry is important across a range of applications, especially in diagnostics. Peptides are attractive receptors for surface immobilization because they are small, cheaper, and generally more robust than other biomolecules (e.g., antibodies), and typically their surface chemistry/orientation is much easier to control. Amongst peptides, the most commonly exploited in this regard is the arginine/glycine/aspartic acid (RGD) tripeptide motif. 8 of the 24 known integrins bind to extracellular membrane proteins in an RGD-dependent manner.1 This promiscuity has led to wide application of the RGD motif as a recognition/capture sequence at cell-capture surfaces.2−5 The platelet integrin, αIIbβ3 (GPIIb/IIIa), spans the plasma membrane of human platelets and is intimately linked to the process of platelet activation and thrombosis.6,7 The integrin family has been comprehensively reviewed.8,9 Fibrinogen, Fg (MW 340 kDa), the primary ligand for αIIbβ3, contains three putative integrin binding sites; an RGD motif within the Aα chain, Aα572−575 (RGDS) and a nonRGD dodecapeptide sequence in the γ chain (C-terminal γ400−411, HHLGGAKQAGDV, alanine/glycine/aspartic acid (AGD)). A second RGD motif also exists, Aα95−98 (RGDF), but is thought to be masked within a coil domain, that precludes its participation in platelet aggregation.10 Both RGD and AGD are implicated in platelet aggregation; however, the mechanistic roles of the two motifs remain © XXXX American Chemical Society
Received: September 18, 2017 Revised: December 7, 2017 Published: December 14, 2017 A
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peptides were purchased from Celtek Peptides, USA. Peptide structure and purity was manufacturer guaranteed, presented in Figures S1 and S2. Phycoerythrin (PE)-labeled mouse antihuman CD62P (λex 488 nm) was purchased from BD Biosciences, USA. Fabrication of Planar Gold and Gold Cavity Array Surfaces. The gold-modified silicon wafer was washed thoroughly with acetone and water before use. Teflon tape was wrapped around half of the gold substrate to expose a 1 cm2 surface area. For planar gold surfaces, a gold film of 100 ± 30 nm thickness was electrochemically deposited. Stock solutions (10 wt %) of commercially obtained PS latex spheres (Bangs Laboratories, USA) with average commercial diameters of 1.73 ± 0.15 μm were diluted to approximately 1 wt % suspension in water. A thin layer of spheres was deposited on the gold-plated silicon wafers, and the solvent evaporated overnight at room temperature. The gold electrolyte solution (Technic Inc., UK) was deoxygenated with nitrogen for 30 min prior to deposition. A fixed potential of −1 V versus an Ag/AgCl (sat. KCl) electrode was applied by a model 660 CH instrument electrochemical workstation. The depth of the spherical cap was controlled through the amount of charge (C) passed in the electrochemical deposition step. Prior to chemical modification, the PS spheres were removed by immersing the array in THF for 30 min at room temperature. All unmodified gold surfaces were washed thoroughly with acetone, water, and dried with nitrogen. All surfaces were characterized by SEM, atomic force microscopy (AFM), cyclic voltammetry (CV), and water contact angle measurements. Chemical Modification of Gold Surfaces. Three surface active thiols were used in this study: the multi-integrin-recognizing peptide (SH)C-Ahx-GRGDS (C26H46N10O11SH), integrin αIIbβ3-specific peptide (SH)C-Ahx-HHLGGAKQAGDV (C59H97N20O18SH), and 1-octanethiol (CH3(CH2)7SH). In the case of planar surface modifications, the SAMs were prepared by immersing the gold substrate, in a 1 μM solution of the thiol for 24 h at room temperature. In the case of the spherical cap gold cavity arrays with 1.6 μm diameter apertures, SAMs were prepared by first sonicating the substrate for 15 min in 1 μM deposition solution, to ensure filling of the cavity interiors, and then the substrates were left in contact with the deposition solution of the thiol for 24 h at room temperature. Prior to cell/platelet adhesion studies, all surfaces were rinsed extensively with buffer before use and incubated immediately (surfaces were not allowed to dry) with cell/platelet suspensions. Electrochemical Analysis of the Modified Gold Surfaces. To confirm the extent of surface coverage of the SAMs, electrochemical desorption of the thiols bound to the gold surfaces was carried out with the modified gold electrode in contact with a 0.1 M solution of sulfuric acid (H2SO4). CV of the unmodified gold array and array functionalized with RGD, AGD, and alkane monolayers was carried out by cycling the electrode between −1.4 and 1.5 V at a scan rate of 0.1 (V/s) on the CH Instrument model 660 work station. Preparation of Washed Human Platelets. Washed platelets (WPs) were prepared as previously described36 from donors who gave informed consent and declared they were medication free for the previous 10 days. The protocol employed has been shown to maintain platelets in their resting state.37,38 Ethical approval was obtained from the RCSI Ethics Committee. Human blood was drawn into a syringe containing acid citrate dextrose (ACD) buffer to prevent coagulation. Modified HEPES platelet buffer (6 mM glucose dextrose D, 0.13 M NaCl, 9 mM NaHCO3, 10 mM sodium citrate, 10 mM HEPES, 3 mM KCl, 0.81 mM KH2PO4, 0.9 mM MgCl2·6H2O) was prepared and adjusted to pH 7.36 with ACD buffer. The blood was centrifuged at 150g for 10 min at room temperature. The PRP (platelet rich plasma) upper phase was transferred to a 50 mL tube. Prostaglandin (1 μM concentration) was added to prevent platelet activation. Platelets were collected by centrifugation at 720g for 10 min. Using 500 μL of platelet buffer, the platelet layer was carefully removed and placed in a 15 mL tube. Platelet counts were measured using a Sysmex XE-2100 hematology analyzer (Sysmex KX Series, Sysmex UK LTD). Platelets were allowed to stand for 1 h. CaCl2 (1.8 mM) was added to the platelets, and the platelets were diluted to 30 ± 2 × 103/μL platelets in platelet buffer immediately before use.
interested in understanding if we could exploit the C-terminal γ400−411, dodecapeptide sequence as an alternative to RGD to selectively address the platelet integrin leading to integrinmediated capture of platelets and not other integrin-containing cells. A neuroblastoma cancer-cell line was chosen because integrins αvβ3 and αvβ5 are expressed in high-risk neuroblastoma.22,23 During angiogenesis, αv integrins are overexpressed on the endothelial cell surface to facilitate the growth and survival of newly forming vessels. Angiogenic integrin αvβ3 and αvβ5 endothelial cell expression are crucial for their survival. These survival pathways can be inhibited via peptide analogues, such as RGD, which blocks the binding site on integrins αvβ3 and αvβ5, inducing apoptosis of the angiogenic endothelial cells.24−28 Platelets are an important diagnostic indicator of cardiovascular health,5,29 they are also indicated as useful biomarkers for some psychiatric disorders30 and play a critical role in cancer metastasis, for example, through tumor cloaking.31,32 Selective capture and analysis of single platelets is therefore of significant interest in diagnostics and surfaces capable of selective capture of platelets are thus attractive in biosensing applications.30−32 We recently described the impact of surface topography on human platelet capture in RGD-modified micro to nanodimensioned cavity arrays. We found that surface curvature can promote or prevent platelet adhesion and that platelets were selectively captured in their resting state on such arrays.33 In this contribution, we evaluate the capacity of selfassembled monolayers (SAMs) of thiolated dodecapeptide: HHLGGAKQAGDV (AGD) to capture platelets as a function of surface topography and compare the extent of integrinmediated capture of human platelets or mammalian cancer cell line at surfaces prepared with RGD and AGD SAMs. Nonadhesive or nonfouling surfaces have exploited alkane thiols with a variety of terminal groups to form inert surfaces, which are resistant to nonspecific protein or cell adhesion; thus adhesion was also compared with hydrophobic thiolated alkane substrates intended to reduce platelet/cell adhesion.34,35 Confocal fluorescence microscopy and scanning electron microscopy (SEM) were used to quantitatively determine the extent of platelet and cell adhesion at planar and micropore cavity array substrates and to evaluate morphological changes to the cells on binding. CD62P (α-granule) and phalloidin (actin) staining was carried out to assess platelet activation status from the extent of α-granule and actin organization of platelets adhered to the various surfaces. Overall, the ability to control and selectively capture platelets over cancer cells is presented in this work, which may prove valuable for platelet and cancer-related diagnostics. In addition, this work also provides fundamental new insights into the specificity of platelet integrin peptide recognition.
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EXPERIMENTAL SECTION
Materials. Silicon wafers coated with a 525 μm thick layer of gold over a 50 Å titanium adhesion layer were purchased from Amsbio, USA. Aqueous gold plating solution was purchased from Technic Inc., UK. Polystyrene (PS) latex spheres of diameter 1.60 ± 0.03 μm were purchased from Bangs Laboratories, USA. Ethanol (99%), 1octanethiol (C8H18S, 95%), 38% paraformaldehyde (PFA), tetramethyl rhodamine B isothiocyanate (TRITC)−phalloidin (λex 540 nm), Hoechst (2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5bi-1H-benzimidazole trihydrochloride hydrate, bisbenzimide), SK-NAS neuroblastoma cell line, calcium chloride, magnesium chloride, and fluoroshield mounting media were purchased from SigmaAldrich, Ireland. C-Ahx-GRGDS and C-Ahx-HHLGGAKQAGDV B
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is a perfect circle and 0.00 is a straight line). Data are means ± standard deviation of three independent measurements using different healthy donors for each investigation; N = 3 for each study: two confocal studies (platelet counts and phalloidin and CD62P staining) and SEM. Prior to the platelet-adhesion assays, accurate platelet counts were also measured using a Sysmex XE-2100 haematology analyzer (Sysmex KX series, Sysmex UK LTD) to ensure that platelet counts were as close as possible to 30 × 103/μL used in these studies, thus strengthening the quantitative aspect of the results presented in this study.
Cell Culture. The SK-N-AS neuroblastoma cancer cell line was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum; 5% nonessential amino acids, L-glutamine; penicillin; and streptomycin. Once cells reached 80−90% confluence, they were detached from the culture using accutase and re-suspended in PBS (supplemented with 1 mM MgCl2 and CaCl2), pH 7.4, to obtain a final cell count of 1 × 103 cells/μL. Platelet/Cell Capture by the Modified Gold Surfaces. The substrate surface area, platelet/cell volume, concentration, incubation time, and temperature were identical for all modified surfaces: 1 cm2, 150 μL volume of 30 ± 2 × 103/μL WPs (4.5 × 106 total platelets) or 1 × 103/μL cells (1.5 × 105 total cells) incubated for 45 min at 37 °C, respectively. Following which the substrates were gently rinsed with PBS buffer prior to staining/fixing as described below. The incubation time (45 min) was chosen based on literature methods (40 min incubation is reported as sufficient for adhesion and full spreading).39 Reversal of Platelet/Cell Binding to Peptide-Modified Gold Surfaces. To investigate the reversibility of platelet/cell adhesion, after platelets/cells had bound to the RGD or AGD-modified surfaces, bound platelets/cells were incubated with 1 mM RGD or AGD in platelet buffer or PBS (pH 7.4) buffer alone for 15 min at 37 °C. On the basis of the reported rapid integrin−RGD binding time of 1−2 min,39 a 15 min incubation time for reversibility studies was deemed more than sufficient for reversibility studies. For cell-adhesion studies, in all cases, PBS was supplemented with 1 mM CaCl2 and MgCl2. All surfaces were washed thoroughly with platelet buffer or PBS prior to SEM and confocal fluorescence microscopy analyses. Scanning Electron Microscopy Analysis. Bound platelets/cells were fixed with 2.5% glutaraldehyde for 2 h at room temperature. Dehydration was carried out via incubation in 5, 10, 20, 40, 60, 80 and 100% acetone for 15−20 min at each stage. The dehydrated platelet/ cell samples were mounted onto carbon surfaces adhered to aluminum SEM stubs. Samples were allowed to air dry for 2 h before sputter coating. Gold films were sputter-coated over the platelet/cell bound arrays using an argon flow, at pressures between 3 and 4 × 10−1 mbar for 2 min at a voltage of approximately 40 mA, resulting in a gold layer of approximately 30 nm thickness. Gold-coated samples were imaged using a Hitachi S-3400N SEM tungsten system instrument. All images were collected under identical conditions at 5.00 kV accelerating voltage unless otherwise stated. Confocal Fluorescence Microscopy Analysis. For platelet staining, live bound platelets were incubated with PE−CD62P (2 mg/ mL stock, 1/100 dilution) for 15 min at 37 °C. CD62P-stained platelets were fixed with 3.8% PFA solution, permeabilized with 0.01% Triton solution for 10 min at room temperature and stained for TRITC−phalloidin (2 mg/mL stock, 1/100 dilution) via incubation for 30 min at room temperature. For cell-staining studies, surface bound cells were washed thoroughly with PBS, fixed with 3.8% PFA solution, permeabilized with 0.01% triton solution for 10 min at room temperature and stained with the nuclear stain, Hoechst (3.7 × 10−4 M) followed by TRITC−phalloidin via incubation for 30 min at room temperature. All stained samples were mounted using fluoroshield mounting media. Luminescence images were recorded on a Zeiss LSM510 meta confocal microscope using a 40× oil immersion objective lens (NA 1.4) unless otherwise stated. Argon ion lasers (405 and 488 nm) were used for Hoechst and PE−CD62P and 540 nm HeNe laser excitation for TRITC−phalloidin imaging. All images, for quantitative platelet-adhesion measurements, covered an area of 200 × 200 μm. Quantitative platelet-adhesion measurements, measuring the number of cells or platelets/cm2, were carried out using ImageJ software (http://rsb.info.nih.gov/ij/). Automated counting of single color images was carried out. The color images (RGB) were converted to greyscale before proceeding, and the threshold was adjusted to highlight the cells/platelets. Binary and watershed programs were applied to accurately separate any touching cells/platelets (a 1 pixel thick line is added where it estimates division should be). As all background noise is eliminated in the previous programs, all particles were analyzed at the default of 0− infinity. Circularity was also kept at the default 0.00−1.00 (where 1.00
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RESULTS AND DISCUSSION Characterization of the Modified Gold Surfaces. The capture substrates were either planar; approximately 100 nm thick electrochemically deposited gold on silicon wafer or 1.61 ± 0.02 μm diameter gold cavity arrays electrodeposited on the planar gold/silicon wafer substrates. The substrates were characterized prior to the assembly of the thiolated monolayers using SEM and AFM (Figures S3 and S4) and were found to have an average root mean square roughness of 12 ± 0.3 and 300 ± 49 nm for the planar gold surface and 1.60 ± 0.02 μm diameter cavity array, respectively. The effect of surface modification on surface hydrophilicity was assessed by contact angle goniometry (Figure S5 and Table S1). The bare (unmodified) gold surfaces were modestly hydrophobic, reflected in a static water contact angle of 61 ± 1° (planar gold) and 79 ± 4° (1.6 μm diameter cavity array). Following 1octanethiol monolayer formation, the water contact angle increased to 92 ± 2° (planar gold) and 110 ± 2° (1.6 μm diameter cavity array). This value is consistent with previous contact angles reported for tightly packed alkane thiol on gold.40,41 C-Ahx-GRGDS and C-Ahx-HHLGGAKQAGDV monolayers rendered the planar substrates hydrophilic with water contact angles of 14 ± 1° and 17 ± 2°, respectively. The former contact angle is consistent with our previously reported value for planar RGD-modified gold surfaces. 42 The introduction of the cavity topography increased hydrophobicity in both cases, with water contact angles of 98 ± 3° and 90 ± 4° for GRGDS and AGD monolayer surfaces, respectively. We have shown that filling of hydrophilic pore arrays of similar dimensions requires sonication of the substrate in aqueous media.43 Similar contact angles have been observed previously in hydrophilic gold cavity array substrates and attributed to the poor wettability of the cavity arrays with smaller diameter pores due to their inefficient filling.33 Voltammetry was performed to confirm monolayer formation on the gold surfaces (Figure S6). For example, the area under the gold oxide reduction peak at +0.7 V decreased by approximately 83%, following alkane monolayer deposition on planar gold surfaces and by approximately 53% for 1.6 μm diameter cavity array surfaces corresponding to a reduction in the real electroactive area from 6.9 × 10−2 to 1.2 × 10−3 cm2 and from 4.1 to 1.9 cm2, respectively. This indicates that the surface coverage of the alkane thiols is relatively high. Furthermore, a reduction peak at a potential of approximately −1.1 V is observed, which is characteristic of thiol desorption from the gold surface.33,44,45 Effect of Surface Modification on Human Cell and Platelet Adhesion and Morphology on Planar Gold Substrates. Platelets, in their resting (nonthrombotic) state, maintain a smooth discoid shape.33,42 On activation, they undergo a morphological change characterized by four stages: dendritic (D), spread-dendritic (SD), spread (S), and fully C
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Figure 1. Bar graphs presenting neuroblastoma cells adhered (%) vs surface modification at unmodified (bare) gold, alkane, RGD- or AGDmodified (A) planar gold, and (B) 1.6 μm-diameter micropore array surfaces. Y-axes of the bar charts are presented on different scales for clarity. (C) SEM images (5.00 kV) of cells adhered to (i,v) unmodified (bare), (ii,vi) alkane, (iii,vii) RGD-, and (iv,viii) AGD-modified surfaces. Approximately, 1 × 105 cells were incubated with the modified surfaces for 45 min at 37 °C. Data are means ± standard deviation of three independent measurements.
Figure 2. Bar graphs presenting platelets adhered (%) vs surface modification and platelet treatment at modified (A) planar gold and (B) 1.6 μm diameter micropore arrays. Surface modifications: approximately 4 × 106 platelets were incubated with unmodified (bare) gold, alkane, RGD-, or AGD-modified surfaces. Platelet postbind treatments: platelets adhered to surfaces were incubated with 1 mM solution of RGD, AGD, or 1 M PBS for 15 min at 37 °C. Y-axes of the bar-charts are presented on different scales for clarity. (C) SEM images (5.00 kV) of platelets adhered to (i,v) unmodified (bare), (ii,vi) alkane, (iii,vii) RGD, and (iv,viii) AGD. Approximately, 4 × 106 platelets were incubated with the modified surfaces for 45 min at 37 °C. Data are means ± standard deviation of three independent measurements. D
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adhesion through the AGD motif is specifically mediated through the integrin αIIbβ3 receptor. In contrast to platelets, the unmodified (bare) gold surfaces (40(±6)%) were as effective as the RGD surface at capturing whole cells. However, the morphology of the captured cells was different, with far more extensive morphological change evident, including extensive flattening, spreading, and filopodial extension at the former. The comparable capture efficiencies but different morphologies observed suggests that non-specific adhesion is occurring at the bare gold (and at AGD monolayer surfaces), where the cells adapt to their environment and remodel the surface through the formation of a complex layer of proteins, for example, fibronectin adsorbed at the surface to subsequently mediate cell−surface interactions. A similar morphology of non-specifically adsorbed cells has been reported previously.53,54 It is also notable that the variance on capture rates over multiple replicate experiments for live cells is considerably greater than for platelets. This may be due to wider diversity of cell surface receptors available as a result of the variation in phases of life cycle across the cells. Effect of Surface Geometry on Human Cell and Platelet Adhesion and Morphology: 1.6 μm Diameter Micropore Arrays. A summary bar graph of cell and platelet adhesion (%) versus surface modification of 1.6 μm diameter micropore arrays is presented in Figures 1B and 2B (the Y-axes for the planar surfaces are expanded for clarity). As for the planar gold surfaces, negligible neuroblastoma cell adhesion was observed at the alkane thiol control; 4(±3)%. Bound cells in the early spreading stages were indicated by their spherical shape and minimal filopodial formation (Figure 1C(i,ii)). However, in contrast to the extensive nonspecific cell capture observed at the planar surfaces, the bare but nanostructured gold surfaces exhibited minimal cell capture 8(±5)%, suggesting significant influence from the surface geometry on cell adhesion. Similarly, negligible numbers of platelets bind to either bare gold or alkane thiol-modified controls, 4(±0.2)% and 5(±0.4)%, respectively. Platelets that did bind were spherical in shape with minimal pseudopodia formation (Figure 2C(i,ii)). Conversely, dramatically enhanced capture of both cells and platelets was observed at the RGD-modified cavity array surfaces, with approximately 70(±5)% of incident cells and 41(±1)% of incident platelets being retained compared to planar RGD-modified surfaces. The capture rates, in the presence of the peptide, had almost doubled in each case with surface structuring. The morphology of the captured cells was relatively uniform in the early spreading stage with some filopodial elongation (Figure 1C(iii)). The morphology of the captured platelet was somewhat less uniform, but the majority was in the early dendritic spreading stage (Figure 2C(iii)). Consistent with observations at planar surfaces, minimal cell adhesion, 8(±4)%, was evident at AGD-modified structured surfaces, whereas enhanced platelet capture, 45(±2)%, was observed at the AGD surface. This observation strongly indicates that the AGD platelet capture is specifically mediated through the integrin αIIbβ3 receptor. Again, the surface structuring promotes platelet adhesion with more than double the capture rate at the structured AGD compared with planar AGD surfaces. The SEM images of cells and platelets bound to unmodified (bare) 1.6 μm diameter gold cavity array surfaces and arrays modified with (ii) alkane thiol, (iii) RGD, and (iv) AGD monolayer surfaces presented in Figures 1 and 2 demonstrate that the geometry in conjunction with surface
spread (FS) and may also form platelet aggregates depending on the conditions.46 Similarly, mammalian cells undergo wellcharacterized morphological change on focal adhesion (FA), which are characterized by three stages. In phase I (cell sedimentation), cells have a smooth round morphology. In phase II (cell flattening and early adhesion), integrin-mediated binding is initiated and cells begin to flatten, and lamellipodia and filopodia, which facilitate their anchoring to the surface, form.5,47 In phase III (spreading and advanced adhesion), receptor aggregation and the accumulation of actin- and integrin-binding proteins in cytoplasmic domains occur, which provide FA points leading to the nucleation of actin microfilaments.5,47 When cell adhesion is advanced, morphological changes including further flattening and spreading of the cell and organization of the cytoskeletal structure occur (actin).48,49 The effect of surface chemistry on capture efficiency and morphology of captured neuroblastoma cells and platelets was investigated by SEM and confocal microscopy. In the latter, the cells/platelets were stained with TRITC−phalloidin. The number of adhered cells/platelets was assessed using Image J software. Significantly, the number of captured cells and platelets and their morphology as measured by SEM and confocal imaging were indistinguishable, indicating that sample preparation for SEM did not adversely affect the cell structure. It is also important to note that all studies were reproducible, and the reported values are representative of n = 3 measurements. Larger scale SEM and confocal images of all studies are presented in the Supporting Information (Figures S7−S10). A summary bar graph reporting cell and platelet adhesion (% captured of total exposed) versus surface chemistry is presented in Figures 1 and 2. Percentage cell and platelet adhesion is expressed in terms of the percentage of bound cells/platelets compared to the 1.5 × 105 total cells or 4.5 × 106 total platelets incubated with the substrate. Data are means ± standard deviation of three independent measurements. Consistent with our previous report, because of the hydrophobicity of the surfaces in both cases, unmodified (bare) gold or alkane thiol-modified gold surfaces presented minimal platelet adhesion; 0.5(±0.1)% and 0.5(±0.3)%, respectively, and any platelets that did bind were in the early stages of spreading (Figure 2C).29 Similarly, cell adhesion was negligible at the alkane thiol control; 11(±9)%, and any cells that did bind had similarly undergone minimal morphological change, Figure 1A. By contrast, both platelets and cells adhered to the RGDmodified gold surfaces. Platelet morphology was moderately uniform with the majority presenting early stage dendritic spreading following 45 min of incubation (Figure 2C). Whole cell adhesion was strongly promoted at the planar surfaces, wherein approximately 40(±9)% of incident cells were retained compared with 17(±0.6)% of incident platelets. The morphology of the captured cells was quite uniform, with cell spreading and filopodial elongation evident (Figure 1C). The relatively high numbers of both bound cells and platelets indicate that the RGD is acting as a generic adhesion ligand.50−52 Notably, neuroblastoma cell adhesion was limited at the AGD-modified surface, at 13(±4)%, that is, the capture rate was comparable to the alkane thiol control, whereas platelet capture was enhanced at the AGD surface compared to the RGD-mediated planar surface at 21(±1)%, suggesting that E
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Figure 3. Confocal luminescence images of (A) SK-N-AS neuroblastoma cancer cells and (B) washed human platelets bound to (i) unmodified (bare) planar gold and (ii) alkane-, (iii) RGD-, and (iv) AGD-modified planar gold stained for Hoechst (blue), PE−CD62P (green), and TRITC− phalloidin (red). Approximately, 1 × 105 cells and 4.5 × 106 platelets were incubated with the modified surfaces for 45 min at 37 °C. Live bound platelets were stained for CD62P (1/100 dilution) for 15 min at 37 °C. Bound cells/platelets were fixed with 3.8% PFA and stained for Hoechst (1/100 dilution) and phalloidin (1/100 dilution) for 30 min at room temperature. Luminescence images were recorded using a 40× oil immersion objective lens (NA 1.4) using 375 nm (Hoechst), 488 nm (CD62P), and 540 nm (phalloidin) laser excitation. Line bars are 5 and 10 μm for the normal and magnified images, respectively. Data are means ± standard deviation of three independent measurements.
platelets. Distinct actin filament structures can be observed in cells and activated platelets such as filopodia, lamellipodia, and thick bundles of filaments resembling stress fibres.69 In platelets, P-selectin, also referred to as CD62P, stored in αgranules in resting platelets, is rapidly directed to the platelet surface and may be excreted upon activation and so is a useful marker for platelet activation. We have previously reported similar structures.42 P-selectin localization is important when studying biomaterials as when a platelet comes in contact with a substrate, its granules amalgamate in a central granulomere that is surrounded by a microtubule ring. Granules then migrate toward the platelet periphery during spreading and activation. Consequently, the localization of P-selectin and actin aids platelet spreading and activation stage determination following adhesion to the modified surfaces. Figure 3 shows representative confocal fluorescence images of cells and platelets captured at (i) bare gold and surfaces modified with (ii) alkane, (iii) RGD, and (iv) AGD. Both cell and platelet capture efficiency and morphology were consistent with the SEM results. Neuroblastoma cell adhesion was enhanced at the RGD monolayer surfaces compared to bare gold, whereas it is inhibited at alkane thiol and AGD-modified surfaces. Cell adhesion at bare gold and AGD-modified surfaces presented stress fibre formation, indicating intermediate cell spreading in both cases, (Figure 3A(i,iv)), whereas alkane thiol and RGD surface capture presented little to no filopodial formation, suggesting early stage cell spreading (Figure 3A(ii,iii)). Consistent with the SEM data, minimal platelet adhesion was observed at the bare (unmodified) gold and alkane thiolmodified surfaces, whereas enhanced adhesion was observed at both the RGD- and AGD-modified surfaces, Figure 3B. In all cases, CD62P is expressed, amalgamated in a central granulomere and phalloidin staining is also centrally located,
chemical composition structuring can be employed to significantly improve platelet-capture efficiency. Consistent with the reported studies in solution, the data presented here clearly indicate that the fibrinogen sequences, RGD, and the dodecapeptide HHLGGAKQAGDV are both highly effective at mediating platelet-surface adhesion mediated by αIIbβ3 integrin.15,17 A wide range of cell responses to different surface topographies has been reported and in summary show that cellular response to topography is influenced not only by the size and density of the features but also by their regularity or periodicity.55−59 For random features, the binding efficiency appears to be cell-type-dependent.60,61 The shape and dimensions of the features are also important.61−64 Substrates presenting grooves and ridges promoted human coronary artery endothelial cell, human coronary artery smooth muscle cells, and human fibroblast cell adhesion and migration along these features with the feature geometry dictating cell morphology.65 Similarly, epithelial cells preferentially adhered to V-shaped grooved surfaces rather than to flat surfaces.66 Preferential bovine aortic endothelial cell (BAEC) adherence to pyramid-shaped surfaces rather than flat surfaces were also noted.56 Conversely, substrates presenting ordered arrays of nanopillars or pits appear to impede human fibroblast adhesion.67,68 In this study, both cell and platelet capture efficiencies were significantly enhanced whilst morphological change/cell spreading was notably reduced at micropore array surfaces compared with planar surfaces. Effect of Surface Geometry and Chemistry on Cell Morphology and Platelet Activation. Selective actin (phalloidin) and nuclear (Hoechst) staining was conducted to gain further insights into the extent of morphological change in the bound cells/platelets. Actin and P-selectin (CD62P) staining was exploited with platelets to assess both the extent of spreading and activation status of the surface-bound F
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Figure 4. Confocal luminescence images of (A) SK-N-AS neuroblastoma cancer cells and (B) washed human platelets bound to (i) unmodified (bare) 1.6 μm diameter gold cavity arrays and (ii) alkane- (iii) RGD-, and (iv) AGD-modified 1.6 μm diameter gold cavity arrays stained for Hoechst (blue), PE−CD62P (green), and TRITC−phalloidin (red). Approximately, 1 × 105 cells and 4.5 × 106 platelets were incubated with the modified surfaces for 45 min at 37 °C. Live bound platelets were stained for CD62P (1/100 dilution) for 15 min at 37 °C. Bound cells/platelets were fixed with 3.8% PFA and stained for Hoechst (1/100 dilution) and phalloidin (1/100 dilution) for 30 min at room temperature. Luminescence images were recorded using a 40× oil immersion objective lens (NA 1.4) using 375 nm (Hoechst), 488 nm (CD62P), and 540 nm (phalloidin) laser excitations. Line bars are 5 and 10 μm for the magnified and larger scale images, respectively. Data are means ± standard deviation of three independent measurements.
GRGDS or C-Ahx-HHLGGAKQAGDV for 15 min at 37 °C was investigated. These investigations provide deep insights into the role of integrins in cell and platelet binding to the peptide-modified surfaces and reversibility of capture. Figures 2 and S11−S13 summarizes the findings. Representative examples of confocal fluorescence images of the cell-modified substrates post peptide treatment are shown in the Supporting Information, Figures S11 and S12. Significantly, neuroblastoma cell adhesion at the planar or structured surfaces was readily reversed by incubation of the cell/platelet capture substrate in solution of either RGD or AGD, Figure S13. The AGD was less effective at the removal of the cells than RGD, but was more effective at removing cells from the structured surfaces than planar substrates. The capacity of AGD to remove the cells was surprising as this peptide showed no affinity as a capture surface for whole cells. Therefore, controls were carried out where adhered cells were incubated with PBS buffer under the same conditions (15 min incubation at 37 °C). It was found that cell adhesion was also reversed under these conditions. It is noteworthy that gentle washing with PBS prior to fixing did not have a significant impact on cell adhesion, that is, extended incubation with buffer was required to remove the cells. Given the dramatically enhanced cell capture efficiency at the RGD surface compared to AGD and thiolated surfaces and differences in morphology induced by cell capture at RGD surfaces compared with nonspecifically binding surfaces, we can conclude that the cell−RGD binding is specific, but the data suggest that kinetics, that is, a fast on and slow off rate may dictate stability of whole cell binding to the RGD surface. By contrast, incubation with PBS buffer alone failed to remove any bound platelets from either AGD- or RGDmodified substrates (Figure S12), indicating irreversible and
suggesting platelets are in the early stages of spreading/ activation (Figure 3B(iii,iv)). To better understand the morphological impact of the micropore array capture surface fluorescence imaging of labeled cells and platelets was conducted. Figure 4A,B shows representative confocal fluorescence images of cells and platelets captured at (i) bare gold surfaces and surfaces modified with (ii) alkane, (iii) RGD, and (iv) AGD. Both cell and platelet capture efficiency and morphology were consistent with SEM. The extent of cell adhesion was comparable to that observed at the modified planar gold surface; however, in agreement with the SEM images, filopodial elongation and actin reorganization did not occur here, indicating that the surface geometry is influencing cell adhesion and morphology. Consistent with observations at planar gold surfaces, captured platelets exhibit comparable CD62P amalgamated in a central granulomere and phalloidin staining at the center of the bound platelets, indicating that in all cases captured platelets are in the early stages of spreading/activation. It is noteworthy that the percentage of platelets adhered to the surfaces is enhanced more than 2-fold at the RGD/AGDmodified micropore arrays (Figure 4B), compared to the RGD/AGD-modified planar surfaces (Figure 3B). Consequently, although it appears that the fluorescence/expression has increased, it is attributed to an increased number of platelets in the image area. In addition, metal surface fluorescence enhancement arising from the underlying gold cavity (reported previously by our group), likely to arise from plasmonic enhancement there rather than increased CD62P expression cannot be ruled out.33 Reversibility of Cell and Platelet Adhesion to Chemically Modified Surfaces. The impact of incubating the cellor platelet-decorated surfaces with a 1 mM solution of C-AhxG
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the number of receptor−ligand pairs increases, the total adhesion strength subsequently increases. On this basis, combined with the prolonged 45 min incubation times utilized in this study (40 min incubation is reported more than adequate for advanced cell adhesion and spreading),39 the cell morphologies presented in Figure 1C suggest that cell adhesion is primarily weak at both the planar and cavity array substrates.5 The significant difference in binding stability between the platelets and cells at RGD-modified surfaces and the specificity of platelet binding to AGD surfaces suggests that either route, RGD modification with microstructuring or exploitation of AGD with microstructuring, should permit selective entrapment of platelets from a sample containing other cell types. In the latter case through postbinding PBS incubation or in the former through selective αIIbβ3-mediated binding. Future studies will examine the prospects of using such approaches with biological samples.
specific platelet integrin-mediated binding at these peptide surfaces. RGD was significantly more effective (compared to AGD) at removing platelets adhered to RGD-and AGDmodified substrates (Figure 2A,B). Incubation with RGD led to, within experimental error, the removal of all bound platelets from both RGD and AGD planar- and cavity-modified surfaces. By contrast, incubation with AGD removed 16% of platelets bound to RGD planar surfaces compared with 94% of platelets bound to the AGD-modified planar surface. For the 1.6 μm-diameter cavity array surfaces, incubation with AGD removed 35% of bound platelets from both RGDand AGD-modified surfaces, further suggesting that platelet integrin αIIbβ3 presents weaker affinity for the AGD motif compared to RGD, Figure 2B. Reversal of cell capture at modified surfaces by competitive exposure to the RGD ligand has been reported for cells previously70 and most recently we have reported the reversal of integrin αIIbβ3-mediated platelet capture via the RGD ligand.42 Crucially, however, this is the first report investigating RGD displacement of AGD-bound platelets. Also, the finding that RGD but not AGD is highly efficient in releasing AGD-bound platelets is strongly consistent with the common binding site model for αIIbβ3.15,17 Both AGD and RGD peptide sequences bind to the αIIbβ3 RGD-binding pocket situated between the αIIb β-propeller and β3 βI domains.15 Thus, the removal of platelets from the substrate through competitive displacement of the immobilized peptide by solution of either peptide might be expected, but the data presented here indicates that the RGD motif has higher affinity for αIIbβ3 at the immobilized platelet compared to AGD. Although there is some disparity in the reported relative affinity for RGD and the dodecapeptide for αIIbβ3, the majority of reports suggest that the binding affinity is greater for RGD.71−74 For example, Bennett, Barsegov et al. recently reported a direct measurement of the affinity of RGD and AGD by optical trap-based force spectroscopy and found that while the affinities of each peptide for αIIbβ3 are comparable, the affinity of RGD is marginally higher and with faster and slower on and off rates, respectively, compared to AGD.71 The difference in extent and stability of cell versus platelet adhesion at the RGD-modified surfaces is particularly notable. Cell adhesion is extensive for the RGD-modified cavity array surfaces, where nearly 80% of incident cells were captured compared with the unmodified gold array (Figure 1B), whereas adhesion is relatively weak. It was reported previously, for nanostructured RGD-modified pillars formed as analogues of FAs (approx. 50 nm spacing) that nanoscale spacing of integrin-binding sites plays a critical role in cell adhesion, spreading, and proliferation.41 As the cells are too large to enter the 1.6 μm pores, the points for adhesion are expected to be confined to the interpore regions at the top surface of the array. Also, indeed, from SEM imaging, the filopodia from the cells are seen to extend along these planar regions. However, whereas this nanostructuring clearly promotes primary binding, the observation that the cells can be eliminated by extended incubation in PBS suggests that the confined top surface topography does not present sufficient planar distance to instigate strong cell adhesion interactions and spreading at these surfaces. Cells in suspension can gravitate (sediment) to within 5 and 8 nm of a substrate in less than 5 min.39 Within a time scale of seconds to minutes, initial cell−substrate interactions are instigated, driven by specific integrin-mediated adhesion, beginning with single receptor−ligand binding. This interaction initiates subsequent receptor−ligand bonds, and as
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CONCLUSIONS Human platelet and cancer cell interfacial adhesion is influenced by both surface chemistry and topography. By combining capture peptide monolayers with controlled surface curvature, one can dramatically promote platelet and cell adhesion. We found that human blood platelets are captured with high efficiency at monolayers presenting both RGD or HHLGGAKQAGDVC ligands. This binding was reversible on treatment with RGD but not AGD suggesting weaker integrin receptor affinity for HHLGGAKQAGDVC compared to RGD. RGD efficiently displaces AGD bound platelets, whereas AGD did not. This is significant and consistent with the common binding site model for αIIbβ3. By contrast, SK-N-AS neuroblastoma cells attached only at RGD-modified surfaces, and adhesion, although specific, was weak (cells could be removed by extended incubation in blank PBS buffer). Captured platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. Our results indicate that using an interplay of both topography and surface chemistry, it may be possible to select cell over platelet adhesion and to maximize adhesion efficiency. This work may provide a convenient route to create selective capture surfaces for platelet and cancer-related diagnostics. Future work will focus on evaluating this possibility using modified surfaces with whole blood and under biorelevant and flow conditions.
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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/acs.langmuir.7b03279. SEM and AFM images of unmodified, electrochemically deposited gold planar and cavity surfaces; images and summary table of water contact angle measurements of all monolayer surfaces; electrochemical desorption spectra for all monolayer surfaces; confocal image example of CD62P and phalloidin-stained activated platelets; and SEM, confocal images, and bar graphs of cell and platelet adhesion reversibility (PDF) H
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kellie Adamson: 0000-0003-2691-6175 Robert J. Forster: 0000-0001-5079-3123 Tia E. Keyes: 0000-0002-4604-5533 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The Health Research Board is gratefully acknowledged as this material is based upon work supported by the Health Research Board funded scholarship program located in the Royal College of Surgeons in Ireland (award no. PHD/2007/11). This material is based on the work also supported by the Science Foundation Ireland under grant nos. [10/IN.1/B3025] and [14/IA/2488], the National Biophotonics and Imaging Platform, Ireland, and funded by the Irish Government’s Programme for Research in Third Level Institutions, Cycle 4, Ireland’s EU Structural Funds Programmes 2007-2013. U.P., R.J.F., and T.E.K. gratefully acknowledge EUFP7 Interreg. Programme under the Celtic Alliance for Nanohealth.
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ABBREVIATIONS Fg, fibrinogen; RGD, arginine/glycine/aspartic acid; AGD, alanine/glycine/aspartic acid; Ahx, aminohexanoic acid; SAM, self-assembled monolayer; PS, polystyrene; TRITC, tetramethylrhodamine B isothiocyanate; PE, phycoerythrin
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