Quantifying Blood Platelet Morphological Changes by Dissipation

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Quantifying Blood Platelet Morphological Changes by Dissipation Factor Monitoring in Multilayer Shells Julien Fatisson,†,‡ Yahye Merhi,‡,§ and Maryam Tabrizian*,†,‡,| Department of Biomedical Engineering, Centre for Biorecognition and Biosensors, Faculty of Dentistry, and McGill Institute for AdVanced Materials, McGill UniVersity, Montreal, Quebec, Canada, and Laboratory of Experimental Pathology, Montreal Heart Institute, UniVersite´ de Montre´ al, Montreal, Quebec, Canada ReceiVed June 4, 2007. In Final Form: September 12, 2007 The ability of electrostatically driven layer-by-layer (LbL) assembly to adapt to the morphological features of a template was explored. Subtle cytoskeletal changes in blood platelets became traceable through energy dissipation monitoring in multilayered shells using microgravimetric measurements. This LbL coating was sequentially deposited on protein-modified chips onto which platelets were adhered. In addition to consequently improving the signal sensitivity, the LbL shell acted in synergy with the cell, allowing the determination and quantification of cytoskeletal changes induced by the specific cell adhesion to the protein-modified chip surface used with a quartz crystal microbalance with dissipation. The difference in cell morphology, as a result of the optimization of specific interactions between the protein layer and cell membrane integrins induced viscoelastic changes in the polyelectrolyte shell, thereby providing quantitative data on platelet conformational changes upon their adhesion to protein-modified chip surface.

Introduction Among the cascade of events leading to an inflammatory response, resulting in blood clotting on biomedical devices, the so-called thrombogenicity, it is well-known that blood platelets play a major role through their adhesion, activation, and aggregation. During these multistep processes, the platelet cytoskeleton gradually changes, affecting the progression of cell adhesion and spreading.1 Numerous studies have been carried out in this area, but the different mechanisms involved are still not fully understood. Many surface biocompatibility studies2-5 have shown that there is a relation between the material characteristics and the cell adhesion along with their conformational state. Besides kinetics of adhesion, cell conformation studies could also be an asset to better understand the conformational changes of a platelet during the adhesion process. Common techniques including electron microscopy allow one to observe cell morphological changes and to extract some qualitative data. However, to better understand the cell adhesion process, new means of analysis are required for real-time in situ cell cytoskeletal modifications. The choice of such analysis seems to be very limited, since it often involves sample alteration steps. Among reported techniques, quartz crystal microbalance with dissipation (QCM-D) has proven to be simple and effective as a real-time nondestructive means of measuring cell adhesion * Corresponding author: Tel: (1) 514 398 8129. Fax: (1) 514 398 7461. E-mail: [email protected]. † Department of Biomedical Engineering, McGill University. ‡ Centre for Biorecognition and Biosensors, McGill University. § Laboratory of Experimental Pathology, Montreal Heart Institute. | Faculty of Dentistry and McGill Institute for Advanced Materials, McGill University. (1) Michelson, A. D. Elsevier Academic Press: San Diego, CA, 2002; 956 pp. (2) Thierry, B.; Winnik, F. M.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2003, 125 (25), 7494-5. (3) Lee, J. H.; Lee, H. B. J. Biomed. Mater. Res. 1998, 41 (2), 304-11. (4) Tsai, W. B.; Grunkemeier, J. M.; McFarland, C. D.; Horbett, T. A. J. Biomed. Mater. Res. 2002, 60 (3), 348-59. (5) Mao, C.; Qiu, Y.; Sang, H.; Mei, H.; Zhu, A.; Shen, J.; Lin, S. AdV. Colloid Interface Sci. 2004, 110 (1-2), 5-17.

and spreading in biological conditions.6-10 Investigations with QCM-D performed with different cell phenotypes revealed that the signals are cell-type dependent.8,9 Frequency and dissipation shifts are very different for neutrophils,6 fibroblasts,8 preosteoblasts,9 or epithelial cells,7 which makes it difficult to extrapolate the results from one cell type to another. This is due to the fact that the cell physiology (morphology and functionality) affects the mechanism of the adhesion (specific and nonspecific). It has also been shown that the frequency shifts can be related to the cell population and their spreading rate, whereas the viscoelasticity of the system induces changes in the energy dissipation factor7,11 (D-factor). However, considering the theory previously described6,11 for the relation between cell physiology and the QCM-D response, some questions about the mechanism leading to cell spreading,7,11 and its detection through the microgravimetric response, remain unanswered. For instance, to what extent does the initial cell contact with the sensor surface change the number of binding integrins, the strength of adhesion, the cytoskeletal changes, and their effect on the D-factor? Consequently, our approach consists of using QCM-D to investigate subtle morphological changes in smaller and/or nonspherical cells, like platelets. This is considered to be very challenging, particularly for the observation of cytoskeletal changes in platelets induced by specific interactions between proteins on the surface and cell receptors upon adhesion.12 To be able to study such a phenomenon, the first step is to amplify the differences among the cell conformations at different stages of platelet adhesion to the surface. The use of viscoelastic multilayers, such as many (6) Nimeri, G.; Fredriksson, C.; Elwing, H.; Liu, L.; Rodahl, M.; Kasemo, B. Colloids Surf. B 1998, 11 (5), 255-264. (7) Fredriksson, C.; Khilman, S.; Kasemo, B.; Steel, D. M. J. Mater. Sci.s Mater. Med. 1998, 9 (12), 785-788. (8) Lord, M. S.; Modin, C.; Foss, M.; Duch, M.; Simmons, A.; Pedersen, F. S.; Milthorpe, B. K.; Besenbacher, F. Biomaterials 2006, 27 (26), 4529-4537. (9) Modin, C.; Stranne, A.-L.; Foss, M.; Duch, M.; Justesen, J.; Chevallier, J.; Andersen, L. K.; Hemmersam, A. G.; Pedersen, F. S.; Besenbacher, F. Biomaterials 2006, 27 (8), 1346-1354. (10) Cans, A.-S.; Hoeoek, F.; Shupliakov, O.; Ewing, A. G.; Eriksson, P. S.; Brodin, L.; Orwar, O. Anal. Chem. 2001, 73 (24), 5805-5811. (11) Fredriksson, C.; Kihlman, S.; Rodahl, M.; Kasemo, B. Langmuir 1998, 14 (2), 248-251. (12) Grinnell, F.; Phan, T. V. Thromb. Res. 1985, 39 (2), 165-71.

10.1021/la7023204 CCC: $40.75 © 2008 American Chemical Society Published on Web 03/07/2008

Quantifying Blood Platelet Morphological Changes

biocompatible polyelectrolyte layers,13-16 has been shown to generate relatively high microgravimetric signals. In addition, a few studies on biological and nonbiological particulate coreshell systems using layer-by-layer (LbL) technique have shown that the dissolution of the core leaves behind a multilayered polyelectrolyte shell having the same morphology and topography as the cell template.17-20 These findings together instigated our interest in integrating the LbL technology and microgravimetric measurement using the D-factor energy, with the hope of studying the morphological changes in three-dimensional biological templates. QCM-D is frequently used to monitor protein adhesion, LbL assembly,21 and film growth on two-dimensional substrates.13,22 The methodology has proven to be a versatile technique to follow layer deposition and also to measure film thickness along with changes in viscoelasticity either through the frequency shift or D-factor energy measurements. To trigger the conformational changes in platelet as a second step of our approach in using QCM-D to study their morphological changes, the chip interface has been modified with appropriate proteins that could be recognized by cell membrane receptors. Knowing that the first step in the primary homeostasis process is the adsorption of plasma proteins,1 which allows blood platelets to adhere and be activated, we took advantage of this event to control the conformational changes during the adhesion process. The QCM-D chip surface was then modified by a mixture of a thrombogenic (human fibronectin, HFN) and a nonthrombogenic (human serum albumin, HSA) proteins to tailor the platelet morphology as a result of their optimal specific interactions between fibronectin and cell membrane integrins.1,12 Due to the fact that platelet adhesion is very complex and many processes are involved in their adhesion, activation, and function, this work is focused only on the platelet primary adhesion process. Moreover, to study the cell adhesion associated with conformational changes as a function of protein composition on the chip while avoiding the platelet activation, we worked in nutrientfree conditions. These conditions allowed us to obtain different cell morphologies, induced by the optimization of specific interactions between the protein biointerface and the cell membrane integrins. The sequential deposition of biocompatible chitosan (CH) and hyaluronic acid (HA) polyelectrolytes on platelets with different morphologies was assessed, and the energy dissipation factors were measured. Materials and Methods 1. Chemicals. Unless otherwise specified, analytical grade of commercially available reagents were used in this work. Chitosan (CH, MW ) 91 kDa), human serum albumin (HSA), and sodium

Langmuir, Vol. 24, No. 7, 2008 3295 dodecyl sulfate (SDS) were purchased from Sigma-Aldrich. Hyaluronic acid (HA, MW ) 215 kDa) was a contribution from Prof. Winnik’s laboratory (Universite´ de Montre´al). Human fibronectin (HFN) was provided by Chemicon International. Paraformaldehyde (PFA) from ACROS Organics was employed to fix blood platelets. Deionized water (Nanopure Diamond system, Barnstead International) was used for solution preparation. Phosphate buffer (PB) (pH ) 7.4, 0.05 mol L-1) was prepared by dissolving NaH2PO4 and Na2HPO4 (Aldrich) in deionized water for the proteins solutions, and phosphate-buffered saline (PBS) was prepared by dissolution of tablets from Sigma in deionized water. 2. Human Platelet Isolation. Fresh venous blood was collected from healthy volunteers, free from medications known to interfere with platelet function at least 10 days before experiments, in accordance with the guideline of the ethical committee of the Montreal Heart Institute. Platelets were then isolated from the whole blood, as previously described,23,24 and their concentration was adjusted to 250 × 106 cells mL-1. They were maintained at room temperature for 30 min before experiments. For result reproducibility and control of adhesion by the protein surface, platelets were suspended in the presence of ethylenediaminetetraacetic acid (EDTA), known to capture the Ca2+ ions nutrient for platelets and prostacyclin, which are necessary to block activation before contact with protein-modified surfaces. 3. Microscopic Studies. Morphological characteristics of blood platelets were investigated with field emission gun scanning electron microscopy (FEG-SEM) (Hitachi S-4700). Protein adsorption was performed on a silica-based chip pretreated in a UV-ozone chamber (Biofore Nanosciences, Inc) by maintaining the surfaces in protein(s) solution during 30 min. Following protein adsorption, the chip surfaces were exposed to platelet suspension for another 30 min. Platelets were then fixed with a solution of PFA 1% in PBS, pH ) 7.4, during 30 min. Surfaces were dehydrated using ethanol/water solution, increasing the ethanol proportion from 30% to 100%, and amyl acetate/ethanol solutions, increasing the amyl acetate proportion from 25% to 100%. Surfaces were gold-palladium coated prior to platelet morphology characterization by FEG-SEM. 4. Quartz Crystal Microbalance Analyses (QCM-D). QCM-D measurements were performed with a Q-Sense D 300 unit (Q-Sense AB, Go¨teborg, Sweden) by monitoring simultaneously the changes in frequencies (∆f) and energy D-factors (∆D) after each deposition step. The QCM-D chip is excited to oscillate in the thickness-shear mode at its fundamental resonance frequency (f ) 5 MHz) and odd overtones (ν ) 3, 5, 7) by applying a radio frequency voltage across the electrodes. Energy dissipation is recorded periodically by computer-controlled disconnection of the oscillating crystal from the circuit. This allowed us to measure the decay time (τ0) of the exponentially dissipated sinusoı¨dal voltage signal over the crystal caused by switching of the voltage applied to the piezoelectric oscillator. The Q-Sense software (QSoft) was then used to acquire the dissipation factor, D, via eq 1: D)

(13) Coche-Guerente, L.; Desbrieres, J.; Fatisson, J.; Labbe, P.; Rodriguez, M. C.; Rivas, G. Electrochim. Acta 2005, 50 (14), 2865-2877. (14) Jourdainne, L.; Arntz, Y.; Senger, B.; Debry, C.; Voegel, J.-C.; Schaaf, P.; Lavalle, P. Macromolecules 2007, 40 (2), 316-321. (15) Schneider, A.; Picart, C.; Senger, B.; Schaaf, P.; Voegel, J.-C.; Frisch, B. Langmuir 2007, 23 (5), 2655-2662. (16) Wittmer Corinne, R.; Phelps Jennifer, A.; Saltzman, W. M.; Van Tassel Paul, R. Biomaterials 2007, 28 (5), 851-60. (17) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baumler, H.; Kiesewetter, H.; Mohwald, H. Chemistry 2002, 8 (23), 5481-5. (18) Leduc, P. R.; Wong, M. S.; Ferreira, P. M.; Groff, R. E.; Haslinger, K.; Koonce, M. P.; Lee, W. Y.; Love, J. C.; McCammon, J. A.; Monteiro-Riviere, N. A.; Rotello, V. M.; Rubloff, G. W.; Westervelt, R.; Yoda, M. Nat. Nanotechnol. 2007, 2 (1), 3-7. (19) Neu, B.; Voigt, A.; Mitlohner, R.; Leporatti, S.; Gao, C. Y.; Donath, E.; Kiesewetter, H.; Mohwald, H.; Meiselman, H. J.; Baumler, H. J. Microencapsul. 2001, 18 (3), 385-95. (20) Peyratout, C. S.; Moewald, H.; Dahne, L. AdV. Mater. 2003, 15 (20), 1722-1726. (21) Decher, G. Science 1997, 277 (5330), 1232-1237. (22) Thierry, B.; Kujawa, P.; Tkaczyk, C.; Winnik, F. M.; Bilodeau, L.; Tabrizian, M. J. Am. Chem. Soc. 2005, 127 (6), 1626-7.

1 2 ) πfτ0 ωτ0

(1)

where f is the resonance frequency and τ0 is the relaxation time constant. These data provided us with information on the adsorption process as well as on certain viscoelastic properties of the adsorbed film. In the case of homogeneous, very thin or quasirigid films, the frequency shift is proportional to the mass uptake per unit area, ∆mf, deduced from the Sauerbrey25 equation (eq 2): -∆ fSauerbrey )

1 ∆mf nC

(2)

where the mass sensitivity, C, is equal to 17.7 ng cm-2 Hz-1 at f (23) Caron, A.; Theoret, J.-F.; Mousa, S. A.; Merhi, Y. J. CardioVasc. Pharmacol. 2002, 40 (2), 296-306. (24) Theoret, J.-F.; Bienvenu, J.-G.; Kumar, A.; Merhi, Y. J. Pharmacol. Exp. Ther. 2001, 298 (2), 658-664. (25) Sauerbrey, G. Z. Phys. 1959, 155, 206-22.

3296 Langmuir, Vol. 24, No. 7, 2008 ) 5 MHz. In order to go beyond the Sauerbrey approximation, the ∆ fν and Dν experimental data are analyzed using the model developed by Voivona et al.26,27 hypothesizing that the film is a homogeneous and isotropic viscoelastic layer. The Q-sense software (QTools) was programmed to model the raw data. Prior to the protein layer deposition, the silica-covered quartz (QSX-303) was cleaned in an UV-ozone chamber during 10 min. The crystal was then mounted in the QCM-D measurement chamber, stabilized at 24.00 ( 0.02 °C, by means of O-rings seals with only one face in contact with the working solution. The frequency shifts and the dissipation values were continuously recorded during adsorption and rinsing steps. Fibronectin and albumin with different mixing ratios were then deposited onto the silica surface, following the same conditions as for the microscopy study. The platelets were left in contact with the protein layer for 30 min, followed by fixation in 1% PFA in PBS at pH ) 7.4 for 30 min. After rinsing with PBS, the LbL buildup of CH and HA was assessed by leaving the modified surface in contact with alternated polyelectrolyte solutions. The modeling of the frequencies and dissipation shifts allowed us to determine the protein layer thickness, for a specific layer density. For this, we used the protein layer density (F) of 1.2 g cm-3 reported by Renner et al.25 and Hook et al.28,29 Since the surface mass (∆m) remains constant, for a given system mass, the equation, ∆m ) dF, could be used to calculate the layer thickness (d) for the corresponding layer density, F. For cell adhesion measurements, the frequency and dissipation shifts (∆ f and ∆D, respectively) were not very reproducible. This has been explained by Fredriksson et al.6,7 to be a result of changes in spatial distribution of cells at the quartz sensor surface among experiments. Since the lateral sensitivity of ∆ f and ∆D is the same,30 plotting the dissipation against the frequency is qualitatively independent of the cell spatial distribution and would also decrease the lack of reproducibility between each measurement. For this reason, this parameter has been used to compare the cell adhesion results. 5. In Situ Layer-by-Layer Deposition. The assembling procedure was performed on clean surfaces using quartz slides. After protein adsorption, followed by platelet adhesion and the rinsing step, the first layer of CH was assembled by injecting into the QCM-D chamber an aqueous solution of positively charged CH (1 mg mL-1 in PBS, pH ) 6) for 15 min and then extensively rinsing with pure buffer solution. The resulting surface was left in contact with an aqueous solution of negatively charged HA (1 mg mL-1 in PBS, pH ) 6) for 15 min, which was then followed by a rinsing step. Alternate contact with CH and HA solutions allowed the generation of multilayered structures that were characterized in situ with the QCM-D. Since the molecular weight of the polyelectrolytes influences the resulting multilayer film,31 a preliminary study was carried out to optimize the QCM-D signal by varying the CH molecular mass, specifically 67, 91, and 145 kDa. The 91 kDa chitosan has been chosen for further experiments. 6. Protein Layer Analysis. The selection of protein concentrations was based on previous studies describing their adsorption.23 After this step, the silica chip was left in contact with 2% SDS overnight. The protein solutions extracted from the chip surfaces were filtered through a Microcon YM-100 000 (Millipore) to separate HFN (500 kDa) from HSA (65 kDa). Solutions were treated with a BCA (bicinchoninic acid) protein assay kit (Pierce Biotechnology Inc.) according to the supplier’s instructions and analyzed with a UV(26) Voinova, M. V.; Jonson, M.; Kasemo, B. Biosens. Bioelectron. 2002, 17 (10), 835-841. (27) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59 (5), 391-396. (28) Renner, L.; Pompe, T.; Salchert, K.; Werner, C. Langmuir 2005, 21 (10), 4571-7. (29) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf. B 2002, 24 (2), 155-170. (30) Rodahl, M.; Kasemo, B. Sens. Actuators B 1996, B37 (1-2), 111-116. (31) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Langmuir 2004, 20 (2), 448-58.

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Figure 1. Plot of the third overtone dissipation factor (Df3) measured for platelet adhesion on a fibronectin-modified quartz sensor, against the corresponding frequency shift, normalized to the overtone number. Table 1. Df Slopes of Stage II Measured for the Platelets’ Adhesion on Different Protein Surfaces protein composition (weight ratio HSA:HFN)

Df slope of stage II (10-6 Hz-1)

HSA only 50:1 7:1 1:3 HFN only

3.22 ( 2.22 1.8 ( 0.41 0.8 ( 0.32 0.71 ( 0.27 0.64 ( 0.18

visible spectrophotometric plate reader (µQuant, Bio-Tek Instruments) to determine the protein concentration.

Results and Discussion Figure 1 shows a typical Df plot for platelet adhesion on fibronectin. It was previously demonstrated that the frequency shift alone cannot be taken as a direct indication of the number of cells attached and of their spreading rate.6,7,11 These studies have also pointed out that measuring the frequency and the dissipation shifts together for cell adhesion did not produce reproducible data. The plot of dissipation against frequency shifts (Df plots) is however believed to be reproducible and unique for every surface used for cell adhesion.11 In our Df plot for platelet adhesion, which was carried out with protein-modified quartz biointerfaces with unfixed cells, stage I corresponds to the initial contact between the quartz sensor and the cells, while stage II describes the platelet adhesion and the subsequent cytoskeletal changes.6 By calculating the Df slope of stage II, it was then possible to speculate on the platelet spreading process during adhesion. Df slopes for the different protein-modified quartz biointerfaces are reported in Table 1. The standard deviation for HSA surfaces remains high, due to the fact that there is no specific interaction between the cell and the albumin. On such a surface, it seems that the platelets could adhere randomly, in a more horizontal or more vertical way while maintaining their discoidal basal conformation. For this reason, the dissipation values for the HSA layer have been discarded for further consideration in the presentation of our data. For the other protein surfaces, the Df slope evolution seems to be decreasing with the increase of specific interactions between platelets and fibronectin. This can be explained by the fact that the platelets tend to form pseudopodia, inducing a higher cell contact area. This enhanced anchorage to the surface increased the rigidity of the system, which led to a lower Df slope. However, Df slopes were not significantly different within protein-modified surfaces, indicating that the subtlety of the platelets’ morphological changes was not sufficient to record any detectable changes in the dissipation values.

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Figure 2. Plots of the Df3 (b), curve 2.1, for a two CH/HA bilayer film, deposited directly on the protein surface, and for the same multilayer buildup on blood platelets (9), curve 2.2, against QCM-D biointerface containing various weight ratios of HSA/HFN. The Df values are called ratios (instead of slopes), since they have been measured at the plateau of polyelectrolyte adsorption.

To amplify the signal, layers of CH and HA have been deposited on the cell surface using the LbL approach to add a new element of measurement in the global viscoelasticity of the system. To preserve the cell morphology and prevent any possible degradation, a fixation step was performed before the LbL deposition. Since the platelets adhesion is carried out under conditions that blocked their activation process, their fixation did not influence the microgravimetric measurements. Figure 2 shows the dissipation measured for the third overtone (as Df ratio, Df3) for a two CH/HA bilayer film against the HSA/HFN ratio in the protein layer. As the LbL technique involves mainly nonspecific electrostatic interactions, the microgravimetric measurements of CH/HA buildup are very reproducible, as has been shown in previous studies.2,22 Therefore, the lateral sensitivity will not affect the LbL multilayer buildup and only the D-factor can be used. However, for consistency and homogeneity of the results, the Df ratio has also been measured at the rinsing step plateau and is presented herein. Moreover, since the LbL is deposited at the cell surface, considering the matter for cell adhesion, the use of Df ratio instead of pure D-factor will allow one to clear the eventual effect of lateral sensitivity. Curve 2.1 is the control, which represents the Df3 data obtained for surfaces modified by (CH/HA)2 in the absence of platelets. It has to be mentioned that the surface analysis with AFM and QCM-D (data not shown) indicated that the initial weight ratios have been maintained after adsorption of proteins on the quartz surfaces. The proteins’ concentrations were high enough to not be influenced by the different adsorption kinetics or by the Vroman’s effect32 in regard to protein displacement.28,33 No variation of dissipation could be recorded in the structure of the films with a change in surface protein composition. The dissipation was in the same range as the one measured for cell adhesion in the absence of LbL film (Table 1). Curve 2.2 represents the Df ratios obtained after LbL deposition of CH and HA films onto platelet surfaces in the presence of HSA/HFN in increasing ratios. Significant variations of the dissipation for Df3 suggest significant changes in system viscoelasticity. Interestingly, Df measurements changed considerably after the deposition of polyelectrolyte on the platelets. The comparison between the Df values for LbL on cells with the controls (chip with LbL without cells or cells without LbL) indicated that the dissipation signal improved substantially. The (32) Turbill, P.; Beugeling, T.; Poot, A. A. Biomaterials 1996, 17 (13), 127987. (33) Vaidya, S. S.; Ofoli, R. Y. Langmuir 2005, 21 (13), 5852-8.

Figure 3. FEG-SEM microphotographs of platelets on a SiO2based QCM-D chip. (A) platelet in basal configuration on a chip surface coated by HSA only, (B) surface coated by HSA:HFN 50:1, (C) surface coated by HSA:HFN 7:1; (D) surface coated by HSA: HFN 1:3, and (E) surface coated by HFN only. Conditions: 5 kV, 10 µA, and magnification of 25 000×.

deposition of the polyelectrolyte shell appeared to act in synergy with the cell to increase the dissipation signal. In spite of the Df values of curve 2.2 obtained for LbL deposition, one cannot ignore the cells underneath and their morphology. The cells contain a considerable amount of water, which renders them very viscoelastic even after fixation. Due to their electrostatic interactions with the polyelectrolyte layers, their own viscoelastic properties will influence the viscoelasticity of the LbL films made of CH and HA. Consequently, the dissipation values reported in curve 2.2 should be considered as the global viscoelasticity of the system synergized by the natural polyelectrolyte multilayered shell.2,22 Since the cells are fixed, their overall global charge remained neutral and does not affect the following LbL deposition as a result of nonspecific electrostatic interactions. Therefore, the differences obtained in Df can be explained by conformational changes rather than by cell membrane integrins rearrangements. To directly compare the dissipation shifts (Figure 2, curve 2.2) with variations in cell conformation, the morphological changes of platelets as a function of surface composition were examined by FEG-SEM. Figures 3and 4show typical FEG-SEM images of the samples on the QCM-D chips containing different protein compositions, where at least 80% of the platelets have a similar morphology. The number of adhered platelets seemed not to be affected by the protein composition at the QCM-D chip biointerface. Generally, an average of 200 × 103 platelets per mm2 ( 10% was found, regardless of the protein layer composition, leaving enough space between cells so that the polyelectrolyte shell deposition could follow the surface morphology without forming a flat pavement on the surface. Although adhesion of platelets should involve different proteins and receptors,1,34,35 the increasing presence of HFN on the quartz biointerface induces changes in platelets cytoskeleton. This is explained by the fact that fibronectin is an (34) Piotrowicz, R. S.; Orchekowski, R. P.; Nugent, D. J.; Yamada, K. Y.; Kunicki, T. J. J. Cell. Biol. 1988, 106 (4), 1359-64. (35) Ill, C. R.; Engvall, E.; Ruoslahti, E. J. Cell. Biol. 1984, 99 (6), 2140-5.

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Figure 5. Plots of the Df3 for a two CH/HA bilayer film deposited on the platelets sited on a QCM-D chip coated with various weight ratios of HSA/HFN, normalized by the Df slopes extracted from Table 1. The variation in Df3 values is related to the changes in platelet morphology.

Figure 4. FEG-SEM microphotographs at magnification of 5000× showing the platelets population on a SiO2-based QCM-D chip. (A) platelet in basal configuration on a chip surface coated only by HSA, (B) surface coated by HSA:HFN 50:1, (C) surface coated by HSA:HFN 7:1, (D) surface coated by HSA:HFN 1:3, and (E) surface coated by HFN only.

adhesive protein. It binds to the platelet surface via the GPIIbIIIa complex, integrin RIIbβ3, and via other receptors.1 The more fibronectin the platelet can encounter, the more specific interactions are promoted. Also, there is a greater chance that the cell will change its cytoskeleton in order to optimize the specific interactions. This enabled us to monitor the platelet morphology on QCM-D chip surfaces with different protein compositions and allowed us to distinguish the differences in energy dissipation signals.22,36 The increase of HFN concentration was not however accompanied with a consistent increase of Df ratio (Figure 2, curve 2.2). The discrepancy in Df values should therefore be explained by the adhesion process itself1 as follows: with the addition of HFN, specific interactions with platelet membrane integrins are triggered (Figure 3-B), cells lose their basal conformation (shown in Figure 3A), and the formation of short pseudopodia is initiated. Pseudopodia tend to float in a wet chamber environment; this results in a more spongy system, therefore, inducing an increase in Df, compared to an albumin-modified QCM-D chip. By increasing the amount of HFN in the protein layer (HSA/HFN 7:1), the pseudopodia elongate and attach firmly to the protein surface (Figure 3C). This makes the interface more rigid and less dissipative, and therefore, a decrease in the Df value occurs. In addition to surface viscoelasticity, which guides the energy dissipation, one has to consider another important parameter, namely, the contact area between the cell and its supporting protein layer. Since the penetration depth of the signal remains the same,6 an increase in the contact area implies that a higher volume should be sensed. The signal will then be damped more rapidly, inducing an increase in the energy dissipation. The effect of the latter is clearly reflected in the Df values measured for HSA/HFN 1:3 and HFN alone. For HSA/HFN 1:3 weight ratio (36) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4 (6), 1564-71.

on the surface (Figure 3D), the formation of new pseudopodia and elongation of existing ones continue to occur, leading to a partial cell spreading. This implies a higher contact area between cell and surface, which increases the energy dissipation when compared to the previous stage. Particularly, this effect becomes more obvious for a surface composed of HFN alone (Figure 3E), where cells are slightly more spread, rendering the system very viscoelastic and pushing Df to a higher value. The energy dissipation was similar for the surface with high HFN contents (surface composed of HFN alone) and the HSA/HFN 50:1 surface. Since both cell dimension and contact area influenced the global viscoelasticity, comparing only the dissipation measured after the polyelectrolyte shell deposition will not permit the discrimination of all the different morphologies. This could be solved by taking into consideration the contact area between the cell and the protein layer, since the Df slopes seem to follow a rationale evolution with respect to the contact area, according to Table 1 results. This allows us to combine the results of Figure 2 and the Df slope values of Table 1 in Figure 5. The curve 2.2 values in Figure 2 for LbL buildup on cells have been normalized toward the same Df slope, by using the corresponding Df value reported in Table 1. Since the values in Table 1 indicate mostly the effect of the contact area of the platelets on the surface, the normalization will eliminate the effect of the contact area on the value reported in curve 2.2. Considering that the cell population remains similar for all surfaces of various protein composition ratios, we can reasonably state that the changes in energy dissipation in the global system, as depicted in Figure 5, could be directly associated with changes in the cell cytoskeleton. This enabled us to monitor the platelet morphology on QCM-D chip surfaces with different protein compositions and allowed us to distinguish the differences in different cell morphologies as a consequences of variation in energy dissipation signals.22,36

Conclusion The novelty of this work is based on the LbL assembly technique used in conjunction with QCM-D. The viscoelastic properties of the LbL film, which acted in synergy with blood platelets, are explored to obtain information on the morphology of these three-dimensional templates. Our improvement brings interesting advantages compared to standard techniques, such as scanning electron microscopy, which is commonly used for morphological studies. The QCM-D methodology does not require sample preparation and dehydration. Biological samples such as cells can be studied in situ in wet conditions. This technique also provides a real-time measurement of the morphological changes within a short time analysis. As a result, this could be considered

Quantifying Blood Platelet Morphological Changes

as a quantitative measurement of platelet morphology and their cytoskeletal changes induced only by their specific adhesion instead of a qualitative analysis, as is the case with microscopic studies. By avoiding the platelet activation, these results implicitly showed that platelet adhesion can induce morphological changes. Consequently, cell conformation could be another relevant parameter to evaluate the cell activity and membrane rearrangements. Finally, the new methodology offers the possibility to follow the viscoelasticity of LbL film on three-dimensional templates by means of QCM-D analysis. This methodology also seems very promising for studying the platelet activation (through following the morphological changes during the activation

Langmuir, Vol. 24, No. 7, 2008 3299

process) in response to different biomaterials and could be a versatile approach to evaluate the material’s thrombogenicity. Acknowledgment. This work is supported by the Canadian Institute of Health Research (CIHR) and the Fonds Que´be´cois de Recherche sur la Nature et les Technologies (FQRNT) through the Centre of Biorecognition and Biosensors. We greatly thank M. Daniel Yacoub, M. Haissam Abou-Saleh, and M. JeanFrancois The´oreˆt for the preparation of the platelets and their help and advice. Ms. Line Mongeon is also greatly acknowledged for her help during the SEM studies. LA7023204