pubs.acs.org/Langmuir © 2010 American Chemical Society
Universal Chemical Gradient Platforms Using Poly(methyl methacrylate) Based on the Biotin-Streptavidin Interaction for Biological Applications Anna Lagunas,*,†,‡ Jordi Comelles,‡ Elena Martı´ nez,†,‡ and Josep Samitier†,‡,§ †
Centro de Investigaci on Biom edica en Red. Bioingenierı´a, Biomateriales y Nanomedicina (Ciber-BBN), C/Marı´a de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain, ‡Institute for Bioengineering of Catalonia (IBEC), C/Baldiri Reixac 10-12, 08028 Barcelona, Spain, and §Department of Electronics, University of Barcelona, c/Martı´ i Franqu es 1, 08028 Barcelona, Spain Received July 1, 2010. Revised Manuscript Received July 29, 2010 This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin-streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PMMA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SAV) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.
Introduction The development of engineered biocompatible surfaces is of particular interest for the design of biomimetic materials. Many strategies for creating functional interfaces with biomimetic characteristics to enhance material-directed cell function have been described.1-3 Biomimetic surfaces are designed to present ligands for specific cell receptors. The way in which these bioactive ligands are presented and the concentration are very important.4-6 In homogeneously modified substrates, a systematic evaluation of the introduced surface properties is very limited. Determining the optimal surface ligand density for cell adhesion or differentiation assays or the correlation between ligand density and cell behavior for drug screening applications usually requires a large number of individual trial experiments. Moreover, for some applications dealing with mimicking the interfacial zones in native tissues, it is very desirable to have substrates available whose physicochemical properties change gradually with distance and may even evolve through time. At the surface level, this can be accomplished by using materials with properties that change in a gradient fashion. *Corresponding author. Tel: (þ34) 93 403 71 77. Fax: (þ34) 93 403 71 81. E-mail:
[email protected]. (1) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487–492. (2) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47–55. (3) Martı´ nez, E.; Lagunas, A.; Mills, C. A.; Rodrı´ guez-Seguı´ , S.; Estevez, M.; Oberhansl, S.; Comelles, J.; Samitier, J. Nanomedicine 2009, 4, 65–82. (4) Massia, S. P.; Hubbell, J. A. J. Cell. Biol. 1991, 114, 1089–1100. (5) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell. Sci. 2000, 113, 1677–1686. (6) Reyes, C. D.; Garcia, A. J. J. Biomed. Mater. Res., Part A 2003, 65, 511–523.
14154 DOI: 10.1021/la102640w
Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. They can be generated by different methods such as controlled diffusion,7-9 gradual immersion of the substrate in a reactive solution,10,11 electrochemical manipulation,12,13 microfluidic devices,14,15 silicone elastomer (SE) stamps,16,17 radio frequency plasma,18 and corona discharge treatments19,20 and also through grafting procedures.21,22 For an extensive review of gradient formation methods, see ref 9. The final objective of most of these methods is the (7) Lieberg, B.; Wirde, M.; Tao, Y. -T.; Tengvall, P.; Gelius, U. Langmuir 1997, 13, 5329–5334. (8) Bhat, R. R.; Fisher, D. A.; Genzer, J. Langmuir 2002, 18, 5640–5643. (9) Genzer, J.; Bhat, R. R. Langmuir 2008, 24, 2294–2317. (10) Baker, B. E.; Kline, N. J.; Treado, P. J.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 8721–8722. (11) Xu, C.; Barnes, S. E.; Wu, T.; Fischer, D. A.; DeLongshamp, D. M.; Batteas, J. D.; Beers, K. L. Adv. Mater. 2006, 18, 1427–1430. (12) Terrill, R. H.; Balss, K. M.; Zhang, Y.; Bohn, P. W. J. Am. Chem. Soc. 2000, 122, 988–989. (13) Jayaraman, S.; Hillier, A. C. Langmuir 2001, 17, 7857–7864. (14) Caelen, I.; Bernard, A.; Juncker, D.; Michel, B.; Heinzelmann, H.; Delamarche, E. Langmuir 2000, 16, 9125–9130. (15) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311–8316. (16) Venkateswar, R. A.; Branch, D. W.; Wheeler, B. C. Biomed. Microdevices 2000, 2, 255–264. (17) Chopi, S.-H.; Zhang Newby, B.-M. Langmuir 2003, 19, 7427–7435. (18) Pitt, W. G. J. Colloid Interface Sci. 1989, 133, 223–227. (19) Lee, J. H.; Kim, H. G.; Khang, G. S.; Lee, H. B.; Jhon, M. S. J. Colloid Interface Sci. 1992, 151, 563–570. (20) Kim, M. S.; Seo, K. S.; Khang, G.; Lee, H. B. Bioconjugate Chem. 2005, 16, 245–249. (21) Wu, T.; Efimenko, K.; Genzer, J. J. Am. Chem. Soc. 2002, 124, 9394–9395. (22) Zhao, B. Langmuir 2004, 20, 11748–11755.
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anchoring of biomolecules in a controlled and gradually changing surface density, but concomitantly, they induce both chemical and physicochemical changes in very relevant surface properties such as wettability and roughness. Therefore, it is difficult to decouple the effects coming from such modifications and unequivocally attribute cell responses to the surface density of the introduced biomolecules. Moreover, in most cases there is a lack of direct quantitative measurement of the density of surface ligands introduced. Poly(methyl methacrylate) (PMMA) is a biocompatible transparent thermoplastic material that has been widely used in biomedical applications.23 Its chemical structure is characterized by pending methyl ester groups along the main chain of the polymer that can be used as reactive points for surface modification.24 Rather than use a nonselective method to introduce functional groups onto a PMMA surface, as in the case of corona discharge treatment,20 we chose the specific alkaline ester hydrolysis for surface functionalization. Matsuda and co-workers have reported the creation of a wettability gradient on a poly(vinylene carbonate) (PVCa) surface via progressive immersion in an alkaline solution to selectively produce a chemical gradient of hydroxyl groups.25 Ester hydrolysis performed under basic conditions constitutes an almost irreversible reaction that can be applied to a broad number of ester-containing polymers and is slow enough to allow accurate time-dependent control. Taking these considerations into account, here a carboxylate concentration gradient was generated on the surface of a PMMA thin film that was spincoated onto a glass substrate by continuous immersion in a sodium hydroxide aqueous solution. The hydrolysis of methyl ester groups can be controlled by modifying the pump dispensing rate, exposure time, experimental temperature, and sodium hydroxide concentration. The biotin-streptavidin complex, with a dissociation constant (Kd) of about 10-14 M, is the strongest noncovalent biological interaction reported.26,27 Moreover, the recognition event takes place fast enough to allow high sensitivity binding assays in short incubation times, thus minimizing side reaction effects.28 In our approach, methyl ester alkaline hydrolysis of PMMA was used for the design of a universal platform of continuously variable concentration gradients of surface-conjugated biomolecules, based on the biotin-SAV recognition for the attachment of any biotinylated molecule in a directed orientation. This platform was comprehensively characterized and a quantitative relationship between sample position and surface streptavidin density established. The suitability of the gradient surface platform for applications typically requiring a variation of molecular concentrations was then tested. As a proof-of-concept, the effects of concentration of the Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Bradykinin, henceforth BK) peptide on cell morphology were evaluated and correlated with the applied drug-dose. BK is a potent vasodilator29,30 that, at the cellular level, induces changes in cell morphology, (23) Ratner, B. D. Biomaterials Science: An Introduction to Materials in Medicine; Academic Press: San Diego, CA, 1996. (24) Hyun, J.; Zhu, Y.; Liebmann-Vinson, A.; Beebe, T. P., Jr.; Chilkoti, A. Langmuir 2001, 17, 6358–6367. (25) Ueda-Yukoshi, T.; Matsuda, T. Langmuir 1995, 11, 4135–4140. (26) Green, N. M. Avidin and Streptavidin. In Avidin-Biotin Technology; Wilcheck, M. B., Edward, A., Eds.; Academic Press: San Diego, CA,1990; Vol. 184, p 51. (27) Holmberg, A.; Blomstergren, A.; Nord, O.; Lukacs, M.; Lundeberg, J.; Uhlen, M. Electrophoresis 2005, 26, 501–510. (28) Piran, U.; Riordan, W. J. J. Immunol. Methods 1990, 133, 141–143. (29) Cuerrier, C. M.; Gagner, A.; Lebel, R.; Gobeil, F., Jr.; Grandbois, M. J. Mol. Recognit. 2009, 22, 389–396. (30) Farmer, S. G.; Burch, R. M. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 511– 536.
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including the formation of peripheral actin microspikes (PAM), membrane ruffles and a reduction in stress fiber formation.31 As BK action occurs through binding to a cell-membrane receptor, the gradient platform described here will provide some benefits when screening dose-dependent drug effects, including shortening the time and diminishing the amount of reagent consumption. The results obtained showed a correlation between the drug surface concentration and cell morphology effects, thus proving that the characteristics of the gradient platform (surface density range and gradient slope) are appropriated for cell studies such as immobilized drug screening.
Materials and Methods Chemicals and Materials. Glass Micro Slides 75 25 mm were purchased from Corning Inc. (NY, USA). Sulfuric acid 95-98%, sodium hydroxide pellets, hydrochloric acid 37%, and ethanol absolute were obtained from Panreac Quı´ mica S. A. U. (Barcelona, Spain). Hydrogen peroxide was supplied by BASF (Barcelona, Spain). (3-Aminopropyl)triethoxysilane, 2-(2-aminoethoxy)ethanol, bovine serum albumin (BSA), FluoromountTM Aqueous Mounting Medium, and the reagents for carboxylic acid group activation -N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N0 -ethyl carbodiimide (EDC)- were purchased from Sigma-Aldrich Quı´ mica S. A. (Madrid, Spain). Poly(methyl methacrylate) 11% solution in anisole (950PMMA A Resist) was obtained from MicroChem Corp. (Newton, MA, USA). EZ-link Amine-PEO3-Biotin and ImmunoPure Streptavidin were from Cultek S. L. (Madrid, Spain). Phosphate-buffered saline (PBS) and cell culture reagents Dulbecco’s Modified Eagle Medium (D-MEM), liquid high glucose, fetal bovine serum (FBS), L-glutamine 200 mM solution in 0,85% NaCl, MEM sodium pyruvate solution 100 mM, penicillin-streptomicin, and TrypLETM express stable trypsin-like enzyme with phenol red and the cell nuclei immunostaining agent Hoechst were obtained from Invitrogen S. A. (Barcelona, Spain). Ammonium chloride and paraformaldehyde (PFA) extra pure DAC were supplied by Merk Sharp&Dohme (Madrid, Spain). Biotin-Bradykinin was purchased from BioNova Cientı´ fica S.L. (Barcelona, Spain) and TRITC-phalloidin for actin immunostaining and saponin were obtained from Fluka (Buchs, Switzerland).
Carboxylate Surface Gradient Formation on Thin PMMA Spin Coating. Precleaned microscope glass slides were
acid cleaned in piranha solution [H2SO4-H2O2 (7:3)] for 10 min, washed thoroughly with Milli-Q water and blown dry using pressurized nitrogen gas to remove excessive moisture. This piranha treatment produces a high hydroxyl group density on the surface of the glass (SiOH).32 A noncovalent interaction is expected among the amino groups on the surface of the glass and the esters in PMMA that favors an increased adhesion between the two phases. Thus, hydroxyl groups on the glass surface will react with the silane (3-aminopropyl)triethoxysilane in a vapor phase self-assembling procedure33 to create an amino-terminated monolayer. The silane-containing slide was baked at 80 °C for 1 h. 950PMMA 11% solution in anisole was spin-coated onto the silane-containing glass surface at room temperature in a Laurell Model WS-400A-6TFM/LITE spinner at 3000 rpm for 30 s and then the slide was heated at 180 °C for 60 s to eliminate excess solvent. Final thickness of the PMMA film is around 2-2.5 μm. A carboxylate surface gradient was then generated by the gradual immersion of the PMMA-glass slide into sodium hydroxide 2 M aqueous solution. Thus, the slide was inserted in a falcon tube connected to a syringe pump filled with the alkaline solution (Figure 1). The pump rate was set to 3 mL/h. After 16 h, the (31) Kozma, R.; Ahmed, S.; Best, A.; Lim, L. Mol. Cell. Biol. 1995, 15, 1942– 1952. (32) Noel, O.; Brogly, M.; Castelein, G.; Schultz, J. Eur. Polym. J. 2004, 40, 965– 974. (33) Halliwell, C. M.; Cass, A. E. G. Anal. Chem. 2001, 73, 2476–2483.
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Figure 1. Schematic representation of the preparation of carboxylic acid gradients on PMMA: custom designed system conveyed to a syringe pump for accurate control when dispensing the alkaline solution. surface carboxylate gradient was neutralized with a 0.1 M solution of hydrochloric acid, then rinsed with Milli-Q water and absolute ethanol, and blown dried with a nitrogen stream. The time that the NaOH solution takes to completely cover the slide length was used to obtain a time versus distance relation (see Supporting Information). This relation was employed to assign a distance to each hydrolysis time. Surface Analysis. Surface chemical analysis of the outermost PMMA layer was performed by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Measurement of the relative amounts of CH3O- fragments remaining (from the nonhydrolyzed groups still on the surface) was performed using a ToF-SIMS IV instrument (ION-TOF, Munster, Germany) operated at a pressure of 5 10-9 mbar (Figure 2). Samples were bombarded with a pulsed bismuth liquid metal ion source (Bi3þ), at 25 keV. The gun was operated with a 20 ns pulse width, 0.3 pA pulsed ion current for a dosage lower than 5 1011 ions/cm2, well below the threshold level of 1 1013 ions/ cm2 generally accepted for static SIMS conditions. Secondary ions were detected by a reflection time-of-flight analyzer, a multichannel plate (MCP) and time-to-digital converter (TDC). Measurements were performed with a typical acquisition time of 20 s, at a TDC time resolution of 200 ps. Charge neutralization was achieved with a low energy (20 eV) electron flood gun, thus no sample conductive coating was needed before the measurements. Secondary ions were extracted with 2 kV voltage and were postaccelerated to 10 keV kinetic energy just before hitting the detector. The maximum mass resolution, R = m/Δm, was around 8000, where m is the target ion mass and Δm is the resolved mass difference at peak half-width. Secondary ion spectra in negative mode were acquired from randomly rastered surface areas of 500 μm 500 μm along the slide. The signal intensity of the CH3Ofragment was normalized against its maximum value and plotted versus distance in order to obtain a qualitative measure of the varying concentration along the slide. Samples were subjected to XPS analysis recorded in a PerkinElmer PHI 5500 Multitechnique System from Physical Electronics (Waltham, MA, USA) with a monochromatic X-ray source (Aluminum KR line of 1486.6 eV energy and 350 W), placed perpendicular to the analyzer axis and calibrated using the 3d5/2 line of Ag with a full width at half-maximum (fwhm) of 0.8 eV. Carbon 1s and oxygen 1s core level scan spectra were recorded for each section (3 sections per sample, with an analysis area of ∼0.5 mm2). The resolution selected for the spectra was 187.5 eV of pass energy and 0.8 eV/step for the general spectra, and 23.5 eV of pass energy and 0.1 eV/step for the spectra of the different elements. All measurements were taken in an ultra high vacuum (UHV) chamber pressure between 7 10-9 and 3 10-8 mbar. When necessary, a low energy electron flood gun (0-3 eV) was used to discharge the samples. Peak fitting was performed using MultiPak V6.0A software from Physical Electronics Inc. (Chanhassen, MN, USA). Peak area of the deconvoluted signal at 287 eV, corresponding to C 1s from the methoxy group in PMMA, was normalized to the total polymer C 1s area and plotted against sample distance. 14156 DOI: 10.1021/la102640w
Lagunas et al. Surface roughness was calculated from atomic force microscopy (AFM) measurements performed on a Dimension 3100 AFM instrument (Veeco Instruments, USA) equipped with a rectangular silicon AFM tip (MikroMasch NSC18/AlBS, spring constant 3.5 N/m, radius of curvature about 10 nm, aluminum backside coating, and 230 μm in length) and operated in tapping mode at room temperature in air. Topographic images obtained from the scanning of 10 10 μm rastered areas along the length of the sample were used for root-mean-square calculations performed with WSxM software (Nanotec Electr onica, Spain).34 At least 7 positions along the sample length of three different substrates were analyzed. Water contact angles (CAs) were measured before the neutralization step by the sessile-drop method at several different positions (at least 10 positions on three independent substrates) along the sample length with an OCA contact angle system (Dataphysics, Germany). Images of the liquid droplets of 1 μL Milli-Q ultrapure water (MilliPore Iberica S.A.U, Spain) in contact with the surface were recorded immediately after droplet stabilization, and the droplet profile was fitted with SCA20 software (Dataphysics, Germany) using an elliptic fitting method. Biotin-Streptavidin Surface Gradient Formation. Chemical modification of the PMMA surface after hydrolysis treatment was carried out according to Scheme 1. After neutralization with HCl solution, carboxylic acid groups were reacted with the amino-terminated EZ-link amine-PEO3biotin compound to create a gradually biotinylated surface. This reaction requires the prior activation of the carboxylic acid moieties. This was performed by placing the slide in contact with a mixture of EDC (73.4 mg, 0.38 mmol) and NHS (8.9 mg, 0.08 mmol) in Milli-Q water (5 mL) at room temperature for 15 min. The slide was washed thoroughly with Milli-Q water and dried with nitrogen gas. For the coupling reaction, an EZ-link aminePEO3-biotin (4.2 mg, 0.01 mmol) solution in Milli-Q water (1 mL) was placed on the top of the NHS-modified gradient surface for 1 h at room temperature. The biotinylated gradient was rinsed several times with Milli-Q water and dried in a stream of nitrogen. Nonreacted positions were blocked by treatment with a 0.1% solution of 2-(2-aminoethoxy)ethanol in PBS for 30 min at room temperature. Then the samples were gently washed with PBS and Milli-Q water and dried in a stream of nitrogen. ImmunoPure streptavidin Texas red was reconstituted following the supplier’s instructions with 0.65 mL of Milli-Q water to a concentration of 1.8 mg/mL and divided into aliquots with a 0.1 mg/mL final concentration. The SAV solution was left for 1 h at room temperature in contact with the biotin gradient in the absence of light. The final SAV-modified PMMA surface was gently rinsed with PBS and stored under moisture to ensure protein structure preservation. The SAV surface gradient was checked by fluorescence microscopy with an Eclipse E1000 upright microscope (Nikon, Netherlands) equipped with a charge-coupled-device (CCD) camera and by working with a green excitation G-2A long-pass emission filter. Images were taken of selected areas along the slide, and they were processed with Matlab 7.0.4.365 (R14) from MathWorks, Inc. to obtain the total image intensity. Results were normalized against the maximum intensity value and plotted versus distance. Streptavidin Surface Density Quantification. Protein coverage was determined by means of surface plasmon resonance (SPR) measurements. SPR occurs under conditions of total internal reflection when an evanescent wave interacts with delocalized surface electrons in a metal film at the interface with a medium of lower refractive index. The displacement in the angle of minimum reflectivity of the incident polarized light is linearly dependent on the changes in the refractive index, and therefore the mass of the protein, in the interfacial region.35 An SPR RT2005 instrument was purchased from Resonant Technologies (RES-TEC) (34) Horcas, I.; Fernandez, R.; Gomez-Rodrı´ guez, J. M.; Colchero, J.; GomezHerrero, J.; Baro, A. M. Rev. Sci. Instrum. 2007, 78, 013705–1-013705-8.
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Figure 2. Characterization of carboxylic acid gradient formation by ToF-SIMS analysis. (a) Plot of the CH3O- fragment (m/z = 31) normalized intensities obtained from selected rastered areas along the slide vs distance (n = 3). (b) Superimposed ToF-SIMS negative ion spectra at the m/z = 31 peak obtained from three of the selected rastered areas along one sample length. Scheme 1. Schematic Representation of SAV Assembly on the Biotin Gradient
refractive index of 1.45 for a homogeneous monolayer of SAV,35,38 the surface coverage can be calculated. Also, SAV surface density A can be calculated using eq 1 A ¼ d
ðn - n0 Þ Dn=Dc
ð1Þ
where A is the adsorbed amount of protein per area and n0 is the refractive index of the solution and with ∂n/∂c = 0.212 mL/g (derived independently by Knoll et al. from refractometric measurements).35
Surface Gradient of Biotin-Bradykinin for Drug Screening Studies. As a proof-of-concept application of the gradient
GmbH (Max Plank Institute for Polymer Research, Germany). SPRchip sensor chips were from GWC Technologies Inc. To emulate the experimental conditions under which a chemical gradient was created in a PMMA surface, a 2% 950PMMA solution in anisole was spin coated onto the gold surface at 4000 rpm for 30 s. The final polymer thickness was around 50 nm, which is thin enough to still ensure sensitive detection of the SPR signal (Supporting Information). A set of samples composed of SPR chips covered with a thin PMMA layer were prepared and modified using different hydrolysis times and then functionalized with biotin as described above. These chips were index matched to the prism and fitted with a 20 μL flow cell connected to a peristaltic pump from Ismatec (Glattbrugg, Switzerland). The baseline in the SPR response was first established with PBS, and then a solution of PBS containing SAV at 100 μg/mL concentration was injected. When the signal indicated coverage saturation, the flow was stopped for 1 h (incubation time) and then restored with PBS solution to remove adsorbed nonspecific protein. The reflected intensity was fitted by means of Fresnel’s equations. The shift in the surface plasmon peak, obtained before and after SAV incubation, was used to calculate the thickness d of the SAV layer (Winspall software, from RES-TEC, Germany). The model was simplified by taking the biotinylated PMMA as a 52-nm-thick single layer with a refractive index of 1.50.36,37 From thickness results and considering a theoretical thickness value of 5 nm and a (35) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F.-J.; Spinke, J. Colloids Surf., A 2000, 161, 115–137. (36) Schmitt, F. J.; Blankenburg, R.; H€aussling, L.; Ringsdorf, H.; Weisenllorn, A. L.; Hansma, P. K.; Leckband, D. E.; Israelachvili, J. N.; Knoll, W. In Synthetic Microstructures in Biological Research; Schnur, J. M., Peckerar, M., Eds.; Plenum Press: New York, 1992; p 147. (37) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012–7019.
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surfaces, they were used to test the effect of BK concentration on cell morphology. For this purpose, the SAV surface gradient on PMMA was modified with biotin-BK. A 12 μg/mL solution in Milli-Q water was placed in contact with the SAV layer for 1 h at room temperature. After this time, samples were washed with PBS and stored under moisture. The NIH/3T3 mouse embryonic fibroblast cell line from passage 15 was expanded for 2 days at 37 °C and 10% CO2 in growth medium. For an increase in cytoskeleton sensitivity toward the drug, a starving process was performed: 24 h before cell culturing, growth medium was replaced by D-MEM supplemented with 0.5% FBS, 1% L-glutamine, 1% penicillin-streptomycin, and 1% sodium pyruvate, and 2 h before cell seeding, cells were maintained in D-MEM supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 1% sodium pyruvate without FBS. BK-gradient slides were preincubated in PBS before cell culturing. NIH/3T3 cells were seeded at a cell density of 660 cells/ cm2. After 90 min of incubation time, nonadherent cells were washed out with PBS. Cells were fixed with 4% PFA in PBS for 20 min and washed three times with PBS, and the remaining free aldehyde groups were then blocked with 50 mM ammonium chloride (20 min at room temperature). After this time, samples were washed twice with PBS and cells were permeabilized with a saponin solution for 10 min at room temperature (0.1% saponin in PBS containing 1% BSA). Actin staining was performed by incubating cells with a solution containing TRITC-phalloidin diluted 1:500 in 1% BSA in PBS from a stock solution of 1 mg/ mL in DMSO. Hoechst was used for nucleus staining (1:1000 in 1% BSA in PBS). Cell morphology effects were checked by fluorescence microscopy with the Eclipse E1000 upright microscope (Nikon, Netherlands) equipped with a charge-coupled-device (CCD) camera and working with a green excitation G-2A long-pass emission filter for actin fiber visualization and with a UV emission filter for the cell (38) Hirashima, A.; Pan, C.; Shinkai, K.; Tomita, J.; Kuwano, E.; Taniguchi, E.; Eto, M.; Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309–315.
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Figure 3. Characterization of the surface chemical composition after hydrolysis by XPS analysis. C 1s signal deconvolution for (a) least hydrolyzed part of the sample and (b) the most hydrolyzed region showing the relative area decrease for component 3, which has been assigned to the methoxy group carbon (inset in a) . nuclei. Images were taken of selected areas along the slide, and they were processed with CellProfiler (www.cellprofiler.org) image-processing software to obtain different morphological parameters: the cell area (Ac), perimeter (Pc), form factor (FF), and solidity (SOL). The FF was calculated using eq 2 FF ¼
4πAc Pc 2
ð2Þ
where Ac and Pc are the cell area and the cell perimeter, respectively. The solidity was calculated using eq 3 SOL ¼
Ac Ah
ð3Þ
where Ah is the convex hull area, defined as the area of the smallest convex polygon circumscribed on the cell silhouette.39
Results and Discussion Hydrolysis gradient formation was checked with ToF-SIMS, XPS, AFM, and CA measurements. Carboxylic acid activation, reaction with amino-derivatized biotin, and SAV incubation led to the formation of a SAV gradient surface. The SAV gradient was characterized by means of fluorescence analysis and quantified with SPR. The surface gradient platform thus designed was tested in drug screening studies for BK effects on cell morphology. Preparation and Characterization of Streptavidin Surface Gradient on PMMA. ToF-SIMS analysis was used to characterize the surface chemical modification after hydrolysis. Because the contact time with the alkaline solution gradually decreases, it is expected that the density of the remaining methyl ester functional groups gradually decreases along the sample length (with distance = 0 being the least hydrolyzed part of the slide). Figure 2a shows the normalized intensity of the methoxy group (CH3O-) fragment obtained from different rastered positions along the slide. The corresponding spectra at the m/z = 31 peak of three of the selected areas are shown in Figure 2b. The methyl ester fragment normalized intensity was higher in the upper part (39) Soltys, Z.; Orzylowska-Sliwinska, O.; Zaremba, M.; Orlowski, D.; Piechota, M.; Fiedorowicz, A.; Janeczko, K.; Oderfeld-Nowak, B. J. Neurosci. Methods 2005, 146, 50–60.
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of the sample where the contact time with the alkaline solution was lower. Despite the intensity variation that clearly appears in Figure 2, SIMS results show only a qualitative effect of the hydrolysis on the polymer surface. To check surface chemical composition variations quantitatively along the gradient surface, XPS analysis was performed. The spectral region corresponding to C 1s shows a broad peak centered at 284.8 eV that can be deconvoluted into four carbon 1s core signals attributed to the different carbon types of the polymer40 and a lower binding energy signal attributed to unspecific residual carbon (Figure 3). Normalization of the C 1s area of the peak centered at 287.6 eV, corresponding to the methoxy group, against the total polymer C 1s area permits us to calculate the extent of the hydrolysis along the sample length: it varies from 18 to 40%. From these results, it can be appreciated that the carboxylic acid surface concentration varies 22% in 75 mm, which is several times smoother than values reported for other gradient formation methods. Whittle et al. obtained around a 27% variation of the chemical composition in their 11 mm length gradients created by plasma polymerization,41 and Hanley and co-workers described a 34% fluorine content increase along a distance of 15 mm of their chemical gradient surfaces created by hyperthermal C3F5þ deposition on PMMA.42 Low slope hydrolysis gradients, such as that reported here, have the unique characteristic of barely modifying the surface physical properties along the sample length. In fact, water contact angle results showed just a slight, gradual decrease from 78 ( 1 to 71 ( 2° when measuring from the least hydrolyzed part of the slide to the most hydrolyzed one. Furthermore, AFM measurements showed negligible roughness variation along the slide distance after hydrolysis, which is less than 0.4 nm, whereas Kim and coworkers reported changes of up to 90 nm in samples treated with corona discharge.20,43 With physical properties remaining insignificantly altered, the effect of a varying concentration along the (40) Louette, P.; Bodino, F.; Pireaux, J.-J. Surf. Sci. Spectra 2005, 12, 69–73. (41) Whittle, J. D.; Barton, D.; Alexander, M. R.; Short, R. D. Chem. Commun. 2003, 1766–1767. (42) Wijesundara, M. B. J.; Fuoco, E.; Hanley, L. Langmuir 2001, 17, 5721– 5726. (43) Kim, M. S.; seo, K. S.; Khang, G.; Lee, H. B. Langmuir 2005, 21, 4066– 4070.
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Figure 4. (a) Selected fluorescent images obtained from 0 to 75 mm along SAV-biotin PMMA slide after SAV Texas red immobilization. The scale bar is 500 μm. (b) Plot of fluorescence intensity of the SAV-Texas-red-modified gradient versus length (n = 4).
sample length will be a consequence only of the chemical modifications introduced. Moreover, low slope gradient surfaces allow for more accurate biomolecule concentration screening. Hydrolysis, in comparison with more efficient methods such as corona discharge19,20 and radio frequency plasma discharge18 treatments, led to the selective formation of carboxylic acid groups on the surface rather than a complex mixture of chemically active functionalities. This ensures that all of the reacted groups on the surface will be able to be functionalized in subsequent steps. Carboxylic acid groups on the hydrolyzed PMMA surface were then derivatized with biotin and fluorescently labeled SAV as depicted in Scheme 1. The interaction of avidin, or related proteins such SAV, with biotin is the most commonly used bioaffinity immobilization approach. Biochemical affinity assays offer oriented homogeneous immobilization of proteins, preserving their structure after binding and thus providing an important advantage over other immobilization techniques.44 The binding of SAV Texas Red to the biotinylated PMMA gradient surfaces was characterized by fluorescence microscopy (Figure 4a). The fluorescence intensity gradually increased in the direction of the longer hydrolysis time, thus showing a gradient in the SAV concentration. Plotting the normalized total intensity of the fluorescence images along the sample length led to the gradient curve shown in Figure 4b. These results show that, in spite of the low hydrolysis reaction conversion values obtained, the protein gradient after functionalization can be well appreciated along the sample. To provide a quantitative measurement of the resultant SAV surface gradient, SPR was used to measure the surface density as a function of hydrolysis time. The schematic model represented in Figure 5a together with Fresnel’s equations were used to fit the experimental resonance curves obtained after SAV incubation. This model assumes that biotinylated PMMA can be treated as a single contributing layer because of the similarities between the refractive indexes of biotin compounds, for which a refractive index of n = 1.50 can be assumed,35 and PMMA, with n = 1.489. In this case, biotin thickness variations can be neglected because of the small contribution that they make to the total layer thickness, which consists mainly of PMMA. From Fresnel’s equations, we obtained the SAV layer thickness, and according to eq 1, SAV surface density A can be calculated for each hydrolysis time (Figure 5b). Assuming a theoretical thickness of d = 5 nm for SAV, the surface coverage can also be inferred (Figure 5c).35,37 In our case, (44) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775–1789.
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a maximum value of 39% was obtained for the most hydrolyzed part of the slide. This is a low surface coverage if we compare it to those reported in the literature, where protein surface coverages of 50-65% have been described for SAV bound to biotinylated SAMs.35,45,46 These results can be explained in terms of the efficiency of the hydrolysis step as noted in the XPS analysis. Nevertheless, this could be advantageous compared to the biotinylated SAMs in the sense that no steric hindrance effects are expected among biotin molecules that affect SAV recognition. If each of the self-assembling molecules carries a biotin moiety, then the recognition and binding of SAV will be severely limited in a densely packed monolayer because of the fact that biotinylated compounds literally block each other. Fitting the SAV surface density experimental data allows us to predict the SAV concentration at any point along the gradient distance (Figure 5b). Moreover, gradient quantification, assuming two free binding sites for each SAV molecule on the surface, permits us to estimate the surface density of any biotinylated compound attached to the designed SAV gradient platform. Proof-of-Concept Application: Biotin-Bradykinin Surface Gradients for Drug Screening Studies. Once produced and characterized, the SAV gradient surfaces were used to test the effects of the BK concentration on cells. BK-induced changes in cell morphology have been extensively studied in solution,29,31 with their effects on filopodia formation being reported to vary in a concentration-dependent manner.31 These alterations in cell morphology are attributed to an underlying cytoskeletal reorganization produced by the drug, and they can be studied by staining the cell actin cytoskeleton. However, the effects of the drug when it is immobilized on a surface and its dependence on the dose applied are largely unknown and constitute the object of this study. For this purpose, SAV gradient platforms were incubated with the biotinylated BK peptide, and NIH/3T3 fibroblasts were seeded onto the BK gradient surfaces at a cell density of 660 cells/ cm2 under serum starving conditions. After 90 min of incubation, the cells were fixed and imaged in phalloidin/Hoechst stain. As can be seen in Figure 6a, cells grown in regions of low BK concentration present a round morphology with peripheral actin microspikes (PAM) and actin staining appears to punctuate and diffuse. In contrast, when moving to areas of higher BK concentration, there is a reduction in the number of PAMs formed and an increase in the number of membrane ruffles and filopodia. To quantify these morphological changes, different form parameters were evaluated, including the solidity (SOL), cell area (Ac), cell perimeter (Pc), and form factor (FF). Among them, the clearest differences were detected for cell area and solidity. Figure 6b,c shows the plots of cell solidity and cell area as a function of distance along the slide. Both parameters show a progressive decrease in mean values with increasing BK concentration that corresponds to cells being less rounded and having an increasing number of filopodia (Figure 6a). Thus, these results indicate that the BK anchored to the engineered surface described here retains its functionality. This is in good agreement with the results published by Kozma et al. where the action of BK is related to the activation of a variety of cell surface receptors.31 Moreover, BK is not only shown to be active once immobilized but also the concentration range achieved with the gradient allows us to observe progressive changes in cell morphology (i.e., mean solidity values varying from 0.74 to 0.65 (Figure 6b)). In fact, from the SPR results, the BK concentration can be estimated to (45) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kabalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387–396. (46) Reiter, R.; Motschmann, H.; Knoll, W. Langmuir 1993, 9, 2430–2435.
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Figure 5. (a) Schematic representation of the model used in SPR measurements for determining SAV coverage. (b) SAV gradient quantification by SPR analysis. Plot of the amount of SAV adsorbed per area. (c) SAV surface coverage against distance, assuming a theoretical thickness of 5 nm.
Figure 6. Effect of BK concentration gradient on cell morphology. (a) Fluorescence microscopy images; overlay of NIH/3T3 fibroblasts stained for cell nuclei (blue) and actin filaments (red) after 90 min of incubation at 37 °C on the BK gradient surface (number of seeded cells = 660 cells/cm2, n = 3). Cell membrane constrictions and filopodia formation are more evident with increasing BK concentration. The scale bar is 50 μm. (b) Cell solidity and (c) cell area variation as a function of distance showing a progressive decrease with increasing BK concentration.
vary from 4.1 to 6.4 ng/cm2 along the sample length. These values are well above the reported BK effectiveness concentration in solution, which has been described to be as low as 10 ng/mL or, translated into surface density values, 1.9 ng/cm2.31 Some recent publications such as the work of Zelzer et al. have suggested that cell behavior (adhesion) on gradient samples and uniform surfaces cannot be directly compared.47 However, low (47) Zelzer, M.; Majani, R.; Bradley, J. W.; Rose, F. R. A. J.; Davies, M. C.; Alexander, M. R. Biomaterials 2008, 29, 172–184.
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slope gradients such as that described here with a BK variation of 0.023 ng/mm2 result in a constant local concentration at distances comparable to cell size. Thus, drug screening results derived from the gradient platform here described could be well correlated to individual surface concentration testing experiments.
Conclusions The production and characterization of a universal gradient platform on PMMA, based on the well-known molecular recognition between SAV and biotin and to which an unlimited variety Langmuir 2010, 26(17), 14154–14161
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of bioactive biotinylated species can be attached, have been described. The progressive hydrolysis of the methyl ester pending groups on PMMA allowed the formation of a selective, highly controllable, low-slope, narrow-concentration-range carboxylic acid gradient. After derivatization with biotin and SAV, SPR was used to find quantitative relationships between actual positions on the gradient surfaces and the SAV surface density. All together, these characteristics offer a versatile platform to which any biotinylated biomolecule can be attached with well-controlled density and orientation. Because the physical properties of PMMA are barely affected because of gradient formation, the surface activity can be attributed mostly to the chemical motifs introduced. Moreover, low-slope gradients allow a more accurate optimal concentration screening that is closer to in vivo conditions, where small variations in concentration could lead to a wide variety of different biological responses. To demonstrate the potential of the gradient platform created, it was applied to the dose-dependent drug screening of the effects of BK on cells. It was found that BK is active after immobilization on the gradient platform and also that the drug molecular surface density and gradient slope were appropriated for the study of dose-dependent effects between cell cytoskeleton alterations and BK concentration. Therefore, the streptavidin gradient platform reported here allows control of the type and degree of specific cell-material
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interactions across a synthetic surface, permitting the simple, high-throughput analysis of cell-biomaterial interactions and the surface screening of directed cell function in a potentially broad range of applications. Acknowledgment. AL. is grateful to the Centro de Investigacion Biomedica en Red en Bioingenierı´ a, Biomateriales y Nanomedicina (CIBER-BBN) for financial backing. E.M. is grateful to the Spanish Ministry of Science and Education for the provision of grants through the I3 system. We acknowledge financial support from the Spanish Ministerio de Ciencia e Innovaci on through the project entitled “Regenerative Stem Cell Therapies for Heart Failure” and from the Science Support Program of the Fundacion Marcelino Botı´ n. Surface characterization was possible thanks to the assistance of Dr. Lorenzo Calvo Barrio from the Surface Analysis Unit at the Serveis Cientı´ ficoTecnics de la Universitat de Barcelona (SCT-UB) and the Plataforma de Nanotecnologia del Parc Cientı´ fic de Barcelona (PCB). Supporting Information Available: A hydrolysis time versus sample distance plot and PMMA layer thickness optimization graph for SPR measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
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