pubs.acs.org/Langmuir © 2010 American Chemical Society
Nanofilm Biomaterials: Localized Cross-Linking To Optimize Mechanical Rigidity and Bioactivity Jennifer A. Phelps,† Samuel Morisse,†,‡ Mathilde Hindie,‡ Marie-Christelle Degat,‡ Emmanuel Pauthe,†,‡ and Paul R. Van Tassel*,† †
Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States, and ‡ERRMECe, Universit e de Cergy-Pontoise, Cergy-Pontoise Cedex 95302, France Received October 15, 2010. Revised Manuscript Received December 1, 2010
Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework;the standard means of increasing a film’s rigidity;can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and bioactive.
I. Introduction Layer-by-layer (LbL) assembly of charged macromolecules is a popular technique for generating nanoscale films for a variety of cell-contacting applications, including sensing, biocatalysis, medical diagnostics, and tissue engineering.1-3 LbL films are easy to fabricate on a variety of flat or irregularly shaped objects (only simple solution exposures are required), are quite conformal even on the nanoscale, are amenable to fine control over physicochemical properties (through choice of polymers, solution conditions, and postformation steps), and allow for the facile immobilization of bioactive species (e.g., proteins, peptides, and nucleic acids).
*To whom correspondence should be addressed. (1) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Biomedical applications of layer-by-layer assembly: From biomimetics to tissue engineering. Adv. Mater. 2006, 18(24), 3203–3224. (2) Picart, C. Polyelectrolyte multilayer films: From physico-chemical properties to the control of cellular processes. Curr. Med. Chem. 2008, 15(7), 685–697. (3) Boudou, T.; Crouzier, T.; Ren, K. F.; Blin, G.; Picart, C. Multiple Functionalities of Polyelectrolyte Multilayer Films: New Biomedical Applications. Adv. Mater. 2010, 22(4), 441–467. (4) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 2003, 4(1), 96–106. (5) Berg, M. C.; Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces. Langmuir 2004, 20(4), 1362–1368. (6) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 2004, 5(2), 284–294. (7) Picart, C.; Elkaim, R.; Richert, L.; Audoin, T.; Arntz, Y.; Cardoso, M. D.; Schaaf, P.; Voegel, J. C.; Frisch, B. Primary cell adhesion on RGD-functionalized and covalently crosslinked thin polyelectrolyte multilayer films. Adv. Funct. Mater. 2005, 15(1), 83–94.
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Mechanical rigidity and bioactivity are two key film properties known to significantly influence the behavior of contacting cells.4-20 Cells tend to attach more efficiently to films of high mechanical (8) Schneider, A.; Francius, G.; Obeid, R.; Schwinte, P.; Hemmerle, J.; Frisch, B.; Schaaf, P.; Voegel, J. C.; Senger, B.; Picart, C. Polyelectrolyte multilayers with a tunable Young’s modulus: Influence of film stiffness on cell adhesion. Langmuir 2006, 22(3), 1193–1200. (9) Schneider, A.; Richert, L.; Francius, G.; Voegel, J. C.; Picart, C. Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films. Biomed. Mater. 2007, 2(1), S45–S51. (10) Wittmer, C. R.; Phelps, J. A.; Saltzman, W. M.; Van Tassel, P. R. Fibronectin terminated multilayer films: protein adsorption and cell attachment studies. Biomaterials 2007, 28, 851–860. (11) Wittmer, C. R.; Phelps, J. A.; Lepus, C. M.; Saltzman, W. M.; Harding, M. J.; Van Tassel, P. R. Multilayer nanofilms as substrates for hepatocellular applications. Biomaterials 2008, 29(30), 4082–4090. (12) Ren, K. F.; Crouzier, T.; Roy, C.; Picart, C. Polyelectrolyte multilayer films of controlled stiffness modulate myoblast cell differentiation. Adv. Funct. Mater. 2008, 18(9), 1378–1389. (13) Berthelemy, N.; Kerdjoudj, H.; Gaucher, C.; Schaaf, P.; Stolz, J. F.; Lacolley, P.; Voegel, J. C.; Menu, P. Polyelectrolyte films boost progenitor cell differentiation into endothelium-like monolayers. Adv. Mater. 2008, 20(14), 2674. (14) Crouzier, T.; Ren, K.; Nicolas, C.; Roy, C.; Picart, C. Layer-By-Layer Films as a Biomimetic Reservoir for rhBMP-2 Delivery: Controlled Differentiation of Myoblasts to Osteoblasts. Small 2009, 5(5), 598–608. (15) Kirchhof, K.; Hristova, K.; Krasteva, N.; Altankov, G.; Groth, T. Multilayer coatings on biomaterials for control of MG-63 osteoblast adhesion and growth. J. Mater. Sci.: Mater. Med. 2009, 20(4), 897–907. (16) Niepel, M. S.; Peschel, D.; Sisquella, X.; Planell, J. A.; Groth, T. pH-dependent modulation of fibroblast adhesion on multilayers composed of poly(ethylene imine) and heparin. Biomaterials 2009, 30(28), 4939–4947. (17) Tsai, W. B.; Chen, R. P. Y.; Wei, K. L.; Chen, Y. R.; Liao, T. Y.; Liu, H. L.; Lai, J. Y. Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions. Acta Biomater. 2009, 5(9), 3467–3477. (18) Semenov, O. V.; Malek, A.; Bittermann, A. G.; Voros, J.; Zisch, A. H. Engineered Polyelectrolyte Multilayer Substrates for Adhesion, Proliferation, and Differentiation of Human Mesenchymal Stem Cells. Tissue Eng., Part A 2009, 15(10), 2977–2990.
Published on Web 12/23/2010
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rigidity, as quantified e.g. by viscoelastic moduli.4,6-9,11,12,18,20 Bioactive films may be achieved through the incorporation of proteins, peptides, or drugs within the film architecture.14,21-29 These bioactive species may serve to enhance cell proliferation or differentiation or induce other types of cellular activity such as hormone or cytokine production. A key challenge is to realize LbL films that are simultaneously mechanically rigid and bioactive. The principal means of increasing a film’s mechanical rigidity, chemical cross-linking of the polymer framework following film formation,6 can significantly impair film bioactivity by (i) diminishing the mobility of bioactive species within the film, thereby inhibiting their interaction with contacting cells,23,26 (ii) suppressing cell-initiated enzymatic film degradation and the concomitant increase in cell access to bioactive species,30,31 and/or (iii) chemically altering embedded biomolecules, affecting their activity.29 We investigate here a strategy to decouple mechanical rigidity and bioactivity, thus potentially enabling LbL films that are both mechanically rigid and bioactive. The idea is to confine chemical cross-linking to the outer region of the film, thus yielding a film with a mechanically rigid outer crust to promote cell attachment and a softer, unperturbed interior to promote bioactive species stability and accessibility to cells. We propose a strategy whereby an “activated” polymer, i.e., capable of forming covalent linkages to other polymers within the film, is added as the final layer (Figure 1). So long as the activated polymer does not penetrate too deeply before the cross-links are formed, then the crosslinking will be confined to the surface region. This approach differs fundamentally from standard LbL film cross-linking where polymers are activated only once inside the film and where cross-linking occurs fairly uniformly throughout the film. (19) Seo, J.; Lee, H.; Jeon, J.; Jang, Y.; Kim, R.; Char, K.; Nam, J. M. Tunable Layer-by-Layer Polyelectrolyte Platforms for Comparative Cell Assays. Biomacromolecules 2009, 10(8), 2254–2260. (20) Hillberg, A. L.; Holmes, C. A.; Tabrizian, M. Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films. Biomaterials 2009, 30(27), 4463–4470. (21) Lvov, Y.; Ariga, K.; Kunitake, T. Layer-by-Layer Assembly of Alternate Protein Polyion Ultrathin Films. Chem. Lett. 1994, No.12, 2323–2326. (22) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Assembly of Multicomponent Protein Films by Means of Electrostatic Layer-by-Layer Adsorption. J. Am. Chem. Soc. 1995, 117(22), 6117–6123. (23) Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J. C.; Ogier, J. Bioactive coatings based on a polyelectrolyte multilayer architecture functionalized by embedded proteins. Adv. Mater. 2003, 15(9), 692– 695. (24) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Bioactive coatings of endovascular stents based on polyelectrolyte multilayers. Biomacromolecules 2003, 4(6), 1564–1571. (25) Benkirane-Jessel, N.; Lavalle, P.; Meyer, F.; Audouin, F.; Frisch, B.; Schaaf, P.; Ogier, J.; Decher, G.; Voegel, J. C. Control of monocyte morphology on and response to model surfaces for implants equipped with anti-inflammatory agents. Adv. Mater. 2004, 16(17), 1507. (26) Benkirane-Jessel, N.; Lavalle, P.; Hubsch, E.; Holl, V.; Senger, B.; Haikel, Y.; Voegel, J. C.; Ogier, J.; Schaaf, P. Short-time timing of the biological activity of functionalized polyelectrolyte multilayers. Adv. Funct. Mater. 2005, 15(4), 648– 654. (27) Jessel, N.; Oulad-Abdeighani, M.; Meyer, F.; Lavalle, P.; Haikel, Y.; Schaaf, P.; Voegel, J. C. Multiple and time-scheduled in situ DNA delivery mediated by beta-cyclodextrin embedded in a polyelectrolyte multilayer. Proc. Natl. Acad. Sci. U.S.A. 2006, 103(23), 8618–8621. (28) Vodouhe, C.; Le Guen, E.; Garza, J. M.; Francius, G.; Dejugnat, C.; Ogier, J.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Control of drug accessibility on functional polyelectrolyte multilayer films. Biomaterials 2006, 27(22), 4149–4156. (29) Schneider, A.; Vodouhe, C.; Richert, L.; Francius, G.; Le Guen, E.; Schaaf, P.; Voegel, J. C.; Frisch, B.; Picart, C. Multifunctional polyelectrolyte multilayer films: Combining mechanical resistance, biodegradability, and bioactivity. Biomacromolecules 2007, 8(1), 139–145. (30) Picart, C.; Schneider, A.; Etienne, O.; Mutterer, J.; Schaaf, P.; Egles, C.; Jessel, N.; Voegel, J. C. Controlled degradability of polysaccharide multilayer films in vitro and in vivo. Adv. Funct. Mater. 2005, 15(11), 1771–1780. (31) Etienne, O.; Schneider, A.; Taddei, C.; Richert, L.; Schaaf, P.; Voegel, J. C.; Egles, C.; Picart, C. Degradability of polysaccharides multilayer films in the oral environment: an in vitro and in vivo study. Biomacromolecules 2005, 6(2), 726–733.
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Figure 1. Schematic of the surface cross-linking idea explored here. In standard cross-linking, a film is exposed to chemical cross-linking agents following layer-by-layer (LbL) assembly, and cross-links form more-or-less uniformly throughout the film. Standard cross-linking generally increases film rigidity, and hence cell attachment, but can suppress bioactivity owing to deactivation or immobilization of embedded biomolecules or inhibition of film biodegradation. In surface cross-linking, chemical cross-linking agents are covalently attached to the terminal-layer polymer prior to its addition to the film. So long as the rate of cross-link formation (with other previously placed polymers) exceeds that of the terminal-layer polymer intrafilm diffusion, cross-links should remain near the outer region of the film. Surface cross-linking yields a mechanically rigid outer skin, to support cell attachment, but leaves the film interior unperturbed, to promote access to bioactive agents.
We employ the polypeptides poly(L-lysine) (PLL) and poly(Lglutamic acid) (PGA) as LbL film constituents; introduce a surface cross-linking strategy in which the terminal film layer is formed by PGA activated via N-hydroxysulfosuccinimide (sulfoNHS); characterize film assembly and cross-linking via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), and laser scanning confocal microscopy (LSCM); and measure the subsequent attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. Our results suggest this strategy to succeed in confining cross-linking to the film surface and to result in increased cell attachment and metabolic activity (compared to native, non-cross-linked films).
II. Materials and Methods II.A. Methods. Quartz Crystal Microbalance with Dissipation Monitoring (QCMD). QCMD is based on a thin quartz crystal sandwiched between a pair of electrodes. The resonant frequency of the crystal, when excited by an ac voltage, depends on the total oscillating mass, including adsorbed macromolecular species and any trapped water. A “soft” (viscoelastic) adsorbed layer will dampen the crystal’s oscillation.32-35 The dissipation may be measured at the fundamental frequency (5 MHz), and at the first three overtone frequencies (n = 3, 5, and 7), and by applying a Voigt model of a viscoelastic medium;specifically, solving the wave (32) Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. QuartzCrystal Microbalance Setup for Frequency and Q-Factor Measurements in Gaseous and Liquid Environments. Rev. Sci. Instrum. 1995, 66(7), 3924–3930. (33) Rodahl, M.; Kasemo, B. Frequency and dissipation-factor responses to localized liquid deposits on a QCM electrode. Sens. Actuators, B 1996, 37(1-2), 111–116. (34) Rodahl, M.; Hook, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Simultaneous frequency and dissipation factor QCM measurements of biomolecular adsorption and cell adhesion. Faraday Discuss. 1997, 107, 229–246. (35) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic acoustic response of layered polymer films at fluid-solid interfaces: Continuum mechanics approach. Phys. Scr. 1999, 59(5), 391–396.
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equation for the shear displacement of the quartz element, a viscoelastic overlayer, and a purely viscous cover medium, with no-slip boundary conditions at the interfaces35;the mass, thickness, elastic shear modulus, and shear viscosity of the adhering film may be determined. (The characteristic shear wave penetration length, ca. 40 μm, is much larger than the film thicknesses investigated here.) Our QCMD instrument (D300, Q-Sense, Sweden) is composed of a parallel plate flow cell whose bottom surface is a QSX 303 Sensor Chip (Q-Sense), consisting of a planar SiO2 coating on a quartz crystal. Silica coated sensor crystals are chosen owing to their hydrophilicity and negative charge (of approximate density -6 μC/cm2 36).
Fourier Transform Infrared Spectroscopy in Attenuated Total Reflection Mode (FTIR-ATR). In situ FTIR spectroscopy in ATR mode is applied using a Bruker Tensor 27 FTIR, with a Pike Veemax II variable angle ATR and a zinc selenide (ZnSe) crystal. The ZnSe crystal has an index of refraction of 2.4 and, assuming a sample refractive index of 1.4, yields an approximate penetration depth of 1-3 μm for the 45° incident angle used here (i.e., much larger than the thicknesses of the films investigated here). To avoid spectral overlap between amide I and II peaks with the O-H bond peak of water, D2O is used as the solvent. Data acquisition involve 96 spectra taken at a 2 cm-1 resolution. A Savitsky-Golay curve smoothing filter of degree 9 is applied to all data.37 Laser Scanning Confocal Microscopy (LSCM). LSCM is an optical imaging technique employing point illumination and a spatial pinhole to eliminate out-of-focus light and enable planar cross-sectional resolution of biological or soft-matter samples. A Biorad model 1024 UV laser confocal microscope system is used, configured with both UV argon ion laser and visible krypton argon lasers and allowing for three-channel LSCM of most commonly used fluorophores.
II.B. Film Assembly and Cross-Linking.
Film Assembly.
A buffer solution consisting of 10 mM Bis-Tris and 150 mM NaCl in deionized H2O is adjusted to a pH of 6 with glacial acetic acid and degassed in an ultrasonic bath for 25 min. A QCMD sensor chip or a ZnSe ATR crystal is washed with 2% Hellmanex (Hellma, Mulheim, Germany), extensively rinsed with deionized water, and connected to a flow cell. A buffer solution is continuously introduced into the flow cell, at a flow rate of 80 μL/min, until a stable baseline is achieved. A 0.4 g/L solution of poly(Llysine) (PLL, Sigma, 70-150 kDa) in buffer is then introduced under flow for 15 min and followed by a buffer rinse under flow of 15 min. (15 min represents a time at least twice that required to reach an adsorption plateau, as shown in Figure 3.) Next, a 0.4 g/ L solution of poly(L-glutamic acid) (PGA, Sigma, 50-100 kDa) in buffer is introduced for 15 min, and a another buffer rinse of 15 min follows. Each subsequent layer is formed via a 15 min adsorption step and a 15 min rinsing step. Full-Film Cross-Linking. Following film formation, a 40 mM 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC)-100 mM N-hydroxysulfosuccinimide (sulfo-NHS) solution in buffer is introduced for 20 min under flow, and then the flow is stopped for 16 h. The system is then rinsed with buffer for 1 h. A schematic of the cross-linking chemistry is shown in Figure 2. Surface Cross-Linking. Activated PGA is obtained by reaction of 40 mM EDC, 100 mM sulfo-NHS with 0.4 g/L PGA for 30 min, followed by addition of 150 mM β-mercaptoethanol (to prevent any further reaction of the EDC). In activated PGA, some of the carboxyl groups are converted to reactive sulfo-NHS esters, capable of ultimately forming amide bonds with free amines (Figure 2). The resulting solution is immediately filtered through a buffer-equilibrated GE Health PD-10 desalting column to remove any unreacted EDC/sulfo-NHS and β-mercaptoethanol. For QCMD and FTIR-ATR experiments, the activated PGA is (36) Rodrigues, F. A.; Monteiro, P. J. M.; Sposito, G. Surface charge density of silica suspended in water-acetone mixtures. J. Colloid Interface Sci. 1999, 211(2), 408–409. (37) Savitzky, A.; Golay, M. J. E. Smoothing þ Differentiation of Data by Simplified Least Squares Procedures. Anal. Chem. 1964, 36(8), 1627.
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Figure 2. Schematic of the EDC/sulfo-NHS cross-linking reaction. A carboxyl group reacts with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) to form an unstable o-acylisourea ester (reaction 1), which then reacts with N-hydroxysulfosuccinimide (sulfoNHS) to form a semistable sulfo-NHS ester (reaction 2). This activated form may then react with a free amine to form an amide cross-link (reaction 3). introduced to a PLL-terminated film for 25 min, the flow is stopped for 16 h, and the system is rinsed with buffer for 25 min. Fluorescence Labeling. Two mL of 4 g/L PLL, 0.1 M NaHCO3 (pH 8.3) is mixed with 10 μL of 10 g/L Alexa Fluor 568 carboxylic acid succinimidyl ester (Invitrogen) in DMSO at 25 °C for 2 h in the dark. AF568-labeled PLL is purified twice using dialysis tubing (D9277, 100 ft, Aldrich) for 12 h in the dark in 0.1 M NaHCO3. Two mL of 4 g/L PGA (either activated, as described above, or native), 0.1 M 2-(N-morpholino)ethanesulfonic acid, and 0.5 M NaCl (pH 5.9) is mixed with 10 μL of 10 g/L Alexa Fluor 488 hydrazide sodium salt (Invitrogen) in DMSO at 25 °C for 2 h in the dark. AF488-labeled PGA is purified twice using dialysis tubing (D9277, 100 ft, Aldrich) for 12 h in the dark in 0.1 M 2-(N-morpholino)ethanesulfonic acid. Purified samples (>99% labeled) are stored at 4 °C in the dark. Samples for LSCM are prepared by dipping #1.5 coverslips into (unlabeled) PLL, PGA, and buffer solutions for the first n - 2 layers, then dipping into a labeled PLL solution for layer n - 1, and finally dipping into a labeled, activated PGA solution for layer n. Adsorption steps are of duration 15 (layers 1 to n - 1) or 25 (layer n) minutes, and rinsing involves three 1 min dips in buffer.
II.C. Osteoblast Attachment and Metabolic Activity Assays. Murine preosteoblastic MC3T3-E1 cells, established as an undifferentiated osteoblastic cell line from normal mouse calvaria, are grown in alpha minimal medium (Invitrogen) supplemented with 10% (v/v) of decomplemented fetal bovine serum (PAA), glutamax (2 mM, Eurobio), penicillin (100 unit/mL, Eurobio), and streptomycin (100 μg/mL, Eurobio). Cells are cultured in 75 cm2 plastic culture flasks and incubated in a humidified incubator (37 °C and 5% CO2). After 3 or 4 days of culture, freshly confluent (or subconfluent) MC3T3-E1 preosteoblast cells are harvested with a trypsin-EDTA solution 1:1 (v/v) and resuspended in complete cell culture medium at 5000 cells/cm2 in a 48-well plate. After 3 and 96 h of culture, MC3T3-E1 preosteoblast cells are visualized using a light microscope (Leica) and optical micrographs are taken using a digital camera. Cells are counted (n = 2 per well), and each condition is averaged. The percentage of adherent cells is calculated in comparison to adherent cells onto cell culture plastic. At 3 and 6 days of culture, an Alamar blue assay is used to assess cell activity.38 Alamar blue is a reagent converted by metabolically active cells into a colorimetric indicator. This assay thus provides an important measure of cellular metabolic activity. In brief, the culture (38) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J. Immunol. Methods 1997, 204(2), 205–208.
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Figure 3. Film thickness (a), elastic shear modulus (b), and shear viscosity (c) versus time during the layer-by-layer assembly of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA), as measured via QCMD. In the case of the surface cross-link film, the 10th layer is composed of sulfoNHS activated PGA, capable of forming chemical cross-links with free amines within the film. In the case of the full cross-link film, after 10 layers, the film is exposed to an EDC-NHS cross-linking solution. medium is aspirated at the desired time, and 200 μL of fresh and complete medium containing 10% v/v Alamar blue (Invitrogen) is added to all wells. Reagent blanks are included as well. Plates are returned to the incubator for 3 h at 37 °C prior to measuring the absorbance at 570 and 600 nm using a spectrophotometric plate reader. Absorbance readings are converted to dye reduction % as per the provider’s instructions. The extent of dye reduction increases with cellular metabolic activity.
III. Results In Figure 3a, we show film thickness during LbL assembly, as determined via QCMD and a Voigt model35 of a viscoelastic film. 1126 DOI: 10.1021/la104156c
We observe an increase in film thickness upon each alternate exposure to PLL and PGA, indicating steady LbL growth. Buffer rinses separating the polyelectrolyte adsorption steps result in weak if any measurable desorption, suggesting the films to be quite stable. Results from two separate experiments are shown; these experiments are identical through the first nine layers. (As evidenced by the difference between the two curves through nine layers, the experimental variability is about 15%.) In one experiment, following 10 polymer layers, EDC/sulfo-NHS chemical cross-linking agents are introduced to the film. A 3-5 nm increase in film thickness results, or roughly 1/2 the thickness of the terminal polyelectrolyte Langmuir 2011, 27(3), 1123–1130
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Table 1. Thickness, Shear Elastic Modulus, and Shear Viscosity for Fully Cross-Linked (Full XL) and Surface Cross-Linked Films (Surface XL), at Various Stages of Assembly, for Two Repeat Experimentsa
experiment
film
thickness (nm)
full XL 1
viscosity (g/(m s))
9 layer 73 10 layer 84 XL 87 Δ (XL-9 layer) 14 Δ (XL-10 layer) 3 full XL 2 9 layer 70 10 layer 80 XL 85 Δ (XL-9 layer) 15 Δ (XL-10 layer) 5 surface XL 1 9 layer 64 10 layer (XL) 73 Δ (XL-9 layer) 9 surface XL 2 9 layer 64 10 layer (XL) 72 Δ (XL-9 layer) 8 a Property differences (Δ) between 10-layer 9- or 10-layer native films are also shown.
elastic modulus (kPa)
3.4 3.9 4.2 0.8 0.3 3.8 4.3 4.5 0.7 0.2 3.1 3.6 0.5 2.9 3.4 0.5 cross-linked
150 170 170 20 0 150 180 180 30 0 130 150 20 120 140 20 films and
layer (Table 1). A buffer rinse following 16 h of exposure to the cross-linking agents results in no detectable change in film thickness. In another experiment, following nine layers, a sulfoNHS-activated PGA layer (prepared as described in section II.B) is added. The thickness increase is quite similar that of a native PGA layer: 8-9 nm for activated versus 10-11 nm for native PGA (Table 1). In Figure 3b,c, we show the shear elastic modulus and shear viscosity during LbL assembly, as determined via QCMD and a Voigt model35 of a viscoelastic film. We observe incremental increases in both quantities during each polyelectrolyte adsorption step. (Experimental variability is again about 15%.) Addition of EDC/sulfo-NHS cross-linking agents causes an increase in viscosity of 0.2-0.3 g/(m s) or somewhat less than the 0.5 g/(m s) increase associated with addition of the last layer (Table 1). Interestingly, addition of the cross-linking agents revealed no measurable increase in shear elastic modulus. Previous studies have also report increased mechanical moduli upon chemical cross-linking.6,7,11,12 Upon addition of a sulfo-NHS-activated PGA to a nine-layer film, we observe 0.5 g/(m s) and 20 kPa increases in viscosity and shear elastic modulus, respectively (Table 1). However, these increases are no larger than those measured upon addition of a native PGA layer. We elaborate on this unexpected result in the Discussion section. In Figure 4, we show FTIR-ATR difference spectra. The difference spectrum between a 10- and 9-layer film serves as a control and demonstrates the incremental IR absorption due to the terminal (PGA) layer. Also shown is the difference spectrum upon placing a terminal layer of sulfo-NHS activated PGA onto a 9-layer film. We note considerable IR absorption over a range of frequencies, compared to the control, and in particular within the amide I and amide II zones (known to be sensitive to the presence of amide bonds).8 Finally, we show a difference spectrum between a 10-layer fully cross-linked film (i.e., subsequently exposed to cross-linking agents, as described in section II.C) and a 9-layer film. Here as well, strong IR absorbance results over a range of frequencies and in particular within the amide I and amide II zones. These difference spectra demonstrate the formation of amide bonds, between free amine and carboxyl groups within the film, upon exposure to standard EDC/sulfo-NHS components and to PGA preactivated with sulfo-NHS. Langmuir 2011, 27(3), 1123–1130
Figure 4. Infrared difference spectra of 10-layer fully cross-linked, surface cross-linked, and native PLL-PGA films. Difference spectra correspond to infrared absorbance minus that of a 9-layer native film.
In Figure 5, we show cross sections of 60-layer films imaged using LSCM. In both cases, the 59th layer is formed from AF568 (red)labeled PLL, and the 60th (and terminal) layer is formed via AF488 (green)-labeled PGA. In one case, the final PGA layer is activated so as to form surface cross-links, and in the other case, native (labeled) PGA is used to form the terminal layer. LSCM images reveal films of thickness ca. 10 μm. In both cases, the labeled PLL is seen to diffuse throughout the film, as observed previously.39 When the terminal layer is composed of activated PGA, we find the green fluorescence to remain principally at the outer region of the film, suggesting the cross-links to remain localized also in this region. (Owing to the optical diffraction limit, it is not possible to measure with precision the width of the zone containing the green-labeled PGA.) In the case of a native PGA terminal layer, we find green fluorescence throughout the film, suggesting the native PGA to diffuse throughout the film (as observed previously39). (Parts of the film where red-labeled PLL and green-labeled PGA coexist appear yellow.) To investigate the influence of surface cross-linked films on contacting cells, we consider the attachment, proliferation, and metabolic activity of MC3T3-E1 preosteoblasts on native films, fully cross-linked films, and surface cross-linked films. In Figure 6, we show optical micrographs following 3 h and 4 days of cell culture. At 3 h, the percent of adherent cells is significantly enhanced from surface cross-linked versus native films and also fully cross-linked versus surface cross-linked films (p < 0.05). In addition, the total cell population at 4 days ranks native film < surface cross-linked film < fully cross-linked film (p < 0.05). These results indicate surface crosslinked films to be intermediate, in terms of cell attachment, between native and fully cross-linked films. In Figure 7, we show results of a cell metabolic assay. At 3 days culture, no differences are noted. However, at 6 days, the overall metabolic activity of cells on fully and surface cross-linked films are significantly increased (p < 0.05) compared to those on native films. Hence, cell activity appears to be comparable on fully and surface cross-linked films and in both cases greater than on native films.
IV. Discussion EDC-NHS chemistry (Figure 2) allows for facile covalent coupling between carboxyl and amine functional groups, as found in the polyelectrolytes used here for LbL assembly.40 Several groups have used this method to increase the mechanical rigidity of LbL films,6-9,11-13,29,41 with typical Young’s modulus values (39) Lavalle, P.; Vivet, V.; Jessel, N.; Decher, G.; Voegel, J. C.; Mesini, P. J.; Schaaf, P. Direct evidence for vertical diffusion and exchange processes of polyanions and polycations in polyelectrolyte multilayer films. Macromolecules 2004, 37(3), 1159–1162. (40) Grabarek, Z.; Gergely, J. Zero-Length Crosslinking Procedure with the Use of Active Esters. Anal. Biochem. 1990, 185(1), 131–135. (41) Richert, L.; Engler, A. J.; Discher, D. E.; Picart, C. Elasticity of native and cross-linked polyelectrolyte multilayer films. Biomacromolecules 2004, 5(5), 1908– 1916.
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Figure 5. Laser scanning confocal microscopy images of 60-layer native and surface cross-linked PLL-PGA films. The 59th layer contains AF568 (red)-labeled PLL, and the 60th (and terminal) layer is formed via AF488 (green)-labeled PGA. The labeled PLL diffuses freely throughout the film. In the surface cross-linked film, the sulfo-NHS-activated PGA remains near the outer surface, while in the native film, the PGA penetrates throughout the film (orange color indicates the presence of both green and red labels). The arrow is of length 10 μm.
Figure 6. Left: optical micrographs of MC3T3-E1 preosteoblast cells following 3 and 96 h culture on native, surface cross-linked, and fully cross-linked PLL-PGA films. Right: percent adhered cells following 3 h and average number of cells per field following 3 and 96 h.
for native and cross-linked films being 3-10 and 100-400 kPa, respectively.8,41,42 In these previous works, cross-linking occurs more or less uniformly throughout the film. The problem with uniform cross-linking is that embedded bioactive species may be deactivated or rendered inaccessible to contacting cells. We propose here the idea to confine cross-linking to the region of the film near to the surface, the hypothesis being that contacting cells will attach strongly to the rigid surface, while bioactive species will be accessible via diffusion to the cell-film interface and/or increased rate of cell-induced film degradation. Our approach involves placement of an activated polymer, i.e., containing covalently attached cross-linking agents, to the film’s terminal layer. Polymers are known to diffuse in the normal (42) Francius, G.; Hemmerle, J.; Ohayon, J.; Schaaf, P.; Voegel, J. C.; Picart, C.; Senger, B. Effect of crosslinking on the elasticity of polyelectrolyte multilayer films measured by colloidal probe AFM. Microsc. Res. Tech. 2006, 69(2), 84–92. (43) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc. Natl. Acad. Sci. U.S.A. 2002, 99(20), 12531–12535. (44) Lavalle, P.; Picart, C.; Mutterer, J.; Gergely, C.; Reiss, H.; Voegel, J. C.; Senger, B.; Schaaf, P. Modeling the buildup of polyelectrolyte multilayer films having exponential growth. J. Phys. Chem. B 2004, 108(2), 635–648. (45) Yoo, P. J.; Zacharia, N. S.; Doh, J.; Nam, K. T.; Belcher, A. M.; Hammond, P. T. Controlling surface mobility in interdiflusing polyelectrolyte multilayers. ACS Nano 2008, 2(3), 561–571.
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Figure 7. Alamar blue assay of 3- and 6-day MC3T3-E1 preosteoblast metabolic activity on native, fully cross-linked, and surface cross-linked PLL-PGA films.
direction within certain LbL films but to remain confined to individual layers (over typical experimental time scales) in others.43-45 The key assumption here is that the polymer in the terminal layer will diffuse relatively slowly compared to the rate of amide bond formation (the latter characterized by a time Langmuir 2011, 27(3), 1123–1130
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scale of ca. 4 min46), thus resulting in cross-links being confined to the outer region of the film. We demonstrate here the formation of amide cross-links during addition of the activated polymer layer (by FTIR-ATR), localization of the activated polymeric species near to the film surface (by LSCM), and enhanced cell attachment and metabolic activity on surface cross-linked films compared to native films. An interesting question is the number and location of the crosslinks formed by the surface cross-linking procedure. FTIR-ATR spectroscopy clearly indicates amide bond formation, but quantitative interpretation of these data is difficult. As an evanescent wave method, it may be possible to employ variable incident angle FTIR-ATR spectroscopy to probe the location of the cross-links. Specifically, an abrupt loss of signal strength may be apparent under a certain threshold evanescent wave depth. Here, we employ LSCM on thicker films to establish that covalent crosslinks occurs primarily in the surface region. However, owing to the optical diffraction limit, it is not possible to resolve the location of the cross-links beyond about 1 μm. We employ here quartz crystal microbalance with dissipation monitoring to measure the mechanical properties of the polymer film. A quartz crystal vibrates at a resonance frequency when subjected to an ac field, and the amount of adsorbed mass scales, to a first approximation, linearly with the change in resonance frequency. In order to obtain information on viscoelastic properties, resonance frequency and energy dissipation data (at the first few overtones) may be used to fit the wave equation describing the shear displacement of the quartz element, a viscoelastic overlayer, and a purely viscous cover medium, with no-slip boundary conditions at the interfaces.35 Film thickness, shear elastic modulus and shear viscosity are the fitting parameters, and they provide important continuous data on film assembly. We find here two unexpected results. First, the viscoelastic moduli exhibit a step-by-step increase similar to that of the film thickness, whereas one would expect these properties to plateau (or perhaps oscillate) following the first few layers. This observation may indicate significant structural changes during the growth of the somewhat thin films analyzed here, as observed previously via AFM.47 Alternatively, there may exist a mechanical modulithickness coupling in this type of data analysis that lacks a physical basis; future work toward better understanding, and possibly improving, this modeling method within the context of LbL films is clearly needed. Second, chemical cross-linking results in only a modest change in the QCMD-calculated mechanical moduli. Introduction of EDC-NHS cross-linking agents yields no measurable change in elastic modulus and only a minor increase in viscosity, and introduction of activated PGA yields increases no bigger than those observed with native PGA. (In all cases, FTIR-ATR data confirm chemical cross-link formation.) Previous full film cross-linking has been shown to yield >10-fold increases in Young’s (i.e., compressive) modulus,8,41,42 so one possibility is that film rigidity is highly anisotropic (e.g., as might occur if full film cross-linking occurred preferentially near the film surface). Also, the wave equation analysis employed here results in the complex shear modulus μ and the density F appearing together as the quantity μ/F. F is fixed during model fitting, but its value may be an underestimate during cross-linking;due to loss of water from the film associated with increased film hydrophobicity;and thus (46) Lomant, A. J.; Fairbanks, G. Chemical Probes of Extended Biological Structures - Synthesis and Properties of Cleavable Protein Cross-Linking Reagent [Dithiobis(Succinimidyl-S-35 Propionate). J. Mol. Biol. 1976, 104(1), 243–261. (47) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Buildup mechanism for poly(L-lysine)/hyaluronic acid films onto a solid surface. Langmuir 2001, 17(23), 7414–7424.
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result in an underestimate of μ as well. Finally, the film thicknessmechanical moduli coupling issue discussed above could be at play here as well. A few other groups have also employed this method of data analysis. Picart et al. report a shear viscosity of PLL/hyaluronic acid films to be of order 100 g/(m s) for a 1 μm thick film.47 This value is considerably larger than values reported here in Table 1 and Figure 3, possibly owing to the thickness of their film (again, touching on the thickness-rigidity issue). Tang et al. report the viscoelastic moduli of 16-layer films composed of poly(acrylic acid) modified so as assemble via click chemical reactions.48 They find elastic modulus and viscosity to vary from 80 to 240 kPa and 2 to 6 g/(m s) upon pH changes from 3.1 and 6.6 and attribute the change to film swelling at higher pH. These values are quite similar in magnitude to those presented here and further illustrate the coupling that may occur between mechanical moduli and film thickness. Another quartz crystal-based method has also been reported, where the local complex shear impedance of the crystal-film interface;defined as the magnitude of the shear stress divided by the magnitude of the shear velocity;is determined from the complex ac impedance of the quartz crystal and related to adsorbed layer viscoelastic properties.49 Galeska et al. use this method and report the shear elastic modulus of a humic acid/ferric ion film to increase linearly with deposition step, with a value of 80 MPa for a 10-layer film.50 This represents a very rigid film (3 orders of magnitude more so than our own!), possibly due to significant film compaction with the small trivalent ferric ions. Calvo et al. report shear elastic modulus and shear viscosity of ca. 1 MPa and 10 g/ (m s), respectively, for films formed by the LbL assembly of glucose oxidase and poly(allylamine) coupled to an osmium complex.51 Salomaki et al. report an elastic shear modulus of 132 MPa for 500 nm thick poly(diallyldimethylammonium chloride)/poly(styrenesulfonate) film52 and 100 kPa for a 100-layer poly(L-lysine)/hyaluronic acid film (the latter being within the range reported here).53 Kujawa et al. report shear elastic moduli of 1000 and 200 kPa, respectively, for LbL films composed of hyaluronic acid/chitosan and hyaluronic acid/phosphorylcholine-modified chitosan and with little variation on film thickness.54 Salomaki et al. show the shear elastic modulus of hyaluronic acid/chitosan films increases from 1.4 to 156 MPa when every other hyaluronic acid layer is substituted with poly(acrylic acid).55 Guzman et al. report the elastic shear modulus of 12-layer poly(diallyldimethylammonium chloride)/ poly(styrenesulfonate) films to be 5 and 15 MPa for films formed (48) Tang, Y. C.; Liu, G. M.; Yu, C. Q.; Wei, X. L.; Zhang, G. Z. Chemical oscillation induced periodic swelling and shrinking of a polymeric multilayer investigated with a quartz crystal microbalance. Langmuir 2008, 24(16), 8929– 8933. (49) Kankare, J. Sauerbrey equation of quartz crystal microbalance in liquid medium. Langmuir 2002, 18(18), 7092–7094. (50) Galeska, I.; Hickey, T.; Moussy, F.; Kreutzer, D.; Papadimitrakopoulos, F. Characterization and biocompatibility studies of novel humic acids based films as membrane material for an implantable glucose sensor. Biomacromolecules 2001, 2(4), 1249–1255. (51) Calvo, E. J.; Forzani, E. S.; Otero, M. Study of layer-by-layer selfassembled viscoelastic films on thickness-shear mode resonator surfaces. Anal. Chem. 2002, 74(14), 3281–3289. (52) Salomaki, M.; Loikas, K.; Kankare, J. Effect of polyelectrolyte multilayers on the response of a quartz crystal microbalance. Anal. Chem. 2003, 75(21), 5895– 5904. (53) Salomaki, M.; Kankare, J. Modeling the growth processes of polyelectrolyte multilayers using a quartz crystal resonator. J. Phys. Chem. B 2007, 111(29), 8509–8519. (54) Kujawa, P.; Schmauch, G.; Viitala, T.; Badia, A.; Winnik, F. M. Construction of viscoelastic biocompatible films via the layer-by-layer assembly of hyaluronan and phosphorylcholine-modified chitosan. Biomacromolecules 2007, 8(10), 3169–3176. (55) Salomaki, M.; Kankare, J. Influence of Synthetic Polyelectrolytes on the Growth and Properties of Hyaluronan-Chitosen Multilayers. Biomacromolecules 2009, 10(2), 294–301.
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at 0.1 and 0.3 M NaCl solutions, respectively.56,57 Taken together, these results suggest the PLL-PGA films described herein to be on the softer side of polyelectrolyte-based LbL films. Also of note is that the shear impedance method appears to be less susceptible to the rigidity-thickness coupling issues plaguing the energy dissipation method (i.e., the method used use). Surface cross-linked films could find application in tissue engineering, where biomaterials of optimal mechanical and chemical properties are needed to promote cell attachment, proliferation, and differentiation. Bone tissue engineering is a particularly apt example, where both mechanical rigidity and bioactivity are of key importance: recent efforts demonstrate the significant influence of mechanical stress and strain on the development of bone and its remodeling58 and the sensitivity of osteoblastic precursors (MC3T3-E1 cells) to adsorbed proteins,59 growth factors,60 and mechanical stress.20,61 Our studies confirm (56) Guzman, E.; Ritacco, H.; Rubio, J. E. F.; Rubio, R. G.; Ortega, F. Saltinduced changes in the growth of polyelectrolyte layers of poly(diallyl-dimethylammonium chloride) and poly(4-styrene sulfonate of sodium). Soft Matter 2009, 5(10), 2130–2142. (57) Guzman, E.; Ritacco, H.; Ortega, F.; Svitova, T.; Radke, C. J.; Rubio, R. G. Adsorption Kinetics and Mechanical Properties of Ultrathin Polyelectrolyte Multilayers: Liquid-Supported versus Solid-Supported Films. J. Phys. Chem. B 2009, 113(20), 7128–7137. (58) Sikavitsas, V. I.; Temenoff, J. S.; Mikos, A. G. Biomaterials and bone mechanotransduction. Biomaterials 2001, 22(19), 2581–2593. (59) Hindie, M.; Degat, M. C.; Gaudiere, F.; Gallet, O.; Van Tassel, P. R.; Pauthe, E. Pre-osteoblasts on poly(L-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption. Acta Biomater. 2011, 7(1), 387–394. (60) Macdonald, M. L.; Rodriguez, N. M.; Shah, N. J.; Hammond, P. T. Characterization of Tunable FGF-2 Releasing Polyelectrolyte Multilayers. Biomacromolecules 2010, 11(8), 2053–2059. (61) Khatiwala, C. B.; Peyton, S. R.; Putnam, A. J. Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells. Am. J. Physiol.: Cell Physiol. 2006, 290(6), C1640–C1650.
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the expected increased attachment, proliferation, and metabolic activity on cross-linked versus native films. In terms of cell attachment and proliferation, we observe surface cross-linked films to perform intermediate between native and fully crosslinked films. In terms of metabolic activity, we observe surface cross-linked films to perform comparably to fully cross-linked films. Perhaps further improvements are possible through a thicker cross-linked “skin” composed of two or more cross-linked layers instead of the one layer proposed here. The benefits in terms of film rigidity would have to be balanced against any suppressed bioactivity of embedded biological species; refining the current strategy toward an optimal degree of surface cross-linking will be the subject of future studies.
V. Conclusion We present a new method toward nanofilm biomaterials that are both mechanically rigid (to promote robust cell adhesion) and bioactive. Our strategy involves terminating the film;formed via layer-by-layer assembly of charged polymers;with an “activated” polymer capable of forming chemical cross-links with previously adsorbed polymers. We demonstrate chemical crosslinks to form and to remain localized toward the outer region of the film and cell attachment and metabolic activity to be significantly enhanced compared to native films. Thus, the current study demonstrates the potential usefulness of surface crosslinked films for cell contacting applications: cells activity is comparable to that on fully cross-linked films, yet the film interior remains soft so as (in principle) to promote cellular interactions with bioactive species. Future studies will investigate the influence of surface cross-linking on the bioactivity of films containing embedded biological agents.
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