Fibronectin terminated multilayer films: Protein adsorption and cell attachment studies
Introduction
Living cells receive important signals through biological recognition events at their surface. Biomaterials capable of mimicking these signals are potentially valuable as tissue-engineering substrates, biosensing surfaces, and drug delivery vehicles. Materials must be capable of signal transmission and also meet stringent structural, mechanical, and degradation requirements—a scenario best realized through a coating approach where bulk material and surface properties are effectively decoupled. Multilayer nanofilms, formed by the layer-by-layer (LbL) method [1], [2], [3], [4], [5], [6], [7], are promising in this regard. LbL assembly occurs through the alternate adsorption of positively and negatively charged polyelectrolytes, and the resultant multilayer films can be rendered bioactive through the adsorption of protein or other biological molecules capable of transmitting signals to contacting cells [8], [9], [10], [11], [12], [13]. Since only physical interactions are involved, LbL assembly represents a simple, controllable, and broadly applicable route to bioactive thin film materials.
Fibronectin (Fn) is an extracellular matrix protein known to promote cell attachment and spreading [14]. The mechanism is thought to involve attachment of α5β1 transmembrane integrin receptors to Fn's cell binding site, located on the 10th type-III repeat module and containing the specific amino acid sequence RGD, as well as to synergy sites located on the 8th and 9th type-III repeats. Thus, materials coated with Fn are promising for a variety of cell-contacting applications. Indeed, a number of studies attest to the enhanced cell attachment and spreading of surfaces coated with Fn, compared to identical materials in the absence of Fn [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36].
Despite the promise of multilayer films in cell-contacting applications, and the proven utility of Fn as a cell adhesion motif, only a few investigations of Fn-coated multilayer films have appeared [37], [38], [39], [40], [41]. Ai et al. showed cerebellar neurons to attach, grow, and differentiate on silicone coated with multilayer films consisting of three bilayers of poly(styrene sulfonate) (PSS)/poly(ethylene imine) (PEI) and four bilayers of Fn/poly(d-lysine) (PDL) [37]. Interestingly, similar cell behavior occurred on silicone coated with a monolayer of PDL without any Fn. Ngankam et al. analyzed Fn adsorption to poly(allylamine hydrochloride) (PAH)/PSS films and found the extent of adsorption to be significant: films of mass 1.1 and 0.5 μg/cm2 were observed on PAH and PSS terminated films, respectively, with considerable surface aggregation present in the latter system [38]. Kreke et al. investigated osteoblast adhesion strength, via a flow-induced detachment assay, onto Fn-coated PAH/heparin sulfate films formed at various values of solution pH [39]. Although Fn adsorption was modest (always less than 0.01 μg/cm2), they observed the force required for cell detachment to scale with quantity of adsorbed Fn. Li et al. found a Fn coating to enhance smooth muscle cell attachment and growth on a PAH/PSS film, and the degree of enhancement to increase with the number of layers [40]. In contrast, Olenych et al. found only a minor influence of adsorbed Fn on the spreading and motility of smooth muscle cells cultured on multilayer films ranging from cell adherent to cell resistant [41].
We investigate here the formation and cell attachment properties of Fn-terminated multilayer films composed of the linear polyelectrolytes poly(l-lysine) (PLL) and dextran sulfate (DS). Films composed of biological polymers, such as polyamino acids and polysaccharides, are ideal for in vivo applications (e.g. tissue engineering) owing to their biological origin and biodegradability. We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to measure the kinetics of LbL assembly and Fn adsorption, and phase contrast optical microscopy to analyze the attachment and spreading of human umbilical endothelial cells (HUVEC) in contact with multilayer films. Our goal is to understand the time-dependent morphological response of a model cell system—in contact with a multilayer film composed of polyelectrolytes of biological origin—in terms of number of polyelectrolyte layers, the charge of the terminal layer, and the presence of Fn.
Section snippets
OWLS
OWLS is a highly sensitive method (precision ∼1 ng/cm2) for measuring macromolecular adsorption kinetics at the solid–liquid interface [42], [43], [44], [45]. Detection is based on excitation of guided modes via a polarized laser light beam directed upon a grating coupler at the surface of an optical waveguide. The mass and thickness of an adsorbed layer can be related to changes in the guided modes through an optical model, such as one assuming an optically uniform adsorbed layer [46]. Solvent
Multilayer film formation
Fig. 2A shows OWLS and QCMD measurements of film mass versus time for the LbL growth of PLL–DS films, as described in Section 2. OWLS provides a measure of the polymer contribution to the film mass (i.e. the “dry mass”); the increase in dry mass is essentially irreversible during both PLL and DS adsorption steps, and plateau levels scale roughly exponentially with layer number. (Schaaf, Voegel, and co-workers have recently linked exponential film scaling to intra-film mobility of at least one
Discussion
We investigate the LbL film formation of a polyamino acid/polysaccharide multilayer film, the adsorption of the matrix protein Fn to films composed of various numbers of layers, and the time-dependent morphological changes of a model cell system (HUVEC) as functions of layer number, charge of terminal layer, and the presence of Fn. The PLL/DS multilayer film grows exponentially with layer number, probably due to the intra-film mobility of PLL, and contains a significant quantity of water, most
Conclusion
We investigate the LbL formation of a multilayer film composed of polyelectrolytes of biological origin; the adsorption of the matrix protein Fn to these films; and the morphological response of a model cell system in terms of number of polyelectrolyte layers, the charge of the terminal polyelectrolyte layer, and the presence of Fn. We find Fn to adsorb more strongly to (positively charged and less hydrated) polycation-terminated films, and for cells to generally spread—to a greater extent and
Acknowledgements
We gratefully acknowledge the National Institutes of Health for financial support through R01-EB00258.
References (85)
- et al.
Buildup of ultrathin multilayer films by a self-assembly process. 3. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces
Thin Solid Films
(1992) - et al.
Layer-by-layer assembled multicomposite films
Curr Opin Colloid Interface Sci
(1998) Recent explorations in electrostatic multilayer thin film assembly
Curr Opin Colloid Interface Sci
(1999)- et al.
Structure and function of fibronectin modules
Matrix Biol
(1996) - et al.
Adsorption of fibronectin and vitronectin onto Primaria(Tm) and tissue-culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial-cells and Bhk-21 fibroblasts
Biomaterials
(1995) - et al.
Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling
Biomaterials
(1999) - et al.
The effect of the surface modification of titanium using a recombinant fragment of fibronectin and vitronectin on cell behavior
Biomaterials
(2005) - et al.
Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries
Biomaterials
(2005) - et al.
Fibronectin's central cell-binding domain supports focal adhesion formation and rho signal transduction
J Biol Chem
(2005) - et al.
Effect of biomaterial surface properties on fibronectin-alpha(5)beta(1) integrin interaction and cellular attachment
Biomaterials
(2006)
Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons
J Neurosci Methods
Modulation of protein adsorption and cell adhesion by poly(allylamine hydrochloride) heparin films
Biomaterials
Integrated optical input grating couplers as biochemical sensors
Sensors Actuators
Output grating couplers on planar optical wave-guides as direct immunosensors
Biosensors Bioelectron
Optical grating coupler biosensors
Biomaterials
Frequency and dissipation-factor responses to localized liquid deposits on a QCM electrode
Sensors Actuators B Chem
Protein adsorption: kinetics and history dependence
J Colloid Interface Sci
Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification
Biomaterials
Endothelial cell—interactions with polyelectrolyte multilayer films
Biomaterials
Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films
Biomaterials
Adhesion of alpha(5)beta(1) receptors to biomimetic substrates constructed from peptide amphiphiles
Biomaterials
Fuzzy nanoassemblies: toward layered polymeric multicomposites
Science
Ultrathin polymer coatings by complexation of polyelectrolytes at interfaces: suitable materials, structure and properties
Macromol Rapid Commun
Form and function in multilayer assembly: new applications at the nanoscale
Adv Mater
Peptide hormone covalently bound to polyelectrolytes and embedded into multilayer architectures conserving full biological activity
Biomacromolecules
Bioactive coatings based on a polyelectrolyte multilayer architecture functionalized by embedded proteins
Adv Mater
Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces
Langmuir
Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping
Langmuir
Primary cell adhesion on RGD-functionalized and covalently crosslinked thin polyelectrolyte multilayer films
Adv Funct Mater
Short-time timing of the biological activity of functionalized polyelectrolyte multilayers
Adv Funct Mater
Effect of the conformation and orientation of adsorbed fibronectin on endothelial-cell spreading and the strength of adhesion
J Biomed Mater Res
Correlation between corneal epithelial-cell outgrowth and monoclonal-antibody binding to the cell-binding domain of adsorbed fibronectin
J Biomed Mater Res
Cell adhesion strength increases linearly with adsorbed fibronectin surface density
Tissue Eng
Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion
J Biomed Mater Res
The role of fibronectin in platelet adhesion to plasma preadsorbed polystyrene
J Biomater Sci Polym Ed
Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion
J Biomed Mater Res Part A
Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates
J Biomed Mater Res Part A
Control of intracellular signaling by modulation of fibronectin conformation at the cell-materials interface
Langmuir
Fibronectin anchorage to polymer substrates controls the initial phase of endothelial cell adhesion
J Biomed Mater Res Part A
ICAM-1 and VCAM-1 expression by endothelial cells grown on fibronectin-coated TCPS and PS
J Biomed Mater Res Part A
Fibronectin adsorption on surface-activated poly(dimethylsiloxane) and its effect on cellular function
J Biomed Mater Res Part A
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