Cyto-mechanoresponsive polyelectrolyte multilayer films

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion.     

Photopatterned nanoporosity in polyelectrolyte multilayer films

We report on spatial control of nanoporosity in polyelectrolyte multilayer (PEM) films using photopatterning and its effects on film optical and adsorption properties. Multilayers assembled from poly(acrylic acid-ran-vinylbenzyl acrylate) (PAArVBA), a photo-cross-linking polymer, and poly(allylamine hydrochloric acid) (PAH) were patterned using ultraviolet light followed by immersion in low pH and then neutral pH solutions to induce nanoporosity in unexposed regions. Model charged small molecules rhodamine B, fluorescein, and propidium iodide and the model protein albumin exhibit increased adsorption to nanoporous regions of patterned PEM films as shown by fluorescence microscopy and radiolabeling experiments. Films assembled with alternating stacks of PAH/poly(sodium-4-styrene sulfonate) (SPS), which do not become nanoporous, and stacks of PAH/PAArVBA were patterned to create nanoporous capillary channels. Interdigitated channels demonstrated simultaneous, separate wicking of dimethyl sulfoxide-solvated fluorescein and rhodamine B. In addition, these heterostack structures exhibited patternable Bragg reflectivity of greater than 25% due to refractive index differences between the nanoporous and nonporous stacks. Finally, the PEM assembly process coupled with photo-cross-linking was used to create films with two separate stacked reflective patterns with a doubling in reflectivity where patterns overlapped. The combined adsorptive and reflective properties of these films hold promise for applications in diagnostic arrays and therapeutics delivery.     

Cell surface engineering with polyelectrolyte multilayer thin films

Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.     

Fabrication of polyelectrolyte multilayer films comprising nanoblended layers

Polyelectrolyte multilayer thin films were prepared via the alternate deposition of poly(allylamine hydrochloride) (PAH) and a blend of poly(acrylic acid) (PAA) and poly(styrenesulfonate) (PSS). When the pH of the blend solution was 3.5, the presence of PAA in this solution significantly increased the total film thickness. With only 10 wt % PAA in the blend adsorption solution, a large increase in film thickness was observed (92 nm cf. 18 nm). It was also demonstrated that the total amount of PSS adsorbed was enhanced by the presence of PAA in the blend solution, showing that the blend solution composition influenced that of the multilayer films. Thin films prepared with nanoblended layers also showed improved pH stability, because they exhibited reduced film rearrangement upon exposure to acidic conditions (pH = 2.5).     

Amperometric uric acid sensors based on polyelectrolyte multilayer films

Uricase (UOx) and polyelectrolyte were used for preparation of a permselective multilayer film and enzyme multilayer films on a platinum (Pt) electrode, allowing the detection of uric acid amperometrically. The polyelectrolyte multilayer (PEM) film composed of poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) were prepared via layer-by-layer assembly on the electrode, functioning as H₂O₂-selective film. After deposition of the permselective film (PAA/PVS)₂PAA, UOx and PAA were deposited via layer-by-layer sequential deposition up to 10 UOx layers to prepare amperometric sensors for uric acid. Current response to uric acid was recorded at +0.6 V vs. Ag/AgCl to detect H₂O₂ produced from the enzyme reaction. The response current increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10⁻⁶-10⁻³ M. The response current and deposited amount of UOx were affected by deposition bath pH and the addition of salt. The deposition of PAA/UOx film prepared in 2 mg ml⁻¹ solution (pH 11) of PAA with NaCl (8 mg ml⁻¹) and 0.1 mg ml⁻¹ solution (pH 8.5) of UOx with borate (100 mM) resulted in an electrode which shows the largest response to uric acid. The response of the sensor to uric acid was decreased by 40% from the original activity after 30 days.     

Modulation of hemocompatibility of polysulfone by polyelectrolyte multilayer films

Polyelectrolyte multilayer (PEM) films have been recently applied to surface modification of biomaterials. Cellular interactions with PEM films consisted of weak polyelectrolytes are greatly affected by the conditions of polyelectrolyte deposition, such as pH of polyelectrolyte solution. Previous studies indicated that the adhesion of several types of mammalian cells to PAH/PAA multilayer films was hindered by low pH and high layer numbers. The objective of this study is to evaluate whether the hemocompatibility of polysulfone can be modulated by deposition of poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayer films. PAH/PAA multilayer films with different layer numbers were assembled onto polysulfone at either pH 2.0 or pH 6.5. The number of platelet adhesion and the morphology of adherent platelets were determined to evaluate hemocompatibility of modified substrates. Compared to non-treat polysulfone, the PEM films developed at pH 2.0 decreased platelet adhesion, while those built at pH 6.5 enhanced platelet deposition. Platelet adhesion was found positively correlated to polyclonal antibodies binding to surface-bound fibrinogen. The extent of platelet spreading was increased with layer numbers of PEM films, suggesting that the adherent platelets on thick PEM films were prone to activation. In conclusion, PAH/PAA films with few layers developed at pH 2.0 possessed better hemocompatibility compared to other substrates.     

Nanomechanical properties of polyaniline and azo polyelectrolyte multilayer films

<正>Nanomechanical properties of multilayer films constructed of polyaniline(PANI) and azobeneze-containing polyelectrolytes(PNACN and PPAPE) were studied by using nanoindentation method.The multilayer films were prepared by the electrostatic layer-by-layer self-assembly through alternately dipping in the polymer solutions.The multilayer films deposited onto the glass slides after proper dry were used for the nanomechanical property testing.The nanomechanical measurement indicated that the PANI/PNACN and PANI/PPAPE multilayers possessed the mean elastic modulus of 5.42 GPa and 4.35 GPa,and hardness of 0.26 GPa and 0.18 GPa,respectively.The nanoscratch properties of the PANI/PNACN and PANI/PPAPE multilayer films were also measured.The critical loads of PANI/PNACN and PANI/PPAPE films were 103.52 mN and 100.59 mN.The degree of electrostatic cross-linking in the multilayers could be altered by exposing the films to aqueous solutions with different pH values.As a result,the modulus and hardness of the multilayer films were changed through the solvent treatment.Both modulus and hardness of the PANI/PNACN films obviously increased after dipping the multilayer films in solutions with pH in a range from 9 to 11.     

Strong response of multilayer polyelectrolyte films to cationic surfactants

The influence of common cationic surfactants on the physical properties of differently composed polyelectrolyte films prepared by the layer-by-layer (LbL) technology was investigated. Free-standing polyelectrolyte films as microcapsules showed a fast, strong response to the addition of less than 1 mM cationic surfactant cetyltrimethylammonium bromide (CeTAB). As a function of the polyelectrolyte composition, the behavior of the capsules varied from negligible changes to complete disintegration via strong swelling. The response of microcapsules consisting of (poly(allylamine hydrochloride)(PAH)/poly(styrene sulfonate)(PSS))(4) was associated with a 5-fold volume increase, a fast switch of permeability, and in the case of fluorescently labeled films a 4-fold increase in fluorescence intensity. The kinetics and strengths of the interaction process were investigated by confocal laser scanning microscopy (CLSM) and fluorescence spectroscopy. Also, the relative stabilities of the polycation/polyanion and surfactant/polyanion complexes were determined. A mechanism was suggested to explain the interactions between the cationic surfactants and polyelectrolyte capsules. The strong response can be exploited in potential applications such as the triggered release of drugs or other encapsulated materials, the fluorescence-based detection of cationic detergents, and a switchable stopper in microchannels. However, the high sensitivity of LbL films to traces of cationic surfactants can also limit their applicability to the encapsulation of drugs or other materials because pharmaceutical or technical formulations often contain cationic surfactants as preservatives such as benzalkonium salts (BAC). It was demonstrated that undesired capsule opening can be effectively prevented by cross-linking the polyelectrolyte multilayers.     

Patterned polyelectrolyte multilayer: surface modification for enhancing selective adsorption

This paper describes the use of surface chemical modification to enhance the difference of the surface charge on a patterned polyelectrolyte multilayer, which can be used for selectively adsorbing functional materials. We fabricated a patterned multilayer by combining the layer-by-layer self-assembly technique and photolithography and taking advantage of the different solubility of polyelectrolyte multilayers of diazo resins (DAR)/poly(acrylic acid) before and after UV irradiation. This patterned surface can be used as a matrix for selective adsorption of small molecular dyes, such as Methylene Blue. However the difference in surface charge on the patterned surface was not enough when we used it to selectively adsorb polystyrene (PS) nanoparticles using electrostatic force as the driving force. Therefore, we modified the patterned surface by interfacial chemistry. After modification, the patterned polyelectrolyte multilayer can be used as a good matrix for selective adsorption of PS nanoparticles with both positive and negative charges.     

Brownian dynamics simulations of polyelectrolyte adsorption onto charged patterned surfaces

The adsorption of single polyelectrolyte molecules onto surfaces decorated with periodic arrays of charged patches was studied using Brownian dynamics simulations. A free-draining, freely jointed bead-rod chain was used to model the polyelectrolyte, and electrostatic interactions were incorporated using a screened Coulombic potential with the excluded volume accounted for by a hard-sphere potential. The simulations predicted that the polyelectrolyte lies close to the adsorbing surface if the patch length, surface charge density, and screening length are sufficiently large. Chain conformations were found to be very sensitive to patch length, patch spacing, and the nature of the charge on adjacent patches. This is due both to the size of the polymer relative to patch length and spacing and to the structure of the electric field near the surface. In some cases, the component of the radius of gyration parallel to the surface can be made smaller than its free-solution value, which is contrary to what is observed for a uniformly charged surface. Isolated charged patches were also considered, and significant adsorption was observed above a critical surface charge density. The results demonstrate how polyelectrolyte conformations can be controlled by the design of the charged patches and may be useful for applications in which adsorbed polyelectrolyte films play a key role.     

Brownian dynamics simulations of polyelectrolyte adsorption onto topographically patterned surfaces

The effect of patterned surface topography on the adsorption of single polyelectrolyte molecules is explored using Brownian dynamics simulations. The polyelectrolyte is modeled as a free-draining, freely jointed bead-rod chain, and electrostatic interactions are incorporated using a screened Coulombic potential with excluded volume interactions accounted for by the repulsive part of a Lennard-Jones potential. Topography consisting of periodically spaced valleys of square cross section separated by flat hills is considered. Chain conformations are characterized for a wide range of valley widths, depths, and spacings as well as for several different types of surface charge distributions. Depending on the parameter values describing the topography, the chains are found to adopt conformations ranging from flat and extended to those associated with bridge-, brush-, or semi-bridge-like structures. The formation of these structures is rationalized on the basis of a free-energy model that takes into account the increase in free energy due to entropic confinement, excluded volume interactions, and chain stretching as well as the decrease in free energy due to bead-surface electrostatic attraction. The results of this work are expected to be useful in designing patterned surface topography to control the conformations of adsorbed polyelectrolyte molecules.     

Study of film structure and adsorption kinetics of polyelectrolyte multilayer films: effect of pH and polymer concentration

The alternate adsorption of polycation poly(allylamine hydrochloride)(PAH) and the sodium salt of the polymeric dye poly(1-[ p-(3'-carboxy-4'-hydroxyphenylazo)benzenesulfonamido]-1,2-ethandiyl)(PCBS) on quartz crystals coated with silica was studied to understand the structural properties and adsorption kinetics of these films using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D), absorbance, and ellipsometry measurements. In-situ deposition of the polycation PAH on QCM crystals was monitored, followed by rinsing with water and then deposition of the polyanion PCBS. The effects of polymer concentration and pH on film structure, composition and adsorption kinetics were probed. The polymers were adsorbed at neutral pH conditions and at elevated pH conditions where PAH was essentially uncharged to obtain much thicker films. The change in the resonant frequency, Deltaf, of the QCM-D showed a linear decrease with the number of bilayers, a finding consistent with absorbance and ellipsometric thickness measurements which showed linear growth of film thickness. By using the Delta f ratios of PCBS to PAH, the molar ratios of repeat units of PCBS to PAH in the bilayer films as determined by QCM-D were approximately 1:1 at polyelectrolyte concentrations 5-10 mM repeat unit, indicating complete dissociation of the ionic groups. The frequency and dissipation data from the QCM-D experiments were analyzed with the Voigt model to estimate the thickness of the hydrated films which were then compared with thicknesses of dry films measured by ellipsometry. This led to estimates of the water content of the films to be approximately 45 wt %. In addition to the QCM-D, some films were also characterized by a QCM which measures only the first harmonic without dissipation monitoring. For the deposition conditions studied, the deposited mass values measured by the QCM's first harmonic were similar to the results obtained using higher harmonics from QCM-D, indicating that the self-assembled polyelectrolyte films were rigid.     

Influence of the nature of the polycation on the adsorption kinetics and on exchange processes in polyelectrolyte multilayer films

The deposition of polyelectrolyte multilayer films (PEMs) appears more and more as a versatile tool to functionalize a broad range of materials with coatings having controlled thicknesses and properties. To increase the control over the properties of such coatings, a good knowledge of their deposition mechanism is required. Since Cohen Stuart et al. (Langmuir 18 (2002) 5607-5612) showed that the adsorption of one polyelectrolyte could induce desorption of polyelectrolyte complexes instead of regular deposition, more and more findings highlight peculiarities in the deposition of such films. Herein we demonstrate that the association of sodium polyphosphate (PSP) as the polyanion and either poly(-L-lysine hydrobromide) (PLL) or poly(allylamine chloride) (PAH) as the polycations may lead to non-monotonous film deposition as a function of time. Complementary, films containing PSP and PLL can be obtained from a (PLL-HA)(n) template films after the exchange of HA (hyaluronic acid) from the sacrificial template by PSP from the solution. This exchange is accompanied by pronounced film erosion. However, when starting from a (PAH-HA)(n) template, the film erosion and exchange due to the contact with PSP is by far less pronounced, nevertheless the film morphology changes. These findings show that the nature of the polycation used to deposit the PEM film may have a profound influence of the film's response to a competing polyanion.     

Theory of polyelectrolyte adsorption onto surfaces patterned with charge and topography

Mean-field theory is used to derive criteria for the adsorption of a weakly charged polyelectrolyte molecule from salt solution onto surfaces patterned with charge and topography. For flat surfaces patterned with periodic arrays of charged patches, the adsorbed layer thickness predicted using mean-field theory and that found by Brownian dynamics simulations are in quantitative agreement in the strong-adsorption regime, which corresponds to sufficiently small kappa or sufficiently large |sigma(eff)q|, where kappa is the inverse Debye screening length, sigma(eff) is an effective surface charge density, and q is the charge on each segment of the polyelectrolyte. Qualitative agreement is obtained in the weak-adsorption regime, and for the case where surfaces are patterned with both charge and topography. For uniformly charged, sinusoidally corrugated surfaces, the theory predicts that the critical temperature required for adsorption can be greater than or less than the corresponding value for a flat surface depending on the relative values of kappa and the corrugation wave number. If the surface charge is also allowed to vary sinusoidally, then adsorption is predicted to occur only when the topography crests have a surface charge opposite to that of the polyelectrolyte. Surfaces patterned with rectangular indentations having charged bottoms which are separated by flat charged plateaus are investigated as well. Adsorption is predicted to occur even when the net surface charge is zero, provided that the plateaus have a charge opposite to that of the polyelectrolyte. If the charge on the plateaus and polyelectrolyte is the same, adsorption may still occur if electrostatic attraction from the indentation bottoms is sufficiently strong.     

Nanocomposite-based lignocellulosic fibers 3: polyelectrolyte adsorption onto heterogeneous fiber surfaces

Wood and natural fibers are composed of multiple polymeric components. Lignin, one primary component, is typically removed to various degrees during paper-making, but is present on thermomechanically isolated fibers. The effect of the residual lignin on the adsorption of poly(diallyldimethylammonium) chloride (PDDA) onto lignocellulosic fibers was investigated under varying solution conditions (electrolyte concentration and pH). Using nitrogen elemental analysis it was shown for the samples containing the highest concentration of lignin, PDDA adsorption to the fibers was reduced for all solution conditions. Upon extracting the alkali-soluble lignin, the samples showed the greatest amount of PDDA adsorption, achieving ~1.6% (w/w), under neutral solution conditions without the presence of added electrolyte. Furthermore, the influence of polyelectrolyte loading and electrokinetic potential on subsequent multilayer formation of PDDA and montmorillonite clay was quantified. It was revealed that electrokinetic potential of fiber after PDDA adsorption, rather than the amount of adsorbed PDDA layer, controlled the subsequent clay adsorption. Significant mass of PDDA/clay (up to ~75% of starting dry fiber mass for only 4 bi-layers) can be adsorbed onto steam-exploded wood fibers through the multilayer assembly process. This paper provides insight into how non-covalent modification of heterogeneous fibrous substrates offers a novel route for the creation of organic/inorganic fiber materials.     

In situ electroacoustic monitoring of polyelectrolyte adsorption onto protein-coated oil droplets

The purpose of this study was to compare the ability of electroacoustic (EA) and microelectrophoresis (ME) techniques for monitoring the adsorption of an anionic polysaccharide onto the surfaces of oppositely charged protein-coated oil droplets. The zeta-potential values determined by the EA technique were in excellent agreement with those determined by the ME technique. Both techniques were able to monitor the adsorption of pectin onto the surfaces of beta-lactoglobulin-coated droplets as a function of pectin concentration and pH. The major advantage of the EA technique was that it could be carried out in situ without having to dilute the emulsions, so that the equilibrium between adsorbed and non-adsorbed polyelectrolyte was not disturbed by dilution. Nevertheless, the good agreement between the zeta-potential values determined by the EA and ME techniques suggested that emulsion dilution did not cause an appreciable change in polysaccharide partitioning for the system used in this study. In summary, the EA technique appears to be a powerful means of monitoring polyelectrolyte adsorption in concentrated colloidal dispersions.     

Direct surface force measurements of polyelectrolyte multilayer films containing nanocrystalline cellulose

Polyelectrolyte multilayer films containing nanocrystalline cellulose (NCC) and poly(allylamine hydrochloride) (PAH) make up a new class of nanostructured composite with applications ranging from coatings to biomedical devices. Moreover, these materials are amenable to surface force studies using colloid-probe atomic force microscopy (CP-AFM). For electrostatically assembled films with either NCC or PAH as the outermost layer, surface morphology was investigated by AFM and wettability was examined by contact angle measurements. By varying the surrounding ionic strength and pH, the relative contributions from electrostatic, van der Waals, steric, and polymer bridging interactions were evaluated. The ionic cross-linking in these films rendered them stable under all solution conditions studied although swelling at low pH and high ionic strength was inferred. The underlying polymer layer in the multilayered film was found to dictate the dominant surface forces when polymer migration and chain extension were facilitated. The precontact normal forces between a silica probe and an NCC-capped multilayer film were monotonically repulsive at pH values where the material surfaces were similarly and fully charged. In contrast, at pH 3.5, the anionic surfaces were weakly charged but the underlying layer of cationic PAH was fully charged and attractive forces dominated due to polymer bridging from extended PAH chains. The interaction with an anionic carboxylic acid probe showed similar behavior to the silica probe; however, for a cationic amine probe with an anionic NCC-capped film, electrostatic double-layer attraction at low pH, and electrostatic double-layer repulsion at high pH, were observed. Finally, the effect of the capping layer was studied with an anionic probe, which indicated that NCC-capped films exhibited purely repulsive forces which were larger in magnitude than the combination of electrostatic double-layer attraction and steric repulsion, measured for PAH-capped films. Wherever possible, DLVO theory was used to fit the measured surface forces and apparent surface potentials and surface charge densities were calculated.     

Absorption of light and heavy water vapours in polyelectrolyte multilayer films

We studied the swelling and the uptake of water (H₂O or D₂O) vapours in polyelectrolyte (PE) multilayer (PEM) samples deposited on solid support (Si wafers) as a function of the isotope nature of the vapour and the charge of the last polymer layer. The samples were prepared with deuterated poly(sodium 4-styrenesulfonate) (dPSS) and poly(allylamine hydrochloride) (PAH). Two types of samples were studied. The sample with a structure Si/PEI/(dPSS/PAH)₆/dPSS was negatively charged. A positively charged sample was PAH terminated and had the structure Si/PEI/(dPSS/PAH)₆. The film thickness and scattering length density were estimated from neutron reflectometry (NR) experiments and the results were complemented with in-situ QCM measurements.We demonstrate that the swelling of PEM in H₂O and D₂O vapours is similar. However, the amount of adsorbed D₂O is around 10% more than the adsorbed H₂O. Such isotope effect correlates well with the rough estimation that the isotope effect usually scales with the difference in the mass density of the different isotope forms of the substances. For precise analysis of the NR data we assumed existence of empty voids in the structure of the PEM. These voids might be filled with “condensed” water when the samples are exposed to water vapors. We show that the layers we studied consist of up to 25% of such voids.We showed that the amount of sorbed water depends on the nature of the last layer which builds the PEM thus confirming the “odd-even effect” already shown in the literature.     

Direct transfer of preformed patterned bio-nanocomposite films on polyelectrolyte multilayer templates

Microarrays containing multiple, nanostructured layers of biological materials would enable high-throughput screening of drug candidates, investigation of protein-mediated cell adhesion, and fabrication of novel biosensors. In this paper, we have examined in detail an approach that allows high-quality microarrays of layered, bionanocomposite films to be deposited on virtually any substrate. The approach uses LBL self-assembly to pre-establish a multilayered structure on an elastomeric stamp, and then uses microCP to transfer the 3-D structure intact to the target surface. For examples, different 3-D patterns containing dendrimers, polyelectrolyte multilayers and two proteins, sADH and sDH, have been fabricated. For the first time, the approach was also extended to create overlaid bionanocomposite patterns and multiple proteins containing patterns. The approach overcomes a problem encountered when using microCP to establish a pattern on the target surface and then building sequential layers on the pattern via LBL self-assembly. Amphiphilic molecules such as proteins and dendrimers tend to adsorb both to the patterned features as well as the underlying substrate, resulting in low-quality patterns. By circumventing this problem, this research significantly extends the range of surfaces and layering constituents that can be used to fabricate 3-D, patterned, bionanocomposite structures. [image in text]     

Controlling primary hepatocyte adhesion and spreading on protein-free polyelectrolyte multilayer films

The development of new methods for fabricating thin films that provide precise control of the three-dimensional topography and cell adhesion could lead to significant advances in the fields of tissue engineering and biosensors. This Communication describes the successful attachment and spreading of primary hepatocytes on polyelectrolyte multilayer (PEM) films without the use of adhesive proteins such as collagen or fibronectin. We demonstrate that the attachment and spreading of primary hepatocytes can be controlled using this layer-by-layer deposition of ionic polymers. In our study, we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated poly(styrene) (SPS) as the polycation and polyanion, respectively, to build the multilayers. Primary hepatocytes attached and spread preferentially on SPS surfaces over PDAC surfaces. SPS patterns were formed on PEM surfaces, either by microcontact printing of SPS onto PDAC surfaces or vice versa, to obtain patterns of primary hepatocytes. PEM is a useful technique for fabricating controlled co-cultures with specified cell-cell and cell-surface interactions on a protein-free environment, thus providing flexibility in designing cell-specific surfaces for tissue engineering applications.