Biochemical functionalization of polymeric cell substrata can alter mechanical compliance.

Biochemical functionalization of surfaces is an increasingly utilized mechanism to promote or inhibit adhesion of cells. To promote mammalian cell adhesion, one common functionalization approach is surface conjugation of adhesion peptide sequences such as Arg-Gly-Asp (RGD), a ligand of transmembrane integrin molecules. It is generally assumed that such functionalization does not alter the local mechanical properties of the functionalized surface, as is important to interpretations of macromolecular mechanotransduction in cells. Here, we examine this assumption systematically, through nanomechanical measurement of the nominal elastic modulus of polymer multilayer films of nanoscale thickness, functionalized with RGD through different processing routes. We find that the method of biochemical functionalization can significantly alter mechanical compliance of polymeric substrata such as weak polyelectrolyte multilayers (PEMs), increasingly utilized materials for such studies. In particular, immersed adsorption of intermediate functionalization reagents significantly decreases compliance of the PEMs considered herein, whereas polymer-on-polymer stamping of these same reagents does not alter compliance of weak PEMs. This finding points to the potential unintended alteration of mechanical properties via surface functionalization and also suggests functionalization methods by which chemical and mechanical properties of cell substrata can be controlled independently.

Patterned superhydrophobic surfaces: toward a synthetic mimic of the Namib Desert beetle.

The present study demonstrates a surface structure that mimics the water harvesting wing surface of the Namib Desert beetle. Hydrophilic patterns on superhydrophobic surfaces were created with water/2-propanol solutions of a polyelectrolyte to produce surfaces with extreme hydrophobic contrast. Selective deposition of multilayer films onto the hydrophilic patterns introduces different properties to the area including superhydrophilicity. Potential applications of such surfaces include water harvesting surfaces, controlled drug release coatings, open-air microchannel devices, and lab-on-chip devices.

Controlled drug release from porous polyelectrolyte multilayers.

Microporous and nanoporous polyelectrolyte multilayer films have been explored as ultrathin coatings for controlled drug release. Ketoprofen and cytochalasin D were successfully loaded into nanoporous films and showed zero-order release kinetics over a period of many days. In addition to homogeneous porous multilayers, heterostructures comprising porous regions stacked alternately with nonporous regions were assembled. The heterostructures behaved as dielectric mirrors, which made it possible to optically monitor the loading process. The effects of varying the number of layers in porous and nonporous regions as well as the pore size on the drug release properties were studied. Nonporous regions in the film had no effect on the release rate or duration of release. The amount of drug released could be tuned by varying the number of layers in the porous regions of films, and the release rate depended on the pore size in the films. Microporous multilayers exhibited a Fickian diffusion of drug that was approximately twice as fast as the corresponding nanoporous films. Finally, cell culture experiments with WT NR6 fibroblasts confirmed that cytochalasin D retained its ability to inhibit mitosis after release from the multilayers.

Tuning compliance of nanoscale polyelectrolyte multilayers to modulate cell adhesion.

It is well known that mechanical stimuli induce cellular responses ranging from morphological reorganization to mineral secretion, and that mechanical stimulation through modulation of the mechanical properties of cell substrata affects cell function in vitro and in vivo. However, there are few approaches by which the mechanical compliance of the substrata to which cells adhere and grow can be determined quantitatively and varied independent of substrata chemical composition. General methods by which mechanical state can be quantified and modulated at the cell population level are critical to understanding and engineering materials that promote and maintain cell phenotype for applications such as vascular tissue constructs. Here, we apply contact mechanics of nanoindentation to measure the mechanical compliance of weak polyelectrolyte multilayers (PEMs) of nanoscale thickness, and explore the effects of this tunable compliance for cell substrata applications. We show that the nominal elastic moduli E(s) of these substrata depend directly on the pH at which the PEMs are assembled, and can be varied over several orders of magnitude for given polycation/polyanion pairs. Further, we demonstrate that the attachment and proliferation of human microvascular endothelial cells (MVECs) can be regulated through independent changes in the compliance and terminal polyion layer of these PEM substrata. These data indicate that substrate mechanical compliance is a strong determinant of cell fate, and that PEMs of nanoscale thickness provide a valuable tool to vary the external mechanical environment of cells independently of chemical stimuli.

Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping.

A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 microm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.

Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces.

A newly discovered class of cell resistant surfaces, specifically engineered polyelectrolyte multilayers, was patterned with varying densities of adhesion ligands to control attachment of mammalian cells and to study the effects of ligand density on cell activity. Cell adhesive patterns were created on cell resistant multilayer films composed of poly(acrylic acid) and polyacrylamide through polymer-on-polymer stamping of poly(allylamine hydrochloride) PAH and subsequent reaction of the amine functional groups with an adhesion ligand containing RGD (Arg-Gly-Asp). These cell patterns demonstrated great promise for long-term applications since they remained stable for over 1 month, unlike ethylene glycol functional surfaces. By changing the stamping conditions of PAH, it was possible to alter the number of available functional groups in the patterned regions, and as a result, control the ligand density. Cell spreading, morphology, and cytoskeletal organization were compared at four different RGD densities. The highest RGD density, approximately 152 000 molecules/microm2, was created by stamping PAH at a pH of 11.0. Lowering the stamping ink pH led to patterns with lower ligand surface densities (83 000 molecules/microm2 for pH 9.0, 53,000 molecules/ microm2 for pH 7.0, and 25 000 molecules/microm2 for pH 3.5). An increasing number of cells attached and spread as the RGD density of the patterns increased. In addition, more cells showed well-defined actin stress fibers and focal adhesions at higher levels of RGD density. Finally, we found that pattern geometry affected cytoskeletal protein organization. Well-formed focal adhesions and cell-spanning stress fibers were only found in cells on wider line patterns (at least 25 microm in width).