Issues of Ligand Accessibility and Mobility in Initial Cell Attachment

Abstract

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).

Microstructure characterization and thermal analysis of hybrid block copolymer α-methoxy-poly(ethylene glycol)-block-poly[ε-(benzyloxycarbonyl)-l-lysine] for biomedical applications

“…PEG and PLL have been covalently combined to form graft [24,25], diblock [26][27][28][29], triblock [30][31][32], and random [33] copolymers and dendrimers [34,35]; some of which have been conjugated to other moieties, such as: folates [36], acids [37,38], sugars [39,40], RGD [41,42], and amphotericin B [43], to mention a few. They have also been used in polyion complex (PIC) formation [27,44,45], and investigated for drug [46,47] and gene [48][49][50] delivery, colloidal lithography [51,52], biomimetic applications [53], photodynamic therapy (PDT) [54,55], antithrombogenicity [56], and as bioimaging agents [57,58].…”

Surface‐Adhered Composite Poly(Vinyl Alcohol) Physical Hydrogels: Polymersome‐Aided Delivery of Therapeutic Small Molecules

“…Coatings can be conjugated with a cell adhesive peptide ( e.g. , RGD [ 35 ] or IKVAV [ 36 ] ) and proteins [ 37 ] to convert surface properties from cell-phobic to cell-philic. Also, for PVA specifi cally, cogelation of PVA with cell adhesive biomacromolecules such as chitosan [ 38 ] or poly(Llysine) and collagen [ 32a ] were considered to render PVA gels cell adhesive.…”

Large‐Scale Protein Arrays Generated with Interferometric Lithography for Spatial Control of Cell‐Material Interactions

“…Focal adhesions are on the order of 10 nm to 10 μm and serve as crucial outside-to-inside signaling gateways that are necessary for proper cell function [2]. The micrometer-and nanometer-scale organization of surface proteins is expected to play a critical role in adhesion complex formation and function [3][4][5][6][7]. Seminal work in the area of cell adhesion studies in Whitesides' laboratory patterned cell adhesive domains on the cellular scale (10s of microns), demonstrating that by controlling the shape and size of the adhesive domain, the shape and degree of physical interaction between the surface and the cell could be controlled [8][9][10][11].…”