Adsorption and Exchangeability of Fibronectin and Serum Albumin Protein Corona on Annealed Polyelectrolyte Multilayers and Their Consequences on Cell Adhesion

Abstract

Cell adhesion is a key process in many physiological and pathological processes and a fundamental issue in biomedical fields.[1–5] Controlling the adhesion of cells to the interface of artificial materials is a basic requirement for the development of scaffolds and medical devices. However, the interaction of materials with cells is complex and often poorly understood.[6] It is known that cells adhere to substrates through adhesion proteins present in the cell media and biological fluids, such as fibronectin (FN), which are sensed by integrins from the cell membrane. Adhesion proteins must adhere first to the material’s surface and cells will follow. The concentration of adhesion proteins is very small in relation to other proteins present in biological fluids, such as hemoglobin or serum albumin, which compete with adhesion proteins for attachment to surfaces and formation of a “protein corona.”[7] Protein conformation should change upon adsorption to different extents based on the protein type and environmental conditions.[8] The attachment and stability of adhesion proteins is highly influenced by the nature of the substrate, including its roughness, surface chemistry, contact angle, and other characteristics, which in turn will play an important role on cell adhesion and growth and on other cell functions such as myogenic differentiation[9] and stem cell differentiation.[10–13] Polyelectrolyte multilayers (PEMs) are fabricated by the so-called layer-by-layer (LbL) technique. The LbL technique makes use of the alternating assembly of oppositely charged polyelectrolytes led by electrostatic interactions.[14] PEMs made from biopolyelectrolytes are very appealing for tissue engineering as they are biocompatible, easy to assemble on charged surfaces, and can incorporate growth factors or other biomolecules assembled in between the layers or on top of the PEM, which can promote cell adhesion, cell mobility, mineralization, tissue regeneration, and other processes. However, biopolyelectrolyte-based PEMs have weak adhesive properties for cells. Picart and co-workers have shown that chemical crosslinking of PEMs improves cell adhesion. Biopolyelectrolyte PEMs are soft, Polyelectrolyte multilayers (PEMs) based on biopolyelectrolytes are highly appealing for the surface engineering of biomaterials and the tuning of cell response and phenotypes for biomedical applications. However, cell adhesion is limited on biopolyelectrolyte PEMs. Thermal annealing provides a simple means to increase or decrease cell adhesion on PEMs. The work presented here aims to understand cellular interactions with annealed PEMs based on the adsorption and exchangeability of two model proteins: fibronectin (FN), an adhesion protein, and bovine serum albumin (BSA), a nonadhesion protein. Protein adsorption and exchangeability are studied on annealed poly-l-lysine (PLL)/sodium alginate (Alg) and chitosan (Chi)/hyaluronic acid (HA) PEMs using [131I] radiolabeled proteins and gamma counting. Upon annealing cell adhesion is enhanced on PLL/Alg multilayers and decreased on Chi/HA multilayers. For PLL/Alg PEMs, annealing increases adsorption of both FN and BSA and reduces exchangeability. For Chi/HA multilayers, annealing increases BSA adsorption but decreases FN deposition, accompanied by a greater exchangeability. Changes in topographic features of deposited proteins on annealed PLL/Alg hint on changes in the 3D structure of the proteins. Circular dichroism shows that FN retains a large β-sheet contribution upon adsorption to both annealed and unannealed PLL/Alg PEMs, also suggesting changes in tertiary structure.