Exploitation of S-Layer Anisotropy: pH-Dependent Nanolayer Orientation for Cellular Micropatterning

Abstract

We have developed a tunable, facile, and reliable cell patterning method using a self-assembled crystalline protein monolayer that, depending on its orientation, can exhibit either cell adhesive (cytophilic) or cell repulsive (cytophobic) surface properties. Our technique exploits, for the first time, the inherent biological anisotropy of the bacterial cell wall protein SbpA capable of interacting with its cytophilic inner side with components of the cell wall, while its outer cytophobic side interacts with the environment. By simply altering the recrystallization protocol from a basic to an acidic condition, the SbpA-protein layer orientation and function can be switched from preventing unspecific protein adsorption and cell adhesion to effectively promote cell attachment, spreading, and proliferation. As a result, the same protein solution can be used to form cell adhesive and repulsive regions over large areas on a single substrate using a simple pH-dependent self-assembly procedure. The reliable establishment of cytophobic and cytophilic SbpA layers allows the generation of well-defined surface patterns that exhibit uniform height (9-10 nm), p4 lattice symmetry with center-to-center spacing of the morphological units of 12 nm, as well as similar surface potential and charge distributions under cell culture conditions. The pH-dependent "orientation switch" of the SbpA protein nanolayer was integrated with micromolding in capillaries (MIMIC) technology to demonstrate its application for cell patterning using a variety of cell lines including epithelial, fibroblast and endothelial cells.

Anisotropic Crystalline Protein Nanolayers as Multi‐Functional Biointerface for Patterned Co‐Cultures of Adherent and Non‐Adherent Cells in Microfluidic Devices

“…[ 22 ] In fact, S-layers have already been proven to be a stable biomaterial under harsh denaturation conditions including treatment with ethanol (<80%), elevated temperatures (<70 °C) and pH shifts (pH > 3). [ 17,23 ] Another advantage of using S-layer proteins as a biointerface is the elimination of unspecifi c binding of biomolecules such as serum proteins and antibodies, thus also preventing unwanted cell-to-surface interactions. This means that it is possible to selectively modify and functionalize the protein nanolayers with affi nity capture probes.…”

“…[ 16 ] Additionally, the pH dependent re-orientation of SbpA monolayers featuring opposing cytophobic or cytophilic surface properties was recently applied for cell patterning using micro molding in capillaries (MIMIC). [ 17 ] Furthermore, specifi c functional domains have already been introduced in S-layer proteins to foster the immobilization of biocatalysts, fl uorescent biomarkers and nanoparticles, while still maintaining their self-assembly properties. [ 18 ] An example of the implementation of specifi c functional domains includes the recombinant fusion protein rSbpA/ZZ, which contains the protein A-binding site for the effi cient capture of antibodies (IgG) and has been used as a specifi c adsorbent in extracorporeal blood purifi cation.…”

Bacterial Surface Layer Proteins: From Moonlighting to Biomimetics: A New Horizonto Lead

“…The outermost envelope possessing oriented self-assembly of proteins are not restricted only to prokaryotic bacterial species but also found in archaea and virus [22]. The comprehensive literature has been documented on the biochemical aspects, self-assembling properties and genetic framework of surface layer proteins but their technical utilization is under the stage of infancy till date [18].…”

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

“…Interestingly, it was shown that a self-assembled layer, depending on whether the inner or outer side is exposed to the aqueous environment, can exhibit against cells in tissue cultures either cell adhesive (cytophilic) or cell repulsive (cytophobic) surface properties, respectively. The different orientation and function of the S-layer protein can simply be achieved by altering the recrystallization protocol from a basic ( pH 9; resulting in an outer smooth cytophobic side) to an acidic ( pH 4; resulting in an inner rough cytophilic surface pattern) condition [105].…”