Nature-Inspired adhesive catecholamine incorporation into biotic/aboitic system and its applications

Abstract

Catechol and catecholamines, found ubiquitously in nature, have attracted significant attention in the field of material science due to its unique properties. Catechol can strongly interact with a variety of organ-ic/inorganic substrates via hydrogen bonding, electrostatic forces, coordination, covalent bonds, and π-π stacking. Another important property of catechol groups is that it can participate in catecholquinone-mediated crosslinking between bio-macromolecules such as polysaccharide and proteins. Also catechol pos-sess the ability to form complex with metal ions and to reduce metal ions due to its significant redox activity. Though the catechol moiety was incorporated in various polymers, surfaces, particles, and biomolecules, there has not been a report of catechol or catecholamine incorporation into biotic system ？ such as viruses, bacteria, and mammalian cells. In this thesis, catechol and catecholamine functional moieties were incorpo-rated into biotic systems and abiotic systems and they were used for various biomaterial applications. In Chapter 2, mussel-inspired adhesive catecholamine was incorporated and displayed on the cell sur-face of Escherichia coli. Mussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-L-phenylalanine (DOPA), lysine, and histi-dine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine on Escherichia coli surfaces using outer membrane protein W as an anchoring motif for the first time. Locali-zation of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence mi-croscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catechola-mine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catechol-amines. The engineered E. coli exhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethyleneterephthalate), poly(urethane), and poly(dimethyl lsiloxane). The uniqueness of this approach utilizing the engineered sticky E. coli is that no chemistry for cell attachment are necessary, and the ability of spontaneous E. coli attachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine displayed sticky E. coli could be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell com-munication systems, and many others. In chapter 3, M13 bacteriophage was engineered to display DOPA on the surface for fabrication of various nanostructure inorganic materials. M13 bacteriophage (phage) was engineered for the use as a versa-tile template for preparing various nanostructured materials via genetic engineering coupled with enzymatic chemical conversions. First, we engineered the M13 phage to display TyrGluGluGlu (YEEE) on the pVIII coat protein and then enzymatically converted the Tyr residue to 3,4-dihydroxyl-l-phenylalanine (DOPA). The DOPA-displayed M13 phage could perform two functions: assembly and nucleation. The engineered phage were able to assemble various noble metals, metal oxides, and semiconducting nanoparticles into one-dimensional arrays. Furthermore, the DOPA-displayed phage triggered the nucleation and growth of gold, silver, platinum, bimetallic cobalt？platinum, and bimetallic iron？platinum nanowires. This versatile phage template enables rapid preparation of phage-based prototype devices by eliminating the screening process, thus reducing effort and time. In chapter 4, new hydrogel system was developed using the stoichiometric dependent reaction of cate-chol/vanadium complexation. In general, mechanical properties and gelation kinetics exhibit a positive corre-lation with the amount of gelation reagents used. Similarly, for catechol-containing hydrogels, which have attracted significant attention, because of their unique dual properties of cohesion and adhesion, increased amounts of cross-linking agents, such as organic oxidants and/or transition metals $(Fe^{3+})$, result in enhanced mechanical strength and more rapid gelation kinetics. Here, we report a new metal？ligand cross-linking chem-istry, inspired by mussels and ascidians that defies the aforementioned conventional stoichiometric concept. When a small amount of vanadium is present in the catechol-functionalized polymer solution (i.e., [V] ≪ [catechol]), organic radicals are rapidly generated which trigger the gelation reaction. However, when a large amount of the ion is added to the same solution (i.e., [V] ≫ [catechol]), the catechol remains chemically intact by coordination which inhibits gelation. Thus, a large amount of cross-linking agent is not required to prepare mechanically strong, biocompatible hydrogels using this system. This new chemistry may provide insight into the biological roles of vanadium and its interaction with catechol-containing molecules (i.e., de-termination of the liquid state versus the solid state). Excess amounts of vanadium ([V] ≫ [catechol]) coor-dinate with catechol, which may result in a liquid state for ascidian blood, whereas excess amounts of cate-chol ([V] ≪ [catechol]) generate an organic radical-mediated chemical reaction, which may result in solid-state conversion of the mussel byssal threads. In chapter 5, bio-inspired artificial biofilm was developed. Biofilms are defined as structured communi-ty of microorganisms enclosed in a self-produced polymetric matrix. The constituent microorganisms profits from the metabolic cooperation along with the protection against environmental stress. The robustness and resilience of biofilms against toxic substances or solvent have made them attractive as biocatalyst. However, the potential of biofilm has not been fully realized due to the complexity of biofilm system. In this study, we have developed artificial biofilm termed BIOMOSAIC (Bio-Inspired Oxygen induced Microbial Organization through Self-Assembly at the air/liquid Interface as Catalyst) film which spontaneously forms when exposed to atmospheric air. The spontaneous BIOMOSAIC film formation process employs unique interfacial reac-tion of catecholamine chemistry. BIOMOSAIC film have highly porous structure with highly dense cell im-mobilization without jeopardizing the cell viability and activity. BIOMOSAIC films with various enzymatic activity were employed in actual bioreactor and showed promising results with reusability.