Silk-collagen-like block copolymers with charged blocks : self-assembly into nanosized ribbons and macroscopic gels

The research described in this thesis concerns the design, biotechnological production, and physiochemical study of large water-soluble (monodisperse) protein triblock-copolymers with sequential blocks, some of which are positively or negatively charged and self-assemble in response to a change in pH or co-assemble in response to oppositely charged polyelectrolytes (including each other). Such molecules displaying controlled self-assembly may lead to new biocompatible nano-structured materials like nano-wires, gels and fibers, with unusual material properties and potential technical and biomedical applications. First, the production and purification as enabling technology is described but the focus of this thesis is on the physiochemical behavior of the produced molecules, including the nano-structures formed, the kinetics of the structure formation and the material properties of the macroscopic gels resulting from the nano-structures.
For producing monodisperse block copolymers with sequential blocks, we chose the natural protein production machinery of living cells, because monodispersity and sequentiality are hallmarks of proteins. First, DNA encoding various polypeptide blocks of the block copolymers was designed. Then, using a modular cloning approach, DNA blocks were built, enlarged to desired block size and connected to form whole genes that encode polypeptide block copolymers. The genes were transferred to the production host, the yeast Pichia pastoris, which when induced, secreted the various protein block copolymers production yields in the gram per liter range, such that various applications of these promising biomaterials become possible. However, choosing this biotechnological approach limited the polymer design to the 20 natural amino acids, but seen the large variation in structure and function of natural proteins, this, in practice, forms hardly any limitation.
Three different nature-inspired poly-peptide blocks were used in the block copolymer designs: two very similar, but oppositely charged silk-like blocks, and one largely uncharged collagen like block. The silk-like blocks (“S”) consist of an octapeptide glycine (G) alanine(A) repeat (GAGAGAGX)24, in which the X position is occupied either by a positively charged histidine residue (H) or a negatively charged glutamic acid residue (E), resulting in an either positively or negatively charged silk-like block: “SH” or “SE” respectively, the theoretical pKa being 7 and 4.3 respectively. These blocks are supposed to self- or co-assemble upon charge neutralization or compensation. The collagen-like blocks (“C”) contains mainly glycine, proline, polar amino acids and a small number of charged residues. It is supposed to form a hydrophilic random coil in most aqueous environments. The four final products, all 802 amino acids long, were either positively charged, 66.1 kDa molecules denoted as SHCCSH and CSHSHC or negatively charged 65.7 kDa molecules denoted as SECCSE and CSESEC. The four different molecules showed different behavior depending on charge and block order. Three aspects of these molecules were studied: nano-structures, kinetics of structure formation, and material properties of pH change induced gels.
Nano-structures, were investigated with a broad range of techniques including: transmission electron microscopy (TEM) cryogenic (cryo-)TEM, atomic force microscopy (AFM), CD spectrometry, small angle X-ray scattering (SAXS) and molecular dynamics modeling (MD). The four different polymers were triggered to form structures and 12 of these self- or co-assembled nano-structures were examined: 1-4) the four pH- induced self-assemblies, 5-7) CSHSHC with poly acrylic acid (PAA), or with a metal bis-ligand supramolecular polymer denoted as Zn-L2(EO)4, or with the (conducting) polythiophene (POWT) that was chemically modified to be zwitterionic, 8) CSESEC with POWT, 9-12) the four possible mixtures of the four different protein block copolymers. Except for the mixtures of protein polymers, which seemed to form kinetically trapped molecular networks, the self- and co-assemblies formed well defined μm long nanoribbons with a hydrophilic C block corona. The core structure, depended on the protein block copolymer used, and on the mode of charge compensation like pH and/or the type of polyelectrolyte used. Interesting features are: the unusual -roll, predicted with MD modeling for the SE block in the self-assembled ribbon core at low pH, the CC middle blocks of SECCSE and SHCCSH forming loops, analogous to flower-like micelles, and the templating of the conductive polymer POWT onto nanoribbons, that might have applications as nanowires.
Kinetics of structure formation was only followed for CSESEC at low pH. CD spectroscopy at 200 nm was used to follow the conformational change associated with ribbon formation of CSESEC in time, in dilute, acidified solutions, revealing a nucleation and growth mechanism for CSESEC ribbons under a critical pH of approximately 4.5. Dynamic light scattering (DLS) measurements revealed that when the pH was increased, the ribbons dissolved and the formed gels melted, but they only did so above pH 5.4 which is much higher than the critical pH of ribbon formation. This pH region in which ribbons do not form, nor dissolve suggests a kinetic barrier to ribbon formation. The purified and freeze-dried CSESEC appeared to contain nuclei from which ribbon growth could start.
To study the effect of block charge and block order, on the mechanical properties of self-assembling block copolymer hydrogels, we tested the physical behavior of CSESEC, SECCSE, and CSHSHC. Dynamic mechanical spectroscopy revealed differences in gelling kinetics and mechanical properties of the three different polymers. Remarkably, the SE containing polymer gels displayed non linear elasticity comparable to that of the actin gels and other biological gels. Consequently we see that like actin gels our gels consist of semi-flexible fibrils (nano-tapes). Moreover, exceptionally high dynamic elasticity moduli, exceeding 40 kPa, were reached already at concentrations as low as 1.5 wt%, without any additional crosslinking agent. Such highly rigid yet dilute gels are rare and sought after. CSHSHC gels were relatively week and formed slowly. Additionally we studied the effect of temperature on the mechanical properties of a CSESEC gel.
The molecules self- and co-assembled into various nano-structures constituting various transparent gels, some of which extremely rigid. These structures and gels have potential biomedical and technological applications. The biotechnological approach for producing these four different monodisperse, sequential block copolymers, yielded amounts that make applications possible and presents an opportunity for the design and production of many more monodisperse and sequential (protein) block copolymers for building other nano-structures.