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In-vitro self-assembly of bacterial pilus toward understanding biological long-range electron transport and the formation of conductive polymers for tissue regeneration

Final Report Summary - CONPILUS (In-vitro self-assembly of bacterial pilus toward understanding biological long-range electron transport and the formation of conductive polymers for tissue regeneration)

The main goal of ConPilus was to improve our understanding of long-range electron transport (ET) across self-assembled biological fibrils and the formation of conductive polymers based on biological materials for applications in tissue engineering.
Nature has developed protein-based systems to conduct electrons over very long distances, where the most studied example is along the pilus of Geobacter sulfurreducens and Shewanella oneidensis, which has drawn much attention in recent years due to its extraordinary ET efficiency. The pilus of the bacteria is composed from a structural protein together with a dense array of deca-heme cytochromes. Though the original aim of ConPilus was to express the proteins of the bacterial pilus and assemble them in vitro, we decided to use a different structural protein, which is commercially available, to significantly increase the starting amount of the protein.
Early on in the beginning of the project we exploited the self-assembly process of bovine serum albumin (BSA) to form protein-based polymers by two different experimental procedures. The first one is the thermally-induced formation of BSA hydrogels by non-covalent entanglement of the BSA fibrils. The second method was electrospinning, in which the BSA was transferred to a non-aqueous solution, and large fibrils of BSA were formed to a shape of a dry mat by applying a high voltage between a needle containing the protein and the substrate. The next step in the project was to characterize the electrical conduction of the newly formed protein-based structure. We demonstrated that BSA mats can adsorb water up to 150% of their weight. During our electrical characterization process, we found that due to the high content of water within the mats, they can be considered as excellent proton conductors with an associated conductivity of around 50 µS/cm. By employing photo-induced proton transfer as well as applying an electric bias to the BSA mats, we could conclude that the protein backbone has also an important role in mediating the protons along the surface of the BSA mat.
The main goal of the project was to explore long-range ET across protein-based structures. In order to enable ET across the BSA mats, they were doped with hemin molecules, as heme is one of the most common ET mediators in nature, in which hemin was non-covalently yet very strongly bound to the BSA. In analogy with solid-state device nomenclature, we refer to this binding as ‘molecular doping’ process, with the selected molecules the ‘dopants’; one might equally characterize this process as the formation of a hybrid or composite material. Following the doping process, we revealed that the measured electrical conduction across the BSA mats increased by 40 folds (to 2 mS/cm). Due to this large conduction we could measure the conductivity across centimeter length scales. We attributed this large increase in conduction to a favorable electron conduction mediated by the hemin molecules. We postulated that the electron conduction was mediated by hopping events between the iron centers within the hemin molecules. Accordingly, we used the Marcus-Hush formalism for an ET process together with the Einstein relation for diffusion of charges to extract the kinetic and energetic parameters associated with the ET process across the hemin-doped BSA mats.
In analogy, we also characterized the electronic properties across the BSA hydrogels before and after doping them with hemin. However, unlike the BSA mats, the water content inside the hydrogel is very large, and the water-associated ionic conduction across the hydrogel is significantly larger than the one across the BSA mats. Accordingly, upon doping the material we witnessed only a mild increase of 5 fold in the measured conductance across the hydrogel.
The end goal of the project was to use the protein-based materials as scaffolds for tissue engineering applications in the field of cardiac cells and neuronal networks. First, we characterized the morphological and mechanical properties of each type of biomaterials, which guided our choice of using the BSA hydrogels for cardiac tissue engineering since they exhibit highly elastic properties that match the ones of the myocardium. On the other side, we decided to use the BSA mats for neuronal networks since the macroscopic fibrillary network of the mat promote the spread of neurons. Following the mechanical and morphological characterization, we plated the cells on the non-doped material. For the cardiac cells (ventricular myocytes) we found that these hydrogels significantly promote the maintenance of the native gene expression profile, with the cells having a distinct Ca2+ transient profile. Furthermore, the hydrogels allow the isotonic contraction of the formed myocardial patch. For the neurons on the non-doped mats we found that they can attach and form networks in a relatively sufficient way. Nevertheless, our most interesting observation was the astonishing improvement of cell attachment and spreading for both cardiac cells and neurons when we plated it on the hemin doped hydrogels and mats, respectively. This attachment resulted in a strong beating hydrogel containing the cardiomyocytes and in a full coverage of neurons on the mats. We are in the progress of analyzing the data and are in preparation of further publications, while in the next stage we tend to pass electric current across the scaffold and investigate its effect on the cellular behavior.
In summary, ConPilus was very successful and achieved its objectives. Serum albumin protein proved to be an exceptional component to construct electrically conductive scaffolds. Moreover, it allows also the easy translation to the human form of the protein. Hence the simple approach presented here to form the scaffold material from an abundant and autologous serum protein makes the hydrogels and mats compelling candidates not only for tissue engineering applications but also for disease modelling and drug testing.