Study of the properties and applications of silk elastin-like recombinamers

Resumen

In the work presented in this thesis, we show the complete construction steps from gene design, gene construction, bioproduction in genetically modified organisms, purification and the characterization of composition and physico-chemical properties of interest for their intended use of three different elastin-like recombinamers (ELRs). At first sight, and simply taking into account the final use/destination of the developed materials, they might seem not to have much in common. However, if we consider their origin, structure and properties, all designed materials do have in common much more than it seems. All of them are elastin-like recombinamers (ELRs), which makes them to share much more things in common, although they might be in a “deeper” plane, inherent to their origin: - They are all protein-based polymers built with sequences found in Nature, such as those found in elastin. - They all have a recombinant origin. This means that all of them are encoded in a DNA sequence built taking advantage of the possibilities the recombinant technologies offer. - They all are built as the fusion of different blocks, each of them with a precisely controlled architecture and function. - Having in common their recombinant origin, composition and block structure they are all highly technological materials which have inherent shared properties: o They are sensitive to external stimuli modifying their soluble / insoluble state as response to such as temperature, pH, saline concentration, etc. o Due to their modular block structure and stimuli responsive nature, the ELRs presented in this thesis are built as “externally activable” amphiphilic molecules. o This stimuli triggered amphiphilicity entails the self-assembly of the ELRs into supramolecular structures. Thus, their self-assembly capacity (this is, their capacity to organize into complex and predesigned structures) can be triggered through and external stimulus, which is absolutely a non-common feature in most of (bio)materials. - Moreover, all three ELRs share in common the presence of another natural-recurring sequence. The B. mori (Bombyx mori) worm silk fibroin sequence has been included in all three ELRs in order to stabilize, through the formation of physical crosslinks, the self-assembled structures. Besides all the compositional and structural points in common of all the ELRs used in this thesis, the final use given to them is quite different. Indeed, developed materials were used as mucoadhesive, with wound healing / dressing purposes or used in the study of peculiar self-assembling properties. These different uses of the ELRs, despite all the shared points in common, reflects the complexity of these biomaterials, where simple changes in their structure imply different properties and applications. In the first objective of this thesis, we have focused on the development of a mucoadhesive ELR. Although the great benefits oral drug administration for gastrointestinal drug delivery, mucoadhesion is a process not completely understood. Moreover, mucoadhesive materials have to deal with a very adverse environment (low pH, large amounts of digestive enzymes, etc.), while interacting with a highly specialized tissue in the adsorption of substances, but also in the protection barrier for pathogens. In addition, while being mucoadhesive, the material has also to be biocompatible. Al all these requirements reveal the complexity when dealing with the development of a mucoadhesive materials. In order to assess the mucoadhesive properties of the designed ELR, we have proven its ability to interact with the mucus, the outer covering layer of the epithelial tissues. To do so, we have performed a large set of in vitro physical characterizations that support our statement. In vitro cell material interactions have proven the absence of toxicity, while specific in vitro epithelial model have demonstrated that the inclusion of a C’-terminal 7 amino acid bioactive sequence enhances the interaction among the material and a gastrointestinal epithelial cell model. In vivo rat experimental model has served to further confirm our hypothesis in a more realistic environment. The second aim of this thesis was to develop of a wound healing / dressing material. Skin is a very important organ that plays a key role in maintaining the homeostatic state of the human body. Wounds are part of human history, as demonstrated by the huge literature written by different cultures since ancient times. Nowadays, wounds are still a great issue of interest, with the same (old) origins such as injuries or war, but also with new origins, such as the world growing diabetes epidemic. Wounds have many requisites for their correct healing, so does a wound dressing material. Hydrogels, and more precisely in situ forming hydrogels, are able of fulfilling many of such requisites. The combination with bioactive substances, such as antibiotics, might be sufficient to develop a suitable wound dressing material. Thus, we have developed a fast hydrogel-forming silk-elastin-like recombinamer (sELR). A crosslinking time course of the sELR was performed and characterized with different techniques in order to understand how does the present silk motifs crosslink the sELR. Moreover, we have studied and modeled the release of an antibiotic from the so formed hydrogels. In addition, we have demonstrated the cytocompatibility of this sELR as well as its non-cell adhesive properties, which are crucial properties for its correct performance under its expected field of use, wound healing / dressing. The last aim of this thesis was trying to understand the self-assembly process of a third sELR. Self-assembly is a ubiquitous process found in Nature, from whom we can learn. Nature has demonstrated that is easier to achieve nanostructures by designing the interactions among the individual components rather than rearranging them. Although, nowadays, a deterministic relation among interactions and final self-assembled structure is not feasible. We have developed a sELR bearing two different interaction mechanisms (by means of hydrophobic and electrostatic interactions). Its self-assembly pathway has been elucidated by means of SEM analysis of the different structures obtained with different sELR solutions. The aim of this chapter is to give some light into the relation among sELR’s structure and supramolecular interactions, sELR concentration in solution and how they influence the final self-assembled structures. The importance of this chapter not only relies in the obtaining of never reported structures, but in the comprehensive study on how they are formed.