Self-assembling nanosystems based on elastin-like recombinamers and antimicrobial peptides for biomedical applications
- Autores: Sergio Acosta Rodríguez
- Directores de la Tesis: José Carlos Rodríguez Cabello (dir. tes.)
- Lectura: En la Universidad de Valladolid ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Raul Machado (presid.), Israel González de Torre (secret.), Óscar Castaño Linares (voc.)
- Programa de doctorado: Programa de Doctorado en Investigación Biomédica por la Universidad de Valladolid
- Materias:
- Enlaces
- Tesis en acceso abierto en: UVADOC
- Resumen
A more thorough understanding of Nature and its building blocks feeds our ability to develop functional biomaterials and vice versa. Understanding the functions of proteins, the most abundant and diverse macromolecules of a cell, has been crucial for the development of advanced biomaterials for biomedical applications. In this sense, the advent of recombinant DNA technology provided a sustainable production method for of natural proteins, but also the engineering of new protein-based materials, including recombinant protein polymers.
Recombinant protein polymers, or recombinamers, are genetically engineered polypeptides based on conserved motifs found in structural proteins. Within this type of biomaterials, we can find elastin-like recombinamers (ELRs). ELRs are polypeptides based on the repetition of the pentapeptide Val-Pro-Gly-X-Gly, found in the hydrophobic domains of tropoelastin that allowed them to mimic its structural and biomechanical properties. Therefore, ELRs are characterized by intrinsic structural disorder, reversible phase transition behavior and excellent biological and mechanical properties. These properties in addition to low-complexity polymeric composition and outstanding tunability, make them an excellent candidate for the study of biological processes and the generation of tailored materials for biomedical applications. Thus, this Thesis is focused on the design, production and processing ELRs, targeting its biomedical application as self-assembled nanosystems and antimicrobial coatings. To this end, different modular designs will be studied using ELRs and antimicrobial peptides (AMPs) as building blocks. AMPs are short cationic peptides with the ability to kill microbes directly or indirectly modulating the host immune system and hence, they constitute one of the most promising alternative to conventional antibiotics in the treatment of drug-resistant infections. A comprehensive review on biomedical applications of AMPs and ELRs is presented in Chapter 1. A great variety of options enables the processing of these biomaterials into gels, coatings or nanostructures for any of a number of biomedical applications, such as extracellular-matrix-mimicking 3D networks for tissue engineering or self-assembled nanocarriers for drug delivery.
In particular, Chapter 2 is focused on the study of the role of charge distribution in self-assembly and phase separation of intrinsically disordered protein polymers (IDPPs). For this purpose, a library of ELRs based on amphiphilic diblocks was designed altering chain length and charge density. Physico-chemical characterization of the nanostructuration in a range of concentrations determined that charge distribution strongly affects self-assembly of IDPPs at different scale lengths. The incorporation of electrostatic repulsion as a variable in the supramolecular organization of ELRs enabled to reach alternative assemblies and to drive liquid-gel phase transition.
Chapter 3 describes the development of a hybrid polypeptide based on AMPs and ELRs. This chapter seeks to explore the usage of AMPs as self-assembling domains (SADs) and to elucidate the interplay between AMPs and ELRs in the self-assembly with the further objective of creating nanoreservoirs for AMPs. The hybrid design consists in an amphiphilic diblock, where the designer peptides GL13K and 1018 were fused to the hydrophilic block. After recombinant biosynthesis, thermal behavior and self-assembly abilities were characterized. The combination of the two functional domains resulted in a multifaceted polypeptide with a dual self-assembly governed by the composition of the AMP-domain. The AMPs triggered the formation of nanofibers that retained the characteristic thermo-responsiveness of the ELRs. Therefore, increasing the temperature, the hierarchical assembly into fibrillar aggregates could be driven. The interplay of the different SADs (ELR or AMP) offers opportunities for the development of new nanocarriers to deliver AMPs and to study the molecular mechanism that control the bactericidal properties of the AMP linked to biopolymers.
Finally, Chapters 4 and 5 present the creation of covalent coatings based on AMPs and ELRs for the prevention of biofilm formation onto biomedical devices. Specifically, Chapter 4 describes the synergistic association of a low-fouling ELR and the antibiofilm designer peptide GL13K. For this purpose, a hybrid polypeptide based on three functional blocks (GL13K, polycationic ELR and a Cys-motif for the selective tethering onto surfaces) was bioproduced. The modular design enabled the covalent immobilization onto model gold surfaces, forming self-assembled monolayers (SAMs). Antibiofilm activity of the SAMs was tested against two staphylococcal strains of medical relevance in the development of nosocomial infections. The combination of the AMP and ELR in the hybrid design (AMP-ELR-coating) provided a synergistic activity increasing the antibiofilm effect of AMP-coatings under static conditions. Additionally, the nanocoatings demonstrated an excellent cytocompatibility against human cells.
In the same vein, Chapter 5 explores the implementation of AMP-ELR for titanium implants. In addition to the recombinant hybrid polypeptide employed in Chapter 4, a second AMP-ELR was developed by chemical derivation attaching the D-enantiomer of the GL13K to the ELR backbone. Commercially pure (grade II) titanium discs were coated using organosilanes as covalent linkers. Then, antibiofilm activity was tested against two different oral biofilm models (monospecies Streptococcus gordonii and an oral microcosm model) under dynamic conditions in a drip flow biofilm reactor (DFBR). Antibiofilm and cytocompatibility assays showed the strong antibiofilm and bactericidal effect of the D-enantiomer when is linked to an ELR with non-toxic side effects against human cells.
In summary, this Thesis provides new insights on the design and fabrication of self-assembled nanosystems based on genetically engineered polymers. Specifically, it describes the physico-chemical and biological characterization of several novel ELRs and hybrid AMP-ELRs and confirms their potential as multivalent platforms for the study of protein self-assembly processes and for the development of advanced biomaterials with antimicrobial properties.
