Novel Hydrogel-forming elastin-like recombinamers for biomedical applications

There is an increasing interest in developing advanced biomaterials with improved biocompatibility and functionality that might find uses in the field of biomedicine, for example in tissue engineering and regenerative medicine (TERM). Nowadays, recombinant polypeptides or polymers are one of the most prominent types of biomaterials, due to their nature. They are obtained through recombinant DNA technology, which allows the controlled biosynthesis of tailored polypeptides that may be designed to include combinations of polymeric amino acid sequences and/or bioactive domains. Within this type of biomaterials we can find elastin-like polypeptides (ELPs), which have also been recently termed elastin-like recombinamers (ELRs), according to their recombinant origin. These ELRs are composed of repetitions of the VPGXG (Val-Pro-Gly-X-Gly) pentapeptide, in which X (guest residue) can be any amino acid expect L-Proline. This composition confers the ELRs a smart behaviour of thermoresponsiveness defined by the so-called Inverse Temperature Transition (ITT) occurring above the Transition Temperature (Tt), which implies a phase transition of the dissolved ELR when this Tt is reached. Therefore, this thermal response is of great interest, since it has been shown to promote the formation of different structures, such as hydrogels that mimic the extracellular matrix, at the physiological temperature, whereas an ELR solution can be easily handled (e.g. injected in vivo) below the Tt. Furthermore, the Tt can be modulated depending on the polarity of the side chain of the amino acid chosen as guest residue. Moreover, if this residue contains functional goups, it can also be used for further chemical modifications in order to achieve, for instance, chemically (covalently) cross-linked hydrogels.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Synthesis and characterization of a silk-elastin-like recombinamer fused to enhanced green fluorescent protein (SELR-EGFP) for the achievement of self-assembled fluorescent nanoparticles and physically cross-linked hydrogels

Se ha realizado el diseño, síntesis y caracterización de un recombinámero de tipo elastina fusionado a la proteína verde fluorescente, con la capacidad de formar distintas estructuras por auto-ensamblado en respuesta a la temperatura, desde nanopartículas a hidrogeles, con una aplicación potencial en el desarrollo de dispositivos biomédicos. Además, se han utilizado técnicas nanotecnológicas para el desarrollo de partículas fluorescentes con capacidad de adhesión e internalización celular.Departamento de Física de la Materia Condensada, Cristalografía y MineralogíaMáster en Nanociencia y Nanotecnología Molecula

Trends in the design and use of elastin-like recombinamers as biomaterials

Producción CientíficaElastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R / BES-2014-069763)Junta de Castilla y León (project VA317P18

FRET-Paired Hydrogel Forming Silk-Elastin-Like Recombinamers by Recombinant Conjugation of Fluorescent Proteins

Producción CientíficaIn the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescens enhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G′ = 60–4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06–0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea (NMP-2014-646075, HEALTH-F4-2011-278557, PITN-GA-2012-317306 and MSCA-ITN-2014-642687), del MINECO (MAT2016-7R8903-R, MAT2016-79435-R, MAT2013-42473-R, MAT2013-41723- and MAT2012-38043), la Junta de Castilla y León (VA244U13 and VA313U14) y el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Recombinant Technology in the Development of Materials and Systems for Soft-Tissue Repair

Producción CientíficaThe field of biomedicine is constantly investing significant research efforts in order to gain a more in-depth understanding of the mechanisms that govern the function of body compartments and to develop creative solutions for the repair and regeneration of damaged tissues. The main overall goal is to develop relatively simple systems that are able to mimic naturally occurring constructs and can therefore be used in regenerative medicine. Recombinant technology, which is widely used to obtain new tailored synthetic genes that express polymeric protein-based structures, now offers a broad range of advantages for that purpose by permitting the tuning of biological and mechanical properties depending on the intended application while simultaneously ensuring adequate biocompatibility and biodegradability of the scaffold formed by the polymers. This Progress Report is focused on recombinant protein-based materials that resemble naturally occurring proteins of interest for use in soft tissue repair. An overview of recombinant biomaterials derived from elastin, silk, collagen and resilin is given, along with a description of their characteristics and suggested applications. Current endeavors in this field are continuously providing more-improved materials in comparison with conventional ones. As such, a great effort is being made to put these materials through clinical trials in order to favor their future use.Ministerio de Industria, Economía y Competitividad (proyectos PRI-PIBAR-2011–1403, MAT2012–38043, MAT2013–42473-R y MAT2013–41723-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA152A12, VA155A12 y VA313U14)CIBER-BBN, y la Junta de Castilla y León y el Instituto de Salud Carlos III mediante el "Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Bioactive scaffolds based on elastin-like materials for wound healing

Producción CientíficaWound healing is a complex process that, in healthy tissues, starts immediately after the injury. Even though it is a natural well-orchestrated process, large trauma wounds, or injuries caused by acids or other chemicals, usually produce a non-elastic deformed tissue that not only have biological reduced properties but a clear aesthetic effect. One of the main drawbacks of the scaffolds used for wound dressing is the lack of elasticity, driving to non-elastic and contracted tissues. In the last decades, elastin based materials have gained in importance as biomaterials for tissue engineering applications due to their good cyto- and bio-compatibility, their ease handling and design, production and modification. Synthetic elastin or elastin like-peptides (ELPs) are the two main families of biomaterials that try to mimic the outstanding properties of natural elastin, elasticity amongst others; although there are no in vivo studies that clearly support that these two families of elastin based materials improve the elasticity of the artificial scaffolds and of the regenerated skin. Within the next pages a review of the different forms (coacervates, fibres, hydrogels and biofunctionalized surfaces) in which these two families of biomaterials can be processed to be applied in the wound healing field have been done. Here, we explore the mechanical and biological properties of these scaffolds as well as the different in vivo approaches in which these scaffolds have been used.Ministerio de Economía, Industria y Competitividad (Projects MAT2015-68901-R, MAT2016-78903-R, PCIN-2015-010)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)European Commission (ELASTISLET N. 646075

Elastin-like recombinamers as smart drug delivery systems

Drug delivery systems that are able to control the release of bioactive molecules and designed to carry drugs to target sites are of particular interest for tissue therapy. Moreover, systems comprising materials that can respond to environmental stimuli and promote self-assembly and higher order supramolecular organization are especially useful in the biomedical field. Suitable biomaterials for this purpose include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin and designed usingrecombinant technologies. The self-assembly and thermoresponsive behaviour of these systems, along with their biodegradability, biocompatibility and well-defined composition as a result of their tailor-made design, make them particularly attractive for drug delivery.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea a través del Fondo Social Europeo (FSE) y el Fondo Europeo de Desarrollo Regional (ERDF), por el del MINECO (MAT2013-41723R, MAT2013-42473-R, MAT2012-38043 y PRI-PIBAR-2011-1403), la Junta de Castilla y León (VA049A11, VA152A12 y VA155A12) y el Instituto de Salud Carlos III bajo el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Nanotechnological Approaches to Therapeutic Delivery Using Elastin-Like Recombinamers

Producción CientíficaThis Review discusses the use of elastin-like polymers and their recombinant version, elastin-like recombinamers, in drug-delivery systems. These macromolecules exhibit a number of interesting properties that are rarely found together in any other family of materials, especially extremely high biocompatibility, high bioactivity and functionality, complex yet fully controlled composition, and stimuli responsiveness. Appropriate design of these molecules opens up a broad range of different possibilities for their use in new therapeutic platforms. The first of these described herein is the use of ELRs in single-molecule devices as therapeutic entities in their own right. Subsequently, we describe how the self-assembly properties of these materials can be exploited to create nanocarriers and, eventually, microcarriers that are able to temporally and spatially control and direct the release of their drug load. Intracellular drug-delivery devices and nanocarriers for treating cancer are among the uses described in that section. Finally, the use of ELRs as base materials for implantable drug depots, in the form of hydrogels, is discussed.Ministerio de Industria, Economía y Competitividad (proyectos PRI-PIBAR-2011–1403, MAT2012–38043, MAT2013–42473-R y MAT2013–41723-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA152A12, VA155A12 y VA313U14)CIBER-BBN, y la Junta de Castilla y León y el Instituto de Salud Carlos III mediante el "Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering

Producción CientíficaHerein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications.Ministerio de Economía, Industria y Competitividad (Projects MAT2015-68901-R, MAT2016-78903-R, PCIN-2015-010)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)European Commission (ELASTISLET No 646075, BIOGEL No 642687

Influence of the Thermodynamic and Kinetic Control of Self‐Assembly on the Microstructure Evolution of Silk‐Elastin‐Like Recombinamer Hydrogels

Complex recombinant biomaterials that merge the self-assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin-like (EL) and silk-like (SL) polypeptides are combined to obtain a silk-elastin-like recombinamer (SELR) with dual selfassembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of β-sheets, stabilized by H-bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in β-sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.Este trabajo forma parte de los proyectos de investigación MAT2016-78903-R, MAT2016-79435-R, RTI2018-096320-B-C22 y DTS19/00162 del Ministerio de Ciencia e Innovación, del proyecto VA317P18 de la Junta de Castilla y León, del proyecto 0624_2IQBIONEURO_6_E del programa Interreg V A España Portugal POCTEP y del Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó