Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure

Producción CientíficaControl over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a 3D-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.European Commission (NMP-2014-646075, PITNGA-2012-317306)Ministerio de Economía, Industria y Competitividad ( grants PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R, MAT2016-79435-R)Junta de Castilla y León (VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Elastin-like polypeptides in drug delivery

Producción CientíficaThe use of recombinant elastin-like materials, or elastin-like recombinamers (ELRs), in drug-delivery applications is reviewed in this work. Although ELRs were initially used in similar ways to other, more conventional kinds of polymeric carriers, their unique properties soon gave rise to systems of unparalleled functionality and efficiency, with the stimuli responsiveness of ELRs and their ability to self-assemble readily allowing the creation of advanced systems. However, their recombinant nature is likely the most important factor that has driven the current breakthrough properties of ELR-based delivery systems. Recombinant technology allows an unprecedented degree of complexity in macromolecular design and synthesis. In addition, recombinant materials easily incorporate any functional domain present in natural proteins. Therefore, ELR-based delivery systems can exhibit complex interactions with both their drug load and the tissues and cells towards which this load is directed. Selected examples, ranging from highly functional nanocarriers to macrodepots, will be presented.Ministerio de Economía, Industria y Competitividad (Project PRI-PIBAR-2011-1403, MAT2012-38043, MAT2013-42473-R and MAT2013- 4 17 23-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. (VA152A12, VA155A12 and VA313U14

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels

Producción CientíficaThe development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.European Commission (NMP-2014-646075, PITN-GA-2012-317306)Ministerio de Economía, Industria y Competitividad (Projects PCIN-2015-010, MAT2015- 68901-R, MAT2016-78903-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

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

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

Recombinant AMP/Polypeptide Self-Assembled Monolayers with Synergistic Antimicrobial Properties for Bacterial Strains of Medical Relevance

Producción CientíficaNosocomial infections are one of the most frequent causes of indwelling biomedical device failure. In this regard, the use of antibiofilm nanocoatings based on antimicrobial peptides (AMPs) is a promising alternative to prevent multiresistant biofilm infections. However, the limitations of chemical production impede the large-scale development of advanced antimicrobial materials that improve the properties of AMPs. Herein, we present a multifunctional modular design for the recombinant coproduction of self-assembled monolayers (SAMs) based on AMPs and elastin-like recombinamers (ELRs), which combine the antimicrobial properties of a designer AMP, GL13K, and low-fouling activity of an ELR in a synergistic manner. The inclusion of a grafting domain intended for oriented tethering onto surfaces allowed the recombinant polymers to be covalently immobilized onto model gold surfaces. The antibiofilm properties against two of the bacterial strains most frequently responsible for indwelling medical device-associated infections, namely Staphylococcus epidermidis and Staphylococcus aureus, were then evaluated. GL13K peptide was found to provide antibiofilm properties to the surface, with these being synergistically enhanced by the antifouling effect of the ELR. This new design offers a promising tool for the development of advanced AMP-based nanocoatings for medical devices with powerful and enhanced features.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (project PCIN-2015-010 / MAT2015-68901-R / MAT2016-78903-R)Junta de Castilla y León (project VA317P18

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

Producción CientíficaA simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out. After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof. Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV - 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic, hydrophobic and mechanical properties.2020-092020-09Comisión Europea (NMP-2014-646075, MSCA-ITN-2014-642687)Ministerio de Economía, Industria y Competitividad (PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R and MAT2016-79435-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. Project VA015U16)Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y Leó

Biomolecular functionalization for enhanced cell–material interactions of poly(methyl methacrylate) surfaces

Producción CientíficaThe integration of implants or medical devices into the body tissues requires of good cell–material interactions. However, most polymeric materials used for these applications lack on biological cues, which enhanced mid- and long-term implant failure due to weak integration with the surrounding tissue. Commonly used strategies for tissue–material integration focus on functionalization of the material surface by means of natural proteins or short peptides. However, the use of these biomolecules involves major drawbacks such as immunogenic problems and oversimplification of the constructs. Here, designed elastin-like recombinamers (ELRs) are used to enhance poly(methyl methacrylate) surface properties and compared against the use of short peptides. In this study, cell response has been analysed for different functionalization conditions in the presence and absence of a competing protein, which interferes on surface–cell interaction by unspecific adsorption on the interface. The study has shown that ELRs can induce higher rates of cell attachment and stronger cell anchorages than short peptides, being a better choice for surface functionalization.5th China-Europe Symposium on Biomaterials in Regenerative Medicine (CESB 2015) Hangzhou, China April 7–10, 2015Ministerio de Industria, Economía y Competitividad (proyectos MAT2008-06887-C03-01, MAT2010-15310, MAT2013-41723-R, MAT2013-42473-R y BES-2009-027524)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA313U14 y VA244U13

Nanogel Formation from Dilute Solutions of Clickable Elastin-like Recombinamers and its Dependence on Temperature: Two Fractal Gelation Modes

Producción CientíficaDiluted, complementary, click-reactive elastin-like recombinamer (ELR) solutions have been prepared and mixed at two different temperatures, one below and one above the characteristic transition temperature (Tt) of these chemically modified ELRs. FTIR measurements, size, aspect ratio, zeta potential, and microrheological measurements have been carried out on the nanostructures formed under these dilute conditions as a way to better understand the relationship between the final macroscopic properties of ELR-based hydrogels and the molecular conditions governing the initial stages of the chemical cross- linking process that occurs, especially its dependence on the preparation temperature relative to Tt. As a result, two different fractal modes of gel formation have been found at the two temperatures studied (above and below Tt). Thus, when the reaction mixture is prepared below Tt, essentially one-dimensional linear nanogels with a high aspect ratio are obtained. In contrast, 3D nanogels are formed above Tt, with spherical shapes predominating. These different structures seem to reflect the two molecular organizations of the single components of the mixture under these conditions, namely extended chains below Tt and a spherical arrangement above Tt. In addition to the interest in these nanogels as models for understanding the formation of microscopic structures and differential macroscopic properties under more conventional hydrogel-formation conditions, these nanogels are of interest because of their thermoresponsiveness and biocompatibility, which provide them with potential uses for drug delivery and other biomedical applications in living systems.Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13

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