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

Intrafibrillar Mineralization of Self-Assembled Elastin-Like Recombinamer Fibrils

Producción CientíficaBiomineralization of bone, a controlled process where hydroxyapatite nanocrystals preferentially deposit in collagen fibrils, is achieved by the interplay of the collagen matrix and noncollagenous proteins. Mimicking intrafibrillar mineralization in synthetic systems is highly attractive for the development of advanced hybrid materials with elaborated morphologies and outstanding mechanical properties, as well as understanding the mechanisms of biomineralization. Inspired by nature, intrafibrillar mineralization of collagen fibrils has been successfully replicated in vitro via biomimetic systems, where acidic polymeric additives are used as analogue of noncollagenous proteins in mediating mineralization. The development of synthetic templates that mimic the structure and functions of collagenous matrix in mineralization has yet to be explored. In this study, we demonstrated that self-assembled fibrils of elastin-like recombinamers (ELRs) can induce intrafibrillar mineralization. The ELRs displayed a disordered structure at low temperature but self-assembled into nanofibrils above its inverse transition temperature. In the presence of the self-assembled ELR fibrils, polyaspartate-stabilized amorphous calcium phosphates preferentially infiltrated into the fibrils and then crystallized into hydroxyapatite nanocrystals with their [001] axes aligned parallel to the long axis of the ELR fibril. As the recombinant technology enables designing and producing well-defined ELRs, their molecular and structural properties can be fine-tuned. By examining the ultrastructure of the self-assembled ELRs fibrils as well as their mineralization, we concluded that the spatial confinement formed by a continuum β-spiral structure in an unperturbed fibrillar structure rather than electrostatic interactions or bioactive sequences in the recombinamer composition played the crucial role in inducing intrafibrillar mineralization.2018-08-01Ministerio de Economía, Industria y Competitividad (Project MAT2013-42473-R and MAT2015-68901R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and VA015U16

A transferrin receptor-binding mucoadhesive elastin-like recombinamer: In vitro and in vivo characterization

Producción CientíficaThe development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro.2021-02-192021-02-19Ministerio de Economía, Industria y Competitividad (Projects PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R, MAT2016-79435-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA015U16)European Commission (NMP-2014-646075

Elastin-like recombinamers: Deconstructing and recapitulating the functionality of extracellular matrix proteins using recombinant protein polymers

Producción CientíficaIn the development of tissue engineering strategies to replace, remodel, regenerate, or support damaged tissue, the development of bioinspired biomaterials that recapitulate the physicochemical characteristics of the extracellular matrix has received increased attention. Given the compositional heterogeneity and tissue-to-tissue variation of the extracellular matrix, the design, choice of polymer, crosslinking, and nature of the resulting biomaterials are normally depended on intended application. Generally, these biomaterials are usually made of degradable or nondegradable biomaterials that can be used as cell or drug carriers. In recent years, efforts to endow reciprocal biomaterial–cell interaction properties in scaffolds have inspired controlled synthesis, derivatization, and functionalization of the polymers used. In this regard, elastin-like recombinant proteins have generated interest and continue to be developed further owing to their modular design at a molecular level. In this review, the authors provide a summary of key extracellular matrix features relevant to biomaterials design and discuss current approaches in the development of extracellular matrix-inspired elastin-like recombinant protein based biomaterials.Comisión Europea (grants MSCA-ITN-2014-ETN-642687 and NMP-2014-646075)Gobierno de España (grants MAT2016-78903-R, MAT2015-68901-R, and RTI2018-096320-B-C22)Junta de Castilla y León (project VA317P18)"Interreg V A España Portugal" Programa operativo de cooperación transfronteriza España Portugal (POCTEP) (grant 0624_2IQBIONEURO_6_E

Coacervation of Elastin-Like Recombinamer Microgels

Producción CientíficaThe investigation of the coacervation (self-aggregation) behavior of biomicrogels which can potentially be used as drug carriers is an important topic, because self-aggregation can not only cause loss of activity, but also toxicity and immunogenicity. To study this effect microgels from elastin-like recombinamer are synthesized using miniemulsion technique. The existence of coacervation for such microgels, at different concentrations and temperatures, is studied and proved by cryo-field emission scanning clectron microscopy (cryo-FESEM), cryo-transmission electron microscopy (cryo-TEM), and by a novel 1H high-resolution magic angle sample spinning (HRMAS), nuclear magnetic resonance (NMR) spectroscopy, and relaxometry methods. The findings by 1H HRMAS NMR spectroscopy and relaxometry show simultaneous processes of volume phase temperature transition and coacervation with different sensitivity for hydrophobic and hydrophilic amino acid side-chains in the microgel. The coacervation process is more evidential by the behavior of glycine α-CH2, 1H NMR peak as compared to the proline β-CH2.2019-12-122019-12-12Ministerio de Industria, Economía y Competitividad (proyectos MAT2013-42473-R y MAT2013-41723-R

Chitosan-recombinamer layer-by-layer coatings for multifunctional implants

Producción CientíficaThe main clinical problems for dental implants are (1) formation of biofilm around the implant—a condition known as peri-implantitis and (2) inadequate bone formation around the implant—lack of osseointegration. Therefore, developing an implant to overcome these problems is of significant interest to the dental community. Chitosan has been reported to have good biocompatibility and anti-bacterial activity. An osseo-inductive recombinant elastin-like biopolymer (P-HAP), that contains a peptide derived from the protein statherin, has been reported to induce biomineralization and osteoblast differentiation. In this study, chitosan/P-HAP bi-layers were built on a titanium surface using a layer-by-layer (LbL) assembly technique. The difference in the water contact angle between consecutive layers, the representative peaks in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), X-ray photoelectron spectroscopy (XPS), and the changes in the topography between surfaces with a different number of bi-layers observed using atomic force microscopy (AFM), all indicated the successful establishment of chitosan/P-HAP LbL assembly on the titanium surface. The LbL-modified surfaces showed increased biomineralization, an appropriate mouse pre-osteoblastic cell response, and significant anti-bacterial activity against Streptococcus gordonii, a primary colonizer of tissues in the oral environmentMinisterio de Economía, Industria y Competitividad (Project MAT2013-42473-R and MAT2015-68901R)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and VA015U16

Histopathological image analysis for breast cancer diagnosis by ensembles of convolutional neural networks and genetic algorithms

One of the most invasive cancer types which affect women is breast cancer. Unfortunately, it exhibits a high mortality rate. Automated histopathological image analysis can help to diagnose the disease. Therefore, computer aided diagnosis by intelligent image analysis can help in the diagnosis tasks associated with this disease. Here we propose an automated system for histopathological image analysis that is based on deep learning neural networks with convolutional layers. Rather than a single network, an ensemble of them is built so as to attain higher recognition rates, which are obtained by computing a consensus decision from the individual networks of the ensemble. A final step involves the optimization of the set of networks that are included in the ensemble by a genetic algorithm. Experimental results are provided with a set of benchmark images, with favorable outcomes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

A double safety lock tumor-specific device for suicide gene therapy in breast cancer

Producción CientíficaThe complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.Fondo Social Europeo - FEDER- Unión Europea (project MP-2014-646075Ministerio de Economía, Industria y Competitividad (project PCIN-2015-010 / MAT2015-68901-R / MAT2016-78903-R)Junta de Castilla y León (project VA317P18

Layer-by-layer biofabrication of coronary covered stents with clickable elastin-like recombinamers

Producción CientíficaCoronary artery disease is the leading cause of death around the world. Endovascular stenting is the preferred treatment option to restore blood flow in the coronary arteries due to the lower perioperative morbidity when compared with more invasive treatment options. However, stent failure is still a major clinical problem, and further technological solutions are required to improve the performance of current stents. Here, we developed coronary stents covered with elastin-like recombinamers (ELRs) by exploiting a layer-by-layer technique combined with catalyst-free click chemistry. The resulting ELR-covered stents were intact after an in vitro simulated implantation procedure by balloon dilatation, which evidenced the elastic performance of the membrane. Additionally, the stents were mechanically stable under high flow conditions, which is in agreement with the covalent and stable nature of the click chemistry crosslinking strategy exploited during the ELR-membrane manufacturing and the successful embedding of the stent. Minimal platelet adhesion was detected after blood exposure in a Chandler loop as shown by scanning electron microscopy. The seeding of human endothelial progenitor cells (EPCs) on the ELR-membranes resulted in a confluent endothelial layer. These results prove the potential of this strategy to develop an advanced generation of coronary stents, with a stable and bioactive elastin-like membrane to exclude the atherosclerotic plaque from the blood stream or to seal coronary perforations and aneurysms, while providing a luminal surface with minimal platelet adhesion and favouring endothelialization.German federal and state governments (project StUpPD_330-18)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R)Junta de Castilla y León (project VA317P18