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

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

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

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

Biomimetic Mineralization of Recombinamer-Based Hydrogels toward Controlled Morphologies and High Mineral Density

Producción CientíficaThe use of insoluble organic matrices as a structural template for the bottom-up fabrication of organic−inorganic nanocomposites is a powerful way to build a variety of advanced materials with defined and controlled morphologies and superior mechanical properties. Calcium phosphate mineralization in polymeric hydrogels is receiving significant attention in terms of obtaining biomimetic hierarchical structures with unique mechanical properties and understanding the mechanisms of the biomineralization process. However, integration of organic matrices with hydroxyapatite nanocrystals, different in morphology and composition, has not been well-achieved yet at nanoscale. In this study, we synthesized thermoresponsive hydrogels, composed of elastin-like recombinamers (ELRs), to template mineralization of hydroxyapatite nanocrystals using a biomimetic polymer-induced liquid-precursor (PILP) mineralization process. Different from conventional mineralization where minerals were deposited on the surface of organic matrices, they were infiltrated into the frameworks of ELR matrices, preserving their microporous structure. After 14 days of mineralization, an average of 78 μm mineralization depth was achieved. Mineral density up to 1.9 g/cm3 was found after 28 days of mineralization, which is comparable to natural bone and dentin. In the dry state, the elastic modulus and hardness of the mineralized hydrogels were 20.3 ± 1.7 and 0.93 ± 0.07 GPa, respectively. After hydration, they were reduced to 4.50 ± 0.55 and 0.10 ± 0.03 GPa, respectively. These values were lower but still on the same order of magnitude as those of natural hard tissues. The results indicated that inorganic−organic hybrid biomaterials with controlled morphologies can be achieved using organic templates of ELRs. Notably, the chemical and physical properties of ELRs can be tuned, which might help elucidate the mechanisms by which living organisms regulate the mineralization process.Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13

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

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

Nanostructured thin coatings from chitosan and an elastin-like recombinamer with acute stimuli-responsive behavior

Publicado em: Materials Science Forum, vols. 730-732; Online available since 2012/Nov/12In the present work, chitosan (CHI) and elastin-like recombinamers (ELRs) were used to conceive nanostructured thin films driven by sequential electrostatic layer-by-layer (LbL), a simple and versatile technique that discards the use of harmful reagents. Two similar ELRs were engineered to contain negatively charged aminoacids and organized and a single monoblock or a triblock. The buildup of the films was monitored in real time using a quartz-crystal microbalance with dissipation monitoring (QCM-D). Wettability transitions were observed from a moderate hydrophobic surface to an extremely wettable upon increasing the temperature to 50 ºC, accompanied by topography changes at the nanoscale as assessed by atomic force microscopy (AFM). Furthermore, the dependence on time for the surface molecular rearrangement was studied for the films with each ELR. The potential of this technology may stimulate the development of devices and biomaterials for biomedical applications in the near future, such as surfaces with tunable and patterned cell adhesion, while the use of ELRs will allow developing polypeptides with biological significance.The authors acknowledge the financial support through Fundação para a Ciência e Tecnologia (FCT, PhD grant SFRH/BD/61126/2009, ‘‘Junta de Castilla y Leon’’ (VA034A09), the MICINN (MAT 2009-14195-C03-03, ACI2009-0890, IT2009-0089, MAT2010-15310, and MAT2010- 15982), the CIBER-BBN and the ‘‘Network Center of Regenerative Medicine and Cellular Therapy of Castilla y León’’.MICINNFundação para a Ciência e a Tecnologia (FCT)Junta de Castilla y LeónCIBER-BBNNetwork Center of Regenerative Medicine and Cellular Therapy of Castilla y Leó