RESUMEN
The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.
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Neoplasias de la Mama , Hidrogeles , Humanos , Femenino , Hidrogeles/farmacología , Hidrogeles/química , Elastina/química , Neoplasias de la Mama/tratamiento farmacológico , Materiales Biocompatibles , Péptidos , Matriz ExtracelularRESUMEN
Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.
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Materiales Biocompatibles , Péptidos , Péptidos/química , Estructura MolecularRESUMEN
More than 260 million surgical procedures are performed worldwide each year. Although sutures and staples are widely used to reconnect tissues, they can cause further damage and increase the risk of infection. Bioadhesives have been proposed as an alternative to reconnect tissues. However, clinical adhesives that combine strong adhesion with cytocompatibility have yet to be developed. In this study, we explored the production of adhesives based on protein-engineered polymers bioinspired by the sequence of elastin (i.e., elastin-like recombinamers, ELRs). We hypothesized that the combination of polyphenols (i.e., tannic acid, TA) and ELRs would produce an adhesive coacervate (ELR+TA), as reported for other protein polymers such as silk fibroin (SF). Notably, the adhesion of ELR alone surpassed that of ELR+TA. Indeed, ELR alone achieved adhesive strengths of 88.8 ± 33.2 kPa and 17.0 ± 2.0 kPa on porcine bone and skin tissues, respectively. This surprising result led us to explore a multicomponent bioadhesive to encompass the complementary roles of elastin (mimicked here by ELR) and silk fibroin (SF), and subsequently mirror more closely the multicomponent nature of the extracellular matrix. Tensile testing showed that ELR+SF achieved an adhesive strength of 123.3 ± 60.2 kPa on porcine bone and excellent cytocompatibility. To express this in a more visual and intuitive way, a small surface of only 2.5 cm2 was able to lift at least 2 kg of weight. This opens the door for further studies focusing on the ability of protein-engineered polymers to adhere to biological tissues without further chemical modification for applications in tissue engineering.
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Elastina , Fibroínas , Adhesivos , Elastina/metabolismo , Fibroínas/farmacología , Adherencias Tisulares , Ingeniería de Tejidos/métodos , Animales , PorcinosRESUMEN
Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)n . The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or ß-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order-disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).
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Elastina , Proteínas Intrínsecamente Desordenadas , Materiales Biocompatibles , Péptidos , Polímeros , Conformación Proteica , Pliegue de ProteínaRESUMEN
Intrinsically disordered protein polymers (IDPPs) have attracted a lot of attention in the development of bioengineered devices and for use as study models in molecular biology because of their biomechanical properties and stimuli-responsiveness. The present study aims to understand the effect of charge density on the self-assembly of IDPPs. To that end, a library of recombinant IDPPs based on an amphiphilic diblock design with different charge densities was bioproduced, and their supramolecular assembly was characterized on the nano-, meso-, and microscale. Although the phase transition was driven by the collapse of hydrophobic moieties, the hydrophilic block composition strongly affected hierarchical assembly and, therefore, enabled the production of new molecular architectures, thus leading to new dynamics that govern the liquid-gel transition. These results highlight the importance of electrostatic repulsion for the hierarchical assembly of IDPPs and provide insights into the manufacture of supramolecular protein-based materials.
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Proteínas Intrínsecamente Desordenadas , Interacciones Hidrofóbicas e Hidrofílicas , Transición de Fase , Polímeros , Electricidad EstáticaRESUMEN
The development of intricate and complex self-assembling structures in the micrometer range, such as biomorphs, is a major challenge in materials science. Although complex structures can be obtained from self-assembling materials as they segregate from solution, their size is usually in the nanometer range or requires accessory techniques. Previous studies with intrinsically disordered proteins (IDPs) have shown that the active interplay of different molecular interactions provides access to new and more complex nanostructures. As such, it is hypothesized that enriching the variety of intra- and intermolecular interactions in a model IDP will widen the landscape of sophisticated intermediate structures that can be accessed. In this study, a model silk-elastin-like recombinamer capable of interacting via three non-covalent interactions, namely hydrophobic, ion-pairing, and H-bonding is built. This model material is shown to self-assemble into complex stable micrometer-sized biomorphs. Variation of the block composition, pH, and temperature demonstrates the necessary interplay of all three interactions for the formation of such complex structures.
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Proteínas Intrínsecamente Desordenadas , Elastina , Interacciones Hidrofóbicas e Hidrofílicas , Morfogénesis , TemperaturaRESUMEN
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 self-assembly. 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 ß-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 ß-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.
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Elastina , Seda , Hidrogeles , Cinética , TermodinámicaRESUMEN
Antimicrobial peptides (AMPs) have attracted great interest as they constitute one of the most promising alternatives against drug-resistant infections. Their amphipathic nature not only provides them antimicrobial and immunomodulatory properties but also the ability to self-assemble into supramolecular nanostructures. Here, we propose their use as self-assembling domains to drive hierarchical organization of intrinsically disordered protein polymers (IDPPs). Using a modular approach, hybrid protein-engineered polymers were recombinantly produced, thus combining designer AMPs and a thermoresponsive IDPP, an elastin-like recombinamer (ELR). We exploited the ability of these AMPs and ELRs to self-assemble to develop supramolecular nanomaterials by way of a dual-assembly process. First, the AMPs trigger the formation of nanofibers; then, the thermoresponsiveness of the ELRs enables assembly into fibrillar aggregates. The interplay between the assembly of AMPs and ELRs provides an innovative molecular tool in the development of self-assembling nanosystems with potential use for biotechnological and biomedical applications.
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Proteínas Intrínsecamente Desordenadas , Nanoestructuras , Elastina , Polímeros , Proteínas Citotóxicas Formadoras de PorosRESUMEN
Diffusion of organic and inorganic molecules controls most industrial and biological processes that occur in a liquid phase. Although significant efforts have been devoted to the design and operation of large-scale purification systems, diffusion devices with adjustable biochemical characteristics have remained difficult to achieve. In this regard, micrometer-scale, bioinspired membranes with tunable diffusion properties have been engineered by covalent cross-linking of two elastin-like recombinamers (ELRs) at a liquid-liquid interface. The covalent approach selected provides the desired ELR-based membranes with structural support, and modulation of the concentration of the polypeptides employed confers direct control of the thickness, pore size, and diffusive properties over a broad range of molecular weights (4-150 kDa). The recombinant and versatile nature of the proteinaceous building blocks employed further paves the way to engineering bioactive motifs within the membrane scaffold, thereby widening their applicability in the biological field.
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Elastina , Polímeros , Química Clic , HidrogelesRESUMEN
Tissue engineering for cartilage repair requires biomaterials that show rapid gelation and adequate mechanical properties. Although the use of hydrogel is the most promising biomaterial, it often lacks in rigidity and anchorage of cells when they are surrounded by synovial fluid while they are subjected to heavy loads. We developed and produced the Silk Elastin-Like co-Recombinamer (SELR), which contains both the physical interaction from elastin motifs and from silk motifs. In the first part of this work, we set up and optimized a preannealing treatment based on the evolution of silk motifs into ß-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new preannealed SELRs (pA(EIS)2-(I5R)6) were characterized with the combination of several experimental techniques (CD, TEM, SEM, and rheology) to provide a deep insight into the material features. Finally, the regeneration properties of the pA(EIS)2-(I5R)6 hydrogel embedded with chondrocytes were evaluated. After 4 weeks of culturing in a standardized and representative ex vivo model, the biochemical and histological analysis revealed the production of glycosaminglycans and collagen. Moreover, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage. Hence, we conclude that the pA(EIS)2-(I5R)6 hydrogel presents improved mechanical properties while conserving the injectability, which leads to successful regeneration of hyaline cartilage in an ex vivo model.
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Cartílago Articular/citología , Condrocitos/citología , Condrogénesis , Elastina/química , Hidrogeles/química , Regeneración , Seda/química , Animales , Materiales Biocompatibles/química , Células Cultivadas , Femenino , Masculino , Reología , Porcinos , Ingeniería de Tejidos/métodosRESUMEN
In 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.
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Elastina/química , Proteínas Fluorescentes Verdes/química , Hidrogeles/química , Seda/química , Animales , Materiales Biocompatibles , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Recombinantes/química , Escifozoos/químicaRESUMEN
Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.
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Materiales Biocompatibles/química , Elastina/química , Cartílago Hialino/crecimiento & desarrollo , Hidrogeles/química , Células Madre Mesenquimatosas/citología , Regeneración , Animales , Fenómenos Biomecánicos , Células de la Médula Ósea/metabolismo , Huesos/metabolismo , Cartílago Articular/patología , Humanos , Imagenología Tridimensional , Imagen por Resonancia Magnética , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Conejos , Reproducibilidad de los Resultados , Ingeniería de Tejidos/métodos , Tomografía Computarizada por Rayos X , Trasplante HeterólogoRESUMEN
The 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 environment.
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Quitosano/química , Materiales Biocompatibles Revestidos/química , Prótesis e Implantes , Antibacterianos/química , Adhesión Celular , Implantes Dentales , Microscopía de Fuerza Atómica , Oseointegración , Osteoblastos/metabolismo , Espectroscopía de Fotoelectrones , Proteínas/química , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Titanio/químicaRESUMEN
Investigation of the aggregation behavior of biohybrid microgels, which can potentially be used as drug carriers, is an important topic, because aggregation not only causes loss of activity, but also toxicity and immunogenicity. To study this effect we synthesized microgels from elastin-like recombinamers (ELRs) using the miniemulsion technique. The existence of aggregation for such biohybrid microgels at different concentrations and temperatures was studied by different methods which include dynamic light scattering (DLS), (1)H high-resolution magic angle sample spinning (HRMAS) NMR spectroscopy, relaxometry and diffusometry. A hysteresis effect was detected in the process of aggregation by DLS as a function of temperature that strongly depends on ELR microgel concentration. The aggregation process was further quantitatively analyzed by the concentration dependence of the (1)H amino-acid residue chemical shifts and microgel diffusivity measured by NMR methods using the population balance kinetic aggregation model.
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Elastina/química , Geles/química , Portadores de Fármacos , Dispersión Dinámica de Luz , Espectroscopía de Resonancia Magnética , TemperaturaRESUMEN
The 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 (1) H high-resolution magic angle sample spinning (HRMAS), nuclear magnetic resonance (NMR) spectroscopy, and relaxometry methods. The findings by (1) H 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 , (1) H NMR peak as compared to the proline ß-CH2 .
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Materiales Biomiméticos/química , Oligopéptidos/química , Materiales Biomiméticos/síntesis química , Portadores de Fármacos , Elastina/química , Emulsiones , Floculación , Geles , Interacciones Hidrofóbicas e Hidrofílicas , Oligopéptidos/síntesis química , Transición de Fase , Temperatura , TermodinámicaRESUMEN
This 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.
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Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Elastina/química , Nanotecnología/métodos , Animales , Humanos , Hidrogeles/química , Proteínas Recombinantes/químicaRESUMEN
In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.
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Antiinfecciosos/química , Antiinfecciosos/farmacología , Bombyx , Elastina/química , Elastina/farmacología , Animales , Línea Celular , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Humanos , Absorción Cutánea/efectos de los fármacos , Absorción Cutánea/fisiología , PorcinosRESUMEN
Elastin-like recombinamer click gels (ELR-CGs) for biomedical applications, such as drug delivery or tissue engineering, have been developed by taking advantage of the click reaction (CuAAC) in the absence of traditional crosslinking agents. ELRs are functionalized with alkyne and azide groups using conventional chemical techniques to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. Hydrogels with moduli in the range 1,000-10,000 Pa have been synthesized, characterized, and tested in vitro against several cell types. The cells embedded into ELR-CGs possessed high viability and proliferation rate. The mechanical properties, porosity and swelling of the resulting ELR-CGs can easily be tuned by adjusting the ELR concentration. We also show that it is possible to replicate different patterns on the hydrogel surface, thus allowing the use of this type of hydrogel to improve applications that require cell guidance or even differentiation depending on the surface topography.
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Materiales Biocompatibles/síntesis química , Química Clic/métodos , Elastina/química , Hidrogeles/síntesis química , Células Madre Mesenquimatosas/fisiología , Ingeniería de Proteínas/métodos , Materiales Biomiméticos/síntesis química , Línea Celular , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Elastina/genética , Elastina/ultraestructura , Humanos , Ensayo de Materiales , Células Madre Mesenquimatosas/citologíaRESUMEN
Developing in vitro models that accurately mimic the microenvironment of biological structures or processes holds substantial promise for gaining insights into specific biological functions. In the field of tissue engineering and regenerative medicine, in vitro models able to capture the precise structural, topographical, and functional complexity of living tissues, prove to be valuable tools for comprehending disease mechanisms, assessing drug responses, and serving as alternatives or complements to animal testing. The choice of the right biomaterial and fabrication technique for the development of these in vitro models plays an important role in their functionality. In this sense, elastin-like recombinamers (ELRs) have emerged as an important tool for the fabrication of in vitro models overcoming the challenges encountered in natural and synthetic materials due to their intrinsic properties, such as phase transition behavior, tunable biological properties, viscoelasticity, and easy processability. In this review article, we will delve into the use of ELRs for molecular models of intrinsically disordered proteins (IDPs), as well as for the development of in vitro 3D models for regenerative medicine. The easy processability of the ELRs and their rational design has allowed their use for the development of spheroids and organoids, or bioinks for 3D bioprinting. Thus, incorporating ELRs into the toolkit of biomaterials used for the fabrication of in vitro models, represents a transformative step forward in improving the accuracy, efficiency, and functionality of these models, and opening up a wide range of possibilities in combination with advanced biofabrication techniques that remains to be explored.
RESUMEN
Despite the remarkable progress in the generation of recombinant elastin-like (ELR) hydrogels, further improvements are still required to enhance and control their viscoelasticity, as well as limit the use of expensive chemical reagents, time-consuming processes and several purification steps. To alleviate this issue, the reactivity of carboxylic groups from glutamic (E) acid distributed along the hydrophilic block of an amphiphilic ELR (coded as E50I60) with amine groups has been studied through a one-pot amidation reaction in aqueous solutions, for the first time. By means of this approach, immediate conjugation of E50I60 with molecules containing amine groups has been performed with a high yield, as demonstrated by the 1H NMR and MALDI-TOF spectroscopies. This has resulted in the preparation of viscoelastic irreversible hydrogels through the "in-situ" cross-linking of E50I60 with another ELR (coded as VKV24) containing amine groups from lysines (K). The rheology analysis demonstrated that the gelation process takes place following a dual mechanism dependent on the ELR concentration: physical cross-linking of I60 block through the hydrophobic interactions, and covalent cross-linking of E50I60 with VKV24 through the amidation reaction. While the chemical network formed between the hydrophilic E50 block and VKV24 ELR preserves the elasticity of ELR hydrogels, the self-assembly of the I60 block through the hydrophobic interactions provides a tunable physical network. The presented investigation serves as a basis for generating ELR hydrogels with tunable viscoelastic properties promising for tissue regeneration, through an ''in-situ", rapid, scalable, economically and feasible one-pot method.