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Elastin polypeptides based on -VPGVG- repeated motifs are widely used in the production of biomaterials because they are stimuli-responsive systems. On the other hand, glycine-rich sequences, mainly present in tropoelastin terminal domains, are responsible for the elastin self-assembly. In a previous study, we have recombinantly expressed a chimeric polypeptide, named resilin, elastin, and collagen (REC), inspired by glycine-rich motifs of elastin and containing resilin and collagen sequences as well. Herein, a three-block polypeptide, named (REC)3, was expressed starting from the previous monomer gene by introducing key modifications in the sequence. The choice was mandatory because the uneven distribution of the cross-linking sites in the monomer precluded the hydrogel production. In this work, the cross-linked polypeptide appeared as a soft hydrogel, as assessed by rheology, and the linear un-cross-linked trimer self-aggregated more rapidly than the REC monomer. The absence of cell-adhesive sequences did not affect cell viability, while it was functional to the production of a material presenting antiadhesive properties useful in the integration of synthetic devices in the body and preventing the invasion of cells.

Producción Científica, The concept of cancer as a systemic disease, and the therapeutic implications of this, has gained special relevance. This concept encompasses the interactions between tumor and stromal cells and their microenvironment in the complex setting of primary tumors and metastases. These factors determine cellular co-evolution in time and space, contribute to tumor progression, and could counteract therapeutic effects. Additionally, cancer therapies can induce cellular and molecular responses in the tumor and host that allow them to escape therapy and promote tumor progression. In this study, we describe the vascular network, tumor-infiltrated immune cells, and cancer-associated fibroblasts as sources of heterogeneity and plasticity in the tumor microenvironment, and their influence on cancer progression. We also discuss tumor and host responses to the chemotherapy regimen, at the maximum tolerated dose, mainly targeting cancer cells, and a multimodal metronomic chemotherapy approach targeting both cancer cells and their microenvironment. In a combination therapy context, metronomic chemotherapy exhibits antimetastatic efficacy with low toxicity but is not exempt from resistance mechanisms. As such, a better understanding of the interactions between the components of the tumor microenvironment could improve the selection of drug combinations and schedules, as well as the use of nano-therapeutic agents against certain malignancies., Ministerio de Ciencia e Innovación y Ministerio de Universidades - CIBER-BBN e ISCIII (DTS19/00162 y PID2019-106386RB-I00)

Cancer has reached pandemic dimensions in the whole world. Although current medicine offers multiple treatment options against cancer, novel therapeutic strategies are needed due to the low specificity of chemotherapeutic drugs, undesired side effects and the presence of different incurable types of cancer. Among these new strategies, nanomedicine arises as an encouraging approach towards personalized medicine with high potential for present and future cancer patients. Therefore, nanomedicine aims to develop novel tools with wide potential in cancer treatment, imaging or even theranostic purposes. Even though numerous preclinical studies have been published with successful preliminary results, promising nanosystems have to face multiple obstacles before adoption in clinical practice as safe options for patients with cancer. In this MiniReview, we provide a short overview on the latest advances in current nanomedicine approaches, challenges and promising strategies towards more accurate cancer treatment.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

One of the main goals in both tissue engineering and regenerative medicine is to design innovative synthetic scaffolds that can simulate and control the communication pathways between cells and the extracellular matrix (ECM). In this context, we describe herein the characterization of protein polymer, a recombinant elastin-like recombinamer (ELR) designed for developing tissue-engineered devices for use in vascular regeneration. This ELR is composed of an elastin-like backbone that contains a fibronectin domain, which provides specific, endothelial cell adhesion, and a protease target domain directed towards specific proteases involved in ECM remodeling. We also compare the specific response of endothelial and fibroblast cells to ELR scaffolds and show that cell adhesion and spreading on this ELR is significantly higher for endothelial cells than for fibroblasts. The reactivity of this polymer and its hydrogels to specific enzymatic degradation is demonstrated in vitro. As with natural elastin, enzymatic hydrolysis of the ELR produces elastin-derived peptides, or "matrikines", which, in turn, are potentially able to regulate important cell activities.

Producción Científica, The 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-2014-646075, Ministerio de Economía, Industria y Competitividad (project PCIN-2015-010 / MAT2015-68901-R / MAT2016-78903-R), Junta de Castilla y León (project VA317P18)

Producción Científica, This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug delivery system based on elastin-like block recombinamers. The DNA recombinant technics allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled, when temperature was increased above 15°C, into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were showed to enter in the cells via clathrin-dependent endocytosis, and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas non-cancerous cells, such as endothelial cells, fibroblasts and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieve successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides in order to modulate multiple cellular processes involved in different diseases., Ministerio de Economía, Industria y Competitividad (PCIN-2015-010, MAT2015-68901-R, MAT2016-79435-R and MAT2016-78903-R), Junta de Castilla y León (VA317P18), European Union (NMP-2014-646075)

Producción Científica, Biomaterials science is one of the most rapidly evolving fields in biomedicine. However, although novel biomaterials have achieved well-defined goals, such as the production of devices with improved biocompatibility and mechanical properties, their development could be more ambitious. Indeed, the integration of active targeting strategies has been shown to allow spatiotemporal control of cell–material interactions, thus leading to more specific and better-performing devices. This manuscript reviews recent advances that have led to enhanced biomaterials resulting from the use of natural structural macromolecules. In this regard, several structural macromolecules have been adapted or modified using biohybrid approaches for use in both regenerative medicine and therapeutic delivery. The integration of structural and functional features and aptamer targeting, although still incipient, has already shown its ability and wide-reaching potential. In this review, we discuss aptamer-functionalized hybrid protein-based or polymeric biomaterials derived from structural macromolecules, with a focus on bioresponsive/bioactive systems., Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional - Fondo Social Europeo (Proyects MAT2016-79435-R, DTS19/00162, and PID2019-106386RB-I00), Junta de Castilla y León (Project VA317P18)

Producción Científica, The 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., 2021-02-19, Ministerio 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)

Producción Científica, This work focuses on improving the effectiveness of current therapies against glaucoma by incorporating self-assembled polymers into the ophthalmic formulation. To that end, we first studied the influence of the dispersing medium on the mechanical performance of self-assembling elastin-like (EL) and silk-elastin-like (SEL) hydrogels by conducting rheological tests. These polymers were subsequently incorporated into the antiglaucoma formulation, which contains timolol maleate (TM) as active ingredient, and in vivo tests, namely adhesion tests and intraocular pressure measurements (IOP), were performed in New Zealand rabbits. An enhanced reduction in IOP due to the presence of the polymers was observed. Moreover, differences in the effectiveness between both EL- and SEL-hydrogels, which can be explained on the basis of the different rheological properties displayed by these two systems, were also encountered. The results point to the potential of this system as a basis for the development of an ophthalmic formulation against glaucoma., European Social Fund (ESF) and European Regional Development Fund (ERDF), Comisión Europea (proyectos NMP-2014-646075, HEALTH-F4-2011-278557, PITN-GA-2012-317306, y MSCA-ITN-2014-642687), Ministerio de Economía, Industria y Competitividad (Projects MAT2015-68901-R, MAT2016-79435-R and MAT2016-78903-R), Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13 y VA313U14), CIBER-BBN, Instituto de Salud Carlos III a través de la "Red de Medicina Regenerativa y Terapia Celular de Castilla y León", con el apoyo del “Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) y la Universidad Nacional de Córdoba (Argentina)

Producción Científica, 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, Ministerio de Economía, Industria y Competitividad (Project (MAT2016-78903-R, MAT2016-9 79435-R, MAT2013-42473-R, MAT2013-41723-R and MAT2012-38043), Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13, VA313U14 and GRS/516/A/10)

A 3D corneal stroma substitute with micro-level patterns was constructed from a stack of 4 micro patterned collagen or collagen-elastin like recombinamer (Col:ELR) blend layers. The transparency of all of the films was quite high with the uncrosslinked (UXL) films and dehydrothermally treated (150 °C, 24 h) Col:ELR films yielding the best results. Human corneal keratocytes (HK) could be attached and proliferated equally well on the single films of Col and Col:ELR. However, for the multilayer constructs the proliferation was higher on Col than on Col:ELR. The cells were found to align along the patterns (microchannels with a 39 μm groove depth, 8 μm groove width, 3.3 μm ridge width, and 54.7° inclination angle) of the films, while no significant alignment was observed on the unpatterned films. The transparency of the seeded Col:ELR films was superior to the Col films over a 30 day incubation period and was quite close to that of a native human cornea. It was concluded that the Col and Col:ELR films and their 3D constructs have significant potential for use as corneal stroma substitutes.

Producción Científica, Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M., Comisión Interministerial de Ciencia y Tecnología (grant CICYT BIO98-0727)

Producción Científica, The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) func- tionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineer- ing: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxya- patite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts., Ministerio de Industria, Economía y Competitividad (proyectos MAT2013-42473-R y MAT2013-41723-R), Ministerio de Ciencia e Innovación (proyectos MAT2012-35556 y MAT2009-14195-C03-02), Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA244U13), Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. VA313U14)

Producción Científica, The search for new and biocompatible materials with high potential for improvement is a challenge in gene delivery applications. A cell type specific vector made of elastin- like recombinamer (ELR) and aptamers has been specifically designed for the intracellular delivery of therapeutic material for breast cancer therapy. A lysine-enriched ELR was constructed and complexed with plasmid DNA to give positively charged and stable polyplexes. Physical character- ization of these polyplexes showed a particle size of around 140 nm and a zeta potential of approximately +40 mV. The incorporation of MUC1-specific aptamers into the polyplexes resulted in a slight decrease in zeta potential but increased cell transfection specificity for MCF-7 breast cancer cells with respect to a MUC1-negative tumor line. After showing the transfection ability of this aptamer-ELR vector which is facilitated mainly by macropinocytosis uptake, we demonstrated its application for suicide gene therapy using a plasmid containing the gene of the toxin PAP-S. The strategy developed in this work about using ELR as polymeric vector and aptamers as supplier of specificity to deliver therapeutic material into MUC1-positive breast cancer cells shows promising potential and continues paving the way for ELRs in the biomedical field., Este trabajo forma parte de los Proyectos de Investigación financiados por la Comisión Europea a través del Fondo Social Europeo (FSE) y de la Consejería de Educación mediante el Fondo Europeo de Desarrollo Regional (ERDF), el MINECO (Proyectos MAT2013-41723-R, MAT2013-42473-R, PRI−PIBAR-2011-1403 y MAT2012-38043), la Junta de Castilla y León (Proyectos VA155A12, VA152A12, and VA244U13), el CIBER-BBN y el Instituto de Salud Carlos III mediante el Centro de Medicina Regenerativa y Terapia Celular de Castilla y León.

Producción Científica, The 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)

Elastin-like recombinamers have grown in popularity in the field of protein-inspired biomimetic materials and have found widespread use in biomedical applications. Modern genetic-engineering techniques have allowed the design of multifunctional materials with an extraordinary control over their architecture and physicochemical properties, such as stimuli-responsiveness, monodispersity, biocompatibility or self-assembly, amongst others. Indeed, these materials are playing an increasingly important role in a diverse range of applications, such as drug delivery, tissue engineering and ‘smart’ systems. Herein, we review some of the most interesting examples of recent advances and progressive applications of elastin-like recombinamers in biomaterial and nano-engineering sciences in recent years.

Introduction With almost 1,4 million new cases each year worldwide, colorectal cancer kills 228.000 Europeans every year. Although new therapies have been discovered, the use of recombinant elastin-based biomaterials could open a new approach in cancer research as polymeric carriers for drug delivery in order to improve the accuracy of the action and reduce the toxicity of chemotherapeutic agents. In this work, we have developed a new elastin-like recombinamer (ELR) fused to a peptide inhibitor of the protein kinase Akt. This polymer self-assembled into nanoparticles, which showed killing ability on colorectal cancer cells (Caco-2). Material and methods Taking advantage of the recombinant DNA technology, the constructs were formed by an amphiphilic backbone and several bioactive sequences, with an Akt inhibitor among others. ELR were produced by E. coli fermentation and, after purification process, were characterised and tested in vitro . Cellular viability assays on cancerous cells were carried out in order to study the killing ability of these nanoparticles. Results and discussions Both physical and chemical characterisation of this novel ELR showed that it is able to self-assemble into nanoparticles with a diameter of 68 nm, which are an effective way to deliver the Akt inhibitor into the cytoplasm, where it will bind to its protein target Akt and stop the kinase activity. With a transition temperature of 18°C and a high negatively-charged surface, nanoparticles perfectly met all requirements for its use as biomedical tools. Moreover, nanoparticles showed increased killing ability on cancerous cells compared to non-cancerous cells. Conclusion Thus, we have designed a new system able to enter into the cancerous cells and inhibit Akt signalling pathway. As an interesting advantage, ELRs have the ability to be modified by adding different molecules, such as aptamers or single chain antibodies, in order to be selectively targeted against cancerous cells. Thus, the action of these nanoparticles will be more accurate so as to achieve a new therapeutic tool for colorectal cancer treatment.

Here, we described a “one-pot” synthesis of smart hybrid materials based on elastin-like recombinamers (ELRs) and gold nanoparticles. Outstandingly, the reduction of auric acid in the presence of ELR Glu15 gave biohybrid Au–Glu15. TEM analysis carried out for Au–Glu15 exhibited nano-sized gold crystals with diameter ranging from 2 to 11 nm. Furthermore, Au–Glu15 promoted the formation of linear arrangements of gold clusters in areas of low particle density. Remarkable was that analogous architectures were obtained in a control experiment carried out with Glu15 and gold nanoparticles synthesized via a citrate reduction route. Therefore, Glu15 promoted the formation of 2D linear arrangements of gold clusters that exhibited interparticle distances in the range from 10 to 40 nm. Notable were the branched nanostructures exhibited by Au–Glu15B obtained for reduced gold–ELR mixtures that exhibited a lower gold ratio. On the other hand, Au–Glu15 exhibited spectroscopic properties (UV–vis absorption) that could be modulated as function of pH and temperature of the environment as result of reversible aggregation–expansion of gold nanoparticles. Thereby, Au–Glu15 displayed remarkable features suitable for the development of stimuli responsive optical sensors and detectors for biological applications that could operate in aqueous media and under a wide range of pH’s.

Multifunctional bioactive chemically cross-linked elastin-like polymers (ELPs) have been prepared as three-dimensional scaffolds for tissue engineering. The salt-leaching/gas-foaming technique was found suitable to prepare highly porous biodegradable hydrogels based on this novel material type. The porosity can be controlled by the amount of sodium hydrogen carbonate incorporated during the cross-linking reaction, whereas the mean pore size is determined by the salt particle size. The gas-foaming process, which involves immersion in a citric acid solution after the cross-linking, facilitates pore interconnectivity and allows a grooved surface essential for cell colonization. Due to the thermoresponsive nature of the ELPs, their physical properties are strongly influenced by the temperature of the aqueous medium. The feasibility to obtain tridimensional scaffolds for tissue engineering has been studied by testing the adhesion and spreading of endothelial cells into the porous ELP hydrogels. The methods and structures described herein provide a starting point for the design and synthesis of macroporous multifunctional elastin-like hydrogels with potential broad applicability.

La matriz extracelular es uno de los principales elementos reguladores de la actividad celular. Los diferentes módulos de las macromoléculas que la componen son capaces de desencadenar señales que activan diferentes rutas intracelulares que organizan las funciones vitales de las células. La ingeniería de tejidos se dedica a desarrollar sistemas capaces de imitar, temporalmente, el comportamiento de la matriz extracelular con objeto de promover la regeneración o el reemplazo de tejidos y órganos dañados, actuando como un soporte atractivo para las células que deben adherirse y crecer sobre ella, hasta reemplazarla por tejido sano. En este trabajo se describe el proceso de diseño y producción de un polímero de tipo elastina que se ha funcionalizado con secuencias bioactivas que añaden actividades específicas al andamio o soporte celular que constituye la elastina. Así,algunos dominios elastoméricos se modificaron con el aminoácido lisina para poder entrecruzar las moléculas de polímero y conseguir matrices. También se incluyó la secuencia REDV, presente el dominio CS5 de la fibronectina humana, como motivo de adhesión celular. Por último, el polímero se funcionalizó con secuencias diana de enzimas proteolíticas para mejorar su bioprocesabilidad. Extracellular matrix (ECM) is a major component for the regulation of cell activity. The different modules of the proteins which constitute the extracellular matrix macromolecules represent for the cells which enter in contact with them, new signals capable of activating several intracellular signaling pathways, resulting in the modulation of numerous cell functions. Tissue engineering tries to develop new materials based on these components as scaffolds for cells to promote their adhesion and growth. In this work, genetic engineering techniques were used to design and biosynthesize an extracellular matrix analogue based in the elastin component. The structural base of our scaffold is an elastin –derived sequence which confers an adequate mechanical behavior. In addition, several domains were included, for adding new bioactivities to this elastin-like polymer (ELP). Some of these elastic domains were modified to contain lysine for cross linking purposes. The polymer also contained periodically spaced fibronectin CS5 domain enclosing the well known cell attachment sequence REDV. Finally, the polymer had target sequences for proteolitic action.

Producción Científica, The 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"

In spite of the enormous possibilities presented by macromolecules for the development of advanced materials with increased functionality, the achievement of functionality is often limited by the randomness associated with polymer synthesis and the exponential increase in technical difficulties encountered in attaining a desired degree of complexity in the molecular design. This paper describes an increasingly important approach to the design of complex and highly functional macromolecules, i.e. the genetic engineering of protein-based macromolecules. The exploitation of the efficient machinery of protein synthesis in living cells opens a route to precisely defined and complex macromolecules. A series of molecular designs with increasing complexity are presented to show how this controlled increase yields materials with increasingly selective and sophisticated multifunctionality. The simplest designs already show interesting mechanical properties, but the adequate introduction of given chemical functions along the polymer chain provides an opportunity to expand the range of properties to smart behavior and self-assembly. Finally, examples are given where the molecular designs further incorporate selected bioactivities in order to develop materials for the most cutting-edge applications in f biomedicine and nanobiotechnology.

The pH-responsive elastin-like polymers, [(PGVGV)2−(PGEGV)−(PGVGV)2]n with n = 5, 9, 15, 30, 45, were obtained using genetic engineering and microbial protein expression. These were intended to stu...

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.

Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.

Multilayered microcapsules of chitosan and biomimetic elastin-like recombinamers (ELRs) were prepared envisaging the intracellular delivery of active agents. Two ELRs containing either a bioactive RGD sequence or a scrambled non-functional RDG were used to construct two types of functionalized polymeric microcapsules, both of spherical shape ∼4μm in diameter. Cell viability studies with human mesenchymal stem cells (hMSCs) were performed using microcapsule/cell ratios between 5:1 and 100:1. After 3 and 72h of co-incubation, no signs of cytotoxicity were found, but cells incubated with RGD-functionalized microcapsules exhibited higher viability values than RDG cells. The internalization efficacy and bioavailability of encapsulated DQ-ovalbumin were assessed by monitoring the fluorescence changes in the cargo. The data show that surface functionalization did not significantly influence internalization by hMSCs, but the bioavailability of DQ-ovalbumin degraded faster when encapsulated within RGD-functionalized microcapsules. The microcapsules developed show promise for intracellular drug delivery with increased drug efficacy., The authors acknowledge financial support from Fundacao para a Ciencia e Tecnologia (Grant SFRH/BD/61126/2009), "Fundo Social Europeu", "Programa Diferencial de Potencial Humano", EU 7th Framework Programme (No. REGPOT-CT2012-316331-POLARIS), the EU through the European Regional Development Fund (ERDF), from the MINECO (MAT-2010-15982, MAT2010-15310, PRI-PI-BAR-2011-1403 and MAT2012-38043), the JCyL (projects VA049A11, VA152A12 and VA155A12), the CIBER-BBN, and the Instituto de Salud Carlos III under the "Network Center of Regenerative Medicine and Cellular Therapy of Castilla and Leon".

In this chapter, we describe two methods for bio-producing recombinant repetitive polypeptide polymers for use in biomedical devices. These polymers, known as elastin-like recombinamers (ELRs), are derived from the repetition of selected amino acid domains of extracellular matrix proteins with the aim of recreating their mechanical and physiological features. The proteinaceous nature of ELRs allows us to make use of the natural biosynthetic machinery of heterologous hosts to express advanced and large polymers or "recombinamers." Despite the essentially unlimited possibilities for designing recombinamers, the production of synthetic genes to encode them should allow us to overcome the difficulties surrounding bioproduction of these non-natural monotonous DNA and protein sequences. The aim of this work is to supply the biotechnologist with fine-tuning methods to biosynthesize advanced self-assembled smart materials.

In this chapter, we describe two methods for bio-producing recombinant repetitive polypeptide polymers for use in biomedical devices. These polymers, known as elastin-like recombinamers (ELRs), are derived from the repetition of selected amino acid domains of extracellular matrix proteins with the aim of recreating their mechanical and physiological features. The proteinaceous nature of ELRs allows us to make use of the natural biosynthetic machinery of heterologous hosts to express advanced and large polymers or "recombinamers." Despite the essentially unlimited possibilities for designing recombinamers, the production of synthetic genes to encode them should allow us to overcome the difficulties surrounding bioproduction of these non-natural monotonous DNA and protein sequences. The aim of this work is to supply the biotechnologist with fine-tuning methods to biosynthesize advanced self-assembled smart materials.

The past few decades have witnessed the development of novel naturally inspired biomimetic materials, such as polysaccharides and proteins. Likewise, the seemingly exponential evolution of genetic-engineering techniques and modern biotechnology has led to the emergence of advanced protein-based materials with multifunctional properties. This approach allows extraordinary control over the architecture of the polymer, and therefore, monodispersity, controlled physicochemical properties, and high sequence complexity that would otherwise be impossible to attain. Elastin-like recombinamers (ELRs) are emerging as some of the most prolific of these protein-based biopolymers. Indeed, their inherent properties, such as biocompatibility, smart nature, and mechanical qualities, make these recombinant polymers suitable for use in numerous biomedical and nanotechnology applications, such as tissue engineering, "smart" nanodevices, drug delivery, and protein purification. Herein, we present recent progress in the biotechnological applications of ELRs and the most important genetic engineering-based strategies used in their biosynthesis.

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruch's membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

In this study a tissue engineering scaffold was constructed from poly(N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 µm groove width, 2 µm ridge width, 20 µm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 °C and 37 °C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 × 104 and 13.2 × 104, respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 × 104 and 12.1 × 104, respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 × 104 and 157.7 × 104, respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions. © 2009 Elsevier Ltd. All rights reserved. BAP-2006-07-02-00-01 Ministerio de Ciencia e Innovación: NAN2004-08538, VA087A06, CB06-01-0003, VA030A08, MAT 2007-66275-C02-01, VA030/08 Devlet Planlama Ürgütü: BAP 08-01-DPT2003K.120920-20 Instituto de Salud Carlos III This study was supported by a grant from METU Graduate School of Natural and Applied Sciences (BAP-2006-07-02-00-01), EU FP6 project BioPolySurf, MICINN (projects MAT 2007-66275-C02-01 and NAN2004-08538), the JCyL (projects VA087A06, VA030/08 and VA030A08), the Ciber-BBN (project CB06-01-0003), and the JCyL and the Instituto de Salud Carlos III under the “Network Center of Regenerative medicine and Cellular Therapy of Castilla and León”and a scholarship to N.O. by the State Planning Organization (BAP 08-01-DPT2003K.120920-20).

To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices.A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox, and ELP-coated. Cells (10(4)) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed.Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers.The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

Publisher Summary Modern biomaterials science is characterized by a growing emphasis on identification of specific design parameters that are critical to performance, and by a growing appreciation of the need to integrate biomaterials design with new insights emerging from studies of cell-matrix interactions, cellular signaling processes, and developmental and systems biology. Elastin-Like Polymers (ELPs) are non-natural polypeptides composed of repeating sequences. They have their origin in the repeating sequences found in the mammalian elastic protein elastin that confers elasticity to structures such as skin and blood vessels. The importance of these polymers reside in the fact that they show a versatile and ample range of interesting properties that are difficult to find together in other materials, and that goes beyond their simple mechanical performance. Certainly, ELPs show a set of properties that places them in an excellent position towards designing advanced polymers for many different applications, including the most cutting edge biomedical and nanobiotechnological uses. Regarding their properties, some of their main characteristics are derived from the natural protein on which they are based.

In spite of the enormous possibilities of macromolecules as key elements in developing advancedmaterials with increased functionality and complexity, the success in this development is often limitedby the randomness associated with polymer synthesis and the exponential increase in technical difficultiescaused by the attempt to reach a sufficiently high degree of complexity in the molecular design.This paper describes a new approach in the design of complex and highly functional macromolecules,the genetic engineering of protein-based macromolecules. The exploitation of the efficient machineryof protein synthesis in living cells opens a path to obtain extremely well-defined and complexmacromolecules. Different molecular designs are presented, with increasing degree of complexity,showing how the controlled increase in their complexity yields (multi)functional materials with moreselect and sophisticated properties. The simplest designs show interesting properties already, butthe adequate introduction of given chemical functions along the polymer chain presents an opportunityto expand the range of properties to enhanced smart behavior and self-assembly. Finally, examplesare given where those molecular designs further incorporate selected bioactivities in order to developmaterials for the most cutting-edge applications in the field of biomedicine and nano(bio)technology.

The self-assembly characteristics of the model genetically engineered elastin-like polymer [(VPGVG)2(VPGEG)(VPGVG)2]15 have been studied in this work. An AFM study of the topology of polymer films deposited from acid and basic solutions on a hydrophobic silicon substrate has been carried out. Under acidic conditions, polymer deposition results in a flat surface with no particular topological features. However, from basic solutions, polymer deposition clearly shows an aperiodic pattern of nanopores ( approximately 70 nm width and separated about 150 nm). This dramatic dependence of film topology on pH is explained in terms of the different polarity of the free gamma-carboxyl group of the glutamic acid. In the carboxylate form, this moiety shows a markedly higher polarity than the rest of the polymer domains and the substrate itself. Under these conditions, the charged carboxylates impede hydrophobic contact with their surroundings, which is the predominant assembly pathway for this type of polymer. The charged domains, along with their hydration sphere, are then segregated from the hydrophobic surroundings giving rise to nanopores.

Genetic engineering techniques were used to design and biosynthesise an extracellular matrix (ECM) analogue. This was designed with a well-defined molecular architecture comprising different functional domains. The structural base is a elastin-derived repeating unit, which confers an adequate elastic characteristic. Some of these elastin domains have been modified to contain lysine; this amino acid can be used for crosslinking purposes. The polymer also contain periodically spaced fibronectin CS5 domains enclosing the well-known cell attachment sequence REDV. Finally, the polymer has target sequences for proteolitic action. These sequences are those found in the natural elastin and are introduced to help in the bioabsorption of the polymer. In addition, these proteolitic sequences were chosen in a way that, after proteolitic action, the released fragments will be bioactive. These fragments are expected to promote cell proliferation activity, angiogenesis and other bioactivities of interest for tissue growing, repairing and healing. After purification, the resulting polymers proved to be of high purity and correct sequence. Glutaraldehyde has shown to be a cross-linking agent for this polymer, yielding insoluble hydrogel matrices. This work is framed in a long term project aimed to exploit the power of genetic engineering for the design and bioproduction of complex ECM analogues showing the rich complexity and multi (bio)functionality of the natural matrix.

Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L. and Miller at rather low concentrations. In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli. The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed. Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal. The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23mgl-1 of bacterial culture. The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein. The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K. The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent.