Engineering responsive and biomimetic material based on elastin-like recombinamers for biomedical application

PROGRAMA DE DOCTORADO EN INVESTIGACIÓN BIOMÉDICA TESIS DOCTORAL: ENGINEERING RESPONSIVE AND BIOMIMETIC MATERIAL BASED ON ELASTIN-LIKE RECOMBINAMERS FOR BIOMEDICAL APPLICATION Presentada por Filippo Cipriani para optar al grado de Doctor/a por la Universidad de Valladolid Dirigida por: José Carlos Rodríguez Cabello Israel González de Torre Gracias a la vida que me ha dado tanto Me ha dado la marcha de mis pies cansados Con ellos anduve ciudades y charcos Playas y desiertos, montañas y llanos. Gracias a la vida – Violeta Parra (1966) ACKNOWLEDGMENTS - AGRADECIMIENTOS - RINGRAZIAMENTI Muchos años después, sobre un avión que conducía desde Florencia a Madrid, el suscrito había de recordar aquella tarde remota en que abrió Google Maps y busco donde estaba Valladolid. Me encuentro sobre el vuelo que va desde Florencia a Madrid y me acuerdo de la primera vez que vine a Valladolid; no sabía dónde estaba y nunca hubiera pensado de vivir casi 4 años en esta ciudad. Pero, si lo pienso bien, tengo que confesar que siempre he tenido un débil por España. No sé, será el Mediterráneo que nos acomuna y nos acerca; también será por el idioma tan sencillo de aprender que seguramente en estos agradecimiento estarán muchos errores. Poco importa, alguien demostró que el 90% de las comunicación nos es verbal y creo que con esto escrito el mensaje llegará aunque haya muchos errores gramaticales. Quiero empezar diciendo que haber tenido la oportunidad de vivir en este país ha sido una gran fortuna y tengo que agradecer muchas persona que han permitido que mi recorrido pasase por aquí. Habría muchos que agradecer, pero intentaré ser los más conciso posible para no aburrir demasiado. En primer lugar quiero agradecer mis directores Carlos y Israel. Muchas gracias por haberme dado esta oportunidad y por haberme guiado a lo largo del camino hasta conseguir el título de Doctor en Investigación Biomédica. Muchas gracias Carlos por tus preciosos consejos, tu enorme conocimiento sobre la ciencia, por tu generosidad y para haberme dado la libertad necesaria para desarrollar recursos que si no nunca habría descubierto. Muchas gracias Israel por tu inmenso ayudo, tu capacidad de aclararme las idea, de animarme en los momentos bajos y por haberme reconducido a las cosas prácticas cuando me estabas perdiendo en mil vueltas mentales. Muchas gracias a Matilde por tu ayuda tanto científica cuando humana, toda las veces que he entrado en tu despacho en momentos críticos, siempre he salido con más fe en mi camino y en mí mismo. Muchas gracias a Javier por tu preciosos consejos sobre el mágico mondo de la Biología Molecular; gracias a Alessandra por tu consejos sobre los experimentos de células y también para los buenos ratos pasados a hablar de la nuestra Italia y de las orígenes Etruscas que tenemos. Gracias a Luis y Menchu porque trabajar con vosotros es un auténtico lujo, donde se aprende lo que es ser científico y trabar con profesionalidad. Gracias a Merche y Irene, que me han dado mucho cariño y sencillamente verlas con esa sonrisa me animaban el corazón. Gracias a Leander y Soraya, mi inseparable compañeros del despacho. Madre mía cuantas horas pasadas untos confrontándonos sobre los problemas y ayudándose para solucionarlos. Nos hemos animado muchísimo, tanto dentro como fuera del laboratorio. Muchas Gracias a Juan y Sofía por todas las risas que nos hemos hechos, por todas las frases épicas que nos han ayudado a tener un ánimo alegre también cuando buscábamos cazar gnomos y no encontrábamos ninguno. Gracias a Tatiana y Sergio por los buenos momentos pasados en muchos congresos; gracias a Ito para sus consejos sobre que canción escuchar para motivarse y sobre los rinconcitos más bonitos de Valladolid; gracias a Arturo y Doriana porque nunca olvidaré mi primer congreso con vosotros en Oporto. Gracias a Fer, Miguel y Marcos porque MAAACCHHOOO este es un cachondeo! (y otros mil dichos que nos han hechos reír mucho). Gracias a Rocío y a Sandra por vuestra ayuda. Rocío eres una crack (como se dice aquí). Siempre lista para ayudar, tanto profesionalmente como humanamente, te mereces lo mejor. Gracias a los nuevos como Diana y Sara, que por mala suerte no hemos tenido la oportunidad de pasar mucho tiempo untos, pero estoy convencido que estaremos en contacto y nos volveremos a ver. Gracias a Sandra por haberla ayudada con su proyecto, te deseo lo mejor por tu trayectoria científica. Gracias también a lo que se han marchados antes que yo: Mohammed, Lorena, Elena, Leticia, Esther, Alicia, Teresa y Dasha, gracias por los buenos momentos pasados untos. También quiero agradecer a Gastón y sus historias sobre Cuba, al final consiguió convencerme a visitarla. Gracias también a todos los colaboradores externos, como los colegas conocidos en las estancias, los médicos de Valladolid y todos los coautores de los artículos. Gracias mis fantásticos compañeros del BIOGEL, tanto los estudiantes cuanto los Seniors, compartir con vosotros este camino profesional ha sido una oportunidad estupenda: agradezco el día que decidí aplicar por este proyecto. He aprendido muchísimo desde cada uno de vosotros, en un sentido común he aprendido lo que significa hacer ciencia y ser científico. A todos vosotros os deseo lo mejor. Terminados los agradecimientos profesionales, quiero agradecer también mi mundo fuera del Doctorado, un mundo donde conseguías recargar las pilas para volver a dar lo mejor. Gracias a todos los amigos conocidos en Valladolid; amigos inolvidable con los cuales hemos creado una conexión especial; nombrarlos todos sería demasiado, por esto me limito a decir un GRACIAS general y que llegue a todos, desde los chicos de la cafetería, pasado por los compañeros de balonmano hasta a quien me cogió en Valladolid como en una Familia. Gracias también a todos los amigos fuera de Valladolid: a lo de España, de Holanda, de Alemania, a mi amigos históricos de Italia, a los nuevos que conocí en los congresos y a lo que conocí en otros rincones del mundo. Gracias a mi Familia, porque no obstante la distancia su ayudo me ha llegado intacto y poderoso. Gracias por todas las veces que habéis venido a visitarnos recordándonos cuanto nos queréis. Que cuando estaba por el mundo conociendo gente de cultura diferente, llevaba conmigo mi mundo y mi raíces y me sentía como si vosotros estuvierais conmigo. 4 años inolvidables! Hechos principalmente de momentos buenos, pero también de momentos difíciles… como el clima de Valladolid. Niebla donde no se ve nada y solo se puede proceder a tentones. Un sol que pega tan fuerte encima de la cara que solo hay que cerrar los ojos y no permitir a las gotas de sudor de entrar. Un frio que penetra los pies porque anden más rápido, de manera de calentarse y alcanzar una meta más lejana. 4 años y mucho más pasado untos! Donde cuando había niebla andabas tu delante de mí; que cuando teníamos el sol en la cara que no se veía nada, estabas tú mirando el camino; y que cuando hacia frio para pararse, me abrazabas y me decías que la meta estaba cerca. Sin ti este reto no hubiera sido posible. Gracias a ti, Sara por todos los momentos que hemos vivido… y que tenemos que vivir. Filippo INDEX ABSTRACT ......................................................................................... 22 INTRODUCTION ............................................................................. 28 PROBLEM STATEMENT AND HYPOTHESIS ...................... 38 OBJECTIVES ...................................................................................... 42 STATE OF THE ART ........................................................................ 46 CHAPTER 1: ELASTIN-LIKE POLYMERS: PROPERTIES, SYNTHESIS AND APPLICATIONS ............................................ 48 1. INTRODUCTION ........................................................................ 49 2. ELASTIN-LIKE RECOMBINAMERs ENGINEERING, BIOPRODUCTION AND DESIGN ................................................................................... 52 2.1. History and evolution of the synthesis of elastin-like recombinamers .......................................................................... 52 1.3.1. Ancient times (the “chemistry ages”) ...................... 52 2.1.2. Modern times (the “recombinant ages”) ................ 52 2.1.3. Contemporary times (the “seamless recursive ages”)……………………………………………………………………………………53 2.2. Hosts for the expression of elastin-like recombinamers……………………………………………………………………….56 2.7. Cell viability assay ................................................................. 203 2.8. In vivo experimental model .................................................. 204 2.9. Gross morphology ................................................................. 205 2.10. Histological analysis ............................................................ 205 2.11. Statistical analysis ............................................................... 206 3. RESULTS .............................................................................. 206 3.1. Rheological characterization ................................................ 206 3.2. Scanning electron microscopy (SEM) ................................... 207 3.3. Cell viability assay ................................................................. 208 3.4. In vivo study results .............................................................. 210 3.4.1. Macroscopic observation of repaired cartilage ................ 210 3.4.2. Histological analysis of repaired cartilage ........................ 211 4. DISCUSSION .............................................................................. 215 5. CONCLUSIONS .......................................................................... 220 Acknowledgments ........................................................................ 221 References .................................................................................... 222 SUPPORTING INFORMATION ....................................................... 228 CHAPTER 5: BICYCLIC RGD PEPTIDES WITH INTEGRIN αvβ3 AND α5β1 AFFINITY PROMOTE CELL ADHESION ON ELASTINLIKE RECOMBINAMERS ........................................................... 236 Abstract ........................................................................................ 237 1. INTRODUCTION ...................................................................... 238 2. MATERIALS AND METHODS ............................................... 240 2.1. ELR biosynthesis, modification and characterization 240 2.2. Reagents and chemicals ............................................. 241 2.3. Peptide synthesis ........................................................ 241 2.4. Synthesis of peptide-cyclooctyne conjugates ........... 242 2.5. Formation of peptide-functionalized ELRs ................ 242 2.6. Analysis of turbidity by UV/Vis-spectroscopy ........... 242 2.7. Adsorption of peptide-functionalized ELRs on TCPS 243 2.8. Contact Angle measurements .................................... 243 2.9. X-ray Photoelectron Spectroscopy (XPS) ................... 243 2.10. Cell culture and cell adhesion assay .......................... 244 2.11. DNA Analysis ............................................................... 244 2.12. 2D Immunofluorescent Staining ................................ 245 2.13. Statistical analysis ...................................................... 245 3. RESULTS .............................................................................. 245 3.1. Selection and synthesis of RGD peptides .................. 245 3.2. ELR functionalization and MALDI-TOF MS analysis ... 248 3.3. Analysis of turbidity by UV/Vis-spectroscopy ........... 251 3.4. Contact Angle .............................................................. 252 3.5. XPS ............................................................................... 253 3.6. Cell-adhesion assay .................................................... 253 3.6.1. Time-dependent cell quantification studies .......... 253 3.6.2. Morphology studies ................................................ 258 4. DISCUSSION ........................................................................ 261 5. CONCLUSIONS .................................................................... 266 Notes ............................................................................................. 266 Acknowledgements ...................................................................... 267 References .................................................................................... 268 SUPPORTING INFORMATION ....................................................... 272 CHAPTER 6: SELECTIVE ENZYMATIC RESPONSIVE SMART- ELRs WITH ALLOSTERIC CONTROL OF RNase A ACTIVITY ............................................................................................................... 284 Abstract ........................................................................................ 285 1. INTRODUCTION ...................................................................... 286 2. MATERIALS AND METHODS ............................................... 288 2.1. ELR biosynthesis and purification .............................. 288 2.2. Smart-ELRs phosphorylation and dephosphorylation…………………… ................................................. 290 2.3. Mass analysis (HPLC-HR-MS) ...................................... 291 2.4. Turbidity analysis ........................................................ 292 2.5. Dynamic Light Scattering (DLS) .................................. 292 2.6. RNase A activity analysis ............................................ 292 2.7. Statistical analysis ....................................................... 293 3. RESULTS .............................................................................. 293 3.1. Smart-ELRs phosphorylation and de-phosphorylation …………………………………………………………………………………293 3.2. Dynamic Light Scattering ............................................ 301 3.3. RNase A activity analysis ............................................ 302 4. DISCUSSION ........................................................................ 304 5. CONCLUSIONS .................................................................... 309 References .................................................................................... 311 SUPPORTING INFORMATION ....................................................... 315 CONCLUSIONS AND FUTURE DIRECTIONS ................... 324 Genetic engineering, bioproduction and characterization of ELRs…………………………………………………………………………..………………324 Development of ELRs-based hydrogel with different gelation mechanisms for osteochondral repair ........................................ 324 In vitro, ex vivo and in vivo evaluation of ELRs-based hydrogel for osteochondral repair .................................................................... 326 Development of a new ELR-peptides hybrid biomaterial .......... 327 A new class of smart-ELRs with allosteric domain ...................... 328 Final remarks ................................................................................ 329 ADDITIONAL .................................................................................. 332 Publications .................................................................................. 332 Conferences .................................................................................. 333 Courses and certifications ............................................................ 335 Marie Curie Fellowship for Early Stage Research Training ......... 336 Awards .......................................................................................... 336 Teaching ........................................................................................ 337 22 ABSTRACT Regenerative Medicine is a well-established field of science that aims to replace, engineer and regenerate human cells, damaged tissues or organs to restore their normal function. This branch of translational research finds a deep interest in the Science of Biomaterials; indeed, the knowledge acquired in that field goes proportionally with the development of novel biomaterials. There is a great need in developing advanced biomaterials capable to fulfil the requirements of stability and bioactivity for their application in biomedicine. Moreover, considering the complexity of the human body, this system needs a certain rate of versatility in order to be tailored to a specific area of application. For all these reasons, recombinant proteins are an interesting approach, in which, elastin-like recombinamers (ELRs) represent one of the most promising biomaterials. ELRs are obtained through DNA recombinant technology, which allows the precise control at the genetic level, affording exquisite control over final protein functionality. ELRs are proteinbased polypeptides that comprise repetitive units of the Val−Pro−Gly−X−Gly (VPGXG)n pentapeptide, in which X (guest residue) could be any amino acid except L-proline. In terms of biomaterial design, ELRs show several outstanding properties. ELRs are inspired by elastin, which is a component of natural extracellular matrix (ECM), showing excellent biocompatibility. One of the most important features of ELRs is that they exhibit thermoresponsiveness; this is due to the change of protein conformation above the so-called transition temperature (Tt), which depends on the amino acid composition of the polymer. Moreover, according to the ELRs design, they can be processed as several supramolecular structures, such as micelles, nanoparticles, films, and hydrogels. The large variety of ELRs, both in terms of structures and bioactivity, permits the application of these protein-based biomaterials to diverse biomedical applications. This Thesis represents a sort of journey towards the exploration of the evolution of ELRs as a powerful tool with great potential in the biomedical field. The first part of this Thesis is dedicated to the description of the history of ELRs from their ancient chemical origin as ELPs (Elastin-like Polypeptides) to the most cutting-edge bioproduction techniques becoming into ELRs. Moreover, it is reported an exhaustive explanation of how ELRs can be processed in many forms (aggregates, fibers, layers, nanoparticles, or hydrogels), 23 giving examples of their great potential in many fields, including drug delivery, tissue engineering, protein purification, anticancer gene therapies, and nanovaccines. Moreover, considering their large interest, one chapter of this Thesis is dedicated to the ELRs as biomaterial forming hydrogels for tissue regeneration and repair. The different mechanisms of gelation are reported, and it is given an overview of the possible applications in tissue engineering, such as osteochondral application, (cardio-)vascular tissue regeneration, and ocular prostheses. The first experimental work of this Thesis is dedicated to the development of novel ELRsbased hydrogel for cartilage repair. 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. In this work, it has been 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 study, it was set up and optimized a preannealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfil 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 glycosaminoglycans and collagen. Finally, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage, which leads to the successful regeneration of hyaline cartilage in an ex vivo model. Not only the physically cross-linked hydrogels have been investigated; indeed, an in situ chemically cross-linked hydrogels have been developed for osteochondral repair. Moreover, another bioactive composition of this biomaterial has been tested; this ELRsbased hydrogel has been designed containing bioactive sequences, such as the well knows adhesion sequences RGD and REDV, and the elastase target domain VGVAPG that 24 provides proteolytic sensitivity to the biomaterial. Compared to the previous study reported in this Thesis, where the ex vivo platform was used, the regeneration properties of the chemically cross-linked ELRs hydrogel were evaluated with an in vivo study. Furthermore, it has been made a comparison between the usage of that biomaterial itself, and the biomaterial embedded with cells (tissue engineering). Both the ELR-based hydrogel alone and the ELR-based hydrogel embedded with rabbit Mesenchymal Stem Cells (rMSCs) were tested for the regeneration of critical subchondral defects in 10 New Zealand rabbits. Thus, cylindrical osteochondral defects were filled with an aqueous solution of ELRs. The animals were sacrificed at 4 months for histological and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality and evaluation of the new matrix in the defects. Although both groups helped cartilage regeneration, the results suggest that the specific composition of the rMSCs-containing hydrogel permitted adequate bone regeneration, whereas the ELR-based hydrogel alone led to an excellent regeneration of hyaline cartilage. In conclusion, the ELR cross-linker solution can be easily delivered and forms a stable, well-integrated hydrogel that supports infiltration and de novo matrix synthesis. As it has been reported above, the aim of this Thesis is to explore the possibilities of ELRs as a powerful tool capable of containing various bioactivities with great potential in the biomedical field. As a further step in the evolutional process towards advanced bioactive ELRs, one objective of this Thesis is to combine these two diametrically opposed approaches in a new hybrid biomaterial. Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In this work, we have combined the strategy of synthetic peptides with the DNA recombinant techniques generating a new hybrid biomaterial. Human umbilical vein endothelial cells (HUVECs) adhesion and proliferation were studied over the ELRs covalently functionalized with each three high-affinity and selectivity αvβ3- and α5β1-binding bicyclic RGD peptides. Next, to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent 31 Glutamic acid – Aspartic acid - Valine) which improve the selectivity for endothelial cells (24). At the same time, the design of ELRs comprising the elastase target domain (human leukocyte elastase I) VGVAPG (L-Valine – Glycine - L-Valine - L-Alanine - L-Proline - Glycine) provides proteolytic sensitivity to the biomaterial (25). In this Thesis is reported the development of novel biomaterials based on ELRs containing the listed sequence in order to evaluate their effectiveness in the most emerging fields. Moreover, generally speaking, it has to be taken into consideration that combining opposite approaches may compensate the drawbacks shown by each one if taken individually. For example, the recombinant synthesis has limitations on the incorporation of non-canonical amino acids and does not allow the formation of cyclized peptides (26). In that sense, the recombinant technique of ELR can be combined with the strategy of designing novel and efficient peptides (27). As a further step in the evolutional process towards advanced bioactive ELRs, one objective of this Thesis is to combine these two diametrically opposed approaches in a new hybrid biomaterial. In Chapter 5 is reported the combination of synthetic peptides with the ELR backbone that can be obtained using copper-free click chemistry (15). A new frontier of ELRs Target complex systems require new approaches capable to combine modular features in multi-capable material. Due to its potentiality, smart materials have gained widespread interest in material science (28). Smart (or stimuli-responsive material) derived from the development of materials that show large conformational changes in response to small environmental stimuli such as temperature, ionic strength, solvent polarity, electric/magnetic field, or light (29). In that sense, ELRs represent a promising tool for the generation of a new frontier of biomaterials. As it has been reported above, the ELRs exhibit several advantages as biomaterials, such as their compatibility, and their thermosensitivity; however, the most important remains the precise control at the genetic level, affording exquisite control over final protein functionality. There are many examples of smart biomaterials based on ELR (smart-ELR); for biomedical and biomimetic applications (30-34). Moreover, recent works have explored a new type of ELRs, which are not only enzyme-responsive but also contain their own activity due to the fusion of selected catalytic domains at the genetic level (35,36). Following this strategy, another aim of the 32 Thesis was to explore a new generation of ELRs. In Chapter 6 is reported the experimental work regarding the design of a new smart ELR containing consensus sequences for enzymatic responsiveness and a catalytic domain for allosteric control. The common ground of this Thesis is the ELRs technology with its outstanding properties; the leitmotiv is represented by the development of engineering material in a sort of journey towards the exploration of new possibilities, mashing approaches and crossing boundaries. 33 References 1. Mason, C. and P. Dunnill, A brief definition of regenerative medicine. Regenerative Medicine, 2008. 3(1): p. 1-5. 2. Migonney V. History of Biomaterials. Biomaterials: John Wiley & Sons, Inc.; 2014. P. 1-10. 3. 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Kinikoglu, B., et al., A smart bilayer scaffold of elastin-like recombinamer and collagen for soft tissue engineering. Journal of Materials Science: Materials in Medicine, 2011. 22(6): p. 1541-1554. 35 34. Cipriani, F., et al., Bicyclic RGD peptides with high integrin αvβ3 and α5β1 affinity promote cell adhesion on elastin-like recombinamers. Biomed Mater, 2019. 35. Du, K., et al., Enhancement of the solubility and stability of d-amino acid oxidase by fusion to an elastin like polypeptide. Journal of Biotechnology, 2015. 212: p. 50-55. 36. Gao, Q., et al., Genetically-modified R-ω-transaminase: purification and self-assembly facilitating interaction with substrate droplets. Biotechnology letters., 2016. 38(3): p. 489-494. 36 37 38 PROBLEM STATEMENT AND HYPOTHESIS The Biomaterial field is a branch of translational research that evolves with technology. Many types of technology have a crucial impact in this scientific field, both for the development of biomaterials and for the application of those biomaterials in the biomedical field. Along the last decades, the scientific community came up with several types of biomaterials from natural sources to synthetic polymers. Despite the large variety of biomaterials, only a few of them are able to show adequate properties of bioactivity and versatility for being applied to different biomedical applications. Nowadays, a partial wrong approach is the tendency to use a “one-type” biomaterial to address diverse biomedical issues. The strategy of developing one type of biomaterial for a large variety of biomedical applications may results successful for some, but unsuccessful for the others. Every issue in the biomedical field needs to be targeted with tailored therapies. One of the biggest challenges of today is to develop tailored biomaterials for specific applications. In this Thesis, we propose to explore the potential of Elastin-like Recombinamers (ELRs) as an engineering responsive and biomimetic material, towards the development and the optimization of tailored solutions for specific biomedical applications. The following points represent the hypothesis of the research planning of this Thesis: - Considering that the DNA recombinant technique allows an exquisite control at the genetic level, we hypothesize to generate several ELRs comprising diverse combinations of bioactive domains. Furthermore, we aim to bio-produced the designed ELRs in Escherichia coli taking advantage of their thermo-responsiveness whereby depending on the composition of the ELRs several structures could be formed. - It is well known that hydrogels based on specifically designed ELRs can be formed by different gelation mechanisms and that these mechanisms influence the mechanical properties of the hydrogels. We hypothesize that the different gelation mechanism (chemical or physical cross-linking) has a crucial influence for 39 the generation of a 3D hydrogel embedded with cells as a successful scaffold for osteochondral repair. - It is well described in the literature that ELR can be modified in order to be reactive for click chemistry reaction. We hypothesized that the strategy of copper-free click chemistry allows the incorporation of non-canonical amino acids and the formation of cyclized peptides, (which represents a limitation shown by the recombinant synthesis) in order to generate a new class of hybrid biomaterials. - The consensus sequence sensible for the kinase/phosphorylase can be included in the ELR composition in combination with the RNAse A catalytic domain in order to verify the allosteric domain regulation. Indeed, new combinations of ELR with bioactive sequences can be explored. 40 47 48 CHAPTER 1 ELASTIN-LIKE POLYMERS: PROPERTIES, SYNTHESIS AND APPLICATIONS José Carlos Rodríguez-Cabello,1 Arturo Ibáñez-Fonseca,1 Filippo Cipriani,2 Leander Poocza,1 Israel González de Torre,1,2 Matilde Alonso1 1 Universidad de Valladolid, BIOFORGE, CIBER-BBN, Valladolid, Spain 2 Technical Proteins NanoBioTechnology (TPNBT) S.L., Valladolid, Spain J.C. Rodríguez-Cabello, A. Ibáñez-Fonseca, F. Cipriani, L. Poocza, I. González de Torre, M. Alonso. Elastin-like Polymers: Properties, Synthesis and Applications. Encyclopedia of Polymer Science and Technology (2017). doi: 10.1002/0471440264.pst656 49 1. INTRODUCTION Etymologically, the term protein comes from the Greek proteios, which means “holding first place”, or could come from the god Proteus which name suggests “primal”, “firstborn” and it is associated to the facility of this God to change his form and opinion. The name itself indicates the crucial role that proteins have in the living beings and the diverse forms in which we can find them in nature. If the importance of water is well known in the composition of the human beings, around 60% in adult males (1), proteins are not less important. In fact, more than 50% of the dry weight of our bodies is formed by proteins. They may have very different functionalities in the living systems, and some of them are large molecules that help to form the structure of our tissues irrespective of their location. In this way, we can find them forming really hard structures like bones, nails, horns or scales but they are also present in softer tissues like liver, muscles or connective tissue. Furthermore, proteins are not only involved in these structural tasks but they play a paramount role in the activation or deactivation of gene expression, or having regulatory functions helping to organize several processes or regulating metabolism or even taking part in muscle contractions. Among these proteins, elastin is one of the most important proteins that can be found composing the extracellular matrix (ECM) which provides structural integrity to the organs and tissues in the living beings. Elastin is not only an important protein within the composition of the ECM, but it also possesses certain features that make it unique. For instance, it is extremely durable and with a very low turnover in healthy tissues, the estimated half-life of this protein is around 70 years (2). Elastin confers elasticity and resilience to many tissues like ligaments, tendons, arteries or lungs among others (3). This elasticity is given by the presence of hydrophobic regions within the structure of the monomers of elastin that tend to aggregate and self-assemble contributing to the polymeric organization of the elastin (4). Along the last decades of the 20th century not few researchers were interested in this self-assemble property and started to explore the synthesis and production of artificial polypeptides based on these hydrophobic domains that conferred such properties to the elastin molecule (5-8). They found that the most frequent fragment of pentapeptides in the structure of the natural elastin was the sequence VPGVG, appearing up to 50 times in a single elastin molecule. It was discovered 50 that synthetic polymers of (VPGVG)n (n ≤ 150) were soluble in water below 25°C but they aggregated suffering a phase transition above this temperature (8). This change in the conformation of the protein leads to a viscoelastic state in which the amount of polymer is around 50% and the other 50% is water. This process is common to all the elastin-like polymers (ELPs) and it is accompanied by a halving of the length of the polymer and a release of a great amount of energy (9). All this process is driven by a change in the structure of the ELP, from an extended conformation below the transition temperature (Tt), to a β-spiral with three units of the basic pentamer VPGVG, forming a type II β-turn per turn of the spiral, above the Tt (5) (Figure 1). The Tt can be tuned by changing the fourth amino acid of the pentamer VPGXG, where X could be any amino acid except proline, because its structure destabilizes the β-turn impeding the correct packing of the chains of the polymer. The Tt of an ELP is clearly influenced by the nature of the guest amino acid on the X position in the sequence (VPGXG)n (apolar residues decrease the Tt, while polar ones increase the value of the Tt) and by the overall polymer length (n). The effect of these two parameters has been deeply investigated and described by Urry and coworkers (10). Moreover, the Tt is sensitive to other external factors as for instance ionic strength, pH, pressure, light or chemical modifications (9). Figure 1. Schematic representation of the conformational change of the ELP and ELR backbone depending on the temperature. 51 Chemical synthesis of various polypeptides based on elastin were successfully obtained by using standard chemical processes as demonstrated by Urry, Prasad and others (11,12), but some problems arise when more complex structures or simply larger polymers want to be synthetized, like problems in purification and polydispersity and, even if these amounts of mixture of products are obtained in small quantities, they can drastically affect the physical properties of the final product (13). The arrival of the recombinant DNA technology opened a new way to design and produce synthetic proteins. The old nomenclature of ELPs was changed to ELRs (elastin-like recombinamers) pointing out the recombinant origin of this new family of elastin-based polymers. With this new approach, some of the problems derived from the old chemical synthesis, like polydispersity, need for organic solvents and the further elimination of their residues, were overcome while a higher control over the amino acid sequence was obtained. Moreover, the purification process changed from classical chemical purification pathways to one based exclusively on the thermal behavior of the ELRs. After some inverse transition cycles (ITC), which imply heating and cooling of the suspension containing ELRs above and below their inverse temperature transition (ITT), they can be easily purified without the need of the addition of chemical agents or solvents that should be removed afterwards. During the last decade, a better control over the recombinant techniques and the use of more specific and accurate enzymes has led to the precise biotechnological processes that are nowadays applied in the production of the ELRs. ELRs are biological polymers that due to their flexibility in the design, their self-assembly properties, easy chemical modification allowing the introduction of many interesting functionalities, versatility to be processed in many forms (aggregates, fibers, layers, nanoparticles or hydrogels) and excellent cyto- and biocompatibility, have a high potential in many fields, from drug-delivery to tissue engineering, including others such as protein purification, anti-cancer gene therapies or nano-vaccines. In the next pages, we will explore the evolution of ELRs from their ancient chemical origin to the most cutting-edge bioproduction techniques, exploring the several hosts that can be used to bioproduce them and their versatility in the design. We will also immerse in the several structures that ELRs can form and their possibilities in the numerous medical fields where they can be applied. 52 2. ELASTIN-LIKE RECOMBINAMERs ENGINEERING, BIOPRODUCTION AND DESIGN 2.1. History and evolution of the synthesis of elastin-like recombinamers 1.3.1. Ancient times (the “chemistry ages”) The finding of repetitive sequences in porcine elastin by Gray et al., in 1973 (14), led to the chemical synthesis of different versions of some of these oligopeptides, being one of them the pentapeptide Val-Pro-Gly-Val-Gly (VPGVG in single-letter amino acid code). There was a lot of enthusiasm to study the conformational properties of these peptides in an attempt to shed light into the features of natural elastin, a polymeric protein that gathered a lot of attention due to its relationship with several diseases (15,16). Hence, Urry’s laboratory began synthetizing elastin-derived peptides and soon became one of the leading research groups in this field. However, the synthetic strategies required complex methods including the use of diverse precursors and solvents, while the overall yield and the length of the polypeptide were very limited (17). Despite the limitations, these synthetic approaches allowed the attaining of VPGVG (poly)pentapeptides to perform conformational studies (17,18), even by covalent cross-linking of these peptides, which also led to morphological studies by scanning electron microscopy (SEM) and to the determination of stress-strain curves (19). Finally, all these early studies resulted in the development of molecular dynamics calculations which gave more information about the secondary structure of the protein and about the backbone torsion angles ψ and φ of the amino acid residues in the polypeptide, both in its relaxed and extended state (20). 2.1.2. Modern times (the “recombinant ages”) By that time, the last years of the 80´s, recombinant DNA technology had arisen as a very promising tool for the biotechnological synthesis of proteins in heterologous hosts, mainly Escherichia coli. Therefore, researchers started to use this technology for the expression of polymeric proteins to overcome the disadvantages of both chemical synthesis and extraction from natural sources. The first biosynthetic strategy for the production of ELRs, 53 in this case fused to silk-like sequences (repetitions of the GAGAGS hexapeptide), was reported by Cappello et al. in 1990 (21). Nevertheless, the molecular biology methods that led to the obtaining of the elastin-like gene were described two years before in a patent by Ferrari et al. (22). This document explains the head-to-tail concatemerization of the elastin-like gene by self-ligation of cohesive DNA ends leading to a final gene encoding the amino acid sequence (VPGVG)160. Hence, this self-ligation method allowed the synthesis of ELR genes with different lengths, although in an uncontrolled manner and without the guarantee of achieving a gene with the desired length. This process was further explained by Tirrell et al. for the genetic engineering and expression of protein polymers in general, also discussing the potential issues derived from the use of the recombinant DNA technology (23). 2.1.3. Contemporary times (the “seamless recursive ages”) After these first steps towards genetic engineering of ELRs, new methods were developed to overcome some disadvantages, like the low number of endonucleases recognizing nonpalindromic cleavage sites needed for the self-ligation in a correct head-to-tail orientation. Hence, some procedures described for general cloning were used in the context of ELRs. This is the case of the “seamless cloning” technique that allowed the cleavage of DNA outside the recognition sequence by the use of the type IIs restriction endonuclease Eam1104I and, therefore, avoided the introduction of extraneous nucleotides (nts) in the cloned sequence (23). This method was first used successfully by Conticello and co-workers for the synthesis of ELR genes, suggesting that it could be a more rapid and efficient system for the bioproduction of protein polymers (25). Nonetheless, they still relied in concatemerization to achieve the desired length of the gene. To overcome this limitation, Meyer and Chilkoti proposed a new method termed “recursive directional ligation” (RDL) (26). In their work, they described the use of two different restriction endonucleases with well-defined features, namely PflMI and BglI, to synthetize ELR genes. These two restriction enzymes leave single-stranded DNA ends upon cleavage that are cohesive one with the other, so one of them can be used to extract the ELR insert, while the other one is used to linearize the cloning vector. Both molecules were then mixed together for ligation to achieve the final construction. The plasmid vector was designed so the restriction sites were maintained after each cloning step, allowing 54 subsequent insertions of ELR-coding genes. As an evolution of this method, Chilkoti and co-workers described a new RDL strategy termed plasmid reconstruction (PRe)-RDL (27). In this case, they introduced type IIs restriction endonucleases to RDL for a more efficient seamless cloning. Moreover, it avoided self-ligation of the vector and nonproductive circularization of the insert by cutting both the insert-donor plasmid and the receptor vector in halves with two different type IIs endonucleases that leave non-complementary overhangs. Therefore, a circular plasmid is only achieved when both insert and vector have been ligated. Despite the great improvement in the genetic engineering of ELRs reached so far, there were still some limitations that should be overcome, like complexity in plasmid design. In this regard, Rodríguez-Cabello et al. described a new method for the easy and rapid generation of ELR gene constructs and their expression in heterologous hosts (28). For this purpose, they relied on the seamless cloning approach through two type IIs endonucleases, namely the aforementioned Eam1104I and SapI. The only difference between them is that SapI recognizes a 7-nt sequence, while Eam1104I recognizes a 6-nt one (GCTCTTC and CTCTTC, respectively), being the latter included in the SapI one. With this strategy, it is possible to engineer a plasmid so it can include two Eam1104I restriction sites, being one of them also a SapI recognition sequence. For this purpose, two commercially available plasmids, one being the pDrive cloning vector and the other one the pET-25b(+) expression vector, were modified by site-directed mutagenesis to exclude inherent Eam1104I and SapI restriction sites and only include the desired ones. On the other hand, it allows controlled concatemerization following the previous guidelines for RDL. Furthermore, it avoids self-ligation in a very simple way by treating the receptor plasmid with a shrimp alkaline phosphatase, hence eluding the need of cutting plasmids in halves as described above. This procedure, named iterative-recursive method, is a good example of how molecular biology methods can be fine-tuned to achieve well-defined repetitive genes coding for protein polymer sequences very efficiently. A schematic representation of the multiple options in ELR design and genetic engineering can be observed in Figure 2. 55 Figure 2. Schematic representation of the different approaches for the design and genetic engineering of protein polymers in general and ELRs in particular. Adapted with permission from (29). 56 2.2. Hosts for the expression of elastin-like recombinamers 2.2.1. Prokaryotic hosts 2.2.2.1 The gold standard: Escherichia coli Like in the case of many other proteins produced by recombinant DNA technology, E. coli was used for the heterologous expression of ELRs in the first place, as is the case for all the works commented above. This is mainly due to the well-studied metabolism and culture conditions of this Enterobacteriaceae. Furthermore, ELRs do not undergo posttranslational modifications and their folding is correctly achieved without the mediation of eukaryotic chaperones or any similar system. Hence, E. coli arose from the beginning as a good host to achieve an optimal expression and yield of ELR biosynthesis. However, although expression was easily achieved for short (30) and long ELRs (31), there was still plenty of room for optimization. First, Guda et al. compared the expression of the gene coding for G-(VPGVG)119-VPGV in Luria Broth (LB) culture medium following induction with isopropylthio-β-D-galactoside (IPTG) and Terrific Broth (TB) without induction (32), and they found that the expression was very much higher in the case of TB culture after 24 hours. This TB medium had shown before a favorable effect on plasmid stability, while the use of lactose in TB made it a good auto-induction medium (33). Other approaches regarding the optimization of ELR bioproduction explored the supplementation of E. coli culture medium with amino acids that are highly repeated in the ELR sequence: glycine, valine, proline, and alanine. In this way, depletion of intracellular amino acid pools in E. coli could be avoided. Therefore, Chow et al. studied the expression of ELR and the final yield in terms of grams of ELR per liter of culture, both with LB culture medium followed by IPTG induction, and with TB medium (34). In this work, the authors showed that the use of glycerol, phosphate buffer and proline as supplements of TB medium enhanced 6-fold the ELR yield compared to the basal TB, from an initial 0.27 g/L to 1.6 g/L, with the subsequent reduction in cost. Furthermore, their results suggest that, surprisingly, supplementation of amino acids other than those abundant in ELRs (asparagine, aspartic acid, glutamine and glutamic acid) also enhanced 63 expressed a library of different SELRs in E. coli cultured in 96-well plates and purified the recombinamers in situ. Then, they performed a physicochemical and mechanical characterization on those SELRs without taking them out of the plates, suggesting that it could be a rapid and powerful tool to elucidate the properties of recombinant materials. In another work, Bracalello et al. designed and produced a chimeric resilin-elastin- collagen-like recombinamer that showed a different self-assembling pattern than any of each protein polymer had shown separately, with great tendency to form higher order fibrillar structures (55). 2.3.3. Fusion of bioactive domains In order to generate extracellular matrix-like materials, cell adhesion motifs have been fused to ELR sequences by genetic engineering as described by different groups. In a first example, Panitch et al. combined a VPGIG repeated sequence with the REDV peptide found in the CS5 region of fibronectin that promotes endothelial cell attachment and spreading, but not smooth muscle cells or platelets (56). In their work, they showed a successful attachment of cells on surfaces coated with the ELR, compared to the control surfaces. Similarly, Girotti et al. fused the REDV sequence to an ELR including lysinecontaining blocks that could be cross-linked to form artificial matrices, as demonstrated (57). Regarding other cell-adhesion sequences, Urry and co-workers used the RGD tripeptide (58) for the first time to successfully enhance cell attachment on ELR-based matrices cross-linked by γ-irradiation (59). However, this first case of RGD modification was performed with chemically synthetized polypeptides. On the other hand, the first example found in the literature of a recombinantly produced ELR including RGD sequences was reported by Liu et al. who compared the cell response between RGD and REDV sequences (60). In their study, they found that RGD promoted a faster attachment while being stronger than in the case of REDV. Most probably because of that finding, many other groups have included RGD domains within ELR molecules since then to improve cell adhesion. In addition to these works regarding cell-adhesion sequences, other motifs have been fused to ELRs to undergo dimerization, therefore modifying the structural properties of 64 their supramolecular assembling. This is described in the work by Fernández-Colino et al., where they showed the fusion of a leucine zipper domain, containing a cysteine residue, to an ELbcR (61). This domain was shown to be able to form dimers stabilized by a disulfide bond, hence allowing the cross-linking of hydrogels based on this zipper-containing ELbcR in a reversible manner, depending on the redox conditions of the system. On the other hand, Zhang et al. designed and bioproduced ELRs including either a SpyTag short polypeptide or a SpyCatcher protein (62). This protein is able to recognize the SpyTag polypeptide undergoing an autocatalytic bond formation between them. The combination of ELRs comprising these Spy sequences resulted in different supramolecular structures that were further characterized. As can be deduced from the above paragraphs, many different sequences can be recombinantly introduced within the backbone of the ELRs depending on the physical, chemical and biological requirements of the final protein and the subsequent application. 2.3.4. Fusion of full-length proteins In order to anticipate the properties of ELRs when fused to different proteins, Chilkoti and co-workers studied the effect of the arrangement of four proteins in the final fusion product, i.e. C-terminus or N-terminus, on the expression levels and yields of purified protein (63). By their results, they could conclude that the yield was higher when the proteins were placed at the C-terminus, and that the specific activity of the fused proteins was higher in that case for three out of four proteins. However, as they comment themselves in the manuscript, these results are applicable only to an ELR with a specific sequence, and to four particular proteins. Additionally, the same group was able to develop a model to predict the effect of hydrophilic proteins on the thermal behavior of ELR fusion proteins, showing that the presence of charged residues is the most important parameter affecting the Tt of the ELR when compared to the ELR itself (64). Many other proteins have been fused to ELRs to date. Some of them used ELRs as tags, taking advantage of their facile purification by ITC (31) to produce recombinant proteins (65) that are finally excised from the ELRs by different methods, namely intein selfcleavage (66), protease-mediated cleavage (67), or by including the self-processing module from Neisseria meningitides FrpC (68). There are many examples in which this 65 approach has been employed in the expression of recombinant proteins in Nicotiana tabacum plants (69), like an anti-human TNF antibody (70), and in tobacco cell suspensions to produce human IL-10 (71). Furthermore, this strategy has been also shown to be successful to produce antimicrobial peptides in E. coli efficiently (72,73). 3. STRUCTURES AND PHYSICAL CHARACTERISTICS OF ELASTIN-LIKE RECOMBINAMERs Materials based on ELRs underlie a high potential, characterized by the extraordinary biocompatibility, tunable mechanical properties and the variety of structures that can be generated (i.e. micelles, nanoparticles, hydrogels, films, and nanofibers). Besides the introduction of bioactive sequences into the ELR-based structures, ELRs can be designed to self-assemble into either micelles, physical hydrogels, nanoparticles, or solvent casted films (Figure 3). Furthermore, reactive cues can be introduced in the sequence to allow chemical modification. Figure 3. The diversity of ELR structures and their sequential origin. 66 3.1. Micelles and nanoparticles The first self-assembling ELRs were achieved with polymers containing blocks of different polarity, inspired by block-copolymers and their related properties, like phase separation, micelle formation, etc. The difference between block-copolymers and amphiphilic ELRs, is that in amphiphilic ELRs the blocks of different polarity retain the ELR pentapeptide sequence (VPGXG), and the changes in polarity are introduced through the X amino acid. The influence of the X amino acid, has been methodically described by Urry (74). As well as block-copolymers, elastin-like block co-recombinamers (ELbcRs) showed the ability to form micelles in solution. The hydrophobic block tends to be embedded in the core, hidden from the water solution, while the hydrophilic block(s) forms the corona exposed to the outer part of the structure. It has been shown that the formation of stable nanoparticles requires a relatively high molecular weight of at least 48 pentapeptides. Furthermore, the particle size and the molecular weight of the ELR are directly related (75). The ITT of the ELRs, depending on the characteristics of the micelles, can either lead to coalescence of micelles into lyotropic gels (76), through polydisperse microparticles (77), or to a simple swelling and deswelling of the micelles, accompanied by size changes. Typical sizes of ELR nanoparticles reported are usually in the range of 10 to 100 nm (78- 83), but due to the temperature sensitivity and related agglomeration, also micro sized particles have been reported (77, 84). The ability to trigger the formation of nanoparticles by the swelling and shrinking kinetics, intensified the research on the tuning of the micelle sizes and the Tt related changes. Several methods have been explored to either stabilize ELR-based micelles. For instance, the concentration of the ELR solution, which has been found to have a clear effect in the particle size (76); another method is the addition of surfactants that can stabilize or destabilize the ELR particles (85); salt concentration is crucial, higher compensation of the charges of the hydrophilic blocks through counter ions can lead to stronger agglomeration of particles (80, 81, 83); and last but not least, the pH affects the stability and size, depending on the chemical properties of the guest amino acid in the fourth position of the basic pentamer (VPGXG) of the hydrophilic block. In the case of cationic amino acids the charge is reduced at high pH, on the other hand, anionic amino acids show the same behavior at low pH (80). Moreover, more specific sensors for changes in salt concentrations have been designed. For instance, the introduction of a 67 calcium selective sequence amplified the influence of calcium concentration, by reducing the transition temperature of the corresponding ELR from 70°C to 35°C (86). Even when the general shape of homogenous micelles is round, it can be tuned to more anisotropic cylindrical shapes by varying the architecture of the ELRs introducing amphiphilic blocks of different sizes in the protein sequence (87). In principle, each ELR has one clear transition temperature, but in the case of amphiphilic ELRs with different blocks, long enough to develop an own intrinsic transition, the effect of temperature on the particles shows two temperature-induced changes. First, the rearrangement of the hydrophobic core, driven by a change in the secondary structure from random coil and β-sheets to type-II β-turns. This change as well corresponds with the exhibition of a cylindrical shape of the particles. This effect is also known as critical micelle temperature (CMT). The second and more prominent change is due to the collapse of the hydrophilic part, which is responsible for the coalescence, agglomeration and precipitation of the micelles (75). Other reported rearrangements describe the change from micelles to vesicles by reorganizations of the hydrophobic blocks. Here, vesicles could be obtained either by an increase of the length of the hydrophilic block, or by addition of another hydrophilic block to a triblock copolymer of the structure hydrophilic-hydrophobic-hydrophilic (48). 3.2. ELR-coatings and films The generation of ELR coatings has a great interest for the creation of either antimicrobial, anti-fibrotic coatings, or for the deposition of a bioinductive layer that allows cellular interaction driving to a good implant integration within the surrounding tissues. In theory, there are two ways to accomplish a coating: physisorption by intermolecular interactions (hydrophobic and/or electrostatic interactions), or a grafting approach by covalent binding (Figure 3). For completion, a third possibility could be possible, which is the creation of elastin-like brushes by a grafting approach, but due to the relatively high molecular weight of ELRs, and the related cost to generate such proteins in a synthetic way, this approach is still a hypothetical option. Additionally, these proteins would not be ELRs since they are not obtained by recombinant techniques so they should be classified as ELPs (8, 19, 88, 89). 68 Physisorption of ELRs has been deeply studied and applied in different works. It was described by Srokowski et al. that longer ELRs formed more stable coatings by physisorption than ELRs with lower MWs (90). Another approach targets on the endothelialization of CoCr alloys, that could be enhanced by physically adsorbed or covalently bond ELRs that bear a REDV sequence (91). ELR coatings also proved to reduce platelet activation and smoothening of surface topographies when coated PTFE substrates by a layer-by-layer approach (92). This was corroborated by other studies that showed that longer ELR sequences not only have a better deposition, but also decrease platelet activation more than short ELRs (90). In an earlier study, the patency time of nonthrombogenic ELR coatings could be more than doubled (93), and thrombus formation reduced (94). Further micelle solutions have been used to adsorb ELRs to surfaces under retention of their nano- and microtopography. For the generation of surfaces that induce bone mineralization nanotopographies, it was performed a coating with an ELR containing a human salivary statherin sequence (95). The coating of previously generated orientated electrospun fibers with ELRs led to a conserved orientation in the scaffold, which guided human vocal fold fibroblasts (96). The transition temperature of the ELRs was also used to enhance the physisorption by thermally induced deposition. Here, surfaces with a low RGD concentration were formed, high enough to allow cell adhesion, but too little to form a cellular monolayer. Thus cells were forced to cluster and semi-functional pseudoislets could be formed (97). The good mechanical performance of SELR hybrids further led to the development of coatings for osteochondral applications (98). Besides the deposition of ELRs in order to induce or avoid attachment of cells or proteins, the ability of the ELRs themselves to influence the microenvironment by temperature changes is of great interest, for example for biosensors, or drug delivery systems. By changing the temperature, features like the wettening of the surface (77), the release kinetics of drugs embedded in the coating (84), or the accessibility of active groups inside the ELR sequence in swelling and deswelling ELR/RGD brushes (99), can be controlled. 69 3.3. ELR-based hydrogels In tissue engineering, hydrogels have been the predominant matrices for application in the human body in the last 40 years (100). A high water content leads to convenient mechanical properties and stimuli-responsiveness, which in many cases chimes with the properties of natural tissues (101,102). ELRs, inspired in a natural protein such as elastin, are excellent candidates for many researchers focused on the generation of artificial matrices that can be used as scaffolds for biomedical applications. The ECM is a very complex system in which the physical properties (elasticity, stiffness), the nanotopography, the presence of signaling molecules, protease-sensitive sites and adhesion domains are of great importance, and gathering as many of these properties as possible is a major requirement that have to be demanded to any material that might be used in tissue engineering. The generation of hydrogels from linear ELRs requires paying special attention to the mechanism that will drive the cross-linking of different ELRs molecules. These cross-linking methods can be either of covalent or physical nature and the position of cross-linking points along the ELR backbone can be fully controlled through genetic engineering to get a precise tuning of the mechanical properties. Polypeptide-based block-copolypeptides, for example, manage to self-assemble into stable hydrogels (103), which can be further stabilized when flanked by protein segments with coiled-coil secondary structure (104, 105). Another approach is the use of recombinant segments of elastin, silk and collagen (106-108). In contrast to general methods for the formation of hydrogels by radically or photo-polymerized acrylate cross-links, ELR hydrogels can be thoroughly controlled (e.g. chain and segment length, number of cross-links per chain). This leads to more homogeneous matrices, and to the reduction of artifacts, which could impair the mechanical properties of the resulting hydrogels (109). Cross-linking mechanisms for ELRs can be as versatile as ELRs themselves: ionic or hydrophobic interactions, reaction of complementary groups, or enzymatically induced cross-links (Figure 4) (110, 111). All these strategies can provide a very tight control over the length and the molecular weight of the proteins by the selection of the cross-linking sites, which usually correspond to lysine groups (112, 113). Besides the control of the cross-linking, aforementioned bioactivities can be introduced into ELRs, so that the matrix is able to interact with the organism, improving the good integration of the generated 70 tissue. The basic ELR sequence (VPGXG) is biocompatible, but lacks cellular adhesion sites. Nevertheless, the possibility of a tailored genetic design permits changes in the transition temperature, integration of protease-sensitive sites (114, 115), cellular adhesion motifs (59, 116), or biological triggers (117-119). Figure 4. Common ELR crosslinking strategies: A) ionic interaction, B) chemical crosslinking, C) hydrophobic interactions, D) enzyme triggered crosslinking. Physical cross-linking of ELRs can be obtained by several strategies. One approach is a cross-linking based on ionic interactions by segments of opposite charge (e.g. cationic and anionic amino acids in the X position of the ELR). Even though ionic interactions are relatively weak, a specific design of complementary charged strands (120, 121) helps to a better strengthening of these interactions which leads to obtain stable ELR hydrogels at room temperature and physiological pH (122). Potentially, the presence of ions could trigger the formation of hydrogels, since some ELRs are sensible to salts concentration changes, especially to chelating ions like calcium (121). More advanced approaches including ELR/chitosan blends, could be stabilized by sodium ions (123), and modification with monosaccharide side chains allowed a complexation with potassium (124). A second way of physical cross-linking is obtained by the introduction of self-assembling motifs, i.e. amphiphilic blocks, or interaction of protein secondary structures (β-sheets, leucine zippers). The self-organization is basically driven by aggregation of different segments. In 71 the case of amphiphilic block ELRs this self-assembling is due to the aggregation process of the hydrophobic segments. On the other hand, the interaction of secondary structures is triggered by the aggregation of β-sheets or pairing of leucine zippers. Hydrophobic blocks are generated when the ELR sequence has several hydrophobic amino acids (like alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr) or methionine (Met)) on the X position. This results in segments of distinct polarity within the same protein chain and leads to mutual repulsion between the different blocks, that tend to segregate. The segregation is locally constrained by the “forced cohabitation” of the blocks within the same ELR chain, resulting in a separation into different domains, which as a consequence, self-assemble into periodic nanostructures (125). Amphiphilic blocks can be designed to form stable hydrogels that even persist in vivo. One way to enhance stability is to create longer sequences with several blocks of alternating polarity (108, 126, 127). The interaction of intermolecular secondary structures, on the other hand, follows the key-lock-principle with the assembly of matching structures of similar polarity. From the tailor perspective, almost any protein secondary structure that is able to form a stable link with itself or contrary groups could be implemented. It has to be considered that in one component systems gelation can occur easily, and solvation is limited. Furthermore, the effect on the ELRs Tt has to be considered. The recent approaches to include physical cross-linking cues into ELRs are inspired by natural silk sequences that enable crystalline-like β-sheets formation with unique mechanical properties (128-132). The repetitive sequence responsible for the β-sheet formation and for the intrinsic strength is the GAGAGS hexapeptide (Bombyx mori silkworm) (133). In this silkworm, the blocks are stabilized by hydrophilic compartments within the protein and a complex mixture of stabilizing agents (134). The integration into ELRs needs to consider the stability of the resulting bonds and the amount of silk domains govern the manipulation of the material. Silk-ELR (SELR) hybrid materials reveal a twostep gelation process when heated above the Tt. The ELR transition occurs first, enabling the thermodynamically driven annealing of silk sequences, hence forming stable physically cross-linked hydrogels in situ (108). SELRs are biocompatible and performed well in in vivo studies (135-137). Moreover, they have been forged into a variety of 72 structures like hydrogels, films, 3D porous matrices and submicron to macroscale fibers (138). Lately, ELRs physically cross-linked by leucine zipper domains (139-145) gained interest. The leucine zippers are capable to dimerize with other leucine zipper domains driven by hydrophobic interactions (139,146-148), and ionic interactions (149-151). On account of its novelty, ELRs with leucine zipper motifs have been less explored than SELRs, and the complete potential remains to be discovered. Nevertheless, the human origin of the domain and the conserved nature of the structure alleviate concerns regarding the biocompatibility. First studies on ELR-Zippers support the biocompatibility and showed enhanced stability when compared to pure amphiphilic block ELRs (61). Furthermore, a recent in vivo study regarding zipper-based scaffolds revealed no foreign body reaction (152). The introduction of amino acids bearing functional groups in the X position can be used for subsequent reactions without losing the pH and temperature sensitivity of the ELRs. The majority of published works used the free ε-amine of lysine residues within the ELR chain for chemical modification due to its reactivity. (153, 154) Regarding the cross-linking process, the MW, concentration and the number of cross-links are important to form stable hydrogels (155). ELRs with high molecular weight have a higher entanglement and are more prone to establish a sufficient number of cross-links to stabilize the hydrogel network. Due to the tailored design of the ELR sequence, the number of cross-linking sites and the segment length between cross-links can be precisely controlled, enabling the tuning of relevant features such as pore size, gelation time, stiffness and degradability. Functionalization of ELRs aims to introduce bioactive sequences, adhesion sites, inhibitors, antibodies or anchor and signaling molecules (156). Degradation can be further controlled through the introduction of protease-sensitive sequences, or through the introduction of labile chemical linkages (157). One drawback of common chemical linkages is that they are reactive under conditions that require organic solvents or other chemical reagents to avoid hydrolysis. To elude removal of this undesired components, the typical cross-linking of hydrogels is based on the reaction of complementary groups (158), enzymatic cross-linking (159-161), condensation reactions (162), or high-energy irradiation (163). The classical cross-linking 79 (91). In this sense, González de Torre et al. have shown the applicability of recently developed ELRs as a coating for vascular stents with the ultimate goal of producing a new endovascular device (165). The ELR applied on the stent gain a full endothelialization in a short time (2 weeks), showing high biocompatibility and a reduced response of the immune system. Finally, another approach by Weber et al. was to generate tissueengineered heart valves (TEHVs) by multi-step injection molding using ELR as a hybrid system with fibrin (228). In 2015, Mata’s group demonstrated the supramolecular interaction between peptides and ELRs to generate complex 3D architectures through a dynamic self-assembly system, forming a stable multilayer membrane. This membrane can be spatiotemporally controlled and can be used to form bioactive tubular scaffolds which may support and lead the growth of different cell lines (229). This tubular morphogenesis could play an important role in tissue engineering applications that requires the formation of tubular structures that usually are complex to obtain in the milli- and micro scale. According to the elastin-like nature of the hydrogel and the high percentage of elastin present in the native chondral matrix, ELR-based hydrogels are likely to simulate the properties of hyaline cartilage (230, 231). The hyaline articular cartilage is a highly specialized tissue characterized by its unique mechanical features and it is formed by a matrix that embeds chondrocytes. Considering that the articular hyaline cartilage does not repair itself and that the generally regenerated fibrocartilage is unable to maintain the biomechanical characteristics of articular cartilage (232, 233), ELR hydrogels could be used as scaffolds for osteochondral tissue engineering. As it has been previously described above, ELRs show thermo-sensitivity, on that regard is possible to form hydrogels stable at body temperature, whenever the transition temperature (Tt) of the ELR is lower than the body temperature. Moreover, ELRs containing bioactive sequences, such as the wellknown RGD cell-adhesion sequence found in fibronectin, which promotes specific cell attachment via integrins (162), are able to form a bioactive scaffold that improves the regenerative potential of the implanted hydrogel. Vila et al. showed how ELR coatings are able to improve the well-known biocompatible and bone regeneration properties of calcium phosphate-based materials (234). 80 Due to the development of novel tissue-engineering methods (52, 235), it has been considered the use of mesenchymal stromal cell (MSC) therapy (236, 237) for the treatment of musculoskeletal lesions (238, 239). For articular defect applications, the use of a hydrogel serves as a vehicle for the MSCs to acquire a 3D structure that could mimic the properties of the ECM providing a cell-friendly environment in order to increase the persistence of the implanted cells at the site of injury. According to ELRs properties, a homogeneous embedding of MSCs in the ELR solution can be achieved at a temperature below Tt, and further applied as a cell-scaffold system for injectable therapies (Figure 5). Figure 5. ELR-based injectable hydrogel for osteochondral applications. Moreover, ELRs might also have beneficial applications in the field of ocular tissue engineering. Cornea wound healing requires cell adhesion and proliferation on a substrate with ligands such as fibronectin, secreted by corneal epithelial cells and stromal fibroblasts during the first steps of corneal wound healing (240). Nevertheless, the ocular surface, unwounded cornea and conjunctiva do not express elastin. Therefore, several metalloproteinases (MMPs) have been described in pathological ocular processes such as dry eye (241) or conjunctivochalasis (242), while some other MMP present at the ocular surface are able to degrade elastin fibers (241). Thus, ELRs are a potential candidate for Bruch´s membrane prosthesis. Martínez-Osorio et al. showed how a blend of ELRs was able to promote epithelial cell adhesion from human conjunctiva-derived primary cells (192). Finally, ELRs application as ocular implants was studied by Srivastasa et al., which confirmed ELRs as a suitable carrier for the transplantation of autologous RPE cells for the treatment of age-related macular degeneration (AMD) (243). 81 4.4. ELRs for surface bio-functionalization The ELRs properties of elasticity and self-assembling allow the formation of a wide range of biomaterial-based constructs such as aggregates (51), films (77), fibers (224), micelles (48, 244), nanoparticles (245), and hydrogels (246). Another advantage of this recombinant biomaterial is the capacity to form hybrid systems with materials having different origins, in order to obtain several morphologies and functional possibilities for a diverse range of applications such as functionalized surfaces, fibers, and drug delivery. Therefore, the recombinant technologies by which ELRs are obtained, allow a perfect control of their sequence, length and stereochemistry; moreover, for functionalized surfaces even a nanometric control of their position displayed by these systems is possible. Chilkoti’s group has created what they refer to as the "Thermodynamically Reversible Addressing of Proteins" (TRAP) (247), where an ELR is covalently micropatterned onto a glass surface obtaining a spatial-temporal system for protein binding that can be applied as a microsensor for detecting single biomolecules in bioanalytical applications. Additionally, different approaches based on controllable properties (stimuli-responsive) of the biofunctionalized surfaces have been studied in order to obtain a cell sheet harvesting system from a culture dish. In this sense Okano et al., have developed a smart surface with PIPAAm polymer (and its derivates) that can switch between a cell-adherent and non-adherent state as a result of a change in temperature (248). Despite this approach led a significant progress in this field (249-251), PIPAAm polymer lacks specific bioactivity, meaning that cellular membrane proteins, such as integrins, are not able to directly bind to the surface. To address this issue, Pierna et al. developed a smart surface system by covalent coupling of tailored ELRs onto glass surfaces by click chemistry methods. This cell sheet harvesting system leads to the exposition of the bioactive RGD motif to the water interface at physiological temperature, producing a cell adherent surface (252) (Figure 6). Na et al. also took advantage of the rapid response to external stimuli of a smart material surface created by adsorption of ELRs for use in cell-based biochips. The smart transition of ELR-based micropatterns between a hydrophilic and a hydrophobic surface glass at Tt allows to revert cell adhesion by way of the incubation temperature (253). 82 Figure 6. Schematic representation of a system based on ELRs for cell sheet harvesting. Layer-by-layer (LbL) techniques are one of the most versatile and easy-to-apply of the numerous surface-modification tools. They are based on the spontaneous adsorption of materials onto a substrate, generally a polymer, biomolecule, or inorganic particle, and allow the sequential formation of a nanostructured film, hence having a great interest in tissue-engineering applications (254-257). Layer-by-layer deposition of alternating ELR- polyelectrolytes generated bioactive surfaces (258). In this work, it was developed a thermoresponsive thin coating by electrostatic self-assembly (ESA); the deposition of an ELR containing the bioactive RGD motif can be exploited for tunable cell adhesion and controlled protein adsorption by nanoscale surface tailoring (77). Another example of biomimetic surface-modification regards the chemical functionalization of ELRs in order to obtain metallic (Commercial pure titanium, Cp Ti) dental implants with osteostimulative capabilities by the covalent immobilization of biomolecules on the Cp Ti (259). Finally, Costa et al. demonstrated the feasibility of LbL synthesis using natural marine-based polysaccharides (chitosan and alginate) and ELRs (260) for possible application in wound dressings and drug-delivery systems (261, 262). Furthermore, ELR molecules can be used for the functionalization of surfaces in order to obtain stronger and faster cell responses on the tissue–biomaterial interface, thus promoting better implant integration than short peptides functionalization; for instance, ELRs have been used to enhance the properties of poly-methylmethacrylate (PMMA) surfaces. Although PMMA has a great usage in several biomedical applications (263-269), this study showed that 83 ELR-functionalized PMMA surfaces can enhance the cellular attachment efficiency and the cell anchorage strength (270). Finally, ELRs were thoroughly investigated as biocompatible vasculogenic surface coatings and smooth muscle cells (SMC) showed enhanced attachment under retention of their contractile phenotype on similar ELR-coated electrospun fibers (271). Abbreviations A Alanine Ala Alanine AMD Age-related macular degeneration APCs Antigen-presenting cells ASTM American Society for Analysis of Materials C Cysteine CMT critical micelle temperature CPPs Cell penetrating peptides D Aspartic Acid DNA Deoxyribonucleic acid E Glutamic Acid E. coli Escherichia coli ECM Extracellular matrix EGFP Enhanced green fluorescent protein ELbcR Elastin-like block co-recombinamer ELP Elastin-like polymer ELR Elastin-like recombinamer ESA Electrostatic self-assembly G Glycine Gly Glycine IL Interleukin Ile Isoleucine IPTG Isopropylthio-β-D-galactoside ITC Inverse transition cycle ITT Inverse temperature transition LB Luria Broth LbL Layer by layer LCST Lower critical solution temperature Leu Leucine M. tuberculosis Mycobacterium tuberculosis Met Methionine MMPs Matrix metalloproteinases MSCs Mesenchymal stromal cells Mw Molecular weight nts Nucleotides 84 P Proline P. pastoris Pichia pastoris PDMAEMA Poly(2-dimethylamino-ethylmethacrylate) PEG Polyethylenglycol PEI Polyethylenimine Phe Phenylalanine PIPAAm poly(N-isopropylacrylamide) PLGA Poly(Lactide-co-Glycolide) PLGA Polylactic-coglycolic acid PLL Polylysine PMMA Polymethyl methacrylate Pre-RDL Plasmid reconstruction-recursive directional ligation Pro Proline PTFE Polytetrafluoroethylene R Arginine RDL Recursive directional ligation RPE Retinal pigment epithelium SELR Silk Elastin like recombinamer SEM Scanning electron microscopy SMCs Smooth muscle cells T Threonine TB Terrific Broth TEHVs Tissue engineering heart valves TNF Tumor necrosis factor TRAP Thermodynamically Reversible Addressing of Proteins Trp Tryptophan Tt Transition temperature Tyr Tyrosine V Valine Val Valine 85 References 1. 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Mateos- Timoneda. Regenerative Biomaterials, (2015). 271. P. H. Blit, K. G. Battiston, M. Yang, J. Paul Santerre and K. A. Woodhouse. Acta Biomaterialia 8, 2493-2503 (2012). 98 99 100 CHAPTER 2 ELASTIN-LIKE MATERIALS FOR TISSUE REGENERATION AND REPAIR José Carlos Rodríguez-Cabello,1,2 Israel González de Torre,1,2 Filippo Cipriani,2 Leander Poocza,1 1 Universidad de Valladolid, BIOFORGE, CIBER-BBN, Valladolid, Spain 2 Technical Proteins NanoBioTechnology (TPNBT) S.L., Valladolid, Spain J.C. Rodríguez-Cabello, I. G. de Torre, F. Cipriani, L. Poocza. Elastin-like materials for tissue regeneration and repair. Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair (2018). doi: 10.1016/B978- 0-08-100803-4.00012-7 101 1. INTRODUCTION 1.1. Elastin-like recombinamers The prerequisites for the use of materials in tissue regeneration include biocompatibility and bioactivity. As such, elastin-like recombinamers (ELRs), which are novel biomaterials inspired by elastin, a component of the natural extracellular matrix (ECM), meet both these requirements (1). Elastin is a fibrous and insoluble protein that constitutes one of the most important structural and functional components of the ECM, allowing for high deformations without damage (2). It is abundant in the lungs (3-7%), skin (2-3%), blood vessels (28-32%) and elastic ligaments (50%) (3), where its elastic properties are essential. Elastin, the main function of which is to provide elasticity to organs and tissues, is an excellent example of how all the properties displayed by biological materials and systems are determined exclusively by the physicochemical properties of the monomers and their sequence (4, 5). Indeed, a single repeated pentapeptide sequence has been shown to be responsible for the elastic behavior in elastin and, as such, forms the basis of all ELRs (6, 12). The most widely studied pentapeptide is (VPGXaaG), namely poly(Val-Pro-Gly-Val-Gly), where Xaa is any natural amino acid except proline. All functional ELRs present a reversible lower critical solution temperature (LCST) in aqueous solution with sharp responsiveness (13). According to Urry’s model, the polymer chains fold hydrophobically and undergo a conformational transition that leads to phase separation above this temperature (14, 15). It has been proven that the amino acid sequence has a significant influence on the LCST of ELRs (16). Thus, substitutions of the amino acid at the fourth position (Xaa) of the pentamer modify the LCST to an extent that depends on the polarity of the amino acid side-chain. The transition temperature of an ELR sequence based on (VPGXG)n can be controlled and adjusted to the desired applications by varying Xaa (hydrophobic AAs decrease Tt and hydrophilic AAs increase it) (16), the segment length n (longer ELR sequences have a lower Tt) (17, 18), concentration (higher ELR concentrations decrease Tt) (17), pH (19) and salt concentration in the selected solvent (20-22). Biotechnology provides us with a powerful set of tools, such as recombinant DNA design (23), that can be used to successfully control the physicochemical features of the amino 102 acid side-chains and their association (24, 25), or to include any protein-based functionality, such as protease active sites, which become important when degradation of the scaffold has to be adjusted to the growth rate of new tissue (26). 1.2. Mechanisms to form ELR matrices for tissueengineering applications The predominant matrices for tissue regeneration, which were also the first to be applied in humans (27), are hydrogels. Indeed, due to their high water content, resemblance to natural tissue, biocompatibility and stimuli-responsiveness, these compounds have attracted increasing interest in the last 40 years (28, 29). However, as traditional methods of hydrogel synthesis lack an exact control over cross-linking points, chain length and sequence, the resulting three-dimensional structure may contain defects that can impair the mechanical properties of the material (30). These problems have been addressed by the development of novel polypeptide-based responsive hydrogels, including blockcopolypeptides (31), or recombinant co-polypeptides flanked by two coiled-coil blocks (32, 33) and recombinant segments of elastin, silk and collagen (34-36). The integration of crosslinking motifs that lead to a stable hydrogel is a prerequisite for the formation of ECM-like matrices from ELRs. This crosslinking can be of either a physical or a covalent nature, with the possible cross-linking mechanisms being as versatile as the ELRs themselves, ranging from ionic and hydrophobic interactions and the reaction of complementary groups to bioinspired protein crosslinks (37, 38). ELRs have the potential to form a material that is both biocompatible and has specific mechanical properties, and can also interact with the body to improve the natural regeneration of tissue. Although the repetitive ELR pentapeptide (VPGXG) itself does not facilitate adhesion, its extraordinary design means that adhesive (RGD) (39, 40) and degradation sequences (41, 42), as well as sequences that allow the inclusion of temperature-related or biological triggers, can readily be integrated (43-45). All these strategies can provide very close control over the length and molecular weight of the proteins by careful selection of the crosslinking sites, which usually correspond to the lysine groups (46, 47). 103 1.3. Physically Cross-linked ELR Hydrogels Several strategies can be applied to prepare physically crosslinked ELR hydrogels. Some of the most popular are described below. 1.3.1. Crosslinking via ionic interactions Ionic crosslinking motifs are based on ELR segments of opposite charge, or the introduction of groups that can chelate multivalent cations into the ELR backbone, which can be further controlled by varying the salt concentration. Peptide sequences with alternating charges and with complementary ionic sites have been shown to perform well in physical crosslinking. Such sequences are classified into different moduli, depending on the size of the equally charged ionic blocks (1-4 amino acids): modulus I, – + – + – + –+; modulus II, – – + + – – + +; modulus III – – – + + +; and modulus IV – – – – + + + +. A modulus I sequence reported by Holmes et al. (48) exhibited salt-induced in situ gelation (49). Ion complexation is obtained by including glutamic and aspartic acid residues in the ELR sequence accompanied by the addition of Ca2+ ions. The resulting hydrogels can be stabilized at room temperature and at physiological pH (50) and are sensitive to chelating agents, which reduces the number of accessible Ca2+ ions. Another approach by Yeo et al. (51) involved the addition of monosaccharides to the side chains of the ELRs, thus allowing them to form stable complexes with potassium. Furthermore, ELR/chitosan blends have been shown to form stable films in the presence of sodium ions (52). 1.3.2. Self-assembly of amphiphilic blocks and graft copolymers Hydrogels can be obtained by aggregation between the hydrophobic segments of multiblock ELR copolymers. The hydrophobic functionalities are provided by alkyl-rich amino acids such as alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), tryptophan (Thp), tyrosine (Tyr) or methionine (Met). The resulting amphiphilic blocks have been shown to be stable in vivo (36, 53, 54). Furthermore, it is common to find protein polymers characterized by alternating blocks of essentially hydrophilic and essentially hydrophobic amino acids. The presence of chemically distinct segments within the same protein chain causes a mutual repulsion between the different blocks, which 104 tend to segregate. However, this segregation is constrained by the “forced cohabitation” of these blocks within the same molecule. As a result, they simply separate into different domains, thereby forming periodic nanostructures (55). 1.3.3. Intermolecular interaction of secondary protein structures Structure design offers the possibility to include almost any sequence that is able to form intramolecular interactions via secondary structures into the ELR backbone using a recombinant approach. The limiting factors in this approach are the need to retain the ELR temperature transition and the fact that complex folding might not occur. One bioinspired approach involved the inclusion of natural silk sequences that are known to form intramolecular beta-sheets which self-assemble into crystalline regions and have unique mechanical properties (56-60). These silk-based materials can be used to form a variety of structures, such as hydrogels, films, 3D porous matrices and submicron to macroscale fibers (61). Furthermore, silk scaffolds have been proven to be biocompatible and to perform well in vivo (62-64). As regards the silkworm (Bombyx mori), the repetitive hydrophobic sequence GAGAGS has been shown to be responsible for the strength of silk fibers (65). These blocks are stabilized by hydrophilic compartments in the protein and a complex mixture of stabilizing proteins (66). Fernandéz-Colino et al. have shown that the GAGAGS sequence can be successfully integrated into an ELR, thereby facilitating in situ gelation of the material and its use as an injectable system (36). A more complex crosslinking has been achieved by using coiled-coil induced oligomerization via leucine zipper motifs, which include sequences that are known to form well-defined secondary structures (67-69). Leucine zippers are characterized by heptad repeating peptide units that form a distorted α-helix, designated as “abcdefg”, where the “a” and “d” positions are occupied by hydrophobic residues such as leucine and “b”, “c”, “e”, “f” and “g” are usually of a hydrophilic nature (see Figure 1) (70). The oligomerization 111 and exert immunomodulatory properties, means that such methods are good candidates for the treatment of musculoskeletal lesions (123, 124). For articular defect applications, the suspension of MSCs needs a scaffold as a cell-carrier in order to acquire a 3D structure that could be re-populated by these cells and to increase the persistence of the implanted cells at the injury site. Moreover, the hydrogels used as vehicles for MSCs should closely mimic the properties of the ECM and provide a cellfriendly environment that supports their regenerative potential. As described previously, elastin-like recombinamers are based on a repetition of the VPGXG pentapeptide found in natural elastin, in which X can be any amino acid except proline. Given the elastin-like nature of the hydrogel and the high percentage of elastin present in the native chondral matrix, ELR-based hydrogels are likely to be similar to hyaline cartilage (125, 126). Furthermore, ELRs show thermo-sensitivity, thus meaning that it should be possible to form hydrogels that are stable at body temperature provided the transition temperature (Tt) of the ELR is lower than body temperature. In addition, as they are soluble at low temperatures, ELRs can be injected at low temperature and rapidly form a gel at physiological temperature, thereby representing a good candidate for use in arthroscopy to reduce the invasiveness of the treatment (Figure 2). This remarkable property permits MSCs to be homogeneously embedded in the ELR solution at a temperature below Tt and to self-assemble into hydrogels above Tt, thus allowing the use of the cell-scaffold system in injectable therapies. Finally, in addition to the significant advantages of ELRs described in this chapter, they can also be designed to contain bioactive sequences, such as the well-known RGD cell-adhesion sequence from fibronectin, which promotes specific cell attachment via integrins (96), in order to achieve a bioactive scaffold that provides a cell-friendly environment, thereby improving the regenerative potential of hydrogel implantation. 112 Figure 2. ELR-based injectable hydrogel for osteochondral applications 3.2. ELRs for (cardio-)vascular tissue regeneration Elastin is an essential extracellular matrix protein in vascular tissue that plays an important biomechanical and biological signaling role. However, as native elastin is insoluble and is difficult to extract from tissues, its use for the manufacture of vascular tissue engineering scaffolds remains relatively rare. ELRs that mimic the structure and function of native tropoelastin represent a practical alternative to the native elastic fiber for vascular applications, especially in cardiovascular implants, where metal-based stents are known to cause in-stent restenosis (ISR) and late thrombosis diseases (107). The most common surgeries in cardiovascular disease are coronary angioplasty and stent insertion (127,128), with stent thrombosis (ST) and ISR being the main reasons for the failure of bare metal stents after implantation. This effect is decreased to less than 10% with the use of drugeluting stents (DES), but remains critical (129), thus emphasizing the need for more efficient coatings. The coating of active ELRs containing adhesion sequences onto stent surfaces is one strategy for recovering a healthy endothelium. However, the bioactive sequences required to selectively promote endothelial growth remain unclear. ELRs have been thoroughly investigated as biocompatible vasculogenic surface coatings (53), with smooth muscle cells (SMC) showing enhanced attachment and retention of their contractile phenotype on similar ELR-coated electrospun fibers (130). It has also been shown that ELR coatings are able to reduce blood platelet activation on synthetic 113 scaffolds (131), which delays the formation of healthy endothelium due to the need for anti-proliferative drugs and leads to late ST, thereby increasing the time patients are required to take anti-platelet therapy (132), thus addressing a negative side-effect of DES. Several studies have also demonstrated that stent surface endothelialization is suitable for inhibiting restenosis and thrombosis (107, 132-134). In addition, ELRs including endothelial adhesion sequences (REDV) have been shown to facilitate endothelialization of CoCr alloys by an early HUVEC cell-adhesion response (107). These findings indicate an involvement of elastin-laminin in the initial SMC/ELR contact (130). de Torre et al. recently used catalyst-free click chemistry to cover stents by injection molding with an in situ gelating ELR system. Full endothelialization was obtained in a flowcontrolled bioreactor and enabled by REDV and RGD sequences in less than two weeks. Minimal platelet adhesion and fibrin adsorption were detected and the ELR-coated stents exhibited mechanical stability even under high flow conditions (89). A more heterogeneous approach by Weber et al. involved generating tissue-engineered heart valves (TEHVs) by multi-step injection molding using an ELR as a hybrid system with fibrin (135). Conditioning of TEHVs in bioreactor systems is necessary to achieve an adequate ECM to withstand the mechanical loads in bodily circulation, with 3D printing (136) and orientated electrospinning (137) also being used in TEHVs to address the stress differences. Since heart valves are elaborate and highly complex structures in the circulatory system, the spatial diversity of this hybrid system was considered to meet the challenges raised by the structural and mechanical anisotropy and optimal function of such valves. Thus, different materials were used for the wall (fibrin) and leaflets (fibrin/ELR) to maintain the functionality of the TEHVs and to address the problems of poor elastogenesis in tissue-engineered constructs (135, 138), which is of particular importance for their long term durability (139). In vivo studies of ELR-based scaffolds with leucine zipper motifs and RGD adhesion sites showed that they were able to support neovascularization without further growth factor treatment and an absence of immune responses, thereby underlining the potential of elastin-like materials for vascular tissue regeneration (80). 114 3.3. ELRs for ocular prostheses Corneal wound healing requires cell adhesion and proliferation, both of which are mediated by the binding of epithelial membrane-bound integrins to substrate ligands, such as fibronectin. The secretion of fibronectin by corneal epithelial cells and stromal fibroblasts increases exponentially in the first steps of corneal wound healing (140). However, the ocular surface, undamaged cornea and conjunctiva do not express elastin. Instead, a “diffusible factor” (141) secreted by the corneal epithelium has been implicated in maintaining the cellular and physiological homeostasis of the ocular surface. Several metalloproteinases (MMP) have been reported to be involved in pathological ocular processes such as dry eye (142) or conjunctivochalasis (143). As such, MMP-3 (Stromelysin 1), MMP-7 (Matrilysin 1), and MMP-9 (Gelatinase B), which are present at the ocular surface and are able to degrade elastin fibers (142), are potential candidates for Bruch’s membrane prostheses. In this regard, Martínez-Osorio et al. (102) showed that an ELR blend containing four different types of building blocks was able to achieve an adequate balance of properties and to foster epithelial cell adhesion from human conjunctivaderived primary cells. This ELR consisted of VPGIG, which has outstanding biocompatibility (99) and appropriate mechanical behaviour (144), a lysine-containing block (VPGKG) for crosslinking purposes, a CS5 human fibronectin REDV domain for efficient cell attachment (145) and an elastase target domain to enhance bioprocessing by naturally occurring enzymes. The resulting biomaterial maintained the growth, phenotype and functional characteristics of the primary cell line and did not exhibit any cytotoxicity. The performance of ELRs as ocular implants was further confirmed by Srivastasa et al. (146), who retained the efficiency of retinal pigment epithelial (RPE) cells and demonstrated that ELRs could be suitable carriers for the transplantation of autologous RPE cells for the treatment of age-related macular degeneration (AMD). 3.4. Other applications of ELRs Even though the excellent and tunable mechanical properties and very low thrombogenicity of these materials make them excellent candidates for applications related to traumatology or cardiovascular diseases, ELRs also have a wide range of possibilities in the wider field of tissue engineering. As noted above, elastin-like 115 recombinamers are a novel peptide-based biomaterial, therefore their full potential remains to be explored. For instance, very few studies have been performed concerning the application of ELRs in neural injuries such as spinal cord injuries, traumatic brain injuries, or nerve transection. As an example, two studies have shown how surfaceadsorbed recombinant protein can be used to modulate the behavior and differentiation of neuronal cells in vitro (147) and the biological activity of neurotrophin-ELR fusion proteins via in vitro culture models (148). As such, the potential applications of ELRs could be many and diverse. Indeed, they are currently being investigated for use in fields as disparate as diabetes treatment or in vitro fertilization. 4. CONCLUSIONS This chapter has described elastin-like recombinamers, a family of novel protein peptide biomaterials obtained using recombinant DNA techniques. Moreover, ELRs meet all the prerequisites of biocompatibility and bioactivity for use in the field of tissue regeneration. Given that the predominant matrices for tissue regeneration are hydrogels, and that ELRs are able to self-assemble into different structures, including hydrogels, these biomaterials play a key role in the development of scaffolds and advanced systems for applications in the biomedical field. As noted previously in this chapter, numerous techniques can be used to form different ELR matrices via physical or covalent crosslinking or by functionalization. Furthermore, the range of tissue-engineering applications for ELRs is very extensive, with recent studies having shown how different types of ELRs can be applied in the most challenging areas of tissue regeneration, such as cardiovascular, ocular prosthesis and osteochondral applications or even in nerve regeneration, diabetes and in vitro fertilization. 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J Mol Biol 1998;279:959– 72. 78. Litowski JR, Hodges RS. Designing heterodimeric two-stranded α-helical coiledcoils: the effect of chain length on protein folding, stability and specificity. J Pept Res 2001;58:477–92. 79. De Crescenzo G, Litowski JR, Hodges RS, O'Connor-McCourt MD. Real-time monitoring of the interactions of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding. Biochemistry 2003;42:1754. 127 128 PLANNING OF RESEARCH 129 In order to address the objectives of this Thesis, several ELRs have been designed and produced. In Table 1 it is reported the amino acid sequence of each ELR, which has a specific composition comprising bioactive sequences. Moreover, in Figure 1 it is reported a scheme of all the ELRs, where the coloured blocks represent the disposition of the bioactive domains of each ELR. Code Name Sequence MW (Da) A (EIS) 2 -(I 5 R) 6 MESLLP-{[(VPGVG) 2 -VPGEG-(VPGVG) 2 ] 10 -(VGIPG) 60 - [V(GAGAGSG)5]2G}-[(VPGIG)5-AVTGRGDSPASS]6V 121012 B VKVx24 MESLLP-VGVPGVG [VPGKG(VPGVG)5]23VPGKGVPGVGVPGVGVPGVGVPGV 60451 C HRGD 6 MGSSHHHHHHSSGLVPRGSHMESLLP-{[(VPGIG) 2 -(VPGKG)- (VPGIG)2]2-AVTGRGDSPASS-[(VPGIG)2-(VPGKG)- (VPGIG)2]2}6V 60660 D REDV MESLLP- [(VPGIG)2VPGKG(VPGIG)2EEIQIGHIPREDVDYHLYP(VPGIG)2VP GKG(VPGIG)2(VGVAPG)3]10V 80813 E TI MESLLP-[(VDLDVPIPGRFDRRVSVAAE(VGIPG) 10 ] 10 V 65075 F TR MESLLP- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]10VETAAAKFERQHMD SSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVC SQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQA NKHIIVACEGNPYVPVHFDASV 78618 130 G RT MESLLPVETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPV NTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRET GSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDAS- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]10V 78618 H TRT MESLLP- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]5VETAAAKFERQHMDS STSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCS QKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQAN KHIIVACEGNPYVPVHFDAS- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]5V 78618 Table 1. ELRs used during the Thesis. Coloured sequences correspond to bioactive sequences as represented in Figure 1. 131 Figure 1. Scheme of all the ELRs used during the Thesis. ELR A has been used in Chapter 3; ELR B has been used in Chapter 4 and Chapter 5; ELRs C and D have been used in Chapter 4; ELRs E, F, G and H have been used in Chapter 6. All the ELRs have been produced and characterized according to the following methods. ELR biosynthesis and purification The gene construction was performed by molecular biology and recombinant DNA technique following standard methods previously described (1, 2). Briefly, the genes encoding the different polypeptides, both ELR and bioactive sequences are synthesized by an external service (NZYTech), and cloned in the pDriveAll plasmid. For the molecular cloning steps, they were used two restriction enzymes without interruptions ("seamless cloning"): EarI and SapI (Thermo Scientific). These two restriction enzymes are type IIS that have endonuclease activity on a sequence contiguous to the recognition, so they are optimal for this type of cloning. Once the final gene construct was obtained, it was extracted from the cloning plasmid and a subcloning was carried out in the expression plasmid pET7, resulting from different modifications on the commercial vector pET-25b 132 (+) (Novagen). Subsequently, this plasmid was used to transform expression strains of Escherichia coli, in particular BLR (DE3) (Novagen). BLR strain was cultured in a 15 L bioreactor (Applikon Biotechnology) under controlled conditions of pH, temperature, agitation and O2 concentration, in order to guarantee a correct bioproduction of the different ELRs. Subsequently, the bacterial cells were disrupted by a mechanical process (disruption model: TS 0.75KW, Constant Systems). Moreover, the ELRs were purified from the rest of cellular content taking advantage of their thermo-sensitivity; the purification process was carried out by several centrifugations preceded by inverse transition cycling (3). The pure ELRs were finally obtained, they were dialyzed and filtered through 0.22 μm filters (Nalgene) to achieve sterile ELR solutions which were then lyophilized (FreeZone 1, LABCONCO) for a better long term preservation. Physical - Chemical characterization of ELRs Analysis of the level of endotoxins The level of endotoxins was determined for all the ELRs used in vivo by the Limulus amebocyte lysate assay (LAL) with the Endosafe-PTS system (Charles River Laboratories). The Endosafe® cartridge technology is a fast and high sensitivity method to perform the analysis of the level of endotoxins (4). The cartridge technology eliminates a significant amount of the raw material and accessories required for traditional LAL methods. The cartridges are pre-loaded with all of the reagents required to perform a LAL test, eliminating the preparation of multiple reagents; thus only the ELRs solution was loaded for the endotoxins analysis. The endotoxin levels of the ELRs used for the in vivo experiments were measured, resulting in a maximum of 2 endotoxin units/mg ELR for the ELRs used at a lower concentration. ELRs used at higher concentrations (up to 300 mg / mL) contained less than 0.01 EU / mg ELR. Thus, even at the highest ELR concentrations, endotoxin levels remained below the limit set by the FDA (EU 20 / biomedical device). Polyacrylamide gel electrophoresis with SDS (in denaturing conditions, SDS-PAGE) SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of polyacrylamide gel electrophoresis, an analytical method for the separation of charged 133 molecules in mixtures by their molecular masses in an electric field. It uses sodium dodecyl sulfate (SDS) molecules to help identify and isolate protein molecules. SDS acts as a surfactant, covering the proteins' intrinsic charge and conferring them very similar charge- to-mass ratios. The electrophoresis in polyacrylamide gels with SDS (5), entails the denaturation of the proteins (ELRs in our case) and the homogeneous distribution of negative charge for a migration dependent on their size. Considering that the migration is not dependent by the charge of the ELR or by the folding of the protein, this methodology allows to know the molecular weight (MW) of the ELRs, as well as the level of purity and degradation thereof. In this Thesis, it was used the vertical electrophoresis system "MiniVE" of Hoefer (Amersham Pharmacia Biotec). For the analysis by electrophoresis, around 1 mg/mL of ELR were loaded, according to the size of the well. After the electrophoresis, the polyacrylamide gels were stained with a 0.3 M solution of copper chloride. This staining works in a negative way; indeed, it does not stain the proteins, but copper interacts electrostatically with the SDS that contains the gel itself. Thus, the area containing the proteins are unstained, which appear as dark bands (6). The images of the gels were taken with the Gel Logic 100 Imaging System (Eastman Kodak) and analyzed with the Kodak 1D Image Analysis (Eastman Kodak) program. Mass Spectrometry (MALDI-TOF) In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation (7). MALDI methodology is a three-step process (Figure 2). First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and then they are accelerated towards the detector (8). The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. Mass spectrometry of the type "Matrix-assisted laser desorption / ionization - 134 Time-of-flight" (MALDI-TOF) allows to know exactly the MW. It is more accurate than the SDS-PAGE technique, hence it can be considered complementary to it. Mass Spectrometry has been carried out in the Laboratory of Instrumental Techniques (LTI) of the University of Valladolid (UVa) in the MALDI-TOF Voyager STR (Applied Biosystems). Figure 2. Schematic representation of the MALDI-TOF spectrometry. Amino acid analysis (HPLC) High-performance liquid chromatography (HPLC) is a technique used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination. HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. The analysis of ELR samples previously hydrolysed by "High-Performance Liquid Chromatography" (HPLC) allows to know the amino acid composition of the ELR molecules. First, ELR are hydrolyzed with 6M HCl, 1% phenol during 2.5 hours at 155˚C, and subsequently dried. Resulting powder is resuspended in 20mM HCl and diluted 1/10 for separation by HPLC. Quantification is done by comparing to standard patrons. A schematic representation of the process can be seen in Figure 3. This method has been carried out in the LTI of the UVa with the Waters 600 gradient HPLC equipment coupled to a Waters 2487 UV detector (Waters). 135 Figure 3. Schematic representation of the experimental set for amino acid analysis. Differential Scanning Calorimetry (DSC) Differential scanning calorimetry, or DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. Both the sample and reference are maintained at the same temperature throughout the experiment. In our case, the sample is the ELR solution dissolved in milliQ water or in saline solution, whereas the reference is the corresponding solution without the ELR. In this Thesis, DSC analysis has been used to measure the Tt of the ELRs. The basic principle underlying this technique is that when the sample undergoes a physical transformation such as phase transitions, more or less heat will need to flow to it than the reference to maintain both at the same temperature. Whether less or more heat must flow to the sample depends on whether the process is exothermic or endothermic. When a phase change or transition occurs, if the amount of heat necessary to be supplied to the ELR solution to increase the temperature is greater than the reference, then it is an endothermic process. This means that the process consumes part of the energy supplied to the system during the transition. The ELRs were dissolved at 50 mg/mL in milliQ water or in PBS, the Tt was measured at different pH values. The experimental procedure consisted of a first isothermal stage at 0 °C for 10 minutes, followed by a heating step, from 0 to 60 °C, at a speed of 5 °C / min. 136 Fourier Transform Infrared Spectroscopy (FTIR) Fourier-transform infrared spectroscopy (FTIR) is a technique that can be used to obtain information about the functional groups of the sample analyzed. The theory behind the technique resides in measuring how much light a sample absorbs at each wavelength. The molecules absorb specific frequencies that are characteristic of their structure. Fouriertransform spectroscopy shines a beam containing many frequencies of light at once and measures how much of that beam is absorbed by the sample. Next, the beam is modified to contain a different combination of frequencies, giving a second data point. This process is rapidly repeated many times over a short timespan. When the frequency of the IR is the same as the vibrational frequency of a bond, the absorption occurs. Afterwards, a computer takes all this data and the examination of the transmitted light reveals how much energy was absorbed at each frequency (or wavelength) revealing the presence of a certain chemical structure. In our case, the pure ELRs in the dry state were analyzed with a Bruker FTIR spectrophotometer (Bruker, USA). For each spectrum, a 512-scan interferogram was collected at single beam absorption mode with a 2 cm-1 resolution within the 4000 - 600 cm-1 region. For each sample, several FTIR absorption spectra were collected. Five measurements were averaged to obtain the final FTIR absorption spectrum of the sample. Residual water vapour absorption was interactively subtracted from the sample spectra. Finally, the spectral calculations were performed by the OPUS (version 4.2) software (MATTSON INSTRUMENT, INC.). Proton nuclear magnetic resonance 1H-NMR Spectroscopy Proton nuclear magnetic resonance (1H-NMR) is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules (9). Nuclear magnetic resonance spectroscopy (NMR) studies the behaviour of certain atomic nuclei in the presence of an external magnetic field. When an atomic nucleus with non-zero total angular momentum is placed in an external electromagnetic field, they interact via the nuclear magnetic dipole moment. The applied magnetic field produces a split of the degenerated energy levels of the nuclear spin, so that transitions between them can be induced as a result of absorption of the adequate electromagnetic radiation. This specific 143 144 CHAPTER 3 CARTILAGE REGENERATION IN PREANNEALED SILK ELASTIN-LIKE CORECOMBINAMERS INJECTABLE HYDROGEL EMBEDDED WITH MATURE CHONDROCYTES IN AN EX VIVO CULTURE PLATFORM Filippo Cipriani,1 Melanie Krüger,2 Israel González de Torre,1,3 Luis Quintanilla Sierra,3 Matilde Alonso Rodrigo,1,3 Linda Kock,2 José Carlos Rodríguez-Cabello,1,3 1 Technical Proteins NanoBioTechnology (TPNBT) S.L., Valladolid, Spain 2 LifeTec Group B.V., Eindhoven, The Netherlands 3 Bioforge, University of Valladolid CIBER-BNN, Paseo de Belén 19, 47001 Valladolid, Spain F. Cipriani, M. Krüger, I. González de Torre, L. Quintanilla Sierra, M. Alonso Rodrigo, L. Kock, J.C. Rodríguez- Cabello. Cartilage Regeneration in Preannealed Silk Elastin-Like Co-Recombinamers Injectable Hydrogel Embedded with Mature Chondrocytes in an Ex Vivo Culture Platform. Biomacromolecules (2018). doi: 10.1021/acs.biomac.8b01211. 145 Keywords Elastin-like Recombinamers; Polymers; Hydrogels; Cartilage repair; Ex vivo culture platform. Abstract 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 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 pre-annealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new pre-annealed 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. 146 1. INTRODUCTION Articular cartilage is central to the proper functioning of synovial joints. It covers the opposing articulating bones and, through its properties of high resiliency and deformability, it protects them from compressive joint loads (1). Moreover, it provides a smooth and gliding surface with a very low coefficient of friction (2). Many people suffer from cartilage degeneration due to genetic abnormalities, trauma, or osteoarthritis (3). One of the main issues in this regard is that articular cartilage possesses limited regeneration ability due to its avascular character, and the fact that only one cell type (chondrocytes) is present (4,5). Because of the absence of self-repair abilities, various surgical interventions and biomaterials have been explored to facilitate regeneration of cells and cartilaginous matrix (5). The physical properties of the extra cellular matrix (ECM) often refer to its rigidity, porosity, insolubility, topography, and other characteristics that are essential for its scaffolding role in supporting tissue structure and integrity, and for its role in migration and anchorage of the cells (6). Moreover, another important parameter to take into account is the permeability of cartilage; it contributes to several tissue functions like the transport of nutrients to chondrocytes, the ability to carry heavy loads, and the maintenance of a lubricating fluid film between opposing articular surfaces (7). Permeability is a measure of the ability of fluid to flow through a porous-permeable material, such as an ECM, and is inversely proportional to the friction drag exerted by the fluid (8). The low permeability of articular cartilage prevents fluid from being quickly squeezed out of the matrix (9). The purpose of surgery is the regeneration of the chondral defects to ultrastructural and biomechanical competent hyaline cartilage. From a scientific point of view, the clinical treatments are limited in their ability to functionally regenerate cartilage defects, as they often result in the formation of fibrotic tissue, which consists mainly of collagen type I and is therefore mechanically inferior to native cartilage. Biomaterials with an elastic modulus in the range of 1-10 kPa are of widespread interest, as many native tissues also have moduli in this range (10,11). The hydrogels developed to 147 repair joint cartilage are more effective when their stress relaxation behavior matches with the native tissue, since such behavior affects load transfer and nutrient transport (12,13). Up to 80 % of articular cartilage wet weight consists of water (14). To replicate this environment, hydrogels have become a popular option for cartilage regeneration in situ and cartilage engineering in vitro (15-17). The purpose of these types of scaffold is not only to provide support for cell attachment and spreading, but also to have mechanical stability at the defect site; although it is important to take into account that, the aim of these scaffolds is not to substitute for the tissue, but to improve cartilage regeneration in order to obtain a mature tissue. Natural polymers such as collagen and hyaluronic acid have some limitations; for instance, the insufficient mechanical integrity and the short lifetime in inflamed defects due to degradation by matrix metalloproteinases (18). From a biological point of view, the major drawback of synthetic polymer hydrogels such as polyglycolic acid (PGA) and polylactide acid (PLA) is that they do not provide specific biological functions (19). Moreover, synthetic polymer hydrogels do not fully recapitulate the chemical and biological features of ECM, considering that they generally regenerated fibro cartilage instead of hyaline cartilage (20). Over the last few decades, recombinant DNA techniques have proven to be very powerful tools for the development of novel protein-based biomaterials that are able to selfassemble into different structures, such as hydrogels (21). These biomaterials include elastin-like recombinamers (ELRs), which are protein based polypeptides that comprise repetitive units of the Val−Pro−Gly−X−Gly (VPGXG)n pentapeptide, in which X (guest residue) could be any amino acid except L-proline. Moreover, they show thermoresponsiveness due to the change of the protein conformation above the so-called transition temperature (Tt), which itself depends on the amino acid composition of the polymer (22). Therefore, taking into account two ELRs with the same amino acid composition except for the guest amino acid, the Tt can be tuned depending on the polarity of the side chain for the guest residue in the X position of the pentapeptide (23, 24). Furthermore, ELRs can be designed so the phase transition occurring above the Tt is translated into a hydrophobically driven self-assembly of the molecules toward supramolecular hydrogels (25). 148 In this work we have used previously described amphiphilic Silk-Elastin-like Recombinamers (SELR) (26, 27) including two types of elastin-like domains, one hydrophilic and the other one hydrophobic. SELR also contains the amino acid sequences derived from other structural proteins like the GAGAGS hexapeptide (G: Glycine, A: Alanine, S: Serine) found in Bombyx mori silk fibroin, hence giving rise to SELR (28). Furthermore, the final sequence also contains the well-known RGD cell-adhesion sequence, which promotes specific cell attachment via integrins that provide a cellfriendly environment (29). This recombinamer contains a dual physical interaction that triggers gel formation, in order to obtain a rapid and stable gel that can be delivered into the area of interest via a simple injection. The elastin motifs have been reported to form elastomeric hydrogels, in which the hydrophilic blocks provide conformational elastic properties, and the hydrophobic blocks form cross-links by hydrophobic aggregation (30, 31). The silk motifs have been reported to be responsible for the supramolecular rearrangement into β-sheets, which increases the moduli of the hydrogels (26). However, the rearrangement of silk motifs into β-sheets with the consequent formation of a fibrillary structure takes time; the long time needed represents a drawback in terms of surgical application for the cartilage environment, which is surrounded by synovial fluid. Furthermore, recent works have demonstrated that physical and structural features of the ECM, such as fibrils, are essential for its scaffolding role in supporting tissue structure and integrity (6, 32). The nanofiber environment plays an essential role in the migration and anchorage of the cells. Considering that one difficulty in nanofiber technology has been the placement of cells within a nanofibrillar structure (33), the purpose of this work is to design a system based on supramolecular self-assembly to form nanofibrillar matrices in situ, around the cells, without cellular damage. This study focuses on the correlation between the elastin motifs and silk motifs, in order to understand how to improve the gelation properties of the hydrogel to obtain a system capable of forming an ECM fibrillary structure directly after injection. We set up and optimized a thermal treatment (pre-annealing treatment), which accelerates the β-sheet formation without losing the injectability of the material. The new pA(EIS)2-(I5R)6 were characterized either with molecular analysis (Circular Dichroism and Transmission Electron Microscopy) or with rheological characterization, in order to investigate the 149 impact of the pre-annealing treatment on the arrangement of the silk motifs into β-sheet conformation. Moreover, the morphology of the hydrogel was checked using Scanning Electron Microscopy in order to verify the interconnected structure and an adequate porosity and permeability. The incorporation of cells into biomaterial scaffolds include multiple aspects in cartilage repair; thus, considering that cells are the driving force of cartilage formation, they can significantly help orchestrate regeneration and overcome some of the limitations of using cells or biomaterials alone (34). The use of mature chondrocytes is based on the premise that native mature cells are best suited to guide regeneration (34). Moreover, the remarkable property of ELRs permits a homogeneous embedding of cells in the ELR solution at a temperature below Tt, while molecules can self-assemble into hydrogels above the Tt, thus allowing the use of the cell-scaffold system in injectable therapies perfectly suitable to the shape of the injured area (35). Therefore, although other ELR- based hydrogels have shown a minimal inflammatory response, confirming its high and extraordinary biocompatibility (36), we performed an in vitro study evaluating the metabolic activity of the chondrocytes embedded in the 3D hydrogel. The potential properties of pA(EIS)2-(I5R)6 hydrogel in cartilage repair were evaluated in an ex vivo osteochondral culture platform (37). The use of bioreactors has some advantages: first, bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment (38). Second, it must be taken into account that cartilage defect models in rodents and mature rabbits show spontaneous self-repair (39). In addition, there are some other disadvantages using an animal model: the limited control over physiological parameters, and the limited possibilities for monitoring and controlling the healing progress from a biological and biomechanical point of view, as well as the high costs of animal care and ethical issues (39, 40). 150 2. MATERIALS AND METHODS 2.1. (EIS)2-(I5R)6 Design Amino acid sequence: MESLLP-{[(VPGVG)2-VPGEG-(VPGVG)2]10-(VGIPG)60- [V(GAGAGSG)5]2G}-[(VPGIG)5-AVTGRGDSPASS]6V The composition of this (EIS)2-(I5R)6 is based on previously synthetized block corecombinamers, which have silk-like motifs (SELR) (26, 41). The original block co-polymer was designed to comprise a hydrophobic block (containing isoleucine as the guest residue) with a low Tt, and a hydrophilic block (containing glutamic acid with a carboxylic group) with a high Tt (27). The final composition was further functionalized to include RGD celladhesion sequences (Figure 1). Initial SELRs were kindly provided by Technical Proteins Nanobiotechnology. Figure 1. Graphical scheme of the composition of (EIS)2-(I5R)6. 2.2. ELR biosynthesis and purification The cloning and molecular biology for gene construction of (EIS)2-(I5R)6 were performed using standard genetic-engineering methods. Production was carried out using recombinant techniques with Escherichia coli as the cell system, as described previously (42-45). Purification was performed using several cooling and heating purification cycles (Inverse Transition Cycling) following centrifugation. 2.3. Pre-Annealing treatment The lyophilized recombinamer was dissolved in ultrapure water at a concentration of 50 mg/ml, and incubated at 37 °C for different time points: 12, 24, 36 and 48 h. The 50 mg/ml concentration for the pre-annealing treatment was selected considering the inability to form a gel even when using a long incubation time (up to 48 h). Afterwards, the solution was frozen and the polymer lyophilized again to finally obtain the pre-annealed SELRs: pA(EIS)2-(I5R)6. The purity and molecular weight of the ELRs were verified by sodium 151 dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy using a Voyager STR apparatus from Applied Biosystems. Amino acid composition analysis was also performed. Additional characterization of ELRs was accomplished using infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) techniques (46) (Supporting Information Figures S1 - S5). 2.4. Circular Dichroism (CD) Circular dichroism is an excellent method for rapidly evaluating the secondary structure and folding of proteins (47). It is known that the ELR conformational state is temperaturedependent as consequence of the ITT (Inverse Temperature Transition) behavior experienced by this class of molecules (41). For performing CD experiments, recombinamers (EIS)2-(I5R)6 and pA(EIS)2-(I5R)6 were dissolved at a final concentration of 1 mg/mL and were kept overnight at 4 °C. Just before performing each measurement, a 1:10 dilution was made. The CD spectrum was acquired using a Jasco J-815 150-S spectrometer (Servicios Centrales de Investigación, University of Almeria). A quartz cuvette with a path length of 0.1 cm was used. The scans were obtained over the wavelength range of 190−260 nm at the experimental temperatures of 4, 37 and 60 °C by acquiring points every 0.5 nm using a scan speed of 50 nm/min. Before each measurement, samples were equilibrated for 15 min. Spectra were corrected by subtraction of the corresponding blank solvent readings. The data was expressed as molar ellipticity [θ], which was calculated as follows: [𝜃𝜃]=𝜃𝜃 𝑑𝑑×𝑀𝑀×10 where θ is the ellipticity, d is the path length (cm) and M is the concentration (mol/L). 2.5. Transmission Electron Microscopy (TEM) Nanostructure formation was checked by TEM. Solutions of (EIS)2-(I5R)6 and pA(EIS)2-(I5R)6 were prepared by dissolving pure, lyophilized products in Milli-Q water to a concentration of 25 μM. These solutions were kept at 4 °C overnight to allow complete dissolution of the proteins. The samples were incubated at 37 °C for 15 min and analyzed directly. TEM 152 measurements were performed using a JEOL JEM-1230 electron microscope operating at 120 kV. The specimens were prepared by placing a drop of the solution on a plasmatreated carbon-coated copper grid, followed by water evaporation at 37 °C. 2.6. Visualization and characterization of the Sol-Gel behavior In order to check the capacity of pA(EIS)2-(I5R)6 to rapidly form hydrogel and to remain stable in an excess of water, the pure recombinamers were dissolved in PBS (Phosphatebuffered saline) at 4 °C for 16 h at the concentrations of 100, 120, 150 and 180 mg/ml. Once the recombinamers were in a liquid state at 4 °C, they were placed inside an oven at 37 °C for 15 min and the Sol-Gel behavior was qualitatively observed tilting the Eppendorf containing the solution. Afterwards the hydrogels were removed and placed in an excess of water at 37 °C. 2.7. Rheological characterization A strain-controlled AR-2000ex rheometer (TA Instruments) was employed to perform rheological experiments by using parallel plates of nonporous stainless steel (diameter = 12 mm). Oscillatory measurements were carried out in shear deformation mode. The volume of the gel was 150 µl, a gap higher than 1000 μm was always reached after the sample relaxed until equilibrium. Measurements were performed at 37 °C, with the sample temperature being controlled and maintained using a Peltier device. Firstly, the solution of pA(EIS)2-(I5R)6 dissolved in PBS was placed over the plate at 37°C, and a time sweep experiment was performed up to 30 min with 1 % strain amplitudes and a frequency of 1 Hz; in this case, in situ gelation took place. Then, once the time sweep was over, two different measurements were carried out sequentially. First, the dynamic shear modulus was measured as a function of strain by a dynamic strain sweep with amplitudes ranging between 0.01% and 20% at a frequency of 1 Hz. This measurement was done to determine the range of strain amplitudes over which the gel exhibited a linear region of viscoelasticity. A second measurement consisted of the dynamic frequency 255 Figure 5. Short-term time-dependent cell quantification (30 min, 4 h, 1 d). Number of cells determined for ELRs containing bicyclic peptides 1a–c (red bars) and 2a–c (green bars), and control RGD peptides 3a–c (blue bars), RGD in the ELR-backbone (P0-RGD), ELR with no RGD peptide (P0), fibronectin coating (P0-FN) and coating with BSA (P0-BSA). The amount of cells was calculated from fluorescence intensities using a calibration curve. All experiments were carried out in triplicate and error bars show standard deviations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). For the short-term study (30min, 4h, 1 d; Figure 5), the peptides included after click modification supported higher levels of cells than the recombinant ELR-RGD (P0-RGD). At 30 min all the ELR-Peptides, except for P3c, showed the maximum statistical difference with P0-RGD. The differences in cell numbers remained high for almost all ELR-Peptides when compared with the P0-RGD for the whole short-term study. In contrast, no significant difference was observed between the recombinant ELR-RGD (P0-RGD) and the 256 ELRs lacking RGD (P0). The second comparison (indicated by a dashed line) shows the differences between the ELR-Peptide groups, in other words bicyclic peptides 1a–c (red bars), 2a–c (green bars), and control RGD peptides 3a–c (blue bars). Essentially no significant differences were found between the bicyclic peptide groups, with the only exception being for the group of non-selective RGD-peptides (3a-c), for which a greater variability and significance were evident. The ELR functionalized with 3a supported cell adhesion most efficiently within this group, with cell counts similar to those for ELRs functionalized with integrin-selective bicycles (1a-c and 2a-c), whereas 3c showed the lowest number of cells among the covalently functionalized ELRs at this time point, albeit still higher than for P0-RGD. The data collected at 4 h are similar to those obtained at 30 min, with all ELR-Peptides except P3c showing the same statistical difference with respect to P0-RGD. The intergroup comparisons were in accordance with the trend observed at 30 min, with the bicyclic peptide groups 1a–c (red bars), 2a–c (green bars) showing no significant differences except for the group of non-selective RGD-Peptides (3a-c). After 1 day, the differences between groups tended to decrease further, although the previous trends were maintained. A slight increase in cell counts at this time point suggests an incipient proliferation, although this is not particularly pronounced at this cell stage. 257 Figure 6. Medium-term time-dependent cell quantification (3 d, 5 d). Number of cells determined for ELRs containing bicyclic peptides 1a–c (red bars) and 2a–c (green bars), and control RGD peptides 3a–c (blue bars), RGD in the ELR-backbone (P0-RGD), ELR with no RGD peptide (P0), fibronectin coating (P0-FN) and coating with BSA (P0-BSA). The number of cells was calculated from fluorescence intensities using a calibration curve. All experiments were carried out in triplicate and error bars show standard deviations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). In the medium-term study (3 d, 5 d; Figure 6), the number of cells practically doubled for almost all conditions, with a similar trend as for the initial adhesion, although with some exceptions. However, the comparison between the ELR-Peptides and P0-RGD appears less clear than for the short-term culture. Thus, despite showing good initial adhesion, P2b proliferation on slowed down over longer time periods. In contrast, the difference between P0-RGD and P0 became more evident. A comparison within the same group revealed statistically significant differences only for P2a-c and P3b - P3c at day 3, and P2a - P2b and P3b - P3c at day 5. Figure 7. Long-term time-dependent cell quantification (7 d, 14 d). Number of cells determined for ELRs containing bicyclic peptides 1a–c (red bars) and 2a–c (green bars), and control RGD peptides 3a–c (blue bars), RGD in the ELR-backbone (P0-RGD), ELR with no RGD peptide (P0), fibronectin coating (P0-FN) and coating with BSA (P0-BSA). The number of cells was calculated from fluorescence intensities using a calibration curve. All experiments were carried out in triplicate and error bars show standard deviations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). 258 Long-term proliferation studies (7 d, 14 d) revealed that HUVEC growth tends to reduce the difference in the ability to promote cell adhesion by the various high-affinity integrinbinding peptides (Figure 7). Thus, after 7 days almost all the bicyclic peptides, i.e. P1a-c, P2a and P2c, showed a significant difference compared with P0-RGD, whereas P3a-c and P2b exhibited no clear difference. No significant difference was found within ELR-Peptide groups. Finally, after 14 days all conditions except the negative control P0-BSA exhibited similar amounts of cells. Indeed, cell counts after 14 days were approximately threefold higher that after culture for 7 days under all conditions. 3.6.2. Morphology studies The ELR-Peptides were also characterized by morphological in vitro studies to investigate cell attachment, cell spreading, cytoskeletal reorganization and formation of focal adhesions. In order to investigate the time-dependent morphology of the cells, they were stained with rhodamin/phalloidin (actin skeleton), DAPI (nuclei) and a fluorescent mAb (vinculin) and examined by fluorescence microscopy (Figures 8 and 9). Figure 8. Immunostaining of actin (red), vinculin (green), and nuclei with DAPI (blue) of HUVEC cells cultured for different time points (30 min, 4 h, 1 d, 3 d, 5 d, 7 d) on ELRs functionalized with bicycles 1a–c (P1a-c) and 2a–c (P2a-c). Scale bars: 100 μm. 259 Figure 9. Immunostaining of actin (red), vinculin (green), and nuclei with DAPI (blue) of HUVEC cells cultured for different time points (30 min, 4 h, 1 d, 3 d, 5 d, 7 d) on ELRs functionalized with controls 3a–c (P3a-c), and comprising RGD in the ELR-backbone (P0-RGD), no RGD peptide (P0), fibronectin coating (P0-FN) and coating with BSA (P0-BSA). Scale bars: 100 μm. Since non-adhered cells were discarded after seeding for 30 minutes, those that remained adhered, although small and spherically shaped, nevertheless showed small protrusions at the periphery or ring-shaped adhesions at the onset of cell culture (30 min) under practically all conditions tested (Supporting Information, Figure S8). At early time points, the focal contacts are circumscribed to the perinuclear zone, whereas at longer times a yellow coloration was found when overlapping actin and vinculin captures. 260 Figure 10. Quantitative image analysis of cell spreading area after 4 h. Cell area has been determined for ELRs containing bicyclic peptides 1a–c (red bars) and 2a–c (green bars), and control RGD peptides 3a–c (blue bars), RGD in the ELR-backbone (P0-RGD), ELR with no RGD peptide (P0) and fibronectin coating (P0-FN). A minimum of 25 images have been analyzed per sample, except for P1c, P2b, P3b-c and P0 where the number of images were less than 25 but in any case more than 10. The results correspond to a single independent experiment. The values are is expressed µm2; the error bars show standard deviations. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). The quantitative image analysis of cell spreading area has been performed after short time incubation (4 h) (Figure 10). P1a-b, P2c and P3a showed the highest statistical difference as compared with P0-RGD, whereas the difference between P3b and P2a was less significant. All the remaining ELR-Peptides (P1c, P2b and P3c) did not exhibit significantly different cell spreading as compared with P0-RGD. In any cases, no significant differences were found within the same peptide group. P2b showed the largest error bars, reflecting the vast population of cells having a different spreading area (Figure 10). In contrast, the negative control P0-BSA does not appear since no cells were present, (Figure 9). The data collected by quantitative image analysis after 4 h are similar to those obtained by cell quantification analysis reported in Figure 5. In both analyses, P2b and P3c showed less difference as compared with P0-RGD. With respect to the cell spreading area analysis, 261 also P3b and P2a showed a less significant difference compared to P0-RGD, whereas for P1c no significant differences were found. When comparing cell spreading areas on ELRs within the same group, the trends observed were in accordance with cell quantification analysis, where no significant differences were shown, except for the group of nonselective RGD-Peptides (3a-c). 4. DISCUSSION The focus of biomaterials research often lies with the biomaterial itself rather than the cell-adhesion sequence (51), especially given that the surface-protein interaction determines the nature of subsequent cell-surface behavior. Herein we have created peptide-functionalized ELRs by conjugating various high-affinity integrin-binding, bicyclic RGD-Peptides to ELRs via copper-free click chemistry in order to overcome the known limitations of recombinant synthesis; for instance, the inability to incorporate noncanonical amino acids and to form cyclized peptides. Furthermore, this strategy allows the bioactive ligand to maintain the flexibility and minimal steric hindrance required for cellular interactions. The purpose of this study was to improve the cell-adhesion and proliferation abilities of the recombinant and biocompatible substrate ELRs by conjugating a very small number (one or two molecules per ELR) of high integrin affinity peptides. The choice of the different conditions and peptides was based on very recent studies by Bernhagen et al., who reported an exhaustive investigation of high-affinity integrin αvβ3- and α5β1-binding bicyclic RGD-Peptides (42, 43). Remarkably, in the same studies the researchers also found that linear GRGDS (non-functionalized version of 3c), which is probably the most common cell-adhesive ligand in hydrogels, showed relatively low integrin αvβ3 and α5β1 affinity, whereas the monocyclic peptide cyclo-KRGDf (nonfunctionalized equivalent to 3b) showed a high affinity for integrins αvβ3 and α5β1. Similarly, the 32-mer knottin-RGD peptide (origin of 3a) published by Kimura et al. (52) non-selectively bound all integrins αvβ3, αvβ5 and α5β1 with high affinity. Finally, a battery of nine RGD-Peptides was successfully synthesized and conjugated with cyclooctyne using an approach in which the integrin-binding sequence was coupled to a peptide linker. Moreover, and discussed above, the solubility in water-based solvents was improved by the inclusion of additional serine residues. All the cyclooctyne-functionalized peptides 262 were conjugated to ELR azides via copper-free click chemistry. Functionalization was carried out as a mono-functionalization (5%) and di-functionalization (10%), and the ELR- Peptides were further analyzed by MALDI-TOF MS. Although MALDI-TOF MS analysis does not allow the degree of functionalization to be determined quantitatively, a comparison of the spectra in Figure 2 reveals different degrees of functionalization depending on the type and amount of peptide applied, thus confirming the successful conjugation of two different quantities of peptides on the ELRs. Furthermore, a turbidity study was performed for all the functionalized ELRs, with a slight shift in Tt to lower values beings observed for all ELR-Peptides in comparison with the non-functionalized ELR. This essentially negligible Tt shift is likely due to the very small quantity of peptide molecules conjugated to the ELRs (one molecule for 5% functionalization and two molecules for 10% functionalization) and to the low molecular weight of the peptides. These data show how this functionalization strategy does not affect the physical properties of the ELR, represented by the Tt value (Supporting Information, Figures S9-S11). The ELRs comprising 5% or 10% peptide functionalization were adsorbed onto TCPS in order to further investigate the cell adhesion behavior and proliferation over these surfaces. XPS and CA analysis were used to confirm the correct adsorption. Thus, XPS analysis revealed a change in the surface composition, as can be seen from Table 3 for ELRs and ELR-Peptides, with a clear enrichment in nitrogen and a reduction in carbon, and oxygen increasing slightly compared with the TCPS surface. These changes in chemical composition confirm an adequate adsorption of ELRs and ELR-Peptides onto TCPS (53-55). CA measurements help to characterize the affinity of a solution or suspension towards a certain surface, with the CA value decreasing as the number and strength of these interactions increases. The adsorption of ELRs onto the surface increases the hydrophilicity with respect to TCPS and, given the XPS results, this is likely due to the enrichment in electronegative atoms like nitrogen and oxygen and to the reduction in carbon. This enrichment in electronegative atoms increases the number and strength of interactions, such as hydrogen bonds, with the aqueous solution. Surprisingly, the ELR containing peptides adsorbed on TCPS was the most hydrophilic surface, with the difference with respect to the other ELRs being significant. This suggests a hydrophilic contribution from the peptides, which likely expose their electronegative atoms 263 outwards, thereby increasing the number and strength of interactions with the aqueous solution. As reported previously, the hydrophilicity of a solid surface is an essential requirement for cell-surface interactions (56) and, in this case, combines with a correct exposure of RGD peptide sequences. HUVECs were chosen to investigate the cell adhesion behavior, proliferation and morphology by culture thereof on ELR and ELR-Peptide adsorbed surfaces. Endothelial cells form part of a highly specialized tissue for vessel formation that provides stable structural support for new vessels. Endothelial cells cover the blood vessels and are metabolically very active, being responsible for maintaining vascular homeostasis. In this study we have synthesized a battery of nine peptides, which can be sub-divided into groups of three with high affinity for αvβ3, α5β1/αvβ3, and multiple integrins (Table 1). According to the literature, the remodeling of blood vessels and concomitant reorganization of the cytoskeleton requires the involvement of integrins (57). In mammals, this family of integrins comprises at least 20 different αβ heterodimers, which are expressed on the surface of endothelial cells (57). Different combinations of integrin subunits on the cell surface allow cells to recognize and respond to a variety of extracellular matrix proteins under different physiological conditions; for example, the αvβ1 and α5β1 fibronectin receptors are highly expressed in quiescent endothelial cells, whereas the αvβ3 fibronectin and vitronectin receptor is expressed only during angiogenesis (57, 58). Herein we have evaluated either the functional avidity or proliferation of HUVECs on ELR and ELR-peptide adsorbed surfaces. Loosely adhered or unbound cells were removed from the surfaces after incubation for 30 min in order to determine the cell fate as a function of the extent of initial attachment to the different surfaces and, therefore, the avidity of the cells for the adhesion sequences exposed. The adhesion behavior was investigated in a short-term study (up to 1 day, Figure 5), which revealed that ELR-bearing peptides supported higher levels of cells than the recombinant ELR-RGD. This difference involves either the bicyclic peptides 1a–c or 2a–c, each of which exhibits high-affinity for αvβ3 and α5β1/αvβ3 integrins, or the control RGD peptides, which exhibit high affinity for multiple integrins 3a–c. It should be noted that the backbone-RGD functionalized ELR (P0-RGD) comprises six RGD motifs per molecule, while the covalently functionalized ELR-Peptides only comprise one RGD moiety per 264 molecule. This suggests either a higher adhesion capacity for the high-affinity integrinbinding peptides to HUVECs or a better exposure of these peptides after protein adsorption. It is well known from the literature that cell attachment is influenced by several factors, one of the most important of which is the RGD concentration (59-61). By creating RGD-Peptides functionalized via copper-free click chemistry, we have improved the cell attachment capacity while reducing the RGD concentration on the surface, although the RGD-concentration function varies for each different bioactive surface and cell line (62, 63). Surprisingly, the adhesion and proliferation results for the different high-affinity integrinbinding peptides used in this study showed no difference when compared with the same ELR-Peptide for the two concentrations selected. This identical behavior for ELRs having 5% and 10% peptide functionalization is likely due to the similar and very low number of peptide molecules (one and two molecules for 5% and 10%, respectively) on the ELR substrate. In contrast, the presence of RGD in the backbone (P0-RGD) did not affect the adhesion behavior of cells in the short-term culture when compared with the ELR lacking RGD (P0). This could be related to the adsorption process of ELR-RGD molecules, which may have not allowed an optimal outwards exposure of the RGD sequence. Overall, the short-term results demonstrate a greater avidity of the cells for the bicycle-functionalized ELRs. According to the cell adhesion and proliferation results obtained for ELR-Peptides functionalized at 5%, the differences observed in the short-term cultures could be explained by considering that signal transduction after interaction with a specific integrin can trigger different or preferential cellular responses (proliferation, migration and/or organization into networks characteristic of early angiogenesis, for example). A comparison within the same groups of ELR-Peptides also revealed clear differences for various peptides at certain time points. However, these differences do not follow a trend as they arise due to the design and synthesis of the different peptides which, although they have the same composition, exhibit different affinities for the integrins expressed on the HUVEC membrane. The PicoGreen® study is in accordance with the morphological study, whereby the focal contacts, which are initially circumscribed to the perinuclear zone, give way to vinculin clustering of activated integrins, as can be inferred from the yellow coloration found when overlapping actin and vinculin captures. In addition, these 271 49. Chen, Y., et al., Validation of a PicoGreen-Based DNA Quantification Integrated in an RNA Extraction Method for Two-Dimensional and Three-Dimensional Cell Cultures. Tissue Engineering Part C: Methods, 2011. 18(6): p. 444-452. 50. Pallarola, D., et al., Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion. Adv Funct Mater, 2014. 24(7): p. 943-956. 51. Hersel, U., C. Dahmen, and H. Kessler, RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials, 2003. 24(24): p. 4385-415. 52. Kimura, R.H., et al., Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and alpha5beta1 integrins with low-nanomolar affinity. Proteins, 2009. 77(2): p. 359-69. 53. Hirano, Y., et al., Cell-attachment activities of surface immobilized oligopeptides RGD, RGDS, RGDV, RGDT, and YIGSR toward five cell lines. Journal of Biomaterials Science, Polymer Edition, 1993. 4(3): p. 235-243. 54. Bearinger, J.P., D.G. Castner, and K.E. Healy, Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression. J Biomater Sci Polym Ed, 1998. 9(7): p. 629-52. 55. Lin, Y.S., et al., Growth of endothelial cells on different concentrations of Gly-Arg-Gly-Asp photochemically grafted in polyethylene glycol modified polyurethane. Artif Organs, 2001. 25(8): p. 617-21. 56. Ma, Z., Z. Mao, and C. Gao, Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids Surf B Biointerfaces, 2007. 60(2): p. 137-57. 57. Short, S.M., G.A. Talbott, and R.L. Juliano, Integrin-mediated signaling events in human endothelial cells. Mol Biol Cell, 1998. 9(8): p. 1969-80. 58. Baranska, P., et al., Expression of Integrins and Adhesive Properties of Human Endothelial Cell Line EA.hy 926. Cancer Genomics - Proteomics, 2005. 2(5): p. 265-269. 59. Kantlehner, M., et al., Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation. Chembiochem, 2000. 1(2): p. 107-14. 60. Jeschke, B., et al., RGD-peptides for tissue engineering of articular cartilage. Biomaterials, 2002. 23(16): p. 3455-63. 61. Danilov, Y.N. and R.L. Juliano, (Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion. Exp Cell Res, 1989. 182(1): p. 186-96. 62. Maheshwari, G., et al., Cell adhesion and motility depend on nanoscale RGD clustering. J Cell Sci, 2000. 113 ( Pt 10): p. 1677-86. 63. Service, R.F., Tissue engineers build new bone. Science, 2000. 289(5484): p. 1498-500. 272 SUPPORTING INFORMATION Differential Scanning Calorimetry (DSC) measurements DSC experiments were performed on a Mettler Toledo 822e DSC with a liquid nitrogen cooler accessory. Both temperature and enthalpy were calibrated with an indium standard at the same experimental conditions used for the studied materials. Water solutions of ELRs at 50 mg mL-1 were prepared at different values of pH. In a typical DSC run, 20 µL of the solution was placed inside a standard 40 µL aluminium pan hermetically sealed. The same volume of water was placed in the reference pan. As for ELR-hydrogel analysis, 20 mg of the hydrated hydrogel was placed in the sample pan. To account for the exact amount of polymer in the assayed hydrogel, the sample was lyophilized and weighted after DSC run. All samples were equilibrated for 10 min at 0ºC inside the sample chamber just before the beginning of each experiment, and then, heated from 0 to 60 ºC at a heating rate of 5ºC/min. The scans were run under a nitrogen atmosphere. Figure S1. DSC graph of HRGD6-N3 showing the experimental Tt in PBS at physiological pH. Integral -9,29 mJ Peak 15,01 °C HRGD6-N3 BF058049A A PBS pH7.58 50 mg/mL, 19.04.2017 19:02:10 HRGD6-N3 BF058049A A PBS pH7.58 50 mg/mL, 0,9650 mg mW 1 min °C0 0 10 20 30 40 50 0246810 12 14 16 ^exo STARe SW 12.00Lab: METTLER 273 Figure S2. DSC graph of VKV-N3 showing the experimental Tt in PBS at physiological pH. Fourier Transform Infrared Spectroscopy (FTIR) FTIR analysis was conducted with a Bruker FTIR spectrophotometer (Bruker, USA). For each spectrum, a 512-scan interferogram was collected at single beam absorption mode with a 2 cm-1 resolution within the 4000- 600 cm-1 region. For each sample several FTIR absorption spectra were collected. Five measurements were averaged to obtain the final FTIR absorption spectrum of the sample. Residual water vapour absorption was interactively subtracted from the sample spectra. Spectral calculations were performed by the OPUS (version 4.2) software (MATTSON INSTRUMENT, INC.). 274 Figure S3. FTIR of HRGD6-N3. Figure S4. FTIR of VKV-N3. Proton nuclear magnetic resonance 1H-NMR Spectroscopy NMR analysis was carried out using a 400 MHz Agilent Technologies equip with an Agilent MR console 400 and a One NMR probe. The measurements were carried out at 298 K with samples of 20–30 mg of the modified elastin like recombinamers, purified and dissolved in DMSO-d6. Chemical shifts (δ) are given in ppm. 275 The no deuterated dimethyl sulfoxide peaks at d ¼ 2.5 ppm and d ¼ 39.51 ppm were used as internal reference for 1H and 13C NMR spectra, respectively. Figure S5. H-NMR spectrum of HRGD6-N3 showing the integration of the peaks corresponding to the different types of hydrogens. 276 Figure S6. H-NMR spectrum of VKV-N3 showing the integration of the peaks corresponding to the different types of hydrogens. Table S1. Molecular weights of all cyclooctyne (BCN)-functionlized RGD peptides as obtained from UPLC/MS analysis. Calculated masses were determined using ChemDraw software. Peptide Sequence Mfound [Da] Mcalc [Da] 1a K(BCN)PPPSG[Abz]SG-C T3 HPQc T3 RGDc T3 2176,78 2176,47 1b K(BCN)PPPSG[Abz]SG-C T3 HPQC T3 RGDc T3 2176,59 2176,47 1c K(BCN)PPPSG[Abz]SG-C T3 HSQC T3 RGDc T3 2166,43 2166,43 2a K(BCN)-PPPSG[Abz]SG-C T3 RGDc T3 AYJC T3 2162,34 2161,50 2b K(BCN)-PPPSG[Abz]SG-C T3 RGDc T3 AWGC T3 2129,34 2128,43 2c K(BCN)-PPPSG[Abz]SG-C T3 RGDc T3 AYaC T3 2119,40 2119,41 3a K(BCN)-PPPSG[Abz]SG-knottin-RGD 4337,85 4336,81 3b cyclo-[K(K(BCN)-PPPSG[Abz]SG)RGDf ] 1648,57 1648,84 3c K(BCN)-PPPSG[Abz]SG-GRGDS 1534,37 1534,65 J: D-Leu 277 278 Figure S7. Time-dependent cell quantification for ELRs containing peptides at 5% and 10%; 1a–c (red bars) and 2a–c (green bars), and control RGD peptides 3a–c (blue bars), at different time points (30 min, 4 h, 1 d, 3 d, 5 d, 7 d, 14 d). All experiments were carried out in triplicate and error bars show standard deviations. There was no statistically significant difference between 5% and 10% at any time point. 279 Figure S8. Some examples of magnified captures of immunostained HUVEC cells seeded on P1a (A), P3a (B), P2c (C); P0-RGD (D), P0-FN (E) and P0-BSA (F) that remained adhered after 30 min of incubation and washing. Actin is stained in red, vinculin in green, and nuclei with DAPI in blue. Small protrusions are marked with white arrows. Scale bars are 50 µm. 280 Figure S9. Turbidity analysis of VKV-N3 and ELRs functionalized with bicycles 1a–c at 5% (P1a-c) and 10% (P1a-c-10). Figure S10. Turbidity analysis of VKV-N3 and ELRs functionalized with bicycles 2a–c at 5% (P2a-c) and 10% (P2a-c-10). 287 that have to be taken into account for the development of smart biomaterial with allosteric domains are the ability to be post-translationally and reversibly modified by enzymatic reaction, and the capacity to exhibit a movable solubility frontier close to the body temperature. The common strategy for the development of enzyme-responsive material is to covalently link enzymatic substrates to amphiphilic copolymers (14, 15). In addition to that strategy, there is also a supramolecular strategy for the preparation of enzyme-responsive biomaterials, which features the non-covalent integration of enzymatic substrates into assemblies (16, 17). A promising and powerful strategy extensively study for the design of bioactive biomaterials is the genetic engineering strategy based on recombinant DNA techniques (18). One of the most important biomaterials taking part in this category is Elastin-like Recombinamers (ELRs). ELRs are protein based polypeptides that comprise repetitive units of the Val−Pro−Gly−X−Gly (VPGXG)n pentapeptide, in which X (guest residue) could be any amino acid except L- proline. ELRs shows several advantages of designing smart biomaterials. First, they exhibit thermo-responsiveness due to the change of the protein conformation above the socalled transition temperature (Tt), which itself depends on the amino acid composition of the polymer (19). ELRs are soluble below their Tt and become insoluble and aggregate at a temperature above their Tt, thereby, ELRs are close to a frontier between solubility and insolubility (20-22). This feature is important considering that gives the possibility to begin the design of temperature triggered self-assembling polymers using a repeated motif that is intrinsically responsive to its environment (23). Second, the genetic engineering method enables biosynthesis of fusion constructs with precise control over chain length and protein position which, makes their sensitivity to the environment highly tuneable (24, 25). Third, the design of the ELR can be engineered in order to be responsive to other triggers in the context of self-assembly (26, 27). Finally, taking advantage from the ability of ELRs to modify the activity of functional proteins, they can be used for the development of natural systems with added allosteric control of their function (28, 29). There are many examples of smart biomaterials based on ELR (smart-ELR); for biomedical and biomimetic applications (30-34), some of them are smart-ELR with enzymeresponsiveness (23, 35-37). Du et al. fused an ELR to the D-amino acid oxidase, thereby increasing the solubility and stability of that enzyme (38). Later, in a paper from the same 288 group, Gao et al. (39) fused ELRs to two enzymes to form ELR-fusion R-ω-transaminase and ELR-fusion D-amino acid oxidase, which, at temperatures above the Tt of the ELR part, assembled to form two-enzyme complexes with significantly improved catalytic efficiency. Herein, we propose a new type of enzymatic responsive smart-ELR with allosteric control of enzymatic activity. The smart-ELR enzyme was designed containing the consensus sequence for the Kinase/Phosphorylase enzyme. Furthermore, it was engineered with the sequence of RNase A which is a type of digestive enzyme used to specifically cleave single-stranded RNA (40). RNase A was chosen as the modular enzyme, considering its relatively small protein structure consisting of only 124 amino acid (41), and its well-known optimal refolding properties (42, 43). Finally, taking into account that the folding of the smart-ELR into the 3D structure can alter the enzymatic activity it was carefully designed three different variants of the smart-ELR according to the position of the RNase A domain relative to the ELR backbone. Finally, the RNase A activity modulated by the enzymatic phosphorylation/de-phosphorylation was evaluated, and a comparison between the different variants has been made. 2. MATERIALS AND METHODS 2.1. ELR biosynthesis and purification The cloning and molecular biology for gene construction were performed using standard genetic-engineering methods (44). In this study, four different smart-ELRs were designed (Figure 1). The four smart-ELR designed differ among each other for the composition of bioactive sequences and for the disposition of those sequences. All of the smart-ELRs were designed containing ten consensus sequence for the Kinase/Phosphorylase enzyme (45); in order to provide a better exposition of the consensus sequence, the consensus sequence was flanked by hydrophilic sites. Moreover, all the smart-ELRs were composed by hydrophobic blocks having Isoleucine as the guest amino acid. Finally, the smart-ELRs was further engineered with the RNase A sequence. According to the position of the RNase A domain relative to the ELR backbone, four different types of smart-ELRs were generated: TI, TR, RT and TRT. 289 TI is composed of the hydrophilic sequence and the hydrophobic block. MESLLP- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]10V TR has the same composition of TI, plus the RNase A sequence at the N-terminal. MESLLP[(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]10VETAAAKFERQHMDSSTSAASSSNYCNQM MKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNC AYKTTQANKHIIVACEGNPYVPVHFDASV RT has the same composition of TR, plus the RNase A sequence located at the C-terminal. MESLLPVETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCS QKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDAS- [(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]10V TRT has the identical composition of TR and RT, but in this case, the RNase A domain was located exactly in the middle of the ELR backbone sequence. MESLLP[(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]5VETAAAKFERQHMDSSTSAASSSNYCNQMM KSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCA YKTTQANKHIIVACEGNPYVPVHFDAS-[(VDLDVPIPGRFDRRVSVAAE(VGIPG)10]5 V Figure 1. Graphical scheme of the smart-ELRs. The hydrophilic sequence containing the consensus sequence is represented by the blue block; according to the key, the consensus sequence is marked in bold, and the Serine residue where occurs the phosphorylation/dephosphorylation is marked in red. The hydrophobic block is represented by the red block. RNase A sequence is represented by the green block. Production was carried out by recombinant techniques using Escherichia coli as the cell system, as described previously (44, 46-48). Purification was performed by several cooling and heating purification cycles (Inverse Transition Cycling) following centrifugation; the ELRs obtained in this manner were dialyzed against MilliQ (MQ) water and lyophilized. 290 The purity and molecular weight (MW) of the ELRs were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Amino acid composition analysis and infrared spectroscopy (FTIR) was also performed (49). The characterization results are provided in the Supporting Information (Figure S1 – S7). 2.2. Smart-ELRs phosphorylation and de-phosphorylation Phosphorylation and de-phosphorylation reactions were performed to modulate the smart-ELR state. The smart-ELRs were dissolved at a concentration of 5 mg/mL either for phosphorylation or de-phosphorylation reaction. Specific buffers were prepared for the two reactions. For the phosphorylation: the phosphorylation buffer was composed by NEBuffer for protein Kinases (PK) 1X (New England BioLabs), Adenosine 5’-Triphosphate (ATP) 200 µM (New England Biolabs), and milliQ water. The concentration of ATP was in excess according to the stoichiometry of the reaction (2,6 times more), in order to guarantee the presence of phosphates groups for complete phosphorylation of the ELRs. The cAMP-dependent Protein Kinase (PKA) catalytic Subunit (New England BioLabs) was added to the solution buffer with a ratio of 5000 Units of enzyme per mL of reaction buffer. The solution was further incubated 3 h at 20 °C (temperature on which the ELRs were at dissolved state) under shaking. For the de-phosphorylation: the dephosphorylation buffer was composed by CutSmart Buffer 1X (New England BioLabs), and milliQ water. The Alkaline Phosphatase, Calf Intestinal (CIP) (New England BioLabs) was added to the solution buffer with a ratio of 100 Units of enzyme per mL of reaction buffer. The solution was further incubated 3 h at 20 °C under shaking. According to the design of the biomaterial, ten phosphate groups can theoretically be transferred/removed to each smart-ELR. After both the reactions, the enzymes (PKA and CIP) were removed in the same way: the solution was heated up to 80 °C for 15 min in order to inactivate and unfold the enzyme. Afterwards, quick centrifugation was performed and the pellet was re-dissolved in MilliQ water at 1 mg/mL O/N at 4 °C under shaking. Then, quick centrifugation at 4 °C was performed to remove the unfolded enzyme from the solution. Finally, the solution went through dialysis and freeze-drying steps. 291 Figure 2. Graphical scheme of the phosphorylation and de-phosphorylation reaction of the smart-ELRs. Ten phosphate groups can theoretically be transferred/removed to each smart-ELR. 2.3. Mass analysis (HPLC-HR-MS) The phosphorylation/de-phosphorylation rate of the four different smart-ELRs was analyzed by High-Performance Liquid Chromatography-High Resolution-Mass spectrometry (HPLC-HR-MS). Mass spectra were performed using an ultra-high-resolution QTOF instrument (MAXIS II, BRUKER, Bremen-Germany). Electrospray ionization source in positive mode was used for all the analyses and the parameters were adjusted as follows: Capillary voltage 3400 V, End plate offset 500 V, in-source Collision Induced Dissociation energy (isCID) 130 eV. Nitrogen was used as nebulizer gas (pressure of 3 Bar) and drying gas (heated to 250°C, flow 4 L/min). The scans of MS spectra were conducted in the m/z range of 1000 to 12000. For accurate high-resolution mass spectrometry (HRMS) external calibration was performed after each chromatographic run by means of a mixture of phosphazenes. Prior to mass detection samples were separated in a HPLC instrument (HP1100 Series, Agilent Technologies) using a Vydac-C4 Protein column (300 Å, 250 mm, 4.6 mm id, 5 mm particle size), the flow rate was set to 0.5 mL/min, solutions of 0.2% trifluoroacetic acid in water (v/v) and 0.1% trifluoroacetic acid in acetonitrile (v/v) were used as mobile phases A and B respectively. For each sample, a 4 mg/mL solution was prepared in DMSO and 100 µL of it was loaded on the column. The separation was achieved by a 25 min linear gradient from 30% to 80% B, afterwards, isocratic conditions were kept for a 60 min total run time. Finally, the MaxEnt algorithm was applied to the 292 protein spectrum to de-convolute the multi-charged signals in order to obtain the neutral intact mass protein. 2.4. Turbidity analysis The Tt of all the ELRs was verified by Turbidity using the Cary Series UV-Vis Spectrophotometer (Agilent Technologies). The analysis for the determination of the Tt shift before and after the phosphorylation and the de-phosphorylation was recorded for ELRs at 5 mg/mL. For all the turbidity analysis (including buffer and concentration dependence experiments), a thermal ramp was carried out at 1 °C/min and the turbidity was recorded at a wavelength of 350 nm. 2.5. Dynamic Light Scattering (DLS) Light scattering measurements were performed using a BI-200SM multi-angle goniometer (Brookhaven Instrument, Holtsville, NY) with a 33mWHe−Ne vertically polarized laser at a wavelength of 632.8 nm and a digital correlator (BI-9000AT). All the ELRs solutions were prepared by dissolving the ELRs in RNase buffer, thus stored at 4 °C overnight to allow the complete dissolution of the smart-ELRs and filtered using 0,45 µm PVDF syringe filter. Afterwards, the samples were introduced into the polystyrene cuvette and stabilized for 5 min at 37 °C to allow supramolecular assembly. DLS measurements were performed to calculate the size distribution (Volume mean (nm)) and polydispersity index (PDI). All the measurements were performed in triplicates. 2.6. RNase A activity analysis The RNase A activity of each smart-ELRs was evaluated either for the phosphorylated or dephosphorylated state. The assay was performed following the general ribonuclease assay using methylene blue (50). The buffer of the assay was adjusted for the RNase A activity. Briefly, the RNase buffer was prepared (50 mM Tris HCl, 250 mM KCl, 3 mM MgCl2, 10 mM DTT); Methylene Blue buffer was obtained dissolving Methylene Blue (Sigma-Aldrich) in RNase buffer at 0,1 mg/mL, whereas RNA solution was obtained dissolving RNA from yeast (Roche) in RNase buffer at 10 mg/mL. RNA solution (100 µL) was mixed with the methylene blue buffer (400 µL) and pre-incubated at 37 °C for 10 min in the dark. Afterwards 500 µL of the ELRs solution (1 mg/mL) was added to reach a final 293 volume of 1 mL and an ELRs concentration of 0,5 mg/mL. Afterwards the solution was incubated at 37 °C for 15 min in the dark, and then the spectrophotometer analysis was performed at 688 nm. 2.7. Statistical analysis Values are expressed as mean ± standard deviation (SD). The data were examined with a one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test. All statistical analyses were performed with GraphPad Prism. A P- value lower than 0.05 was considered statistically significant. 3. RESULTS 3.1. Smart-ELRs phosphorylation and de-phosphorylation The design of our smart-ELR was composed by the inclusion of ten (de-)phosphorylation consensus sequence regularly distributed along the ELR, moreover, the consensus domain was flanked by a hydrophilic sequence for better accessibility to the Kinase (PKA) and Phosphatase (CIP). Finally, the Isoleucine was selected as a guest amino acid in order to have a counter hydrophobic-part tailoring our smart-ELR for mild conditions. Furthermore, according to the position of RNase A sequence in relation to the ELR backbone, three different versions of the smart-ELRs were obtained by DNA recombinant techniques (TR, RT, TRT) (Figure 1). The phosphorylation/de-phosphorylation rate of all the smart-ELRs was analyzed by HPLCHR-MS. Mass Spectrometry spectra are shown in Supporting Information (Figure S8-S11). In table 1 there are reported the values of all the smart-ELRs in different states, where Native means before phosphorylation, Phosphorylated means after phosphorylation, and De-Phosphorylated means after De-phosphorylation. 294 HPLC-HR-MS (Da) Native Phosphorylated Dephosphorylated Δ Native - Phosphorylated TI 65077 65873 65075 796 TR 78611 79410 78612 799 RT 78612 79412 78610 800 TRT 78609 79411 78613 802 Table 1. HPLC-HR-MS of all the smart-ELRs in different states. Native: before Phosphorylation; Phosphorylated: after Phosphorylation; De-Phosphorylated: after De-phosphorylation. Mass values are expressed in Dalton (Da). In the case of TI, the mass measured in the native state is 65077 Da; whereas the phosphorylated state showed a mass of 65873 Da; finally, the de-phosphorylated TI mass was 65075 Da. The difference between the Native and the Phosphorylated Mass of TI was 796 Da, which coincides with the difference in MW recorded between the Phosphorylated and the De-phosphorylated state of TI (798 Da). On the other hand, the mass values recorded for TR, RT and TRT in their native state are higher than the mass of TI in the native state, due to the presence of RNase A sequence. These values were 78611 Da for TR, 78612 Da for RT and 78609 Da for TRT; whereas after the phosphorylation it was recorded 79410 Da, 79412 Da and 79411 Da, for TR, RT and TRT respectively. As it was recorded for TI, also for TR, RT and TRT the difference between the Native and the Phosphorylated Mass was in around 800 Da, which coincides, also for the last ones, with the difference recorded between the Phosphorylated and the de-Phosphorylated state. The thermally driven aggregation of the ELRs was investigated using the turbidity approach. The increment in temperature beyond the critical point results in a sharp increase in turbidity (51, 52). The Tt is defined as the temperature at 50% maximal turbidity (51). Turbidity was measured as a function of the temperature of ELR solutions to investigate the aggregation temperature. The turbidity analysis was performed for all smart-ELRs in order to verify the smart-ELR state modulation by the enzymatic phosphorylation and de-phosphorylation. As it has reported above, a thermal ramp was recorded before and after the phosphorylation (Figure 4, red lines), and before and after the de-phosphorylation (Figure 4, black lines). All the smart-ELRs showed a sharp peak in 295 turbidity when they turned to aggregate states; this subsequently stabilized over the entire temperature range analyzed, with a maximum optical density (OD) of around 2.5. In all the cases, a clear shift in temperature was recorded. The difference in Tt for TI between before and after phosphorylation was of 15 °C, passing from 26 °C to 41 °C. Whereas for TR, RT and TRT the Tt shift was around 12 °C in all the cases. The smart-ELRs dissolved in the phosphorylase buffer showed lower Tt compared to the kinase buffer due to the different saline composition (Figure 4, red dashed line compared to the black continuous line). The difference in Tt between the smart-ELRs before and after dephosphorylation was in all the cases less evident than after the phosphorylation. After the de-phosphorylation, TI showed a Tt shift of 13 °C; whereas for TR, RT and TRT the Tt shift was around 8 °C. Although the delta (Δ) in Tt shift recorded after the de-phosphorylation was lower than after the phosphorylation, the Tt of the de-phosphorylated smart-ELRs was similar to the Tt of the native smart-ELRs in all the cases. Indeed, for TR, RT and TRT the Tt before phosphorylation (Figure 4 B, C, D red continuous lines) coincides with the Tt after the de-phosphorylation (Figure 4 B, C, D black dashed lines). The turbidity analysis for TI before phosphorylation (Figure 4 A red continuous lines) showed a Tt slightly higher (26 °C) than the Tt after the de-phosphorylation (24 °C) (Figure 4 A black dashed lines). 296 303 phosphorylated state of ELRs (P) is represented with blue bars; whereas the dephosphorylated state of ELRs (D) is represented with green bars. Figure 7. RNA quantification of the solutions containing the different smart-ELRs dissolved in RNase buffer at 0,5 mg/mL after 15 min of incubation in the darkness at 37 °C. Control: black bar (RNase buffer solution without any smart-ELR); grey bars: smart-ELRs in native state (TI-N, TR-N, RT-N, TRT-N); blue bars: phosphorylated smart-ELRs (TI-P, TR-P, RT-P, TRT-P); green bars: dephosphorylated smart-ELRs (TI-D, TR-D, RT-D, TRT-D). For all the measurements, the absorbance was recorded at a wavelength of 688 nm. All experiments were carried out in triplicate and error bars show standard deviations (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). The comparison between the control and all the rest of the conditions showed statistical difference with all the smart-ELRs at the different state except for TI-P, RT-D and TRT-D. This may be due to the presence of the polymer, that affected by default the measurement at 688 nm. In order to make the bar charts clearer, the RNA quantification outcomes have been clustered by the smart-ELR type (TI, TR, RT, TRT) in different states. The comparison within each group of smart-ELRs has revealed that no difference in RNA quantification was recorded between the different states of TI. The same trend was 304 presented by TRT smart-ELR, where no difference in RNA quantification was recorded between the native, phosphorylated and de-phosphorylated state. On the other hand, the absorbance recorded for the TR-P solution after 15 min of incubation revealed a significant decrease in the amount of RNA when compared with TR-N and TR-D. Comparing the phosphorylated state (TR-P) with the de-phosphorylated state (TR-D), it was recorded a reduction in RNA amount of 17% on average. Moreover, no significant difference was recorded between TR-N and TR-D. The RT smart-ELRs presented similar values than TR. Indeed, a significant reduction (20%) in RNA amount was recorded for the RT-P compared with the RT-D. In addition, no significant difference was recorded comparing RT-N and RT-D. 4. DISCUSSION There is a great need of new biomaterials capable to undergo a reversible change in response to small variations in solution conditions, showing a fine modulation, such as allosteric control (53). The aim of the study was to develop an enzymatic responsive smart-ELR with allosteric control of the enzymatic activity, which, allow the communication between the biological environment and the material. The allosteric control was given by the consensus sequence which, was selectively recognized by Kinase and Phosphatase enzymes for phosphorylation/de-phosphorylation. It has already been demonstrated that the enzymatic (de-)phosphorylation which, is commonly used in biology to alter structural features of proteins, can also modulate the Tt and induce gelation of other polymers kind (54-57). Indeed, the change in hydrophobicity is the key to the subsequent conformational rearrangements shows by the hydrophobic domains. The increase in polarity by phosphorylation moves the frontier of solubility to a higher temperature, as the complementary effect of de-phosphorylation moves the frontier back. Furthermore, considering that smart-biomaterials are commonly developed as enzymatic substrates rather than playing an active role in an enzymatic reaction, RNase A was chosen as a single domain protein to confer a catalytic enzyme-responsiveness. As it is reported above, the native enzyme was modified at the genetic level by adding at the amino or carboxyl end the ELR-counterpart containing (de-)phosphorylation consensus sites. Thus, it was obtained an enzymatic responsive smart-ELR with allosteric control of 305 RNAse A activity. Finally, three different variants of smart-ELRs were obtained by DNA recombinant techniques (TR, RT, TRT), where the position of RNase A domain change in relation to the ELR backbone sequence. The first step towards the development of the smart-ELR with allosteric control was to verify that TI had complete responsiveness to the Kinase/Phosphatase. Mass spectrometry is an excellent method for detecting protein molecular weight and quantifying its change. Protein phosphorylation events are detected by increases in amino-acid residue mass of +80 Da, which report the addition of HPO3 (58). As shown in Table 1, the precise HPLC-HR-MS analysis for TI revealed a difference between TI before and after phosphorylation of around 800 Da in molecular weight. The incorporated consensus sequence was specifically recognized by the enzyme(s); the phosphorylation occurred completely, indeed, ten phosphate groups (which corresponds to 800 Da) were transferred above the ten consensus sequences. Also, the (de-)phosphorylation occurred completely, indeed the difference between the TI phosphorylated and TI dephosphorylated was again of around 800 Da, demonstrating the complete reversibility of the ELR state. These results showed optimal accessibility by the enzyme(s) to the substrate, due to the adequate exposition by the hydrophilic loop flanking the consensus sequence. Furthermore, the same results were obtained for the three smart-ELRs containing the RNase A sequence (TR, RT, TRT), where the difference between the phosphorylated and the de-phosphorylated state was again around 800 Da. The presence of the RNase A domain does not prevent the recognition of the consensus sequence by the Kinase/Phosphatase enzyme(s). Moreover, the different position of the RNase A domain does not avoid the complete transferral of ten phosphate groups, meaning that the consensus sequence keeps optimal accessibility to the enzyme even when linked to the RNase A domain. Finally, the complete phosphorylation and de-phosphorylation of the smart-ELRs were accomplished in all the cases; indeed, the theoretical number of phosphate groups that can be transferred was fulfilled in all the cases, showing a 100% effectiveness of the system developed. All the smart-ELRs before and after (de-)phosphorylation were further analyzed by turbidity (Figure 4). First, it is important to notice that, both reactions were performed at a lower temperature (20 °C) than the Tt of the ELRs, in order to guarantee the efficiency 306 of the reactions. Although, the turbidity analysis revealed an evident shift in Tt between the smart-ELR before and after the (de-)phosphorylation in all the cases, for TI the Δ Tt was slightly higher than for the smart-ELRs (TR, RT, TRT). This is due to the presence of RNase A domain, which, increment the size of the protein, reducing the impact of the ten phosphates groups over the Tt shift. Moreover, no difference in Δ Tt was recorded between TR, RT and TRT, demonstrating that the position of the RNase A sequence does not affect the Tt. In any case, the turbidity corroborated the reversibility of the system validated by mass spectrometry, showing the capacity of all the smart-ELRs to be driven back and forth between association (insolubility) and dissociation (solubility) by selective enzymatic activity. The smart-ELR demonstrated to be enzyme-responsive, the allosteric domains were able to switch between association and dissociation as a consequence of polarity changes caused by post-translational modification (phosphorylation). Moreover, also the effect of the saline buffers was observed; in fact, all the smart-ELRs dissolved in the phosphorylase buffer showed lower Tt compared to the kinase buffer (Figure 4). The effect of saline buffer on the Tt was highlighted by the turbidity analysis performed dissolving TI in milliQ (Figure 5A). As it has been extensively studied, an increase in saline concentration has a significant effect on the Tt (59). Due to the absence of salts, the contribution of the phosphate groups was more evident for the milliQ solution, indeed, the Δ Tt recorded in milliQ was much higher than the Tt shift recorded in saline buffer. Finally, in all the turbidity curves it can be observed a shoulder for the smart-ELRs around 15 °C. ELRs formed aggregates with a significant increase recorded at OD 350 nm, due to their conformational change and self-organization. This behavior was already reported by García-Arévalo et al., where a similar ELR containing Isoleucine as the guest amino acid was used (48). These low OD values indicate that neither of the ELRs forms the typical coacervate state (60), rather, the small absorbance increase suggests the formation of nanoaggregates (23, 61). Pattaniak et al., have already demonstrated how the Tt of an ELR could be modulated by (de-)phosphorylation; however, they developed a system capable to be only partially phosphorylated, showing the effectiveness of 23% (54). In this work, we have developed an optimal design for the selective enzyme-responsive smart-ELR, with the ability to be completely (de-)phosphorylated, showing the effectiveness of 100%. As it is reported in 307 the literature, the polymer concentration has a significant effect on the altering of Tt (19). This correlation was also demonstrated for our system, where decreasing the concentration of smart-ELR it corresponded an increase in Tt (Figure 5B). In the light of this, a certain concentration was fixed in order to evaluate the modulation of the ELRs from the dissolved state (phosphorylated smart-ELR), to aggregate state (dephosphorylated smart-ELR) and viceversa at mild conditions. The turbidity behaviour for all the smart-ELRs dissolved at 0,5 mg/mL in RNase buffer (Figure 6) demonstrated that 37 °C was a temperature in which, smart-ELRs in the native and de-phosphorylated state are aggregated, and smart-ELRs in the phosphorylated state are dissolved. Moreover, the Δ Tt recorded in RNase buffer is much higher than the one recorded in Kinase or Phosphatase buffer. As it has been explained above, this difference depends on the concentration of the polymer and on the saline buffer of the solution. Moreover, the Tt shift of the smart-ELRs appeared slightly different comparing TR and RT with TRT; indeed, the lower concentration of polymers highlighted the small difference in self-assembly behavior. Finally, all the smart-ELRs containing RNase A domain showed a less sharply curved due to higher MW, especially for the phosphorylated state, which also showed higher Tt. The formation of aggregates was further corroborated by DLS analysis. These measurements showed that smart-ELRs in the native and dephosphorylated state formed aggregate at 37 °C, whereas all the smart-ELRs in the phosphorylated state were dissolved, indeed no aggregates were recorded. Furthermore, the DLS analysis revealed a decrease in volume aggregates between the native TR, RT and TRT and the de-phosphorylated ones; this unexpected result can be due to some sort of reorganization after the (de- )phosphorylation reactions between the RNase A sequence and the ELR backbone, since it appeared only for TR, RT and TRT and not for TI (Table 2). A possible explanation for the unexpected smaller aggregates after the (de-)phosphorylation could be that there would be some ionic rearrangements between the ELRs backbone and the RNase domain during the phosphorylation reaction; this new charges interaction favours a tighter aggregation when the de-phosphorylation occurred. Finally, the outcomes showed above demonstrated that all the smart-ELRs assemblies were responsive to the enzymatic activities. Upon the addition of PKA, the multiple-charged ATP molecules were hydrolyzed 308 into ADP molecules and phosphate groups were transferred to the serine residues. The supra-amphiphilic self-assembled aggregates underwent disassembly upon addition of PKA. On the other hand, upon the addition of CIP, the phosphate groups were removed from the serine residue, causing the supra-amphiphilic self-assembled aggregates. As shown above, the system was completely reversible (Figure 2). The allosteric control of the RNase A activity was analyzed monitoring the RNA quantification of the solutions in mild conditions. These results revealed a different behaviour of the smart-ELRs. TI does not contain the RNase A domain; indeed, it has shown no RNase A activity. Either the phosphorylated TI (TI-P) or de-phosphorylated TI (TI-D) does not have any impact over the RNA quantification, meaning that the state of the polymer does not have any cleavage activity over the RNA in the solution. On the other hand, the three variants of smart-ELR containing the RNase domain (TR, RT and TRT) showed a different RNase A activity comparing the phosphorylated state with the dephosphorylated state. Both TR-P and RT-P exhibited a significant reduction in RNA quantification, showing a gained catalytic activity due to the selective phosphorylation. The conformation changes caused by the phosphorylation distorted the overall 3D structure of the hybrid ELR-enzyme altering its efficiency. The complete phosphorylation allowed to the TR-P and RT-P to be in its dissolved state, where the RNase A domain resulted to be free to active. The reduction in RNA amount of 17% in the case of TR and 20% in the case of RT demonstrated the capacity of our system to switch from an inactive state (de-phosphorylated) to an active state (phosphorylated) by a selective enzymatic activity performed by Kinase/Phosphatase. Contrary, TRT exhibited no significant difference between the dissolved state (TRT-P) and aggregate state (TRT-D). In this case, the RNase A catalytic site remained inactive even when the state of the smart-ELR was modulated by Kinase/Phosphatase. This phenomenon could be due to a sort of steric hindrance, where the accessibility of the RNA molecule to the catalytic site was blocked by the flanked ELR backbone, or perhaps because of the catalytic site of RNase A is stretched by the ELR backbone altering the performance of the catalytic site (Figure 8). It is well known that, when enzymatic domains are incorporated into polymer assemblies, the accessibility of the substrate to the enzyme becomes susceptible to several factors, such as the affinity and the permeability of the assemblies to the enzymes, and other 309 factors that can affect the kinetics of the enzyme-responsive behaviour (62-65). In this study, the accessibility of the substrate (RNA) to the catalytic site of RNase A is selectively regulated by the phosphorylation-responsive behavior. Therefore, we have demonstrated that the sequence design of the biomaterial has a crucial influence on the allosteric regulation of the enzymatic activity. Figure 8. Schematic illustration of TR, RT and TRT allosteric control over the RNase A activity. 5. CONCLUSIONS In this study, we have developed a smart biomaterial based on Elastin-like Recombinamers with allosteric control of RNase A activity. We have obtained an optimal design for the Kinase/Phosphatase-responsive behaviour, whereby, the 100% of phosphorylation of the smart-ELR was responsible to move the frontier of solubility. This system has demonstrated to be completely reversible, moving back and forth between association (insolubility) and dissociation (solubility) by (de-)phosphorylation. The allosteric regulation of the RNAse A activity was successfully demonstrated; the RNase A catalytic activity depended by the assembled state of the biomaterial, which, is selectively regulated by the phosphorylation-responsive sites in the assemblies. Moreover, the 310 design principles of phosphatase-involved self-assembling systems can stimulate the development of other smart self-assembling systems responding to other important enzymes. 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Nawroth, J.F., et al., Maleimide-Functionalized Poly(2-Oxazoline)s and Their Conjugation to Elastin-Like Polypeptides. Macromolecular Bioscience, 2016. 16(3): p. 322-333. 33. Kinikoglu, B., et al., A smart bilayer scaffold of elastin-like recombinamer and collagen for soft tissue engineering. Journal of Materials Science: Materials in Medicine, 2011. 22(6): p. 1541- 1554. 319 Figure S8. HPLC-HR-MS Spectra for TI-N (red spectra), TI-P (blue spectra) and TI-D (green spectra). 320 Figure S9. HPLC-HR-MS Spectra for TR-N (blue spectra), TR-P (light green spectra) and TR-D (black spectra). 321 Figure S10. HPLC-HR-MS Spectra for RT-N (red spectra), RT-P (green spectra) and RT-D (blue spectra). 322 Figure S11. HPLC-HR-MS Spectra for TRT-N (dark green spectra), TR-P (light green spectra) and TR-D (purple spectra). 323 324 CONCLUSIONS AND FUTURE DIRECTIONS Genetic engineering, bioproduction and characterization of ELRs In this Thesis, it has been shown the development of several ELRs, each one containing bioactive sequences for tailored biomedical application. The hyphosesis to generate several ELRs comprising diverse combinations of bioactive domains was demonstrated. In terms of cell adhesion, RGD and REDV bioactive sequences have been included in the ELRs composition. For hydrogel-forming ELRs the amino acid sequences GAGAGS hexapeptide found in Bombyx mori silk fibroin was included in the ELR sequence giving stability to the hydrogel. Moreover, in the case of ELRs-based hydrogel for osteochondral repair an elastase target domain was also included in order to permit the replacement of the scaffold for the regenerated tissue. Furthermore, it was developed a series of ELRs containing the consensus sequence sensible for the kinase/phosphorylase in combination with the RNase A catalytic domain for allosteric domain’s regulation. Characterization techniques such as SDS-PAGE and MALDI-TOF confirmed the theoretical MW of the ELRs. Moreover, the Tt was assessed through DSC for all the ELRs; confirming that it was below the physiological temperature for the ELRs forming hydrogels when injected in vivo. 1H- NMR and HPLC for amino acid analysis assessed the purity of each batch of the different ELRs, while the FTIR analysis showed good agreement of the theoretical composition of the ELR with the experimental functional groups analyzed. Finally, the analysis of endotoxins ensured the purity of the ELRs for in vivo application. Development of ELRs-based hydrogel with different gelation mechanisms for osteochondral repair An important part of this Thesis focusses on the development of bioactive hydrogel for Tissue Engineering (TE) application. We hypothesized that the gelation mechanism had a crucial influence for the generation of a 3D hydrogel embedded with cells for osteochondral repair. Different ELRs-based hydrogels for osteochondral repair having two 325 different gelation mechanisms of hydrogels have been produced, either by chemical (covalent bonds) or by physical cross-linking. In both cases, injectable hydrogels were obtained. In the case of the chemically cross-linked hydrogel, the ELRs concentration selected demonstrated adequate mechanical properties for the osteochondral application. In the case of physically cross-linked hydrogels, the pre-annealing treatment applied to the Silk Elastin co-Recombinamer has shown to improve the mechanical properties of the hydrogel, guarantying fast gelation and the presence of a fibrillary structure directly after injection of the hydrogel. For both scenarios, SEM analysis showed a homogeneous porous environment with an interconnected structure. Although the ELR- based hydrogels developed in this Thesis had different gelation mechanisms, in both cases, the rheological study demonstrated how the concentration of the hydrogel could modulate the mechanical properties. For instance, according to the ELRs composition and its MW, the chemically cross-linked hydrogel allows the gelation of the hydrogel at a lower concentration than the physically cross-linked hydrogel; this thereby allows the cells embedded in the hydrogel matrix to have more space available, and also to play with a higher amount of cells for the formation of the 3D matrix. On the other hand, the physically cross-linked hydrogel does not contain covalent bonds and considering the slower process of gelation, this could provide a better environment for cells embedding. In conclusion, both gelation mechanisms demonstrated immediate gelation, conferring the benefit of being an injectable scaffold. Moreover, both systems offer tuneable features, such as the possibility to choose between a range of hydrogel’s concentration and cell’s density, which give the advantage of making the therapy as much tailored as possible. For instance, moving forward in the field of Tissue Engineering for osteochondral repair, it could be formed a scaffold which comprises both proposed hydrogels; it could be designed a double layer hydrogel containing a chemically cross-linked hydrogel for the bone area, whereas a physically cross-linked hydrogel could support the regeneration of cartilage layer. 326 In vitro, ex vivo and in vivo evaluation of ELRs-based hydrogel for osteochondral repair The ELRs-based hydrogels containing different bioactive sequences were tested for the osteochondral application. The physically cross-linked hydrogel containing the bioactive sequence RGD which supports cell adhesion via integrins was embedded with chondrocytes. This biomaterial has demonstrated the capacity to form a high cells density 3D hydrogel and to be delivered into the area of interest. The biocompatibility was proved by in vitro study; the addition of the silk allows to make hydrogels with a lower concentration, leading to larger pores, which is most likely responsible for better cell spreading, and proliferation. The regeneration capacities for cartilage repair were evaluated using an ex vivo culture platform. After four weeks of culturing, the hydrogel embedded with chondrocytes exhibited remarkable advantages; such as the de novo ECM formation, the absence of fibro-cartilage and the production of hyaline cartilage. On the other hand, the chemically cross-linked hydrogel was designed in order to contain the listed bioactive sequences: RGD and REDV to support cell adhesion via integrins and improve the selectivity for endothelial cells, and VGVAPG which provides proteolytic sensitivity to the biomaterial. This ELRs-based hydrogel was further embedded with MSCs and the biocompatibility was proved by an in vitro study, whereas the regeneration properties were evaluated by an in vivo study. Femoral bone defects were created in New Zealand rabbits, which were subsequently filled with the hydrogel embedded with MSCs and the hydrogel itself; then, after four months the samples were extracted and the regeneration was assessed by different methods. This ELR-hydrogel has been demonstrated to have an adequate composition; indeed it has shown a right ratio of bioactive sequences exhibiting a good balance between the degradation rate and adhesion behaviour, allowing for the colonization of chondrocytes with optimal secretion of extracellular matrix-collagen type II. Finally, it was demonstrated that the specific composition of this hydrogel allowed a faster bone regeneration when embedded with rMSCs compared to the injection of the hydrogel alone. On the other hand, the comparison of the regeneration between the hydrogel embedded with MSCs and the hydrogel itself has shown an excellent cartilage repair without the need for cellular 327 implantation. We demonstrated that the gelation mechanism has a crucial influence for the generation of a successful scaffold for osteochondral repair, especially when embedded with cells. In conclusion, the outcomes collected using the different hydrogel’s compositions with or without the cellular contribution open up other possibilities in design and combination of hydrogels, perhaps creating a scaffold composed by different layers, each one having its own composition, gelation mechanism, and cellular contribution. Development of a new ELR-peptides hybrid biomaterial The hypothesis that the strategy of copper-free click chemistry allows the incorporation of non-canonical amino acids and the formation of cyclized peptides on ELRs was demonstrated. A new hybrid material comprising the recombinant technique of ELRs and the strategy of the synthesis of bicyclic peptides was developed. Indeed, ELRs were covalently functionalized with each three high-affinity and selectivity αvβ3- and α5β1- binding bicyclic RGD peptides and with various integrin-binding benchmark peptides. The covalent functionalization was validated by MALDI-TOF analysis, guarantees flexibility and minimal steric hindrance for interactions with cellular integrins. Moreover, the objective to explore the potentiality of the new hybrid biomaterial for biomedical application was achieved; indeed, after the adsorbtion of the ELR-Peptides over tissue culture plate surface (TCPS), the adhesion capacity of HUVECs was evaluated. The in vitro studies of ELRs have shown that covalent RGD-functionalization of ELRs via copper-free click reaction is more efficient for inducing integrin-mediated cell adhesion and proliferation than the recombinant synthesis of ELRs comprising RGD as part of their backbone. The in vitro studies of ELRs functionalized with high-affinity integrin αvβ3- and α5β1-binding RGD bicycles represent an interesting alternative to promote fast cell adhesion on 2D biomaterial surfaces compared with well-known linear or monocyclic RGD peptides. In conclusion, a new system based on two diametrically opposed strategies was developed, demonstrating that combining approaches could be a new manner to explore new frontiers. In this case, we believe that ELRs functionalized with integrin-selective 328 RGD-bicycles have a great potential to evaluate cell-adhesion behavior and tailor high integrin peptides for specific biomedical applications. A new class of smart-ELRs with allosteric domain As a final stage of this journey, in this Thesis we have explored a new class of ELRs. A smart biomaterial based on ELRs with allosteric control of RNase A activity was developed. Firstly, the consensus sequence phosphorylation site was introduced and regularly distributed (ten times) along the ELR sequence. The HPLC-HR-MS analysis demonstrated the ability to fully phosphorylate/de-phosphorylate the ELRs, and the reversibility of this system. Furthermore, the turbidity analysis demonstrated an evident shift in Temperature transition (Tt) value due to the transfer/removal of the phosphate groups. Secondly, taking advantage of the recombinant technique, the Ribonuclease A catalytic domain (RNase A) was fused to the smart-ELR at the genetic level. According to the position of RNase A relative to the ELR backbone, three variants of the smart-ELR were produced and all of them demonstrated the capacity to fully phosphorylated/de-phosphorylated. The DLS and turbidity analysis performed at 37 °C, at the mild condition for a selected concentration, showed that the system was completely reversible, moving back and forth between association (insolubility) and dissociation (solubility) by (de-)phosphorylation. Finally, the RNase A catalytic activity showed dependency by the state of the biomaterial. Indeed, the RNase A activity was selectively enhanced by the phosphorylation of the ELR consensus sequences. The allosteric regulation of the RNase A activity was successfully demonstrated, showing that the design of the sequence has a central role in the activity of the selected enzyme. However, a next step would be to find a finer methodology to assess the allosteric regulation of the RNase A activity. In conclusion, the allosteric control demonstrated for the RNase A activity represents an initial proof-of-concept for a new class of responsive macromolecules, the design principles of phosphatase-involved self-assembling systems can stimulate the development of other smart self-assembling systems responding to other important enzymes. 335 ORAL & POSTER COMMUNICATION: - Elastin-Like Recombinamers bioactive hydrogel in tissue engineering for osteochondral repair. Advanced Materials for Biomedical Applications (AMBA) Congress. Ghent (Belgium). POSTER COMMUNICATION: - A new class of SMART Elastin-Like Recombinamer with enzyme responsiveness. International Symposium on Bioinspired macromolecular systems (ISBMS) Congress. Aveiro (Spain). FLASH TALK & POSTER COMMUNICATION: - “Dual” SMART Elastin-Like Recombinamer. 2016 BIOGEL Conference. Ringberg Castle (Germany). ORAL COMMUNICATION: - Elastin-like Recombinamers (ELRs): From Bioinspired Motifs to Biomedical Application. POSTER COMMUNICATION: - Self-assembled injectable ECM hydrogels from Elastin-like Co-Recombinamers biomaterials. Courses and certifications - Time and Project Management. Bpmsat (Spain). - Statistical Validation and Experimental Design. Biostatech (Spain). - Leadership and coaching techniques. University of Valladolid (Spain). - Research ethics and integrity. Organized by BIOGEL project. - Mechanical properties of the biomaterials. Radboud University. Nijmegen (the Netherlands). - Biomimetic hydrogels. Organized by BIOGEL project. 336 - Skills you really need when you apply for a job. Organized by BIOGEL project. - The Route to Academia: How to fail successfully. Organized by BIOGEL project. - Hydrogel Imaging. Austrian Institute of Technology (AIT). (Wien) Austria. - In vitro, ex vivo, and in vivo models. LifeTec Group. (Eindhoven) the Netherlands. - Synthesis of Biomaterials. University of Thessaloniki. Thessaloniki (Greece). - Critical thinking for public discourse. University of Amsterdam (The Netherlands). - Certification “In vivo experimentation for the design and realization of animal trials”. University of Salamanca (Spain). - The Whole Scientist. Jackson Laboratory. Bar Harbor (USA). Marie Curie Fellowship for Early Stage Research Training Attendance of the listed progress meetings with scientific communication: - Network Meeting at TPNBT, 14th December, 2015. Valladolid (Spain). - Network Meeting at Radboud University, 22th June, 2016. Nijmegen (the Netherlands). - Network Meeting at University of Valladolid, 12th December, 2016. Valladolid (Spain). - Network Meeting organized by the Centre for Research & Technology Hellas (CERTH), 14th June, 2017. Poros (Greece). - Network Meeting at Austrian Institute of Technology (AIT), 30th January, 2018. (Wien) Austria. - Network Meeting at DWI-Leibniz Institute for Interactive Materials, 25th July, 2018. (Aachen) Germany. - Network Meeting at DWI-Leibniz Institute for Interactive Materials, 14th December, 2018. Aachen (Germany). Awards Chair-Elect TERMIS Student and Young Investigator Section- Europe (SYISEU). Organization of SYIS activities at TERMIS congresses. TERMIS-SYIS provides a platform for the "next generation" of scientists and engineers in the field of tissue engineering and regenerative medicine to interact. Best Social Outreach at FIRM conference 2017 (Girona, Spain). 337 SYIS-TERMIS Debate Competition Winner at TERMIS-EU Congress 2017 (Davos, Switzerland). In concordance with the TERMIS 2017 theme on ‘Personalised Therapies for Regenerative Medicine’, the topic of the debate: “Will 3D-Printing or Decellularised organs rescue the donor-based organ scarcity for transplantation?” Teaching Supervision of the scientific training of the student Sandra Ramos at the University of Valladolid (Spain). The Workshop title: "POLÍMEROS RECOMBINANTES TIPO ELASTINA PARA APLICACIONES APLICACIONES BIOMÉDICAS".