Добірка наукової літератури з теми "Hydrogels à base de peptides"

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.

Hydrogels are a class of soft biomaterials and the material of choice for a myriad of biomedical applications due to their biocompatibility and highly tunable mechanical and biochemical properties. Specifically, light-mediated thiol-norbornene click reactions between norbornene-modified macromers and di-thiolated crosslinkers can be used to form base hydrogels amenable to spatial biochemical modifications via subsequent light reactions between pendant norbornenes in the hydrogel network and thiolated peptides. Macromers derived from natural sources (e.g., hyaluronic acid, gelatin, alginate) can cause off-target cell signaling, and this has motivated the use of synthetic macromers such as poly(ethylene glycol) (PEG). In this study, commercially available 8-arm norbornene-modified PEG (PEG-Nor) macromers were reacted with di-thiolated crosslinkers (dithiothreitol, DTT) to form synthetic hydrogels. By varying the PEG-Nor weight percent or DTT concentration, hydrogels with a stiffness range of 3.3 kPa–31.3 kPa were formed. Pendant norbornene groups in these hydrogels were used for secondary reactions to either increase hydrogel stiffness (by reacting with DTT) or to tether mono-thiolated peptides to the hydrogel network. Peptide functionalization has no effect on bulk hydrogel mechanics, and this confirms that mechanical and biochemical signals can be independently controlled. Using photomasks, thiolated peptides can also be photopatterned onto base hydrogels, and mesenchymal stem cells (MSCs) attach and spread on RGD-functionalized PEG-Nor hydrogels. MSCs encapsulated in PEG-Nor hydrogels are also highly viable, demonstrating the ability of this platform to form biocompatible hydrogels for 2D and 3D cell culture with user-defined mechanical and biochemical properties.

The usage of short peptide-based polysaccharide hydrogels for tissue engineering was discussed in this review. It explained the drawbacks of employing short peptide-based polysaccharide hydrogels as tissue regeneration scaffolds, while highlighting their benefits. In this review, we first gave a brief overview of short peptide-based polysaccharide hydrogel design process. Then, we provided additionally detailed information of the hydrogels with categorized polysaccharides (hyaluronic acid, dextran, chitosan, alginate, and agarose). We also explained the bioactive short peptides Arg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV), and Tyr-Ile-Gly-Ser-Arg (YIGSR) that were used to modify these polysaccharide hydrogels in order to enhance cell behaviors, including survival, adhesion, proliferation, and migration. Their applications in tissue engineering were also demonstrated and summarized in this review.

In light of the increasing levels of antibiotic resistance, nanomaterials and novel biologics are urgently required to manage bacterial infections. To date, commercially available self-assembling peptide hydrogels have not been studied extensively for their ability to inhibit micro-organisms relevant to tissue engineering sites such as dental root canals. In this work, we assess the biocompatibility of dental pulp stem/stromal cells with commercially available multicomponent peptide hydrogels. We also determine the effects of dental pulp stem/stromal cell (DPSC) culture in hydrogels on growth factor/cytokine expression. Furthermore, to investigate novel aspects of self-assembling peptide hydrogels, we determine their antimicrobial activity against the oral pathogens Staphylococcus aureus, Enterococcus faecalis, and Fusobacterium nucleatum. We show that self-assembling peptide hydrogels and hydrogels functionalized with the adhesion motif Arg-Gly-Asp (RGD) are biocompatible with DPSCs, and that cells grown in 3D hydrogel cultures produce a discrete secretome compared with 2D-cultured cells. Furthermore, we show that soluble peptides and assembled hydrogels have antimicrobial effects against oral pathogens. Given their antibacterial activity against oral pathogens, biocompatibility with dental pulp stem/stromal cells and enhancement of an angiogenic secretome, multicomponent peptide hydrogels hold promise for translational use.

Peptide-based hydrogels (PHGs) are biocompatible materials suitable for biological, biomedical, and biotechnological applications, such as drug delivery and diagnostic tools for imaging. Recently, a novel class of synthetic hydrogel-forming amphiphilic cationic peptides (referred to as series K), containing an aliphatic region and a Lys residue, was proposed as a scaffold for bioprinting applications. Here, we report the synthesis of six analogues of the series K, in which the acetyl group at the N-terminus is replaced by aromatic portions, such as the Fmoc protecting group or the Fmoc-FF hydrogelator. The tendency of all peptides to self-assemble and to gel in aqueous solution was investigated using a set of biophysical techniques. The structural characterization pointed out that only the Fmoc-derivatives of series K keep their capability to gel. Among them, Fmoc-K3 hydrogel, which is the more rigid one (G’ = 2526 Pa), acts as potential material for tissue engineering, fully supporting cell adhesion, survival, and duplication. These results describe a gelification process, allowed only by the correct balancing among aggregation forces within the peptide sequences (e.g., van der Waals, hydrogen bonding, and π–π stacking).

Hydrogel biomaterials mimic the natural extracellular matrix through their nanofibrous ultrastructure and composition and provide an appropriate environment for cell–matrix and cell–cell interactions within their polymeric network. Hydrogels can be modified with different proteins, cytokines, or cell-adhesion motifs to control cell behavior and cell differentiation. Collagens are desirable and versatile proteins for hydrogel modification due to their abundance in the vertebrate extracellular matrix and their interactions with cell-surface receptors. Here, we report a quick, inexpensive and effective protocol for incorporation of natural, synthetic and recombinant collagens into Fmoc-based self-assembling peptide hydrogels. The hydrogels are modified through a diffusion protocol in which collagen molecules of different molecular sizes are successfully incorporated and retained over time. Characterization studies show that these collagens interact with the hydrogel fibers without affecting the overall mechanical properties of the composite hydrogels. Furthermore, the collagen molecules incorporated into the hydrogels are still biologically active and provide sites for adhesion and spreading of human fibrosarcoma cells through interaction with the α2β1 integrin. Our protocol can be used to incorporate different types of collagen molecules into peptide-based hydrogels without any prior chemical modification. These modified hydrogels could be used in studies where collagen-based substrates are required to differentiate and control the cell behavior. Our protocol can be easily adapted to the incorporation of other bioactive proteins and peptides into peptide-based hydrogels to modulate their characteristics and their interaction with different cell types.

Poly(ethylene glycol) (PEG)-based synthetic hydrogels based on Michael-type addition reaction have been widely used for cell culture and tissue engineering. However, recent studies showed that these types of hydrogels were not homogenous as expected since micro domains generated due to the fast reaction kinetics. Here, we demonstrated a new kind of method to prepare homogenous poly(ethylene glycol) hydrogels based on Michael-type addition using the side chain amine-contained short peptides. By introducing such a kind of short peptides, the homogeneity of crosslinking and mechanical property of the hydrogels has been also significantly enhanced. The compressive mechanical and recovery properties of the homogeneous hydrogels prepared in the presence of side chain amine-contained short peptides were more reliable than those of inhomogeneous hydrogels while the excellent biocompatibility remained unchanged. Furthermore, the reaction rate and gelation kinetics of maleimide- and thiol-terminated PEG were proved to be significantly slowed down in the presence of the side chain amine-contained short peptides, thus leading to the improved homogeneity of the hydrogels. We anticipate that this new method can be widely applied to hydrogel preparation and modification based on Michael-type addition gelation.

AbstractSelf-assembled hydrogels from 9-fluorenylmethoxycarbonyl-modified diphenylalanine (Fmoc-FF) peptides were evaluated as potential vehicles for drug delivery. During self-assembly of Fmoc-FF, high concentrations of indomethacin (IDM) drugs were shown to be incorporated into the hydrogels. The β-sheet arrangement of peptides was found to be predominant in Fmoc-FF–IDM hydrogels regardless of the IDM content. The release mechanism for IDM displayed a biphasic profile comprising an initial hydrogel erosion-dominated stage followed by the diffusion-controlled stage. Small amounts of polyamidoamine dendrimer (PAMAM) added to the hydrogel (Fmoc-FF 0.5%–IDM 0.5%–PAMAM 0.03%) resulted in a more prolonged IDM release compared with Fmoc-FF 0.5%–IDM 0.5% hydrogel. Furthermore, these IDM-loaded hydrogels demonstrated excellent thixotropic response and injectability, which make them suitable candidates for use as injectable self-healing matrices for drug delivery.

Multicomponent hydrogels (HGs) based on ultrashort aromatic peptides have been exploited as biocompatible matrices for tissue engineering applications, the delivery of therapeutic and diagnostic agents, and the development of biosensors. Due to its capability to gel under physiological conditions of pH and ionic strength, the low molecular-weight Fmoc-FF (Nα-fluorenylmethoxycarbonyl-diphenylalanine) homodimer is one of the most studied hydrogelators. The introduction into the Fmoc-FF hydrogel of additional molecules like protein, organic compounds, or other peptide sequences often allows the generation of novel hydrogels with improved mechanical and functional properties. In this perspective, here we studied a library of novel multicomponent Fmoc-FF based hydrogels doped with different amounts of the tripeptide Fmoc-FFX (in which X= Cys, Ser, or Thr). The insertion of these tripeptides allows to obtain hydrogels functionalized with thiol or alcohol groups that can be used for their chemical post-derivatization with bioactive molecules of interest like diagnostic or biosensing agents. These novel multicomponent hydrogels share a similar peptide organization in their supramolecular matrix. The hydrogels’ biocompatibility, and their propensity to support adhesion, proliferation, and even cell differentiation, assessed in vitro on fibroblast cell lines, allows us to conclude that the hybrid hydrogels are not toxic and can potentially act as a scaffold and support for cell culture growth.

Thixotropy is a fascinating feature present in many gel systems that has garnered a lot of attention in the medical field in recent decades. When shear stress is applied, the gel transforms into sol and immediately returns to its original state when resting. The thixotropic nature of the hydrogel has inspired scientists to entrap and release enzymes, therapeutics, and other substances inside the human body, where the gel acts as a drug reservoir and can sustainably release therapeutics. Furthermore, thixotropic hydrogels have been widely used in various therapeutic applications, including drug delivery, cornea regeneration and osteogenesis, to name a few. Because of their inherent biocompatibility and structural diversity, peptides are at the forefront of cutting-edge research in this context. This review will discuss the rational design and self-assembly of peptide-based thixotropic hydrogels with some representative examples, followed by their biomedical applications.

I. Tema y objetivos de la tesis Debido a la estrecha relación entre el auto-ensamblaje de péptidos y proteínas con las enfermedades de tipo amiloide,(1) entender los principios que gobiernan este fenómeno resulta de gran interés en la actualidad. El uso de pequeños péptidos como modelos o inhibidores del proceso de fibrilación es considerado una de las mejores aproximaciones debido a su facilidad de síntesis, versatilidad y biocompatibilidad.(2) Se conoce además que la organización jerárquica de estas fibrillas peptídicas da lugar a menudo a redes auto-ensambladas que retienen el disolvente dando lugar a geles supramoleculares.(3) Estos materiales soportados mediante interacciones no covalentes presentan multitud de propiedades incluyendo reversibilidad, auto-reparación o sensibilidad frente a diferentes estímulos físico-químicos que hacen de ellos unos materiales extremadamente útiles en campos tan diferentes como la biomedicina, la catálisis o la ciencia de materiales.(4) Teniendo en cuenta estas consideraciones durante el transcurso de la presente tesis doctoral se han desarrollado nuevos péptidos anfipáticos con tendencia a la formación de hojas-ß para: - Profundizar en los fundamentos del auto-ensamblaje proporcionando un mayor entendimiento del complejo proceso de fibrilación. - Estudiar la habilidad de estos compuestos como hidrogelantes supramoleculares para utilizarlos como catalizadores o materiales que respondan a estímulos. II. Resultados y discusión 1) Diseño y síntesis de los hidrogelantes supramoleculares El primer paso para alcanzar los objetivos previamente mencionados consiste en el diseño y la síntesis de los compuestos que posteriormente van a ser estudiados como hidrogelantes. En nuestro caso se han sintetizado tres familias de compuestos anfipáticos derivados de péptidos cuya estructura general se describe a continuación: - Tetrapéptidos básicos formados por L-fenilalanina (F) y L-aspártico (D) Compuestos por dos fenilalaninas y dos residues de ácido aspártico protegidos por un grupo Cbz y una cadena alifática en el extremo C- y -N terminal respectivemente. La tendencia de dichos péptidos anfipáticos a la formación de hojas ß ha sido previamente demostrada en multitud de ocasiones y en nuestro caso, las variaciones sistemáticas en la secuencia de aminoácidos nos permitirán estudiar el papel que desempeña el balance hidrofóbico/hidrofílico en el proceso de fibrilación. Es necesario mencionar que ambos extremos de la cadena peptídica han sido bloqueados usando una pequeña cadena alifática y un grupo protector de tipo benciloxicarbonilo en el extremo C- y N-terminal respectivamente para evitar ionizaciones adicionales y simplificar de ese modo el análisis del proceso de agregación. - Tetrapéptidos básicos formados por L-fenilalanina (F) y L-lisina (K) De modo semejante a los tetrapéptidos descritos en el párrafo anterior se han diseñado adicionalmente dos tetrapéptidos básicos que contienen lisina para estudiar su habilidad como hidrogelantes y posteriormente estudiar su coagregación con sus análogos tetrapeptídicos que contienen aspártico. - Tripéptidos anfifílicos formados por L-prolina (P), L-fenilalanina (F) y L-ácido glutámico (E) Finalmente una familia de tripéptidos anfifílicos que contienen L-prolina como fragmento funcional ha sido diseñada para su posterior estudio de agregación en agua tamponada y como catalizadores de condensaciones aldólicas. La síntesis de todos ellos ha sido llevada a cabo mediante una metología por pasos en disolución y los compuestos fueron obtenidos sin dificultad y con buenos rendimientos. Además la purificación se realizó de manera sencilla sin necesidad de cromatografía. Sin embargo, debido a la creciente utilización de fase sólida para la síntesis de derivados peptídicos se ha decidido probar también dicha metodología en la síntesis de uno de nuestros compuestos estableciendo finalmente ventajas e inconvenientes de cada metodología para nuestros propósitos en particular. Así pues se ha establecido que la síntesis en fase sólida podría ser de gran utilidad para fabricar pequeñas cantidades de compuesto destinadas a una evaluación inicial de sus propiedades. La síntesis en disolución sin embargo, podría ser utilizada para la obtención a mayor escala de aquellos que resulten más convenientes en función de dicha evaluación inicial. También resulta de interés recalcar que durante la síntesis se ha descubierto una inesperada labilidad del grupo Z (benziloxicarbonilo) en condiciones básicas muy suaves. Dicho fenómeno ha sido estudiado para una familia mucho más amplia de compuestos llegándose a la conclusión de que una asistencia anquimérica adicional proporcionada por un puente de hidrógeno entre el carbamato y una amida adyacente resulta fundamental para la desprotección de este grupo.(5) 2) Tetrapéptidos ácidos formados por D y F Afortunadamente todos los compuestos sintetizados han dado lugar a hidrogeles utilizando el cambio de pH como estímulo externo. Dichos hidrogeles han sido estudiados minuciosamente utilizando técnicas como la resonancia magnética nuclear, dicroísmo circular, microscopía de transmisión y de barrido, espectroscopia de infrarojo, fluorescencia, etc. Mediante este completo estudio se comprobó el efecto que causaba la disposición de los aminoácidos en esta cadena ya que dan lugar a redes fibrilares de morfologías y propiedades muy distintas. La combinación de los resultados experimentales con los modelos teóricos propuestos nos ha permitido observar el papel que tanto las interacciones hidrófobicas como aromáticas desempeñan en el proceso de agregación disponiendo bloques segregados de aminoácidos polares y no polares para los mejores hidrogelantes. Continuando con los hidrogeles tetrapeptídicos se ha llevado a cabo un estudio referente a la termodinámica y cinética del proceso de fibrilación, a menudo aspectos no considerados durante la descripción de nuevos hidrogelantes. Se han determinado para ello constantes de solubilidad y acidez detectándose un aumento general del valor de pKa (en comparación con el pKa del ácido aspártico en disolución) a consecuencia de la agregación que permite la obtención de hidrogeles a valores de pH cercanos a la neutralidad. Estas diferencias de pKa están directamente relacionadas con la termodinámica de agregación. Por otro lado los estudios cinéticos nos han permitido detectar un periodo de inducción previo a la agregación para todos los compuestos aunque con diferencias significativas entre ellos. Dicho periodo contiene el tiempo necesario para alcanzar el valor de pH que da lugar a fibrilación (termodinámica) y además otro período en el cual se encuentran presentes especies metastables. Mediante experimentos de fluorescencia se ha detectado que el paso determinante en el proceso de fibrilación resulta ser la formación de dímeros probablemente debido a la energía de activación asociada a la reorganización estructural. Como consecuencia del conocimiento tanto termodinámico como cinético de estos sistemas se ha conseguido obtener geles metaestables en presencia de otros compuestos que forman agregados termodinámicamente más estables pero que permanecen en disolución (self-sorting cinético). Teniendo en cuenta el papel que los péptidos pequeños desempeñan tanto como modelos o inhibidores de la agregación amiloide se ha decido probar la interacción entre los tetrapéptidos ácidos y el péptido amiloide Ab1-40 obteniendo resultados satisfactorios para al menos dos de los compuestos que parecen funcionar como semillas (núcleos) para la agregación del péptido amiloide. Comparando nuestros resultados con otros previamente publicados resulta importante destacar que nuestros compuestos son de pequeño tamaño (cuatro aminoácidos) y además no poseen ningún fragmento contenido en dicha proteína. 3) Tetrapéptidos básicos formados por K y F Tras el estudio de los tetrapéptidos ácidos se han sintetizado dos análogos tetrapépticos básicos (7-8) que contienen lisina (K) en lugar de ácido aspártico (D). Además de formar geles en agua básica se ha logrado la coagregación a valores de pH neutros con los compuestos 1 y 2 utilizando para ello interacciones no covalentes. La metodología utilizada para ello ha sido mediante la inyección de sus componentes por separado en disolución dando lugar a hidrogeles homogéneos y transparentes con características idóneas para potenciales aplicaciones biológicas. Utilizando el mismo concepto se han desarrollado además geles multi-componente capaces de mantener su estabilidad macroscópica en todo el rango de pH. Dicho comportamiento es debido al intercambio que se establece entre redes co-agregadas de dos componentes a pH neutro y redes formadas por un solo compuesto a valores ácidos o básicos de pH. La singularidad de este sistema ha sido aprovechada para la liberación controlada de algunos colorantes teniendo en cuenta por un lado la estructura molecular de dichos compuestos y por otro lado las características particulares que presentan las diferentes redes co-ensambladas. - 4) Tripéptidos anfifílicos formados por P, F y E Por último, las propiedades catalíticas de la prolina en sitemas autoensamblados han sido ampliamente estudiadas, en colaboración con el profesor Ashkenasy de la Universidad Ben Gurinon (Israel), atendiendo a los precedentes publicados por el grupo.(6) Para ello se han utilizado los compuestos de prolina anteriormente descritos en reacciones de condensación aldólica mostrando una clara influencia de la hidrofobicidad proporcionada por la cadena alifática del extremo C-terminal. Aquellos compuestos que formaron redes fibrilares en agua resultaron activos en la formación de enlaces C-C mientras que los compuestos que permanecieron en disolución resultaron inactivos. Estos sistemas catalíticos demuestran pues la emergencia de propiedades catalíticas debidas al autoensamblaje. Además el orden en la secuencia de aminoácidos influyó en los resultados obtenidos sugiriendo la formación de sitios catalíticos de diferente accesibilidad. Dichos compuestos sirvieron además de modelos para decapéptidos de secuencia semejante que resultaron también ser activos en reacciones aldólicas. Finalmente y tras conocer las propiedades catalíticas des estos compuestos se probó su efectividad en la obtención de productos biológicamente más relevantes de tipo carbohidratos. III. Conclusiones En resumen, durante el transcurso de la presente tesis doctoral se han diseñado y sintetizado con éxito nuevos hidrogelantes basados en péptidos anfipáticos de pequeño tamaño. Ambas metodologías empleadas para ello, tanto en disolución como en fase sólida han dado lugar a resultados satisfactorios pudiendo obtener los productos con elevado grado de pureza y suficiente en cantidad. El estudio del proceso de agregación desarrollado sobre todo para los tetrapéptidos ácidos ha permitido poner de manifiesto lo importante que resulta el balance hidrofóbico/hidrofílico en el modo de agregación y propiedades de los sistemas finales. Profundizar en la termodinámica y cinética de este proceso ha permitido además discernir la etapa limitante del mismo y utilizar dicho conocimiento para la elaboración de sistemas multi-componente más complejos basados en especies termodinámicamente metaestables. Dichos estudios resultan de gran utilidad considerando que los péptidos son considerados buenos modelos de la agregación amiloide y que los fenómenos que aquí se presentan pueden observarse e incluso magnificarse en sistemas proteicos de mayor tamaño. Por otro lado el co-ensamblaje entre dos compuestos opuestamente cargados ha permitido obtener geles cuya estabilidad se extiende a todo el rango de pH. Dichos sistemas resultan de gran utilidad biológica puesto que pueden formarse a pH neutro sin necesidad de calentar o aplicar ningún otro estímulo externo simplemente mediante la mezcla de sus componentes por separado en disoluciones acuosas. Además se ha logrado modular la liberación de ciertos colorantes atendiendo a la estructura del mismo y de la red fibrilar que lo contiene. Finalmente la aparición de propiedades catalíticas debidas al auto-ensamblaje ha sido demostrada para derivados anfifílicos que contienen prolina como fragmento funcional. La aparición de nuevas propiedades a consecuencia del autoensamblaje así como el papel fundamental que él mismo desempeña en la formación de compartimentos aislados confiere relevancia prebiótica a estos pequeños sistemas peptídicos agregados. Los resultados obtenidos contribuyen pues al mejor entendimiento de este complejo proceso que representa la fibrilización. IV. Referencias 1. a) Knowles, T. P. J.; Vendruscolo, M.; Dobson, C. M., The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014, 15, 384-396; b) Hamley, I. W., The amyloid ß peptide: A chemist's perspective role in Alzheimer's and fibrillization. Chem. Rev. 2012, 112, 5147-5192. 2. a) Lakshmanan, A.; Cheong, D. W.; Accardo, A.; Di Fabrizio, E.; Riekel, C.; Hauser, C. A. E., Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis. Proc. Natl. Acad. Sci. 2013, 110, 519-524; b) Gazit, E., Self assembly of short aromatic peptides into amyloid fibrils and related nanostructures. Prion 2007, 1, 32-35; c) Hamley, I. W., Peptide fibrillization. Angew. Chem. Int. Ed. 2007, 46, 8128-8147; d) Gazit, E., Mechanisms of amyloid fibril self-assembly and inhibition. FEBS J. 2005, 272, 5971-5978. 3. a) Dasgupta, A.; Mondal, J. H.; Das, D., Peptide hydrogels. Rsc Advances 2013, 3, 9117-9149; b) Jonker, A. M.; Löwik, D. W. P. M.; van Hest, J. C. M., Peptide- and Protein-Based Hydrogels. Chem. Mater. 2012, 24, 759-773. 4. Escuder, B.; Miravet, J.F., Functional Molecular Gels. RSC 2013; pp 001-319. 5. Tena-Solsona, M.; Angulo-Pachón, C. A.; Escuder, B.; Miravet, J. F., Mechanistic insight into the lability of the benzyloxycarbonyl (Z) group in N-protected peptides under mild basic conditions. Eur. J. Org. Chem. 2014, 3372-3378. 6. a) Berdugo, C.; Miravet, J. F.; Escuder, B., Substrate selective catalytic molecular hydrogels: the role of the hydrophobic effect. Chem. Commun. 2013, 49, 10608-10610; b) Rodríguez-Llansola, F.; Miravet, J. F.; Escuder, B., A supramolecular hydrogel as a reusable heterogeneous catalyst for the direct aldol reaction. Chem. Commun. 2009, 7303-7305.

Les hydrogels sont des réseaux 3D retenant de grandes quantités d'eau. Biocompatibles, ils sont utilisés pour la délivrance de médicaments. Dans le but de développer des hydrogels antibactériens, cette thèse présente deux études basées sur l'utilisation d'un tripeptide phosphorylé protégé par un fluorénylméthoxycarbonyle (Fmoc), qui peut s'auto-assembler en hydrogel. Dans la première étude, différentes conditions de préparation (pH, sel, présence de polysaccharide) ont été étudiées pour former un hydrogel autocicatrisant et antibactérien libérant un antibiotique, le florfénicol. Dans la seconde étude, des stratégies de synthèse peptidiques et de phosphoramidites en phase solide ont été combinées pour ajouter le florfénicol au phosphate de tyrosine protégé par le Fmoc via un phosphodiester, clivable par des nucléases produites par des bactéries. Des résultats encourageants ont montré la formation du composé ciblé, ouvrant la voie au design d'un peptide antibactérien auto-défensif
Hydrogels are 3D networks of fibers that retain large amounts of water when swollen. Due to their biocompatibility, they are increasingly used for drug delivery. To develop antibacterial peptide-based hydrogels, this dissertation presents two studies based on the use of a fluorenylmethoxycarbonyl (Fmoc)-protected phosphorylated tripeptide that can self-assemble into a hydrogel. In the first study, different preparation conditions (pH, salt, presence of polysaccharide) were investigated to obtain a self-healing and antibacterial hydrogel capable of releasing an antibiotic, florfenicol. In the second study, a solid-phase peptide and phosphoramidite synthesis strategies were combined to add florfenicol to the Fmoc-protected tyrosine phosphate via a phosphodiester, which can be cleaved by nucleases produced by bacteria. Encouraging results showed the formation of the targeted compound, paving the way for the design of a self-defensive antibacterial peptide

The intervertebral disc (IVD), situated between adjoining vertebrae, consists of the gelatinous nucleus pulposus (NP) in the centre surrounded by the tougher annulus fibrosus (AF). Its main roles are to distribute loads and to act as joints. With aging, degenerative disc disease (DDD) occurs due to an imbalance in anabolic and catabolic events in the IVD, which results in a loss of function. Lower back pain (LBP) affects 84% of people at some point in their lifetime and is strongly associated with DDD. Current LBP treatments have limited long term efficacy and are symptomatic rather than curative. Cell-based therapies are regarded to hold great potential for the treatment of DDD as it has been hypothesised that they could regenerate the damaged tissue and alleviate LBP. A number of natural and synthetic biomaterials have been investigated as NP tissue engineering scaffolds with varying results. In this study, a self assembling peptide hydrogel (SAPH) was investigated for its potential as a cell carrier and/or scaffold for NP tissue engineering. SAPHs display the advantages of natural polymer hydrogels such as biocompatibility and biodegradability whilst combining the advantages of synthetic materials such as controlled structural and mechanical propertiesCharacterisation determined that the SAPH nanofibrous architecture had features that were of similar scale to extracellular matrix (ECM) components of the human NP. The mechanical properties of the SAPH could be optimised to closely match the native tissue. The system could shear thin and self-heal making the system ideally suited to delivery via minimally invasive procedure. The three dimensional (3D) culture of bovine NP cells (bNPCs) in the SAPH demonstrated that the NP phenotype could be restored after de-differentiation during monolayer culture. Gene expression results demonstrated that ‘traditional’ and ‘novel’ NP markers were highly expressed throughout in vitro culture. Cell viability was high, cell population remained stable and bNPCs adopted the characteristic rounded morphology of native NPCs. Finally, type II collagen and aggrecan, the main ECM components of the NP, were deposited with increasing production over culture period. Growth differentiation factor 6 (GDF-6) has been identified as the most promising current growth factor for inducing discogenic differentiation from human bone marrow mesenchymal stem cell (h-BMMSCs). After samples were stimulated with GDF-6, gene expression results confirmed that a NP-like phenotype could be induced with high expression of ‘traditional’ and ‘novel’ NP markers. Cell viability was high, cell population remained stable and NP associated ECM components were deposited with cells displaying a rounded morphology. Interestingly, when h-BMMSCs were cultured without GDF-6, it was strongly suggested that spontaneous discogenic differentiation occurred after culture in the SAPHs as ‘traditional’ and ‘novel’ NP markers were highly expressed, morphology was comparable to native NPCs and type II collagen and aggrecan were deposited extracellularly. If these findings were accurate then this is the first study to demonstrate that a NP-like phenotype could be induced from MSCs without use of an exogenous growth factor or a discogenic bioactive motif. Despite exciting and novel results, further work is required to confirm the potential of SAPHs for NP tissue engineering scaffolds.

Several studies exist in the literature which utilise either dendritic (covalent) or self-assembling (non-covalent) strategies to achieve multivalent binding to a biological target, but rarely are the two explored together. Herein, we compare and contrast dendritic and self-assembling approaches to organise a multivalent array of ligands to bind the protein, integrin αvβ3. In the first instance, linear RGD (Arg-Gly-Asp) peptides were covalently attached to first and second generation dendritic frameworks, and a positive (monovalent) and negative control were synthesised. A fluorescence polarisation (FP) competition assay was used to quantify the binding. The first generation dendron (2.16) was the most effective binder, with an EC50 of 125 μM (375 μM per peptide unit); significantly better than the monovalent ligand (2.19), the binding of which could not be quantified in our assay, even at 1 mM concentration; whilst the second generation dendron (2.17) was somewhat less effective, indicating that there is an optimum number of ligands that can be displayed before the multiple ligand array becomes disadvantageous to the binding. To explore the non-covalent approach, the linear RGD peptide was conjugated to either a single hydrophobic C12 aliphatic chain (3.2), an aromatic pyrene (3.4), a C22 aliphatic chain (3.6), or two C12 aliphatic chains (3.18), which gave rise to amphiphilic peptides which were able to self-assemble to differing degrees and into markedly different morphologies, as shown by a Nile Red encapsulation study and TEM imaging. Spherical micelles were formed by amphiphiles 3.2 and 3.4, whereas 3.6 and 3.18 produced cylindrical, rod-like micelles. Compounds 3.2 and 3.4 were the most effective integrin binders at concentrations of 200 μM and 110 μM, respectively, whilst 3.6 and 3.18 failed to produce a quantifiable binding concentration. The results therefore show that not only does the micellar self-assembly approach yield multivalent ligand displays with improved efficiency of binding compared with the dendritic method, but the morphology of the self-assembled system can also be detrimental to the recognition of the protein, at least in our FP assay and using purified integrin in solution. Finally, we report on a family of linear RGD peptide conjugate hydrogelators. Of particular interest was the novel bolaamphiphile C12-[urea-RGD]2 (4.4), comprised of linear RGD peptide head groups connected to either end of a hydrophobic C12 aliphatic chain via urea linkages. The molecule undergoes thermoreversible chiral self-assembly in water and generates a sample-spanning nanofibrous gel network, as determined by circular dichroism spectroscopy and TEM/SEM imaging, respectively. Gels were formed at a minimum gel concentration (MGC) of 0.06 wt % (0.6 mg/ml, 0.5 mM), one of the lowest MGCs reported and represents a “super” hydrogel. We report on its responsiveness (breakdown) towards charge dense basic anions such as phosphate and acetate, but its stability in the presence of more charge diffuse halide and nitrate anions. Furthermore, we demonstrate the passive diffusion of encapsulated low MW additives out of the gel phase, whereas high MW, protein-sized molecules remain trapped within the fibrous network.

Thesis (M.Ch.E.)--University of Delaware, 2008.
Principal faculty advisors: Joel P. Schneider, Dept. of Chemistry & Biochemistry; and Eric M. Furst, Dept. of Chemical Engineering. Includes bibliographical references.

AbstractIn this chapter, we will thoroughly discuss characterization techniques used to elucidate the exact structure and define properties of peptide-based nanomaterials. In particular we divide methods into: Quality control performance (mass spectroscopy and high-performance liquid chromatography. Spectroscopy (Fourier transform infrared spectroscopy, Raman spectroscopy, circular and linear dichroism, nuclear magnetic resonance and fluorescence spectroscopy). Microscopy (scanning and transmission electron microscopies, atomic force microscopy, optical and polarized light microscopy). Scattering (small angle X-ray and neutron scattering, X-ray diffraction). Bulk structures (mainly hydrogels) rheological characterization. The methodology is described for molecular structures, self-assembled nanostructures and aggregates, as well as hybrid, composite and/or conjugated nanomaterials and their bulk forms. Both common, as well as more exotic versions of all methods are presented in the context of peptide-based nanomaterials. Where utilized, examples of combinatorial use of techniques are demonstrated. Representative studies accompany the discussion and usefulness of all presented methods.

Functional foods are gaining traction as a source of peptides possessing hydrogelation properties. Analysis of peptides (n=429) in egg white protein hydrolysates resulted in the identification of six peptides: IFYCPIAIM, NIFYCPIAIM, VLVNAIVFKGL, YCPIAIMSA, MMYQIGLF, and VYSFSLASRL as prominent self-assembly candidates based on prediction of their aggregation-prone segments. The objective of this study was to characterize the hydrogel formed via self-assembly of the peptides. Of the six peptides studied, NIFYCPIAIM and MMYQIGLF showed promising self-assembly and hydrogelation properties. Thioflavin T kinetics indicated that NIFYCPIAIM possesses the strongest self-assembly property, confirmed by dynamic light scattering which indicated the largest average particle diameter was achieved after 24 hours. Rheological characterization indicated that all six peptides possessed viscoelastic pseudoplastic properties and some were able to regain some level of viscosity following the exertion of shear stress. Finally, transmission electron microscopy of the six peptides showed the development of fibrillar structures of varying morphologies after 24 hours. The remarkable difference in self-assembly and hydrogelation properties of NIFYCPIAIM, IFYCPIAIMSA and YCPIAIMSA, which share a common sequence YCPIAIM, indicate the importance of amino acid sequence in the formation and property of peptide hydrogels. Identification of the egg white-derived peptides with hydrogelation properties shows a promising future for the use of functional foods in applications of drug delivery systems and tissue engineering, in the food, pharmaceutical, cosmetics, and biomedical sectors.

Self-assembly of β-sheet forming peptides into filaments has drawn great interests in biomedical applications [1,2]; Hydrogels formed by filaments self-assembled from de novo designed peptides possess potential applications for cell culture scaffolds [3]. On the other hand, peptides derived from amyloidogenic proteins in neurodegenerative diseases such as Alzheimer’s and Parkinson’s also form similar β-sheet filaments in vitro. They share little sequence homology, yet filaments formed by these self-assembling peptides commonly have the cross-β structure, the key signature of the amyloid fibril. Detailed structural information of the self-assembled β-sheet filaments has been limited partly due to the difficulty in preparing ordered filament samples, and it has been only recently that solid-state nuclear magnetic resonance and x-ray techniques have revealed their molecular structure at the atomic level [4,5]. Although molecular structures of amyloid fibrils are becoming available, physical principles governing their self-assembly and the properties of the filaments are not well-understood, for which computational as well as theoretical approaches are desirable [6].

Recent research in our group has shown that artificial cell membranes formed at the base of a hair-like structure can be used to sense air flow in a manner similar to the mechanotransduction processes found in mammalian hair cells. Our previous work demonstrated that a single artificial hair cell can be formed in an open substrate. However, that study also motivated the need to develop fully-encapsulated devices that feature arrays of hair-cells. Since the transduction element in this concept is an artificial cell membrane, or lipid bilayer, this work investigates two parallel substrate designs for providing encapsulation and a method for forming arrays of bilayers. In one effort, a flexible substrate with internal compartments for hosting the biomolecules and mating cap are constructed and experimentally characterized. The regulated attachment method (RAM) is used to form interface bilayers within the sealed device. Capacitance measurements of the sealed interface bilayer show that the sealing cap slightly compresses the bottom insert and reduces the size of the enclosed bilayer. Single channel measurements of alamethicin peptides further verify that the sealed device, which is also leak-proof under water, can be used to detect the insertion and gating activity of transmembrane proteins in the membrane. The second effort pursued herein is the fabrication and initial testing of a method to form arrays of interface bilayers by using anchored hydrogel pads that support curved aqueous lenses in oil. In this fashion, the configuration of the array does not require manipulating droplets, but instead depends on the arrangement of the built-in gels used to support the aqueous lenses. As with RAM, mechanical force is used to promote contact of adjacent aqueous lenses held in the flexible substrate. Initial tests show that gel-supported lenses can be used for forming multiple lipid bilayers within the device and that these interfaces can be interrogated individually or collectively using an electrode switching circuit.

Articular cartilage has a limited regenerative capacity, and there exist no methodologies to restore structure and function after damage or degeneration. This has focused intense work on cell-based therapies for cartilage repair, with considerable literature demonstrating that chondrocytes in vitro and in vivo can generate cartilage-like tissue replacements. However, use of primary cells is limited by the amount and quality of autologous donor cells and tissue. Multipotential mesenchymal stem cells (MSCs) derived from bone marrow offer an alternative cell source for cartilage tissue engineering. MSCs are easily accessible and expandable in culture, and differentiate towards a chondrocyte-like phenotype with exposure to TGF-β [1]. For example, we have shown that bovine MSCs undergo chondrogenic differentiation and mechanical maturation in agarose, self-assembling peptide, and photocrosslinkable hyaluronic acid (HA) hydrogels [2]. HA hydrogels are particularly advantageous as they are biologically relevant and easily modified to generate a range of hydrogel properties [3]. Indeed, bovine MSCs show a strong dependence of functional outcomes on the macromer density of the HA gel [4]. To further the clinical application of this material, the purpose of this study was to investigate functional chondrogenesis of human MSCs in HA compared to agarose hydrogels. To carry out this study, juvenile bovine and human MSCs were encapsulated and cultured in vitro in HA and agarose hydrogels, and cell viability, biochemical, biomechanical, and histological properties were evaluated over 4 weeks of culture.

In the present work the physical-chemical and energetic properties of the molecules of hydrogels about chitosan crosslinking with genipin were studied in the adsorption process of Metformine and Glibenclamide. The analysis was done by means of PM6 model in order to obtain the necessary data base to establish their potential use as transdermal controlled release systems. Currently, the drugs (metformine and glibenclamide) are administered orally, however, there are already several oral medications that have been presented as transdermal administration systems (SAT), with great advantages, such as patient comfort and zero order release. Drugs before mentioned, are especially important in chronic diseases such as type 2 diabetes.

The article discusses new data obtained in the study of the functions of a conservative peptide of cabbage plants, which is encoded by the microRNA156a. Comparative analysis of the nucleotide sequences of the promoter regions of these genes allowed us to identify a highly conserved 42-residue block located before the starting point of pri-miR156a transcription at a distance of 210-260 base pairs. It was found that promoter fragments containing a highly con-served block have a significantly higher ability to bind miPEP156a in vitro. We carried out mutagenesis of a highly conserved promoter block in its central part, which includes a tetramer of TG dinucleotides. It has been shown that the introduction of mutations into the promoter tetramer of TG dinucleotides significantly reduces the affinity of the promoter DNA to miPEP156a. The miPEPs revealed in plants have been found only in dicotyledons. The question of how miPEPs are distributed in other plant taxa is very important for understanding the evo-lutionary origin of such micropeptides. As an initial approach, we searched for taxonomically conservative miPEPs in mosses, since microRNAs have been studied in a great detail in the case of Physcomitrium patens moss. For two genes in the region preceding the Ppt-pre-miR160a sequence, rather short open reading frames were found that encoded peptides having a clear similarity of amino acid sequences in the central region. Importantly, such highly con-served peptide block homologous to that encoded by Ppt-miPEP160a gene was detected in short proteins encoded in pri-miR160a in almost 20 Bryopsida mosses.