Self-assembling peptides have emerged as new nanobiomaterials in the areas of nanoscience and biomedical engineering. In this category are self-assembling, ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to a unique combination of amphiphilicity and ionic complementarity. These peptides can self-assemble into stable nanostructures or macroscopic membranes that can withstand conditions of high temperature, extreme pH, many digesting enzymes and denaturation agents. Moreover, they exhibit good biocompatibility with various cultured mammalian cells, and do not have detectable immune responses when introduced into animals. These properties make them ideal materials for tissue scaffolding, regenerative medicine and drug delivery. This thesis focuses on the utilization of self-assembling peptides for hydrophobic anticancer drug delivery. The hydrophobic anticancer agent ellipticine was selected as a model drug. The studies include: (i) characterization of the photophysical properties of ellipticine in different environments; (ii) study of the formation of peptide-ellipticine complexes and the release kinetics; (iii) investigation of the cellular toxicity of the complexes and ellipticine uptake; (iv) study of the peptide sequence effect on the complex formation and in vitro delivery. Prior to applying ellipticine to the peptide-based delivery system, the fundamental studies on the effect of solution conditions, especially solvent polarity and hydrogen bonding, on the fluorescence of ellipticine were carried out. Ultraviolet (UV) absorption and fluorescence emission of ellipticine were found to be solvent/environment dependent. The absorption and emission maxima shifted to higher wavelengths (red shift) with increased solvent polarity. Large Stokes’ shifts were due to intramolecular charge transfer (ICT), which was enabled by large solvent polarity and hydrogen bonding of ellipticine with the solvents. The photophysical response of ellipticine to changes in solvent polarity and hydrogen bond formation could be used to infer the location of ellipticine in a heterogeneous medium, such as liposomes and cultured cells. EAK16-II, a model self-assembling peptide, was found to be able to stabilize ellipticine in aqueous solution. The equilibration time required to form peptide-ellipticine complex suspensions was found to be peptide concentration-dependent and related to the peptide critical aggregation concentration (CAC, ~0.1 mg/mL). With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be obtained in the complexes. The release kinetics of ellipticine from the complex into egg phosphatidylcholine (EPC) vesicles (cell membrane mimics) was also affected by the peptide concentration used in the drug formulation. A higher peptide concentration resulted in a faster transfer rate, in relation to the size of the resulting complexes. Subsequent cellular studies on two cancer cell lines, A549 and MCF-7, showed that the complexes with protonated ellipticine were more effective against both cell lines, but their dilutions were not very stable. In addition, it was found that ellipticine uptake in both cell lines was very fast and through direct membrane permeation. Three peptides, EAK16-II, EAK16-IV and EFK16-II, either having a different charge distribution (EAK16-II vs. EAK16-IV) or hydrophobicity (EAK16-II vs. EFK16-II), were tested for the complexation and in vitro delivery of ellipticine. It was found that EAK16-II and EAK16-IV were able to stabilize protonated or crystalline ellipticine depending on the peptide concentration; EFK16-II, on the other hand, could stabilize neutral ellipticine molecules and ellipticine (micro)crystals. The viability results showed that the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the states of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Self-assembling peptides have emerged as new nanobiomaterials in the areas of nanoscience and biomedical engineering. In this category are self-assembling, ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to a unique combination of amphiphilicity and ionic complementarity. These peptides can self-assemble into stable nanostructures or macroscopic membranes that can withstand conditions of high temperature, extreme pH, many digesting enzymes and denaturation agents. Moreover, they exhibit good biocompatibility with various cultured mammalian cells, and do not have detectable immune responses when introduced into animals. These properties make them ideal materials for tissue scaffolding, regenerative medicine and drug delivery. This thesis focuses on the utilization of self-assembling peptides for hydrophobic anticancer drug delivery. The hydrophobic anticancer agent ellipticine was selected as a model drug. The studies include: (i) characterization of the photophysical properties of ellipticine in different environments; (ii) study of the formation of peptide-ellipticine complexes and the release kinetics; (iii) investigation of the cellular toxicity of the complexes and ellipticine uptake; (iv) study of the peptide sequence effect on the complex formation and in vitro delivery. Prior to applying ellipticine to the peptide-based delivery system, the fundamental studies on the effect of solution conditions, especially solvent polarity and hydrogen bonding, on the fluorescence of ellipticine were carried out. Ultraviolet (UV) absorption and fluorescence emission of ellipticine were found to be solvent/environment dependent. The absorption and emission maxima shifted to higher wavelengths (red shift) with increased solvent polarity. Large Stokes’ shifts were due to intramolecular charge transfer (ICT), which was enabled by large solvent polarity and hydrogen bonding of ellipticine with the solvents. The photophysical response of ellipticine to changes in solvent polarity and hydrogen bond formation could be used to infer the location of ellipticine in a heterogeneous medium, such as liposomes and cultured cells. EAK16-II, a model self-assembling peptide, was found to be able to stabilize ellipticine in aqueous solution. The equilibration time required to form peptide-ellipticine complex suspensions was found to be peptide concentration-dependent and related to the peptide critical aggregation concentration (CAC, ~0.1 mg/mL). With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be obtained in the complexes. The release kinetics of ellipticine from the complex into egg phosphatidylcholine (EPC) vesicles (cell membrane mimics) was also affected by the peptide concentration used in the drug formulation. A higher peptide concentration resulted in a faster transfer rate, in relation to the size of the resulting complexes. Subsequent cellular studies on two cancer cell lines, A549 and MCF-7, showed that the complexes with protonated ellipticine were more effective against both cell lines, but their dilutions were not very stable. In addition, it was found that ellipticine uptake in both cell lines was very fast and through direct membrane permeation. Three peptides, EAK16-II, EAK16-IV and EFK16-II, either having a different charge distribution (EAK16-II vs. EAK16-IV) or hydrophobicity (EAK16-II vs. EFK16-II), were tested for the complexation and in vitro delivery of ellipticine. It was found that EAK16-II and EAK16-IV were able to stabilize protonated or crystalline ellipticine depending on the peptide concentration; EFK16-II, on the other hand, could stabilize neutral ellipticine molecules and ellipticine (micro)crystals. The viability results showed that the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the states of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

<p>Peptides are a fundamental building-block of living systems and play crucial roles at both functional and structural level. Therefore, they have attracted increased attention as a platform to design and engineer new self-assembled systems that span the nano-to-meso scales. The rules of peptide design and folding enable the construction of suitable building-blocks to develop soft materials for biomaterial applications. Herein we present the use of the alpha-helical secondary structure to create two distinct structural motifs, namely coiled-coils and helical bundles. These peptide components can differ in size and incorporate a host of different functional moieties, the effects of which are described through their hierarchical assembly. </p> <p>First, we describe the self-assembly of coiled coil oligomers (trimer and tetramer) of the GCN4 leucine zipper peptide. The trimeric coiled coil was modified with varying number of aromatic groups (one to three) along each helical backbone, to facilitate higher order assemblies into banded nano- to micron-sized structures, the formation of which could be controlled reversibly as a function of pH. In addition, the electrostatic and aromatic interactions of the peptide material were harnessed for non-covalent binding of small drug molecules, followed by their subsequent pH-triggered release. Furthermore, these nanostructures are compatible with MCF-7 breast cancer cells, making them suitable drug-delivery agents for chemotherapeutics. In the absence of aromatic modifications, the coiled-coil trimer assembles into higher-order nanotubes that can be harnessed for selective encapsulation of high molecular weight biomolecules. With an increase in oligomerization from three to four, along with a single aromatic group modification on each helix, the tetrameric coiled-coil mutant successfully demonstrates a metal-assisted two-tier structural assembly into microbarrels and spheres.</p> <p>Second, we present the higher-order assembly of short tetrameric and pentameric helical bundle proteins, covalently stabilized by a belt of disulfide bridges, with metal-binding ligands at each helix termini. The addition of metals like Zn(II) and Cu(II) promote the assembly of the bundles into a 3D globular matrix, which upon thermal annealing transforms into microspheres. Additionally, these microspheres also demonstrate the metal-assisted inclusion of His-tagged fluorophores. Thus, peptide-based materials can be constructed by self-assembly of alpha-helical building blocks into systems with sophisticated, diverse morphologies and dynamic chemical properties, that can be further modulated to enhance performance for medical applications. </p>

The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.

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Le cartilage a une capacité de régénération très limitée car il n'est pas vascularisé. Laréparation de ce tissu est un défi et les techniques chirurgicales actuelles sont insatisfaisantes à longterme. Le cartilage est donc un bon candidat pour l'ingénierie tissulaire. La transplantation dechondrocytes autologues (TCA) a été la première thérapie cellulaire développée en rhumatologie maiscette procédure implique une amplification des cellules qui aboutit à une perte du phénotypechondrocytaire (perte de l'expression du collagène de type II, protéine majoritaire du cartilage), auprofit d'un phénotype fibroblastique (caractérisé par l'expression du collagène de type I, retrouvé dansles tissus fibreux). La TCA conduit donc à une greffe de chondrocytes dédifférenciés produisant unfibrocartilage, dont les propriétés mécaniques sont inférieures à celles du cartilage articulaire.Aujourd'hui, les agences de santé au niveau international s'accordent pour dire que cette procédurenécessite d'être améliorée, par un meilleur contrôle du phénotype cellulaire et l'utilisation debiomatériaux pour mieux combler les lésions articulaires. Il s'agit donc de passer de la thérapiecellulaire à l'ingénierie tissulaire du cartilage.L'objectif de nos travaux a été d'évaluer la capacité d'un gel innovant de peptides autoassemblants,l'hydrogel IEIK13, à jouer le rôle de support pour des chondrocytes humains afin qu'ilsproduisent une matrice cartilage sous l'action de facteurs chondrogéniques. L'objectif visé a été lacréation d'un gel cartilage implantable par arthroscopie. Le défi a été de surmonter la dédifférenciationdes chondrocytes inhérente à leur amplification et incontournable pour augmenter le réservoircellulaire. L'amplification de chondrocytes humains a été réalisée en présence de FGF-2 et d'insuline(cocktail FI) puis leur redifférenciation a été induite en gel IEIK13 sous l'action de BMP-2, d'insuline etd'hormone T3 (cocktail BIT). C'est la combinaison sélective des deux cocktails qui permet la séquencedédifférenciation-redifférenciation. Le phénotype des chondrocytes et la nature de la matriceextracellulaire synthétisée en gel ont été évalués dans un premier temps in vitro, par des analyses dePCR en temps réel, Western-blots et d'immunohistochimie. Dans un second temps, nous avonstransplanté le gel cartilage dans des lésions articulaires de genou d'un modèle original de primate nonhumain(singe cynomolgus), un type de gros animal dont la posture et le fonctionnement desarticulations s'apparentent à l'homme. Nos études d'imagerie non invasive (telle qu'elle est pratiquéechez l'homme) et immunohistochimiques trois mois après implantation montrent une réparationsatisfaisante des lésions, en comparaison avec les lésions laissées non comblées. L'ensemble de nosrésultats montre pour la première fois que l'hydrogel IEIK13 est un biomatériau favorable pourreconstruire le cartilage et que le primate non-humain est un modèle préclinique unique pour évaluerl'efficacité de l'ingénierie tissulaire du cartilage / Cartilage is not vascularized and presents poor capacity of self-regeneration. Repairing thistissue is a challenge and current surgical techniques are not satisfactory in the long term. Cartilage isthus a good candidate for tissue engineering. Autologous chondrocyte transplantation (ACT) was thefirst cell therapy developed for cartilage repair. This procedure implies amplification of cells whichresults in chondrocyte dedifferentiation (loss of expression of type II collagen, the major protein ofcartilage and acquisition of expression of type I collagen, the major protein found in fibrous tissues).Thus, ACT results in implantation of fibroblastic cells producing fibrocartilage with biomechanicalproperties inferior to native articular cartilage. The international health agencies agree that ACT needsto be improved with better control of the chondrocyte phenotype and use of biomaterials. Therefore,cell therapy of cartilage needs to move towards tissue engineering of cartilage.The objective of our study was to evaluate the capacity of an innovative self-assemblingpeptide (IEIK13) to support cartilage matrix production by human chondrocytes. Our goal was to createa cartilage gel that can be implanted by arthroscopy. A main challenge was to meet the problem ofchondrocyte dedifferentiation induced by cell amplification necessary to increase the cellularreservoir. Amplification of human chondrocytes was performed in the presence of FGF-2 and insulin(cocktail FI), and redifferentiation was subsequently induced in IEIK13 gel with BMP-2, insulin, andtriiodothyronine T3 (cocktail BIT). The specific combination of these two cocktails alloweddedifferentiation-redifferentiation of chondrocytes. The status of the chondrocyte phenotype and thenature of the extracellular matrix secreted in gel were first assessed in vitro by real-time PCR, Westernblottingand immunhostochemistry analyses. With a view of clinical application, we then transplantedIEIK13-engineered cartilages into defects created in knees of an original model of non-human primate(cynomolgus monkey), a type of large animal whose anatomy and biomechanics mimic human. Ournon-invasive imaging analyses and our inmmunohistochemical studies performed three months afterimplantation show correct reparation of the lesions, in comparison with the defects left untreated.Altogether, our results demonstrate for the first time that IEIK13 is a suitable biomaterial for cartilagerepair and that cynomolgus monkey represents a unique preclinical model to evaluate efficiency ofcartilage tissue engineering.

Acute Lymphoblastic Leukemia (ALL) is one of the most common type of hematologic malignancy in children, characterized by an excessive proliferation of unfunctional immature lymphoblasts in the blood and the bone marrow, which leads to a range of severe blood-related complications. Given the remarkable increase in the prevalence of leukemia in the past 20 years, there has been a particular interest in the development of in vitro experimental models for cancer research. Ultra-short self-assembling peptides have shown to be a promising class of synthetic biomaterials due to their biocompatibility, tunable mechanical properties, and the possibility of controlling the scaffold composition. The objective of this study was to create a bioactive but well-defined synthetic 3D model of the bone marrow (BM) microenvironment for the simulation of ALL using biofunctionalized ultrashort self-assembling peptide scaffolds. Different bioactive motifs derived from integral extracellular matrix (ECM) constituents that are known to enhance cell-matrix adhesion, including RGDS from fibronectin, YIGSR from laminin, and GFOGER from collagen, were incorporated into the parent peptide IIZK. These peptides demonstrated to be capable of generating stable hydrogel structures composed of fibrous porous networks, each with unique nanofiber morphology and mechanical properties. All the peptide scaffolds that were investigated in this study exhibited optimal characteristics concerning the cytocompatibility of multiple BM niche cells, including human bone marrow mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), and patient derived ALL cells. The suitability of the scaffolds as drug screening platforms was evaluated, demonstrating their potential as versatile tools for the assessment of drug efficacy.

En aquesta Tesi, un nou biomaterial de disseny composat per seqüències peptídiques repetitives i amfifíliques, que per autoensamblatge forma xarxes de nanofibres (i hidrogels), AcN-RADARADARADARADA-CONH2, s´ha utilitzat com a anàleg de la matriu extracel·lular per al manteniment, proliferació i diferenciació cel·lular. Aquest pèptid s'ha funcionalititzat amb motius biològicament actius procedents de proteïnes de la matriu extracel·lular incloent laminina-1 i colàgen IV. El scaffold peptídic autoensamblant RAD16-I i els seus derivats biològicament actius s´han caracteritzat i provat utilitzant diferents sistemes cel·lulars com pot ser les cèl·lules d'aorta humanes (HAEC), hepatocits madurs i la línea progenitora de fetge (Lig-8). La proteòlisi d'aquest pèptid s'ha avaluat utilitzant tripsina com a enzim proteolític, i els fragments resultants s'han analitzat per MALDI-TOF i AFM. Així mateix, la segona generació de biomaterials basats en el RAD16-I s'ha provat tant amb HAEC com amb hepatocits madurs. Amb aquests sistemes hem demostrat que el desenvolupament d'una matriu biomiètica reforça, a la vegada que manté, les funcions específiques de cada teixit. En particular, els resultats obtinguts en diferenciació, proliferació i manteniment de la funció cel·lular utilitzant pèptids sintètics autoensamblants són comparables amb els resultats que s'obtenen utilitzant matrius biològiques (Colàgen I i Matrigel). Això indica que els nostres anàlegs de la matriu extracel·lular poden substituir als materials naturals, i suggereix l'ús d'aquests materials intel·ligents amb capacitat instructiva en aplicacions terapèutiques. Així mateix s'ha provat que l'ús d'aquests pèptids auto-ensamblants és eficient en la construcció d'un nínxol de cèl·lules mare. Hem sigut capaços de controlar la cinètica cel·lular (de simètrica a assimètrica) induint diferenciació funcional, a la vegada que es mantenia una petita proporció de cèl·lules no diferenciades. Aquests resultats indiquen clarament que hem sigue capaços d'obtenir un nínxol on cèl·lules primitives (Lig-8) es diferencien adquirint funcions d'hepatocits madurs. Hem desenvolupat una plataforma de biomaterials que es podrien utilitzar per la funcionalització amb innumerables biomolècules amb capacitat d'induir processos biològics com la diferenciació, proliferació i funció metabòlica. Aquests biomaterials, preveiem que tindran un gran impacte a l'àrea terapèutica i biología regenerativa. / En esta Tesis, un nuevo biomaterial de diseño compuesto por secuencias peptídicas repetitivas y amfifílicas que por autoensamblaje forma redes de nanofibras (e hidrogeles), AcN-RADARADARADARADA-CONH2 (RAD16-I), se ha utilizado como análogo de la matriz extracelular para el mantenimiento, proliferación y diferenciación celular. Este péptido se ha funcionalizado con motivos biológicamente activos procedentes de proteínas de la matriz extracelular incluyendo laminina-1 y colágeno IV. El scaffold peptídico autoensamblante RAD16-I y sus derivados biológicamente activos se han caracterizado y probado utilizando diferentes sistemas celulares como puede ser células endoteliales de aorta humanas (HAEC), hepatocitos maduros y la línea progenitora de hígado Lig-8. La proteólisis de este péptido se ha evaluado utilizando tripsina como enzima proteolítico, y los fragmentos resultantes se han analizado por MALDI-TOF y AFM. Asimismo, la segunda generación de biomateriales basados en el RAD16-I se ha probado tanto con HAEC como hepatocitos maduros. Con estos sistemas hemos demostrado que el desarrollo de una matriz biomimética refuerza a la vez que mantiene las funciones específicas de cada tejido. En particular, los resultados obtenidos en diferenciación, proliferación y mantenimiento de la función celular utilizando los péptidos sintéticos auto-ensamblantes son comparables con los resultados que se obtienen usando matrices biológicas (Colágeno I y Matrigel). Esto indica que nuestros análogos de la matriz extracelular pueden reemplazar a los materiales naturales, y sugiere el uso de estos materiales inteligentes con capacidad instructiva en aplicaciones terapéuticas. Asimismo, se ha probado que el uso de estos péptidos auto-ensamblantes es eficiente en la construcción de un nicho de células madre. Hemos sido capaces de controlar la cinética celular (de simétrica a asimétrica) induciendo diferenciación funcional, a la vez que se mantenía una pequeña proporción de células no diferenciadas. Estos resultados indican claramente que hemos sido capaces de obtener un nicho donde células primitivas (Lig-8) se diferencian adquiriendo funciones de hepatocitos maduros. Hemos desarrollado una plataforma de biomateriales que se podrían utilizar para la funcionalización con innumerables biomoléculas con capacidad de inducir procesos biológicos como la diferenciación, proliferación y función metabólica. Estos biomateriales preveemos que tendrán un gran impacto en el área terapéutica y biología regenerativa. / In this Thesis, a new designed biomaterial made out of short repetitive amphiphilic peptide sequence AcN-RADARADARADARADA-CONH2 (RAD16-I) that self-assembles forming nanofiber networks (hydrogel scaffold) has been used as synthetic extracellular matrix analog for cell maintenance, proliferation and differentiation. This peptide has been functionalized with biological active motifs from extracellular matrix proteins including laminin-1 and collagen IV. The prototypic self-assembling peptide scaffold RAD16-I and its biologically active derivatives have been characterized and tested using several cellular systems such as human aortic endothelial cells (HAEC), mature hepatocytes and a putative liver progenitor cell line, Lig-8. The proteolysis of the peptide RAD16-I has been evaluated using trypsin as a proteolytic enzyme and the resulting fragments have been analyzed by MALDI-TOF and AFM. Moreover the second generation of RAD16-I-based biomaterials have been tested using HAEC and mature hepatocytes. With these systems we have shown that the development of a biomimetic matrix enhances as well as maintain tissue-specific functions. In particular, the results obtained in cell differentiation, proliferation and maintenance of cell function using the synthetic self-assembling peptide matrices, are comparable with the results obtained using natural biological matrices counterparts (Collagen-I and Matrigel). This indicates that our extracellular matrix analogs can replace the use of naturally-derived materials and suggests the use of these smart biomaterials with instructive capacity for cells in therapeutics. Moreover, the use of the self-assembling peptide RAD16-I in the recreation of a stem-cell niche proved to be highly efficient. We were able to control stem-cell kinetics (from symmetric to assymetric) inducing functional differentiation while maintaining a small proportion of undifferentiated cells. This striking results clearly indicate that we were able to obtain a stem-cell niche where primitive cells (Lig-8) undergo differentiation acquiring mature hepatic functions. We have developed a biomaterial platform that can be used for functionalization with innumerable biomolecules, with capacity to induce biological processes like differentiation, control of proliferation, metabolic function, etc. These biomaterials will have a strong impact in therapeutics and regenerative biology.