Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.Nanomedicine: an integrative approac

Engineering of Thermoresponsive Diblock Elastin-like Polypeptides

La thèse présentée porte sur l’ingénierie de diblocs de polypeptides à base de motifs élastine (ELPs) susceptibles de s’auto-assembler sous forme de nanoparticules après modification chimique de certains résidus. La stratégie inclue le développement de diblocs d’ELPs composés d’un bloc hydrophobe comportant l’isoleucine en position hôte, fusionné à l’extrémité N-terminale avec un bloc contenant des résidus méthionine chimiosélectivement modifiables. Des modifications du groupement thioéther permettent en effet l’hydrophilisation du segment ELP correspondant ainsi que l’introduction de groupements réactifs. Une première génération d’ELPs diblocs a été développée par génie génétique, production recombinante chez Escherichia coli et caractérisée. Le résidu cystéine à l’extrémité C-terminale a été modifié pour contrôler la monodispersité et introduire des fluorochromes, tandis que les résidus méthionine ont été modifiés pour changer l’équilibre hydrophile/hydrophobe et introduire des groupements réactifs. L’auto-assemblage des diblocs non-modifiés et post-modifiés a été étudiée par des mesures de turbidité et diffusion dynamique de la lumière. Ces méthodes ont mis en évidence une transition thermale de chaînes solubles en agrégats de taille micronique. Pour permettre la formation particules de taille nanométrique, une deuxième génération d’ELPs diblocs a été conçue grâce à l’application d’un modèle empirique. La deuxième génération d’ELPs diblocs forme effectivement des nanoparticules après modification chimiosélective des résidus méthionines. Ces structures pourront potentiellement contribuer au développement de nanoformulations à base d’ELPs.The present work focuses on the engineering of diblock elastin-like polypeptides (ELPs) that thermally assemble into nanostructures after the application of chemical modifications. The strategy involved the development of diblock ELPs composed of a hydrophobic isoleucine-containing block fused at its N-terminal end to a block containing residues amenable to chemoselective modifications, namely methionine. This particular residue was employed because the orthogonal modification of its thioether group allows for the change of the hydrophilic/lipophilic balance of the diblock ELP and the possible simultaneous grafting of functional ligands. A first generation of diblock ELPs was therefore designed by means of molecular clonings, produced in E. coli, and characterized by chemical methods to further monitor post-modifications. The chemical modifications were applied at the C-terminal cysteine to control the system monodispersity and introduce fluorescent probes, and also at methionine in order to change the hydrophilic/lipophilic balance and introduce reactive groups. The self-assembly of the non-modified and post-modified ELPs was monitored by means of turbidimetry, nanoparticle tracking analysis and dynamic light scattering, which showed that these sequences possessed a transition from monomers to aggregates. To access nanoparticle formation, a second generation of diblock ELPs was developed, the design of which was based on theoretical modeling. The second generation diblocks self-assembled into nanoparticles by means of methionine post-modifications. It is expected that these sequences will contribute to the development of diblock ELP-based nano-formulations

Design de Polypeptides Thermosensibles à Base de Motifs Élastine

The present work focuses on the engineering of diblock elastin-like polypeptides (ELPs) that thermally assemble into nanostructures after the application of chemical modifications. The strategy involved the development of diblock ELPs composed of a hydrophobic isoleucine-containing block fused at its N-terminal end to a block containing residues amenable to chemoselective modifications, namely methionine. This particular residue was employed because the orthogonal modification of its thioether group allows for the change of the hydrophilic/lipophilic balance of the diblock ELP and the possible simultaneous grafting of functional ligands. A first generation of diblock ELPs was therefore designed by means of molecular clonings, produced in E. coli, and characterized by chemical methods to further monitor post-modifications. The chemical modifications were applied at the C-terminal cysteine to control the system monodispersity and introduce fluorescent probes, and also at methionine in order to change the hydrophilic/lipophilic balance and introduce reactive groups. The self-assembly of the non-modified and post-modified ELPs was monitored by means of turbidimetry, nanoparticle tracking analysis and dynamic light scattering, which showed that these sequences possessed a transition from monomers to aggregates. To access nanoparticle formation, a second generation of diblock ELPs was developed, the design of which was based on theoretical modeling. The second generation diblocks self-assembled into nanoparticles by means of methionine post-modifications. It is expected that these sequences will contribute to the development of diblock ELP-based nano-formulations.La thèse présentée porte sur l’ingénierie de diblocs de polypeptides à base de motifs élastine (ELPs) susceptibles de s’auto-assembler sous forme de nanoparticules après modification chimique de certains résidus. La stratégie inclue le développement de diblocs d’ELPs composés d’un bloc hydrophobe comportant l’isoleucine en position hôte, fusionné à l’extrémité N-terminale avec un bloc contenant des résidus méthionine chimiosélectivement modifiables. Des modifications du groupement thioéther permettent en effet l’hydrophilisation du segment ELP correspondant ainsi que l’introduction de groupements réactifs. Une première génération d’ELPs diblocs a été développée par génie génétique, production recombinante chez Escherichia coli et caractérisée. Le résidu cystéine à l’extrémité C-terminale a été modifié pour contrôler la monodispersité et introduire des fluorochromes, tandis que les résidus méthionine ont été modifiés pour changer l’équilibre hydrophile/hydrophobe et introduire des groupements réactifs. L’auto-assemblage des diblocs non-modifiés et post-modifiés a été étudiée par des mesures de turbidité et diffusion dynamique de la lumière. Ces méthodes ont mis en évidence une transition thermale de chaînes solubles en agrégats de taille micronique. Pour permettre la formation particules de taille nanométrique, une deuxième génération d’ELPs diblocs a été conçue grâce à l’application d’un modèle empirique. La deuxième génération d’ELPs diblocs forme effectivement des nanoparticules après modification chimiosélective des résidus méthionines. Ces structures pourront potentiellement contribuer au développement de nanoformulations à base d’ELPs

Peptide-Based Electrospun Fibers: Current Status and Emerging Developments

Electrospinning is a well-known, straightforward, and versatile technique, widely used for the preparation of fibers by electrifying a polymer solution. However, a high molecular weight is not essential for obtaining uniform electrospun fibers; in fact, the primary criterion to succeed is the presence of sufficient intermolecular interactions, which function similar to chain entanglements. Some small molecules able to self-assemble have been electrospun from solution into fibers and, among them, peptides containing both natural and non-natural amino acids are of particular relevance. Nowadays, the use of peptides for this purpose is at an early stage, but it is gaining more and more interest, and we are now witnessing the transition from basic research towards applications. Considering the novelty in the relevant processing, the aim of this review is to analyze the state of the art from the early 2000s on. Moreover, advantages and drawbacks in using peptides as the main or sole component for generating electrospun nanofibers will be discussed. Characterization techniques that are specifically targeted to the produced peptide fibers are presented

A switchable self-assembling and disassembling chiral system based on a porphyrin-substituted phenylalanine-phenylalanine motif

Artificial light-harvesting systems have until now not been able to self-assemble into structures with a large photon capture cross-section that upon a stimulus reversibly can switch into an inactive state. Here we describe a simple and robust FLFL-dipeptide construct to which a meso-tetraphenylporphyrin has been appended and which self-assembles to fibrils, platelets or nanospheres depending on the solvent composition. The fibrils, functioning as quenched antennas, give intense excitonic couplets in the electronic circular dichroism spectra which are mirror imaged if the unnatural FDFD-analogue is used. By slightly increasing the solvent polarity, these light-harvesting fibres disassemble to spherical structures with silent electronic circular dichroism spectra but which fluoresce. Upon further dilution with the nonpolar solvent, the intense Cotton effects are recovered, thus proving a reversible switching. A single crystal X-ray structure shows a head-to-head arrangement of porphyrins that explains both their excitonic coupling and quenched fluorescence.Funding Agencies|European Commission [229927]; University of Crete; PHOTOPEPMAT, ARISTEIA II Action, action Education and Lifelong Learning [3941]; European Union (the European Social Fund); AngioMatTrain, Marie Curie Industry Initial Training Network (ITN), call FP7-PEOPLE ITN [317304]; SERC (Swedish e-Science Research Center); DFG-Center for Functional Nanostructures (CFN); Helmholtz STN programme; FP7 Future and Emerging Technologies for Energy Efficiency project PEPDIODE [256672]; CNRS; Aix-Marseille University through the A*MIDEX project EtNA; Region Provence-Alpes Cote dAzur through project NanoMosaique; Region Provence-Alpes Cote dAzur through project CaliGraph</p

Nanoparticles based on natural, engineered or synthetic proteins and polypeptides for drug delivery applications

Medicine formulations at the nanoscale, referred to as nanomedicines, have managed to overcome key challenges encountered during the development of new medical treatments and entered clinical practice, but considerable improvement in terms of local efficacy and reduced toxicity still need to be achieved. Currently, the fourth-generation of nanomedicines is being developed, employing biocompatible nanocarriers that are targeted, multifunctional, and stimuli-responsive. Proteins and polypeptides can fit the standards of an efficient nanovector because of their biodegradability, intrinsic bioactivity, chemical reactivity, stimuli-responsiveness, and ability to participate in complex supramolecular assemblies. These biomacromolecules can be obtained from natural resources, produced in heterologous hosts, or chemically synthesized, allowing for different designs to access suitable carriers for a variety of drugs. To enhance targeting or therapeutic functionality, additional chemical modifications can be applied. This review demonstrates the potential of polypeptide and protein materials for the design of drug delivery nanocarriers with a special focus on their preclinical evaluation in vitro and in vivo.Développement de polymersomes avec possibilité de suivi par imagerie et activation à distance pour la libération de composés d’intérêt thérapeutique dans des tissus profondsDéveloppement de squelettes polypeptidiques recombinants pour la synthèse de glycoconjugués multivalents parfaitement définisNanomedicine: an integrative approac