Thermoresponsive Core-cross-linked Nanoparticles from HA-b-ELP Diblock Copolymers.

Stabilization against the dilution-dependent disassembly of self-assembled nanoparticles is a requirement for in vivo application. Herein, we propose a simple and biocompatible cross-linking reaction for the stabilization of a series of nanoparticles formed by the self-assembly of amphiphilic HA-b-ELP block copolymers, through the alkylation of methionine residues from the ELP block with diglycidyl ether compounds. The core-cross-linked nanoparticles retain their colloidal properties, with a spherical core-shell morphology, while maintaining thermoresponsive behavior. As such, instead of a reversible disassembly when non-cross-linked, a reversible swelling of nanoparticles' core and increase of hydrodynamic diameter are observed with lowering of the temperature.

Protocells Featuring Membrane-Bound and Dynamic Membraneless Organelles.

Living cells, especially eukaryotic ones, use multicompartmentalization to regulate intra- and extracellular activities, featuring membrane-bound and membraneless organelles. These structures govern numerous biological and chemical processes spatially and temporally. Synthetic cell models, primarily utilizing lipidic and polymeric vesicles, have been developed to carry out cascade reactions within their compartments. However, these reconstructions often segregate membrane-bound and membraneless organelles, neglecting their collaborative role in cellular regulation. To address this, we propose a structural design incorporating microfluidic-produced liposomes housing synthetic membrane-bound organelles made from self-assembled poly(ethylene glycol)-block-poly(trimethylene carbonate) nanovesicles and synthetic membraneless organelles formed via temperature-sensitive elastin-like polypeptide phase separation. This architecture mirrors natural cellular organization, facilitating a detailed examination of the interactions for a comprehensive understanding of cellular dynamics.

Polypeptide- and Protein-Based Conjugate Nanoparticles via Aqueous Ring-Opening Polymerization-Induced Self-Assembly (ROPISA).

Protein-polymer conjugates and polymeric nanomaterials hold great promise in many applications including biomaterials, medicine, or nanoelectronics. In this work, the first polymerization-induced self-assembly (PISA) approach performed in aqueous medium enabling protein-polymer conjugates and nanoparticles entirely composed of amino acids is presented by using ring-opening polymerization (ROP). It is indeed shown that aqueous ring-opening polymerization-induced self-assembly (ROPISA) can be used with protein or peptidic macroinitiators without prior chemical modification and afford the simple preparation of nanomaterials with protein-like property, for example, to implement biomimetic thermoresponsivity in drug delivery.

Transcription-Factor-Induced Aggregation of Biomimetic Oligonucleotide-b-Protein Micelles.

Polymeric micelles and especially those based on natural diblocks are of particular interest due to their advantageous properties in terms of molecular recognition, biocompatibility, and biodegradability. We herein report a facile and straightforward synthesis of thermoresponsive elastin-like polypeptide (ELP) and oligonucleotide (ON) diblock bioconjugates, ON-b-ELP, through copper-catalyzed azide-alkyne cycloaddition. The resulting thermosensitive diblock copolymer self-assembles above its critical micelle temperature (CMT ∼30 °C) to form colloidally stable micelles of ∼50 nm diameter. The ON-b-ELP micelles hybridize with an ON complementary strand and maintain their size and stability. Next, we describe the capacity of these micelles to bind proteins, creating more complex structures using the classic biotin-streptavidin pairing and the specific recognition between a transcription factor protein and the ON strand. In both instances, the micelles are intact, form larger structures, and retain their sensitivity to temperature.

Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Photooxidation Responsive Elastin-Like Polypeptide Conjugates for Photodynamic Therapy Application.

Stimuli-responsive recombinant elastin-like polypeptides (ELPs) are artificial protein polymers derived from the hydrophobic domain of tropoelastin that have attracted significant interest for drug delivery and tissue engineering applications. In the present study, we have conjugated a photosensitizer (PS) to a hydrophobic methionine-containing ELP scaffold, which upon reaction with singlet oxygen (1O2) is transformed into a hydrophilic sulfoxide derivative facilitating the disassembly of photosensitizer-delivery particles during the photodynamic therapy (PDT) process. A peripherally substituted carboxy-Zn(II)-phthalocyanine derivative (TT1) bearing a carboxyl group directly linked to the Pc-ring, and presenting an absorption maximum around 680 nm, was selected as PS which simultaneously acted as a photooxidation catalyst. A TT1-ELP[M1V3-40] conjugate was prepared from ELP[M1V3-40] modified with an alkyne group at the N-terminal chain end, and from TT1-amide-C3-azide by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. This innovative model photooxidation sensitive PS delivery technology offers promising attributes in terms of temperature-controlled particle formation and oxidation-triggered release, narrow molar mass distribution, reproducibility, scalability, non-immunogenicity, biocompatibility, and biodegradability for pharmaceutical applications in an effort to improve the clinical effectiveness of PDT treatments.

Elastin-like Polypeptide-Based Bioink: A Promising Alternative for 3D Bioprinting.

Three-dimensional (3D) bioprinting offers a great alternative to traditional techniques in tissue reconstruction, based on seeding cells manually into a scaffold, to better reproduce organs' complexity. When a suitable bioink is engineered with appropriate physicochemical properties, such a process can advantageously provide a spatial control of the patterning that improves tissue reconstruction. The design of an adequate bioink must fulfill a long list of criteria including biocompatibility, printability, and stability. In this context, we have developed a bioink containing a precisely controlled recombinant biopolymer, namely, elastin-like polypeptide (ELP). This material was further chemoselectively modified with cross-linkable moieties to provide a 3D network through photopolymerization. ELP chains were additionally either functionalized with a peptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS) or combined with collagen I to enable cell adhesion. Our ELP-based bioinks were found to be printable, while providing excellent mechanical properties such as stiffness and elasticity in their cross-linked form. Besides, they were demonstrated to be biocompatible, showing viability and adhesion of dermal normal human fibroblasts (NHF). Expressions of specific extracellular matrix (ECM) protein markers as pro-collagen I, elastin, fibrillin, and fibronectin were revealed within the 3D network containing cells after only 18 days of culture, showing the great potential of ELP-based bioinks for tissue engineering.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Thermosensitive Vesicles from Chemically Encoded Lipid-Grafted Elastin-like Polypeptides.

Biomimetic design to afford smart functional biomaterials with exquisite properties represents synthetic challenges and provides unique perspectives. In this context, elastin-like polypeptides (ELPs) recently became highly attractive building blocks in the development of lipoprotein-based membranes. In addition to the bioengineered post-translational modifications of genetically encoded recombinant ELPs developed so far, we report here a simple and versatile method to design biohybrid brush-like lipid-grafted-ELPs using chemical post-modification reactions. We have explored a combination of methionine alkylation and click chemistry to create a new class of hybrid lipoprotein mimics. Our design allowed the formation of biomimetic vesicles with controlled permeability, correlated to the temperature-responsiveness of ELPs.

Design of Polysaccharide-b-Elastin-Like Polypeptide Bioconjugates and Their Thermoresponsive Self-Assembly.

The advantageous biological properties of polysaccharides and precise stimuli-responsiveness of elastin-like polypeptides (ELPs) are of great interest for the design of polysaccharide- and polypeptide-based amphiphilic block copolymers for biomedical applications. Herein, we report the synthesis and characterization of a series of polysaccharide-block-ELP copolymers, containing two biocompatible and biodegradable blocks coupled via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The resulting bioconjugates are capable of self-assembling into well-defined nanoparticles in aqueous solution upon raising the solution temperature above a specific transition temperature (Tt)-a characteristic of the ELP moiety. To the best of our knowledge, this is the first study where polysaccharides were combined with a stimuli-responsive ELP for the preparation of thermosensitive self-assemblies, providing insight into novel pathways for designing bioinspired stimuli-responsive self-assemblies for biomedical applications.

Dynamic Spatial Formation and Distribution of Intrinsically Disordered Protein Droplets in Macromolecularly Crowded Protocells.

Elastin-like polypeptides (ELPs) have been proposed as a simple model of intrinsically disordered proteins (IDPs) which can form membraneless organelles by liquid-liquid phase separation (LLPS) in cells. Herein, the behavior of fluorescently labeled ELP is studied in cytomimetic aqueous two-phase system (ATPS) encapsulated protocells that are formed using microfluidics, which enabled confinement, changes in temperature, and statistical analysis. The spatial organization of ELP could be observed in the ATPS. Furthermore, changes in temperature triggered the dynamic formation and distribution of ELP-rich droplets within the ATPS, resulting from changes in conformation. Proteins were encapsulated along with ELP in the synthetic protocells and distinct partitioning properties of these proteins and ELP in the ATPS were observed. Therefore, the ability of ELP to coacervate with temperature can be maintained inside a cell-mimicking system.

Hyaluronic-Acid-Presenting Self-Assembled Nanoparticles Transform a Hyaluronidase HYAL1 Substrate into an Efficient and Selective Inhibitor.

In this study, an original method of macromolecular design was used to develop a hyaluronidase-1 (HYAL1) inhibitor from its principal substrate, hyaluronic acid (HA). HA-based nanoparticles (HA-NP) were obtained by copolymer self-assembly and their effects on HYAL1 activity were investigated by combining different analytical tools. Compared to HA, HA-NP exhibited an enhanced stability against HYAL1 degradation while maintaining its interaction with the HA receptors CD44 and aggrecan. HA-NP displayed a strong and selective inhibition of HYAL1 activity and retarded the hydrolysis of higher-molar-mass HA in solution. A co-nanoprecipitation process was used to formulate a range of hybrid nanoparticle samples, which demonstrated the specificity and efficiency of HA-NP in HYAL1 inhibition.

Aqueous Ring-Opening Polymerization-Induced Self-Assembly (ROPISA) of N-Carboxyanhydrides.

Reported here is the first aqueous ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs) using α-amino-poly(ethylene oxide) as a macroinitiator to protect the NCA monomers from hydrolysis through spontaneous in situ self-assembly (ISA). This ROPISA process affords well-defined amphiphilic diblock copolymers that simultaneously form original needle-like nanoparticles.

Self-Assembly of PEG-b-PTMC Copolymers: Micelles and Polymersomes Size Control.

Poly(ethylene glycol)45-b-poly(trimethylene carbonate)n PEG45-b-PTMCn diblock copolymers were synthesized with five different PTMC degrees of polymerization (n = 38, 96, 144, 170, 332) and their self-assembly properties in water were studied using a manual nanoprecipitation procedure. We confirmed that the copolymer's hydrophilic weight fraction (fPEG) is controlling nanoparticles morphology. We determined that the PEG45-b-PTMC96 with fPEG ≈ 17% is the optimal hydrophilic fraction for the stabilization of well-defined unilamellar vesicles with a membrane thickness of δ ≈ 14.6 nm. Maintaining this fraction constant and modulating the overall molar mass of the block copolymers allowed the establishment of a power law of [Formula: see text] which provides a robust correlation between the molar mass of PTMC and vesicles' membrane thickness. Finally, we proved that controlling nanoprecipitation's conditions by microfluidics allowed fine-tuning and control of the nanoparticles size and polydispersity index while maintaining their shape with a perfect batch-to-batch reproducibility.

Self-Assembly of Stimuli-Responsive Biohybrid Synthetic- b-Recombinant Block Copolypeptides.

The synthesis and original thermoresponsive behavior of hybrid diblock copolypeptides composed of synthetic and recombinant polypeptides are herein reported. A thermoresponsive recombinant elastin-like polypeptide was used as a macroinitiator to synthesize a range of poly( l-glutamic acid)- block-elastin-like polypeptide (PGlu- b-ELP) diblock copolypeptides with variable PGlu block lengths. Their temperature-triggered self-assembly in water and in phosphate-buffered saline (PBS) was investigated at the macroscopic scale using complementary techniques such as turbidimetry, dynamic and static light scattering, small-angle neutron scattering, and at the molecular scale by 1H NMR and circular dichroism (CD). In deionized water, PGlu- b-ELP copolypeptides showed one transition from free soluble chains below the transition temperature ( Tt) of the ELP block to macroscopic aggregates above the Tt. In contrast, in PBS, four successive regimes were observed upon increasing temperature: below the Tt, copolypeptides were soluble, above the Tt, large aggregates appeared and fell apart into discrete and defined spherical nanoparticles at a temperature named critical micellization temperature (CMT), before finally reaching an equilibrium. During the last regime, neutron scattering experiments revealed that the micelle-like structures underwent a densification step and expelled water from their core. In addition, 1H NMR and CD experiments revealed, in deionized water, the formation of type II ß-turns into the ELP block upon temperature increase. These ß-turns are known to participate in the intrinsic thermoresponsive behavior of the ELPs. In contrast, in PBS, circular dichroism measurements showed an attenuation of folded structure during the self-assembly phase, leading to less cohesive aggregates able to reorganize into nanoparticles at the CMT.

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains.

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation.

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.

Characterisation of hydration and nanophase separation during the temperature response in hydrophobic/hydrophilic elastin-like polypeptide (ELP) diblock copolymers.

To understand the complex nanoscale dehydration process during the lower critical solution temperature (LCST) based inverse phase transition of a class of thermoresponsive biopolymers, diblock elastin-like polypeptides (ELPs) were investigated by spin probing continuous wave electron paramagnetic resonance (CW EPR) spectroscopy. The diblock copolymers composed of a hydrophobic block and a hydrophilic block showed different mechanisms of a temperature-driven phase transition. While the phase transition temperature is a function of the hydrophobic mass fraction of the diblock ELPs, the hydrophilic block length determines the molecular structure of the polymer aggregates formed above the transition temperature. When the weight ratio of hydrophilic block length to hydrophobic block length is greater than or equal to 0.3, the polymer aggregates consist of a hydrophobic core and a hydrophilic corona. The interface of these two regions become permeable at temperatures above the transition temperature. In case of smaller ratios, the aggregating hydrophobic parts of the polymer enclose the hydrated hydrophilic blocks, that are too small to form a hydrophilic corona, leading to bigger and less dense aggregates of higher polarity.

Tuning Sizes, Morphologies, and Magnetic Properties of Monocore Versus Multicore Iron Oxide Nanoparticles through the Controlled Addition of Water in the Polyol Synthesis.

The polyol route is a versatile and up-scalable method to produce large batches of iron oxide nanoparticles with well-defined structures and magnetic properties. Importance of parameters such as temperature and reaction time, heating profile, nature of the polyol solvent or organometallic precursors on nanostructure and properties has already been described in the literature. Yet, the crucial role of water in the forced hydrolysis pathway has never been reported, despite its mandatory presence for nanoparticle production. This communication investigates the influence of the water amount and temperature at which it is injected in the reflux system for either a pure polyol solvent system or a mixture with poly(hydroxy)amine. Distinct morphologies of nanoparticles were thereby obtained, from ultra-ultra-small smooth spheres down to 4 nm in diameter to larger ones up to 37 nm. Well-defined multicore assemblies with narrow grain size dispersity termed nanoflowers were also synthesized. A diverse and large library of samples was obtained by manipulating the nature of solvents and the amount of added water while keeping all other parameters constant. The different morphologies lead to magnetic nanoparticles suitable for important biomedical applications such as magnetic hyperthermia, magnetic resonance imaging (MRI) contrast agent, or both.

Design and self-assembly of PBLG-b-ELP hybrid diblock copolymers based on synthetic and elastin-like polypeptides.

The precision synthesis and self-assembly of amphiphilic copolypeptides containing a recombinant elastin-like polypeptide (ELP) block used as a macroinitiator for the ring opening polymerization (ROP) of γ-benzyl-l-glutamate (γ-BLG NCA) are herein presented. The molecular weight of the resulting PBLG-b-ELP block copolypeptides was precisely controlled without the use of complex initiator or demanding experimental setup. Diblock copolypeptides were obtained with an excellent control of the polymerization highlighted by the dispersity below 1.04. These amphiphilic hybrid synthetic/recombinant copolypeptides were self-assembled in water and the nanoparticles obtained were characterized by a combination of dynamic light scattering and electron microscopy. A variety of morphologies, namely polymersomes, inter-connected worm-like micelles and spherical micelles, were evidenced depending on the hydrophilic ratio of the diblocks as well as the self-assembly procedure.