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.

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.

Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

An Elastin-Derived Composite Matrix for Enhanced Vascularized and Innervated Bone Tissue Reconstruction: From Material Development to Preclinical Evaluation.

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix is developed, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation leads to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, longitudinal micro-CT analysis is performed, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration.

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.

Group-Transfer Polymerization-Induced Self-Assembly (GTPISA) in Non-polar Media: An Organocatalyzed Route to Block Copolymer Nanoparticles at Room Temperature.

Polymerization-induced self-assembly (PISA) enables the synthesis at large scale of a wide variety of functional nanoparticles. However, a large number of works are related to controlled radical polymerization (CRP) methods and are generally undertaken at elevated temperatures (>50 °C). Here is the first report on methacrylate-based nanoparticles fabricated by group transfer polymerization-induced self-assembly (GTPISA) in non-polar media (n-heptane). This GTPISA process is achieved at room temperature (RT) using 1-methoxy-1-(trimethylsiloxy)-2-methylprop-1-ene (MTS) and tetrabutylammonium bis-benzoate (TBABB) as initiator and organic catalyst, respectively. Under these conditions, well-defined metal-free and colorless diblock copolymers are produced with efficient crossover from the non-polar stabilizing poly(lauryl methacrylate) (PLMA) block to the non-soluble poly(benzyl methacrylate) (PBzMA) segment. The resulting PLMA-b-PBzMA block copolymers simultaneously self-assemble into nanostructures of various sizes and morphologies. GTPISA in non-polar solvent proceeds rapidly at RT and avoids the use of sulfur or halogenated compounds or metallic catalysts associated with the implementation of CRP methods, thus expanding the potential of PISA formulations for applications in non-polar environments.

Controlling polymersome size through microfluidic-assisted self-assembly: Enabling 'ready to use' formulations for biological applications.

The self-assembly of poly(ethylene glycol)-block-poly(trimethylene carbonate) PEG-b-PTMC copolymers into vesicles, also referred as polymersomes, was evaluated by solvent displacement using microfluidic systems. Two microfluidic chips with different flow regimes (micromixer and Herringbone) were used and the impact of process conditions on vesicle formation was evaluated. As polymersomes are sensitive to osmotic variations, their preparation under conditions allowing their direct use in biological medium is of major importance. We therefore developed a solvent exchange approach from DMSO (Dimethylsulfoxide) to aqueous media with an osmolarity of 300 mOsm L-1, allowing their direct use for biological evaluation. We evidenced that the organic/aqueous solvent ratio does not impact vesicle size, but the total flow rate and copolymer concentration have been observed to influence the size of polymersomes. Finally, nanoparticles with diameters ranging from 76 nm to 224 nm were confirmed to be vesicles through the use of multi-angle light scattering in combination with cryo-TEM (Cryo-Transmission Electron Microscopy) characterization.

Control of Enzyme Reactivity in Response to Osmotic Pressure Modulation Mimicking Dynamic Assembly of Intracellular Organelles.

In response to variations in osmotic stress, in particular to hypertonicity associated with biological dysregulations, cells have developed complex mechanisms to release their excess water, thus avoiding their bursting and death. When water is expelled, cells shrink and concentrate their internal bio(macro)molecular content, inducing the formation of membraneless organelles following a liquid-liquid phase separation (LLPS) mechanism. To mimic this intrinsic property of cells, functional thermo-responsive elastin-like polypeptide (ELP) biomacromolecular conjugates are herein encapsulated into self-assembled lipid vesicles using a microfluidic system, together with polyethylene glycol (PEG) to mimic cells' interior crowded microenvironment. By inducing a hypertonic shock onto the vesicles, expelled water induces a local increase in concentration and a concomitant decrease in the cloud point temperature (Tcp ) of ELP bioconjugates that phase separate and form coacervates mimicking cellular stress-induced membraneless organelle assemblies. Horseradish peroxidase (HRP), as a model enzyme, is bioconjugated to ELPs and is locally confined in coacervates as a response to osmotic stress. This consequently increases local HRP and substrate concentrations and accelerates the kinetics of the enzymatic reaction. These results illustrate a unique way to fine-tune enzymatic reactions dynamically as a response to a physiological change in isothermal conditions.

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.

Improving aqueous solubility of paclitaxel with polysarcosine-b-poly(γ-benzyl glutamate) nanoparticles.

New stealth amphiphilic copolymers based on polysarcosine (PSar) rather than poly(ethylene glycol) (PEG) have gained more attention for their use as excipients in nanomedicine. In this study, several polysarcosine-b-poly(γ-benzyl glutamate) (PSar-b-PGluOBn) block copolymers were synthesized by ring opening polymerization (ROP) of the respective N-carboxyanhydrides (NCAs) and were characterized by Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR) and size-exclusion chromatography (SEC). Copolymers had different PGluOBn block configuration (racemic L/D, pure L or pure D), degrees of polymerization of PSar between 28 and 76 and PGluOBn between 9 and 93, molar masses (Mn) between 5.0 and 24.6 kg.mol-1 and dispersities (D) lower than 1.4. Nanoparticles of PSar-b-PGluOBn loaded with paclitaxel (PTX), a hydrophobic anti-cancer drug, were obtained by nanoprecipitation. Their hydrodynamic diameter (Dh) ranged from 27 to 118 nm with polydispersity indexes (PDI) between 0.01 and 0.20, as determined by dynamic light scattering (DLS). Their morphology was more spherical for copolymers with a racemic L/D PGluOBn block configuration synthesized at 5 °C. PTX loading efficiency was between 63 and 92 % and loading contents between 7 and 15 %. Using PSar-b-PGluOBn copolymers as excipients, PTX apparent water-solubility was significantly improved by a factor up to 6600 to 660 µg.mL-1.

Emerging opportunities in bioconjugates of Elastin-like polypeptides with synthetic or natural polymers.

Nature is an everlasting source of inspiration for chemical and polymer scientists seeking to develop ever more innovative materials with greater performances. Natural structural proteins are particularly scrutinized to design biomimetic materials. Often characterized by repeat peptide sequences, that together interact by inter- and intramolecular interactions and form a 3D skeleton, they contribute to the mechanical properties of individual cells, tissues, organs, and whole organisms. (Numata, K. Polymer Journal 2020, 52, 1043-1056) Among them elastin, and its main repeat sequences, have been a source of intense studies for more than 50 years resulting in the specific research field dedicated to elastin-like polypeptides (ELPs). These are currently widely investigated in different applications, namely protein purification, tissue engineering, and drug delivery, and some technologies based on ELPs are currently explored by several start-up companies. In the present review, we have summarized pioneering contributions on ELPs, progress made in their genetic engineering, and understanding of their thermal behavior and self-assembly properties. Considered as intrinsically disordered protein polymers, we have finally focused on the works where ELPs have been conjugated to other synthetic macromolecules as covalent hybrid, statistical, graft, or block copolymers, highlighting the huge opportunities that have still not been explored so far.

Efficient Gene Delivery of Tailored Amphiphilic Polypeptides by Polyplex Surfing.

Within this study, an amphiphilic and potentially biodegradable polypeptide library based on poly[(4-aminobutyl)-l-glutamine-stat-hexyl-l-glutamine] [P(AB-l-Gln-stat-Hex-l-Gln)] was investigated for gene delivery. The influence of varying proportions of aliphatic and cationic side chains affecting the physicochemical properties of the polypeptides on transfection efficiency was investigated. A composition of 40 mol% Hex-l-Gln and 60 mol % AB-l-Gln (P3) was identified as best performer over polypeptides with higher proportions of protonatable monomers. Detailed studies of the transfection mechanism revealed the strongest interaction of P3 with cell membranes, promoting efficient endocytic cell uptake and high endosomal release. Spectrally, time-, and z-resolved fluorescence microscopy further revealed the crucial role of filopodia surfing in polyplex-cell interaction and particle internalization in lamellipodia regions, followed by rapid particle transport into cells. This study demonstrates the great potential of polypeptides for gene delivery. The amphiphilic character improves performance over cationic homopolypeptides, and the potential biodegradability is advantageous toward other synthetic polymeric delivery systems.

Memory Effect in Thermoresponsive Proline-based Polymers.

We report that synthetic polymers consisting of L-proline monomer units exhibit temperature-driven aggregation in water with unprecedented hysteresis. This protein-like behavior is robust and governed by the chirality of the proline units. It paves the way to new processes, driven by either temperature or ionic strength changes, such as a simple "with memory" thermometer.

Mannose-based surfactant as biofunctional nanoemulsion stabilizer.

The development and implementation of new amphiphiles based on natural resources rather than petrochemical precursors is an essential requirement due to their feedstock depletion and adverse environmental impacts. In addition, the use of bio-based surfactants can provide unique characteristics and improve the properties and versatility of the colloidal systems in which they are applied, such as emulsions. Here, the emulsification properties of a synthesized biocompatible mannose-based surfactant were investigated. Its behavior was evaluated in the presence of four different natural oils (castor, sunflower, olive and soybean) as well as two different aqueous phases (pure water and phosphate-buffered saline). The results highlighted its interest as surfactant in O/W nanoemulsions for all tested oil and aqueous phases, using a low-energy preparation protocol and relatively low surfactant concentrations. Furthermore, the mannose groups present on the polar head of the surfactant and adsorbed on the surface of the emulsion droplets were shown to retain their native biological properties. The specific mannose-concanavalin A binding was observed in vitro by the designed nanoemulsions, revealing the biorecognition properties of the surfactant and its potential applicability as a nanocarrier.

Aqueous synthesis and self-assembly of bioactive and thermo-responsive HA-b-ELP bioconjugates.

The design of synthetic (bio)macromolecules that combine biocompatibility, self-assembly and bioactivity properties at the molecular level is an intense field of research for biomedical applications such as (nano)medicine. In this contribution, we have designed and synthesized a library of bioactive and thermo-responsive bioconjugates from elastin-like polypeptides (ELPs) and hyaluronic acid (HA) in order to access bioactive self-assembled nanoparticles. These were prepared by a simple synthetic and purification strategy, compatible with the requirements for biological applications and industrial scale-up. A series of 9 HA-b-ELP bioconjugates with different compositions and block lengths was synthesized under aqueous conditions by strain-promoted azide-alkyne cycloaddition (SPAAC), avoiding the use of catalysts, co-reactants and organic solvents, and isolated by a simple centrifugation step. An extensive physico-chemical study was then performed on the whole library of bioconjugates in an attempt to establish structure-property relationships. In particular, the determination of the critical conditions for thermally driven self-assembly was carried out upon temperature (CMT) and concentration (CMC) gradients, leading to a phase diagram for each of these bioconjugates. These parameters and the size of nanoparticles were found to depend on the chemical composition of the bioconjugates, namely on the respective size of individual blocks. Understanding the mechanism underlying this dependency is a real asset for designing more effective experiments: with key criteria defined (e.g. concentration, temperature, salinity, and biological target), the composition of the best candidates can be rationalized. In particular, four of the bioconjugates (HA4.6k-ELPn80 or n100 and HA24k-ELPn80 or n100) were found to self-assemble into well-defined spherical core-shell nanoparticles, with a negative surface charge due to the HA block exposed at the surface, a hydrodynamic diameter between 40 and 200 nm under physiological conditions and a good stability over time at 37 °C. We therefore propose here a versatile and simple design of smart, controllable, and bioactive nanoparticles that present different behaviors depending on the diblocks' composition.

Design and Self-Assembly of Sugar-Based Amphiphiles: Spherical to Cylindrical Micelles.

Sugar-based amphiphiles are a relevant natural alternative to synthetic ones due to their biodegradable properties. An understanding of their structure-assembly relationship is needed to allow the concrete synthesis of suitable derivatives. Here, four different mannose-derivative surfactants are characterized by pendant drop, dynamic light scattering, small-angle X-ray scattering, cryotransmission electron microscopy, and molecular dynamics techniques in aqueous media. Measurements denote how the polysaccharide average degree of polymerization (DP¯) and the addition of a hydroxyl group to the hydrophobic tail, and thus the presence of a second hydrophilic moiety, affect their self-assembly. A variation in the DP¯ of the amphiphile has no effect in the critical micelle concentration in contrast to a change in the hydrophobic molecular region. Moreover, high-DP¯ amphiphiles self-assemble into spherical micelles irrespective of the hydroxyl group presence. Low-DP¯ amphiphiles with only one hydrophilic moiety form cylindrical micelles, while the addition of a hydroxyl group to the tail leads to a spherical shape.