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.

Self-Healing Transparent Poly(dimethyl)siloxane with Tunable Mechanical Properties: Toward Enhanced Aging Materials for Space Applications.

When exposed to the geostationary orbit, polymeric materials tend to degrade on their surface due to the appearance of cracks. Implementing the self-healing concept in polymers going to space is a new approach to enhancing the lifespan of materials that cannot be replaced once launched. In this study, the elaboration of autonomous self-healing transparent poly(dimethylsiloxane) (PDMS) materials resistant to proton particles is presented. The PDMS materials are functionalized with various compositions of urea and imine moieties, forming dynamic covalent and/or supramolecular networks. The hydrogen bonds induced by the urea ensure the formation of a supramolecular network, while the dynamic covalent imine bonds are capable of undergoing exchange reactions. Materials with a broad range of mechanical properties were obtained depending on the composition and structure of the PDMS networks. As coating applications in a spatial environment were mainly targeted, the surface properties of the polymer are essential. Thus, percentages of scratch recovery were determined by AFM. From these data, self-healing kinetics were extracted and rationalized based on the polymer structures. The obtained data were in good agreement with the relaxation times determined by rheology in stress relaxation experiments. Moreover, the accelerated aging of materials under proton irradiation, simulating a major part of the geostationary environment, revealed a strong limitation or disappearance of cracks while keeping the transparency of the PDMS. These promising results open routes to prepare new flexible autonomous polymeric materials for space applications.

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.

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.

Switchable Lipids: From Conformational Switch to Macroscopic Changes in Lipid Vesicles.

It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC, 2H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG2000) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.

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.

Hybrid polymer/lipid vesicles: Influence of polymer architecture and molar mass on line tension.

Hybrid polymer/lipid vesicles are self-assembled structures that have been the subject of an increasing number of studies in recent years. They are particularly promising tools in the development of cell membrane models because they offer the possibility to fine-tune their membrane structure by adjusting the distribution of components (presence or absence of "raft-like" lipid domains), which is of prime importance to control their membrane properties. Line tension in multiphase membranes is known to be a key parameter on membrane structuration, but remains unexplored, either experimentally or by computer modeling for hybrid polymer/lipid vesicles. In this study, we were able to measure the line tension on different budded hybrid vesicles, using a micropipette aspiration technique, and show the influence of the molar mass and the architecture of block copolymers on line tension and its consequences for membrane structuration.

Spatiotemporal Dynamic Assembly/Disassembly of Organelle-Mimics Based on Intrinsically Disordered Protein-Polymer Conjugates.

Design of reversible organelle-like microcompartments formed by liquid-liquid phase separation in cell-mimicking entities has significantly advanced the bottom-up construction of artificial eukaryotic cells. However, organizing the formation of artificial organelle architectures in a spatiotemporal manner within complex primitive compartments remains scarcely explored. In this work, thermoresponsive hybrid polypeptide-polymer conjugates are rationally engineered and synthesized, resulting from the conjugation of an intrinsically disordered synthetic protein (IDP), namely elastin-like polypeptide, and synthetic polymers (poly(ethylene glycol) and dextran) that are widely used as macromolecular crowding agents. Cell-like constructs are built using droplet-based microfluidics that are filled with such bioconjugates and an artificial cytoplasm system that is composed of specific polymers conjugated to the IDP. The distinct spatial organizations of two polypeptide-polymer conjugates and the dynamic assembly and disassembly of polypeptide-polymer coacervate droplets in response to temperature are studied in the cytomimetic protocells. Furthermore, a monoblock IDP with longer length is concurrently included with bioconjugates individually inside cytomimetic compartments. Both bioconjugates exhibit an identical surfactant-like property, compartmentalizing the monoblock IDP coacervates via temperature control. These findings lay the foundation for developing hierarchically structured synthetic cells with interior organelle-like structures which could be designed to localize in desired phase-separated subcompartments.

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 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.

Membrane reinforcement in giant hybrid polymer lipid vesicles achieved by controlling the polymer architecture.

The physical properties of membranes of hybrid polymer lipid vesicles are so far relatively unknown. Since their discovery a decade ago, many studies have aimed to show their great potential in many fields of application, but so far, few systematic studies have been carried out to decipher the relationship between the molecular characteristics of the components (molar mass, chemical nature, and architecture of the copolymer), the membrane structure and its properties. In this work, we study the association of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(dimethylsiloxane)-b-poly(ethylene oxide) (PDMS-b-PEO) diblock copolymers of different molar masses in giant hybrid vesicles and establish a complete phase diagram of the membrane structure. We also measured the mechanical properties of the giant hybrid unilamellar vesicle (GHUV) through micropipette aspiration at different lipid/polymer compositions. Thanks to a previous work using triblock PEO-b-PDMS-b-PEO copolymers, we were able to reveal the effect of the architecture of the block copolymer on membrane structure and properties. Besides, the association of diblock copolymers PDMS-b-PEO and POPC leads to the formation of hybrid vesicles with unprecedented membrane toughness.

Switchable Lipid Provides pH-Sensitive Properties to Lipid and Hybrid Polymer/Lipid Membranes.

Blending amphiphilic copolymers and lipids constitutes a novel approach to combine the advantages of polymersomes and liposomes into a new single hybrid membrane. Efforts have been made to design stimuli-responsive vesicles, in which the membrane's dynamic is modulated by specific triggers. In this investigation, we proposed the design of pH-responsive hybrid vesicles formulated with poly(dimethylsiloxane)-block-poly(ethylene oxide) backbone (PDMS36-b-PEO23) and cationic switchable lipid (CSL). The latter undergoes a pH-triggered conformational change and induces membrane destabilization. Using confocal imaging and DLS measurements, we interrogated the structural changes in CSL-doped lipid and hybrid polymer/lipid unilamellar vesicles at the micro- and nanometric scale, respectively. Both switchable giant unilamellar lipid vesicles (GUV) and hybrid polymer/lipid unilamellar vesicles (GHUV) presented dynamic morphological changes, including protrusions and fission upon acidification. At the submicron scale, scattered intensity decreased for both switchable large unilamellar vesicles (LUV) and hybrid vesicles (LHUV) under acidic pH. Finally, monitoring the fluorescence leakage of encapsulated calcein, we attested that CSL increased the permeability of GUV and GHUV in a pH-specific fashion. Altogether, these results show that switchable lipids provide a pH-sensitive behavior to hybrid polymer/lipid vesicles that could be exploited for the triggered release of drugs, cell biomimicry studies, or as bioinspired micro/nanoreactors.

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

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.

Large and Giant Unilamellar Vesicle(s) Obtained by Self-Assembly of Poly(dimethylsiloxane)-b-poly(ethylene oxide) Diblock Copolymers, Membrane Properties and Preliminary Investigation of their Ability to Form Hybrid Polymer/Lipid Vesicles.

In the emerging field of hybrid polymer/lipid vesicles, relatively few copolymers have been evaluated regarding their ability to form these structures and the resulting membrane properties have been scarcely studied. Here, we present the synthesis and self-assembly in solution of poly(dimethylsiloxane)-block-poly(ethylene oxide) diblock copolymers (PDMS-b-PEO). A library of different PDMS-b-PEO diblock copolymers was synthesized using ring-opening polymerization of hexamethylcyclotrisiloxane (D3) and further coupling with PEO chains via click chemistry. Self-assembly of the copolymers in water was studied using Dynamic Light Scattering (DLS), Static Light Scattering (SLS), Small Angle Neutron Scattering (SANS), and Cryo-Transmission Electron Microscopy (Cryo-TEM). Giant polymersomes obtained by electroformation present high toughness compared to those obtained from triblock copolymer in previous studies, for similar membrane thickness. Interestingly, these copolymers can be associated to phospholipids to form Giant Hybrid Unilamellar Vesicles (GHUV); preliminary investigations of their mechanical properties show that tough hybrid vesicles can be obtained.

Polypeptide Nanoparticles Obtained from Emulsion Polymerization of Amino Acid N-Carboxyanhydrides.

Polypeptide nanoparticles were obtained by the miniemulsion polymerization of S-(o-nitrobenzyl)-l-cysteine (NBC) N-carboxyanhydride (NCA). Through process optimization, reaction conditions were identified that allowed the polymerization of the water sensitive NCA to yield nanoparticles of about 220 nm size. Subsequent UV-irradiation of the nanoparticle emulsions caused the in situ removal of the nitrobenzyl group and particle cross-linking through disulfide bond formation accompanied by the shrinkage of the particles.

Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) and outer monolayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 µm2 s-1 at 25 °C and D = 2.3 ± 0.7 µm2 s-1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

Liposomes in Polymersomes: Multicompartment System with Temperature-Triggered Release.

Multicompartmentalization is a key feature of eukaryotic cells, allowing separation and protection of species within the membrane walls. During the last years, several methods have been reported to afford synthetic multicompartment lipidic or polymeric vesicles that mimic biological cells and that allow cascade chemical or enzymatic reactions within their lumen. We hereby report on the preparation and study of liposomes in polymersomes (LiPs) systems. We discuss on the loading and coloading of lipidic nanovesicles made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (diC15-PC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) inside the lumen of giant poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) polymersomes. These LiPs systems were characterized by confocal microscopy and UV-visible spectroscopy. We further demonstrate that we can achieve controlled sequential release of dyes from diC15-PC and DPPC liposomes at defined temperatures inside the giant PBut-b-PEO polymersomes. This controlled release could be used as a means to initiate cascade reactions on demand in confined microreactors.

Polymersome Popping by Light-Induced Osmotic Shock under Temporal, Spatial, and Spectral Control.

The light-triggered, programmable rupture of cell-sized vesicles is described, with particular emphasis on self-assembled polymersome capsules. The mechanism involves a hypotonic osmotic imbalance created by the accumulation of photogenerated species inside the lumen, which cannot be compensated owing to the low water permeability of the membrane. This simple and versatile mechanism can be adapted to a wealth of hydrosoluble molecules, which are either able to generate reactive oxygen species or undergo photocleavage. Ultimately, in a multi-compartmentalized and cell-like system, the possibility to selectively burst polymersomes with high specificity and temporal precision and to consequently deliver small encapsulated vesicles (both polymersomes and liposomes) is demonstrated.

Modulation of phase separation at the micron scale and nanoscale in giant polymer/lipid hybrid unilamellar vesicles (GHUVs).

Phase separation in giant polymer/lipid hybrid unilamellar vesicles (GHUVs) has been described over the last few years. However there is still a lack of understanding on the physical and molecular factors governing the phase separation in such systems. Among these parameters it has been suggested that in analogy to multicomponent lipid vesicles hydrophobic mismatches as well as lipid fluidity play a role. In this work, we aim to map a global picture of phase separation and domain formation in the membrane of GHUVs by using various copolymers based on poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) (PEO) with different architectures (grafted, triblock) and molar masses, combined with phospholipids in the fluid (POPC) or gel state (DPPC) at room temperature. From confocal imaging and fluorescence lifetime imaging microscopy (FLIM) techniques, the phase separation into either micro- or nano-domains within GHUVs was studied. In particular, our systematic studies demonstrate that in addition to the lipid/polymer fraction or the lipid physical state, important factors such as line tension at lipid polymer/lipid boundaries can be finely modulated by the molar mass and the architecture of the copolymer and lead to the formation of stable lipid domains with different sizes and morphologies in such GHUVs.