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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nano-encapsulation of plitidepsin: in vivo pharmacokinetics, biodistribution, and efficacy in a renal xenograft tumor model.

PURPOSE: Plitidepsin is an antineoplasic currently in clinical evaluation in a phase III trial in multiple myeloma (ADMYRE). Presently, the hydrophobic drug plitidepsin is formulated using Cremophor®, an adjuvant associated with unwanted hypersensitivity reactions. In search of alternatives, we developed and tested two nanoparticle-based formulations of plitidepsin, aiming to modify/improve drug biodistribution and efficacy. METHODS: Using nanoprecipitation, plitidepsin was loaded in polymer nanoparticles made of amphiphilic block copolymers (i.e. PEG-b-PBLG or PTMC-b-PGA). The pharmacokinetics, biodistribution and therapeutic efficacy was assessed using a xenograft renal cancer mouse model (MRI-H-121 xenograft) upon administration of the different plitidepsin formulations at maximum tolerated multiple doses (0.20 and 0.25 mg/kg for Cremophor® and copolymer formulations, respectively). RESULTS: High plitidepsin loading efficiencies were obtained for both copolymer formulations. Considering pharmacokinetics, PEG-b-PBLG formulation showed lower plasma clearance, associated with higher AUC and Cmax than Cremophor® or PTMC-b-PGA formulations. Additionally, the PEG-b-PBLG formulation presented lower liver and kidney accumulation compared with the other two formulations, associated with an equivalent tumor distribution. Regarding the anticancer activity, all formulations elicited similar efficacy profiles, as compared to the Cremophor® formulation, successfully reducing tumor growth rate. CONCLUSIONS: Although the nanoparticle formulations present equivalent anticancer activity, compared to the Cremophor® formulation, they show improved biodistribution profiles, presenting novel tools for future plitidepsin-based therapies.

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.

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.

Encapsidation of RNA-polyelectrolyte complexes with amphiphilic block copolymers: toward a new self-assembly route.

Amphiphilic block copolymers are molecules composed of hydrophilic and hydrophobic segments having the capacity to spontaneously self-assemble into a variety of supramolecular structures like micelles and vesicles. Here, we propose an original way to self-assemble amphiphilic block copolymers into a supported bilayer membrane for defined coating of nanoparticles. The heart of the method rests on a change of the amphiphilicity of the copolymer that can be turned off and on by varying the polarity of the solvent. In this condition, the assembly process can take advantage of specific molecular interactions in both organic solvent and water. While the concept potentially could be applied to any type of charged substrates, we focus our interest on the design of a new type of polymer assembly mimicking the virus morphology. A capsid-like shell of glycoprotein-mimic amphiphilic block copolymer was self-assembled around a positively charged complex of siRNA and polyethyleneimine. The process requires two steps. Block copolymers first interact with the complexes dispersed in DMSO through electrostatic interactions. Next, the increase of the water content in the medium triggers the hydrophobic effect and the concomitant self-assembly of free block copolymer molecules into a bilayer membrane at the complex surface. The higher gene silencing activity of the copolymer-modified complexes over the complexes alone shows the potential of this new type of nanoconstructs for biological applications, especially for the delivery of therapeutic biomolecules.

Supramolecular structure characterization of cellulose II nanowhiskers produced by acid hydrolysis of cellulose I substrates.

Cellulose II nanowhiskers (CNW-II) were produced by treatment of microcrystalline cellulose with sulfuric acid by both controlling the amount of H(2)SO(4) introduced and the time of addition during the hydrolysis process. The crystalline structure was confirmed by both XRD and (13)C CP-MAS NMR spectroscopy. When observed between crossed polarizers, the cellulose II suspension displayed flow birefringence and was stable for several months. The CNW-II nanowhiskers were significantly smaller than the cellulose I nanowhiskers (CNW-I) and had a rounded shape at the tip. The CNW-II average length and height were estimated by AFM to be 153 ± 66 and 4.2 ± 1.5 nm, respectively. An average width of 6.3 ± 1.7 nm was found by TEM, suggesting a ribbon-shape morphology for these whiskers. The average dimensions of the CNW-II elementary crystallites were estimated from the XRD data, using Scherrer's equation. A tentative cross-sectional geometry consistent with both XRD and NMR data was then proposed and compared with the geometry of the CNW-I nanowhiskers.

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.

Nanogels based on poly(vinyl acetate) for the preparation of patterned porous films.

The use of poly(vinyl acetate) (PVAc) nanogels for the fabrication of patterned porous surfaces is described. These nanogels were synthesized by controlled radical cross-linking copolymerization (CRCC) involving a xanthate-mediated reversible addition-fragmentation chain transfer (RAFT) mechanism. This synthesis methodology allowed for the preparation of nanogels based on PVAc with a controlled constitutive chain length and average numbers of chains and cross-links. Solutions of these branched polymers were prepared in THF with a fixed amount of water and spin coated onto a surface of graphite. The surface porosity of corresponding films was observed by atomic force microscopy (AFM). Compared with linear PVAc homologues with a degree of polymerization (DP) sufficiently high to favor the formation of porous structures (DP = 50), a sharper and better defined porosity was observed with nanogels, the constitutive chains of which had the same DP. For nanogels differing only in their cross-link density, the pores were smaller and better defined in the case of the higher cross-link density, suggesting an enhanced stabilization of the water droplets during film formation. To explain these observations, it is postulated that PVAc nanogels can behave as compact particles providing steric stabilization of water droplets, which is referred to as a Pickering effect. The coalescence of water droplets would be better prevented as the cross-link density of the nanogels increases, resulting in a smaller size pore.

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.