AIE Polymer Micelle/Vesicle Photocatalysts Combined with Native Enzymes for Aerobic Photobiocatalysis.

Biocatalytic transformation has attracted increasing attention in the green synthesis of chemicals due to the diversity of enzymes, their high catalytic activities and specificities, and environmentally benign conditions. Most redox enzymes in nature are dependent on nicotinamide cofactors like ß-nicotinamide adenine dinucleotide (NAD+)/reduced nicotinamide adenine dinucleotide (NADH). The use of solar energy, especially visible light, in the regeneration of cofactors through the combination of photocatalysis and biocatalysis provides an extraordinary opportunity to make complete green processes. However, the combination of photocatalysts and enzymes has been challenged by the rapid degradation and deactivation of the enzymatic material by photogenerated reactive oxygen species (ROS). Here, we design core-shell structured polymer micelles and vesicles with aggregation-induced emission (AIE) as visible-light-mediated photocatalysts for highly stable and recyclable photobiocatalysis under aerobic conditions. NAD+ from NADH can be efficiently regenerated by the photoactive hydrophobic core of polymer micelles and the hydrophobic membrane of polymer vesicles, while the enzymatic material (glucose 1-dehydrogenase) is screened from the attack of photogenerated ROS by the hydrophilic surface layer of polymer colloids. After at least 10 regeneration cycles, the enzyme keeps its active state; meanwhile, polymer micelles and vesicles maintain their photocatalytic activity. These polymer colloids show the potential to be developed for the implementation of industrially relevant photobiocatalytic systems.

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids.

Stimuli-responsive polymersomes formed by amphiphilic block copolymers have attracted substantial attention as smart and robust containers for drug delivery and nano/microreactors. Biosourced amphiphilic diblock copolypeptoids were developed that can self-assemble into oxidation-responsive unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species which can be the hydrogen peroxide or the singlet oxygen produced by light-activation of a photosensitizer with spatiotemporal control. Polysarcosine (PSar, also called poly(N-methyl glycine)) was selected as the hydrophilic block because of its resistance to protein adsorption and low toxicity, similar to poly(ethylene glycol) (PEG). We designed and synthesized poly(N-3-(methylthio)propyl glycine) as the hydrophobic block. Its polyglycine backbone is the same as that of PSar, and especially, its hydrophobic N-substituents, thioether side chains, can be oxidized to hydrophilic sulfoxides. These oxidation-responsive polymersomes entirely based on N-substituted poly(amino acid)s were biocompatible as confirmed by cell viability tests and may find applications in drug delivery, biosensing, biodetection, and nano/microreactors.

Poly(ε-caprolactone)-block-polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermoresponsive Self-Assembly.

Biocompatible amphiphilic block copolymers composed of polysarcosine (PSar) and poly(ε-caprolactone) (PCL) were synthesized using ring-opening polymerization of sarcosine N-thiocarboxyanhydride initiated by oxyamine-ended PCL and characterized by NMR, SEC, and DSC. Self-assembling of two triblock copolymers PSar8-b-PCL28-b-PSar8 (CS7) and PSar16-b-PCL40-b-PSar16 (CS10) in dilute solution was studied in detail toward polymersome formation using thin-film hydration and nanoprecipitation techniques. A few giant vesicles were obtained by thin-film hydration from both copolymers and visualized by confocal laser scanning microscope. Unilamellar sheets and nanofibers (with 8-10 nm thickness or diameter) were obtained by nanoprecipitation at room temperature and observed by Cryo-TEM. These lamellae and fibrous structures were transformed into worm-like cylinders and spheres (D∼30-100 nm) after heating to 65 °C (>Tm,PCL). Heating CS10 suspensions to 90 °C led eventually to multilamellar polymersomes (D∼100-500 nm). Mechanism II, where micelles expand to vesicles through water diffusion and hydrophilic core forming, was proposed for polymersome formation. A cell viability test confirmed the self-assemblies were not cytotoxic.

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography.

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.

Multilamellar liposomes entrapping aminosilane-modified maghemite nanoparticles: "magnetonions".

4.6 nm-sized aminosilane-modified maghemite (γ-Fe(2)O(3)) nanoparticles (aMNPs) were synthesized and encapsulated into onion-type multilamellar vesicles of soybean phosphatidylcholine (90%mol) and monoolein (10%mol). The magnetic multilamellar vesicles were obtained by shearing lipids with an aqueous dispersion of the preformed aMNPs (ferrofluid). The influence of ferrofluid concentration on the stability of the constitutive lamellar phase and the resulting dispersed onions was analyzed by small-angle X-ray diffraction (SAXD) and cryo-TEM imaging, respectively. When [Fe(III)] <60 mM, stable, magnetic onions were produced with aMNPs inserted inside onion water compartments as isolated or aggregated particles. Encapsulation efficiencies were measured by EPR spectroscopy and magnetic measurements: much higher values (up to 75%) than unilamellar liposomes were found. The deduced aMNP-to-onion ratio increased with ferrofluid concentration before reaching a maximal value of ca. 45 as confirmed by cryo-TEM imaging. When [Fe(III)] >60 mM, uni- or oligolamellar vesicles in addition to onions formed, probably because of a two-phase separation between an aMNP-rich phase and an aMNP-containing lamellar phase as revealed by SAXD.

Cryo-electron tomography of nanoparticle transmigration into liposome.

Nanoparticle transport across cell membrane plays a crucial role in the development of drug delivery systems as well as in the toxicity response induced by nanoparticles. As hydrophilic nanoparticles interact with lipid membranes and are able to induce membrane perturbations, hypothetic mechanisms based on membrane curvature or hole formation have been proposed for activating their transmigration. We report on the transport of hydrophilic silica nanoparticles into large unilamellar neutral DOPC liposomes via an internalization process. The strong adhesive interactions of lipid membrane onto the silica nanoparticle triggered liposome deformation until the formation of a curved neck. Then the rupture of this membrane neck led to the complete engulfment of the nanoparticle. Using cryo-electron tomography we determined 3D architectures of intermediate steps of this process unveiling internalized silica nanoparticles surrounded by a supported lipid bilayer. This engulfing process was achieved for a large range of particle size (from 30 to 200 nm in diameter). These original data provide interesting highlights for nanoparticle transmigration and could be applied to biotechnology development.

Magnetic multilamellar liposomes produced by in situ synthesis of iron oxide nanoparticles: "magnetonions".

We report the formation of magnetic onion-type multilamellar vesicles. Iron oxide nanoparticles (np's) were synthesized inside lipidic multilamellar vesicles by coprecipitation of vesicle-encapsulated Fe(2+) and Fe(3+) ions induced by HO(-) diffusion through vesicle lamellae. The iron ion encapsulation efficiency of onions was measured by potentiometry and UV-vis absorbance spectroscopy. Its high value (75 +/- 5% for both Fe(2+) and Fe(3+)) ensures an intravesicular synthesis, as confirmed by cryo-transmission electron microscopy (TEM) imaging. The as-grown nanoparticles are characterized by X-ray diffraction analysis and TEM, and magnetic onions are imaged by cryo-TEM. The np size, controlled by temperature and time, ranges from 3 to 6 nm and is shown to be a key parameter for onion stability.

Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study.

We prepared and characterized polymersomes functionalized with nanobodies (VHHs) on the basis of biocompatible, biodegradable and FDA-approved poly(ethylene glycol)-block-poly(ϵ-caprolactone) (PEG-b-PCL). Fluorescein isothiocyanate (FITC) and N-beta-maleimidopropyl-oxysuccinimide ester were allowed reacting with H2N-PEG-b-PCL to produce FITC and maleimide (Mal) functionalized copolymers, Mal-PEG-b-PCL and FITC-PEG-b-PCL. A mixture of MeO-PEG-b-PCL, Mal-PEG-b-PCL and FITC-PEG-b-PCL was used to prepare polymersomes by thin film hydration and nanoprecipitation methods. Morphological studies by cryogenic transmission electron microscopy (Cryo-TEM) showed that the nanoparticles exhibited predominantly vesicular structures (polymersomes). Their mean diameters measured by dynamic light scattering were around 150 nm and the zeta-potentials around -1 mV at pH 7.4. The nanoparticles were functionalized with either anti-HER2 (VHH1) or anti-GFP (VHH2) nanobodies using maleimide-cysteine chemistry. Their particle size and zeta-potential increased slightly after nanobody-functionalization. The specific binding of VHH-functionalized polymersomes and control nanoparticles towards HER2 positive breast cancer cells was analyzed by flow cytometry and confocal microscopy. The collected results represent the first report which experimentally demonstrates that VHH1-functionalized PEO-b-PCL polymersomes can target specifically breast cancer cells expressing HER2 receptors. The detailed morphological and cell-binding studies described herein pave the way for future in vivo studies to evaluate the feasibility to use such nanoparticles for targeted drug delivery.

Structure of artificial and natural VE-cadherin-based adherens junctions.

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell-cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1-EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin-catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.