Tear of lipid membranes by nanoparticles.

Health concerns associated with the advent of nanotechnologies have risen sharply when it was found that particles of nanoscopic dimensions reach the cell lumina. Plasma and organelle lipid membranes, which are exposed to both the incoming and the engulfed nanoparticles, are the primary targets of possible disruptions. However, reported adhesion, invagination and embedment of nanoparticles (NPs) do not compromise the membrane integrity, precluding direct bilayer damage as a mechanism for toxicity. Here it is shown that a lipid membrane can be torn by small enough nanoparticles, thus unveiling mechanisms for how lipid membrane can be compromised by tearing from nanoparticles. Surprisingly, visualization by cryo transmission electron microscopy (cryo-TEM) of liposomes exposed to nanoparticles revealed also that liposomal laceration is prevented by particle abundance. Membrane destruction results thus from a subtle particle-membrane interplay that is here elucidated. This brings into a firmer molecular basis the theorized mechanisms of nanoparticle effects on lipid bilayers and paves the way for a better assessment of nanoparticle toxicity.

Adaptation of Bacillus thuringiensis to Plant Colonization Affects Differentiation and Toxicity.

The Bacillus cereus group (Bacillus cereus sensu lato) has a diverse ecology, including various species that are vertebrate or invertebrate pathogens. Few isolates from the B. cereus group have however been demonstrated to benefit plant growth. Therefore, it is crucial to explore how bacterial development and pathogenesis evolve during plant colonization. Herein, we investigated Bacillus thuringiensis (Cry-) adaptation to the colonization of Arabidopsis thaliana roots and monitored changes in cellular differentiation in experimentally evolved isolates. Isolates from two populations displayed improved iterative ecesis on roots and increased virulence against insect larvae. Molecular dissection and recreation of a causative mutation revealed the importance of a nonsense mutation in the rho transcription terminator gene. Transcriptome analysis revealed how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage over single cells when colonizing plants, creating a trade-off between swimming and multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity. IMPORTANCE Biologicals-based plant protection relies on the use of safe microbial strains. During application of biologicals to the rhizosphere, microbes adapt to the niche, including genetic mutations shaping the physiology of the cells. Here, the experimental evolution of Bacillus thuringiensis lacking the insecticide crystal toxins was examined on the plant root to reveal how adaptation shapes the differentiation of this bacterium. Interestingly, evolution of certain lineages led to increased hemolysis and insect larva pathogenesis in B. thuringiensis driven by transcriptional rewiring. Further, our detailed study reveals how inactivation of the transcription termination protein Rho promotes aggregation on the plant root in addition to altered differentiation and pathogenesis in B. thuringiensis.

Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Phage-Mediated Control of Flavobacterium psychrophilum in Aquaculture: In vivo Experiments to Compare Delivery Methods.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g-1) or by irreversible immobilization (8.3 × 107 PFU g-1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg-1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml-1; 3rd bath: ∼105 PFU ml-1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish-1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.

First multi-reactive polysaccharide-based transurf to produce potentially biocompatible dextran-covered nanocapsules.

A multi-reactive polysaccharide-based transurf (acting both as macro-Chain Transfer Agent and stabilizer) was used to confine RAFT polymerization of methyl methacrylate (MMA) at the oil/water (o/w) miniemulsion interface. Dithiobenzoate groups and hydrophobic aliphatic side chains were introduced onto dextran, conferring it both transfer agent properties and ability to stabilize direct miniemulsion of MMA in the presence of a biocompatible oil, used as co-stabilizer. Because of their amphiphilic character, transurfs were initially adsorbed at the (o/w) interface and their reactive sites mediated RAFT polymerization via the R-group approach. PMMA-grafted dextran glycopolymers were consequently produced at the o/w interface, thus leading to dextran coverage/PMMA shell/oily core nanocapsules (NCs) as evidenced by Cryo-TEM analyses. The influence of dextran-based transurf chemistry and oil amount on MMA RAFT polymerization control was investigated. Positive preliminary results on NCs cytotoxicity suggest the potential of these objects for biomedical applications.

Microfluidic-Assisted Production of Size-Controlled Superparamagnetic Iron Oxide Nanoparticles-Loaded Poly(methyl methacrylate) Nanohybrids.

In this paper, superparamagnetic iron oxide nanoparticles (SPIONs, around 6 nm) encapsulated in poly(methyl methacrylate) nanoparticles (PMMA NPs) with controlled sizes ranging from 100 to 200 nm have been successfully produced. The hybrid polymeric NPs were prepared following two different methods: (1) nanoprecipitation and (2) nanoemulsification-evaporation. These two methods were implemented in two different microprocesses based on the use of an impact jet micromixer and an elongational-flow microemulsifier. SPIONs-loaded PMMA NPs synthesized by the two methods presented completely different physicochemical properties. The polymeric NPs prepared with the micromixer-assisted nanoprecipitation method showed a heterogeneous dispersion of SPIONs inside the polymer matrix, an encapsulation efficiency close to 100 wt %, and an irregular shape. In contrast, the polymeric NPs prepared with the microfluidic-assisted nanoemulsification-evaporation method showed a homogeneous dispersion, an almost complete encapsulation, and a spherical shape. The properties of the polymeric NPs have been characterized by dynamic light scattering, thermogravimetric analysis, and transmission electron microscope. In vitro cytotoxicity assays were also performed on the nanohybrids and pure PMMA NPs.

Functionalizing Nanoemulsions with Carboxylates: Impact on the Biodistribution and Pharmacokinetics in Mice.

Efficiency of drug administration is related to the inhibition of adverse effects, and can be improved by drug targeting through lipid nanocarriers encapsulation. Targeting technology generally goes along with the nanocarrier functionalization that can be surface modification and/or ligand grafting. The great advantage of nanoemulsions is their loading capability and the possibilities to encapsulate several entities in a single droplet, however, the decoration of the lipid droplets with strongly anchored reactive functions is challenging. This study proposes a reliable and innovative method to functionalize lipid droplets, based on the lipophilic polymer poly(maleic anhydride-alt-1-octadecene), solubilized in the droplet core, and able to hydrolyze at the oil/water interface. Interfacial chemistry and physicochemical properties of nanodroplets are characterized. In vitro studies reveal that the presence of carboxylates at interface has a strong impact on the interactions with cells, as the internalization of functionalized droplets is much higher than control ones. This difference is confirmed with longitudinal computed tomography studies in mice after i.v. administration, strongly impacting the pharmacokinetics and biodistributions. This work establishes the proof-of-concept of a new method for functionalizing lipid droplets and demonstrates that surface modification can have a significant impact on their interaction with cells, pharmacokinetics, and biodistribution.

A new method for the formulation of double nanoemulsions.

Double emulsions are very attractive systems for many reasons; the most important of these are their capacity to encapsulate hydrophilic and lipophilic molecules simultaneously in a single particle and their potentiality to protect fragile hydrophilic molecules from the continuous phase. Double emulsions represent a technology that is widely present down to the micrometer scale; however, double nanoemulsions, with their new potential applications as nanomedicines or diagnosis agents, currently present a significant challenge. In this study, we propose an original two-step approach for the fabrication of double nanoemulsions with a final size below 200 nm. The process consists of the formulation of a primary water-in-oil (w1/O) nanoemulsion by high-pressure homogenization, followed by the re-emulsification of this primary emulsion by a low-energy method to preserve the double nanostructure. Various characterization techniques were undertaken to confirm the double structure and to evaluate the encapsulation efficiency of a small hydrophilic probe in the inner aqueous droplets. Complementary fluorescence confocal and cryo-TEM microscopy experiments were conducted to characterize and confirm the double structure of the double nanoemulsion.

Keratin made micro-tubes: The paradoxical thermal behavior of cortex and cuticle.

Keratin micro-tubes were obtained by heating medullated keratin fibres to temperatures above 230°C under nitrogen atmosphere, when, as documented by microscopy, the cortex (the core of the fibre) melts from the medulla outwards, followed by pyrolysis of the material through the remaining solid cuticle (shell) layer. The resulted hollow tubes from fibres void of cortical material keep the external cuticle structure, as shown by AFM investigation, and the moisture sorption properties of the initial keratin fibre. Despite similar amino-acid compositions of cuticle and cortex the two morphological components differ significantly in their thermal behaviour, which appears to be a "cortex-cuticle thermal stability paradox".

Microfluidic conceived Trojan microcarriers for oral delivery of nanoparticles.

In this study, we report on a novel method for the synthesis of poly(acrylamide) Trojan microparticles containing ketoprofen loaded poly(ethyl acrylate) or poly(methyl acrylate) nanoparticles. To develop these composite particles, a polymerizable nanoemulsion was used as a template. This nanoemulsion was obtained in an elongational-flow micromixer (µRMX) which was linked to a capillary-based microfluidic device for its emulsification into micron range droplets. Downstream, the microdroplets were hardened into Trojan particles in the size range of 213-308 µm by UV initiated free radical polymerization. The nanoemulsion size varied from 98 -132 nm upon changes in surfactant concentration and number of operating cycles in µRMX. SEM and confocal microscopy confirmed the Trojan morphology. Under SEM it was observed that the polymerization reduced the size of the nanoemulsion down to 20-32 nm for poly(ethyl acrylate) and 10-15 nm for poly(methyl acrylate) nanoparticles. This shrinkage was confirmed by cryo-TEM studies. We further showed that Trojan microparticles released embedded nanoparticles on contact with suitable media as confirmed by transmission electron microscopy. In a USP phosphate buffer solution of pH 6.8, Trojan microparticles containing poly(ethyl acrylate) nanoparticles released 35% of encapsulated ketoprofen over 24h. The low release of the drug was attributed to the overall low concentration of nanoparticles and attachment of some of nanoparticles to the poly(acrylamide) matrix. Thus, this novel method has shown possibility to develop Trojan particles convieniently with potential to deliver nanoparticles in the gastrointestinal tract.

Supercritical carbon dioxide assisted silicon based finishing of cellulosic fabric: a novel approach.

Usage of supercritical carbon dioxide as a medium for finishing cotton fabrics with modified dimethylsiloxane polymers terminated with silanol groups was investigated, different cross-linkers namely 3-isocyanatepropyltriethoxysilane (IPES) and tetraethylorthosilicate (TEOS) were used for covalently bonding between silicon and cellulose. The presence and the amount of PDMS compounds on the treated fabrics were characterized by FT-IR. Qualitative and quantitative information on the distribution of the silicon molecules across the fibre cross section was provided by SEM/EDX analysis and Confocal Raman microscopy (CRM) respectively. The results confirm that all fibres treated with PDMS and IPES have larger silicon amounts than those treated with TEOS. SC-CO2 medium provides good coating of cotton surface with a 3D network of DMS compound and cross linker, and leads to forming highest DMS concentration in a layer between 1 and 2µ under the surface of cotton fabrics.

Suitability of Confocal Raman microscopy for monitoring the penetration of PDMS compounds into cotton fibres.

PDMS compound was chosen as a molecule-model for investigating the diffusion of silicon products into cotton bulk. The study demonstrates the suitability of Confocal Raman microscopy (CRM) to monitor the distribution of poly(dimethylsiloxane) (PDMS) molecules into cotton fibres. Different molecular weights of PDMS compounds were used in two different solvents (water and hexane) at various temperatures (25, 50 and 60°C). The surfaces of the fibres were studied with scanning electron microscopy and Confocal Raman microscopy was run to detect the PDMS on the surface and in the bulk of treated fabrics. We concluded that all PDMS compounds, irrespectively their molecular weights and the silicon oil infiltrate into cotton fibre. The penetration is strongly dependent on the solvent used. Water proved suitable for assisting the infiltration of low and medium molecular weight PDMS, at elevated temperatures. High molecular weight PDMS infiltrates better from hexane and at room temperature than from water.

The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin.

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in vivo conditions. In short, the alpha-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.