Isabelle Pignot-Paintrand

Elaboration of chitosan-coated nanoparticles loaded with curcumin for mucoadhesive applications.

Polycaprolactone (PCL) nanoparticles decorated with a mucoadhesive polysaccharide chitosan (CS) containing curcumin were developed aiming the buccal delivery of this drug. These nanoparticles were prepared by the nanoprecipitation method using different molar masses and concentrations of chitosan and concentrations of triblock surfactant poloxamer (PEO-PPO-PEO), in order to optimize the preparation conditions. Chitosan-coated nanoparticles showed positive surface charge and a mean particle radius ranging between 114 and 125 nm, confirming the decoration of the nanoparticles with the mucoadhesive polymer, through hydrogen bonds between ether and amino groups from PEO and CS, respectively. Dynamic Light Scattering (DLS) studies at different scattering angles and concentrations have shown that the nanoparticles are monodisperse (polydispersity indices were lower than 0.3). The nanoparticle systems were also examined with Nanoparticle Tracking Analysis (NTA), and the results were in good agreement with those obtained by DLS. Colloidal systems showed mean drug content about 460 μg/mL and encapsulation efficiency higher than 99%. Finally, when coated with chitosan, these nanoparticles show a great ability to interact with mucin indicating also their suitability for mucoadhesive applications.

Multilayer assembly of hyaluronic acid/poly(allylamine): control of the buildup for the production of hollow capsules.

The objective of this work was to investigate the formation of hollow microcapsules composed of hyaluronic acid (HA) and poly(allylamine) (PAH) by layer-by-layer adsorption on CaCO 3 microparticles and subsequent core removal by addition of chelating agents for calcium ions. We found that the molecular weight of HA as well as the HA solution concentration used during deposition are crucial parameters influencing the multilayer structure. Whereas the effect of molecular weight of HA was mainly attributed to the porous structure of the template which allows penetration of polyelectrolytes when their size is below the maximum pore size of the template ( approximately 60 nm), that of the concentration of the HA solution was related to the intrinsic properties of the polysaccharide. Indeed, as shown by quartz crystal microbalance with dissipation monitoring as well as electron microscopy techniques, the latter leads to dense structures for concentrations from five to ten times the critical overlap concentration during adsorption. Such conditions were found to be favorable for the formation of hollow shells. Regarding conditions for core dissolution, we demonstrated the possibility to use either ethylenediaminetetraacetic acid (EDTA) or citric acid as chelating agents. However, in some cases, it was necessary to chemically cross-link the shell to maintain its integrity.

The plastid division proteins, FtsZ1 and FtsZ2, differ in their biochemical properties and sub-plastidial localization

Plastid division in higher plants is morphologically similar to bacterial cell division, with a process termed binary fission involving constriction of the envelope membranes. FtsZ proteins involved in bacterial division are also present in higher plants, in which the ftsZ genes belong to two distinct families: ftsZ1 and ftsZ2. However, the roles of the corresponding proteins FtsZ1 and FtsZ2 in plastid division have not been determined. Here we show that the expression of plant FtsZ1 and FtsZ2 in bacteria has different effects on cell division, and that distinct protein domains are involved in the process. We have studied the assembly of purified FtsZ1 and FtsZ2 using a chemical cross-linking approach followed by PAGE and electron microscopy analyses of the resulting polymers. This has revealed that FtsZ1 is capable of forming long rod-shaped polymers and rings similar to the bacterial FtsZ structures, whereas FtsZ2 does not form any organized polymer. Moreover, using purified sub-plastidial fractions, we show that both proteins are present in the stroma, and that a subset of FtsZ2 is tightly bound to the purified envelope membranes. These results indicate that FtsZ2 has a localization pattern distinct from that of FtsZ1, which can be related to distinct properties of the proteins. From the results presented here, we propose a model for the sequential topological localization and functions of green plant FtsZ1 and FtsZ2 in chloroplast division.

Self-Assembly of Amphiphilic Glycoconjugates into Lectin-Adhesive Nanoparticles

This work describes the synthesis and self-assembly of carbohydrate-clicked rod-coil amphiphilic systems. Copper-catalyzed Huisgen cycloaddition was efficiently employed to functionalize the hydrophilic extremity of PEG-b-tetra(p-phenylene) conjugates by lactose and N-acetyl-glucosamine ligands. The resulting amphiphilic systems spontaneously self-assembled into nanoparticles when dissolved in aqueous media, as evidenced by dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The formation of highly monodisperse micelles having a mean diameter of 10 nm was observed for systems containing a PEG 900 core, and a decrease in the hydrophilic moiety (PEG 600) led to the formation of vesicles with a broader size distribution. The presence of carbohydrate residues on the surfaces of the micelles and their ability to establish specific interactions with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were further highlighted by light-scattering measurements, thus confirming the attractive applications of such sugar micelles in biosensor devices.

Polyelectrolyte multilayer nanoshells with hydrophobic nanodomains for delivery of Paclitaxel.

Efficient and effective delivery of poorly water-soluble drug molecules, which constitute a large part of commercially available drugs, is a major challenge in the field of drug delivery. Several drugs including paclitaxel (PTX) which are used for cancer treatment are hydrophobic, exhibit poor aqueous solubility and need to be delivered using an appropriate carrier. In the present work, we engineered PTX-loaded polyelectrolyte films and microcapsules by pre-complexing PTX with chemically modified derivative of hyaluronic acid (alkylamino hydrazide) containing hydrophobic nanocavities, and subsequent assembly with either poly(l-lysine) (PLL) or quaternized chitosan (QCHI) as polycations. The PTX loading capacity of the films was found to be dependent on number of layers in the films as well as on the initial concentration of PTX pre-complexed to hydrophobic HA, with a loading capacity up to 5000-fold the initial PTX concentration. The films were stable in physiological medium and were degraded in the presence of hyaluronidase. The PTX-loaded microcapsules were found to decrease the viability and proliferation of MDA MB 231 breast cancer cells, while unloaded microcapsules did not impact cell viability. All together, our results highlight the potential of hyaluronan-based assemblies containing hydrophobic nanodomains for hydrophobic drug delivery.

Hydrophobic Shell Loading of Biopolyelectrolyte Capsules

A versatile method to selectively encapsulate water-insoluble molecules in the nanoshell of layer-by-layer capsules made exclusively of polysaccharides is described. This relies on the very high affinity of hydrophobic molecules for an alkylated hyaluronic acid used as a polyanionic partner of a chitosan derivative for the capsule synthesis. The hydrophobic molecules entrapped in the nanoshell can be delivered intracellularly in dendritic cells.

Developmentally regulated association of plastid division protein FtsZ1 with thylakoid membranes in Arabidopsis thaliana.

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.

Photochemical crosslinking of hyaluronic acid confined in nanoemulsions: towards nanogels with a controlled structure

We present the preparation of nanogels made of hyaluronic acid (HA) with a well-controlled structure. To this end, HA precursors with polymerizable methacrylate groups (HA-MA) were confined within water-in-oil nanoemulsion droplets as nanoreactors and further photopolymerized under UV. Particular attention was paid to the preparation of a stable nanoemulsion template with a homogeneous droplet size. Upon UV irradiation of the emulsion containing HA-MA, crosslinked HA-MA particles with a well-defined size were obtained. Moreover, by varying the photopolymerization conditions, i.e. the number of received photons, we could control the conversion rate of the polymerization, as proved by 1H-NMR. Nanogels with controlled cross-linking densities were thus obtained. Not only could their crosslinking densities be controlled by the number of incident photons, but also by the degree of methacrylation (DM) of HA-MA derivatives. In addition, the swelling properties of the nanogels depended on external factors, showing their pH and ionic strength responsiveness. We show that these structures were highly biocompatible, stable under storage and enzymatically biodegradable, which opens the route for their application as drug delivery systems. Finally, insulin was loaded in the nanogels and its pH-dependent release was demonstrated. This versatile method of nanogel preparation, which can be applied to every type of hydrophilic precursor, offers a potential synthetic route to design other types of fully biocompatible drug delivery systems.

Self-assembled carbohydrate-based micelles for lectin targeting

Biocompatible low-polydispersity micelles designed for lectin targeting have been prepared by spontaneous self-assembly in water of macromolecular glycosylated amphiphiles. Propargyl-β-lactoside and N-acetyl-β-D-glucosaminide were conjugated by copper-catalyzed Huisgen cycloaddition to azide-terminated PEG 900 stearate. Upon dissolution in water, the resulting amphiphiles immediately self-assemble into highly regular micelles having a mean diameter of 10 nm. Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and Small-Angle X-ray Scattering (SAXS) were used to investigate the structure of the self-assembled saccharidic amphiphiles micelles. The presence of the carbohydrate epitopes on the surface of the micelles and their bioavailability for lectin targeting were also demonstrated by light scattering measurements. Specific interaction of the GlcNac and Lac residues with Wheat Germ Agglutinin (WGA) and Peanut Agglutinin (PNA) respectively, unveils potential applications of such carbohydrate-derived surfactants as simple and site-specific vectorization systems for drug delivery.

Seeing structures and measuring properties with transmission electron microscopy images: A simple combination to study size effects in nanoparticle systems

We report on a method to measure the mean inner potential (V0) using transmission electron microscopy. It is based on phase retrieval from a focus series and has allowed to measure V0 as a function of the size for a system of gold nanoparticles. It comes out that V0 increases for particles below 2 nm. The focus series being carried out in conditions close to the high-resolution ones, structural information can be directly obtained. The high-resolution images have revealed that significant structural change occurs below the 2 nm size, which should be related to the V0 increase.