Sylviane Lesieur

Vesicle reconstitution from lipid-detergent mixed micelles.

The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid-detergent systems. The mechanisms involved in both the lipid self assembly and the micelle-vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle-vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle-vesicle transition.

Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch+Apc+/1638N mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and β-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of β-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by β-catenin nuclear translocation after 15 days. As a consequence, increased expression of β-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic β-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.

Characterization of loaded liposomes by size exclusion chromatography.

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.

Multicompartment lipid cubic nanoparticles with high protein upload: millisecond dynamics of formation.

Membrane shapes, produced by dynamically assembled lipid/protein architectures, are crucial for both physiological functions and the design of therapeutic nanotechnologies. Here we investigate the dynamics of lipid membrane-neurotrophic BDNF protein complexes formation and ordering in nanoparticles, with the purpose of innovation in nanostructure-based neuroprotection and biomimetic nanoarchitectonics. The kinetic pathway of membrane states associated with rapidly occurring nonequilibrium self-assembled lipid/protein nanoarchitectures was determined by millisecond time-resolved small-angle X-ray scattering (SAXS) at high resolution. The neurotrophin binding and millisecond trafficking along the flexible membranes induced an unusual overlay of channel-network architectures including two coexisting cubic lattices epitaxially connected to lamellar membrane stacks. These time-resolved membrane processes, involving intercalation of discrete stiff proteins in continuous soft membranes, evidence stepwise curvature control mechanisms. The obtained three-phase liquid-crystalline nanoparticles of neurotrophic composition put forward important advancements in multicompartment soft-matter nanostructure design.

Small-Angle X-ray Scattering Investigations of Biomolecular Confinement, Loading, and Release from Liquid-Crystalline Nanochannel Assemblies

This Perspective explores the recent progress made by means of small-angle scattering methods in structural studies of phase transitions in amphiphilic liquid-crystalline systems with nanochannel architectures and outlines some future directions in the area of hierarchically organized and stimuli-responsive nanochanneled assemblies involving biomolecules. Time-resolved small-angle X-ray scattering investigations using synchrotron radiation enable monitoring of the structural dynamics, the modulation of the nanochannel hydration, as well as the key changes in the soft matter liquid-crystalline organization upon stimuli-induced phase transitions. They permit establishing of the inner nanostructure transformation kinetics and determination of the precise sizes of the hydrophobic membraneous compartments and the aqueous channel diameters in self-assembled network architectures. Time-resolved structural studies accelerate novel biomedical, pharmaceutical, and nanotechnology applications of nanochannel soft materials by providing better control of DNA, peptide and protein nanoconfinement, and release from diverse stimuli-responsive nanocarrier systems.

Preparation of amphiphilic cyclodextrin nanospheres using the emulsification solvent evaporation method. Influence of the surfactant on preparation and hydrophobic drug loading

Abstract In this study we verified the feasibility of preparing nanospheres from an amphiphilic 2,3-di- O -hexanoyl- γ -cyclodextrin ( γ CDC 6 ) using the emulsification solvent evaporation method. This preparation method consists in emulsifying an organic phase containing the cyclodextrin in an aqueous phase containing Pluronic F68 as surfactant. The influence of the process parameters, i.e. surfactant concentration and initial γ CDC 6 content, on the characteristics of nanosphere preparation, as well as on the nanosphere loading of a hydrophobic drug, progesterone, was evaluated. Cyclodextrin nanospheres presenting a mean diameter varying from 50 to 200 nm were obtained, even in the presence of low surfactant concentration. The formation of colloidal particles in these conditions was associated with the amphiphilic properties of the cyclodextrin derivative. However the partitioning of the γ CDC 6 molecule between the organic and aqueous phases was observed as being a function of surfactant concentration in the continuous phase. This partitioning was related to the formation of very small aggregates of the order 10 to 20 nm, probably Pluronic F68/ γ CDC 6 mixed micelles as evidenced by the micrographs obtained by TEM. In the case of the nanospheres loaded with progesterone, the partitioning of the drug between the dispersed phase containing the cyclodextrin and the continuous aqueous phase containing Pluronic F68/ γ CDC 6 aggregates was also demonstrated. The drug content found in the final nanospheres ranged from 4 to 5% (w/w) of the carrier. Finally, dilution experiments were carried out to evaluate the stability of the drug particle association.

Partition coefficient of a surfactant between aggregates and solution: application to the micelle-vesicle transition of egg phosphatidylcholine and octyl beta-D-glucopyranoside.

The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.

DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics

Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG2000) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle-nanoparticle structural transition and lipoplex formation involving tightly packed pDNA.

Protein entrapment in PEGylated lipid nanoparticles

Defining appropriate delivery strategies of therapeutic proteins, based on lipid nanoparticulate carriers, requires knowledge of the nanoscale organization that determines the loading and release properties of the nanostructured particles. Nanoencapsulation of three cationic proteins (human brain-derived neurotrophic factor (BDNF), α-chymotrypsinogen A, and histone H3) was investigated using anionic nanoparticle (NP) carriers. PEGylated lipid NPs were prepared from self-assembled liquid crystalline phases involving monoolein and eicosapentaenoic acid. Inclusion of the antioxidant α-tocopherol favoured the preparation of stealth hexosome carriers. The purpose of the present work is to reveal the structural features of the protein-loaded lipid nanocarriers by means of high resolution small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). The obtained results indicate that protein entrapment is concentration-dependent and may significantly modify the inner liquid crystalline structure of the lipid nanocarriers through changes in the interfacial curvature and hydration.

Neurotrophin delivery using nanotechnology

Deficits or overexpression of neurotrophins cause neurodegenerative diseases and psychiatric disorders. These proteins are required for the maintenance of the function, plasticity and survival of neurons in the central (CNS) and peripheral nervous systems. Significant efforts have been devoted to developing therapeutic delivery systems that enable control of neurotrophin dosage in the brain. Here, we suggest that nanoparticulate carriers favoring targeted delivery in specific brain areas and minimizing biodistribution to the systemic circulation should be developed toward clinical benefits of neuroregeneration. We also provide examples of improved targeted neurotrophin delivery to localized areas in the CNS.