Mechanical induction of the tumorigenic ß-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 ß-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 ß-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by ß-catenin nuclear translocation after 15 days. As a consequence, increased expression of ß-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 ß-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.

Mesoporous self-assembled nanoparticles of biotransesterified cyclodextrins and nonlamellar lipids as carriers of water-insoluble substances.

Soft mesoporous hierarchically structured particles were created by the self-assembly of an amphiphilic deep cavitand cyclodextrin ßCD-nC10 (degree of substitution n = 7.3), with a nanocavity grafted by multiple alkyl (C10) chains on the secondary face of the ßCD macrocycle through enzymatic biotransesterification, and the nonlamellar lipid monoolein (MO). The effect of the non-ionic dispersing agent polysorbate 80 (P80) on the liquid crystalline organization of the nanocarriers and their stability was studied in the context of vesicle-to-cubosome transition. The coexistence of small vesicular and nanosponge membrane objects with bigger nanoparticles with inner multicompartment cubic lattice structures was established as a typical feature of the employed dispersion process. The cryogenic transmission electron microscopy (cryo-TEM) images and small-angle X-ray scattering (SAXS) structural analyses revealed the dependence of the internal organization of the self-assembled nanoparticles on the presence of embedded ßCD-nC10 deep cavitands in the lipid bilayers. The obtained results indicated that the incorporated amphiphilic ßCD-nC10 building blocks stabilize the cubic lattice packing in the lipid membrane particles, which displayed structural features beyond the traditional CD nanosponges. UV-Vis spectroscopy was employed to characterize the nanoencapsulation of a model hydrophobic dimethylphenylazo-naphthol guest compound (Oil red) in the created nanocarriers. In perspective, these dual porosity carriers should be suitable for co-encapsulation and sustained delivery of peptide, protein or siRNA biopharmaceuticals together with small molecular weight drug compounds or imaging agents.

Identification of large channels in cationic PEGylated cubosome nanoparticles by synchrotron radiation SAXS and Cryo-TEM imaging.

Extra-large nanochannel formation in the internal structure of cationic cubosome nanoparticles results from the interplay between charge repulsion and steric stabilization of the lipid membrane interfaces and is evidenced by cryogenic transmission electron microscopy (Cryo-TEM) and synchrotron radiation small-angle X-ray scattering (SAXS). The swollen cubic symmetry of the lipid nanoparticles emerges through a shaping transition of onion bilayer vesicle intermediates containing a fusogenic nonlamellar lipid. Cationic amphiphile cubosome particles, thanks to the advantages of their liquid crystalline soft porous nanoarchitecture and capability for multi-drug nanoencapsulation, appear to be of interest for the design of mitochondrial targeting devices in anti-cancer therapies and as siRNA nanocarriers for gene silencing.

Development of nanoparticles based on amphiphilic cyclodextrins for the delivery of active substances.

Although amphiphilic cyclodextrin derivatives (ACDs) serve as valuable building blocks for nanomedicine formulations, their widespread production still encounters various challenges, limiting large-scale manufacturing. This work focuses on a robust alternative pathway using mineral base catalysis to transesterify ß-cyclodextrin with long-chain vinyl esters, yielding ACD with modular and controlled hydrocarbon chain grafting. ACDs with a wide range of degrees of substitution (DS) were reliably synthesized, as indicated by extensive physicochemical characterization, including MALDI-TOF mass spectrometry. The influence of various factors, including the type of catalyst and the length of the hydrocarbon moiety of the vinyl ester, was studied in detail. ACDs were assessed for their ability to form colloidal suspensions by nanoprecipitation, with or without PEGylated phospholipid. Small-angle X-ray scattering and cryo-electron microscopy revealed the formation of nanoparticles with distinct ultrastructures depending on the DS: an onion-like structure for low and very high DS, and reversed hexagonal organization for DS between 4.5 and 6.1. We confirmed the furtivity of the PEGylated versions of the nanoparticles through complement activation experiments and that they were well tolerated in-vivo on a zebrafish larvae model after intravenous injection. Furthermore, a biodistribution experiment showed that the nanoparticles left the bloodstream within 10 h after injection and were phagocytosed by macrophages.

Quatsomes: vesicles formed by self-assembly of sterols and quaternary ammonium surfactants.

Thermodynamically stable nanovesicle structures are of high interest for academia and industry in a wide variety of application fields, ranging from preparation of nanomaterials to nanomedicine. Here, we show the ability of quaternary ammonium surfactants and sterols to self-assemble, forming stable amphiphilic bimolecular building-blocks with the appropriate structural characteristics to form in aqueous phases, closed bilayers, named quatsomes, with outstanding stability, with time and temperature. The molecular self-assembling of cholesterol and surfactant cetyltrimethylammonium bromide (CTAB) was studied by quasi-elastic light scattering, cryogenic transmission electron microscopy, turbidity (optical density) measurements, and molecular dynamic simulations with atomistic detail, upon varying the cholesterol-to-surfactant molar ratio. As pure species, CTAB forms micelles and insoluble cholesterol forms crystals in water. However, our molecular dynamic simulations reveal that the synergy between CTAB and cholesterol molecules makes them self-assemble into bimolecular amphiphiles and then into bilayers in the presence of water. These bilayers have the same structure of those formed by double-tailed unimolecular amphiphiles.

Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase with large water channels. Time-resolved X-ray diffraction (XRD) scans allowed us to detect metastable intermediate and coexisting structures and monitor the temperature-induced phase sequences of mixed systems containing glycerol monooleate, a soluble protein macromolecule, and an interfacial curvature modulating agent. These observed states correspond to the stages of the growth of the nanofluidic channel network. With the application of a thermal stimulus, the system becomes progressively more ordered into a double-diamond cubic lattice formed by a bicontinuous lipid membrane. High-resolution freeze-fracture electronic microscopy indicates that nanodomains are induced by the inclusion of proteins into nanopockets of the supramolecular cubosomic assemblies. These results contribute to the understanding of the structure and dynamics of functionalized self-assembled lipid nanosystems during stimuli-triggered LC phase transformations.

Earliest stage of the tetrahedral nanochannel formation in cubosome particles from unilamellar nanovesicles.

Studies of nonequilibrium lipid polymorphism at the nanoscale contribute to the in-depth understanding of the structural pathways for formation of aqueous channels and emerging of channels-network ordering in liquid-crystalline (LC) nanovehicles. We present experimental structural evidence for the smallest tetrahedral-type lipid membrane aggregate, which involves completely formed nanochannels and occurs as an early intermediate state during the bilayer vesicle-to-cubosome particle transition. Nanovehicles are generated from a self-assembled lipid mixture and studied by means of high-resolution cryogenic transmission electron microscopy (cryo-TEM) and synchrotron radiation small-angle X-ray scattering (SAXS). The investigated lipid membrane composition allows for the stabilization of long-lived intermediates throughout the unilamellar vesicle-to-cubosome nanoparticle (NP) transformation at ambient temperature. The observed small cubosomic particles, with well-defined water channels, appear to be precursors of larger cubic membrane structures, thus confirming the theoretical modeling of nanochannel-network growth in diamond-type cubic lipid particles. The reported structural findings, highlighting that bilayer vesicle membrane packing and fusion are required for nanochanneled cubosome particle formation, are anticipated to advance the engineering of small lipid NPs with controllable channels for biomolecular loading and release.

SAXS investigation of a cubic to a sponge (L3) phase transition in self-assembled lipid nanocarriers.

The encapsulation and release of peptides, proteins, nucleic acids, and drugs in nanostructured lipid carriers depend on the type of the self-assembled liquid-crystalline organization and the structural dimensions of the aqueous and membraneous compartments, which can be tuned by the multicomponent composition of the systems. In this work, small-angle X-ray scattering (SAXS) investigation is performed on the 'melting' transition of the bicontinuous double diamond cubic phase, formed by pure glycerol monooleate (MO), upon progressive inclusion of varying fractions of pharmaceutical-grade glycerol monooleate (GO) in the hydrated system. The self-assembled MO/GO mixtures are found to form diamond (Pn3m) inverted cubic, inverted hexagonal (H(II)), and sponge (L(3)) phases at ambient temperature in excess of aqueous medium without heat treatment. Mixing of the inverted-cubic-phase-forming MO and the sponge-phase-forming GO components, in equivalent proportions (50/50 w/w), yields an inverted hexagonal (H(II)) phase nanostructured carrier. Scattering models are applied for fitting of the experimental SAXS patterns and identification of the structural changes in the aqueous and lipid bilayer subcompartments. The possibility of transforming, at ambient temperature (20 °C), the bicontinuous cubic nanostructures into inverted hexagonal (H(II)) or sponge (L(3)) mesophases may facilitate novel biomedical applications of the investigated liquid crystalline self-assemblies.

Interaction of n-octyl ß,D-glucopyranoside with giant magnetic-fluid-loaded phosphatidylcholine vesicles: direct visualization of membrane curvature fluctuations as a function of surfactant partitioning between water and lipid bilayer.

The present study deals with the morphological modifications of giant dioleoyl phosphatidylcholine vesicles (DOPC GUVs) induced by the nonionic surfactant n-octyl ß,D-glucopyranoside at sublytic levels, i.e., in the first steps of the vesicle-to-micelle transition process, when surfactant inserts into the vesicle bilayer without disruption. Experimental conditions were perfected to exactly control the surfactant bilayer composition of the vesicles, in line with former work focused on the mechanical properties of the membrane of magnetic-fluid-loaded DOPC GUVs submitted to a magnetic field. The purpose here was to systematically examine, in the absence of any external mechanical constraint, the dynamics of giant vesicle shape and membrane deformations as a function of surfactant partitioning between the aqueous phase and the lipid membrane, beforehand established by turbidity measurements from small unilamellar vesicles.

The effect of magnetic targeting on the uptake of magnetic-fluid-loaded liposomes by human prostatic adenocarcinoma cells.

Interactions of magnetic-fluid-loaded liposomes (MFL) with human adenocarcinoma prostatic cell line PC3 were investigated in vitro. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 180 nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5 mol.% of distearylphosphatidylcholine poly(ethylene glycol)(2000) (DSPE-PEG(2000)) in the vesicle bilayer. The association processes with living cells, including binding and effective internalization, were followed versus time at two levels. On one hand, the lipid vesicles labeled by 1 mol.% of rhodamine-marked phosphatidylethanolamine were imaged by confocal fluorescence microscopy. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. This allowed modeling of MFL uptake kinetics as a two-step process involving first binding adsorption onto the outer cell membrane followed by subsequent internalization. Capture efficiency was significantly improved by guiding MFL in the near vicinity of the cells by means of a 0.29-T external magnet developing a magnetic field gradient close to 30 mT/mm. Double detection of lipids by fluorescence tracking and of iron oxide by magnetophoresis showed excellent correlation. This demonstrated that MFL associate with tumor cells as intact vesicle structures which conserve their internal content.

Physico-chemical investigation of asymmetrical peptidolipidyl-cyclodextrins.

A new class of amphiphilic peptidolipidyl-cyclodextrins is reported. The derivatives are chiral due to the presence of an L-leucine in the spacer arm that links a saccharide moiety and a grafted, saturated hydrocarbon chain. Self-assembly properties of the peptidolipidyl-cyclodextrins are characterized by quasi-elastic light scattering, turbidity and UV-visible absorption measurements. NMR experiments give insight into the intermolecular dipolar interactions as a function of temperature and concentration. N-dodecyl-N alpha-(6 I-amidosuccinyl-6 1-deoxy-cyclomaltoheptaose)-L-leucine (1) is poorly soluble in aqueous media. N-dodecyl-N(alpha)-(6 I-amidosuccinyl-6 I-deoxy-2 I,3 I-di-O-methyl-hexakis-(2 II-VII,3 II-VII,6 II-VII-tri-O-methyl)-cyclomaltoheptaose)-L-leucine (2) is found to be more soluble and self-assembles into stable supramolecular colloidal aggregates with nanometric dimensions above a critical aggregation concentration (CAC). It has a propensity for solubilization of hydrophobic species revealing a micellar-like behavior, which is compared to that of the non-ionic detergent octyl glucoside. On the contrary, compound 1 precipitates in a crystalline phase beyond its water solubility limit, and it does not display any solubilizing capacity. The observed behavior corroborates at the molecular level with the NMR results.

A scalable process to produce lipid-based compartmented Janus nanoparticles with pharmaceutically approved excipients.

In the field of nanotechnologies, theranostic approaches and fixed-dose combination products require the development of innovative carriers able to co-encapsulate several entities of interest. This communication describes the preparation and characterization of lipid-based Janus compartmented nanoparticles. They were successfully prepared using a scalable process with pharmaceutically approved excipients. The analysis of the microscopic structure and supramolecular organization demonstrated the formation of two physico-chemically different compartments enabling the co-administration at once of both liposoluble and hydrosoluble active pharmaceutical ingredients.

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

Influence of polymer behaviour in organic solution on the production of polylactide nanoparticles by nanoprecipitation.

The aim of this study was to define the parameters determining an optimized yield of monodisperse, nanosized particles after nanoprecipitation of a biodegradable polymer, with a view to industrial scale-up the process. Poly(d,l)-lactides (PLAs) from a homologous series of different molar masses were nanoprecipitated at different initial polymer concentrations from two organic solvents, acetone and tetrahydrofuran (THF), into water without surfactant according to a standardized procedure. Quasi-elastic light scattering and gel permeation chromatography with universal detection were used respectively to size the particles and to determine the molar mass distribution of the polymeric chains forming both nanoparticles and bulk aggregates. The intrinsic viscosity of the polymers as a function of molar mass and solvent were determined by kinematic viscosity measurements in organic solutions. High yields of small nanoparticles were obtained with polymers of lower molar mass (22600 and 32100 g/mol). For a given polymer concentration in organic solution, the particle diameter was always lower from acetone than from THF. For initial molar masses higher than 32100 g/mol, only dilute organic solutions gave significant yields of nanoparticles. Furthermore, polymer mass fractionation occurred with increasing initial molar mass and/or concentration: the nanoparticles were formed by polymeric chains of molar masses significantly lower than the average initial one. In general, nanoparticle production was satisfactory when the initial organic solution of polymer was in the dilute rather than the semi-dilute regime. Moreover, acetone, which acted as a theta solvent for PLA, always led to smaller particles and better yields than THF.

New nanoparticles obtained by co-assembly of amphiphilic cyclodextrins and nonlamellar single-chain lipids: Preparation and characterization.

This work aimed at preparing new nanoscale assemblies based on an amphiphilic bio-esterified ß-cyclodextrin (ß-CD), substituted at the secondary face with n-decanoic fatty acid chains (ß-CD-C10), and monoolein (MO) as new carriers for parenteral drug delivery. Stable binary (ß-CD-C10/MO) and ternary (ß-CD-C10/MO/stabilizer) nanoscale assemblies close to 100nm in size were successfully prepared in water by the solvent displacement method. The generated nanoparticles were fully characterized by dynamic light scattering, transmission electron microscopy, small-angle X-ray scattering, residual solvent analysis, complement activation and the contribution of each formulation parameter was determined by principal component analysis. The ß-CD-C10 units were shown to self-organize into nanoparticles with a hexagonal supramolecular packing that was significantly modulated by the molar ratio of the constituents and the presence of a steric or electrostatic stabilizer (DOPE-PEG2000 or DOPA/POPA, respectively). Indeed, nanoparticles differing in morphology and in hexagonal lattice parameters were obtained while the co-existence of multiple mesophases was observed in some formulations, in particular for the ß-CD-C10/MO/DOPA and ß-CD-C10/MO/POPA systems. The mixed ß-CD-C10/MO/DOPE-PEG2000 nanoparticles (49:49:2 in mol%) appeared to be the most suitable for use as a drug delivery system since they contained a very low amount of residual solvent and showed a low level of complement C3 activation.

Formulation and in vitro efficacy of liposomes containing the Hsp90 inhibitor 6BrCaQ in prostate cancer cells.

6BrCaQ is a promising anti-cancer agent derived from novobiocin, which has been shown to inhibit Hsp90. 6BrCaQ was loaded into nanometer-scaled phospholipid vesicles (liposomes) suitable for drug delivery to solid tumors. The effective incorporation of the drug within the phospholipid bilayer was investigated by differential scanning calorimetry. Liposomal 6BrCaQ showed good activity on PC-3 cell lines in vitro in terms of apoptosis induction and cell growth arrest in G2/M. Liposomes containing 6BrCaQ were also shown to slow down migration of PC-3 cells in presence of chemokine ligand 2 and to synergize with doxorubicin. Several Hsp90 targeting molecules like geldanamycin induce accumulation of Hsp70, leading to cytoprotection and often correlated with poor prognosis. In this study, we did not report any Hsp70 induction after treatment with liposomal 6BrCaQ but a decrease in Hsp90 and CDK-4 protein expression, indicating an effect on the chaperon machinery. Liposomal encapsulation of 6BrCaQ revealed promising anti-cancer effects and a better understanding of its mechanism of action.

Liposome retention in size exclusion chromatography.

BACKGROUND: Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. RESULTS: Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. CONCLUSION: Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography.

Physico-chemical characterization of polysaccharide-coated nanoparticles.

A series of amphiphilic copolymers (PCL-DEX) made of poly(epsilon-caprolactone) (PCL) side chains grafted onto a dextran (DEX) backbone, was used to modify the surface of PCL nanoparticles. PCL-DEX nanoparticles were prepared by a technique derived from emulsion-solvent evaporation. The purpose of the present study was to investigate the DEX coating (quantification, conformation, mobility) in order to better understand particle surface-protein interactions. The DEX coating was deeply examined using different complementary methods: zeta potential measurement, specific degradation of the DEX shell by dextranase, energy-filtering transmission electron microscopy coupled to image-spectrum electron energy-loss spectroscopy, electronic paramagnetic resonance, high performance size exclusion chromatography as well as nonspecific bovine serum albumin adsorption. All our data together supported a core-shell structure of the nanoparticles, DEX moieties constituting the external coating. The amount of DEX located on the nanoparticle surface was estimated to 70%. The organisation of the shell including chains density and mobility was found to be dramatically influenced by DEX molar mass. The steric repulsion conferred by the presence of DEX at the surface of the nanoparticles decreased the adsorption of albumin. The nanoparticle-protein interaction was, however, greatly influenced by the polysaccharide conformation onto the surface.

Evaluation of buccal methyl-beta-cyclodextrin toxicity on human oral epithelial cell culture model.

Cyclodextrins, especially methylated beta-cyclodextrins offer several advantages for drug delivery which include improved drug solubilization, protection against physicochemical and enzymatic degradation, as well as a potential for absorption improvement. However, little or no data are available for their use as drug penetration enhancer via the buccal route. This study focuses on the toxicity of randomly methylated beta-cyclodextrin (RAMEB) on buccal mucosa using a reconstituted human oral epithelium model composed of TR 146 cells. Toxicity of RAMEB on TR 146 cells was evaluated by measuring cell viability (MTT assay) and membrane damages followed by LDH release after single and repeated exposures to RAMEB solutions. Inflammatory effects of RAMEB are also considered by measuring expression of interleukin-1alpha and are supported by histological examination. The present results indicate that 10% RAMEB results in cytotoxic and inflammatory effects depending on time exposure, whereas 2% and 5% RAMEB do not induce tissue damages even after 5 days of repeated exposures. Therefore, the highly water-soluble RAMEB is thought to be a safe candidate as an excipient for buccal mucosal drug delivery.

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.