Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

ζ-potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0). Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan-decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan-hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.     

Hydrodynamic and molecular characteristics of graft copolymers of chitosan with acrylamide

The conformational properties of macromolecules of chitosan and its copolymers with acrylamide in a mixed solvent 0.33 M CH₃COOH + 0.3 M NaCl have been investigated by means of translation diffusion and viscometry. The copolymer macromolecules in a solvent suppressing polyelectrolyte effects possess a higher intracoil density (ρ_av = 0.010 g/cm³) than the chitosan macromolecules (ρ_av = 0.006 g/cm³), even though the hydrodynamic radius R _h of chitosan is smaller by a factor of ～1.5.     

Resealing of Polyelectrolyte Capsules after Core Removal

Poly(styrene sulfonate) and poly(allylamine hydrochloride) layers have been adsorbed supplementarily on polyelectrolyte capsules. The permeability of the original capsules consisting of four layer pairs was of the order of 10^–5 m/s for fluorescein. They were also permeable for macromolecules. Polyelectrolyte layers adsorbed afterwards reduced the permeability by three orders of magnitude for small molecules. These findings are interpreted as a resealing of pores, induced by the osmotic stress during fabrication.     

Entrapment of α‐Chymotrypsin into Hollow Polyelectrolyte Microcapsules

Stable hollow polyelectrolyte capsules were produced by the layer‐by‐layer assembling of non‐biodegradable polyelectrolytes – poly(allylamine) and poly(styrenesulfonate) on melamine formaldehyde microcores followed by the core decomposition at low pH. A proteolytic enzyme, α‐chymotrypsin, was encapsulated into these microcapsules with high yields of up to 100%. The encapsulation procedure was based on the protein adsorption onto the capsule shells and on the pH‐dependent opening and closing of capsule wall pores. The protein in the capsules retained a high activity, and thermo‐ and storage stability. The nanostructured polyelectrolyte shell protected the proteinase from a high molecular weight inhibitor. Such enzyme‐loaded capsules can be used as microreactors for biocatalysis.     

Graphite Based Microsensors Developed for the Electrochemical Determination of L‐Tyrosine from Pharmaceutical Samples

Two electrochemical sensors were proposed for the determination of L‐tyrosine from pharmaceutical capsules. The electrochemical sensors were based on plain graphite paste and chitosan modified graphite paste. Working conditions, e. g. pH, supporting electrolytes have been optimized. The results revealed for both electrodes very low limits of quantification (LOQ) (0.18 mg L^?1 and 0.0018 mg L^?1) for plain graphite paste based sensors and for chitosan modified paste sensors respectively. A higher sensitivity of 7.95×10^?10 A mg L^?1 was obtained for the sensor based on plain graphite paste (G). The uniformity content test showed that L‐Tyrosine can be recovered in pharmaceutical capsules with an average value higher than 98.00 % and a relative standard deviation (RSD%) value less than 1.00 % (N=5).     

Motion‐Based,High‐Yielding,and Fast Separation of Different Charged Organics in Water

We report a self‐propelled Janus silica micromotor as a motion‐based analytical method for achieving fast target separation of polyelectrolyte microcapsules, enriching different charged organics with low molecular weights in water. The self‐propelled Janus silica micromotor catalytically decomposes a hydrogen peroxide fuel and moves along the direction of the catalyst face at a speed of 126.3 μm s^?1. Biotin‐functionalized Janus micromotors can specifically capture and rapidly transport streptavidin‐modified polyelectrolyte multilayer capsules, which could effectively enrich and separate different charged organics in water. The interior of the polyelectrolyte multilayer microcapsules were filled with a strong charged polyelectrolyte, and thus a Donnan equilibrium is favorable between the inner solution within the capsules and the bulk solution to entrap oppositely charged organics in water. The integration of these self‐propelled Janus silica micromotors and polyelectrolyte multilayer capsules into a lab‐on‐chip device that enables the separation and analysis of charged organics could be attractive for a diverse range of applications.     

Forming Lipid Bilayer Membrane Arrays on Micropatterned Polyelectrolyte Film Surfaces

A novel method of forming lipid bilayer membrane arrays on micropatterned polyelectrolyte film surfaces is introduced. Polyelectrolyte films were fabricated by the layer‐by‐layer technique on a silicon oxide surface modified with a 3‐aminopropyltriethoxysilane (APTES) monolayer. The surface pK_a value of the APTES monolayer was determined by cyclic voltammetry to be approximately 5.61, on the basis of which a pH value of 2.0 was chosen for layer‐by‐layer assembly. Micropatterned polyelectrolyte films were obtained by deep‐UV (254 nm) photolysis though a mask. Absorbed fluorescent latex beads were used to visualize the patterned surfaces. Lipid bilayer arrays were fabricated on the micropatterned surfaces by immersing the patterned substrates into a solution containing egg phosphatidylcholine vesicles. Fluorescence recovery after photobleaching studies yielded a lateral diffusion coefficient for probe molecules of 1.31±0.17 μm² s^?1 in the bilayer region, and migration of the lipid NBD PE in bilayer lipid membrane arrays was observed in an electric field.     

Diffusion Through Membranes of the Polyelectrolyte Complex of Chitosan and Alginate

The swelling of membranes of the polyelectrolyte complex (PEC) between chitosan and alginate shows a similar pattern to that of other PECs. However, if the swelled membranes are dried, a second swelling process is seen which exhibits Fickian behavior. The apparent activation energy was estimated to be 32.8 kJ · mol^?1. The release rate of model solutes was highly dependent on their molecular weight and the pH of the medium.

Arrhenius type plot of the temperature dependence of the apparent diffusion coefficients for the membrane of the polyelectrolyte complex between chitosan and alginate in water.     

Layer-by-layer films from hyaluronan and amine-modified hyaluronan

Hyaluronan is a polysaccharide that is increasingly investigated for its role in cellular adhesion and for the preparation of biomimetic matrices for tissue engineering. Hyaluronan gels are prepared for application as space fillers, whereas hyaluronan films are usually obtained by adsorbing or grafting a single hyaluronan layer onto a biomaterial surface. Here, we examine the possibility to employ the layer-by-layer technique to deposit thin films of cationic-modified hyaluronan (HA+) and hyaluronan (HA) of controlled thicknesses. The buildup conditions are investigated, and growth is compared to that of other polyelectrolyte multilayer films containing either HA as the polyanion or HA+ as the polycation. The films could be formed in a low ionic strength medium but are required to be cross-linked prior to contact with a physiological medium. NIH3T3 fibroblasts were perfectly viable on self-assembled hyaluronan films with, however, a preference for hyaluronan ending films. These findings point out the possibility to tune the thickness of thin hyaluronan films at the nanometer scale. Such architectures could be employed for investigating cell/substrate interactions or for functionalizing biomaterial surfaces.     

Development of polyelectrolyte complexes of chitosan and phosphotungstic acid as pervaporation membranes for dehydration of isopropanol

Using a solution technique, chitosan-based polyelectrolyte complexes (PECs) were developed as pervaporation membranes by incorporating phosphotungstic acid (PTA). The resulting membranes were characterized by Fourier transform infrared spectroscopy (FTIR), wide-angle X-ray diffraction (WAXD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). Membranes were tested for their ability to separate water–isopropanol mixtures by pervaporation in the temperature range of 30–50 °C. The experimental results demonstrated that both flux and selectivity were increased simultaneously with increasing PTA content in the membrane. The permeation flux of pure chitosan membrane was increased dramatically from 4.13 to 11.70 × 10⁻² kg/m² h and correspondingly its separation factor was increased from 4490 to 11,241 and then decreased to 7490 at 30 °C for 10 mass% of water in the feed. The total flux and flux of water were found to be almost overlapping particularly for PECs membranes, suggesting that these could be used effectively to break the azeotropic point of water–isopropanol mixtures. From the temperature dependency of diffusion and permeation values, the Arrhenius activation parameters were estimated and discussed in the context of membranes efficiency. The pure chitosan and a small amount of PTA-incorporated PECs membranes exhibited positive heat of sorption while other PECs membranes exhibited negative heat of sorption, giving exothermic contribution.     

New-chitosan characterization and its bioassay in different salinity solutions using <Emphasis Type="Italic">Artemia salina</Emphasis> as bio tester

New chitosan powder extracted from eggs capsules of Rapana venosa and commercial chitosan powder were analyzed by spectral (UV–Vis) and color methods. The novelty of this study consists in the first time toxicity evaluation of the chitosan extracted from the walls of Rapana venosa eggs capsules. The evaluation was focused on the cytological behaviour of the new-chitosan particles in two different salinity conditions (3 and 35‰), at different concentrations, on bio tester organisms. The aim of this study was to determine the biological effect of the chitosan easily extracted from new marine sources and with a low financial effort. The experimental studies were performed by exposure of Artemia salina larvae, naupliar stage in saline chitosan solutions. The larvae were picked-up in 24 h time, after hatching and they were placed in the experimental recipients of 1 mL volume capacities, three repetitions were made for each test. The bioassay effects were recorded at 24 and 48 h after experiment started. The toxicity was indicated by the registration of larvae mortality, probably, induced by the cell growth inhibitions. The obtained results showed that the high toxicity effects could be observed at low concentrations of chitosan, in law salinity solutions. Due to this cytotoxicity variability, chitosan solutions could be useful in a wide range of applications (antifouling, biomedical or sewage cleaning).     

胶束增强型聚电解质胶囊的结构研究

研究了胶束增强型聚电解质(PAH/PSS和PADA/PSS)胶囊在不同溶液环境中的形貌变化,发现这种新型的胶囊具有迥异于传统聚电解质胶囊的囊壁结构;研究了二维聚电解质复合膜与模板溶解液中嵌段共聚物PS-b-PAA胶束之间的相互作用,发现胶束层可以通过静电力与聚电解质胶囊囊壁相互作用.同时,模拟模板溶出后聚电解质胶囊内部的环境条件,研究了嵌段共聚物胶束在胶囊内部的存在状态及其在透析过程中的变化规律,认为共聚物可以通过疏水作用沉积于聚电解质复合膜的内壁,并通过Ca2+离子的桥联作用稳定,也就是在聚电解质复合膜层基础上又形成了一层胶束层.即这种胶束增强型聚电解质微胶囊的囊壁是由聚电解质层和胶束层所形成的双层结构.用这种双层结构模型,我们合理解释了胶囊在高盐离子浓度下的形貌变化.     

Diffusion of Oligonucleotides from within Iron‐Cross‐Linked,Polyelectrolyte‐Modified Alginate Beads: A Model System for Drug Release

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe³⁺‐cross‐linked and polyelectrolyte‐doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross‐linking, that is, the absence of a surface‐layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well‐defined component in drug‐release systems and for the oligonucleotide‐release transduction steps in drug‐delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.     

Ordering in Bio-Polyelectrolyte Chitosan Solutions

The scaling of the polyelectrolyte scattering peak in chitosan solutions, as deduced from the relation q_max∼c_p^α was studied by synchrotron SAXS as a function of the charge density of the polymer. We observe a variation in the α exponent corresponding to the limit of the ionic condensation, by varying the degree of acetylation of the polymer. The nature of the solution medium also affects the polyelectrolyte peak, and it is shown that in alcoholic/water mixtures, the lower dissociation of the acid induces a lower charge density, thus influencing the polyelectrolyte ordering.     

Formation and properties of positively charged colloids based on polyelectrolyte complexes of biopolymers

Formation of colloids based on polyelectrolyte complexes (PECs) was mainly studied with synthetic polyelectrolytes. In this study, we describe the elaboration of positively charged PEC particles at a submicrometer level obtained by the complexation between two charged polysaccharides, chitosan as polycation and dextran sulfate (DS) as polyanion. The complexes were elaborated by dropwise addition of default amounts of DS to excess chitosan. Quasi-elastic light scattering was used to investigate in detail the influence of the characteristics of components (chain length, degree of acetylation) and parameters linked to the reaction of complexation (molar mixing ratio, ionic strength, concentration in polymer) on the sizes and polydispersity of colloids. Chain length of chitosan is the major parameter affecting the dimensions of the complexes, high molar mass chitosans leading to the largest particles. Variations of hydrodynamic diameters of PECs with the molar mass of chitosan are consistent with a mechanism of particle formation through the segregation of the neutral and then hydrophobic blocks of the polyelectrolyte complexed segments. Resulting particles display probably a structure constituted by a neutral core surrounded by a chitosan shell ensuring the colloidal stabilization. Such a structure was evidenced by measurements of electrophoretic mobilities revealing that the positive charge of particles was decreasing with pH, in relation with the neutralization of excess glucosamine hydrochloride moieties.     

Evaluation of retention and release processes of two antibiotics from the biocompatible core-shell microparticles

By dropwise addition of a chitosan solution into different non-solvent, such as: 1 N and 2 N NaOH as well as 1 N NaOH: C₂H₅OH mixture (2:1, v/v) at temperature of 25 °C and 50 °C under stirring, the spherical pure chitosan microparticles were performed. As solvents for chitosan was used 0.1 N acetic acid or 0.1 N HCl. The immersion of the pure chitosan microparticles in hyaluronan solution led to complex microparticles, namely chitosan microparticles covered by a hyaluronan layer. For all the microparticles performed the behaviours in the retention process of two antibiotics: chloramphenicol succinate sodium salt and cefotaxime sodium salt were analyzed. Also, the study shows the release behaviour of cefotaxime sodium salt by the microparticles loaded with this drug. Among the microparticles performed a type of complex microparticles can be considered a suitable drug delivery system for cefotaxime. These microparticles were performed by dropwise addition of chitosan solution in 0.1 N acetic acid into the 1 N NaOH: C₂H₅OH (2:1, v/v) non-solvent at 20 °C for 3 h, followed by their washing up to alkalinity loss and the immersion in hyaluronan solution of 10 g/L concentration for 24 h.     

Colloid Stabilization by an Oppositely Charged Polysaccharide: Mechanism of Interaction and Interface Studied with Synchrotron X-Rays

The liposome surface is modeled by a 2-D lipid monolayer made of behenic acid forming a negatively charged interface. The properties at the air/liquid interface were examined by pressure-area isotherms in a Langmuir trough introducing diluted chitosan solution in the subphase. X-ray reflectivity of the interface was measured in the same conditions in order to determine the layer thickness of the chitosan adsorbed on the behenic acid monolayer formed on water. Influence of pH of the subphase and molecular weight of adsorbed chitosan was investigated. All these results allow confirming that the charge and the stability of the lipid layer are controlled by the nature of the polyelectrolyte and the nature of the medium. In particular, the use of biocompatible charged polysaccharides is of interest for biomedical applications.     

Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film

An amperometric pesticides inhibition biosensor has been developed and used for determination of pesticides in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/polystyrensulfonate) were coated on the glass carbon electrode. Then, acetylcholinesterase was immobilized on the electrode based on surface-treated nanoporous ZrO₂/chitosan composite film as immobilization matrix. As a modified substrate, acetylthiocholine was hydrolysed by acetylcholinesterase and produced thiocholine which can be oxidized at +700?mV vs. SCE. Pesticides inhibit the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to acetylthiocholine within 9.90?×?10^?6 to 2.03?×?10^?3?M. The electrode provided a linear response over a concentration range of 6.6?×?10^?6 to 4.4?×?10^?4?M for phoxim with a detection limit of 1.3?×?10^?6?M, over a range of 1.0?×?10^?8 to 5.9?×?10^?7?M for malathion, and over a range of 8.6?×?10^?6 to 5.2?×?10^?4?M for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample.     

Polyelectrolyte complex materials consisting of antibacterial and cell-supporting layers

The characterization of a polyelectrolyte complex material comprised of two biopolymers, a chitosan upper layer and a gellan gum under layer, is reported. It is shown that the upper layer of chitosan with incorporated levofloxacin displays an antibacterial activity, while the under layer of a gellan gum/TiO(2) composite supports the growth of fibroblastic cells.     

Synthesis of binary polyelectrolyte/inorganic composite capsules of micron size

Different approaches for the synthesis of binary polyelectrolyte/inorganic layered composite capsules of micron size are described. As the polyelectrolyte part of the composite, a poly(styrene sulfonate)/poly(allylamine hydrochloride) complex was taken; the inorganic component was composed of magnetic nanoparticles (Fe₃O₄, CoFe₂O₄, MnFe₂O₄, ZnFe₂O₄), insulator nanoparticles (rare-earth fluorides) or metal nanoparticles (Ag). An inner inorganic layer was formed inside the hollow polyelectrolyte capsule via chemical or photochemical reaction in a spatially restricted capsule volume. The inorganic nanophase synthesized was characterized by transmission electron microscopy, scanning electron microscopy, and wide angle X-ray scattering techniques and weakly crystallized particles 6–9 nm in diameter were detected, presumably attached to the inner side of the capsule shell. Polyelectrolyte capsules filled with ferrite (magnetite) particles possess substantial magnetic activity and are easily manipulated in water solution by an external magnetic field.