Films of end-grafted hyaluronan are a prototype of a brush of a strongly charged, semiflexible polyelectrolyte with intrinsic excluded volume

Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the organization and function of pericellular coats and extracellular matrices in vertebrates, and that is becoming increasingly popular in biomedical applications. To gain insight into the physical phenomena that govern the mechanical behavior of HA assemblies, we have studied the response of films of end-grafted HA to compression over a large range of ionic strength. Compression forces were measured as a function of the absolute distance between a colloidal probe and the planar surface on which the HA film was constructed, using a combined atomic force microscopy and reflection interference contrast microscopy setup. The HA films were well-defined in the sense that they are made of chains with a narrow size distribution that are grafted at controlled density to a solid support. Detailed comparison of the experimental data with analytical expressions derived from polymer and polyelectrolyte brush theory reveals that films of end-grafted HA behave as strongly charged polyelectrolyte brushes. To quantitatively reproduce the experimental data, intrinsic excluded volume interactions and chain stiffness of the polymer backbone must be taken into account. At low ionic strength, chains become almost fully stretched. In our experimental system, several micrometer thick films are formed that reach a hydration of up to 99.98%, and the brush thickness decreases by more than 5-fold with increasing ionic strength. More generally, the study provides quantitative theoretical predictions for the film thickness and compressive response as a function of HA length, grafting density and ionic strength.     

Hydrophilic monolayer formation of adsorbed cationic starch and cationic hydroxyethyl cellulose derivatives on polyester surfaces

Cationic starch, cationic cellulose derivatives, and hydrophobically modified cationic cellulose were physically adsorbed from aqueous solution onto oppositely charged hydrophobic polyester (poly(ethylene terephthalate)) fabric and nonwoven, and this resulted in hydrophilic surface properties. Surface coverage of the polysaccharides occurred primarily by strong electrostatic interactions, and the surface characteristics were evaluated by measuring the time required for a water droplet to be absorbed into the polyester material as well as by electron spectroscopy for chemical analysis (ESCA). From a comparison of the adsorption characteristics we assess the polysaccharide-dependent and substrate-dependent adsorption behavior and discuss the similarities and differences in the hydrophilic properties and wettability observed. In particular, the temperature of the cationic polysaccharide solutions in which the substrate was immersed, the configuration of the polymer in solution, and the presence of hydrophobic substituents on the cationic moiety have a considerable effect on the polysaccharide affinity and its adsorption on the surface, irrespective of the substrate type (fabric or nonwoven). We also evaluate the relative contribution of the polyelectrolyte molecular weight, concentration in solution, and degree of charge density along the polymer chain which determine the range of interactions and alter surface hydroplilicity dependent on the type of substrate.     

Effect of polyelectrolyte adsorption on lateral distribution and dynamics of anionic lipids: a Monte Carlo study of a coarse-grain model

We employ Monte Carlo simulations to investigate the interaction between an adsorbing linear flexible cationic polyelectrolyte and a ternary mixed fluid membrane containing neutral (phosphatidylcholine, PC), monovalent (phosphatidylserine, PS), and multivalent (phosphatidylinositol, PIP₂) anionic lipids. We systematically explore the influences of polyelectrolyte chain length, polyelectrolyte charge density, polyelectrolyte total charge amount, and salt solution ionic strength on the static and dynamic properties of different anionic lipid species. Our results show that the multivalent PIP₂ lipids dominate the polyelectrolyte–membrane interaction and competitively inhibit polyelectrolyte–PS binding. When the total charge amount of the polyelectrolyte is less than that of the local oppositely charged PIP₂ lipids, the polyelectrolyte can drag the bound multivalent lipids to diffuse on the membrane, but cannot interact with the PS lipids. Under this condition, the diffusion behaviors of the polyelectrolyte closely follow the prediction of the Rouse model, and the polyelectrolyte chain properties determine the adsorption amount, concentration gradients, and hierarchical mobility of the bound PIP₂ lipids. However, when the total charge amount of the polyelectrolyte is larger than that of the local PIP₂ lipids, the polyelectrolyte further binds the PS lipids around the polyelectrolyte–PIP₂ complex to achieve local electrical neutrality. In this condition, parts of the polyelectrolyte desorb from the membrane and show faster mobility, and the bound PS presents much faster mobility than the segregated PIP₂. This work provides an explanation for heterogeneity formation in different anionic lipids induced by polyelectrolyte adsorption.     

Polyelectrolytes at the endothelial cell surface.

It is recalled that the tension in a stretched polyelectrolyte chain mechanically compensates both the coulomb interaction and the hydrostatic pressure increase around the chain in a compromise which minimises the free energy and keeps water chemical potential constant throughout. Stretching strongly favors parallel cylinder nematic order in polyelectrolyte brushes on a surface or in the slit between two surfaces when the polyelectrolyte chains function as bridges. Strong, stiffly stretched chains result when the molarity of the fixed charge distribution is larger than the molarity of the neutral salt solution with which the brushes are in equilibrium. The relevance of these two systems to the endothelial cells which cover the walls of blood vessels is discussed.     

Adsorption of a strong polyelectrolyte to model lignin surfaces

The adsorption of a strong, highly charged cationic polyelectrolyte to a kraft lignin thin film was investigated as a function of the adsorbing solution conditions using the quartz crystal microbalance. The polyelectrolyte, PDADMAC, with a molecular weight of 100 kDa and one cationic charge group per monomer, was adsorbed to the anionically charged lignin film in the pH range 3.5-9.5 in electrolyte solution of 0.1 to 100 mM NaCl. At low pH, the adsorbed amount of PDADMAC was minimal, however, this increased as a function of increasing pH. Indeed, the surface excess increased significantly at about pH 8.5, where ionization of the phenolic groups on the lignin macromolecule may be expected. Furthermore, at this elevated pH, the adsorbed amount of PDADMAC decreased as the ionic strength of the solution increased above 1 mM. This is due to the competitive adsorption of counterions to the lignin surface and indicates that the adsorption of PDADMAC to lignin is of a pure electrosorption nature.     

Calculation of the Electrophoretic Mobility of a Particle Bearing Bound Polyelectrolyte Using the Nonlinear Poisson-Boltzmann Equation

A numerical method for determining the electrophoretic mobility of a polyelectrolyte-coated particle is presented. The particle surface is modeled as having a permeable layer of polyelectrolyte molecules anchored to its surface. Fluid flow within the polyelectrolyte layer is subject to Stokes drag arising from the polyelectrolyte segments. The method allows arbitrary distribution of polymer segments and charge density normal to the surface to be used. The hydrodynamic plane of shear may also be varied. The potential profile is determined by a numerical solution to the nonlinearized Poisson-Boltzmann equation. The potential profile is then used in a numerical solution to the Navier-Stokes equation to give the required mobility. The use of the nonlinearized Poisson-Boltzmann equation extends the results to higher charge density/lower ionic strength conditions than previous treatments. The surface potentials and mobilities for three limiting charge distributions are compared for both the linear and nonlinear treatments to delimit the range of validity of the linear treatment. The utility of the numerical, nonlinear treatment is demonstrated by an improved fit to the electrophoretic mobility of human erythrocytes as a function of ionic strength in the range 10 to 150 mM.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Adsorption of charged amphiphiles to oppositely charged polysaccharides—A study of the influence of polysaccharide structure and hydrophobicity of the amphiphile molecule

The binding of three different amphiphiles (with different hydrophobic characters) to oppositely charged polyelectrolytes (κ-carrageenan, dextran sulphate, alginate, and hyaluronan) is investigated. It is shown that the degree of binding is related not only to the hydrophobicity of the amphiphile and to the charge density of the polyelectrolyte, but also to the flexibility of the polyion. Furthermore, the cooperativity in the binding of amphiphile to polyelectrolyte was observed in all cases except for hyaluronan, which showed a very weak adsorption isotherm. The effect of polyelectrolyte concentration on the adsorption isotherm was also investigated for dextran sulphate and alginate. The effect of concentration was found to be weak. © 1997 John Wiley & Sons, Inc. Biopoly 41: 765–772, 1997     

Polyelectrolyte/amphiphile interaction studied by surface tension measurements

The interaction of κ-carrageenan with three positively charged drug molecules with amphiphile character has been examined using surface tension measurements. The surface tension was measured by the pendant drop method which makes possible the determination at an apparent steady state which is important for polymeric systems. The results are compared with adsorption isotherms from dialysis equilibrium. The surface tension data, show that the presence of κ-carrageenan in the amphiphile solutions leads to an increased and pronounced lowering of the surface tension in a low concentration range of amphiphile. It is also shown that not only the hydrophobicity of the amphiphile but also the structure of the polyelectrolyte (charge density and helix-coil structure) largely determine the extent of interaction.     

Mechanisms of polyelectrolyte enhanced surfactant adsorption at the air-water interface

Chitosan, a naturally occurring cationic polyelectrolyte, restores the adsorption of the clinical lung surfactant Survanta to the air-water interface in the presence of albumin at much lower concentrations than uncharged polymers such as polyethylene glycol. This is consistent with the positively charged chitosan forming ion pairs with negative charges on the albumin and lung surfactant particles, reducing the net charge in the double-layer, and decreasing the electrostatic energy barrier to adsorption to the air-water interface. However, chitosan, like other polyelectrolytes, cannot perfectly match the charge distribution on the surfactant, which leads to patches of positive and negative charge at net neutrality. Increasing the chitosan concentration further leads to a reduction in the rate of surfactant adsorption consistent with an over-compensation of the negative charge on the surfactant and albumin surfaces, which creates a new repulsive electrostatic potential between the now cationic surfaces. This charge neutralization followed by charge inversion explains the window of polyelectrolyte concentration that enhances surfactant adsorption; the same physical mechanism is observed in flocculation and re-stabilization of anionic colloids by chitosan and in alternate layer deposition of anionic and cationic polyelectrolytes on charged colloids.     

Influence of polyelectrolyte chemical structure on their interaction with lipid membrane of zwitterionic liposomes

In this paper we extend our previous experimental work on interaction between polyelectrolytes and liposomes. First, the adsorption of chitosan and alkylated chitosan (cationic polyelectrolytes) with different alkyl chain lengths on lipid membranes of liposomes is examined. The amount of both chitosans adsorbed remains the same even if more alkylated polysaccharide has to be added to get saturation if compared with unmodified chitosan. It is demonstrated that alkyl chains do not specifically interact with the lipid bilayer and that electrostatic interaction mechanism governs the chitosan adsorption. The difference observed between unmodified and alkylated chitosans behavior to reach the plateau can be interpreted in terms of a competition between electrostatic polyelectrolyte adsorption on lipid bilayer and hydrophobic autoassociation in solution (which depends on the alkyl chain length). Second, interaction of liposomes with hyaluronan (HA) and alkylated hyaluronan (anionic polyelectrolytes) is analyzed. The same types of results as discussed for chitosan are obtained, but in this case, autoassociation of alkylated HA only occurs in the presence of salt excess. Finally, a first positive layer of chitosan is adsorbed on the lipid membrane, followed by a second negative layer of HA at three different pHs. This kind of multilayer decoration allows the control of the net charge of the composite vesicles. A general conclusion is that whatever the pH and, consequently, the initial charge of the liposomes, chitosan adsorption gives positively charged composite systems, which upon addition of hyaluronan, give rise to negatively charged composite vesicles.     

Light scattering,CD, and ligand binding studies of ferrihemoglobin–polyelectrolyte complexes

Quasi‐elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL or greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intra‐polymer complex) with apparent diameter approximately 2.5 times that of the protein‐free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin–polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin–poly(2‐acrylamide‐2‐methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte‐free protein, but minimal change is induced by complexation with an AMPS‐based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer. © 1999 John Wiley & Sons, Inc. Biopoly 50: 153–161, 1999     

The surface charge of human glutaraldehyde-fixed erythrocytes

We measured the number of charged residues at the surface of fresh human erythrocytes after fixation with glutaraldehyde by polyelectrolyte titration using polycations of different chemical composition and various molecular weights. Independent of the reagents used, the number was (8.5 +/- 1.5) X 10(8) negatively charged residues per cell. The surface charge density of 6.3 e/nm2 is considerably higher than that calculated from the electrophoretic mobility for which the surface charge density is calculated to be 0.09 e/nm2.     

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of μ-bioreactors. The spherical μ-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of μ-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.     

Large and giant vesicles "decorated" with chitosan: effects of pH, salt or glucose stress, and surface adhesion

This paper describes the behavior of large and giant unilamellar vesicles (LUVs and GUVs, respectively) in the presence of chitosan, a positively charged polyelectrolyte. Variation of the zeta-potential of LUVs as a function of chitosan concentration is studied for two different molecular weights (MW) after a preliminary study devoted to pH and salt effects on zeta-potential in order to discriminate among the effects of protons, salt, and chitosan concentrations. The difference observed between pH and salt effects on the one hand and chitosan on the other allows us to conclude there is a strong LUV-chitosan interaction. In presence of chitosan, the zeta-potential of LUVs becomes positive and two distinct regimes of variation are suggested and interpreted as follows: a first step consists of chitosan adsorption flat on the membrane (independent of MW) followed by a possible reorganization of the polymer of higher molecular weight on the surface, giving rise to loops. Then a comparative observation of the effect of pH and salt by optical microscopy is made on naked and chitosan-decorated GUVs. Results further confirm a membrane-chitosan interaction and are interpreted in the light of the results obtained for LUVs in terms of both electrostatic and hydrophobic interaction. A large majority of decorated vesicles remain stable down to pH = 1 while in the absence of chitosan they burst quickly at pH between 2 and 3. Osmotic pressure and net charge change due to addition of HCl results in a decrease in the diameter of the decorated vesicles, which remain spherical while forming tubes of lipids. In presence of NaCl, a higher resistance of decorated vesicles is also evidenced (they are stable for NaCl concentrations up to 10-1 M while naked vesicles burst when [NaCl] is between 10-2 and 10-3 M). At higher salt concentration, aggregation of decorated vesicles occurs, which is attributed to the screening of electrostatic repulsions between vesicles covered by the positively charged chitosan. Finally, adhesion of vesicles on a positively charged surface is investigated. In absence of chitosan, the vesicles immediately burst when they come in contact with the surface. On the contrary, suspension of chitosan-vesicles remain stable down to pH = 1.5. Under gentle flow vesicles move: they do not adhere on the substrate, probably due to the repulsion between positively adsorbed charged chitosan and substrate; spherical deflation occurs, but in this case daughter vesicles are formed instead of lipid tubes.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.     

Charge patch attraction and reentrant condensation in DNA-liposome complexes

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.     

Interactions of voltage-sensing dyes with membranes. II. Spectrophotometric and electrical correlates of cyanine-dye adsorption to membranes.

The adsorption to bilayer membranes of the thiadicarbocyanine dyes, diSCn(5), has been studied as a function of the membrane's surface-charge density, the aqueous ionic strength, and the length (n) of the hydrocarbon side chain of the dye. "Probe" measurements in planar bilayers, microelectrophoresis of liposomes, and measurement of changes in dye absorbance and fluorescence in liposomes were used to study dye adsorption to membranes. These measurements indicated that the membrane:water partition coefficient for the dye monomer increases with the length of the hydrocarbon side chain. However, the formation of large aggregates in the aqueous phase also increases with increasing chain length and ionic strength so that the actual dye adsorbing to the membrane goes through a maximum at high but not at low ionic strengths. More dye adsorbs to negatively charged than neutral membranes. Membrane-bound dye spectra were easily resolved in negatively charged liposomes where it was observed that these dyes could exist as monomers, dimers, and large aggregates. For diSC1(5) a spectral peak was observed at low but not high ionic strengths (i.e. the conditions in which this dye appears to form voltage-gated channels) corresponding to small aggregates which appeared to adsorb to the membrane. Finally, the adsorption of these dyes to membranes results in more positive electrostatic potentials composed primarily of dye-induced "boundary" potentials and somewhat less of "double-layer" potentials.     

Molecular dynamics simulation of isothermal crystallisation of polymer chains around single polymer lamella

The isothermal crystallisation of polyethylene (PE) chains around single PE lamella in vacuum is investigated by molecular dynamic simulation. The crystallisation process is analysed in terms of the orientational order parameters, principal moments of inertia for the simulated systems. The effects of charge interactions between the polymer chains and lamella are discussed. It is found that the crystallisation process for uncharged systems can be divided into three stages: (1) adsorption, (2) orientation and (3) arrangement. The single polymer lamella changes a little during the three stages. PE chains are arranged parallel to the chain direction of the stems in the crystalline state. When considering the effect of charge interactions between the polymer chains and lamella, a different crystallisation process appears. The single polymer lamella is affected by the charged polymer chains.     

Protein adsorption at polymer-grafted surfaces: comparison between a mixture of saliva proteins and some well-defined model proteins

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, beta-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.