Interaction between chitosan and bovine lung extract surfactants

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 degrees C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of "low" chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. <5 mJ/m2), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of "high" chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. >15 mJ/m2). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from "low" to "high" chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of "patches" of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.     

Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113 kDa and 213 kDa. The other two additives were cationic peptides, polylysine 50 kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive-surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50 μl/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive-surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500 μl/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant).     

Interaction between chitosan and bovine lung extract surfactants

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 °C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of “low” chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. < 5 mJ/m²), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of “high” chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. > 15 mJ/m²). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from “low” to “high” chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of “patches” of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.     

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.     

Effect of budesonide and salbutamol on surfactant properties.

The objective of this study was to evaluate the in vitro effect of budesonide and salbutamol on the surfactant biophysical properties. The surface-tension properties of two bovine lipid extracts [bovine lipid extract surfactant (BLES) and Survanta] and a rat lung lavage natural surfactant were evaluated in vitro by the captive bubble surfactometer. Measurements were obtained before and after the addition of a low and high concentration of budesonide and salbutamol. Whereas salbutamol had no significant effect, budesonide markedly reduced the surface-tension-lowering properties of all surfactant preparations. Surfactant adsorption (decrease in surface tension vs. time) was significantly reduced (P < 0.01) at a high budesonide concentration with BLES, both concentrations with Survanta, and a low concentration with natural surfactant. At both concentrations, budesonide reduced (P < 0.01) Survanta film stability (minimal surface vs. time at minimum bubble volume), whereas no changes were seen with BLES. The minimal surface tension obtained for all surfactant preparations was significantly higher (P < 0.01), and the percentage of film area compression required to reach minimum surface tension was significantly lower after the addition of budesonide. In conclusion, budesonide, at concentrations used therapeutically, adversely affects the surface-tension-lowering properties of surfactant. We speculate that it may have the same adverse effect on the human surfactant.     

Disparate effects of two phosphatidylcholine binding proteins, C-reactive protein and surfactant protein A, on pulmonary surfactant structure and function

C-reactive protein (CRP) and surfactant protein A (SP-A) are phosphatidylcholine (PC) binding proteins that function in the innate host defense system. We examined the effects of CRP and SP-A on the surface activity of bovine lipid extract surfactant (BLES), a clinically applied modified natural surfactant. CRP inhibited BLES adsorption to form a surface-active film and the film's ability to lower surface tension (gamma) to low values near 0 mN/m during surface area reduction. The inhibitory effects of CRP were reversed by phosphorylcholine, a water-soluble CRP ligand. SP-A enhanced BLES adsorption and its ability to lower gamma to low values. Small amounts of SP-A blocked the inhibitory effects of CRP. Electron microscopy showed CRP has little effect on the lipid structure of BLES. SP-A altered BLES multilamellar vesicular structure by generating large, loose bilayer structures that were separated by a fuzzy amorphous material, likely SP-A. These studies indicate that although SP-A and CRP both bind PC, there is a difference in the manner in which they interact with surface films.     

Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency.

Pulmonary surfactant is a mixture of phospholipids ( approximately 90%) and surfactant-associated proteins (SPs) ( approximately 10%) that stabilize the lung by reducing the surface tension. One proposed mechanism by which surfactant is altered during acute lung injury is via direct oxidative damage to surfactant. In vitro studies have revealed that the surface activity of oxidized surfactant was impaired and that this effect could be overcome by adding SP-A. On the basis of this information, we hypothesized that animals receiving oxidized surfactant preparations would exhibit an inferior physiological and inflammatory response and that the addition of SP-A to the oxidized preparations would ameliorate this response. To test this hypothesis, mechanically ventilated, surfactant-deficient rats were administered either bovine lipid extract surfactant (BLES) or in vitro oxidized BLES of three doses: 10 mg/kg, 50 mg/kg, or 10 mg/kg + SP-A. When instilled with 10 mg/kg normal surfactant, the rats had a significantly superior arterial Po2 responses compared with the rats receiving oxidized surfactant. Interestingly, increasing the dose five times mitigated this physiological effect, and the addition of SP-A to the surfactant preparation had little impact on improving oxygenation. There were no differences in alveolar surfactant pools and the indexes of pulmonary inflammation between the 10 mg/kg dose groups, nor was there any differences observed between either of the groups supplemented with SP-A. However, there was significantly more surfactant and more inflammatory cytokines in the 50 mg/kg oxidized BLES group compared with the 50 mg/kg BLES group. We conclude that instillation of an in vitro oxidized surfactant causes an inferior physiological response in a surfactant-deficient rat.     

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.     

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.     

Reactive oxygen species inactivation of surfactant involves structural and functional alterations to surfactant proteins SP-B and SP-C

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects.     

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

Lung surfactant (LS) is an extra-cellular lipid-protein system responsible for maintaining low surface tension in the lung and alveolar stability. Serum proteins cause dysfunction of this material, e.g. in adult respiratory distress syndrome (ARDS). BLES is a clinically used LS consisting of most of the lipids and associated proteins from bovine lung lavage. Aqueous phases of BLES at 30% and 70% hydration, with and without 5% by weight of bovine serum albumin (BSA), calculated on the amount of lipids, were studied using X-ray diffraction during cooling from 42 to 5 degrees C. The diffraction curves are consistent with a transition from a lamellar liquid crystalline phase to a gel phase transition at cooling in the interval 30-20 degrees C. The long-spacings correspond to a reduction of the bilayer thickness during this transition. The wide-angle region shows a peak at 4.1 A below 25 degrees C, which is characteristic of the hexagonal chain packing of the gel phase. The perturbation of the bilayers by the presence of BSA seems to induce a significant decrease of the bilayer thickness. Calculations on the observed limits of swelling (taking place in the range 50-60%) indicate that BSA is closely associated with the BLES bilayers, probably due to electrostatic interaction with the cationic surfactant proteins SP-B and SP-C. This study show that the LS lipid structural organizations are extremely susceptible to small amounts of serum albumin, which may have implications in surfactant related lung disease and clinical applications of surfactant therapy.     

Effect of cholesterol on the biophysical and physiological properties of a clinical pulmonary surfactant

Pulmonary surfactant is a complex mixture of lipids and proteins that forms a surface-active film at the air-water interface of alveoli capable of reducing surface tension to near 0 mN/m. The role of cholesterol, the major neutral lipid component of pulmonary surfactant, remains uncertain. We studied the physiological effect of cholesterol by monitoring blood oxygenation levels of surfactant-deficient rats treated or not treated with bovine lipid extract surfactant (BLES) containing zero or physiological amounts of cholesterol. Our results indicate no significant difference between BLES and BLES containing cholesterol immediately after treatment; however, during ventilation, BLES-treated animals maintained higher PaO2 values compared to BLES+cholesterol-treated animals. We used a captive bubble tensiometer to show that physiological amounts of cholesterol do not have a detrimental effect on the surface activity of BLES at 37 degrees C. The effect of cholesterol on topography and lateral organization of BLES Langmuir-Blodgett films was also investigated using atomic force microscopy. Our data indicate that cholesterol induces the formation of domains within liquid-ordered domains (Lo). We used time-of-flight-secondary ion mass spectrometry and principal component analysis to show that cholesterol is concentrated in the Lo phase, where it induces structural changes.     

Constrained sessile drop as a new configuration to measure low surface tension in lung surfactant systems.

Existing methodology for surface tension measurements based on drop shapes suffers from the shortcoming that it is not capable to function at very low surface tension if the liquid dispersion is opaque, such as therapeutic lung surfactants at clinically relevant concentrations. The novel configuration proposed here removes the two big restrictions, i.e., the film leakage problem that is encountered with such methods as the pulsating bubble surfactometer as well as the pendant drop arrangement, and the problem of the opaqueness of the liquid, as in the original captive bubble arrangement. A sharp knife edge is the key design feature in the constrained sessile drop that avoids film leakage at low surface tension. The use of the constrained sessile drop configuration in conjunction with axisymmetric drop shape analysis to measure surface tension allows complete automation of the setup. Dynamic studies with lung surfactant can be performed readily by changing the volume of a sessile drop, and thus the surface area, by means of a motor-driven syringe. To illustrate the validity of using this configuration, experiments were performed using an exogenous lung surfactant preparation, bovine lipid extract surfactant (BLES) at 5.0 mg/ml. A comparison of results obtained for BLES at low concentration between the constrained sessile drop and captive bubble arrangement shows excellent agreement between the two approaches. When the surface area of the BLES film (0.5 mg/ml) was compressed by about the same amount in both systems, the minimum surface tensions attained were identical within the 95% confidence limits.     

Pulmonary surfactant function is abolished by an elevated proportion of cholesterol

A molecular film of pulmonary surfactant strongly reduces the surface tension of the lung epithelium-air interface. Human pulmonary surfactant contains 5-10% cholesterol by mass, among other lipids and surfactant specific proteins. An elevated proportion of cholesterol is found in surfactant, recovered from acutely injured lungs (ALI). The functional role of cholesterol in pulmonary surfactant has remained controversial. Cholesterol is excluded from most pulmonary surfactant replacement formulations, used clinically to treat conditions of surfactant deficiency. This is because cholesterol has been shown in vitro to impair the surface activity of surfactant even at a physiological level. In the current study, the functional role of cholesterol has been re-evaluated using an improved method of evaluating surface activity in vitro, the captive bubble surfactometer (CBS). Cholesterol was added to one of the clinically used therapeutic surfactants, BLES, a bovine lipid extract surfactant, and the surface activity evaluated, including the adsorption rate of the substance to the air-water interface, its ability to produce a surface tension close to zero and the area compression needed to obtain that low surface tension. No differences in the surface activity were found for BLES samples containing either none, 5 or 10% cholesterol by mass with respect to the minimal surface tension. Our findings therefore suggest that the earlier-described deleterious effects of physiological amounts of cholesterol are related to the experimental methodology. However, at 20%, cholesterol effectively abolished surfactant function and a surface tension below 15 mN/m was not obtained. Inhibition of surface activity by cholesterol may therefore partially or fully explain the impaired lung function in the case of ALI. We discuss a molecular mechanism that could explain why cholesterol does not prevent low surface tension of surfactant films at physiological levels but abolishes surfactant function at higher levels.     

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

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.     

Effect of chitosan on the electrophoretic motility of thymocytes

An increase of contact interaction between macrophages and thymocytes in the presence of polysaccharide chitosan was suggested to be due to the change of surface charge of interacting cells. It was found that incubation of thymocytes in the presence of chitosan leads to the change of their charge in the positive direction. The measurement of electrophoretic mobility of the cells was carried out on " Parmoquant 2" (Carl Zeiss Jena, DDR). The change of electrophoretic mobility increases with decrease of pH, increase of chitosan concentration and decrease of the cell concentration in the medium. This phenomenon is probably due to absorption of positively charged molecules of chitosan on negatively charged cell surface.     

Effect of rare earth cations on bactericidal activity of anionic surfactants

Summary Rare earth metal cations are antibacterially synergistic with anionic surfactants, yielding mixtures that have bactericidal activity against a variety of gram-positive and gram-negative bacteria at minimum concentrations ranging from 16 to 125 g/ml. Uptake of surfactant byEscherichia coli increases in the presence of lanthanum, suggesting that the role of rare earth metal cations is to reduce the net negative surface charge on the bacteria, thereby increasing the affinity between the negatively charged surfactant and the bacterial surface.