Calcium-induced phospholipid ordering depends on surface pressure

The effect of sodium and calcium ions on zwitterionic and anionic phospholipids monolayers is investigated using vibrational sum-frequency generation in conjunction with surface pressure measurements and fluorescence microscopy. Sodium ions only subtly affect the monolayer structure, while the effect of calcium is large and depends strongly on the surface pressure. At low surface pressures (approximately 5 mN/m), the presence on Ca2+ results in the unexpected appearance of ordered domains. For pressures between approximately 5 and approximately 25 mN/m, Ca2+ ions induce disorder in the monolayer. For pressures exceeding 25 mN/m, calcium cations expand the monolayer, while simultaneously ordering the lipid chains. Interestingly, effects are similar for both zwitterionic lipids and negatively charged lipids. In both vibrational sum-frequency generation and surface tension measurements, the molecular signature of the association of Ca2+ with the lipids is evident from Ca2+-induced changes in the signals corresponding to area changes of 4 A2/lipid-precisely the surface area of a Ca2+ ion, with evidence for a change in lipid Ca2+ complexation at high pressures.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Infrared reflection absorption spectroscopy coupled with Brewster angle microscopy for studying interactions of amphiphilic triblock copolymers with phospholipid monolayers

Novel water-soluble amphiphilic triblock copolymers poly(glycerol monomethacrylate)-b-poly(propylene oxide)-b-poly(glycerol monomethacrylate) (PGMA-b-PPO-b-PGMA) were synthesized because of their expected enhanced ability to interact with biological membranes compared to the well-known poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-PPO-b-PEO) block copolymers. Their bulkier hydrophilic PGMA blocks might induce a disturbance in the packing of liquid-crystalline lipid bilayers in addition to the effect caused by the hydrophobic PPO block alone. To gain a better insight into the polymer-membrane interactions at the molecular level, the adsorption kinetics and concomitant interactions of (PGMA14)(2-)PPO(34) with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were monitored using infrared reflection absorption spectroscopy (IRRAS) coupled with Brewster angle microscopy (BAM) and surface pressure (pi) measurements. The maximum penetration surface pressure of ca. 39 mN/m suggests that (PGMA14)(2-)PPO(34) is able to insert into lipid monolayers even above the so-called monolayer-bilayer equivalent pressure of 30-35 mN/m. Copolymer adsorption to a liquid-expanded DPPC-d62 monolayer proceeds in a two-step mechanism: (i) initially only the more hydrophobic PPO middle block penetrates the lipid monolayer; (ii) following the liquid-expanded-liquid-condensed (LE-LC) phase transition, the bulky PGMA hydrophilic blocks are dragged into the headgroup region as the PPO block inserts further into the fatty acid region. The adsorption kinetics is considerably faster for DMPC-d54 monolayers due to their higher fluidity. Copolymer adsorption to an LC-DPPC-d62 monolayer leads to a change in the monolayer packing by forcing the lipid alkyl chains into a more vertical orientation, their tilt angle with respect to the surface normal being reduced from initially 30 degrees +/- 3 degrees to 18 degrees +/- 3 degrees. BAM images rule out macroscopic phase separation and show that coalescence of DPPC-d62 LC domains takes place at relatively low surface pressures of pi > or = 23 mN/m, suggesting that (PGMA14)(2-)PPO (34) partitions into both LE as well as LC domains.     

Thermodynamic characteristics and Langmuir-Blodgett deposition behavior of mixed DPPA/DPPC monolayers at air/liquid interfaces

The role of dipalmitoylphosphatic acid (DPPA) as a transfer promoter to enhance the Langmuir-Blodgett (LB) deposition of a dipalmitoylphosphatidylcholine (DPPC) monolayer at air/liquid interfaces was investigated, and the effects of Ca2+ ions in the subphase were discussed. The miscibility of the two components at air/liquid interfaces was evaluated by surface pressure-area per molecule isotherms, thermodynamic analysis, and by the direct observation of Brewster angle microscopy (BAM). Multilayer LB deposition behavior of the mixed DPPA/DPPC monolayers was then studied by transferring the monolayers onto hydrophilic glass plates at a surface pressure of 30 mN/m. The results showed that the two components, DPPA and DPPC, were miscible in a monolayer on both subphases of pure water and 0.2 mM CaCl2 solution. However, an exception occurs between X(DPPA)=0.2 and 0.5 at air/CaCl2-solution interface, where a partially miscible monolayer with phase separation may occur. Negative deviations in the excess area analysis were found for the mixed monolayer system, indicating the existence of attractive interactions between DPPA and DPPC molecules in the monolayers. The monolayers were stable at the surface pressure of 30 mN/m for the following LB deposition as evaluated from the area relaxation behavior. It was found that the presence of Ca2+ ions had a stabilization effect for DPPA-rich monolayers, probably due to the association of negatively charged DPPA molecules with Ca2+ ions. Moreover, the Ca2+ ions may enhance the adhesion of DPPA polar groups to a glass surface and the interactions between DPPA polar groups in the multilayer LB film structure. As a result, Y-type multilayer LB films containing DPPC could be fabricated from the mixed DPPA/DPPC monolayers with the presence of Ca2+ ions.     

嵌入花生酸镉单层中的卟啉-紫精分子聚集行为的研究

本文制备了卟啉-紫精与花生酸镉混合LB膜, 用紫外可见光谱研究了膜中卟啉基团的聚集及取向, X射线衍射说明混合LB膜具有层状有序的周期结构。扫描电镜结果表明: 通过调节膜的表面压可使聚集成"微畴"的卟啉-紫精均匀分布在花生酸镉单分子层中, 随膜表面压的增大, 小的"微畴"相互连接形成更大的"微畴"。电子衍射说明混合膜中两组份分相存在, 且都为六角对称的有序结构。     

Memory effects of monolayers and vesicles formed by the non-ionic surfactant, 2C(18)E(12)

The behaviour of monolayers and bilayers formed by the dialkyl chain non-ionic surfactant, 1,2-di-O-octadecyl-rac-glycerol-3-omega-methoxydodecaethylene glycol (2C(18)E(12)) in water at 297 K has been investigated. Using a surface film balance (or Langmuir trough) the compression-expansion cycle of the 2C(18)E(12) monolayer was found to be reversible when compressed to surface pressures (pi) less than 42 mN m(-1). Compression of 2C(18)E(12) monolayer to pi greater than 42 mN m(-1) above this resulted in a considerable hysteresis upon expansion with the pi remaining high relative to that obtained upon compression, suggesting a time/pressure dependent re-arrangement of 2C(18)E(12) molecules in the film. Morphology of the 2C(18)E(12) monolayer, investigated using Brewster angle microscopy, was also found to depend upon monolayer history. Bright, randomly dispersed domains of 2C(18)E(12) of approximately 5 mum in size were observed during compression of the monolayer to pi less than 42 mN m(-1). At pi of 42 mN m(-1) and above, the surfactant film appeared to be almost completely 'solid-like.' Regardless of the extent of compression of the monolayer film, expansion of the film caused formation of chains or 'necklaces' of individual surfactant domains, with the extent of chain formation dependent upon pressure of compression of the monolayer and the length of time held at that pressure. Irreversible effects on 2C(18)E(12) vesicle size were also seen upon temperature cycling the vesicles through their liquid-crystalline phase transition temperature with vesicles shrinking in size and not returning to their original size upon standing at 298 K for periods of more than 24 h. No comparable hysteresis, time, pressure or temperature effects were observed with the monolayer or vesicles formed by the corresponding phospholipid, disteaorylphosphatidylcholine, under identical conditions. The effects observed with 2C(18)E(12) are attributed to the ability of the polyoxyethylene head group to dehydrate and intrude into the hydrophobic chain region of the mono- and bilayers. These studies have important implications for the use of the vesicles formed by 2C(18)E(12) as drug delivery vehicles.     

Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes

The hydrolytic activity of secretory phospholipase A(2) (PLA(2)) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA(2) on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA(2) molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA(2)s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA(2) decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA(2)s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA(2)s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA(2)s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA(2) activity and the mobilization of its substrate and product to facilitate the catalytic process.     

Evaluation of antitubercular drug insertion into preformed dipalmitoylphosphatidylcholine monolayers

Insertion profiles of antitubercular drugs isoniazid (INH), rifampicin (RFM) and ethambutol (ETH) into dipalmitoylphosphatidylcholine (DPPC) membrane models were evaluated by Langmuir monolayer technique. Maximum drug insertion into DPPC monolayer was observed with rifampicin with a surface pressure increase (Δπ_max) in the range of 21–33 mN/m depending upon rifampicin concentration. Isoniazid had minimal insertion resulting in a lower Δπ_max of about 2–3 mN/m, suggestive of minimal interactions between INH and DPPC. Ethambutol surface pressure increment on insertion resulted in an intermediate rise in the Δπ_max (6–10 mN/m). Antitubercular drug combination in the ratio of 2 mM:0.7 mM:4.5 mM for INH:RFM:ETH, attained Δπ_max between 25 and 33 mN/m. Insertion profiles similar to rifampicin were exhibited by the antitubercular drug mixture suggestive of predominant rifampicin insertion into the DPPC monolayer. The extent of drug insertion into the DPPC monolayer is suggestive of the drug penetration potential into biological membranes in vivo. Higher RFM Δπ_max is suggestive of excellent cell membrane penetration, which explains broad reach of the drug to all the organs including the cerebrospinal fluid while lower Δπ_max of INH suggests poor membrane penetration restricting the entry of the drug in different biological membranes. DPPC membrane destabilization was observed at higher antitubercular drug concentrations indicated by the negative slopes of the surface pressure–time curves. This may correlate with the dose related toxic effects observed in tuberculosis affected patients. Drug insertion studies offer a potential tool in understanding the pharmacotoxicological behavior of the various pharmacological agents.     

Effects of surface pressure on the structure of the monolayer formed at the air/water interface by a non-ionic surfactant

The monolayer formed at an air/water interface by the synthetic non-ionic surfactant, 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-methoxydodecakis (ethylene glycol)) (2C18E12) has been characterized using Langmuir trough measurements, Brewster angle microscopy (BAM), and neutron reflectometry. The BAM and reflectometry studies were performed at four different surface pressures (pi) in the range 15-40 mN/m. The BAM studies (which give information on the in-plane organisation of the surfactant layer) demonstrate that the 2C18E12 molecules are arranged on the water surface to form distinct, approximately circular, 5 microm diameter domains. As the surface pressure is increased these domains retain their size and shape but are made progressively more close-packed, such that the monolayer is made more or less complete at pi=40 mN/m. The neutron reflectometry measurements were made to determine the structure of the interfacial surfactant layer at pi=15, 28, 34 and 40 mN/m, providing information on the thickness of the 2C18E12 alkyl chains', head groups' and associated solvent distributions (measured along the surface normal), along with the separations between these distributions, and the effective interfacial area per molecule. Partial structure factor analyses of the reflectivity data show that the effective interfacial area occupied decreases from 217 A2 per 2C18E12 molecule at pi=15 mN/m down to 102 A2 at pi=40 mN/m. There are concomitant increases in the widths of the surfactant's alkyl chains' and head groups' distributions (modelled as Gaussians), with the former rising from 12 A (at pi=15 mN/m) up to 19 A (at pi=40 mN/m) and the latter rising from 13 A (at pi=15 mN/m) up to 24 A (at pi=40 mN/m). The compression of the monolayer is also shown to give rise to an increased surface roughness, some of which is due to the thermal roughness caused by capillary waves, but with a significant contribution also coming from the intrinsic/structural disorder in the monolayer. At all surface pressures studied, the alkyl chains and head groups of the 2C18E12 are found to exhibit a significant overlap, and this increases with increasing pi. Given the various trends noted on how the structure of the 2C18E12 monolayer changes as a function of pi, we extrapolate to consider the structure of the monolayer at pi>40 mN/m (making comparison with its single chain (CnEm) counterparts) and then relate these findings to the observations recorded on the structure and solute entrapment efficiency of 2C18E12 vesicles.     

Penetration and interactions of the antimicrobial peptide, microcin J25, into uncharged phospholipid monolayers

Microcin J25 forms stable monolayers at the air-water interface showing a collapse at a surface pressure of 5 mN/m, 220 mV of surface potential, and 6 fV per squared centimeter of surface potential per unit of molecular surface density. The adsorption of microcin J25 from the subphase at clean interfaces leads to a rise of 10 mN/m in surface pressure and a surface potential of 220 mV. From these data microcin appears to be a poor surfactant per se. Nevertheless, the interaction with the lipid monolayer further increase the stability of the peptide at the interface depending on the mode in which the monolayer is formed. Spreading with egg PC leads to nonideal mixing up to 7 mN/m, with hyperpolarization and expansion of components at the interface, with a small excess free energy of mixing caused by favorable contributions to entropy due to molecular area expansion compensating for the unfavorable enthalpy changes arising from repulsive dipolar interactions. Above 7 mN/m microcin is squeezed out, leaving a film of pure phospholipid. Nevertheless, the presence of lipid at 10 and 20 mN/m stabilize further microcin at the interface and adsorption from the subphase proceeds up to 30 mN/m, equivalent to surface pressure in bilayers.     

Relationships between equilibrium spreading pressure and phase equilibria of phospholipid bilayers and monolayers at the air-water interface

The intricate interplay between the bilayer and monolayer properties of phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) phospholipids, in relation to their polar headgroup properties, and the effects of chain permutations on those polar headgroup properties have been demonstrated for the first time with a set of time-independent bilayer-monolayer equilibria studies. Bilayer and monolayer phase behavior for PE is quite different than that observed for PC and PG. This difference is attributed to the characteristic biophysical PE polar headgroup property of favorable intermolecular hydrogen-bonding and electrostatic interactions in both the bilayer and monolayer states. This characteristic hydrogen-bonding ability of the PE polar headgroup is reflected in the condensed nature of PE monolayers and a decrease in equilibrium monolayer collapse pressure at temperatures below the monolayer critical temperature, T(c) (whether above or below the monolayer triple point temperature, T(t)). This interesting phenomena is compared to equilibrated PC and PG monolayers which collapse to form bilayers at 45 mN/m at temperatures both above and below monolayer T(c). Additionally, it has been demonstrated by measurements of the equilibrium spreading pressure, pie, that at temperatures above the bilayer main gel-to-liquid-crystalline phase-transition temperature, T(m), all liquid-crystalline phospholipid bilayers spread to form monolayers with pie around 45 mN/m, and spread liquid-expanded equilibrated monolayers collapse at 45 mN/m to form their respective thermodynamically stable liquid-crystalline bilayers. At temperatures below bilayer T(m), PC and PG gel bilayers exhibit a drop in bilayer pi(e) values < or =0.2 mN/m forming gaseous monolayers, whereas the value of pic of spread monolayers remains around 45 mN/m. This suggests that spread equilibrated PC and PG monolayers collapse to a metastable liquid-crystalline bilayer structure at temperatures below bilayer T(m) (where the thermodynamically stable bilayer liquid-crystalline phase does not exist) and with a surface pressure of 45 mN/m, a surface chemical property characteristically observed at temperatures above bilayer T(m) (monolayer T(c)). In contrast, PE gel bilayers, which exist at temperatures below bilayer T(m) but above bilayer T(s) (bilayer crystal-to-gel phase-transition temperature), exhibit gel bilayer spreading to form equilibrated monolayers with intermediate pie values in the range of 30-40 mN/m; however, bilayer pie and monolayer pic values remain equal in value to one another. Contrastingly, at temperatures below bilayer T(s), PE crystalline bilayers exhibit bilayer pie values < or =0.2 mN/m forming equilibrated gaseous monolayers, whereas spread monolayers collapse at a value of pic remaining around 30 mN/m, indicative of metastable gel bilayer formation.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Lateral and vertical nanophase separation in Langmuir-Blodgett films of phospholipids and semifluorinated alkanes

It has recently been found that monodisperse surface micelles (hemimicelles) were formed in Langmuir monolayers of the semifluorinated alkane C8F17C16H33 (F8H16) after transfer onto silicon wafers. Grazing incidence X-ray diffraction studies have demonstrated that compression of mixed Langmuir monolayers made from combinations of dipalmitoyl phosphatidylethanolamine (DPPE) and diblock F8H16 in various molar ratios resulted in the complete expulsion of the diblock molecule at high surface pressure. F8H16 then formed a second layer on top of a DPPE-only monolayer, demonstrating a novel type of reversible, pressure-induced, vertical phase separation. Using atomic force microscopy and X-ray reflectivity, we show now that mixed DPPE/F8H16 (1:1.3) Langmuir-Blodgett films transferred onto silicon wafers below 10 mN m(-1) are laterally phase separated and consist of domains of F8H16 surface micelles in coexistence with a monolayer of DPPE. The density of the network of F8H16 surface micelles increases when the surface pressure of transfer increases. Around 10 mN m(-1), the F8H16 surface micelles start to glide on the DPPE monolayer, progressively overlying it, until total coverage is achieved.     

Halofantrine-phospholipid interactions: monolayer studies.

The antimalarial agent halofantrine penetrates dipalmitolylphosphatidylcholine (DPPC) monolayers resulting in an increase in surface pressure and an expansion in area occupied by the lipid components of the monolayer. This phenomenon is observed at concentrations (0.05-0.2 microm) of halofantrine that have no surface activity. Penetration increases with drug concentration and is greatest at low initial surface pressures of the monolayer. A critical surface pressure of the DPPC monolayer has been determined from constant area and constant pressure conditions. The magnitude of these values support the hypothesis that halofantrine readily penetrates the DPPC monolayers. The presence of cholesterol in the DPPC monolayer hampers penetration and a lower critical surface pressure is obtained under such conditions. Even then, a slower rate of penetration is observed only in monolayers maintained at high initial surface pressures (10, 15 mN/m), corresponding to the liquid condensed phase of the monolayer, and not at low surface pressures (2.5, 5.0 mN/m). These results help to give a better understanding of the dynamics of the halofantrine-phospholipid interaction as well as the pharmacodynamic character of the drug.     

Modulation of lysozyme charge influences interaction with phospholipid vesicles

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.     

Effect of Ester Moiety on Structural Properties of Binary Mixed Monolayers of Alpha-Tocopherol Derivatives with DPPC

Phospholipid membranes are ubiquitous components of cells involved in physiological processes; thus, knowledge regarding their interactions with other molecules, including tocopherol ester derivatives, is of great importance. The surface pressure–area isotherms of pure α-tocopherol (Toc) and its derivatives (oxalate (OT), malonate (MT), succinate (ST), and carbo analog (CT)) were studied in Langmuir monolayers in order to evaluate phase formation, compressibility, packing, and ordering. The isotherms and compressibility results indicate that, under pressure, the ester derivatives and CT are able to form two-dimensional liquid-condensed (LC) ordered structures with collapse pressures ranging from 27 mN/m for CT to 44 mN/m for OT. Next, the effect of length of ester moiety on the surface behavior of DPPC/Toc derivatives’ binary monolayers at air–water interface was investigated. The average molecular area, elastic modulus, compressibility, and miscibility were calculated as a function of molar fraction of derivatives. Increasing the presence of Toc derivatives in DPPC monolayer induces expansion of isotherms, increased monolayer elasticity, interrupted packing, and lowered ordering in monolayer, leading to its fluidization. Decreasing collapse pressure with increasing molar ratio of derivatives indicates on the miscibility of Toc esters in DPPC monolayer. The interactions between components were analyzed using additivity rule and thermodynamic calculations of excess and total Gibbs energy of mixing. Calculated excess area and Gibbs energy indicated repulsion between components, confirming their partial mixing. In summary, the mechanism of the observed phenomena is mainly connected with interactions of ionized carboxyl groups of ester moieties with DPPC headgroup moieties where formed conformations perturb alignment of acyl chains, resulting in increasing mean area per molecule, leading to disordering and fluidization of mixed monolayer.     

Patterning of magnetic bimetallic coordination nanoparticles of Prussian blue derivatives by the Langmuir-Blodgett technique

We report a novel method to prepare patterns of nanoparticles over large areas of the substrate. This method is based on the adsorption of the negatively charged nanoparticles dispersed in an aqueous subphase onto a monolayer of the phospholipid dipalmitoyl-l-α-phosphatidylcholine (DPPC) at the air-water interface. It has been used to prepare patterns of nanoparticles of Prussian blue analogues (PBA) of different size (K(0.25)Ni[Fe(CN)(6)](0.75) (NiFe), K(0.25)Ni[Cr(CN)(6)](0.75) (NiCr), K(0.25)Ni[Co(CN)(6)](0.75) (NiCo), Cs(0.4)Co[Cr(CN)(6)](0.8) (CsCoCr), and Cs(0.4)Co[Fe(CN)(6)](0.9) (CsCoFe)). The behavior of DPPC monolayer at the air-water interface in the presence of the subphase of PBA nanoparticles has been studied by the compression isotherms and Brewster angle microscopy (BAM) images. Atomic force microscopy (AFM) of the transferred films on mica substrates shows that patterns of the nanoparticles are observed for a 10(-4) M concentration of the subphase, based on the nanoparticle precursors, at surface pressures between 1 and 6 mN/m and transfer velocities from 10 to 80 mm/min. Vertical, horizontal, or tilted fringes of the nanoparticles with respect to the transfer direction can be obtained depending on the transfer velocity and surface pressure.     

A monolayer study on interactions of docetaxel with model lipid membranes

Docetaxel (DCT) is an antineoplastic drug for the treatment of a wide spectrum of cancers. DCT surface properties as well as miscibility studies with l-alpha-dipalmitoyl phosphatidylcholine (DPPC), which constitutes the main component of biological membranes, are comprehensively described in this contribution. Penetration studies have revealed that when DCT is injected under DPPC monolayers compressed to different surface pressures, it penetrates into the lipid monolayer promoting an increase in the surface pressure. DCT is a surface active molecule able to decrease the surface tension of water and to form insoluble films when spread on aqueous subphases. The maximum surface pressure reached after compression of a DCT Langmuir film was 13 mN/m. Miscibility of DPPC and DCT in Langmuir films has been studied by means of thermodynamic properties as well as by Brewster angle microscopy (BAM) analysis of the mixed films at the air-water interface, concluding that DPPC and DCT are miscible and they form non-ideally mixed monolayers at the air-water interface. Helmholtz energies of mixing revealed that no phase separation occurs. In addition, Helmholtz energies of mixing become more negative with decreasing areas per molecule, which suggests that the stability of the mixed monolayers increases as the monolayers become more condensed. Compressibility values together with BAM images indicate that DCT has a fluidizing effect on DPPC monolayers.     

Infrared reflection-absorption spectroscopy and polarization-modulated infrared reflection-absorption spectroscopy studies of the aequorin langmuir monolayer

The Langmuir monolayer of aequorin and apoaequorin was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS techniques. The alpha-helices in the aequorin Langmuir monolayer were parallel to the air-water interface at zero surface pressure. When the surface pressure increased to 15 mN.(m-1), the alpha-helices became tilted and the turns became parallel to the air-water interface. As for apoaequorin, the alpha-helices were also parallel to the air-water interface at 0 mN.m(-1). However, the alpha-helix became tilted and the turns became parallel to the air-water interface quickly at 5 mN.m(-1). With further compression of the apoaequorin Langmuir monolayer, the orientation remained the same. The different behaviors of aequorin and apoaequorin at the air-water interface were explained by the fact that aequorin formed dimers at the air-water interface but apoaequorin was a monomer. It is more difficult for a dimer to be tilted by the compression of the Langmuir monolayer.