Liposomes bearing platelet proteins: a model for surface functions studies.

An improved procedure for the direct transfer of membrane proteins from human platelets to liposomes involving the treatment of platelets with linolenic acid was developed. The transfer of platelet proteins to liposomes prepared from the mixture of L-alpha-dimyristoyl-phosphatidylcholine/sphingomyelin in the molar ratio 80/20 appeared to be significantly enhanced compared with liposomes prepared from the same components mixed in other ratios. A wide range of platelet proteins was transferred, the most important being GPIb (170 kDa), GPIIb/IIIa (135 and 110 kDa). GPIV (90 kDa), GPIX (24 kDa) and the serotonin transporter (68 kDa). The recognition interactions between these proteoliposomes and specific protein antibodies clearly indicate that the non-invasive procedure used in this study ensured the reproducible transfer of platelet proteins without essentially altering their original conformation. The obtained results reveal also that the affinity of proteoliposomes to bind paroxetin was virtually the same as that of the native serotonin transporter. These results provide an indication of the possible use of such proteoliposomes as models to study at the molecular level the interaction of these proteins with their ligands.

Molecular organization of the human serotonin transporter at the air/water interface.

The serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10,400 and 7,100 A(2)/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7,100 A(2)/molecule was found to be approximately equal to 84 A and coincided well with the theoretical value estimated from the calculations based on the dimensions of alpha-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths d(z)=83--85 A and d(y) or d(x)=41--47 A.

Ligand interaction with the purified serotonin transporter in solution and at the air/water interface.

The purified serotonin transporter (SERT) was spread at the air/water interface and the effects both of its surface density and of the temperature on its interfacial behavior were studied. The recorded isotherms evidenced the existence of a stable monolayer undergoing a lengthy rearrangement. SERT/ligand interactions appeared to be dependent on the nature of the studied molecules. Whereas an unrelated drug (chlorcyclizine) did not bind to the spread SERT, it interacted with its specific ligands. Compared to heterocyclic drugs, for which binding appeared to be concentration-dependent, a 'two-site' mechanism was evidenced for pinoline and imipramine.

Adsorption of bovine submaxillary mucin on silicone contact lenses grafted with poly(vinyl pyrrolidone).

Bovine submaxillary mucin is considered to be an analogue of the high molecular protein present in the conjunctival mucus. This mucin was isolated from fresh salivary glands and acetylated with [1-14C]acetic anhydride. In situ adsorption of the bovine submaxillary mucin on silicone contact lenses ungrafted and grafted with poly(vinyl pyrrolidone) was performed for the first time using an original radiotracer technique. The results show that the adsorbed amounts of mucin are higher on grafted samples and that thick layers are adsorbed when mucin concentration in the bulk solution is increased. Desorption experiments reveal that in addition to the tightly adsorbed protein layer, a loosely bound mucin layer of the same thickness exists on grafted and ungrafted silicones.

Role of phospholipid lining on respiratory mucus clearance by cough.

Phospholipid lining, present at the respiratory mucus-mucosa interface, may have an important role in the protective function of the airways by its abhesive properties and may also facilitate mucus transport. To mimic respiratory mucus-mucosa interface, monolayers of three different forms of phosphatidylglycerol (PG) have been deposited on glass slides by the Langmuir-Blodgett technique. Mucus adhesion and clearance by cough of mucus on these PG-coated or noncoated surfaces have been analyzed and compared, using frog respiratory mucus as "normal" mucus. Among the three PG types studied, the phosphatidylglycerol distearoyl, which is the phospholipid with the longest saturated fatty acid chain, was found to significantly improve the mucus cough clearance by decreasing the mucus work of adhesion compared with the noncoated surfaces. On the other hand, phosphatidylglycerol dipalmitoyl did not improve mucus cough clearance although it decreased mucus adhesion, and phosphatidylglycerol dioleyl did not improve either mucus cough clearance or mucus adhesion.

Fucosyled neoglycolipids: synthesis and interaction with a phospholipid.

The interfacial behavior of the neoglycolipids formed of Guerbet alcohol (G(28)) bound to a triethylene glycol spacer (E(3)) and to a sugar moiety (alpha- and beta-fucose) spread at the air/water interface has been studied under dynamic conditions of compression. Although the alpha (alpha-FucE3G28)- and beta-fucose (beta-FucE3G28) derivatives possessed the same chemical structure, the positioning of the sugar moiety relative to the whole molecule had a significant influence on the organization of neoglycolipid molecules in the spread monolayers. Thus, beta-fucose molecules exhibited higher compressibilities and larger molecular areas than a alpha/beta (84/16%) mixture (alpha(84)-FucE3G28). The comparison of the compressional behavior of the fucose derivatives with that of Guerbet alcohol in the absence and in the presence of the triethylene glycol spacer shows that the presence of the E(3) chain is necessary to stabilize the lipid at the interface and that the incorporation of a sugar moiety into the molecule resulted in an important expansion of a monolayer. Despite their different interfacial behaviors, the two sugar derivatives formed ideal mixtures when cospread at the air/water interface. Conversely, in the presence of a phospholipid, such as DMPC, repulsive interactions were observed and appeared to be stronger for DMPC/alpha(84)-FucE3G28 mixed monolayers. The membrane fluidity of DMPC liposomes bearing the studied amphiphilic molecules was assessed by fluorescence depolarization measurements. The results reveal that whereas G(28) was deeply inserted into the liposome bilayers, the presence of a E(3) chain and of a sugar moiety in these bilayers induced a transfer of the amphiphilic derivatives from the hydrophobic core towards polar headgroups of phospholipid molecules.

The effect of the surface free energy of pharmaceutical tablets on liquid penetration.

The surface free energies (gamma SV) of the integral and partial tablet formulations of an antiarrhythmic drug--cibenzoline succinate, have been assessed by contact angle measurements using high-viscosity polyols and Neumann's equation of state. Independent measurements of penetration for these liquids into the tablets yielded pore size values through the use of the Washburn equation. The role of different constituents of the formulations are analysed in terms of their influence upon the free surface energy of the tablets and penetration rates. The relation between (gamma SV) and penetration rates yields for a series of liquids two threshold values: (gamma SV)s 'start' and (gamma SV)r, 'rapid', which define respectively the beginning of the measurable penetration and the rapid penetration.

Monolayer studies on poly(isobutylcyanoacrylate)-ampicillin association.

Surface pressure-area isotherms (pi-A) of poly(isobutylcyanoacrylate) monolayers with or without glucose and dextran as polymerization adjuvants used in the preparation of nanoparticles have been derived from measurements made at the air-water interface with the subphase pH at 2.7, 5.5 or 8.8. The isotherms were characteristic of the expanded type of polymer monolayer curves, yielding unusually low limiting area values compared with those of other known poly(acrylate) derivatives. This behaviour may be explained by the folding of polar moieties of the polymer groups in the water subphase. Ampicillin incorporated during preparation of nanoparticles had a negligible effect on the general behaviour of adjuvant-free monolayers, but a significant one on the adjuvant-loaded polymer monolayer system which showed an increase in surface area throughout the compression cycle, as compared with the surface area of the adjuvant-free polymer system although the collapse pressure was practically the same at 67 mN m-1.

Wettability of polymers by mucin aqueous solutions.

The wettability of poly(methyl methacrylate) and polyethylene by water and aqueous mucin solutions have been studied by sessile drop and under-water captive air bubble contact angles, respectively. From the sessile drop and octane under-water contact angles the polymer-water interfaces have been characterized in terms of works of adhesion and acid-base (polar) interactions. A large water-air contact angle hysteresis observed with poly(methyl methacrylate) surfaces has been attributed to side-chain beta relaxations of polymer ester methyl groups. The wettabilities of the polymers by mucin aqueous solutions have been studied as a function of protein concentration and related to the surface tensions. A positive slope of adhesion tension vs surface tension line, characteristic of polar surfaces, was found with poly(methyl methacrylate). By contrast, a change in the slope, explained as a change in mucin relative adsorption densities at solid/liquid and solid/vapour interfaces, was observed with polyethylene. This adhesion tension behavior appeared to be in agreement with previous data we have published concerning the quantity and state of mucin which are adsorbed to polymers characterized by different surface properties.

[Stabilization and function of liposomes]. / Stabilisation et fonctionnalisation des liposomes.

Liposomes stability can be improved by covering them with polysaccharide derivatives. The anchoring mechanism of these derivatives into the lipid membrane has been studied and explained. In order to improve liposome specificity, protein transfer from erythrocytes and platelets into liposome membrane has been carried out. The effect of a newly developed phospholipid, DDPC, on the efficiency and the selectivity of protein transfer is reported.

Influence of a neoglycolipid and its PEO-lipid moiety on the organization of phospholipid monolayers.

The surface properties of the neoglycolipid (GlcNAcE(3)G(28)) and of its PEO-lipid (E(3)G(28)) moiety mixed with phospholipids (dipalmitoylphosphatidylcholine, DPPC; distearoylphosphatidylcholine, DSPC; diarachidoylphosphatidylcholine, DAPC; and dibehenoylphosphatidylcholine, DBPC) were studied in Langmuir monolayers at various mixture compositions and surface pressures. The pi-A isotherms of the pure compounds revealed that because of the presence of the sugar group in its molecule, GlcNAcE(3)G(28) collapsed at a higher surface pressure and occupied a larger molecular area than the PEO-lipid moiety. It was also observed that the presence of the PEO-lipid (E(3)G(28)) in the mixtures triggered a strong alteration of both phospholipid pi-A isotherm profiles and surface diffraction spectra, an indication that the disordering of the initially structured phospholipid monolayers took place. Unlike E(3)G(28), GlcNAcE(3)G(28) did not disorganize phospholipid monolayers but generated a partial segregation of the film-forming components. The calculated excess free energies of mixing (DeltaG(exc)) for GlcNAcE(3)G(28)-phospholipid mixtures enabled us to predict the stability of such systems.

Competitive adsorption of albumin against collagen at solution-air and solution-polyethylene interfaces.

The adsorption of human serum albumin (HSA) from the binary mixtures with collagen was monitored at solution-air and solution-polyethylene interfaces by the in situ measurements. The results clearly demonstrate that on both interfaces albumin is the only adsorbing protein within a large collagen solution concentration range. At the albumin concentration equal to 0.005 mg/mL, the presence of collagen in solution results in the enhancement of albumin adsorption at solution-air interface relative to its adsorption from the single protein system. The same phenomenon is manifested at the solution-polyethylene interface, although the increase in albumin adsorption at this interface occurs at the albumin concentration equal to 0.01 mg/mL. These results are attributed to the lowering in the solution-air and solution-polyethylene interfacial tensions, and thus to the increase in the spreading characteristics of albumin in the presence of collagen molecules. The desorption experiments carried out with a buffer solution on polyethylene surfaces reveal the irreversibility of adsorbed albumin from both the single and the binary mixtures with collagen. When after 20 h of adsorption from the solutions containing albumin only, collagen was added to these solutions or when the samples after that period of time were first rinsed with a buffer and then with a collagen solution, the amounts of albumin remaining at the surfaces were in both cases reduced by one-half.

The interaction of plasma proteins with polymers. I. Relationship between polymer surface energy and protein adsorption/desorption.

Adsorption of bovine albumin, gamma-globulin and fibrinogen from phosphate buffer solution (pH = 7.5) onto several polymer films was studied using the radioiodinated proteins (125I). The kinetics of desorption of the proteinated polymer films in bovine plasma was determined. Contact angle measurements on these same polymers allowed the calculation of dispersive (WA d) and polar (Ip) components of the polymer-protein solution system. Results from these measurements show that the nondispersive-dispersive force balance at the polymer-protein solution interface, expressed by the Ip/WA d ratio, is an important factor for binding of proteins on polymer surfaces. The purity of fibrinogen and the cleaning procedures for the polymer surfaces influence the absolute values of proteins adsorbed on polymer surfaces.

[Insulin-phospholipid interactions. A study in monolayers by measuring surface potential]. / Interactions phospholipides-insuline. Etude en couches monomoléculaires par des mesures de potentiel de surface.

Surface potential (delta V) measurements were performed to assess information on insulin penetration/interaction with dipalmitoylphosphatidylcholine (DPPC) monolayers spread at the water-air interface. The results reveal existence of the threshold surface density value of spread lipid molecules (7.5 x 10(13) molecules/cm2) above which none penetration of insulin molecules occurs. Surface potential data clearly indicate also that insulin penetration/interaction with DPPC monolayers is enhanced in the presence of the second studied constituent of these monolayers in the order DPPC + stearylamine greater than DPPC + cholesteryl betainate greater than DPPC + cholesterol greater than DPPC. The results clearly indicate the existence of two types of phospholipid-insulin interactions namely: adsorption and penetration.

Complete denture retention. Part II: Wettability studies on various acrylic resin denture base materials.

The objective of this study was to characterize in vitro selected acrylic resin denture base materials by water-contact angle measurements. The sessile drop method and the underwater-bubble method were used. The results obtained from these measurements are discussed in terms of contact angle and polymer-water work of adhesion hysteresis. On polished heat-polymerized samples this hysteresis results from the reorientation of superficial polymer chains. The combined effect of increased sample roughness and of the entrapment of water droplets in the pores of material gives rise to the highest contact-angle hysteresis observed on sand-abraded samples. On the basis of physical analysis of the mechanism involved in complete denture retention, developed in Part I of this work, it is believed that the sand-abraded material is the most convenient for the retention of the complete denture.

Native and Hydrophobically Modified Human Immunoglobulin G at the Air/Water Interface.

The adsorption of human immunoglobulin G (IgG) at the air/water interface was monitored both by the in situ radiotracer technique using [(14)C] labeled IgG and by surface tension measurements. The results reveal that adsorption of IgG from single protein systems displays bimodality due to molecular rearrangements at the interface. Above the threshold value of 1.5x10(-2) mg/ml solution concentration, adsorbed IgG molecules reoriented from the side-on to the end-on configuration. The existence of a lag time which did not appear in Gamma=f(t) curves, was observed in Pi=f(t) relationships at low protein concentrations and was due to the limits of the surface pressure technique to detect protein adsorption. The adsorption of native IgG was also carried out in the presence of a hydrophobized IgG obtained by grafting capryloyl residues to its lysine groups by reaction with N-hydroxysuccinimide ester of caprylic acid, which yielded 19 covalently bound alkyl chains to the IgG molecule (19C(8)-IgG). This modified IgG exhibited enhanced adsorption at the air/water interface, as manifested by its increased adsorption efficiency relative to the native protein. Sequential and competitive adsorption experiments from binary mixtures of native IgG and 19C(8)-IgG clearly demonstrate that the displacement of the native protein from the air/water interface strongly depended on the manner of how 19C(8)-IgG and native IgG competed with each other. When the two proteins competed simultaneously, 19C(8)-IgG predominantly occupied the available area but when native IgG was adsorbed first, for 2 h, the sequentially adsorbed 19C(8)-IgG was incapable of substantially displacing it from the interface. Copyright 2001 Academic Press.

Surface characterization of heparin-complexing poly(amido amine) chains grafted on polyurethane and glass surfaces.

Poly(amido-amine) chains grafted onto polyurethanes and glass form stable complexes with heparin yielding potential nonthrombogenic surfaces. The characterization of the surfaces, and the product of each chemical reaction including final heparinized surfaces, has been studied by contact angle data and scanning electron microscopy (SEM). Air in water, octane in water, and drop-on-plate contact angle data were used to estimate surface (gamma sv) and interfacial (gamma sw) free energies. Solid-water work of adhesion (Wa) and its dispersive (Wda) and polar (Ipsw) components were calculated for all studied surfaces. It has been found that the viscosity of polyurethane solution used for film casting influences wetting properties of these films. It has also been found that a direct correlation exists between the Ipsw/Wda values and the degree of coverage of the surfaces by cellular deposits after their exposure to platelet-rich plasma. Final heparinized polyurethane and glass materials are hydrophilic, their Ipsw/Wda ratio is high, and little or no cellular deposit is observed on their surfaces.

Collagen at interfaces. I. In situ collagen adsorption at solution/air and solution/polymer interfaces.

Collagen was isolated from rat tail tendons and acetylated with 1-14C acetic anhydride. In situ adsorption of this collagen from a buffer solution (pH = 2.7) was measured at the interfaces to air, polyethylene and polyethylene grafted with poly(maleic acid), respectively. The kinetics of adsorption were recorded for all surfaces studied and the corresponding diffusion coefficients for collagen in solution with various protein concentrations were calculated. The desorption of collagen from polymer surfaces was also studied. These experiments reveal the existence of both a reversibly and an irreversibly adsorbed collagen layer on the polymers tested. The desorption/adsorption ratio for the polyethylene is higher than that for the grafted polyethylene indicating stronger interactions of collagen with the grafted surface than with the non-modified polyethylene.

Collagen at interfaces. II: Competitive adsorption of collagen against albumin and fibrinogen.

Adsorption of chemically radiolabeled [14C] collagen from binary mixtures with albumin or fibrinogen was studied on the solution/air and solution/polyethylene interfaces and revealed the preferential adsorption of albumin. This phenomenon is confirmed by the data of surface tension measurements of single protein, collagen-albumin, and collagen-fibrinogen solutions. Desorption experiments clearly show that more irreversibly adsorbed collagen was found on polyethylene surfaces when adsorption was performed from collagen-fibrinogen than from collagen-albumin solutions. The combined adsorption-desorption and the surface tension data show that competitive adsorption of collagen at the hydrophobic surfaces is strongly influenced by the surface tension properties of the proteins in solution.