Studies of a serum albumin-liposome complex as a model lipoprotein membrane

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicular, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associate with lipid.Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 Å. The same lamellar separation in the positively charged system is 108 Å. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 Å while that for the positively charged lipoprotein system is 91 Å. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer.Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 Å spacing, characteristic of hexagonal packing of the hydrocarbon chains.A classical light scattering technique is to used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose < arabinose < malonamide < glycerol). K⁺ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Structural Study of the Interaction Between the Mitochondrial Presequence of Cytochrome c Oxidase Subunit IV and Model Membranes

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of -helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.     

LBL structured chitosan-layered silicate intercalated composites based fibrous mats for protein delivery

Organic rectorite (OREC) was used to prepare intercalated composites with chitosan. The negatively charged cellulose acetate (CA) fibrous mats were modified with multilayers of the positively charged chitosan or chitosan-OREC intercalated composites and the negatively charged bovine serum albumin (BSA) via electrostatic layer-by-layer (LBL) self-assembly technique. The morphology and protein delivery properties of the resultant samples were investigated by regulating the number of deposition bilayers, the outermost layer and the composition of coating bilayers. The thickness of LBL films coated CA mats increased as the number of bilayers was increased and OREC was added. X-ray photoelectron spectroscopy indicated that chitosan and OREC were deposited on CA fibers. Small angle X-ray diffraction patterns showed that OREC was intercalated by chitosan. The in vitro BSA encapsulation and release experiments demonstrated that OREC could affect the degree of protein loading capacity and release efficiency of the LBL films coating.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Structure and phase behaviour of dimyristoylphosphatidic acid/poly(L-lysine) systems

Summary

Differential scanning calorimetry (DSC) and X-ray diffraction studies on (DMPA)/poly(L-lysine) systems are reported. DSC studies revealed that addition of poly(L-lysine) to DMPA bilayers raises the gel to liquid-crystalline phase transition of the systems, and that this effect depends on the molecular weight of the poly(L-lysine). Small-angle X-ray diffraction measurements showed that, in the liquid-crystalline phase, the lamellar spacing of a DMPA/short-poly(L-lysine) (～4000 mol. wt.) system is shorter than that of a DMPA/long-poly(L-lysine) (～22 000 mol. wt.). In this connection wide-angle X-ray diffraction measurements indicate that the long-poly(L-lysine) adopts a β-sheet conformation on the DMPA bilayers in both the gel and the liquid-crystalline phases, but the short-poly(L-lysine) adopts this conformation only on gel phase DMPA bilayers. We found that the spacings of the hydrocarbon chain packing in a DMPA bilayer in the gel phase increases with temperature, while the spacing between neighbouring polypeptide chains in long-poly(L-lysine) in the β-sheet conformation remains almost constant. These observations indicate that the positively charged lysine residues are structurally independent of the negatively charged head groups of the phospholipid. On the basis of the present results we propose a model to explain the elementary behaviour of extrinsic membrane proteins in biomembranes.     

Encapsulation of hemoglobin in phospholipid liposomes: characterization and stability

Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.     

Serum-induced leakage of negatively charged liposomes at nanomolar lipid concentrations

An enzyme inhibition assay was developed to determine methotrexate-gamma-aspartate leakage from liposomes at lipid concentrations as low as 43 nM phospholipid. When negatively charged liposomes prepared with phosphatidylglycerol/cholesterol 67:33 or phosphatidylinositol/cholesterol 67:33 were incubated in 10% (v/v) newborn calf serum, they leaked over 90% of their contents in 2 min. In contrast, liposomes prepared from phosphatidylcholine/cholesterol 67:33 leaked 18% of their contents under the same conditions. The amount of negative charge required to induce liposome leakage was determined by preparing liposomes with varying amounts of phosphatidylglycerol and phosphatidylcholine. Extensive leakage was observed only from liposomes prepared with greater than 50 mol of phosphatidylglycerol per 100 mol of phospholipid. The effect of the phase transition temperature on leakage of negatively charged liposomes in 10% (v/v) serum was investigated by using a series of phosphatidylglycerols with varying acyl chain lengths. Liposomes prepared from distearoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol leaked less than 18% of their contents in 10% serum, whereas liposomes prepared with dilauroylphosphatidylglycerol or unsaturated lipids leaked more than 70% of their contents. Lipoprotein removal from serum followed by treatment with lipid to remove residual apoproteins reduced the leakage from phosphatidylglycerol liposomes in 10% serum. Phosphatidylglycerol liposomes leaked 73% in the presence of human low-density lipoproteins, but only 29% in the presence of bovine apolipoprotein A-I, and 25% in the presence of human high-density lipoproteins. Phosphatidylglycerol/cholesterol and phosphatidylserine/cholesterol liposomes leaked 67% in 4 mg/mL bovine serum albumin purified by cold ethanol extraction. The leakage of liposomes in albumin solutions could be substantially reduced by treating the albumin with lipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

胆固醇对脂双层结构影响的SAXS和STM研究

用小角X射线散射（SAXS)和扫描隧道显微镜（STM）技术分别研究了模拟生物膜脂质体的结构以及胆固醇对生物膜双层结构的影响。结果表明,在扫描隧道显微镜照片中,磷脂分子在石墨表面形成规则的二维点状排列图像;磷脂胆固醇脂质体在石墨表面形成规则的二维波纹状排列图像。用小角X射线散射研究结果表明,DPPC脂质体是片层相结构,DPPC＋Chol脂质体是复相片层结构,DPPE＋Chol脂质体是片层立方相结构,DPPC＋DPPE＋Chol脂质体是立方六角形相结构。     

The effect of surface charges of liposomes in immunopotentiation

The purpose of this study was to establish the effect of surface charges of liposomes on its adjuvant activity to an entrapped protein antigen. The immune responses of rabbits immunized subcutaneously with lysozyme entrapped in neutral negatively and positively charged liposomes and compared with complete Freund's adjuvant (CFA), showed positively charged liposomes to be a better adjuvant than neutral, negatively charged liposomes and even CFA. This was true for solid liposomes also. Interestingly, injection of positively charged liposomes led to the formation of granulomas at the sites of immunization, which was not observed with neutral and negatively charged liposomes.     

A Quantitative Approach to Membrane Binding of Human Ubiquitous Mitochondrial Creatine Kinase Using Surface Plasmon Resonance

We have evaluated surface plasmon resonance with avidin-biotin immobilized liposomes tocharacterize membrane binding of ubiquitous mitochondrial creatine kinase (uMtCK). Whilethe sarcomeric sMtCK isoform is well known to bind to negatively charged phospholipids,especially cardiolipin, this report provides the first experimental evidence on the membraneinteraction of an uMtCK isoform. Qualitative measurements showed that liposomes containing16% (w/w) cardiolipin bind octameric as well as dimeric human uMtCK and also cytochromec, but not bovine serum albumin. Quantitative parameters could be derived only for themembrane interaction of octameric human uMtCK using an improved analytical approach.Association and dissociation kinetics of octameric uMtCK fit well to a model for heterogeneousinteraction suggesting two independent binding sites. Rate constants of the two sites differedby one order of magnitude, while their affinity constants were both about 80–100 nM. Thedata obtained demonstrate that surface plasmon resonance with immobilized liposomes is asuitable approach to characterize the binding of peripheral proteins to a lipid bilayer and thatthis method yields consistent quantitative binding parameters.     

Effects of bilayer surface charge density on molecular adsorption and transport across liposome bilayers

Second harmonic generation (SHG) was used to study both the adsorption of malachite green (MG), a positively charged organic dye, onto liposomes of different lipid compositions, and the transport kinetics of MG across the liposome bilayer in real time. We found that the dye adsorption increased linearly with the fraction of negatively charged lipids in the bilayer. Similarly, the transport rate constant for crossing the bilayer increased linearly with the fraction of charged lipid in the bilayer.     

Bilayer dimensions and hydration of glycolipids

X-ray diffraction measurements are available on a wide range of glycolipid multilamellar assemblies in excess water, but not at the defined water contents that are needed to derive bilayer dimensions. For lamellar crystalline phases and gel phases with untilted chains, or where the tilt angle is known, the cross-sectional area per chain from wide-angle diffraction can be used to determine the area per lipid molecule at the bilayer surface. Using the lipid molecular volume from densitometry, it is then possible to obtain the bilayer thickness and hence, from the lamellar repeat spacing, the water layer thickness and degree of hydration of the lipid polar groups. This is done here by using the available data for bilayer-forming diacyl and dialkyl glycosylglycerols, and for certain glycosphingolipids. The lamellar crystalline phases of these glycolipids are largely anhydrous, and the degree of hydration of the lamellar gel phases is much lower than that of the corresponding phosphoglycerolipid gel phases. A point of current uncertainty is whether the chains in the gel phases of diacyl glycoglycerolipids are appreciably tilted, unlike their dialkyl counterparts.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.     

Preparation and characteristics of novel porous hydrogel films based on chitosan and glycerophosphate

In this study, we prepared thermosensitive hydrogels by adding α-β-glycerophosphate (α-β-GP) to chitosan (CS) solutions. Then the hydrogels were dried to form films at room temperature. Scanning electron microscope (SEM) revealed that the hydrogel films had rough surfaces and porous cross-sections. Compared with pure chitosan films, the CS/GP hydrogel films showed better elasticity and lower tensile strength. Contact angle studies indicated that all these materials have good hydrophilicity. The CS/GP hydrogel films exhibited higher protein adsorption against both negatively charged protein (bovine serum albumin) and positively charged protein (lysozyme) than pure chitosan films. The results of MTT assay performed with the extracts of the CS/GP hydrogel films revealed the films had nontoxicity. The mouse embryonic fibroblast cells cultured on the CS/GP hydrogel films had good spreading and no apparent impairment of cell morphology. The results indicated that the CS/GP hydrogel film could be a promising candidate biomaterial for biomedicine applications.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Multiwalled carbon nanotubes-ceramic electrode modified with substrate-selective imprinted polymer for ultra-trace detection of bovine serum albumin

This study describes the synthesis of a new class of substrate-selective molecularly imprinted polymer. This involved tetraethylene glycol 3-morpholin propionate acrylate (functional monomer) and bovine serum albumin (template) for polymerization in aqueous condition, using "surface grafting-from" approach directly on a vinyl exposed multiwalled carbon nanotubes-ceramic electrode. The analyte recapture at pH 6.8 in aqueous environment simultaneously involved hydrophobically driven hydrogen bonds and ionic interactions between negatively charged bovine serum albumin and positively charged imprinted nanofilm. The selectively encapsulated bovine serum albumin first gets reduced at -0.9V and then oxidized within the cavity, without getting stripped off, to respond a differential pulse voltammetry signal. The limit of detection [0.42ngmL(-1) (3σ, RSD≤1.02%)] obtained was free from any cross-reactivity and matrix complications in aqueous, pharmaceutical, serum, and liquid milk samples. The proposed sensor can be used as a practical sensor for ultra-trace analysis of bovine serum albumin in clinical settings.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.     

Efficacy of antifreeze protein types in protecting liposome membrane integrity depends on phospholipid class

Antifreeze proteins have been reported to be capable of maintaining the membrane integrity of cold sensitive mammalian cells when exposed to hypothermic temperatures. However the mechanism(s) whereby these proteins exert this protective effect is unknown. The present study used liposomes as a model system to examine the nature of the interactions between four antifreeze (glyco)protein types (AFP I, II, III and AFGP) and albumin, with lipid membranes. Fluorescein isothiocyanate labelling indicated that all of the proteins bound to the three liposome types (dielaidoylphosphatidylcholine (DEPC), dielaidoylphosphatidylethanolamine (DEPE) and dielaidoylphosphatidylglycerol (DEPG)). AFGP was found to be highly effective at preventing leakage from all three liposome compositions as they were cooled through their phase transition temperatures. This was not the case for the other proteins. All four antifreeze types prevented zwitterionic DEPC liposomes from leaking as they were cooled through their phase transition temperature. However, albumin was equally as effective, indicating that this capacity was not unique to antifreeze proteins. All of the proteins, except AFGP, induced the negatively charged DEPG liposomes to leak prior to cooling, and were less effective than AFGP in preventing phase transition leakage from DEPE liposomes. It is proposed that many proteins, including antifreeze proteins, can protect zwitterionic liposomes, such as DEPC, by binding to the lipid bilayer thereby maintaining the ordered structure of the membrane during phase transition. However, when the membrane contains a negatively charged polar group, such as with DEPE and DEPG, proteins, although bound to them, may not be able to maintain sufficient membrane organization to prevent leakage during phase transition or, they may gain entry into the lipid bilayer, disrupt the structure and induce leakage. These results imply that the efficacy of antifreeze proteins in the cold protection of mammalian cells will not only depend on protein structure, but also on the lipid composition of the cell membrane.