Polyelectrolyte/amphiphile interaction studied by surface tension measurements

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

Exploration of orally available calpain inhibitors: peptidyl alpha-ketoamides containing an amphiphile at P3 site

A novel series of dipeptidyl alpha-ketoamide derivatives with amphiphile was designed and synthesized as water-soluble calpain inhibitors. The introduction of amphiphiles at the P3 site increased water solubility without loss of membrane permeability and provided the oral available inhibitors. Extension of the ethylene glycol chain at the P3 site led to an improvement in persistence of plasma levels. In particular, introduction of a combination of a diethylene glycol methyl ether moiety at the P3 site, a phenylalanine residue at the P1 site and a cyclopropyl moiety at the P' site was the most effective modification for an increase in plasma drug exposure.     

Partition of protein (lipase) through insoluble complex with amphiphiles

Summary An insoluble complex of soluble protein (lipase fraction) and commercially available amphiphile was prepared. The extraction of protein from complex revealed that the protein interacted with amphiphile was specific. The capacity of a protein to form the insoluble complex depended on its solubility and on amphiphile used in preparing solution. It suggested that the commercially available amphiphiles might be effective for partitioning soluble protein.     

Membrane components can modulate the substrate specificity of protein kinase C

The cationic amphiphile, cholesteryl-3-carboxyamidoethylene-trimethylammonium iodide, can alter the substrate specificity of protein kinase C (PKC). The phosphorylation of histone catalyzed by PKC requires the binding of the enzyme to phospholipid vesicles. This cationic amphiphile reduces both the binding of PKC to lipid and as a consequence its rate of phosphorylation of histone. In contrast, PKC bound to large unilamellar vesicles (LUVs) composed of 50 mol % POPS, 20 mol % POPC, and 30 mol % of this amphiphile catalyzes protamine sulfate phosphorylation by an almost 4 fold greater rate. This activation requires phosphatidylserine (PS) and is inhibited by Ca²⁺. The extent of activation is affected by the time of incubation of PKC with LUVs. This data suggests a novel mechanism by which PKC-dependent signal transduction pathways may be altered by altering the protein targets of this enzyme.     

Structural characterization of bacteriophage M13 solubilization by amphiphiles

The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.     

Kinetics and thermodynamics of lipid amphiphile exchange between lipoproteins and albumin in serum

We have examined the kinetics and thermodynamics of the exchange of a fluorescent amphiphile derived from a phospholipid, NBD-DMPE, between serum albumin and the serum lipoproteins of high density (HDL2 and HDL3), LDL, and VLDL. Binding of the fluorescent lipid amphiphile to bovine serum albumin is characterized, at 35 degrees C, by an equilibrium binding constant of approximately 3 x 10(6) M(-1) and a characteristic time < or = 0.1 s. Association of NBD-DMPE with the lipoprotein particles, if considered as a partitioning of amphiphile monomers between the aqueous phase and the lipoprotein particles, is characterized by an equilibrium partition coefficient between 10(5) and 10(6), being highest for LDL and lowest for HDL. The association of NBD-DMPE monomers with lipoprotein particles can be described by insertion rate constants on the order of 10(5) M(-1) s(-1) for VLDL and LDL and 10(4) M(-1) s(-1) for HDL. The desorption rate constants are on the order of 10(-5) s(-1) for all particles. The study was performed as a function of temperature between 15 and 35 degrees C. This permitted the calculation of the equilibrium thermodynamic parameters (deltaG(o), deltaH(o), and deltaS(o)) as well as the activation parameters (deltaG++(o), deltaH++(o), and deltaS++(o)) for the insertion and desorption processes. The association equilibrium is dominated by the entropic contribution to the free energy in all cases. The results are discussed in relation to phospholipid and amphiphile exchange phenomena involving the lipoproteins.     

Self‐assembly of pH and calcium dual‐responsive peptide‐amphiphilic hydrogel

Peptide‐based hydrogels have gained much interest for biomedical applications as a result of their biocompatibility. Herein, we reported a synthetic pH‐sensitive and calcium‐responsive peptide‐amphiphilic hydrogel. The sequences of the peptide amphiphiles were derived from the repeat‐in‐toxin (RTX) motif. At a certain peptide‐amphiphile concentration, self‐assembly was accompanied by the formation of a rigid, viscoelastic hydrogel at low pH or the presence of calcium ions. Circular dichroism spectra showed that the peptide amphiphiles adopted beta‐sheet structure. Meanwhile, as revealed by transmission electron microscopy, the peptide‐amphiphile self‐assembly was accompanied by the formation of long interconnected nanofibrillar superstructure. Material properties of the resulting peptide‐amphiphile hydrogel were characterized using oscillatory sheer rheology, and the storage modulus (G′) was found to be one order of magnitude higher than the loss modulus (G″), indicating a moderately rigid viscoelastic material. Furthermore, with systematical residue substitution, it was found that the aspartic acid within the repeat‐in‐toxin sequence of peptide amphiphiles was responsible for the pH and calcium selectivity. The environmental responsiveness, secondary structure, morphology, and mechanical nature of the peptide‐amphiphile hydrogel make it a possible material candidate for biomedical and engineering application. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Nutritionally variant streptococcal serotype I antigen. Characterization as a lipid-substituted poly(ribitol phosphate)

Initial characterization of the nutritionally variant streptococcal serotype I amphipathic polymer indicated an estimated m.w. of 360,000 for the aggregated form of the molecule, whereas the dissociated form had an estimated m.w. of 38,000 based on dextran m.w. standards. In addition, SDS-PAGE indicated an amphiphile with a stepladder appearance made up of several components with m.w. larger than the major electrophoresis components of LPS or lipoteichoic acid. Chemically, the serotype I amphiphile appeared to be a lipid-substituted poly(ribitol phosphate) with galactose and alanine substitution on the ribitol phosphate backbone. This report represents the first characterization of a bacterial amphiphile with a chemical composition and structure as proposed here. As is the case with other bacterial amphiphiles, the nutritionally variant streptococcal serotype I amphiphile was found both intra- and extracellularly. Finally, immunofluorescence studies demonstrated that the amphiphile was expressed on the cell surface.     

Preparation of gemini-type amphiphiles bearing cyclitol head groups and their application as high-performance modifiers for lipases

Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N'-[iminobis(trimethylene)]bisquinamide, prepared from iminobispropylamine and quino-1,5-lactone, and dialkyl N-(3-carboxypropanoyl)-L-glutamates as polar and hydrophobic components, respectively. Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL) are co-lyophilized with these synthesized gemini-type amphiphiles, and their transesterification activities in organic solvents are evaluated. The modified PCL and CRL prepared by using each amphiphile showed highly enhanced and moderately enhanced enzyme activity, respectively. These results are discussed in terms of the increased preferential exclusion of the hydrophilic heads of the amphiphile and of the topological view of the amphiphile.     

Formation of complexes between DNA and cationic amphiphile molecules by the fluorescent probe method

The formation of complexes of DNA with dodecylamine, dodecyltrimethylammonium, tetradecyltrimethylammonium, and hexadecyltrimethylammonium was studied using a fluorescent probe pyrene. The dependences of the spectral parameters of the hydrophobic pyrene probe on the concentration of the cationic amphiphile in the presence and absence of DNA were obtained and analyzed. It is shown that, in the absence of DNA, these dependences exhibit only one S-shaped region, which corresponds to the micelle formation of the amphiphile, whereas in the presence of DNA there are two S-shaped regions, which indicates the cooperative formation of two types of DNA-cationic amphiphile complexes. For each of the four cationic amphiphiles, the critical concentrations for the micelle formation in the absence of DNA (C0) and the concentrations at which the first (Cd1) and the second complex with DNA are formed were determined. It was found that the Cd1 value is 15-40 times lower than C0. The Cd1 value does not depend on DNA concentration and is determined only by the length of the hydrocarbon chain and the structure of the amphiphile ionic fragment. The Cd1 value increases as the length of the aliphatic chain decreases and upon replacement of mobile hydrogen atoms in the ammonium fragment by methyl groups. It was shown that hydrophobic clusters of amphiphile arising upon complex formation with DNA play the role of cross-links promoting DNA aggregation, or DNA compactization in the case of dilute solution of high-molecular weight DNA. The structures of the first and second DNA-cationic amphiphile complexes are proposed, and the mechanism and nature of interactions that determine their formation are discussed.     

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.     

An improved tripod amphiphile for membrane protein solubilization

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a "tripod amphiphile," that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.     

Characterization of the Streptococcus adjacens group antigen structure.

Serological classification of bacteria requires the presence of an antigen unique to the organism of interest. Streptococci are serologically differentiated by group antigens, many of which are carbohydrates, although some are amphiphiles. This report describes the chemical characterization of the Streptococcus adjacens group antigen structure. Previous studies demonstrated that the amphiphile contained phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids. Phosphodiester bonds present in the purified group antigen were identified as part of a poly(ribitol phosphate), since ribitol phosphate was the only organic phosphate detected after acid hydrolysis. Hydrofluoric acid cleavage of the phosphodiester bonds generated oligosaccharide repeating units. Gas chromatography-mass spectrometric analysis of the methylated, acetylated oligosaccharide suggested that the repeating unit is a trisaccharide of Galp beta 1-3Galp beta 1-4GalNac with N-acetylgalactosamine attached in beta-linkage to either the number two or the number four carbon of ribitol. The lipid- and carbohydrate-substituted poly(ribitol phosphate) of the S. adjacens group antigen therefore is a unique amphiphile structure, differing in its repeating-unit structure from the polyglycerophosphate structure of the more common gram-positive amphiphile lipoteichoic acid.     

Interaction of amphiphiles with integral membrane proteins. I. Structural destabilization of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols, and fatty amines

The effect of model amphiphiles on the structural stability of the anion exchange protein (band 3) of the human erythrocyte membrane was studied by differential scanning calorimetry. The concentration of membranes, as well as the concentration, head group, alkyl chain length, degree of unsaturation, and double bond configuration of a variety of alkane derivatives were all varied in a systematic way. The depression of the denaturation temperature of band 3 per unit membrane concentration of the amphiphile was then determined in order to quantitate the potency of each drug. Saturated fatty acids of chain length C8 to C24 displayed a monotonic decrease in potency up to C20, followed by a dramatic diminution in potency at C22 and C24. Unsaturation caused only minor increases in the abilities of fatty acids to perturb the anion exchanger, and surprisingly, there was neither a trend for the number of double bonds nor a significant cis-trans distinction. Arachidonic acid, as an exception, was much more effective than any other amphiphile in destabilizing band 3. Fatty acids were about three times more potent than fatty amines and fatty alcohols; however, the enhanced partitioning of the latter into the membrane compensated at certain membrane/buffer ratios for its reduced intrinsic potency. A quantitative model interpretation of the data is presented in an accompanying paper.     

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

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

Compaction of DNA in an anionic micelle environment followed by assembly into phosphatidylcholine liposomes

A difficult problem concerning the interaction of DNA with amphiphiles of opposite charge above their critical micelle concentration is the propensity for aggregation of the condensed DNA complexes. In this study, this problem was addressed by attenuating amphiphile charge density within a cholate micelle environment. The amphiphile consisted of a cationic peptide, acetyl-CWKKKPKK-amide, conjugated to dilaurylphosphatidylethanolamine. In the presence of cholate, multiple equivalents of cationic charge were required to bring about the completion of DNA condensation. At the end point of condensation, stable, soluble DNA–micelle complexes were formed, which by dynamic light scattering exhibited apparent hydrodynamic diameters between 30 and 60 nm. Aggregation, as measured by static light scattering at 90° and by turbidity, was not observed until further additions of peptide–lipid conjugate were made beyond the end point of DNA condensation. Liposome complexes containing the non-aggregated, compacted DNA were formed by adding dioleoylphosphatidylcholine followed by removing the cholate by dialysis. The resulting complexes were distributed within a narrow density range, the DNA was quantitatively assembled into the liposomes, and liposomes without DNA were not detected. Small particles were formed with a mean hydrodynamic diameter of 77 nm. The liposomal DNA showed complete retention of its supercoiled form and no detectable sensitivity to DNase (25 U/10 µg DNA, 1.5 h, 37°C). The use of an anionic, dialyzable amphiphile to attenuate charge interactions between DNA and cationic amphiphiles is a useful technology for the quantitative assembly of compacted DNA into conventional liposomes, with complete protection against nuclease activity.     

Dual modulation of protein kinase C activity by sphingosine.

Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N'-cholesteryl amido-ethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted with caution because of this dual role of the amphiphile.     

Lipoplex formation under equilibrium conditions reveals a three-step mechanism

Cellular transfection can be accomplished by the use of synthetic amphiphiles as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties of the amphiphile/gene complex is crucial. Here, we have studied the interaction between a plasmid and cationic amphiphiles, using a monolayer technique, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the plasmids, interacting with the monolayer, display a strong tendency of orientational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex formed is determined by the supercoiled size of the plasmid, and calculations reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphiphile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bulk phase, where complex assembly is triggered by mixing amphiphilic vesicles and plasmids, a similar wrapping process is observed. However, in this case, imperfections in this process may give rise to a partial exposure of plasmids, i.e., part of the plasmid is not covered with a layer of amphiphile. We suggest that these exposed sites may act as nucleation sites for massive lipoplex clustering, which in turn may affect transfection efficiency.     

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile and their application for immunosensor engineering

Langmuir-Blodgett (LB) technique appears to be quite suitable for generating biospecific surfaces and it has potential application for fabricating biosensors. In this work, mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile have been transferred onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. In order to obtain stable LB films, the influences of different parameters - type of amphiphile, surface pressure and pH - on the properties of mixed IgG/amphiphile monolayer, were investigated. Electrochemical properties of the engineered immunosensor have been measured by impedimetric spectroscopy. The immunosensor obtained exhibits a high sensitivity and a good specificity in a linear dynamic range from 200 to 1000 ng ml(-1).