A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Polymer − surfactant vesicular complexes in aqueous medium

The introduction of ionic single-tailed surfactants to aqueous solutions of EO18BO10 [EO = poly(ethylene oxide), BO = poly(1,2-butylene oxide), subscripts denote the number of repeating units] leads to the formation of vesicles, as probed by laser scanning confocal microscopy. Dynamic light scattering showed that the dimensions of these aggregates at early stages of development do not depend on the sign of the surfactant head group charge. Small-angle X-ray scattering (SAXS) analysis indicated the coexistence of smaller micelles of different sizes and varying polymer content in solution. In strong contrast to the dramatic increase of size of dispersed particles induced by surfactants in dilute solution, the d-spacing of corresponding mesophases reduces monotonically upon increasing surfactant loading. This effect points to the suppression of vesicles as a consequence of increasing ionic strength in concentrated solutions. Maximum enhancements of storage modulus and thermal stability of hybrid gels take place at different compositions, indicating a delicate balance between the number and size of polymer-poor aggregates (population increases with surfactant loading) and the number and size of polymer−surfactant complexes (number and size decrease in high surfactant concentrations).

PEGylated amyloid peptide nanocontainer delivery and release system

A micellar nanocontainer delivery and release system is designed on the basis of a peptide-polymer conjugate. The hybrid molecules self-assemble into micelles comprising a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide previously studied in our group forms helical ribbons based on a beta-sheet motif and contains beta-amino acids that are excluded from the beta-sheet structure, thus being potentially useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied, enzymatic degradation using alpha-chymotrypsin leads to selective cleavage close to the PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated form. The release of monomeric peptide is useful because aggregation of the released peptide into beta-sheet amyloid fibrils is not observed. This concept has considerable potential in the targeted delivery of peptides for therapeutic applications.

Heterosite effects in novel heteronuclear clusters [OS2Ru(CO)(11)(PPh3)] and Os2Ru(CO)(10)(2-acetylpyridine-N-isopropylimine)]

A new synthetic route towards the mixed-metal cluster [OS2Ru(CO)(12)] is described together with the syntheses of its PPh3 and iPr-AcPy (iPr-AcPy = 2-acetylpyridine-N-isopropylimine) derivatives. The molecular structures of the novel clusters [Os2Ru(CO)(11)(PPh3)] and [Os2Ru(CO)(10)(iPr-AcPy)] were determined on the basis of crystalline solid solutions of the Os2Ru and corresponding Os-3 species. The structures reveal that coordination of the Lewis bases occurs exclusively at the ruthenium site of [Os2Ru(CO)(12)], which is in agreement with density functional theory (DFT) calculations on several structural isomers of these compounds. According to the time-dependent DFT results, the lowest optically accessible excited state of [Os2Ru(CO)(10)(iPr-AcPy)] has a prevailing sigma(Ru-Os-2)pi*(iPr-AcPy) character, with a partial sigma sigma*(Ru-Os-2) contribution. In weakly coordinating 2-chlorobutane, the excited state has a lifetime tau = 10.4 +/- 1.2 ps and produces biradicals considerably faster than observed for [Os-3(CO)10(iPr-AcPy) (tau = 25.3 +/- 0.7ps)]. In coordinating acetonitrile, the excited state of [Os2Ru(CO)(10)(iPr-AcPy)] decays mono-exponentially with a lifetime tau = 2.1 +/- 0.2 ps. In contrast to [Os-3(CO)(10)(iPr-AcPy)] that forms biradicals as the main primary photoproduct even in strongly coordinating solvents, zwitterion formation from the solvated lowest excited state is observed for the heterometallic cluster. This is concluded from time-resolved absorption studies in the microsecond time domain. Due to the lower tendency of the coordinatively unsaturated Ru+(CO)(2)(iPr-AcPy-/0) moiety to bind a Lewis base, the heteronuclear biradical and zwitterionic photoproducts live significantly shorter than their triosmium counterparts. The influence of the weaker Os-2-Ru(iPr-AcPy) bond on the redox reactivity is clearly reflected in very reactive radical anions formed upon electrochemical reduction of [Os2Ru(CO)(10)(iPr-AcPy)]. The dimer [-OS(CO)(4)-Os(CO)(4)-Ru(CO)(2)(iPr-AcPy)](2)(2-) is the only IR-detectable intermediate reduction product. The dinuclear complex [Os-2(CO)(8)](2-) and insoluble [Ru(CO)(2)(iPr-AcPy)](n), are the ultimate reduction products, proving fragmentation of the OS2Ru core.

Effect of headgroup size, charge, and solvent structure on polymer−micelle interactions, studied by molecular dynamics simulations

We performed atomistic molecular dynamics simulations of anionic and cationic micelles in the presence of poly(ethylene oxide) (PEO) to understand why nonionic water-soluble polymers such as PEO interact strongly with anionic micelles but only weakly with cationic micelles. Our micelles include sodium n-dodecyl sulfate (SDS), n-dodecyl trimethylammonium chloride (DTAC), n-dodecyl ammonium chloride (DAC), and micelles in which we artificially reverse the sign of partial charges in SDS and DTAC. We observe that the polymer interacts hydrophobically with anionic SDS but only weakly with cationic DTAC and DAC, in agreement with experiment. However, the polymer also interacts with the artificial anionic DTAC but fails to interact hydrophobically with the artificial cationic SDS, illustrating that large headgroup size does not explain the weak polymer interaction with cationic micelles. In addition, we observe through simulation that this preference for interaction with anionic micelles still exists in a dipolar "dumbbell" solvent, indicating that water structure and hydrogen bonding alone cannot explain this preferential interaction. Our simulations suggest that direct electrostatic interactions between the micelle and polymer explain the preference for interaction with anionic micelles, even though the polymer overall carries no net charge. This is possible given the asymmetric distribution of negative charges on smaller atoms and positive charges oil larger units in the polymer chain.

The adsorption geometry and chemical state of lysine on Cu{110}

Chemisorbed layers of lysine adsorbed on Cu{110} have been studied using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. XPS indicates that the majority (70%) of the molecules in the saturated layer at room temperature (coverage 0.27 ML) are in their zwitterionic state with no preferential molecular orientation. After annealing to 420 K a less densely packed layer is formed (0.14 ML), which shows a strong angular dependence in the characteristic π-resonance of oxygen K edge NEXAFS and no indication of zwitterions in XPS. These experimental results are best compatible with molecules bound to the substrate through the oxygen atoms of the (deprotonated) carboxylate group and the two amino groups involving Cu atoms in three different close packed rows. This μ4 bonding arrangement with an additional bond through the !-amino group is different from geometries previously suggested for lysine on Cu{110}.

Self-assembly of amphiphilic peptides

The self-assembly of amphiphilic peptides is reviewed. The review covers surfactant-like peptides with amphiphilicity arising from the sequence of natural amino acids, and also peptide amphiphiles (PAs) in which lipid chains are attached to hydrophilic peptide sequences containing charged residues. The influence of the secondary structure on the self-assembled structure and vice versa is discussed. For surfactant-like peptides structures including fibrils, nanotubes, micelles and vesicles have been reported. A particularly common motif for PAs is beta-sheet based fibrils, although other structures have been observed. In these structures, the peptide epitope is presented at the surface of the nanostructure, providing remarkable bioactivity. Recent discoveries of potential, and actual, applications of these materials in biomedicine and bionanotechnology are discussed.

Photopolymerization of Pluronic F127 diacrylate: a colloid-templated polymerization

Pluronic F127 diacrylate (F127DA) is a bifunctional acrylate and as such it should in principle produce macroscopically cross-linked materials; however, its photopolymerization in water does not lead to 3D-extended hydrogels. The main species present after photopolymerization appear to be cross-linked micelles, which indicates that the micellar morphology of F127DA has a template effect on the polymerization. The structural analogy causes the physical state of precursor and polymerized materials to be very similar for a wide range of concentrations (5–25% wt) and temperatures (10–37 °C). Also the long-range morphology of F127DA appears to have a template effect: samples photopolymerized in a micellar gel state and redispersed at high concentration (25% wt) show a long-range organization that depended on the concentration and therefore on the order of the precursor.

Salt-induced hydrogelation of functionalised-dipeptides at high pH

Addition of divalent cations to a solution of a naphthalene-diphenylalanine that forms worm-like micelles at high pH results in the formation of a rigid, self-supporting hydrogel

Comparison of the rheology of mozzarella-type curd made from buffalo and cows’ milk

Curd rheology and calcium distribution in buffalo and cows’ milk, were compared at their natural pH and during acidification (pH 6.5–5.6). Buffalo milk displays a curd structure and rheology different from that of cows’ milk and the casein-bound calcium, as well as the contents of fat, protein and calcium, are also higher. Due to these higher amounts of casein-bound calcium, the overall curd strength with buffalo milk (as indicated by the dynamic moduli) was higher, at similar pH values, than those of equivalent gels produced from cows’ milk. The curd rheology was adversely affected at lower pH (5.8–5.6) in both of the milk types, due to the loss of casein-bound calcium from casein micelles. The degree of solubilisation of calcium in buffalo milk during acidification is quite different from that observed in cows’ milk with a lower proportion of the calcium being solubilised in the former. The maximum curd firmness was obtained at pH 6.0 in both milk types. For both species, these rheological and micellar changes were qualitatively the same but quantitatively different, due to the different milk compositions.

Effect of water-soluble polymers, polyethylene glycol and poly(vinylpyrrolidone),on the gelation of aqueous micellar solutions of Pluronic copolymer F127

The micellization of F127 (E98P67E98) in dilute aqueous solutions of polyethylene glycol (PEG6000 and PEG35000) and poly(vinylpyrrolidone) (PVP K30 and PVP K90) is studied. The average hydrodynamic radius (rh,app) obtained from the dynamic light scattering technique increased with increase in PEG concentration but decreased on addition of PVP, results which are consistent with interaction of the micelles with PEG and the formation of micelles clusters, but no such interaction occurs with PVP. Tube inversion was used to determine the onset of gelation. The critical concentration of F127 for gelation increased on addition of PEG and of PVP K30 but decreased on addition of PVP K90. Small-angle X-ray scattering (SAXS) was used to show that the 30 wt% F127 gel structure (fcc) was independent of polymer type and concentration, as was the d-spacing and so the micelle hard-sphere radius. The maximum elastic modulus (G0 max) of 30 wt% F127 decreased from its value for water alone as PEG was added, but was little changed by adding PVP. These results are consistent with the packed-micelles in the 30 wt% F127 gel being effectively isolated from the polymer solution on the microscale while, especially for the PEG, being mixed on the macroscale.

Interactions of surfactants (edge activators) and skin penetration enhancers with liposomes

Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with ipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (Tm). Surfactants also removed the lipid pre-transition but reduced Tm and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower Tm. The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced Tm and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The Tm and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2 mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.

Influence of a non-ionic amphiphilic copolymer on the self-assembly of a peptide amphiphile that forms nanotapes

The influence of a non-ionic polymeric surfactant on the self-assembly of a peptide amphiphile (PA) that forms nanotapes is investigated using a combination of microscopic, scattering and spectroscopic techniques. Mixtures of Pluronic copolymer P123 with the PA C16-KTTKS in aqueous solution were studied at a fixed concentration of the PA at which it is known to self-assemble into extended nanotapes, but varying P123 concentration. We find that P123 can disrupt the formation of C16- KTTKS nanotapes, leading instead to cylindrical nanofibril structures. The spherical micelles formed by P123 at room temperature are disrupted in the presence of the PA. There is a loss of cloudiness in the solutions as the large nanotape aggregates formed by C16-KTTKS are broken up, by P123 solubilization. At least locally, b-sheet structure is retained, as confirmed by XRD and FTIR spectroscopy, even for solutions containing 20 wt% P123. This indicates, unexpectedly, that peptide secondary structure can be retained in solutions with high concentration of non-ionic surfactant. Selfassembly in this system exhibits slow kinetics towards equilibrium, the initial self-assembly being dependent on the order of mixing. Heating above the lipid chain melting temperature assists in disrupting trapped non-equilibrium states.

Surface chemistry of glycine on Pt{111} in different aqueous environments

Adsorption of glycine on Ptf111g under UHV conditions and in different aqueous environments was studied by XPS (UHV and ambient pressure) and NEXAFS. Under UHV conditions, glycine adsorbs in its neutral molecular state up to about 0.15 ML. Further deposition leads to the formation of an additional zwitterionic species, which is in direct contact with the substrate surface, followed by the growth of multilayers, which also consist of zwitterions. The neutral surface species is most stable and decomposes at 360 K through a multi-step process which includes the formation of methylamine and carbon monoxide. When glycine and water are co-adsorbed in UHV at low temperatures (< 170 K) inter-layer diffusion is inhibited and the surface composition depends on the adsorption sequence. Water adsorbed on top of a glycine layer does not lead to significant changes in its chemical state. When glycine is adsorbed on top of a pre-adsorbed chemisorbed water layer or thick ice layer, however, it is found in its zwitterionic state, even at low coverage. No difference is seen in the chemical state of glycine when the layers are exposed to ambient water vapor pressure up to 0.2 Torr at temperatures above 300 K. Also the decomposition temperature stays the same, 360 K, irrespective of the water vapor pressure. Only the reaction path of the decomposition products is affected by ambient water vapor.

Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process

The aim of this work was to investigate the lipopeptides aggregation behavior in single and mixed solutions in a wide range of concentrations, in order to optimize their separation and purification following the two-step ultrafiltration process and using large pore size membranes (up to MWCO = 300 kDa). Micelle size was determined by dynamic light scattering. In single solutions of lipopeptide both surfactin and mycosubtilin formed micelles of different size depending on their concentration, micelles of average diameter = 5–105 nm for surfactin and 8–18 nm for mycosubtilin. However when the lipopeptides were in the same solution they formed mixed micelles of different size (d = 8 nm) and probably conformation to that formed by the individual lipopeptide, this prevents their separation according to size. These lipopeptides were purified from fermentation culture by the two-step ultrafiltration process using different MWCO membranes ranging from 10 to 300 kDa. This led to their effective rejection in the first ultrafiltration step by membranes with MCWO = 10–100 kDa but poor rejection by the 300 KDa membrane. The lipopeptides were recovered at 90% purity (in relation to protein) and with 2.34 enrichment in the permeate of the second ultrafiltration step with the 100 KDa membrane upon addition of 75% ethanol.