Non Inflammatory Boronate Based Glucose-Responsive Insulin Delivery Systems

Boronic acids, known to bind diols, were screened to identify non-inflammatory cross-linkers for the preparation of glucose sensitive and insulin releasing agglomerates of liposomes (Agglomerated Vesicle Technology-AVT). This was done in order to select a suitable replacement for the previously used cross-linker, ConcanavalinA (ConA), a lectin known to have both toxic and inflammatory effects in vivo. Lead-compounds were selected from screens that involved testing for inflammatory potential, cytotoxicity and glucose-binding. These were then conjugated to insulin-encapsulating nanoparticles and agglomerated via sugar-boronate ester linkages to form AVTs. In vitro, the particles demonstrated triggered release of insulin upon exposure to physiologically relevant concentrations of glucose (10 mmoles/L–40 mmoles/L). The agglomerates were also shown to be responsive to multiple spikes in glucose levels over several hours, releasing insulin at a rate defined by the concentration of the glucose trigger

Thermotropic phase behavior and headgroup interactions of the nonbilayer lipids phosphatidylethanolamine and monogalactosyldiacylglycerol in the dry state

<p>Abstract</p> <p>Background</p> <p>Although biological membranes are organized as lipid bilayers, they contain a substantial fraction of lipids that have a strong tendency to adopt a nonlamellar, most often inverted hexagonal (H_II) phase. The polymorphic phase behavior of such nonbilayer lipids has been studied previously with a variety of methods in the fully hydrated state or at different degrees of dehydration. Here, we present a study of the thermotropic phase behavior of the nonbilayer lipids egg phosphatidylethanolamine (EPE) and monogalactosyldiacylglycerol (MGDG) with a focus on interactions between the lipid molecules in the interfacial and headgroup regions.</p> <p>Results</p> <p>Liposomes were investigated in the dry state by Fourier-transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). Dry EPE showed a gel to liquid-crystalline phase transition below 0°C and a liquid-crystalline to H_IItransition at 100°C. MGDG, on the other hand, was in the liquid-crystalline phase down to -30°C and showed a nonbilayer transition at about 85°C. Mixtures (1:1 by mass) with two different phosphatidylcholines (PC) formed bilayers with no evidence for nonbilayer transitions up to 120°C. FTIR spectroscopy revealed complex interactions between the nonbilayer lipids and PC. Strong H-bonding interactions occurred between the sugar headgroup of MGDG and the phosphate, carbonyl and choline groups of PC. Similarly, the ethanolamine moiety of EPE was H-bonded to the carbonyl and choline groups of PC and probably interacted through charge pairing with the phosphate group.</p> <p>Conclusions</p> <p>This study provides a comprehensive characterization of dry membranes containing the two most important nonbilayer lipids (PE and MGDG) in living cells. These data will be of particular relevance for the analysis of interactions between membranes and low molecular weight solutes or soluble proteins that are presumably involved in cellular protection during anhydrobiosis.</p

Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket

Differential scanning calorimetry and X-ray diffraction studies of the thermotropic phase behavior of the diastereomeric di-tetradecyl-beta-D-galactosyl glycerols and their mixture.

We have investigated the thermotropic phase behavior of aqueous dispersions of the 1,2- and 2,3-di-O-tetradecyl-1(3)-O-(beta-D-galactopyranosyl)-sn- glycerols and their diastereomeric mixture using differential scanning calorimetry and low-angle and wide-angle x-ray diffraction. Upon heating, unannealed aqueous dispersions of these compounds all exhibit a lower temperature, moderately energetic phase transition at approximately 52 degrees C and a higher temperature, weakly energetic phase transition at approximately 63 degrees C, both of which are reversible on cooling. X-ray diffraction measurements identify these events as the L beta (or L' beta)/L alpha and L alpha/HII phase transitions, respectively. The structures of the L beta, L alpha, and HII phases of these lipids, as determined by x-ray diffraction measurements, are identical within the error bars for all of these lipids. On annealing below the L beta/L alpha phase transition temperature, the L beta phase converts to an Lc phase at a rate which is strongly dependent on the chirality of the glycerol backbone (1,2-sn > 1,2-rac > 2,3-sn). The temperature of the phase transition from the Lc phase seen on reheating is also dependent on the glycerol chirality. In addition, the nature of the Lc phase changes on subsequent heating in the 1,2-sn and 1,2-rac lipids, but we have not been able to detect this Lc1/Lc2 phase transition by calorimetry. However, wide-angle x-ray diffraction measurements indicate that these Lc phases differ mostly in their hydrocarbon chain packing modes. The Lc2 phase does not appear to be present in the 2,3-sn compound, suggesting that its formation is not favored in this diastereomeric isomer. These observations are discussed in relation to the effect of glycerol chirality on the molecular packing of these glycolipids, particularly on hydrogen bonding and hydration in the interfacial region of the bilayer

Measuring the composition-curvature coupling in binary lipid membranes by computer simulations

The coupling between local composition fluctuations in binary lipid membranes and curvatureaffects the lateral membrane structure. We propose an efficient method to compute the composition-curvature coupling in molecular simulations and apply it to two coarse-grained membrane models—aminimal, implicit-solvent model and the MARTINI model. Both the weak-curvature behavior that istypical for thermal fluctuations of planar bilayer membranes as well as the strong-curvature regimecorresponding to narrow cylindrical membrane tubes are studied by molecular dynamics simulation.The simulation results are analyzed by using a phenomenological model of the thermodynamics ofcurved, mixed bilayer membranes that accounts for the change of the monolayer area upon bending.Additionally the role of thermodynamic characteristics such as the incompatibility between thetwo lipid species and asymmetry of composition are investigated.Fil: Barragán Vidal, I. A.. Georg August Universität, Gottingen; AlemaniaFil: Rosetti, Carla Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Pastorino, Claudio. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Müller, M.. Georg August Universität, Gottingen; Alemani

X-ray diffraction structures of some phosphatidylethanolamine lamellar and inverted hexagonal phases.

X-ray diffraction is used to solve the low-resolution structures of fully hydrated aqueous dispersions of seven different diacyl phosphatidylethanolamines (PEs) whose hydrocarbon chains have the same effective chain length but whose structures vary widely. Both the lower-temperature, liquid-crystalline lamellar (L(alpha)) and the higher-temperature, inverted hexagonal (H(II)) phase structures are solved, and the resultant internal dimensions (d-spacing, water layer thickness, average lipid length, and headgroup area at the lipid-water interface) of each phase are determined as a function of temperature. The magnitude of the L(alpha) and H(II) phase d-spacings on either side of the L(alpha)/H(II) phase transition temperature (T(h)) depends significantly on the structure of the PE hydrocarbon chains. The L(alpha) phase d-spacings range from 51.2 to 56.4 A, whereas those of the H(II) phase range from 74.9 to 82.7 A. These new results differ from our earlier measurements of these PEs (Lewis et al., Biochemistry, 28:541-548, 1989), which found near constant d-spacings of 52.5 and 77.0-78.0 A for the L(alpha) and H(II) phases, respectively. In both phases, the d-spacings decrease with increasing temperature independent of chain structure, but, in both phases, the rate of decrease in the L(alpha) phase is smaller than that in the H(II) phase. A detailed molecular description of the L(alpha)/H(II) phase transition in these PEs is also presented