Fabrizia Foglia

On the hydration of the phosphocholine headgroup in aqueous solution

The hydration of the phosphocholine headgroup in 1,2-dipropionyl-sn-glycero-3-phosphocholine (C(3)-PC) in solution has been determined by using neutron diffraction enhanced with isotopic substitution in combination with computer simulation techniques. The atomic scale hydration structure around this head group shows that both the -N(CH(3))(3) and -CH(2) portions of the choline headgroup are strongly associated with water, through a unique hydrogen bonding regime, where specifically a hydrogen bond from the C-H group to water and a strong association between the water oxygen and N(+) atom in solution have both been observed. In addition, both PO(4) oxygens (P=O) and C=O oxygens are oversaturated when compared to bulk water in that the average number of hydrogen bonds from water to both X=O oxygens is about 2.5 for each group. That water binds strongly to the glycerol groups and is suggestive that water may bind to these groups when phosophotidylcholine is embedded in a membrane bilayer.

Interaction of Amphotericin B with Lipid Monolayers

Langmuir isotherm, neutron reflectivity, and Brewster angle microscopy experiments have been performed to study the interaction of amphotericin B (AmB) with monolayers prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and mixtures of this lipid with cholesterol or ergosterol to mimic mammalian and fungal cell membranes, respectively. Isotherm data show that AmB causes a more pronounced change in surface pressure in the POPC/ergosterol system than in the POPC and POPC/cholesterol systems, and its interaction with the POPC/ergosterol monolayer is also more rapid than with the POPC and POPC/cholesterol monolayers. Brewster angle microscopy shows that, in interaction with POPC monolayers, AmB causes the formation of small domains which shrink and disappear within a few minutes. The drug also causes domain formation in the POPC/cholesterol and POPC/ergosterol monolayers; in the former case, these are formed more slowly than is seen with the POPC monolayers and are ultimately much smaller; in the latter case, they are formed rather more quickly and are more heterogeneous in size. Neutron reflectivity data show that the changes in monolayer structure following interaction with AmB are the same for all three systems studied: the data are consistent with the drug inserting into the monolayers with its macrocyclic ring intercalated among the lipid acyl chains and sterol ring systems, with its mycosamine moiety colocalizing with the sterol hydroxyl and POPC head groups. On the basis of these studies, it is concluded that AmB inserts in a similar manner into POPC, POPC/cholesterol, and POPC/ergosterol monolayers but does so with differing kinetics and with the formation of quite different in-plane structures. The more rapid time scale for interaction of the drug with the POPC/ergosterol monolayer, its more pronounced effect on monolayer surface pressure, and its more marked changes as regards domain formation are all consistent with the drugs selectivity for fungal vs mammalian cell membranes.

Neutron diffraction studies of the interaction between amphotericin B and lipid-sterol model membranes

Over the last 50 years or so, amphotericin has been widely employed in treating life-threatening systemic fungal infections. Its usefulness in the clinic, however, has always been circumscribed by its dose-limiting side-effects, and it is also now compromised by an increasing incidence of pathogen resistance. Combating these problems through development of new anti-fungal agents requires detailed knowledge of the drugs molecular mechanism, but unfortunately this is far from clear. Neutron diffraction studies of the drugs incorporation within lipid-sterol membranes have here been performed to shed light on this problem. The drug is shown to disturb the structures of both fungal and mammalian membranes, and co-localises with the membrane sterols in a manner consistent with trans-membrane pore formation. The differences seen in the membrane lipid ordering and in the distributions of the drug-ergosterol and drug-cholesterol complexes within the membranes are consistent with the drugs selectivity for fungal vs. human cells.

Structural Studies of the Monolayers and Bilayers Formed by a Novel Cholesterol-Phospholipid Chimera

Langmuir isotherm, neutron reflectivity, and small angle neutron scattering studies have been conducted to characterize the monolayers and vesicular bilayers formed by a novel chimeric phospholipid, ChemPPC, that incorporates a cholesteryl moeity and a C-16 aliphatic chain, each covalently linked via a glycerol backbone to phosphatidylcholine. The structures of the ChemPPC monolayers and bilayers are compared against those formed from pure dipalmitoylphoshatidylcholine (DPPC) and those formed from a 60:40 mol % mixture of DPPC and cholesterol. In accord with previous findings showing that very similar macroscopic properties were exhibited by ChemPPC and 60:40 mol % DPPC/cholesterol vesicles, it is found here that the chimeric lipid and lipid/sterol mixture have very similar monolayer structures (each having a monolayer thickness of ∼26 Å), and they also form vesicles with similar lamellar structure, each having a bilayer thickness of ∼50 Å and exhibiting a repeat spacing of ∼65 Å. The interfacial area of ChemPPC, however, is around 10 Å(2) greater than that of the combined DPPC/cholesterol unit in the mixed lipid monolayer (viz., 57 ± 1 vs 46 ± 1 Å(2), at 35 mN·m(-1)), and this difference in area is attributed to the succinyl linkage which joins the ChemPPC steroid and glyceryl moieties. The larger area of the ChemPPC is reflected in a slightly thicker monolayer solvent distribution width (9.5 vs 9 Å for the DPPC/cholesterol system) and by a marginal increase in the level of lipid headgroup hydration (16 vs 13 H(2)O per lipid, at 35 mN·m(-1)).

Small-angle neutron scattering studies of the effects of amphotericin B on phospholipid and phospholipid-sterol membrane structure

Small-angle neutron scattering (SANS) studies have been performed to study the structural changes induced in the membranes of vesicles prepared (by thin film evaporation) from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations and different aggregation states of the anti-fungal drug, amphotericin B (AmB). In the majority of the experiments reported, the lipid vesicles were prepared with the drug added directly to the lipid dispersions dissolved in solvents favouring either AmB monomers or aggregates, and the vesicles then sonicated to a mean size of ~100 nm. Experiments were also performed, however, in which micellar dispersions of the drug were added to pre-formed lipid and lipid-sterol vesicles. The vesicles were prepared using the phospholipid palmitoyloleoylphosphatidylcholine (POPC), or mixtures of this lipid with either 30 mol% cholesterol or 30 mol% ergosterol. Analyses of the SANS data show that irrespective of the AmB concentration or aggregation state, there is an increase in the membrane thickness of both the pure POPC and the mixed POPC-sterol vesicles-in all cases amounting to ~4 Å. The structural changes induced by the drugs insertion into the model fungal cell membranes (as mimicked by POPC-ergosterol vesicles) are thus the same as those resulting from its insertion into the model mammalian cell membranes (as mimicked by POPC-cholesterol vesicles). It is concluded that the specificity of AmB for fungal versus human cells does not arise because of (static) structural differences between lipid-cholesterol-AmB and lipid-ergosterol-AmB membranes, but more likely results from differences in the kinetics of their transmembrane pore formation and/or because of enthalpic differences between the two types of sterol-AmB complexes.

Water dynamics in Shewanella oneidensis at ambient and high pressure using quasi-elastic neutron scattering

Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth’s deep biosphere and biotechnology applications. Here we applied QENS to study water transport in Shewanella oneidensis at ambient (0.1 MPa) and high (200 MPa) pressure using H/D isotopic contrast experiments for normal and perdeuterated bacteria and buffer solutions to distinguish intracellular and transmembrane processes. The results indicate that intracellular water dynamics are comparable with bulk diffusion rates in aqueous fluids at ambient conditions but a significant reduction occurs in high pressure mobility. We interpret this as due to enhanced interactions with macromolecules in the nanoconfined environment. Overall diffusion rates across the cell envelope also occur at similar rates but unexpected narrowing of the QENS signal appears between momentum transfer values Q = 0.7–1.1 Å−1 corresponding to real space dimensions of 6–9 Å. The relaxation time increase can be explained by correlated dynamics of molecules passing through Aquaporin water transport complexes located within the inner or outer membrane structures.

Neutron Scattering Studies of the Effects of Formulating Amphotericin B with Cholesteryl Sulfate on the Drug's Interactions with Phospholipid and Phospholipid-Sterol Membranes

Langmuir surface pressure, small-angle neutron scattering (SANS), and neutron reflectivity (NR) studies have been performed to determine how formulation of the antifungal drug amphotericin B (AmB), with sodium cholesteryl sulfate (SCS)-as in Amphotec-affects its interactions with ergosterol-containing (model fungal cell) and cholesterol-containing (model mammalian cell) membranes. The effects of mixing AmB in 1:1 molar ratio with cholesteryl sulfate (yielding AmB-SCS micelles) are compared against those of free AmB, using monolayers and bilayers formed from palmitoyloleoylphosphatidylcholine (POPC) in the absence and presence of 30 mol % ergosterol or cholesterol, in all cases employing a 1:0.05 molar ratio of lipid:AmB. Analyses of the (bilayer) SANS and (monolayer) NR data indicate that the equilibrium changes in membrane structure induced in sterol-free and sterol-containing membranes are the same for free AmB and AmB-SCS. Stopped-flow SANS experiments, however, reveal that the structural changes to vesicle membranes occur far more rapidly following exposure to AmB-SCS vs free drug, with the kinetics of these changes varying with membrane composition. With POPC vesicles, the structural changes induced by AmB-SCS become apparent only after several minutes, and equilibrium is reached after ∼30 min. The corresponding onset of changes in POPC-ergosterol and POPC-cholesterol vesicles, however, occurs within ∼5 s, with equilibrium reached after 10 and 120 s, respectively. The rate of insertion of AmB into POPC-sterol membranes is thus increased through formulation as AmB-SCS. Moreover, the differences in monolayer surface pressure and SANS structure-change equilibration times suggest significant rearrangement of AmB within these membranes following insertion. The reduced times to equilibrium for the POPC-ergosterol vs POPC-cholesterol systems are consistent with the known differences in affinity of AmB for these two sterols, and the reduced time to equilibrium for AmB-SCS interaction with POPC-ergosterol membranes vs that for free AmB is consistent with the reduced host toxicity of Amphotec.