Structure Dependence of Pyridine and Benzene Derivatives on Interactions with Model Membranes.

Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, 1H one-dimensional NMR and 1H-1H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.

Correlation of insulin-enhancing properties of vanadium-dipicolinate complexes in model membrane systems: phospholipid langmuir monolayers and AOT reverse micelles.

We explore the interactions of V(III) -, V(IV) -, and V(V) -2,6-pyridinedicarboxylic acid (dipic) complexes with model membrane systems and whether these interactions correlate with the blood-glucose-lowering effects of these compounds on STZ-induced diabetic rats. Two model systems, dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers and AOT (sodium bis(2-ethylhexyl)sulfosuccinate) reverse micelles present controlled environments for the systematic study of these vanadium complexes interacting with self-assembled lipids. Results from the Langmuir monolayer studies show that vanadium complexes in all three oxidation states interact with the DPPC monolayer; the V(III) -phospholipid interactions result in a slight decrease in DPPC molecular area, whereas V(IV) and V(V) -phospholipid interactions appear to increase the DPPC molecular area, an observation consistent with penetration into the interface of this complex. Investigations also examined the interactions of V(III) - and V(IV) -dipic complexes with polar interfaces in AOT reverse micelles. Electron paramagnetic resonance spectroscopic studies of V(IV) complexes in reverse micelles indicate that the neutral and smaller 1:1 V(IV) -dipic complex penetrates the interface, whereas the larger 1:2 V(IV) complex does not. UV/Vis spectroscopy studies of the anionic V(III) -dipic complex show only minor interactions. These results are in contrast to behavior of the V(V) -dipic complex, [VO2 (dipic)](-) , which penetrates the AOT/isooctane reverse micellar interface. These model membrane studies indicate that V(III) -, V(IV) -, and V(V) -dipic complexes interact with and penetrate the lipid interfaces differently, an effect that agrees with the compounds' efficacy at lowering elevated blood glucose levels in diabetic rats.

Gel formulation containing mixed surfactant and lipids associating with carboplatin.

The interaction of amphiphilic molecules such as lipids and surfactants with the hydrophilic drug carboplatin was investigated to identify suitable self-assembling components for a potential gel-based delivery formulation. (1) H-NMR Studies in sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT, AOT)-based reverse micelles show that carboplatin associates and at least partially penetrates the surfactant interface. Langmuir monolayers formed by dipalmitoyl(phosphatidyl)choline are penetrated by carboplatin. Carboplatin was found to also penetrate the more rigid monolayers containing cholesterol. A combined mixed surfactant gel formulation containing carboplatin and cholesterol for lymphatic tissue targeting was investigated for the intracavitary treatment of cancer. This formulation consists of a blend of the surfactants lecithin and AOT (1 : 3 ratio), an oil phase of isopropyl myristate, and an aqueous component. The phases of the system were defined within a pseudo-ternary phase diagram. At low oil content, this formulation produces a gel-like system over a wide range of H(2) O content. The carboplatin release from the formulation displays a prolonged discharge with a rate three to five times slower than that of the control. Rheological properties of the formulation exhibit pseudoplastic behavior. Microemulsion and Langmuir monolayer studies support the interactions between carboplatin and amphiphilic components used in this formulation. To target delivery of carboplatin, two formulations containing cholesterol were characterized. These two formulations with cholesterol showed that, although cholesterol does little to alter the phases in the pseudo-ternary system or to increase the initial release of the drug, it contributes significantly to the structure of the formulation under physiological temperature, as well as increases the rate of steady-state discharge of carboplatin.

Investigating lipid interactions and the process of raft formation in cellular membranes using ToF-SIMS.

There is an increased interest in how lipids interact with each other, especially in the lateral separation of lipids into coexisting liquid phases as this is believed to be an attribute of raft formation in cell membranes. ToF-SIMS has shown itself to be an excellent tool for investigating cellular and model membrane systems and will be perhaps the most powerful one for investigating raft formation. Results from our laboratory show the capability of ToF-SIMS at identifying unequivocally the content of coexisting liquid lipid phases. Using supported lipid monolayers we find that the inclusion of dipalmitoylphosphatidylethanolamine (DPPE) to a homogeneous dipalmitoylphosphatidylcholine (DPPC)/cholesterol phase results in the formation of cholesterol-rich domains [A.G. Sostarecz, C.M. McQuaw, A.G. Ewing, N. Winograd, J. Am. Chem. Soc. 126 (2004) 13882]. Also, for DPPE/cholesterol systems a single homogeneous DPPE/cholesterol phase is formed at ~50 mol% cholesterol, whereas DPPC/cholesterol systems form a single phase at 30 mol% cholesterol [C.M. McQuaw, A. Sostarecz, L. Zheng, A.G. Ewing, N. Winograd, Langmuir 21 (2005) 807]. Currently we are exploring the incorporation of sphingomyelin into phospholipid-cholesterol mixtures in an effort to gain a better understanding of its role in raft formation.

Lateral heterogeneity of dipalmitoylphosphatidylethanolamine-cholesterol Langmuir-Blodgett films investigated with imaging time-of-flight secondary ion mass spectrometry and atomic force microscopy.

To better understand the influence of cholesterol (CH) on dipalmitoylphosphatidylethanolamine (DPPE), Langmuir-Blodgett (LB) model membranes of DPPE with varying amounts of cholesterol were imaged by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). Cholesterol has a condensing effect on DPPE that at low cholesterol concentrations results in lateral heterogeneity of the LB monolayer. At 4:1 DPPE/CH, islands of DPPE/CH phase exist with a connected DPPE phase. As the concentration of cholesterol is increased, the percolation threshold is crossed and the DPPE/CH phase islands connect to separate the DPPE phase (2:1 DPPE/CH). Finally, at 50 mol % cholesterol a single homogeneous DPPE/CH phase LB monolayer exists. ToF-SIMS of the DPPE/CH phase provides a lower ion signal for the characteristic lipid fragments and substrate apparently owing to the higher molecular density induced by cholesterol. AFM data indicate that the DPPE/CH phase is lower in height than the DPPE phase. As phosphatidylethanolamine is predominant in the inner lipid leaflet of cellular membranes, this work has implications for the understanding of cholesterol domains in the inner leaflet of cells.

Depth profiling of Langmuir-Blodgett films with a buckminsterfullerene probe.

Bombardment with C60+ primary ions of monolayer and multilayer barium arachidate Langmuir-Blodgett (LB) films is investigated. The behavior of cluster versus atomic (Ga+) bombardment is monitored by the barium-cationized arachidate ion (mass-to-charge ratio (m/z) 449) and a characteristic fragment ion (m/z 209) using 1-, 7-, and 15-layer model systems. The removal rate of material from the films is shown to be on the order of several hundred molecules per C60 impact, a value 100-fold larger than Ga+ impact. The enhancement in secondary ion yield is also shown to be larger for the 15-layer film (400x) than for the monolayer film (100x). Moreover, most of the increase in yield is shown to be associated with ejection of sputtered species rather than an increase in ionization probability. High yields associated with cluster bombardment are also shown to be amenable to depth profiling experiments in which the two ions can be monitored as the film is being removed. In this modality, chemical damage associated with bombardment is removed before it can accumulate on the surface. Due to the similarity of fatty acid LB films to cellular membranes, these results suggest that C60+ primary ion beams may improve the prospects for TOF-SIMS studies of biological systems.

Phosphatidylethanolamine-induced cholesterol domains chemically identified with mass spectrometric imaging.

Coexisting liquid phases of model membrane systems are chemically identified using imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). The systems studied were Langmuir-Blodgett (LB) model membranes of cholesterol (CH) with two different phospholipids, one a major component in the outer plasma membrane bilayer leaflet (dipalmitoylphosphatidylcholine (PC)) and the other a major component in the inner leaflet (dipalmitoylphosphatidylethanolamine (PE)). Binary mixtures of CH with each of the phospholipids were investigated, as well as a ternary system. A single homogeneous phase is evident for PC/CH, whereas both systems containing PE show lateral heterogeneity with phospholipid-rich and CH-rich regions. The interaction between CH and the two phospholipids differs due to the disparity between the phospholipid headgroups. Imaging TOF-SIMS offers a novel opportunity to chemically identify and differentiate the specific membrane locations of CH and phospholipid in membrane regions without the use of fluorescent dyes. This unique imaging method has been used to demonstrate the formation of micrometer-size CH domains in phosphatidylethanolamine-rich systems and is further evidence suggesting that CH may facilitate transport and signaling across the two leaflets of the plasma membrane.

Influence of molecular environment on the analysis of phospholipids by time-of-flight secondary ion mass spectrometry.

Understanding the influence of molecular environment on phospholipids is important in time-of-flight secondary ion mass spectrometry (TOF-SIMS) studies of complex systems such as cellular membranes. Varying the molecular environment of model membrane Langmuir-Blodgett (LB) films is shown to affect the TOF-SIMS signal of the phospholipids in the films. The molecular environment of a LB film of dipalmitoylphosphatidylcholine (DPPC) is changed by varying the film density, varying the sample substrate, and the addition of cholesterol. An increase in film density results in a decrease in the headgroup fragment ion signal at a mass-to-charge ratio of 184 (phosphocholine). Varying the sample substrate increases the secondary ion yield of phosphocholine as does the addition of proton-donating molecules such as cholesterol to the DPPC LB film. Switching from a model system of DPPC and cholesterol to one of dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol demonstrates the ability of cholesterol to also mask the phospholipid headgroup ion signal. TOF-SIMS studies of simplistic phospholipid LB model membrane systems demonstrate the potential use of these systems in TOF-SIMS analysis of cells.