Jenifer Thewalt

From Lanosterol to Cholesterol: Structural Evolution and Differential Effects on Lipid Bilayers

Cholesterol is an important molecular component of the plasma membranes of mammalian cells. Its precursor in the sterol biosynthetic pathway, lanosterol, has been argued by Konrad Bloch (Bloch, K. 1965. Science. 150:19-28; 1983. CRC Crit. Rev. Biochem. 14:47-92; 1994. Blonds in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. Yale University Press, New Haven, CT.) to also be a precursor in the molecular evolution of cholesterol. We present a comparative study of the effects of cholesterol and lanosterol on molecular conformational order and phase equilibria of lipid-bilayer membranes. By using deuterium NMR spectroscopy on multilamellar lipid-sterol systems in combination with Monte Carlo simulations of microscopic models of lipid-sterol interactions, we demonstrate that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquid-ordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. We also discuss the biological relevance of our results, in particular in the context of membrane domains and rafts.

The Effect of Ceramide on Phosphatidylcholine Membranes: A Deuterium NMR Study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cers chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.

The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo.

Triggered release of liposomal contents following tumor accumulation and mild local heating is pursued as a means of improving the therapeutic index of chemotherapeutic drugs. Lysolipid-containing thermosensitive liposomes (LTSLs) are composed of dipalmitoylphosphatidylcholine (DPPC), the lysolipid monostearoylphosphatidylcholine (MSPC), and poly(ethylene glycol)-conjugated distearoylphosphatidylethanolamine (DSPE-PEG(2000)). We investigated the roles of DSPE-PEG(2000) and lysolipid in the functional performance of the LTSL-doxorubicin formulation. Varying PEG-lipid concentration (0-5 mol%) or bilayer orientation did not affect the release; however, lysolipid (0-10 mol%) had a concentration-dependent effect on drug release at 42 degrees C in vitro. Pharmacokinetics of various LTSL formulations were compared in mice with body temperature controlled at 37 degrees C. As expected, incorporation of the PEG-lipid increased doxorubicin plasma half-life; however, PEG-lipid orientation (bilayer vs. external leaflet) did not significantly improve circulation lifetime or drug retention in LTSL. Approximately 70% of lysolipid was lost within 1 h postinjection of LTSL, which could be due to interactions with the large membrane pool of the biological milieu. Considering that the present LTSL-doxorubicin formulation exhibits significant therapeutic activity when used in conjunction with mild heating, our current study provided critical insights into how the physicochemical properties of LTSL can be tailored to achieve better therapeutic activity.

Phase Behavior of an Equimolar Mixture of N-Palmitoyl-d-erythro-sphingosine, Cholesterol, and Palmitic Acid, a Mixture with Optimized Hydrophobic Matching

The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 degrees C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.

Fatty Acids Influence “Solid” Phase Formation in Models of Stratum Corneum Intercellular Membranes

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skins barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cers small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.

Computational and experimental approaches for investigating nanoparticle-based drug delivery systems.

Most therapeutic agents suffer from poor solubility, rapid clearance from the blood stream, a lack of targeting, and often poor translocation ability across cell membranes. Drug/gene delivery systems (DDSs) are capable of overcoming some of these barriers to enhance delivery of drugs to their right place of action, e.g. inside cancer cells. In this review, we focus on nanoparticles as DDSs. Complementary experimental and computational studies have enhanced our understanding of the mechanism of action of nanocarriers and their underlying interactions with drugs, biomembranes and other biological molecules. We review key biophysical aspects of DDSs and discuss how computer modeling can assist in rational design of DDSs with improved and optimized properties. We summarize commonly used experimental techniques for the study of DDSs. Then we review computational studies for several major categories of nanocarriers, including dendrimers and dendrons, polymer-, peptide-, nucleic acid-, lipid-, and carbon-based DDSs, and gold nanoparticles. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Modeling lipid-sterol bilayers: applications to structural evolution, lateral diffusion, and rafts.

Publisher Summary This chapter describes the off-lattice models for lipid–cholesterol and lipid–lanosterol bilayers. The difference in behavior between the two sterols was modeled on the basis of their specific molecular characteristics and in terms of their differential interactions with lipid molecules. The Metropolis Monte Carlo method and the algorithm used in the MMC simulations of the model are analyzed. The simulated equilibrium phase diagrams for the lipid–cholesterol and the lipid–lanosterol membranes are shown. It is found that the small modification in the lipid–sterol interaction strength leads to considerable differences in the overall topologies of the two-phase diagrams. To characterize quantitatively, the differential effects of the two sterols on the physical properties of lipid–sterol bilayer membranes, the calculated conformational order parameter is presented. The lipid tracer diffusion coefficient in a model lipid–cholesterol binary mixture was calculated as a function of cholesterol concentration and temperature.

A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholines sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohols long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.

Interaction of a charged polymer with zwitterionic lipid vesicles.

The interaction between polyethylenimine (PEI) and phospholipid bilayers plays an important role in several biophysical applications such as DNA transfection of target cells. Despite considerable investigation into the nature of the interaction between PEI and phospholipid bilayers, the physical process remains poorly understood. In this paper, we study the impact of PEI on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles as a function of salt concentration using several techniques including dynamic (DLS) and static (SLS) light scattering, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). At low salt concentration, vesicles aggregate, leading to the formation of stable clusters whose final size depends on the PEI concentration. At high salt concentration the system does not aggregate; DSC and NMR data reveal that the PEI penetrates into the bilayer, and SLS measurements are consistent with PEI crossing the bilayer. The transfectional ability of PEI is discussed in terms of these results.

Deuterium nuclear magnetic resonance study of the interaction of branched chain compounds (phytanic acid, phytol) with a phospholipid model membrane

Deuterium nuclear magnetic resonance (2H-NMR) spectra have been determined for 50 wt% aqueous dispersions of 1-palmitoyl(stearoyl)-2-[2H31]palmitoyl-sn-glycero-3-phosphocho lin e (PC-d31) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid over the temperature range -5-55 degrees C. Concentration effects of the isoprenoid compounds are also reported. First moments (M1) and order parameters were calculated from the spectra. 20 Mol% of either branched chain compound causes an approximate 9% increase in the mean order parameter SCD. Significant effects are seen on the PC-d31 phase behavior. 20 Mol% of either branched chain compound causes the gel to liquid crystalline onset temperature (Ts) to drop to 28 degrees C from 38 degrees C for PC-d31 alone, as seen from the temperature dependent M1 values. The melting range ([Tl--Ts]) is congruent to 1.5 degrees C for PC-d31 and congruent to 11 degrees C for PC-d31 containing 20 mol% of the branched chain compounds. This is in direct contrast to their straight chain analogues, hexadecanol and palmitic acid, which have been shown to elevate the phase transition temperature. The isoprenoid compounds cause significant disruption of the gel phase, forcing nearest neighbor phospholipid chains apart. Transverse relaxation times (T2e, the time constant for decay of the quandrupolar echo) have been determined over the temperature range -5-50 degrees C. Possible explanation for the effect of the isoprenoid compounds on the dynamic structure of phospholipids in the bilayer are proffered.