Simple Lipid Research Articles

Lipid formulation as a drug carrier for drug delivery.

In recent years, a Drug Delivery System (DDS), a preparative approach attracts the attention in the development of new drugs. DDS focuses on the regulation of the in vivo dynamics, such as absorption, distribution, metabolism, and elimination, thereby improving the effectiveness and the safety of the drugs by an applicable use of drug preparation technologies. A conventional intravenous dosage form of Amphotericin B (AmB), Fungizone, is the most effective clinically available for treating fungal infections. However, the clinical efficacy of AmB is limited by its adverse effects. Several lipid formulations, such as Liposomal AmB (L-AmB), AmB lipid complex (ABLC), and AmB colloidal dispersion (ABCD), with reduced side effects have been developed. These formulations are reported to have excellent safety and efficacy. However, comparable efficacy can be achieved only when they are administered at high doses than AmB. One of the problems of using these formulations is that they are easily taken up by the reticuloendothelial system (RES). An artificial lipoprotein-like particles, a novel drug carrier Lipid Nano-Sphere (LNS), which is 25 - 50 nm in size and is composed of phospholipids and simple lipid. LNS show a higher plasma concentration of drugs and lower uptake by RES-tissue different forms other lipid base drug carriers. In vitro and in vivo, LNS incorporating AmB, NS-718, shows reduced toxicity, while maintaining activity against fungi. LNS have a unique characteristic as an effective carrier of AmB for treatment of fungal infection.

Anchor dependency for non-glycerol based cationic lipofectins: mixed bag of regular and anomalous transfection profiles.

Although detailed structure-activity, physicochemical and biophysical investigations in probing the anchor influence in liposomal gene delivery have been reported for glycerol-based transfection lipids, the corresponding investigation for non-glycerol based simple monocationic transfection lipids have not yet been undertaken. Towards this end, herein, we delineate our structure-activity and physicochemical approach in deciphering the anchor dependency in liposomal gene delivery using fifteen new structural analogues (lipids 1-15) of recently reported non-glycerol based monocationic transfection lipids. The C(14) analogues in both series 1 (lipids 1-6) and series 2 (lipids 7-15) showed maximum efficiency in transfecting COS-1 and CHO cells. However, the C(12) analogue of the ether series (lipid 3) exhibited a seemingly anomalous behavior compared with its transfection efficient C(10) and C(14) analogues (lipids 2 and 4) in being completely inefficient to transfect both COS-1 and CHO cells. The present structure-activity investigation also convincingly demonstrates that enhancement of transfection efficiencies through incorporation of membrane reorganizing unsaturation elements in the hydrophobic anchor of cationic lipids is not universal but cell dependent. The strength of the interaction of lipids 1-15 with DNA was assessed by their ability to exclude ethidium bromide bound to the DNA. Cationic lipids with long hydrophobic tails were found, in general, to be efficient in excluding EtBr from DNA. Gel to liquid crystalline transition temperatures of the lipids was measured by fluorescence anisotropy measurement technique. In general (lipid 2 being an exception), transfection efficient lipids were found to have their mid transition temperatures at or below physiological temperatures (37 degrees C).

Design, Synthesis, and Transfection Biology of Novel Cationic Glycolipids for Use in Liposomal Gene Delivery

The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

Assessment of the retention properties of poly(vinyl alcohol) stationary phase for lipid class profiling in liquid chromatography

Potentialities of polymerized vinyl alcohol on silica gel were assessed for class separation of simple lipids, sphingolipids, glyceroglycolipids and phospholipids by high-performance liquid chromatography. A screening of pure solvents in binary gradient elution and a chemometric approach was used to define a rugged two segment linear gradient formed from four solvents for total lipid class separation. Triethylamine and formic acid were added in all mobile phase components for acidic phospholipid separation and evaporative light scattering response enhancement. Simple analytical procedures are described for the analysis of complex lipid materials.

Stability and behavior of a comb-graft copolymer stabilizing a thin oil emulsion film

Amphiphilic polymers are able to adsorb at liquid/liquid interfaces and provide stability for emulsions. Studies on thin liquid films provide a way to measure forces between two approaching interfaces which are responsible for emulsion and foam stability. This paper begins with a brief review of some of the work which examine the effects of polymeric surfactants on stabilizing thin films. Then, a more detailed look at the effects of a graft–comb copolymer on stabilizing an oil-in-water film is given. The stabilizing polymeric surfactant (“polysoap”) comprised a polydimethylsiloxane backbone with hydrophobic alkyl and hydrophilic ethylene/propylene oxide grafts. Electrical compressive stresses were imposed on the films, and their thicknesses were determined from measurements of capacitance and optical interference. Also, larger compressive stresses were applied in order to rupture the films and observe film breakdown behavior. The films were remarkably thick and compressible compared with films formed from simple surfactant or lipid systems. The film properties were relatively insensitive to the surfactant concentration and moderately sensitive to polymeric surfactant purity. The observed thicknesses are shown not to arise from interfacial electrostatic effects or van der Waals forces but from steric interactions. The observed thicknesses are consistent either with strongly stretched chains adsorbed at the interface or with multichain aggregate structures at the interface. The exact mechanism is still unclear. Copyright © 2001 John Wiley & Sons, Ltd.

Voltammetric and spectroscopic studies on methyl green and cationic lipid bound to calf thymus DNA

DNA interaction with cationic lipids promises to be a versatile and effective synthetic transfection agent. This paper presents the study on binding of a simple artifical cationic lipid, cetyltrimethylammonium bromide (CTAB), to calf thymus DNA (CT DNA) prior to the condensation process, taking methyl green (MG) as a probe. The results show that the CTAB binds to DNA through electrostatic interaction forming a hydrophobic complex, thus changing the micro-environment of duplex of DNA, so the binding state of MG and DNA is changed, and a complex CTAB-CT DNA-MG is formed. This fact suggests a new way to mediate the conformation of molecular assemblies of DNA and lipids.

Molecular dynamics simulation studies of lipid bilayer systems.

The main structural element of biological membranes is a liquid-crystalline lipid bilayer. Other constituents, i.e. proteins, sterols and peptides, either intercalate into or loosely attach to the bilayer. We applied a molecular dynamics simulation method to study membrane systems at various levels of compositional complexity. The studies were started from simple lipid bilayers containing a single type phosphatidylcholine (PC) and water molecules (PC bilayers). As a next step, cholesterol (Chol) molecules were introduced to the PC bilayers (PC-Chol bilayers). These studies provided detailed information about the structure and dynamics of the membrane/water interface and the hydrocarbon chain region in bilayers built of various types of PCs and Chol. This enabled studies of membrane systems of higher complexity. They included the investigation of an integral membrane protein in its natural environment of a PC bilayer, and the antibacterial activity of magainin-2. The latter study required the construction of a model bacterial membrane which consisted of two types of phospholipids and counter ions. Whenever published experimental data were available, the results of the simulations were compared with them.

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.

Perturbation of Lipid and Protein Structure by General Anesthetics: How Little Is Too Little?

Assistant Professor of Anesthesia Harvard Medical SchoolAssistant AnesthetistMassachusetts General HospitalDepartment of AnesthesiaBoston, Massachusetts 02114Raines@etherdome.MGH.harvard.edu.To the Editor:—Lipid-based theories of general anesthetic action have long endured because numerous studies have shown that the in vivo pharmacology of an anesthetic correlates remarkably well with its ability to perturb the structural properties of simple lipid bilayers. The Meyer-Overton correlation between anesthetic potency and hydrophobicity, the inactivity of nonanesthetic long chain alcohols and highly halogenated volatile compounds (nonimmobilizers), and pressure reversal have all been demonstrated in studies using protein-free lipid bilayers. 1–6Nevertheless, a most persuasive and often mentioned argument against lipid-based theories is that at clinically relevant concentrations, anesthetics induce only small perturbations in lipid bilayer structure. 7,8For example, halothane reduces the order parameter (increase the “fluidity”) of lipid bilayers by only 1% at clinically relevant concentrations. 9An equivalent reduction in order parameter may be obtained by raising the temperature of the bilayer by less than 1°C. Similarly, halothane reduces the transition temperature between a lipid bilayer’s liquid and gel phases by only 0.5°C at anesthetic concentrations and by only 5°C even at 10 times the minimum aveolar concentration (MAC). 10I was, therefore, very interested to read the study by Johansson et al. quantifying the effects of isoflurane and halothane on structural properties of bovine serum albumin, a lipid-free protein model used in mechanistic studies of anesthetic action. 11What did their studies show? At approximately 1 MAC, isoflurane and halothane increased the fluorescence anisotropy of bovine serum albumin by 1%. An equivalent reduction was obtained by raising the temperature of bovine serum albumin by approximately 1° C. Similarly, isoflurane and halothane raised the transition temperature between the folded and unfolded states of bovine serum albumin by less than 1°C at anesthetic concentrations and by only 3–4°C even at 10 times MAC. Studies of anesthetic binding to other protein models have been similarly unable to demonstrate significant anesthetic-induced changes in protein structure. 12,13Thus, anesthetics induce similarly small changes in the structural properties of lipids and proteins. For consistency, shouldn’t we now conclude that such insensitivity argues strongly against a protein site of anesthetic action?The inability to detect significant anesthetic-induced structural changes in either lipid or protein model systems highlights the practical (and obvious) limitations of such studies: we can only measure what we can measure. Fluorescence anisotropy, denaturation temperature, phase transition temperature, and order parameter have been used by biophysicists for many years as indicators of lipid bilayer and protein structure in large part because they are easily quantitated. There is no compelling theoretical reason to believe that changes in these properties directly accounts for the functional effects of anesthetics on relevant targets in the central nervous system. In fact, it seems quite likely that the anesthetic state results from changes in other lipid and/or protein physical properties that are not so easily measured.

Lipid Compositions of Seven Species of Genus Pichia, Methanol-Utilizing Yeast

Lipid compositions of 7 species (10 strains) of the genus Pichia, methanol-utilizing yeast were determined and found to be 3.4 to 10.4 percent lipid in dry cells. Cellular fatty acids were predominantly oleic and linoleic acids. The yeast strains were those containing either several or only a few percent α-linolenic acid in cellular lipid. Dry cells of Pichia minuta contained more than 10 percent α-linolenic acid distributed in phospholipid more than in simple lipids and glycolipids.For growing strains, the most suitable methanol content in the medium was 1 percent and optimum incubation temperature, 25°C. Lower temperature increased α-linolenic acid content in cellular lipid.

Solid phase lipid synthesis (SPLS) for construction of an artificial glycolipid library

A simple solid phase lipid synthesis (SPLS) is proposed for construction of an artificial glycolipid library; this method is so convenient and flexible that we have efficiently constructed a glycolipid library; some of these glycolipids formed into stable bilayer aggregates in aqueous solution, suggesting that the glycolipids obtained by SPLS are useful as a suitable model of naturally occurring glycolipids.

Lipid composition of the extracellular matrix of Botrytis cinerea germlings

Six simple lipid classes (mono-, di- and tri-acylglycerols, free fatty acids, free fatty alcohols and wax esters) were identified by TLC in the extracellular matrix of Botrytis cinerea germlings and the molecular components of each class were characterized using GC-MS. The relative amounts of fatty acids and fatty alcohols within each lipid class were determined by GC-FID. Over all the lipid classes, the most abundant saturated fatty acids were palmitic (ca. 30%) and stearic acid (ca. 22%). Palmitoleic and oleic acids made up ca. 21% and 24% (respectively) of the free fatty acids, while erucic (ca. 4.1%) and linoleic (ca. 3.6%) acids were the most abundant unsaturated fatty acids in the acylglycerides. The acylglycerides also contained almost 35% long chain fatty acids (C20:0 to C28:0). Six fatty acids were identified which had odd-numbered carbon chain lengths (C15:0, C17:0, C19:0, C21:0, C23:0 and C25:0). Of these, pentacosanoic acid made up almost 14% of the fatty acids in the acylglycerides. Three methyl-branched chain fatty acids, namely isopalmitic, isoheptadecanoic and anteisopalmitic, were identified in the ECM, all in small amounts. Of the fatty alcohols identified, only palmityl and stearyl alcohols were found in the free form (ca. 57% and 43%, respectively) but arachidyl alcohol (ca. 47%) and 1-octacosanol (ca. 30%) were the most abundant fatty alcohols found in the wax ester fraction.

Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia

Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [ 3H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg 2+-stimulated) and independent (Mn 2+-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro- D- myo-inositol (3-F-Ins) and 1-deoxy-1-F- scyllo-Ins (1-F- scyllo-Ins), which are considered potent inhibitors of Mn 2+-stimulated headgroup exchange enzyme, inhibited the incorporation of [ 3H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn 2+-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F- scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [ 3H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [ 3H]PI and [ 3H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis.

How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?

Although transport of long-chain free fatty acids (FFAs) into cells is often analyzed in the same way as glucose transport, we argue that the transport of the lipid-soluble amphipathic FFA molecule must be viewed differently. The partitioning of FFAs into phospholipid bilayers and their interfacial ionization are particularly relevant to transport. We summarize new data supporting the diffusion hypothesis in simple lipid bilayers and in plasma membranes of cells. Along with previous supporting data, the new data indicate that transport of FFAs through membranes could occur rapidly by flip-flop of the un-ionized form of the FFA. It appears that, at least for the adipocyte, passive diffusion guarantees fast entry and exit of FFAs at both low and high concentrations. Although there are several candidate proteins for the membrane transport of FFAs, most of these proteins have other established functions. Thus, unlike the glucose transporters, these proteins would not be single-function proteins. Definitive proof of their function as FFA transporters awaits their reconstitution into simple model systems.

Effect of lipid unsaturation on the binding of native and a mutant form of cytochrome b5 to membranes.

The partitioning of native cytochrome b5 and a mutant form, where Trp-108 and Trp-112 were both replaced by Leu, into small unilamellar lipid vesicles was examined. The vesicles were made from phosphatidylcholines containing mono- and di-unsaturated acyl chains. As these amphipathic proteins self-associate in aqueous solution, the binding was not monitored by a simple lipid titration experiment but by an exchange assay using fluorescence quenching by brominated lipids. Each protein had a greater affinity for lipids containing mono-unsaturated chains than for vesicles containing di-unsaturated chains, and the affinities of both proteins increased in buffers of higher ionic strength. The native protein had a higher affinity than the mutant protein for all vesicles; the ratio of the affinities was relatively constant at approximately 30. This corresponds to a difference in the free energy of partitioning of 2 kcal mol(-)(1). The fluorescence quantum yields of both proteins were much lower in lipids with di-unsaturated chains whereas a similar lowering was not seen with a simple Trp compound. These data suggest that the decreased membrane hydrophobicity seen by the proteins in di-unsaturated membranes is not an inherent property of the bilayer but is induced by the insertion of the protein. Further, the similar behavior of the two proteins suggests this modulation is not sensitive to the amino acid side chains of the inserted domain.

Novel series of non-glycerol-based cationic transfection lipids for use in liposomal gene delivery.

A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery reagents were better than that of lipofectamine, a widely used transfection agent in cationic lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a 1:1:0.3 mol ratio of DHDEAB (N, N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide):cholesterol:HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower than 1 suggests that the amphiphile-DNA complex may have net negative charge. Our results reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge of the lipid-DNA complex need not always be positive. In addition, our transfection results also imply that favorable hydrogen-bonding interactions between the lipid headgroups and the cell surface of biological membranes may have some role for improving the transfection efficiency in cationic lipid-mediated gene delivery.

Two-Microelectrode Voltage Clamp of Xenopus Oocytes: Voltage Errors and Compensation for Local Current Flow

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The most common method for their electrophysiological investigation is the two-microelectrode voltage clamp technique. The quality of voltage clamp recordings obtained with this technique is poor when membrane currents are large and when rapid charging of the membrane is desired. Detailed mathematical modeling of the experimental setup shows that the reasons for this weak performance are the electrical properties of the oocytes and the geometry of the setup. We measured the cytosolic conductivity to be ∼5 times lower than that of the typical bath solution, and the specific membrane capacitance to be ∼6 times higher than that of a simple lipid bilayer. The diameter of oocytes is typically ∼1 mm, whereas the penetration depth of the microelectrodes is limited to ∼100 μm. This eccentric current injection, in combination with the large time constants caused by the low conductivity and the high capacitance, yields large deviations from isopotentiality that decay slowly with time constants of up to 150 μs. The inhomogeneity of the membrane potential can be greatly reduced by introducing an additional, extracellular current-passing electrode. The geometrical and electrical parameters of the setup are optimized and initial experiments show that this method should allow for faster and more uniform control of membrane potential.

Hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bacteria in vitro.

OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens...

Structure and phase transitions in Langmuir monolayers

Lipid monolayers on the surface of water have been studied for over a hundred years, but in the last decade there has been a dramatic evolution in our understanding of the structures and phase transitions of these systems, driven by new experimental techniques and theoretical advances. In this review, dense monolayers of simple lipids are described in detail, including structures revealed by x-ray-diffraction experiments, computer simulations, molecular models, and a phenomenological theory of phase transitions. The effects of chirality and the structures of phospholipid monolayers are considered. Open questions and possible approaches to finding answers are discussed.

Composition and enzymatic activity of the extracellular matrix secreted by germlings of botrytis cinerea.

Germlings of Botrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260-265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) and myo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis of p-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.