Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Effect of lysophosphatidylcholine on behavior and structure of phosphatidylcholine liposomes

Differential scanning calorimetry (DSC), fluorescence polarization and X-ray diffraction were per-formed to investigate the kinetics of the micellar to the lamellar phase transition of dipalmitoylphosphatidylcholine/1-palmitoylphosphatidylcholine (16:0 LPC/DPPC) liposomes at gel phase. With a 16:0 LPC concentration up to 27 mol% only the sharp main transition with relatively high enthalpy (△H) values of DPPC was observed. Increasing 16 : 0 LPC concentration, the phase transition was broadened and the transition enthalpy was decreased and finally totally disappeared. The fluorescence probes of 3AS, 9AS, 12AS, and 16AP were employed, respectively, to detect the mo-bility of various sites of carbon chains of DPPC or 16:0 LPC/DPPC liposomes. It was shown that DPPC liposomes formed in the absence of 16:0 LPC always had a fluidity gradient in both gel and liquid-crystalline phase, while in the presence of 14.1 mol% and 27.0 mol% 16:0 LPC in the mixtures, the fluidity gradient tended to disappear below 40℃:     

Effects of diacylglycerol on the structure and phase behaviour of non-bilayer forming phospholipid

The phase behaviour of mixed aqueous dispersions of the monomethyl derivative of dioleoylphosphatidylethanolamine and dipalmitoylglycerol has been characterised by X-ray diffraction, differential scanning calorimetry and freeze-fracture electron microscopy for mixtures containing dipalmitoylglycerol in the concentration range 0-20 mol%. Dispersions prepared at temperatures where the phospholipid exhibits a liquid-crystalline lamellar phase show that dipalmitoylglycerol is completely phase separated into aggregates of stable crystal phase (beta'-phase). Heating mixed dispersions results in transformation of lamellar into hexagonal-II structure commencing at approximately 45 degrees C. This temperature coincides with a disappearance of beta'-phase of DPG which becomes incorporated into hexagonal-II phase. The pure phospholipid is transformed upon cooling from hexagonal-II into characteristic cubic phases; the formation of cubic phase is prevented by the presence of dipalmitoylglycerol and mixed dispersions initially form a lamellar liquid-crystalline phase in which the lipids are phase separated. The X-ray and thermal data suggest that relatively small domains of metastable crystal phase (alpha-phase) of DPG form initially on cooling and these subsequently coalesce and transform to beta'-phase.     

Structures of lipid-DNA complexes: supramolecular assembly and gene delivery

Recently, there has been a flurry of experimental work on understanding the supramolecular assemblies that are formed when cationic liposomes (CLs) are mixed with DNA. From a biomedical point of view, CLs (vesicles) are empirically known to be carriers of genes (sections of DNA) in nonviral gene delivery applications. Although viral-based carriers of DNA are presently the most common method of gene delivery, nonviral synthetic methods are rapidly emerging as alternative carriers, because of their ease of production and nonimmunogenicity (viral carriers very often evoke an undesirable and potentially lethal immune response). At the moment, cationic-lipid-based carriers have emerged as the most popular nonviral method to deliver genes in therapeutic applications, for example, CL carriers are used extensively in clinical trials worldwide. However, because the mechanism of transfection (the transfer of DNA into cells by CL carriers, followed by expression) of CL--DNA complexes remains largely unknown, the measured efficiencies are, at present, very low. The low transfection efficiencies of current nonviral gene delivery methods are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels. Recently, work has been carried out on determining the supramolecular structures of CL--DNA complexes by the quantitative technique of synchrotron X-ray diffraction. When DNA is mixed with CLs (composed of mixtures of cationic DOTAP and neutral DOPC lipids), the resulting CL--DNA complex consists of a multilamellar structure (L(alpha)(C)) comprising DNA monolayers sandwiched between lipid bilayers. The existence of a different columnar inverted hexagonal (H(II)(C)) phase in CL--DNA complexes was also demonstrated using synchrotron X-ray diffraction. Ongoing functional studies and optical imaging of cells are expected to clarify the relationship between the supramolecular structures of CL--DNA complexes and transfection efficiency.     

Structure Relationship of Cationic Lipids on Gene Transfection Mediated by Cationic Liposomes

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.Key words: cationic lipids, cationic liposomes, gene transfection     

Cationic O-ethylphosphatidylcholines and their lipoplexes: phase behavior aspects,structural organization and morphology

Ethylphosphatidylcholines, positively charged membrane lipid derivatives in which the anionic charge of the phosphate oxygen has been eliminated by ethylation, are promising nonviral, metabolizable transfection agents. We studied in detail the phase behavior, structural organization and morphology of the ethylphosphatidylcholines and their lipoplexes. Unlike the other phospholipids, dehydration does not change the melting transition temperature of O-ethyl-dipalmitoylphosphatidylcholine (EDPPC). Neither does an isoelectric amount of DNA, when added to the EDPPC aqueous dispersion. This is ascribed to the inability of EDPPC to form hydrogen bonds because of its headgroup modification. Similarly to its parent lipid DPPC, EDPPC displays a subtransition at 15 degrees C in its differential scanning calorimetry (DSC) heating scans after prolonged low-temperature incubation. The cooling behavior of the O-ethylphosphatidylcholines is sensitive to the thermal prehistory and the ionic strength. Different aggregate morphologies in the solid and the liquid-crystalline phases-respectively lamellar sheets and vesicles, as documented by light microscopy-are considered responsible for the cooling pattern. The interconversion between these morphologies is slow or even kinetically hindered, however, increasing the ionic strength to physiological values facilitates the conversion. The interdigitated chain arrangement of EDPPC gel phase tolerates incorporation of DNA between the bilayers. The minimum observed separation between the DNA strands is approximately 30-32 A, at DNA/lipid molar ratio > or =1. Formation of lipoplexes with DNA ordered in a 1-D lattice sandwiched between interdigitated lipid bilayers is reported for the first time.     

Nonionic Surfactant Vesicles Composed of Novel Spermine-Derivative Cationic Lipids as an Effective Gene Carrier In Vitro

In the present study, nonionic surfactant vesicles (niosomes) formulated with Span 20, cholesterol, and novel synthesized spermine-based cationic lipids with four hydrocarbon tails in a molar ratio of 2.5:2.5:1 were investigated as a gene carrier. The effects of the structure of the cationic lipids, such as differences in the acyl chain length (C14, C16, and C18) of the hydrophobic tails, as well as the weight ratio of niosomes to DNA on transfection efficiency and cell viability were evaluated in a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The niosomes were characterized both in terms of morphology and of size and charge measurement. The formation of complexes between niosomes and DNA was verified with a gel retardation assay. The transfection efficiency of these cationic niosomes was in the following order: spermine-C18 > spermine-C16 > spermine-C14. The highest transfection efficiency was obtained for transfection with spermine-C18 niosomes at a weight ratio of 10. Additionally, no serum effect on transfection efficiency was observed. The results from a cytotoxicity and hemolytic study showed that the cationic niosomes were safe in vitro. In addition, the cationic niosomes showed good physical stability for at least 1 month at 4°C. Therefore, the cationic niosomes offer an excellent prospect as an alternative gene carrier.     

New structural model for mixed-chain phosphatidylcholine bilayers

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP)

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.     

Structure and phase behaviour of dimyristoylphosphatidic acid/poly(L-lysine) systems

Summary

Differential scanning calorimetry (DSC) and X-ray diffraction studies on (DMPA)/poly(L-lysine) systems are reported. DSC studies revealed that addition of poly(L-lysine) to DMPA bilayers raises the gel to liquid-crystalline phase transition of the systems, and that this effect depends on the molecular weight of the poly(L-lysine). Small-angle X-ray diffraction measurements showed that, in the liquid-crystalline phase, the lamellar spacing of a DMPA/short-poly(L-lysine) (～4000 mol. wt.) system is shorter than that of a DMPA/long-poly(L-lysine) (～22 000 mol. wt.). In this connection wide-angle X-ray diffraction measurements indicate that the long-poly(L-lysine) adopts a β-sheet conformation on the DMPA bilayers in both the gel and the liquid-crystalline phases, but the short-poly(L-lysine) adopts this conformation only on gel phase DMPA bilayers. We found that the spacings of the hydrocarbon chain packing in a DMPA bilayer in the gel phase increases with temperature, while the spacing between neighbouring polypeptide chains in long-poly(L-lysine) in the β-sheet conformation remains almost constant. These observations indicate that the positively charged lysine residues are structurally independent of the negatively charged head groups of the phospholipid. On the basis of the present results we propose a model to explain the elementary behaviour of extrinsic membrane proteins in biomembranes.     

Structure and properties of totally synthetic galacto- and gluco-cerebrosides

The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides.     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).     

Transfection activity of binary mixtures of cationic o-substituted phosphatidylcholine derivatives: the hydrophobic core strongly modulates physical properties and DNA delivery efficacy

A combination of two cationic lipid derivatives having the same headgroup but tails of different chain lengths has been shown to have considerably different transfection activity than do the separate molecules. Such findings point to the importance of investigating the hydrophobic portions of cationic amphiphiles. Hence, we have synthesized a variety of cationic phosphatidylcholines with unusual hydrophobic moieties and have evaluated their transfection activity and that of their mixtures with the original molecule of this class, dioleoyl-O-ethylphosphatidylcholine (EDOPC). Four distinct relationships between transfection activity and composition of the mixture (plotted as percent of the new compound added to EDOPC) were found, namely: with a maximum or minimum; with a proportional change; or with essentially no change. Relevant physical properties of the lipoplexes were also examined; specifically, membrane fusion (by fluorescence resonance energy transfer between cationic and anionic lipids) and DNA unbinding (measured as accessibility of DNA to ethidium bromide by electrophoresis and by fluorescence resonance energy transfer between DNA and cationic lipid), both after the addition of negatively charged membrane lipids. Fusibility increased with increasing content of second cationic lipid, regardless of the transfection pattern. However, the extent of DNA unbinding after addition of negatively charged membrane lipids did correlate with extent of transfection. The phase behavior of cationic lipids per se as well as that of their mixtures with membrane lipids revealed structural differences that may account for and support the hypothesis that a membrane lipid-triggered, lamellar-->nonlamellar phase transition that facilitates DNA release is critical to efficient transfection by cationic lipids.     

Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.     

O-Alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Studies of the structure and organization of cationic lipid bilayer membranes: calorimetric, spectroscopic, and x-ray diffraction studies of linear saturated P-O-ethyl phosphatidylcholines

Differential scanning calorimetry, x-ray diffraction, and infrared and (31)P-nuclear magnetic resonance ((31)P-NMR) spectroscopy were used to examine the thermotropic phase behavior and organization of cationic model membranes composed of the P-O-ethyl esters of a homologous series of n-saturated 1,2-diacyl phosphatidylcholines (Et-PCs). Differential scanning calorimetry studies indicate that on heating, these lipids exhibit single highly energetic and cooperative endothermic transitions whose temperatures and enthalpies are higher than those of the corresponding phosphatidylcholines (PCs). Upon cooling, these Et-PCs exhibit two exothermic transitions at temperatures slightly below the single endotherm observed upon heating. These cooling exotherms have both been assigned to transitions between the liquid-crystalline and gel phases of these lipids by x-ray diffraction. The x-ray diffraction data also show that unlike the parent PCs, the chain-melting phase transition of these Et-PCs involves a direct transformation of a chain-interdigitated gel phase to the lamellar liquid-crystalline phase for the homologous series of n > or = 14. Our (31)P-NMR spectroscopic studies indicate that the rates of phosphate headgroup reorientation in both gel and liquid-crystalline phases of these lipids are comparable to those of the corresponding PC bilayers. However, the shape of the (31)P-NMR spectra observed in the interdigitated gel phase indicates that phosphate headgroup reorientation is subject to constraints that are not encountered in the non-interdigitated gel phases of parent PCs. The infrared spectroscopic data indicate that the Et-PCs adopt a very compact form of hydrocarbon chain packing in the interdigitated gel phase and that the polar/apolar interfacial regions of these bilayers are less hydrated than those of corresponding PC bilayers in both the gel and liquid-crystalline phases. Our results indicate that esterification of PC phosphate headgroups results in many alterations of bilayer physical properties aside from the endowment of a positively charged surface. This fact should be considered in assessing the interactions of these compounds with naturally occurring lipids and with other biological materials.     

Properties of Palmitoyl Phosphatidylcholine, Sphingomyelin, and Dihydrosphingomyelin Bilayer Membranes as Reported by Different Fluorescent Reporter Molecules

The properties of vesicle membranes prepared from 16:0-SM, 16:0-DHSM, or DPPC were characterized using steady-state and time-resolved fluorescence spectroscopy and different fluorescent reporter molecules. The acyl-chain region was probed using free and phospholipid-bound 1,6-diphenyl-1,3,5-hexatriene. 16:0-DHSM was found to be the more ordered than both DPPC and 16:0-SM 5°C below and above melting temperature. Interfacial properties of the phospholipid bilayers were examined using 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan), 6-propionyl-2-dimethyl-amino-naphthalene (Prodan), and dansyl-PE. Laurdan and Prodan reported that the two sphingomyelin (SM) membrane interfaces were clearly different from the DPPC membrane interface, whereas the two SM membrane interfaces had more similar properties (both in gel and liquid-crystalline phase). Prodan partition studies showed that membrane resistance to Prodan partitioning increased in the order: 16:0-SM < DPPC < 16:0-DHSM. The degree to which dansyl-PE is exposed to water reflects the structural properties of the membrane-water interface. By comparing the lifetime of dansyl-PE in water and deuterium oxide solution, we could show that the degree to which the dansyl moiety was exposed to water in the membranes increased in the order: 16:0-SM < DPPC < 16:0-DHSM. In conclusion, this study has shown that DHSM forms more ordered bilayers than acyl-chain matched SM or phosphatidylcholine, even in the liquid-crystalline state.