Nonlamellar Phases Research Articles

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 the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59–80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and 31P and 2H solid-state NMR spectroscopy. SP-B 59–80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B 59–80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B 59–80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B 59–80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL 4, a peptide mimetic of SP-B which was originally designed using SP-B 59–80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.

Effect of Cations on the Structure of Bilayers Formed by Lipopolysaccharides Isolated from Pseudomonas aeruginosa PAO1

The asymmetric outer membrane of Gram-negative bacteria contains lipopolysaccharides (LPSs) which contribute significantly to the bacterium's surface properties and play a crucial role in regulating membrane permeability. We report on neutron diffraction studies performed on aligned, self-assembled bilayers of Na-, Ca-, and Mg-salt forms of LPS isolated from Pseudomonas aeruginosa PAO1. From the one-dimensional neutron scattering length density profiles we find that water penetrates Ca2+-LPS bilayers to a lesser extent than either Na+- or Mg2+-LPS bilayers. This differential water penetration could have implications as to how small molecules permeate the outer membrane of Gram-negative bacteria and, possibly, how nonlamellar phases are formed.

Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes

Daptomycin is a cyclic anionic lipopeptide with an antibiotic activity that is completely dependent on the presence of calcium (as Ca 2+). In a previous study [Jung et al., 2004. Chem. Biol. 11, 949–957], it was concluded that daptomycin underwent two Ca 2+-dependent structural transitions, whereby the first transition was solely dependent on Ca 2+, while the second transition was dependent on both Ca 2+ and the presence of negatively charged lipids that allowed daptomycin to insert into and perturb bilayer membranes with acidic character. Differences in the interaction of daptomycin with acidic and neutral membranes were further investigated by spectroscopic means. The lack of quenching of intrinsic fluorescence by the water-soluble quencher, KI, confirmed the insertion of the daptomycin Trp residue into the membrane bilayer, while the kynurenine residue was inaccessible even in an aqueous environment. Differential scanning calorimetry (DSC) indicated that the binding of daptomycin to neutral bilayers occurred through a combination of electrostatic and hydrophobic interactions, while the binding of daptomycin to bilayers containing acidic lipids primarily involved electrostatic interactions. The binding of daptomycin to acidic membranes led to the induction of non-lamellar lipid phases and membrane fusion.

Influence of the Lamellar Phase Unbinding Energy on the Relative Stability of Lamellar and Inverted Cubic Phases

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (QII) phases at temperatures between the lamellar (Lα) and inverted hexagonal (HII) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct Lα/HII phase transitions and may form intermediate QII phases only after the temperature is cycled repeatedly across the Lα/HII phase transition temperature, TH, or when the HII phases are cooled from T>TH. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the Lα phase bilayers, gu, that serves to stabilize the Lα phase relative to the nonlamellar phases. The planar Lα phase bilayers must separate for a QII phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of QII phase from Lα at temperatures near the Lα/QII transition temperature (TQ). Using gu and elastic constant values typical of unsaturated PEs, we show that gu is sufficient to make TQ>TH for the latter lipids. Such systems would display direct Lα→HII transitions, and a QII phase might only form as a metastable phase upon cooling of the HII phase. The gu values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase TQ by only a few degrees, consistent with observations of these systems. This influence of gu also rationalizes the effect of some aqueous solutes to increase the rate of QII formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring TQ and on measuring QII phase unit cell dimensions, respectively. In view of the effect of gu on TQ that we describe here, the latter method, which does not depend on the value of gu, is preferable.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C(95) on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C(95) had no detectable effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C(95). Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C(95). These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.

Interaction of the C-Terminal Region of the G γ Protein with Model Membranes

Heterotrimeric G-proteins interact with membranes. They accumulate around membrane receptors and propagate messages to effectors localized in different cellular compartments. G-protein-lipid interactions regulate G-protein cellular localization and activity. Although we recently found that the G βγ dimer drives the interaction of G-proteins with nonlamellar-prone membranes, little is known about the molecular basis of this interaction. Here, we investigated the interaction of the C-terminus of the G γ 2 protein (P γ -FN) with model membranes and those of its peptide (P γ ) and farnesyl (FN) moieties alone. X-ray diffraction and differential scanning calorimetry demonstrated that P γ -FN, segregated into P γ -FN-poor and -rich domains in phosphatidylethanolamine (PE) and phosphatidylserine (PS) membranes. In PE membranes, FN increased the nonlamellar phase propensity. Fourier transform infrared spectroscopy experiments showed that P γ and P γ -FN interact with the polar and interfacial regions of PE and PS bilayers. The binding of P γ -FN to model membranes is due to the FN group and positively charged amino acids near this lipid. On the other hand, membrane lipids partially altered P γ -FN structure, in turn increasing the fluidity of PS membranes. These data highlight the relevance of the interaction of the C-terminal region of the G γ protein with the cell membrane and its effect on membrane structure.

On the correlation between phase evolution of lipoplexes/anionic lipid mixtures and DNA release

We investigated the structural evolution of three lipoplex formulations when interacting with anionic lipids by synchrotron small angle x-ray diffraction, while the extent of DNA release from lipoplexes by anionic lipids was evaluated by gel electrophoresis. Lipoplexes formed lamellar phases when mixed with anionic dioleoylphosphatidylglycerol (DOPG), while promoting the formation of nonbilayer structures when mixed with anionic dioleoylphosphatidic acid (DOPA). However, lipoplexes exhibited a virtually identical extent of DNA release when mixed with DOPG or DOPA. Thus, the recently proposed correlation between the formation of nonlamellar phases in lipoplex/anionic lipid mixtures and the increase of DNA release does not seem to exist.

Nonlamellar Phases in Asymmetric Rod−Coil Block Copolymers at Increased Segregation Strengths

A new phase consisting of rectangular rod nanodomains packed onto a hexagonal lattice is observed in rod−coil block copolymers at the limit of both large volume fraction asymmetry and large geometrical asymmetry between the rod and coil. In moderately segregated poly(alkoxyphenylenevinylene-b-isoprene) (PPV-b-PI), an order−order transition is observed between hexagonal and lamellar phases for polymers near the phase boundary, and the lamellar phase is observed at high temperatures. The domain spacings of polymers in the lamellar phase collapse on to a simple scaling relationship where domain spacing is proportional to molecular weight. The proportionality constant is equal to the statistical segment length of the PPV rod block, suggesting that the angle between the rod director and the lamellar interface is nearly 90°. At higher temperatures, the block copolymers transition from ordered to nematic to isotropic states, with the intermediate nematic phase being observed for all coil fractions studied. A three-dimensional phase diagram shows the microphase and liquid crystalline transitions in rod−coil block copolymers as a function of temperature, geometrical asymmetry, and coil fraction.

D-72 Self Assembly and High Pressure Behavior of Non-Lamellar Phospholipid Phases On Solid Supports Studied with Gisaxd

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Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, ∼ 40–45 °C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer “frustration” which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a “frustrated” bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex–membrane interactions and DNA release energetics.

The thermotropic phase behaviour and phase structure of a homologous series of racemic β- d-galactosyl dialkylglycerols studied by differential scanning calorimetry and X-ray diffraction

The thermotropic phase behaviour of aqueous dispersions of some synthetic 1,2-di- O-alkyl-3- O-(β- d-galactosyl)- rac-glycerols ( rac-β- d-GalDAGs) with both odd and even hydrocarbon chain lengths was studied by differential scanning calorimetry (DSC), small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction. DSC heating curves show a complex pattern of lamellar (L) and nonlamellar (NL) phase polymorphism dependent on the sample's thermal history. On cooling from 95 °C and immediate reheating, rac-β- d-GalDAGs typically show a single, strongly energetic phase transition, corresponding to either a lamellar gel/liquid-crystalline (L β/L α) phase transition ( N ≤ 15 carbon atoms) or a lamellar gel/inverted hexagonal (L β/H II) phase transition ( N ≥ 16). At higher temperatures, some shorter chain compounds ( N = 10–13) exhibit additional endothermic phase transitions, identified as L/NL phase transitions using SAXS/WAXS. The NL morphology and the number of associated intermediate transitions vary with hydrocarbon chain length. Typically, at temperatures just above the L α phase boundary, a region of phase coexistence consisting of two inverted cubic (Q II) phases are observed. The space group of the cubic phase seen on initial heating has not been determined; however, on further heating, this Q II phase disappears, enabling the identification of the second Q II phase as P n 3 ¯ m (space group Q 224). Only the P n 3 ¯ m phase is seen on cooling. Under suitable annealing conditions, rac-β- d-GalDAGs rapidly form highly ordered lamellar-crystalline (L c) phases at temperatures above ( N ≤ 15) or below ( N = 16–18) the L β/L α phase transition temperature ( T m). In the N ≤ 15 chain length lipids, DSC heating curves show two overlapping, highly energetic, endothermic peaks on heating above T m; corresponding changes in the first-order spacings are observed by SAXS, accompanied by two different, complex patterns of reflections in the WAXS region. The WAXS data show that there is a difference in hydrocarbon chain packing, but no difference in bilayer dimensions or hydrocarbon chain tilt for these two L c phases (termed L c1 and L c2, respectively). Continued heating of suitably annealed, shorter chain rac-β- d-GalDAGs from the L c2 phase results in a phase transition to an L α phase and, on further heating, to the same Q II or H II phases observed on first heating. On reheating annealed samples with longer chain lengths, a subgel phase is formed. This is characterized by a single, poorly energetic endotherm visible below the T m. SAXS/WAXS identifies this event as an L c/L β phase transition. However, the WAXS reflections in the di-16:0 lipid do not entirely correspond to the reflections seen for either the L c1 or L c2 phases present in the shorter chain rac-β- d-GalDAGs; rather these consist of a combination of L c1, L c2 and L β reflections, consistent with DSC data where all three phase transitions occur within a span of 5 °C. At very long chain lengths ( N ≥ 19), the L β/L c conversion process is so slow that no L c phases are formed over the time scale of our experiments. The L β/L c phase conversion process is significantly faster than that seen in the corresponding rac-β- d-GlcDAGs, but is slower than in the 1,2- sn-β- d-GalDAGs already studied. The L α/NL phase transition temperatures are also higher in the rac-β- d-GalDAGs than in the corresponding rac-β- d-GlcDAGs, suggesting that the orientation of the hydroxyl at position 4 and the chirality of the glycerol molecule in the lipid/water interface influence both the L c and NL phase properties of these lipids, probably by controlling the relative positions of hydrogen bond donors and acceptors in the polar region of the membrane.

Gene delivery by cationic lipids: in and out of an endosome

Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required.

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.

An intracellular lamellar-nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents.

Two cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: ethyl esters of oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). The former was 50 times more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1/C10-EPC relative to C18:0/C10-EPC (and to many other cationic lipoids) was suggested by the phases that evolved when these lipoids were mixed with negatively charged membrane lipid formulations. The saturated C18:0/C10-EPC remained lamellar in mixtures with biomembrane-mimicking lipid formulations [e.g., dioleoyl-phosphatidylcholine/dioleoyl-phosphatidylethanolamine/dioleoyl-phosphatidylserine/cholesterol at 45:20:20:15 (wt/wt)]; in contrast, the unsaturated C18:1/C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures, which took place at physiological temperatures, approximately 37 degrees C. As is well known, lipid vehicles exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Moreover, nonlamellar phase-forming compositions are frequently highly fusogenic. Indeed, FRET experiments showed that C18:1/C10-EPC exhibits lipid mixing with negatively charged membranes that is several times more extensive than that of C18:0/C10-EPC. Thus, C18:1/C10-EPC lipoplexes are likely to easily fuse with membranes, and, as a result of lipid mixing, the resultant aggregates should exhibit extensive phase coexistence and heterogeneity, thereby facilitating DNA release and leading to superior transfection efficiency. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for transfection success and suggest a strategy for the rational design of superior cationic lipid carriers.

Elastic curvature constants of lipid monolayers and bilayers

Bending elasticity is an important property of lipid vesicles, non-lamellar lipid phases and biological membranes. Experimental values of the mean curvature moduli, k c, of lipid bilayers and of the monolayer leaflets of inverted hexagonal (H II) phases of lipids are tabulated here for easy reference. Experimental estimates of the Gaussian curvature modulus, k ¯ c , are also included. Consideration is given to the relation between the bending moduli of bilayers and the constituent monolayer leaflets. Useful mathematical relations involving the bending moduli and spontaneous curvature are summarized.

Barrier Dysfunction and Pathogenesis of Neutral Lipid Storage Disease with Ichthyosis (Chanarin–Dorfman Syndrome)

Neutral lipid storage disease with ichthyosis (NLSDI; Chanarin-Dorfman syndrome) is an ichthyosiform syndrome, often associated with mutations in a lipid hydrolase, CGI-58. The presence of oil red O-positive, neutral lipid droplets in tissue biopsies, and/or in leukocytes on blood smears, coupled with a constellation of multisystem abnormalities and a pruritic ichthyosiform erythroderma, are together diagnostic of NLSDI. We investigated the pathogenesis of the ichthyosiform erythroderma in patients from three unrelated kindreds with a clinical diagnosis of NLSDI. Basal permeability barrier function and stratum corneum (SC) integrity were abnormal, but barrier recovery rates were faster than normal, as in atopic dermatitis. The basal barrier abnormality was linked to the secretion of lipid micro-inclusions, first segregated within lamellar bodies (LB), which then form a non-lamellar phase within the SC interstices, shown by combined ruthenium tetroxide post-fixation and lipid-retaining resin-white embedding. With colloidal lanthanum nitrate perfusion, excess water/solute movement was restricted to the SC interstices, and further localized to non-lamellar domains. Phase separation of excess stored lipid provides a unifying pathogenic mechanism not only for NLSDI, but also in several other inherited ichthyosiform disorders of lipid metabolism, such as recessive X-linked ichthyosis and type 2 Gaucher's disease.

Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH range as a consequence of an increased protonation state. In the same way, lipoplexes made with these amphiphiles exhibit a lamellar morphology at physiological pH and a non-lamellar phase at acidic pH. In this study, we confirm that the gemini surfactants are able to form complexes with plasmid DNA at physiological pH and are able to transfect efficiently CHO cells in vitro. Out of the five compounds tested here, two of these amphiphiles, GS1 and GS2, led to 70% of transfected cells with a good cell survival. These two compounds were tested further for in vivo applications. Because of their lamellar organisation, these lipoplexes exhibited a good colloidal stability in salt and in serum at physiological pH compatible with a prolonged stability in vivo. Indeed, when injected intravenously to mice, these stable lipoplexes apparently did not substantially accumulate, as inferred from the observation that transfection of the lungs was not detectable, as examined by in vivo bioluminescence. This potential of avoiding 'preliminary capture' in the lungs may, thus, be further exploited in developing devices for specific targeting of gemini lipoplexes.

“Sponge” Nanoparticle Dispersions in Aqueous Mixtures of Diglycerol Monooleate, Glycerol Dioleate, and Polysorbate 80

Lipid nanoparticles of nonlamellar lyotropic phases have a wide solubilizing and encapsulating spectrum for a range of substances thanks to their nanostructured interior featuring both lipophilic and hydrophilic domains. As a consequence, these systems have emerged as promising drug delivery systems in various pharmaceutical and diagnostic applications. Here we present the phase behavior and dispersion properties of a novel three-component lipid system composed of diglycerol monooleate (DGMO), glycerol dioleate (GDO), and polysorbate 80 (P80) which shows several advantageous features relating to drug delivery applications including: spontaneous dispersion formation with a narrow size distribution and tunable particle phase-structure. The obtained phase diagram shows the presence of lamellar (L(alpha)), hexagonal (H(2)), and reverse bicontinuous cubic (V(2)) liquid crystalline phases and an inverse micellar (L(2)) solution. A particularly interesting observation is the presence of a phase region where two liquid phases coexist, most likely the L(2) and L(3) ("sponge phase"). These two phase structures appear also to coexist in the submicron particles formed in the dilute water region, where the L(3) element appears to stabilize nanoparticles with inner L(2) structure. Increasing the fraction of the dispersing P80 component results in the growth of the more water rich L(3) "surface phase" at the expense of the size of the inner L(2) core.