Destabilization of cationic lipid vesicles by an anionic hydrophobically modified poly(N-isopropylacrylamide) copolymer: a solid-state 31P NMR and 2H NMR study.

The effect of binding PNIPAM-Py-Gly, a copolymer of N-isopropylacrylamide, N-[4-(1-pyrenyl)butyl]-N-n-octadecylacrylamide and N-glycydyl-acrylamide, on membrane stability in cationic multilamellar vesicles (MLVs) was examined using solid-state phosphorus (31P) and deuterium (2H) nuclear magnetic resonance (NMR) spectroscopy. For MLVs of composition n-octadecyldiethylene oxide (ODEO)+cholesterol (CHOL)+1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+dimethyldioctadecylammonium bromide (DODAB) (molar ratios 75:10.5:10.5:4), PNIPAM-Py-Gly induced a complete conversion from a bilayer-type 31P NMR spectrum to one characteristic of lipids undergoing isotropic motional averaging, indicating the existence of regions of high local membrane curvature. This response was sustained even at elevated temperatures. For MLVs of composition POPC+1,2-dioleoyloxy-3-(trimethylammonio)-propane (DOTAP), only at high levels of DOTAP and ionic strength did PNIPAM-Py-Gly induce even a partial conversion to an isotropic-type 31P NMR spectrum. At lower pH this effect was diminished. Raising the temperature eliminated the isotropic 31P NMR spectral component, and this effect was not reversible upon returning to room temperature. 2H NMR spectroscopy of headgroup-deuterated DOTAP and POPC confirmed the 31P NMR results, but did not provide specific surface electrostatic information. We conclude that the binding of PNIPAM-Py-Gly to phospholipid-based vesicles is dominated by electrostatic attraction between cationic lipids and the polymer's glycine residues. At high binding levels, the polymer assumes a collapsed conformation at the surface, resulting in regions of high local curvature of the lipid assembly. For ODEO-based liposomes, these effects are magnified by the additional contribution of hydrogen bonding to the strength of polymer binding.

Evolution of cystic and adnexal tumors identified by echography.

The authors present their experience in detecting volume and echostructure alterations of the ovary in 14,525 women examined echographically and clinically. They analyzed 499 adnexal tumors and observed after clinical follow-up and echography that 60.6% of the cystic-septa tumors had involuted spontaneously. Percentage of spontaneous resolution was higher in small-diameter tumors, avoiding unnecessary surgery.

2H NMR detection of transmembrane potential-driven tetraphenylphosphonium transbilayer redistribution.

2H NMR of specifically choline-deuterated phosphatidylcholine incorporated into giant unilamellar vesicles (GUVs), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) plus 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) plus cholesterol (CHOL), was shown to detect a transmembrane potential-driven redistribution of the potential-sensitive, surface- binding dye tetraphenylphosphonium (TPP+) across the GUV lipid bilayer. The method is based on resolving differences in the surface charge at the inner versus the outer monolayer of the vesicle's bilayer using the so-called 2H NMR "molecular voltmeter" technique. A mathematical model to describe the 2H NMR results was derived by combining the Nernst, Boltzmann, Langmuir, and Gouy-Chapman equations with the established sensitivity of deuterium quadrupolar splittings from choline- deuterated POPC to surface electrostatic charge effects. This model identified experimental factors likely to yield enhanced sensitivity and resolution of the inner versus outer monolayer surface charges via 2H NMR. The predictions of the model were then confirmed experimentally. The improvement in resolution resulting from these studies removes a major hindrance to the general exploitation of 2H NMR for monitoring transbilayer surface charge asymmetrics.

Detection and quantification of asymmetric lipid vesicle fusion using deuterium NMR.

It is demonstrated that deuterium nuclear magnetic resonance (2H NMR) spectroscopy can be used to detect and to quantify fusion between anionic giant unilamellar vesicles (GUVs) and cationic small unilamellar vesicles (SUVs). The sensitivity to fusion relies on the conformational response of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to changes in membrane surface electrostatic charge due to lipid mixing upon fusion. This conformational change is reported in the 2H NMR spectrum as a change in the quadrupolar splitting from choline-deuterated POPC. GUVs were composed of varying molar ratios of the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), plus cholesterol (CHOL), plus POPC. SUVs were composed of the cationic lipid 1,2-dioleoyloxy-3-(dimethylammonio)-propane (DODAP), plus POPC with or without 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). Using a quantitative model that relates the 2H NMR quadrupolar splitting to the mole fractions of cationic, anionic, and neutral lipids in the vesicle membrane, it was possible to deduce the extent of fusion between the two oppositely-charged vesicle populations directly from the quadrupolar splitting. SUVs composed of DODAP + POPC + POPE (40/40/20) fused 100% with GUVs composed of POPC + CHOL + POPG (60/30/10). Removing POPE from the SUVs reduced the extent of fusion, as did reducing the POPG content of the GUVs.

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.

2H NMR and polyelectrolyte-induced domains in lipid bilayers.

2H NMR studies of polyelectrolyte-induced domain formation in lipid bilayer membranes are reviewed. The 2H NMR spectrum of choline-deuterated phosphatidylcholine (PC) reports on any and all sources of lipid bilayer surface charge, since these produce a conformation change in the choline head group of PC, manifest as a change in the 2H NMR quadrupolar splitting. In addition, homogeneous and inhomogeneous surface charge distributions are differentiated. Adding polyelectrolytes to lipid bilayers consisting of mixtures of oppositely charged and zwitterionic lipids produces 2H NMR spectra which are superpositions of two Pake sub-spectra: one corresponding to a polyelectrolyte-bound lipid population and the other to a polyelectrolyte-free lipid population. Quantitative analysis of the quadrupolar splittings and spectral intensities of the two sub-spectra indicate that the polyelectrolyte-bound populations is enriched with oppositely charged lipid, while the polyelectrolyte-free lipid population is correspondingly depleted. The same domain-segregation effect is produced whether cationic polyelectrolytes are added to anionic lipid bilayers or anionic polyelectrolytes are added to cationic lipid bilayers. The 2H NMR spectra permit a complete characterization of domain composition and size. The anion:cation ratio within the domains is always stoichiometric, as expected for a process driven by Coulombic interactions. The zwitterionic lipid content of the domains is always statistical, reflecting the systems tendency to minimize the entropic cost of demixing charged lipids into domains. Domain formation is observed even with rather short polyelectrolytes, suggesting that individual polyelectrolyte chains aggregate at the surface to form "superdomains". Overall, the polyelectrolyte bound at the lipid bilayer surface appears to lie flat along the surface and to be essentially immobilized through its multiple electrostatic contacts.

Polyelectrolyte-induced domains in lipid bilayer membranes: the deuterium NMR perspective.

Domain formation in lipid bilayer membranes can occur through electrostatic interactions between charged lipids and oppositely charged polyelectrolytes, such as proteins or polynucleic acids. This review describes a novel method for examining such domains in lipid bilayers, based on 2H NMR spectroscopy. The 2H NMR spectrum of choline-deuterated phosphatidylcholine is sensitive to, and reports on, lipid bilayer surface charge. When a charged lipid bilayer is exposed to an oppositely charged polyelectrolyte, the latter binds electrostatically to the bilayer surface and attracts charged lipids into its vicinity. The resulting inhomogeneous charge distribution produces overlapping 2H NMR subspectra arising from phosphatidylcholine within charge-enriched versus charge-depleted regions. Such spectral details as the quadrupolar splittings and the relative intensities of the subspectra permit a complete analysis of the domain composition, size, and, within limits, lifetime. Using 2H NMR, domain formation in lipid bilayer membranes can be observed with both cationic and anionic polyelectrolytes, whether of natural or synthetic origin. Domain size and composition prove to be sensitive to the detailed chemical structure of both the polyelectrolyte and the charged lipids. Within the domains there is always a stoichiometric anion/cation binding ratio, indicating that the polyelectrolyte lies flat on the membrane surface. The amount of phosphatidylcholine within the domain varies as a function of its statistical availability, in accordance with the predictions of a recent thermodynamic model of domain formation. When the molecular weight of the polyelectrolyte is varied, the domain size alters in accordance with the predictions of classical polymer physics. As expected for a predominantly electrostatic phenomenon, the observed domains dissipate at high ionic strength.