Crystalline Phase Transition Temperature Research Articles

Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks

Apolipoprotein mediated formation of nanodisks was studied in detail using apolipophorin III (apoLp-III), thereby providing insight in apolipoprotein–lipid binding interactions. The spontaneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles occured only in a very narrow temperature range at the gel–liquid–crystalline phase transition temperature, exhibiting a net exothermic interaction based on isothermal titration calorimetry analysis. The resulting nanodisks were protected from proteolysis by trypsin, endoproteinase Glu-C, chymotrypsin and elastase. DMPC solubilization and the simultaneous formation of nanodisks were promoted by increasing the vesicle diameter, protein to lipid ratio and concentration. Inclusion of cholesterol in DMPC dramatically enhanced the rate of nanodisk formation, presumably by stabilization of lattice defects which form the main insertion sites for apolipoprotein α-helices. The presence of fully saturated acyl chains with a length of 13 or 14 carbons in phosphatidylcholine allowed the spontaneous vesicle solubilization upon apolipoprotein addition. Nanodisks with C13:0-phosphatidylcholine were significantly smaller with a diameter of 11.7±3.1nm compared to 18.5±5.6nm for DMPC nanodisks determined by transmission electron microscopy. Nanodisk formation was not observed when the phosphatidylcholine vesicles contained acyl chains of 15 or 16 carbons. However, using very high concentrations of lipid and protein (>10mg/ml), 1,2,-dipalmitoyl-sn-glycero-3-phosphocholine nanodisks could be produced spontaneously although the efficiency remained low.

Dynamic Structure of Bombolitin II Bound to Lipid Bilayers as Revealed by Solid-state NMR and Molecular-Dynamics Simulation

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state 31P and 13C NMR spectroscopy. 31P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T c) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T c. 13C NMR spectra of site-specifically 13C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers.

AFM surface morphology and friction force studies of microscale domain structures of binary phospholipids

We have studied friction forces on binary mixtures of phospholipid monolayer films. The phospholipid monolayer films have been prepared via the Langmuir–Blodgett (LB) technique on mica. The two-component phospholipids are distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). At 25 °C the LB monolayer films give the gel-state DSPC domains surrounded by the liquid crystalline DLPC matrix. The friction forces measured on the DSPC domain region are significantly greater than those on the DLPC matrix region at this temperature. An increased temperature results in a decreased friction of the DSPC domain region, and above the gel-to-liquid crystalline phase transition temperature of DSPC, the difference in the friction forces measured on the two phospholipids becomes negligible. This means that the phase state is a key factor in determining friction forces on the phospholipid monolayer films.

Effects of temperature, acyl chain length, and flow-rate ratio on liposome formation and size in a microfluidic hydrodynamic focusing device

Microfluidic hydrodynamic focusing of an alcohol–lipid mixture into a narrow fluid stream by two oblique buffer streams provides a controlled and reproducible method of forming phospholipid bilayer vesicles (i.e., liposomes) with relatively monodisperse and specific size ranges. Previous work has established that liposome size can be controlled by changing the relative and absolute flow rates of the fluids. In other previous work, a kinetic (non-equilibrium) theoretical description of the detergent dilution liposome formation method was developed, in which planar lipid bilayer discs aggregate until they become sufficiently large to close into spherical liposomes. In this work, we show that an approximation of the kinetic theory can help explain liposome formation for our microfluidic method. This approximation predicts that the liposome radius should be approximately proportional to the ratio of the membrane bending elasticity modulus to the line tension of the hydrophobic edges of the lipid bilayer disc. In combination with very fast microfluidic mixing, this theory enables a new method to measure the ratio of the elasticity modulus to the line tension of membranes. The theory predicts that the temperature should change the liposome size primarily as a result of its effect on the ratio of the membrane bending elasticity modulus to the line tension, in contrast to previous work on microdroplet and microbubble formation, which showed that the effect of temperature on droplet/bubble size was primarily due to viscosity changes. In agreement with theory, most membrane compositions form larger liposomes close to or below the gel-to-liquid crystalline phase transition temperature, where the membrane elasticity modulus is much larger, and they have a much smaller dependence of size on temperature far above the transition temperature, where the membrane elasticity modulus is relatively constant. Other parameters modulated by the temperature (e.g., viscosity, free energy, and diffusion coefficients) appear to have little or no effect on liposome size, because they have counteracting effects on the lipid aggregation rate and the liposome closure time. Experiments are performed using phospholipids with varying hydrophobic acyl chain lengths that have phase transition temperatures ranging from −1 °C to 55 °C, so that the temperature dependence is examined below, above, and around the transition temperature. In addition, the effect of IPA stabilizing the edges of the bilayer discs can be examined by comparing the liposome sizes obtained at different flow-rate ratios. Finally, polydispersity is shown to increase as the median liposome size increases, regardless of whether the change in size is due to changing temperature or flow-rate ratio.

Propyl paraben-induced changes in dipalmitoyl phosphatidylethanolamine vesicles

Abstract This article reports the influence of the preservative, propyl paraben (PPB), on the phase transition and dynamics of dipalmitoyl phosphatidylethanolamine (DPPE) vesicles both in multilamellar vesicular (MLV) and unilamellar vesicular (ULV) forms using DSC and (1H and 31P) NMR. DSC results indicate that the mechanism by which PPB interacts with DPPE vesicles is similar in both forms. Addition of PPB to DPPE dispersion results in lowering of the gel to liquid crystalline phase transition temperature (T m) and consequently increases DPPE headgroup fluidity. At high PPB concentration, additional transitions are observed whose intensity increases with increasing PPB concentration. DSC and NMR data indicate that the PPB molecules get intercalated between the DPPE headgroups as the polar group of the PPB molecules interacts with the polar group of PE, and the alkyl chain of PPB penetrates into the acyl chain region. The interesting finding with MLV is that the gel phase of DPPE in the presence of PPB, ...

Interaction of amphiphilic derivatives of chitosan with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)

The interaction of chitosan and its N-dodecyl and poly(ethylene glycol) derivatives with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles was studied to evaluate the influence of molecular architecture of the polymers on the liposomes. The study was carried out in aqueous solution using differential scanning microcalorimetry (DSC) and dynamic light scattering. The interaction of these polymers with DPPC vesicles altered the gel–liquid crystalline phase transition temperature and decreased both the enthalpy (ΔH) and cooperativity of the phase transition. The results obtained indicate that perturbations in the vesicles surface and the incorporation of chitosan and its derivatives into the lipid bilayer upon polysaccharides interaction are responsible for the formation of large vesicles.

A comparative study of the effect of cholesterol on bicelle model membranes using X-band and Q-band EPR spectroscopy

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters ( S mol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.

A Novel Fluorescent Probe That Senses the Physical State of Lipid Bilayers

Cell membrane lipids and proteins are heterogeneously distributed in the membrane plane. In recent years, much attention has been paid to the heterogeneous distribution of the lipid components, particularly the formation of cholesterol-rich domains that are thought to be important in signaling processes. This has led to renewed interest in the phase diagrams of complex lipid mixtures, such as three-component mixtures containing phospholipids and cholesterol. We report here a novel fluorescent probe (NBD-R595) that is useful for exploring the phase behaviors of one-, two-, and three-component large unilamellar vesicles. In one-component fluid-phase membranes, the probe has the expected spectral characteristic of monomeric 7-nitrobenzo-2-oxa-1,3-diazol, with a fluorescence maximum of 540 nm when excited at 470 nm. But below the gel-to-liquid crystalline phase transition temperature, an additional emission peak appears at ∼610 nm, because of Förster resonance energy transfer from NBD-R595 monomers to NBD-R595 Jelley aggregates of limited size formed by the association of 7-nitrobenzo-2-oxa-1,3-diazol moieties. This may be the first report of Förster resonance energy transfer from a single fluorophore in two different physical states. In a test of the probe, we found NBD-R595 to be remarkably sensitive to the molar composition of large unilamellar vesicles formed from cholesterol, distearoylphosphatidylcholine, and dioleoylphosphatidylcholine.

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.

Interaction of Na 2B 12H 11SH with dimyristoyl phosphatidylcholine liposomes

Previous investigations have revealed that the boron cluster compound Na 2B 12H 11SH (BSH) is very potent in causing major structural rearrangements of and leakage from phosphatidylcholine liposomes. This somewhat unexpected finding is interesting from a fundamental point of view and may also constitute the basis of future important pharmaceutical/medical applications of BSH. In order to further explore the BSH-lipid interaction, we have studied the effects caused by BSH on dimyristoyl phosphatidylcholine (DMPC) liposomes. Cryo-transmission electron microscopy showed that BSH induces aggregation, membrane rupture and increasing wall thickness of the liposomes. Differential scanning calorimetry revealed a BSH dependent shift of the gel to liquid crystalline phase transition temperature of DMPC. The zeta potential of the liposomes decreases with increasing BSH concentrations, and an apparent dissociation constant of 0.23 mM was found. BSH caused leakage of liposome-encapsulated carboxyfluorescein; leakage was higher at 23 °C (near the phase transition temperature) than at 15 °C and 37 °C. It induced lipid mixing only at very high concentrations.

New Lipid Family That Forms Inverted Cubic Phases in Equilibrium with Excess Water: Molecular Structure−Aqueous Phase Structure Relationship for Lipids with 5,9,13,17-Tetramethyloctadecyl and 5,9,13,17-Tetramethyloctadecanoyl Chains

With a view to discovering a new family of lipids that form inverted cubic phases, the aqueous phase behavior of a series of lipids with isoprenoid-type hydrophobic chains has been examined over a temperature range from -40 to 65 degrees C by using optical microscopy, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering) techniques. The lipids examined are those with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17-tetramethyloctadecanoyl chains linked to a series of headgroups, that is, erythritol, pentaerythritol, xylose, and glucose. All of the lipid/water systems displayed a "water + liquid crystalline phase" two-phase coexistence state when sufficiently diluted. The aqueous phase structures of the most diluted liquid crystalline phases in equilibrium with excess water depend both on the lipid molecular structure and on the temperature. Given an isoprenoid chain, the preferred phase consistently follows a phase sequence of an H II (an inverted hexagonal phase) to a Q II (an inverted bicontinuous cubic phase) to an L alpha (a lamellar phase) as A* (cross-section area of the headgroup) increases. For a given lipid/water system, the phase sequence observed as the temperature increases is L alpha to Q II to H II. The present study allowed us to find four cubic phase-forming lipid species, PEOC 18+4 [mono- O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol], beta-XylOC 18+4 [1- O-(5,9,13,17-tetramethyloctadecyl)-beta- d-xylopyranoside], EROCOC 17+4 [1- O-(5,9,13,17-tetramethyloctadecanoyl)erythritol], and PEOCOC 17+4 [mono- O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol]. The values of T K (hydrated solid-liquid crystalline phase transition temperature) of the cubic phase-forming lipids are all below 0 degrees C. Quantitative analyses of the lipid molecular structure-aqueous phase structure relationship in terms of the experimentally evaluated "surfactant parameter" allow us to rationally select an optimum combination of hydrophilic/hydrophobic part of a lipid molecule that will form a desired phase in a desired temperature range.

Effect of Polyacrylic Acid on Phase State of Lipids and Diffusion in Lipid-Water System

Lipid vesicles interacting with polyanions are promising for controlled drug delivery. However, different aspects of the interaction of these polymers with lipids are far from complete understanding. In this work we studied the influence of polyacrylic acid (PAA) with small concentrations (1–4 mol%) on the change of the phase state, lateral diffusion of these lipids in lamellar phase and transmembrane water diffusion in macroscopically oriented bilayers of lipid-water systems formed by dimiristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine. Measurements were performed by 31P nuclear magnetic resonance (NMR) spectroscopy and the 1H NMR technique with a pulsed field gradient. It was found that the presence of PAA does not change the lamellar structure of the system. However, a part of bilayers changes their originally flat geometry and forms vesicles with a higher surface curvature. Macroscopic orientation of bilayers disappeared. For DMPC the presence of PAA leads to a shift of the gel-to-liquid crystalline phase-transition temperature to higher temperatures. An increase of PAA concentration leads to a monotonous decrease in the lateral diffusion coefficient of lipids that is caused, probably, by the ordering of lipids in bilayers. The transbilayer diffusion coefficient of water increases in the presence of PAA, but it depends slightly on the PAA concentration. An increase of pH leads to a change of the lipid lateral and transbilayer diffusion coefficients to the values typical for a pure bilayer.

In vitro characterization of phosphatidylglyceroglycerol‐based thermosensitive liposomes with encapsulated 1H MR T1‐shortening gadodiamide

Thermosensitive liposomes (TSL) with encapsulated proton (1H) magnetic resonance (MR) contrast agents have been proposed for noninvasive online temperature monitoring during tumor treatment using chemotherapy combined with hyperthermia (HT). The technique exploits the fact that water exchange between the TSL interior and exterior is increased and/or the encapsulated 1H MR contrast agent is released near the gel-to-liquid crystalline phase transition temperature (Tm) of TSL and thus shortens the 1H MR relaxation time of tissue. In this work, newly developed, phosphatidylglyceroglycerol (DPPGOG)-based TSL with encapsulated 1H MR longitudinal relaxation time (T1)-shortening gadodiamide (Gd-DTPA-BMA) were characterized in vitro by measuring the temperature dependence of the T1 of these gadodiamide-containing DPPGOG-TSL samples between 30 and 50 degrees C. The measurements revealed that the T1 nonlinearly slightly decreased with increasing temperature from 30 to 37 degrees C, mainly due to increased water exchange between the gadodiamide-containing DPPGOG-TSL interior and exterior with the exception of negligible gadodiamide release. This implies that gadodiamide-containing DPPGOG-TSL were stable at temperatures < or =37 degrees C, which was also confirmed by an independent stability study. From 37 to 44 degrees C, the T1 nonlinearly markedly decreased with increasing temperature since encapsulated gadodiamide was rapidly released. Above 44 degrees C, gadodiamide was completely released and the T1 was directly proportional to temperature while heated from 44 to 50 degrees C and cooled from 50 to 30 degrees C, respectively. Additionally, gadodiamide release was theoretically quantified and this calculated concentration was consistent with the actually released amount directly obtained from the cooling course of empty DPPGOG-TSL with completely released gadodiamide.

Structural polymorphism of hydrated ether-linked dimyristyl maltoside and melibioside

The structural polymorphism of two selected disaccharide glycolipids with a maltose (DMMA) and a melibiose (DMME) carbohydrate headgroup linked to dimyristyl alkyl chains were investigated by FTIR-spectroscopy, differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and film-balance measurements. The compounds displayed thermotropic multilamellar phases. In the gel phase, DMMA formed also a crystalline phase of orthorhombic symmetry, and DMME an interdigitated phase. The gel to liquid crystalline phase transition temperature T c of DMMA depended on the storage and hydration conditions, a precooled sample having a T c around 45 °C, and a freshly prepared sample around 33 °C. In contrast, the phase transition temperature for the gel to liquid crystalline phase of DMME was always found at 24 °C. Surface pressure isotherms of the lipids on water and buffer showed that DMMA covers only a small surface area (∼35 Å 2) whereas DMME requires 50 Å 2 of space on the surface. Films of DMMA can be compressed up to a maximum compressibility Π max of 54 mN m −1 whereas the tilted DMME forms less stable films with Π max of 34 mN m −1. These different structural characteristics reflect the different conformations of the disaccharide head groups. The presence of the α1 → 4 linked maltose head group in DMMA and an α1 → 6 linked melibiose head group in DMME induces geometrical structures ranging from a slightly wedge-shaped towards a more tilted structure, and as a consequence of Israelachvilis packing model, to the formation of different phases. In addition, the structural constraints of DMME allow the formation of a phase with interdigitated hydrocarbon chains.

Memory effects of monolayers and vesicles formed by the non-ionic surfactant, 2C 18E 12

The behaviour of monolayers and bilayers formed by the dialkyl chain non-ionic surfactant, 1,2-di- O-octadecyl- rac-glycerol-3- ω-methoxydodecaethylene glycol (2C 18E 12) in water at 297 K has been investigated. Using a surface film balance (or Langmuir trough) the compression–expansion cycle of the 2C 18E 12 monolayer was found to be reversible when compressed to surface pressures ( π) less than 42 mN m −1. Compression of 2C 18E 12 monolayer to π greater than 42 mN m −1 above this resulted in a considerable hysteresis upon expansion with the π remaining high relative to that obtained upon compression, suggesting a time/pressure dependent re-arrangement of 2C 18E 12 molecules in the film. Morphology of the 2C 18E 12 monolayer, investigated using Brewster angle microscopy, was also found to depend upon monolayer history. Bright, randomly dispersed domains of 2C 18E 12 of approximately 5 μm in size were observed during compression of the monolayer to π less than 42 mN m −1. At π of 42 mN m −1 and above, the surfactant film appeared to be almost completely ‘solid-like.’ Regardless of the extent of compression of the monolayer film, expansion of the film caused formation of chains or ‘necklaces’ of individual surfactant domains, with the extent of chain formation dependent upon pressure of compression of the monolayer and the length of time held at that pressure. Irreversible effects on 2C 18E 12 vesicle size were also seen upon temperature cycling the vesicles through their liquid–crystalline phase transition temperature with vesicles shrinking in size and not returning to their original size upon standing at 298 K for periods of more than 24 h. No comparable hysteresis, time, pressure or temperature effects were observed with the monolayer or vesicles formed by the corresponding phospholipid, disteaorylphosphatidylcholine, under identical conditions. The effects observed with 2C 18E 12 are attributed to the ability of the polyoxyethylene head group to dehydrate and intrude into the hydrophobic chain region of the mono- and bilayers. These studies have important implications for the use of the vesicles formed by 2C 18E 12 as drug delivery vehicles.

Influence of Perfluorinated Compounds on the Properties of Model Lipid Membranes

The influence of selected perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS), on the structure and organization of lipid membranes was investigated using model membranes-lipid monolayers and bilayers. The simplest model--a lipid monolayer--was studied at the air-water interface using the Langmuir-Blodgett technique with surface pressure and surface potential measurements. Lipid bilayers were characterized by NMR techniques and molecular dynamics simulations. Two phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), characterized by different surface properties have been chosen as components of the model membranes. For a DPPC monolayer, a phase transition from the liquid-expanded state to the liquid-condensed state can be observed upon compression at room temperature, while a DMPC monolayer under the same conditions remains in the liquid-expanded state. For each of the two lipids, the presence of both PFOA and PFOS leads to the formation of a more fluidic layer at the air-water interface. Pulsed field gradient NMR measurements of the lateral diffusion coefficient (DL) of DMPC and PFOA in oriented bilayers reveal that, upon addition of PFOA to DMPC bilayers, DL of DMPC decreases for small amounts of PFOA, while larger additions produce an increased DL. The DL values of PFOA were found to be slightly larger than those for DMPC, probably as a consequence of the water solubility of PFOA. Furthermore, 31P and 2H NMR showed that the gel-liquid crystalline phase transition temperature decreased by the addition of PFOA for concentrations of 5 mol % and above, indicating a destabilizing effect of PFOA on the membranes. Deuterium order parameters of deuterated DMPC were found to increase slightly upon increasing the PFOA concentration. The monolayer experiments reveal that PFOS also penetrates slowly into already preformed lipid layers, leading to a change of their properties with time. These experimental observations are in qualitative agreement with the computational results obtained from the molecular dynamics simulations showing a slow migration of PFCs from the surrounding water phase into DPPC and DMPC bilayers.

Use of a passive equilibration methodology to encapsulate cisplatin into preformed thermosensitive liposomes

A conventional, cholesterol-containing liposome formulation of cisplatin has demonstrated insignificant activity in clinical trials, due in part, to insufficient release of encapsulated content following localization within solid tumors. For this reason, the development of a triggered release liposome formulation is desirable. In this report, cisplatin was encapsulated into lysolipid-containing thermosensitive liposomes (LTSL) using a novel technique, which relies on the equilibration of cisplatin across the liposomal membrane at temperatures above the gel-to-liquid crystalline phase transition temperature ( T C) of the bulk phospholipid. Mild heating and drug loading into LTSL did not induce morphological changes of the liposomes. In vitro data demonstrated that >95% of encapsulated cisplatin was released from LTSL within 5 min following mild heating at 42 °C, while <5% was released at 37 °C. Under similar conditions, lysolipid-free thermosensitive liposomes exhibited 70% release of cisplatin at 42 °C, and cholesterol-containing liposomes exhibited negligible drug release at 42 °C. The pharmacokinetic profiles of LTSL- and TSL-cisplatin indicated that these formulations were rapidly eliminated from circulation (terminal t 1/2 of 1.09 and 2.83 h, respectively). The therapeutic utility of LTSL-cisplatin formulation will be based on strategies where hyperthermia is applied prior to the administration of the liposomal drug—a strategy similar to that used in the clinical assessment of LTSL-doxorubicin formulation.

Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Effect of propyl paraben on the dipalmitoyl phosphatidic acid vesicles

The effect of the preservative propyl paraben (PPB) on the phase transition and dynamics of dipalmitoyl phosphatidic acid (DPPA)-buffer (pH 7.4/9.3) vesicles has been studied using DSC and ( 1H and 31P) NMR. These investigations were carried out with DPPA dispersion in both multilamellar vesicular (MLV) and unilamellar vesicular (ULV) forms. DSC results indicate that the mechanism by which PPB interact with the DPPA vesicles is similar in MLV and ULV and is independent of pH of the buffer used to form the dispersion. However, for a given concentration of PPB, the perturbation in DPPA bilayer is more when the dispersion is prepared in buffer pH 7.4. PPB affected both the thermotropic phase transition and the molecular mobility of the DPPA membrane. In the presence of PPB, the gel to liquid crystalline phase transition temperature ( T m ) of the DPPA vesicles decreases hence increases membrane fluidity due to reduced headgroup–headgroup interaction. For all concentrations, the PPB molecules seem to get intercalated between the polar groups of the phospholipids with its alkyl chain penetrating into the co-operative region. At high PPB concentration, additional transitions are observed whose intensity increases with increasing PPB concentration. The large enthalpy values obtained at high PPB concentration suggest that presence of PPB makes the DPPA bilayer more ordered (rigid). The interesting finding obtained with MLV is that the stable gel phase of DPPA-buffer (pH 9.3/7.4) system in the presence of high PPB concentration becomes a metastable gel phase, this metastable gel phase on equilibration at 25 °C or when cooled to −20 °C transforms to a stable crystalline phase(s). The intensity of this new phase(s) increases with increasing PPB concentration. However, the transition temperatures of these new phases are not significantly changed with increasing PPB concentration. The effect of inclusion of cholesterol in the PPB-free and PPB-doped DPPA dispersion was also studied.

Structure of Mixed Micelles Formed in PEG-Lipid/Lipid Dispersions

Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPE-PEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.