Development and modeling of arsenic-trioxide–loaded thermosensitive liposomes for anticancer drug delivery

In this article, a novel delivery system for the anticancer drug, arsenic trioxide (ATO), is characterized. The release of ATO from DPPC liposomes with MPPC lysolipid incorporated into the bilayer was measured. Upon heating the liposomes to 37°C, there was a 15–25% release over 24 hours. The ATO release from the DPPC and DPPC:MPPC (5%) systems leveled off after 10 hours at 37°C, whereas the DPPC:MPPC (10%) liposomes continue to release ATO over the 24-hour time span. Upon heating the liposomes rapidly to 42°C, the release rate was substantially increased. The systems containing lysolipids exhibited a very rapid release of a significant amount of arsenic in the first hour. In the first hour, the DPPC:MPPC (5%) liposomes released 40% of the arsenic and the DPPC:MPPC (10%) liposomes released 55% of the arsenic. Arsenic release from pure DPPC liposomes was comparable at 37 and 42°C, indicating that the presence of a lysolipid is necessary for a significant enhancement of the release rate. A coarse-grained molecular dynamics (CGMD) model was used to investigate the enhanced permeability of lysolipid-incorporated liposomes and lipid bilayers. The CG liposomes did not form a gel phase when cooled due to the high curvature; however, permeability was still significantly lower below the liquid-to-gel phase-transition temperature. Simulations of flat DPPC:MPPC bilayers revealed that a peak in the permeability did coincide with the phase transition from the gel to LC state when the lysolipid, MPPC, was present. No pores were observed in the simulations, so it is unlikely this was the permeability-enhancing mechanism.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Effects of hydrostatic pressure on the molecular structure and endothermic phase transitions of phosphatidylcholine bilayers: a Raman scattering study

The temperature dependences of the Raman spectra of aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were monitored at different but constant pressures between 1 and 1210 bar. The changes observed in these Raman spectra are discussed in terms of the effects of high pressure on the phase state and molecular structure of lipid bilayers. It is demonstrated that the temperature of the endothermic gel to liquid-crystal phase transition, as well as the temperature of the pretransition, increases linearly with increasing hydrostatic pressure. The dTm/dP values obtained from a wide range of pressures are 20.8 degrees C X kbar-1 for DPPC and 20.1 degrees C X kbar-1 for DMPC. The dTp/dP value for DPPC is 16.2 degrees C X kbar-1. It is also shown that the volume change that occurs at the gel to liquid-crystal transition is not constant; i.e., d delta Vm/dP decreases by 6.2% (DPPC) or 6.3% (DMPC) per kilobar pressure. The volume change at the pretransition is also pressure dependent; the d delta Vp/dP value of DPPC decreases by 4.7% per kilobar pressure.     

Effect of stereoconfiguration on ripple phases (P beta') of dipalmitoylphosphatidylcholine

Mixtures of sn-1 (D) and sn-3 (L) enantiomers of fully hydrated dipalmitoylphosphatidylcholine (DPPC) were studied with differential scanning calorimetry and freeze-fracture microscopy. The pretransition temperature of racemic mixtures of DPPC was 1.8 C degrees below that of either pure sn-1 or sn-3 enantiomers, which had similar pretransition temperatures. The main transition temperature of racemic mixtures was also depressed, but to a lesser extent, 0.8 C degrees. Freeze-fracture images of liposomes of sn-1, sn-3, and racemic mixtures of DPPC frozen from the P beta' phase showed well-defined ripples of wavelength 13 nm. Lipid stereoconfiguration had no effect on ripple wavelength, configuration or amplitude, or on the number and nature of surface defects.     

Lectin-mediated agglutination of liposomes containing glycophorin: Effects of acyl chain length

Glycophorin from human erythrocytes has been incorporated into liposomes of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The thermal properties of unsonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42±22 (DMPC), 197±28 (DPPC) and 240±64 (DSPC). The initial rates of agglutination of sonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 by wheat germ agglutinin in the concentration range 0–7 μM have been measured over a range of temperature. Below the gel-to-lamellar phase transition (T_c) the rates of agglutination increase with acyl chain length in the sequence DMPC < DPPC < DSPC. Agglutination is found to be second order in liposome concentration and is completely reversed on saturation of the wheat germ agglutinin-binding sites by N-acetylglucosamine. Agglutination rates decrease with increasing temperature below T_c and are largely independent of temperature above T_c. The results are discussed in relation to the clustering of glycophorin in the phospholipid bilayers and its effect on binding and subsequent interliposomal bridge formation by wheat germ agglutinin.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Thermolabile Liposomes with a High Fusion Efficacy at 42°C can be Made of Dipalmitoylphosphatidylcholine/Fatty Alcohol Mixtures in the Molar Ratio of 1/2

Abstract

Liposomes made of dipalmitoylphosphatidylcholine (DPPC²), dipalmitoyl-phosphatidylglycerol (DPPG), and different long-chain fatty alcohols were investigated with respect to their colloidal stability, chain-melting phase transition temperature, and temperature dependent inter-vesicle fusion. In particular, the practical usefulness of the stoichiometric 1/2 (mol/mol) mixtures of the phospholipids and fatty alcohols, mainly elaidoyl alcohol (EL-OH) were studied. The mole fraction of DPPG in the bilayers of such vesicles affects crucially the colloidal stability of the resulting lipid suspensions; at least 15 mol-% of DPPG (relative to DPPC) must be incorporated into the bilayers in order to make the liposome suspension colloidally sufficiently stable at room temperature. The corresponding DPPC/DPPG/EL-OH (0.85/0.15/2) mixed lipid vesicles undergo a lamellar-gel to inverted hexagonal (H_IT) phase transition at 52.7°C, however, and then fuse and aggregate massively. The related phase transition temperature of the DPPC/DPPG/palmitelaidoyl alcohol (0.85/0.15/2) mixture is 48.4°C. This indicates that the chain-melting phase transition temperature of the investigated lipid mixtures is rather sensitive to the alcohol chain-length. This transition temperature is independent, however, of the bulk proton concentration in the pH region between 4.9 and 7.2. Stoichiometric 1/2 mixtures of phospholipids and EL-OH have a high propensity for the inter-vesicle fusion at 42°C and neutral pH. The reason for such fusion 10°C below the lamellar-to-nonlamellar phase transition temperature are the defects that are generated during the chain-melting of the (partly segregated) phospholipid component at 42°C; the proximity of the lamellar to non-lamellar phase transition temperature of the phospholipid/fatty alcohol (1/2) complex at 52°C also plays an important role.     

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of stereoconfiguration on ripple phases (Pβ′) of dipalmitoylphosphatidylcholine

Mixtures of sn-1 ( ) and sn-3 ( ) enantiomers of fully hydrated dipalmitoylphosphatidylcholine (DPPC) were studied with differential scanning calorimetry and freeze-fracture microscopy. The pretransition temperature of racemic mixtures of DPPC was 1.8 C° below that of either pure sn-1 or sn-3 enantiomers, which had similar pretransition temperatures. The main transition temperature of racemic mixtures was also depressed, but to a lesser extent, 0.8 C°. Freeze-fracture images of liposomes of sn-1, sn-3, and racemic mixtures of DPPC frozen from the P_β′ phase showed well-defined ripples of wavelength 13 nm. Lipid stereoconfiguration had no effect on ripple wavelength, configuration or amplitude, or on the number and nature of surface defects.     

Interaction of melittin with model membranes: effect on the size and permeability of liposomes

The influence of melittin, a monomer devoid of the phospholipase activity, on the size and permeability of liposomes from egg lecithin (PC), dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) has been investigated by the methods of fluorescence spectroscopy, quasi-elastic light scattering and freeze-fracture electron microscopy. While studying calcein release from liposomes under the influence of melittin it has been shown that binding of melittin with a bilayer is a fast process which depends on the concentration lipid: protein (Ri) ratio as well as on the phase state of the lipid. The lipids being in the liquid-crystalline forms (PC and DMPC) are characterized by a more rapid release of the dye-stuff from liposomes than DPPC vesicles being in gel state with the same Ri. Under the influence of different melittin concentrations heterogeneity of the system and its medium hydrodynamic size of particles at first increases (100 less than or equal to Ri less than 500) due to their fusion and then these parameters decrease to the initial values.     

Effect of 2,4-dichlorophenol on DPPC/water liposomes studied by X-ray and freeze-fracture electron microscopy

The effect of 2,4-dichlorophenol (DCP) was studied on the fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)--water liposomes. The structure and the thermotropic phase behaviour of the liposomes was examined in the presence of DCP (DCP/DPPC molar ratio, varied from 2x10(-2) up to 1) using small- and wide-angle X-ray scattering (SAXS, WAXS) and freeze-fracture electron microscopy. The structural behaviour of the DPPC/DCP/water system was strongly dependent on the concentration of the DCP. In the pretransition range the DCP molecules (at 2x10(-2) DCP/DPPC molar ratio) induced the interdigitated phase beside the parent (gel and rippled gel) phases, locally which can be form at higher DCP concentration. When the DCP/DPPC molar ratio was increased the pretransition disappeared and the main transition was shifted to lower temperatures. In the molar ratio range from 2x10(-1) up to 5x10(-1), a coexistence of different phases was observed in the wide temperature range from 20 up to 40 degrees C. With a further increase of the DCP/DPPC molar ratio (6x10(-1) to 1) only the interdigitated gel phase occurred below 25 degrees C. A schematic phase diagram of DPPC/DCP/water system was constructed to summarise the results.     

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℃:     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

Successive phase-transition phenomena and phase diagram of the phosphatidylcholine-water system as revealed by differential scanning calorimetry

A completely dehydrated dipalmitoylphosphatidylcholine (DPPC) was prepared with dehydration under high vacuum and at a temperature above its main transition temperature. Thermal analyses on about forty different samples of the DPPC-water system indicated that the main transition temperature decreased stepwise with an increase in the water content to the limiting temperature at 42.6°C, reflecting the thermal behaviors of a total of five endothermic peaks. The pretransition appeared at a water content above 17 g%, and the predominant role of ‘newly incorporated water’ between the bilayers of DPPC molecules at the pretransition was made evident.     

Comparison of steady-state fluorescence polarization and urea permeability of phosphatidylcholine and phosphatidylsulfocholine liposomes as a function of sterol structure

The well-known reduction in the permeability properties of liposomes of dimyristoylphosphatidylcholine (DMPC) by sterols has also been demonstrated for its sulfonium analog (DMPSC) in which the N+(CH3)3 group of choline is replaced by S+(CH3)2. We have now compared the effects of 25 mol% 24-methylenecholesterol and cholesterol on the initial rates of urea permeation into dipalmitoyl-PC (DPPC) and dipalmitoyl-PSC (DPPSC) liposomes above the gel-to-liquid-crystalline phase transition temperature and found a greater reduction with 24-methylenecholesterol/DPPSC than with cholesterol/DPPSC liposomes but little difference between the two sterols in DPPC liposomes. Fluorescence polarization studies, using diphenylhexatriene as a probe, show that polarization (P) values are considerably higher in DMPSC liposomes containing 20 and 30 mol% 24-methylenecholesterol than in DMPC liposomes containing 20 and 30 mol% cholesterol. Higher P values were also obtained in DMPSC liposomes containing other 24-alkyl-substituted sterols (beta-sitosterol, ergosterol and campesterol) than in DMPC liposomes containing the same sterols. Reduced permeability rates in PSC liposomes containing 24-alkyl-substituted sterols are correlated with higher polarization values, reflecting an increased degree of order and/or motion in these liposomes compared with liposomes from the corresponding PC. These results suggest that alkyl substitution at C-24 of the sterol molecule results in tighter interactions with the sulfonium analog of PC than with PC.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.