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The physical properties of hydrated multilamellar sample of 1,2-dimyristamido-1,2-deoxyphosphatidylcholine (DDPC) were investigated by means of differential scanning calorimetry (DSC), static X-ray diffraction, and simultaneous DSC and X-ray diffraction. The DDPC is a synthetic sphingomyelin analogue and has two amide bonds in its hydrophobic parts. This paper reports on metastable phase behavior of the hydrated DDPC sample. By cooling from a chain-melted state at the rates of greater than 4 degrees C min(-1), hydrated DDPC bilayers form a metastable gel phase. In the gel phase, the hydrophobic chains are tilted with respect to the bilayer normal, as like the gel phase of glycero-phosphatidylcholines. By heating, the metastable gel phase is transformed in to a stable phase associated with an exothermic heat event at 18.3 degrees C (DeltaH=14.6 kJ mol(-1)) and then the stable phase is transformed into a liquid-crystalline phase at 25.6 degrees C (DeltaH=42 kJ mol(-1)). The incubation at 17 degrees C for more than 1 h also induces the formation of the stable phase. In the stable phase, the hydrophobic chains are packed into highly ordered crystal-like structure. However, the X-ray diffraction pattern of the stable phase suggested that the entire DDPC molecules do not form a two-dimensional molecular ordered lattice, differing from normal subgel phase of glycero-phosphatidylcholines. The structure and phase behavior of DDPC revealed by the present study are discussed from the viewpoint of hydrogen bonds.

Direct fusion between Jurkat cell and a liposome modified with poly(ethylene oxide)-bearing lipid (PEO-lipid) was examined using diphtheria toxin fragment A (DTA) as the probe. Only the DTA-loaded liposome modified with PEO-lipid(n = 32) (n is the number of ethylene oxide units) exerted significant cytotoxicity against Jurkat cells, while liposomes lacking either the PEO-lipid or DTA did not. Liposomes modified by the PEO-lipid with shorter PEO chain(n = 5 or 15) did not show any cytotoxicity, irrespective of their DTA-loading. The cytotoxicity was observed even in the presence of cytochalasin B, an inhibitor of endocytosis. Judging from these results, we concluded that the PEO-lipid(n = 32)-modified liposome directly fused with plasma membrane of Jurkat cell.

Egg PC liposome as reconstituted with poly(ethylene oxide)-bearing lipid (coded as PEO-lipid (n = 15)) was remarkably endocytosed by Jurkat cell, which was a lymphoblastoma derived from human T cell. To confirm the endocytosis, two kinds of fluorescent probes (FITC-dextran and octadecyl rhodamine B) were employed. The former was loaded in the aqueous phase of the liposome, while the latter was embedded in the liposomal membrane. Both probes were found coincidentally at the same site in the cytosol, clearly suggesting that whole liposome entered the cell. The endocytosis was most obvious when PEO-lipid (n = 15) was employed above 50 mol%. FITC-Dextran entered the cell was found small dots in the cell, not dispersive. Even when octadecyl rhodamine B was used, no membrane fluorescence was observed at all. The uptake closely related to the cell metabolism as affected by the culture temperature and serum in the incubation medium. Furthermore, the addition of cytochalasin B completely prohibited the cell uptake of liposomes.

The direct transfer of membrane proteins from human platelets to the liposomal fraction was examined, particularly in relation to platelet activation during the process. The incorporation of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), in the interacting liposome considerably enhanced the efficiency of the protein transfer. The transfer proceeded with neither significant activation nor lysis of the platelet, and the activation of the platelet with thrombin did not affect the amount of the transferred proteins. A wide range of platelet membrane proteins was transferred, and they were almost comparable to those in a sample prepared by glycerol lysis/centrifugation. In addition, they included the major surface glycoproteins GPIIb and GPIIIa without noticeable contamination of soluble cytosol proteins. The protein transfer method is a one-pot process and clearly more convenient than the conventional 'extract and reconstitute' approach. These results strongly support the use of the transfer process, especially with DDPC, as an alternative to the conventional detergent-solubilization or the solvent-extraction methods for preparation of samples of platelet membrane proteins.

The synthesis and characterization of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), are described. DDPC has two amide bonds instead of ester bonds of regular lecithins such as 1,2-dimyristoylphosphatidylcholine (DMPC). In differential scanning calorimetry (DSC) measurements, DDPC gave two endothermic peaks: one was at 18.0 degrees C (delta H = 10.74 kJ.mol-1) and the other at 23.0 degrees C (delta H = 12.91 kJ.mol-1). The former peak was sharp and considered to be the phase transition of the hydrocarbon region, while the latter was assigned to the melt of the hydrogen-belt formed by the amide groups of DDPC. Addition of DDPC to DMPC made the DMPC membrane less fluid in the region close to the surface, and significantly increased the reconstitution efficiency of glycophorin into the membrane. This effect of DDPC was much larger than that of naturally occurring lipid, sphingomyelin.

Membrane dynamics of dimyristoylphosphatidylcholine (DMPC) lipid bilayer which contains glycophorin with an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), was investigated by 2H-NMR technique. For this purpose, both DMPC and DDPC were deuterated at the position of the 8th carbon atom of their acyl chains. Comparing, with DMPC bilayers, DDPC bilayers showed larger deuterium quadrupole splitting (delta nu rho) by approx. 2 kHz. This was explicable in terms of the stabilization of the membrane due to the formation of a strong hydrogen bonding in bilayers. Addition of glycophorin to the DMPC or DDPC single bilayers caused an increase in the delta nu rho value. The delta nu rho value of DMPC/DDPC mixed lipid bilayer was smaller than that of each single lipid bilayer DDPC in the DDPC/DMPC mixed bilayer was not phase-separated but homogeneously distributed. In glycophorin-reconstituted DMPC-d4/DDPC mixed bilayers, the delta nu rho of DMPC-d4 was almost identical to that of the simple DMPC-d4 bilayer. On the other hand, the delta nu rho of DDPC-d4 in the DMPC/DDPC-d4 mixed bilayer increased significantly upon the reconstitution of glycophorin. Judging from these data, we concluded that, in the DDPC/DMPC mixed bilayer which contains glycophorin, DMPC simply behaves as the matrix lipid, while DDPC surrounds glycophorin and certainly plays a role of the boundary lipid.

To understand the role of omega-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1, 2-di-11-cyclohexylundecanoyl-L-alpha-phosphatidylcholine (11CYPC), 1,2-di-13-cyclohexyltridecanoyl-L-alpha-phosphatidylcholine (13CYPc), and 1-13-cyclohexyltridecanoyl-2-11-cyclohexylundecanoyl-L-alpha- phosphatidylcholine (1-13CY-2-11CYPC) were prepared and the steady-state fluorescence anisotropy of 1, 6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the omega-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolysis of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamellar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.

Liposomes labeled with [14C]coenzyme Q10 in the lipid bilayer were coated with various polysaccharide derivatives, i.e., palmitoyl conjugates of pullulan, pullulan phosphate, amylopectin, amylopectin phosphate and amylopectin sulfate. The kinetics of disposition and the tissue distribution of [14C]coenzyme Q10 after intravenous injection of the liposomes into guinea pigs were investigated. Lung uptake of radioactivity after injection of the O-palmitoyl amylopectin- and O-palmitoyl amylopectin phosphate-coated liposomes was 5- and 3-times higher, respectively, at 30 min after injection than that of the conventional liposomes. For doubly labeled liposomes with [3H]inulin and [14C]coenzyme Q10, the 3H/14C ratios in the lung, spleen and heart were similar to one another. Urinary excretion of [3H]inulin encapsulated in O-palmitoyl amylopectin-coated liposomes was much lower than that of unencapsulated [3H]inulin. These observations suggest that the O-palmitoyl amylopectin-coated liposomes are rather stable in vivo and are taken up into tissues in the intact form.

Physiochemical damage of egg phosphatidylcholine liposomes, caused by the salts of three bile acids, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid, has been investigated. Of the three bile salts, that of chenodeoxycholic acid was the most destructive, and the effect of the damage was examined by monitoring the induced 6-carboxyfluorescein release from the liposomes. For all three of the bile salts and under the experimental conditions, the minimum (effective) concentrations causing the 6-carboxyfluorescein release were below their critical micelle concentrations. In the case of the salt of chenodeoxycholic acid, the presence of cholesterol in the liposomal bilayers did not show any significant effect on the induced 6-carboxyfluorescein release, while, for the salts of ursodeoxycholic acid and cholic acid, the presence of cholesterol tended to depress the release. Permeation of bile salts into the membranes of liposomal bilayers made these membranes more fluid, and this fluidity was monitored by measuring the change in fluorescence polarization using 1,6-diphenylhexatriene entrapped in the liposomes. Coating the liposomes with polysaccharides, to make them more hydrophobic, led to their easier lysis by the bile salts.

An improved methodology for providing a more stable and targetable drug carrier has been developed. This method involves the synthesis of a newly designed immunoliposome by coating the outermost surface of large oligolamellar vesicles of egg phosphatidylcholine with the polysaccharide pullulan, modified to carry both cholesterol, as the hydrophobic anchor, and the monoclonal antibody fragment (anti-sialosyl Lewis X, IgMs) as the sensory device. Compared with the binding of pullulan-coated liposomes, that of this immunoliposome to specific cells in vitro was significantly increased by factors of 447 to PC-9 and 295 to KATO-III, but only by a factor of 148 to the less specific cell, 3LL. This strong and specific binding of the immunoliposome to the cell surface of PC-9 was also confirmed by a fluorescence-microscopic investigation using the immunoliposome, which bore the hydrophobic fluorescent probe, terbium trisacetylacetonate, in the liposomal membrane.

The interaction and reaction between liposomal membrane and a functional detergent, N-hexadecyl-N-(imidazol-4-yl)methyl-N,N-dimethylammonium chloride hydroperchlorate (Im-I), have been investigated in conjunction with the leakage of bromothymol blue encapsulated as a marker in the bilayers of liposomes. Im-I carries an imidazole moiety and was expected to behave as a simple lipase model. The reaction with Im-I significantly enhanced the leakage of bromothymol blue encapsulated in the egg lecithin and dipalmitoyl phosphatidylcholine liposomes. During the course of reaction with Im-I, the formation of acyl-imidazole intermediate was clearly identified, which was certainly connected with the bromothymol blue release. From various kinetic results on bromothymol blue release and acyl-imidazole formation, it has been suggested that the bromothymol blue release from liposomal bilayer may be caused by the local and instantaneous decomposition of lipids when Im-I penetrates into the bilayer. However, it has also been demonstrated that the immediate reconstruction of liposomes retains the barrier function to protect against the further release of bromothymol blue.

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.

Autoxidation of egg phosphatidylcholines and polyunsaturated fatty acids, arachidonic and linoleic acids, has been investigated in homogeneous and liposomal membrane systems. In order to monitor the very initial stage of the radical chain mechanism of the autoxidation, an improved method using 1,1-diphenyl-2-picrylhydrazyl radical was newly implemented and used with the regular thiobarbituric acid test to determine the peroxides of polyunsaturated fatty acids. Autoxidation of polyunsaturated fatty acids or lipids was significantly enhanced in liposomal bilayers compared to that in a bulk homogeneous solution. In liposomal bilayers, the reaction could be controlled by membrane fluidity, which was confirmed by the fluorescence polarization technique using 1,6-diphenylhexatriene and dansylhexadecylamine. Even vitamin E esters such as the acetate and the pivalate could depress effectively the autoxidation of egg phosphatidylcholines in bilayer systems, which supported Lucy's proposal (Diplock, A.T. and Lucy, J.A. (1973) FEBS Lett. 29, 205-210) about the importance of the side-chain of vitamin E as an antioxidant.