The phase behaviour of dispersions of Bis-Azo PC: photoregulation of bilayer dynamics via lipid photochromism.

A phospholipid, 1,2-bis(4-(n-butyl)phenylazo-4'-phenylbutyroyl)phosphatidylcholine (Bis-Azo PC), has been synthesised and shown to form stable bilayer vesicles. Light-scattering measurements and differential scanning calorimetry show that a dispersion of the lipid has a cooperative phase transition at a similar temperature to that of dipalmitoylphosphatidylcholine, which Bis-Azo PC resembles in overall size. The phase behaviour of Bis-Azo PC has been investigated by fluorescence spectroscopy and using a series of spin-labelled fatty acid probes. Fluorescence measurements using chlorophyll a as probe sense the onset of the cooperative phase transition, but this is not clearly revealed by any of the spin probes tested. Hysteresis in the phase transition is detected both by light scattering measurements and by fluorescence spectroscopy. No transition is observed for a lipid analogue having a palmitic acid chain and a single azo-containing substituent. Bis-Azo PC is reversibly photochromic, isomerising on exposure to ultraviolet light to a photostationary state mixture where cis isomer predominates. Electron microscopy shows that photoisomerisation decreases average vesicle size, and light scattering and calorimetry demonstrate that the cooperative phase transition is abolished. Illumination with visible light establishes a new photostationary state where trans isomer predominates, and the phase transition is restored. The ability to modulate bilayer phase behaviour reversibly has possible application to relaxation studies of bilayer membrane function, and to drug delivery research.

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase.

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.

Lytic effects of melittin and delta-haemolysin from Staphylococcus aureus on vesicles of dipalmitoylphosphatidylcholine.

The effects of the lytic peptides, melittin and delta-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid 'burst' of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by delta-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for delta-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of delta-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, delta-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles.

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4'-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent 'donor' and 1-(4'-carboxyethyl)-6-(4"-nitro)diphenyl-trans-1,3,5-hexatriene as 'acceptor'. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20 degrees C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with less than 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.

The use of a phospholipid analogue of diphenyl-1,3,5-hexatriene to study melittin-induced fusion of small unilamellar phospholipid vesicles.

A phospholipid analogue incorporating the diphenyl-1,3,5-hexatriene (DPH) chromophore has been synthesized. The compound has been shown to have similar fluorescence properties to DPH itself but, unlike DPH, is unable to exchange freely through solution when incorporated as probe in a subset of phospholipid vesicles of given composition. The non-exchangeability of this probe has been exploited to study the fusion of phospholipid vesicles to form larger structures. The peptide melittin was used to initiate fusion, and it was shown that vesicles which had been induced to fuse by heating in the presence of melittin would not fuse with subsequently added vesicles.

Light-induced fusion of liposomes with release of trapped marker dye is sensitised by photochromic phospholipid.

Liposomes have been prepared from dipalmitoylphosphatidylcholine containing small amounts of a synthetic photochromic phospholipid, 'Bis-Azo PC'. In the dark, these are stable at room temperature, and contents do not significantly leak over weeks. Photoisomerisation results in immediate release of trapped marker, and in liposome fusion to form larger structures. Fusion has been detected using a fluorescence polarisation assay, and confirmed by electron microscopy. In mixtures, fusion occurs between 'photochromic' liposomes and those of pure lipid. Bis-Azo PC contains two photochromic acyl chains; analogues bearing a single photochromic chain appear to have little effect on bilayer permeability after isomerisation. Photo-induced leakage and liposome fusion suggest possible applications for localised drug delivery as an adjunct to phototherapy. The ability to non-invasively trigger fusion processes should be useful in fundamental studies of membrane interactions. We believe this to be the first report of photo-induced fusion to date.

Liposome fusion and lipid exchange on ultraviolet irradiation of liposomes containing a photochromic phospholipid.

A photochromic phospholipid, 1,2-bis[4-4(4-n-butylphenylazo) phenylbutyroyl] phosphatidylcholine (Bis-Azo PC) has been incorporated into liposomes of gel- and liquid-crystalline- phase phospholipids. Liposomes of gel-phase phospholipid are stable in the presence of the trans photostationary state Bis-Azo PC and can encapsulate fluorescent marker dye. On photoisomerization to the cis photostationary state, trapped marker is rapidly released. Liposomes containing Bis-Azo PC can rapidly fuse together after UV isomerization, this process continuing in the dark. Exposure to white light causes reversion of Bis-Azo Pc to the trans form and halts dye leakage and vesicle fusion. Both unilamellar and multilamellar liposomes are able to fuse together on UV exposure. On UV photolysis, liposomes containing Bis-Azo PC do not fuse with a large excess of unlabeled liposomes, but transfer of Bis-Azo PC can be demonstrated spectrophotometrically. Vesicles of pure gel-phase lipid containing trapped marker dye but initially no Bis-Azo PC become leaky as a result of this lipid transfer. Liposomes composed of liquid-crystalline-phase phosphatidylcholine- containing Bis-Azo PC neither leak trapped marker no fuse together on photolysis, nor do liquid-crystalline-phase liposomes fuse with gel-phase liposomes under these conditions. These results are discussed together with some possible applications of liposome photodestabilization.

Biocompatible surfaces using methacryloylphosphorylcholine laurylmethacrylate copolymer.

Many materials used in the medical device industry were not originally developed for these applications. In general, these materials elicit adverse biologic responses when in contact with body fluids such as blood, and the mechanisms of the response of blood to an artificial surface are well characterized. Protein adsorption, platelet adhesion, and activation of the coagulation pathway can subsequently lead to thrombus formation with grave clinical consequences in the absence of anticoagulant. However, the use of anticoagulants can result in complications. In recent years various approaches for overcoming these problems by improvement of the biocompatibility of materials have been advocated. One approach is that of biomembrane mimicry, whereby the surface of a material is coated with a derivative of phosphorylcholine (PC). PC is the major lipid head group component found in the outer surface of biologic cell membranes. In this paper, the application of PC coatings to a range of materials is discussed together with characterization of the surfaces using in vitro biocompatibility tests. Studies of fibrinogen and platelet binding have shown significant reductions in adsorption of these components to various PC coated materials relative to uncoated controls. Materials tested, amongst others, include PVC, polyethylene, polycarbonate, and nylon. The stability of the PC coatings has been studied using radiolabeled derivatives. Results using several materials show that physiadsorbed PC coatings are extremely stable, thus making the coatings suitable for use in a wide variety of medical applications. Extensive biologic evaluations to assess the toxicologic profile of PC derivatives and coated devices have also been carried out and in all tests the materials have been shown to be nontoxic, thus making them suitable for human use. Ex vivo animal and human studies performed support the in vitro data.

Phosphorylcholine coating of ePTFE reduces platelet deposition and neointimal hyperplasia in arteriovenous grafts.

In an attempt to reduce platelet deposition and inhibit neointimal hyperplasia, we evaluated the effect of coating expanded polytetrafluoroethylene (ePTFE) grafts with phosphorylcholine (PC), a lipid found in animal cell membranes, in a dog model of femoral arteriovenous (AV) grafts. Eight mongrel dogs underwent placement of a PC-coated femoral AV graft on one side and an untreated control graft on the contralateral side. Platelet deposition was measured by autologous 111Indium-labeling and scintillation camera imaging analysis. Platelet deposition on the PC-coated grafts at 30 and 90 min. was 9.32 +/- 4.35 x 10(9) and 10.00 +/- 4.38 x 10(9), respectively, as compared with 10.26 +/- 4.36 x 10(9) and 11.64 +/- 5.08 x 10(9) platelet deposition on control grafts (P < 0.05). All grafts were patent at 4 weeks. There was a significant reduction of neointimal area at both arterial (0.07 +/- 0.05 mm2) and venous (0. 18 +/- 0.09 mm2) anastomoses in the treated grafts as compared with arterial (0.15 +/- 0.05 mm2) and venous (0.43 +/- 0.22 mm2) anastomoses in the control grafts (P < 0.05). In addition, neointimal cell proliferation assayed by bromodeoxyuridine (BrdU) incorporation was reduced in both arterial (2.05 +/- 0.81%) and venous (3.25 +/- 0.17%) anastomoses of treated grafts compared with arterial (3.12 +/- 1.23%) and venous (5.36 +/- 1.18%) anastomoses of control grafts (P < 0.05). These data demonstrated that PC coating of ePTFE grafts significantly reduced platelet deposition, anastomotic neointimal hyperplasia, and neointimal cell proliferation in a dog model of AV grafts. This may represent a new strategy for prolonging hemodialysis graft patency.

Phosphorylcholine coating of ePTFE grafts reduces neointimal hyperplasia in canine model.

This study attempts to prevent neointimal hyperplasia by coating the graft luminal surface with a derivative of phosphorylcholine (PC), thereby providing a biocompatible surface with the assumption of limiting pannus tissue ingrowth from the graft anastomoses. Bilateral carotid artery bypass grafts were placed in six dogs using expanded polytetrafluoroethylene (ePTFE). In each animal, one carotid arterial-arterial conduit was constructed using a graft having a PC coating over the entire luminal surface of the graft. On the contralateral side, uncoated graft served as a control. The processed specimens were analyzed for graft neointimal area and neointimal thickness. Cell proliferation was assessed by staining for bromodeoxyuridine (BrdU) incorporation. All grafts were patent except one control graft that was occluded at 4 weeks. There was a significant reduction in the anastomotic graft neointimal area between the treated and control groups (0.27 +/- 0.17 mm2 versus 0.53 +/- 0.13 mm2, respectively; p = 0.008). Furthermore, the BrdU labeling index in the graft neointimal tissues was significantly smaller (p < 0.001) in the treated group (2.64 +/- 0.77%) as compared with the control group (5.07 +/- 0.83%). These data demonstrate that PC coating of ePTFE significantly reduces graft neointimal hyperplasia and cell proliferation in a canine carotid artery bypass model. The application of PC within the ePTFE graft effectively blocks tissue ingrowth from the adjacent native vessel, thereby preserving the anastomosis luminal diameter.