Postsynthetic Photocontrol of Giant Liposomes via Fusion-Based Photolipid Doping.

We report on photolipid doping of giant unilamellar vesicles (GUVs) via vesicle fusion with small unilamellar photolipid vesicles (pSUVs), which enables retroactive optical control of the membrane properties. We observe that vesicle fusion is light-dependent, if the phospholipids are neutral. Charge-mediated fusion involving anionic and cationic lipid molecules augments the overall fusion performance and doping efficiency, even in the absence of light exposure. Using phosphatidylcholine analogs with one or two azobenzene photoswitches (azo-PC and dazo-PC) affects domain formation, bending stiffness, and shape of the resulting vesicles in response to irradiation. Moreover, we show that optical membrane control can be extended to long wavelengths using red-absorbing photolipids (red-azo-PC). Combined, our findings present an attractive and practical method for the precise delivery of photolipids, which offers new prospects for the optical control of membrane function.

Light-Controlled Lipid Interaction and Membrane Organization in Photolipid Bilayer Vesicles.

Controlling lateral interactions between lipid molecules in a bilayer membrane to guide membrane organization and domain formation is a key factor for studying and emulating membrane functionality in synthetic biological systems. Here, we demonstrate an approach to reversibly control lipid organization, domain formation, and membrane stiffness of phospholipid bilayer membranes using the photoswitchable phospholipid azo-PC. azo-PC contains an azobenzene group in the sn2 acyl chain that undergoes reversible photoisomerization on illumination with UV-A and visible light. We demonstrate that the concentration of the photolipid molecules and also the assembly and disassembly of photolipids into lipid domains can be monitored by UV-vis spectroscopy because of a blue shift induced by photolipid aggregation.

Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles.

Giant unilamellar vesicles (GUVs) represent a versatile model system to emulate the fundamental properties and functions associated with the plasma membrane of living cells. Deformability and shape transitions of lipid vesicles are closely linked to the mechanical properties of the bilayer membrane itself and are typically difficult to control under physiological conditions. Here, we developed a protocol to form cell-sized vesicles from an azobenzene-containing phosphatidylcholine (azo-PC), which undergoes photoisomerization on irradiation with UV-A and visible light. Photoswitching within the photolipid vesicles enabled rapid and precise control of the mechanical properties of the membrane. By varying the intensity and dynamics of the optical stimulus, controlled vesicle shape changes such as budding transitions, invagination, pearling, or the formation of membrane tubes were achieved. With this system, we could mimic the morphology changes normally seen in cells, in the absence of any molecular machines associated with the cytoskeleton. Furthermore, we devised a mechanism to utilize photoswitchable lipid membranes for storing mechanical energy and then releasing it on command as locally usable work.

Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release.

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lß phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lß phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

Molecular-Level Modifications Induced by Photo-Oxidation of Lipid Monolayers Interacting with Erythrosin.

Incorporation into cell membranes is key for the action of photosensitizers in photomedicine treatments, with hydroperoxidation as the prominent pathway of lipid oxidation. In this paper, we use Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as cell membrane models to investigate adsorption of the photosensitizer erythrosin and its effect on photoinduced lipid oxidation. From surface pressure isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) data, erythrosin was found to adsorb mainly via electrostatic interaction with the choline in the head groups of both DOPC and DPPC. It caused larger monolayer expansion in DOPC, with possible penetration into the hydrophobic unsaturated chains, while penetration into the DPPC saturated chains was insignificant. Easier penetration is due to the less packed DOPC monolayer, in comparison to the more compact DPPC according to the monolayer compressibility data. Most importantly, light irradiation at 530 nm made the erythrosin-containing DOPC monolayer become less unstable, with a relative surface area increase of ca. 19%, in agreement with previous findings for bioadhesive giant vesicles. The relative area increase is consistent with hydroperoxidation, supporting the erythrosin penetration into the lipid chains, which favors singlet oxygen generation close to double bonds, an important requirement for photodynamic efficiency.

Modeling kinetics and equilibrium of membranes with fields: milestoning analysis and implication to permeation.

Coarse graining of membrane simulations by translating atomistic dynamics to densities and fields with Milestoning is discussed. The space of the membrane system is divided into cells and the different cells are characterized by order parameters presenting the number densities. The dynamics of the order parameters are probed with Milestoning. The methodology is illustrated here for a phospholipid membrane system (a hydrated bilayer of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) lipid molecules). Significant inhomogeneity in membrane internal number density leads to complex free energy landscape and local maps of transition times. Dynamics and distributions of cavities within the membrane assist the permeation of nonpolar solutes such as xenon atoms. It is illustrated that quantitative and detailed dynamics of water transport through DOPC membrane can be analyzed using Milestoning with fields. The reaction space for water transport includes at least two slow variables: the normal to the membrane plane, and the water density.

Giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer.

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

Peroxidation of polyunsaturated phosphatidyl-choline lipids during electroformation.

Giant unilamellar vesicles (GUVs) have been utilized both as model systems to study the physico-chemical properties of biomembranes and as host materials for investigating biological processes in microbioreactors. GUVs are commonly formed by an electroformation technique. However, there is a concern that the electric fields applied during electroformation can peroxidize lipid acyl chains, thereby altering the phospholipid composition and material properties of the synthesized vesicles. Here in this paper, we report the effect of electroformation on the extent of peroxidation of a number of polyunsaturated phosphatidyl-choline lipids (PULs). Specifically, we detected peroxidation byproducts (malonaldehydes and conjugated dienes) of the following lipids utilizing UV/Vis spectroscopy: dilinoleoyl phosphatidyl-choline (DLPC) (di-18:2 PC), dilinolenoyl phosphatidyl-choline (DNPC) (di-18:3 PC), diarachidonoyl phosphatidyl-choline (DAPC) (di-20:4 PC), and didocosaheexaenoyl phosphatidyl-choline (DHA) (di-22:6 PC). The results indicate that PC PULs lipids are prone to peroxidation, with increasing unsaturation levels leading to higher levels of peroxidation byproducts. The levels of peroxidation byproducts of DAPC were found to depend linearly on the strength of the electric field, indicating that the observed effects were due to the applied electric field. Lipid peroxidation can affect a number of important membrane properties, including domain formation and mechanical stability. Thus, alteration of the chemical composition of polyunsaturated lipids (PULs) by the electroformation technique can potentially complicate the interpretation of experimental studies that utilize GUVs composed of PULs.

Scoparone inhibits ultraviolet radiation-induced lipid peroxidation.

Antioxidant capabilities of scoparone, the component of Artemisia scoparia and other medicinal plants, against lipid peroxidation induced by ultraviolet radiation or Fenton reaction have been analyzed. Lipid peroxidation was monitored by measuring the absorption spectra of the conjugated dienes and quantified by the Klein oxidation index. Obtained results imply that scoparone is a very efficient inhibitor of ultraviolet radiation-induced lipid peroxidation and damage.

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer.

The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes.

Molecules analogous to biological and synthetic lipids have been prepared conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10(3)-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.

The optical activity and circular dichroic spectra of diacetylenic phospholipid polymers.

Phospholipids (phosphatidylcholines) which contain a diacetylene group in a single acyl chain and within both acyl chains have been synthesized. Upon irradiation with ultraviolet light, both types of lipid crosslink via the diacetylene groups to produce coloured polymers. The colour arises from the conjugated double and triple bonds which make up the polymer backbone. These phospholipid polymers can exhibit optical activity, as shown by their circular dichroic spectra. The optical activity is thought to stem from asymmetric packing of the polydiacetylene chains, a packing of one particular screw sense being favoured by the chiral glycerol moiety of the lipid. The presence of an intrinsic membrane protein within the liposome structure affects the CD spectra of polymer produced by irradiation.

Lipid oxidation and gel to liquid-crystalline transition temperatures of synthetic polyunsaturated mixed-acid phosphatidylcholines.

Synthetic preparations of the polyunsaturated phosphatidylcholines 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) and 1-stearoyl-2-alpha-linolenoyl-sn-glycero-3-phosphocholine (SLnPC) were observed to undergo autooxidation sometimes during synthesis and also on storage. Oxidation was also induced by treatment of unoxidized SLPC with ultraviolet irradiation. Oxidation was estimated by thin layer chromatographic, fatty acid and ultraviolet spectral analyses. With limited oxidation, the gel to liquid-crystalline transition temperatures of aqueous dispersions of these lipids were seen to increase. Extensive oxidation led to a reduction in the enthalpies of the transitions. The increases in transition temperatures were consistent with the presence of conjugated double bonds, as shown by increased absorption at 230 nm, and trans double bonds, in the oxidized lipids leading to the creation of more rigid domains within the bilayer. Some of the changes in the transitions, especially the decreasing enthalpy after extensive oxidation, may have occurred because of the presence of small amounts of lysophosphatidylcholine and other oxidation intermediates or breakdown products seen by thin layer analysis. Thermograms of mixtures of unoxidized SLPC with amounts of lysostearoylPC found in the oxidized samples showed, however, that lysoPC likely did not contribute significantly to the increase in transition temperatures. Thin layer analysis suggested that the presence of cross-linked products could have contributed to the observed thermotropic properties.

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.

Interaction of cytochrome c with liposomes: covalent labeling of externally bound protein by the fluorescent probe, azidonaphthalenedisulfonic acid, enclosed in the inner aqueous compartment of unilamellar vesicles.

The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) +/- 20 mol% dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isoelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.

Kinetics of radiation- and cytochrome c-induced modifications in liposomes analysed by FT-Raman spectroscopy.

Fourier transform Raman spectroscopy on artificial lipid membranes was used to study radiation-induced peroxidation processes as a function of time after radiation exposure. The time dependent intensity changes of the Raman lines of various C=C bondings were compared to results obtained by measuring conjugated dienes and by the thiobarbituric acid test for malondialdehydes. The results show that mainly the cis C=C bonds of the lipid chains are involved and, therefore, indicate that gamma-radiation induces conformational changes in the lipid chain while the mobility of the lipid chains is reduced. New Raman bands can be assigned to aldehyde products induced at the end of the peroxidation process. The immediate decrease of the =CH vibration lines was directly correlated with the formation of conjugated C=C double bonds suggesting that these vibration lines are in contrast to the C=C lines solely Raman active, when isolated C=C bonds are present. Cytochrome c (ox.) incorporated into the bilayer of the artificial membranes induced autooxidation processes not influenced by gamma-radiation. It was observed that cytochrome c (ox.)-induced changes of the relative intensity of the C=C bonds differ from those induced by gamma-radiation. These results of cytochrome c together with the inhibitory effects of the antioxidant alpha-tocopherol suggest that the radical species involved in the cytochrome c induced process might be different from the free radicals involved in the gamma-radiation-induced process.

Stable nitroxide radicals protect lipid acyl chains from radiation damage.

The present study focused on protective activity of two six-membered-ring nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-Tempo (Tempol), against radiation damage to acyl chain residues of egg phosphatidylcholine (EPC) of small unilamellar vesicles (SUV). SUV were gamma-irradiated (10-12 kGy) under air at ambient temperature in the absence and presence of nitroxides. Acyl chain composition of the phospholipids before and after irradiation was determined by gas chromatography. Both Tempo and Tempol effectively and similarly protected the acyl chains of EPC SUV, including the highly sensitive polyunsaturated acyl chains, C20:4, C22:5, and C22:6. The conclusions of the study are: (a) The higher the degree of unsaturation in the acyl chain, the greater is the degradation caused by irradiation. (b) The fully saturated fatty acids palmitic acid (C16) and stearic acid (C18) showed no significant change in their levels. (c) Both Tempo and Tempol provided similar protection to acyl chain residues. (d) Nitroxides' lipid-bilayer/aqueous distribution is not validly represented by their n-octanol/saline partition coefficient. (e) The lipid-bilayer/aqueous partition coefficient of Tempo and Tempol cannot be correlated with their protective effect. (f) The nitroxides appear to protect via a catalytic mode. Unlike common antioxidants, such as alpha-tocopherol, which are consumed under irradiation and are, therefore, less effective against high radiation dose, nitroxide radicals are restored and terminate radical chain reactions in a catalytic manner. Furthermore, nitroxides neither yield secondary radicals upon their reaction with radicals nor act as prooxidants. Not only are nitroxides self-replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers a powerful strategy to protect liposomes, membranes, and other lipid-based assemblies from radiation damage.

The effect of the chemical structure of the phospholipid polymer on fibronectin adsorption and fibroblast adhesion on the gradient phospholipid surface.

The interaction between biocomponents and the polyethylene (PE) surface modified with poly[omega-methacryloyloxyalkyl phosphorylcholine (MAPC)] was considered taking into account the surface characteristics, i.e., density, mobility, and orientation of the poly(MAPC). The PE surface, grafted gradually with the poly(MAPC) was prepared by corona irradiation method. The amount of peroxide produced on the PE surface which was determined with 1,1-diphenyl-2-picryl-hydrazyl, increased with an increase in the energy of the corona. The surface density of the poly(MAPC) was increased with an increase in the amount of the peroxides produced by the corona irradiation. The orientation and mobility of the poly(MAPC) grafted on the PE surface was evaluated with 1,6-diphenyl-1,3,5-hexatriene. The orientation of the poly[6-methacryloyloxyhexyl phosphorylcholine (MHPC)] which has six methylene chains between the phospholipid polar group and the backbone was higher than that of other poly(MAPC)s. The mobility of the poly(MAPC) decreased with an increase in the methylene chain length in the MAPC unit. The fibronectin adsorption on the gradient PE sheet grafted with poly(MAPC) was determined with enzyme-labeled immunoassay. The amount of adsorbed fibronectin on the PE grafted with poly[2-methacryloyloxyethyl phospohorylcholine(MPC)] and poly(MHPC) decreased with an increase in their surface density. Especially, the PE sheet grafted with the poly(MHPC) was effectively reduced compared with other poly(MAPC)s. On the poly[10-methacryloyloxydecyl phosphorylcholine (MDPC)], there is a minimum amount of adsorbed fibronectin. The fibronectin adsorption pattern on the PE sheet grafted with poly(MAPC) was quite different from the chemical structure of the MAPC unit. The human normal diploid fibroblasts (WI-38 cells) were cultured on the gradient PE sheet grafted with poly(MAPC) changing the concentration of seeded WI-38 cells. The adhesion behavior of the WI-38 cells was different depending on the concentration of the seeded WI-38 cells. When the concentration was low, the number of the adherent WI-38 cells had the same tendency as fibronectin adsorption. The gradient PE sheet grafted with the poly(MHPC) effectively reduced WI-38 cells adhesion even when the concentration of the WI-38 cells was high. The biocompatibility of polymer surfaces can be improved by highly oriented phosphorylcholine group.

Gamma-irradiation effects on phosphatidylcholine multilayer liposomes: calorimetric, NMR, and spectrofluorimetric studies.

Chemical and structural modifications in multilayer liposomes of synthetic phosphatidylcholine induced by gamma irradiation are investigated with different techniques. Fluorescence anisotropy of the DPH probe and differential scanning calorimetry reveal a broadening of the main lipid transition and the disappearance of pretransition. Fluorescence anisotropy is shown to be higher in the irradiated sample and particularly so at low temperatures. NMR and TLC results show that lysolecithin and palmitic acid are formed with a consequent change in bilayer organization. The possibility that these modifications may account for the permeability variations observed in irradiated natural membranes is discussed.