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.

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.

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.

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.

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.

Light- and temperature-responsive liposomes incorporating cinnamoyl Pluronic F127.

Light- and temperature-responsive liposomes were prepared by immobilizing cinnamoyl Pluronic F127 (CP F127) on the surface of egg phosphatidylcholine liposomes. CP F127 was prepared by a condensation reaction, and the molar ratio of cinnamoyl group to Pluronic F127 was calculated to be 1:1.4 on (1)H NMR spectrum. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of a UV light (254 nm, 6 W). CP F127 decreased the absolute value of the zeta potential of liposome possibly because it can shift the hydrodynamic plane away from the liposome surface. The size of liposome decorated with CP F127, measured on a dynamic light scattering machine and observed on a TEM, was larger than that of bare liposome. The liposome bearing CP F127 seemed to fuse and aggregate each other. The liposome released calcein, a fluorescence dye, in response to a UV irradiation, possibly because the photo-dimerization of cinnamoyl group perturbs the liposomal membrane. Moreover, the liposome released the dye in response to a temperature change, possible due to the phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer with liposomal membrane.

Development of 'leaky' liposome triggered by radiation applicable to a drug reservoir and a simple radiation dosimeter.

Radiation effects on permeability of liposomes with different lipid compositions and the content ratios were investigated using X-rays. Liposomes constituted from an unsaturated phosphatidylcholine (PC), a saturated PC, and cholesterol were selected. As a result, we found that a PC with bis-allylic hydrogen, particularly dilinoleoyl PC (DLOPC), is essential for the radiation sensitivity in permeability, and it can be controlled by changing DLOPC content and dose rate. These characteristics are contributive to a novel pharmaceutical and a new-conceptual radiation dosimeter.

Gold-embedded photosensitive liposomes for drug delivery: triggering mechanism and intracellular release.

Liposomes embedded with gold nanoparticles show light-triggered contents release. We investigated the mechanism of the light-induced changes and functionality of the light-induced release in the cells. The real time small angle X-ray scattering (SAXS) analysis revealed time-dependent phase transitions in distearoylphosphatidylcholine (DSPC)/dipalmitoylphosphatidylcholine (DPPC) liposomes upon heating. Similar changes were observed when gold nanoparticle-embedded liposomes were exposed to the UV light: gold nanoparticles absorb light energy and transfer it to heat, thereby causing lipid phase transition from gel phase to rippled phase, and further to fluid phase. Without UV light exposure the gold nanoparticles did not affect the liposomal bilayer periodicity. The light-triggered release of hydrophilic fluorescent probe (calcein) from the gold nanoparticle-loaded liposomes was demonstrated with fluorescence-activated cell sorting after liposome internalization into the ARPE-19 cells. The liposome formulations did not decrease the cell viability in vitro. In conclusion, the light-triggered release from the liposomes is functional in the cells, and the release is triggered by thermal phase changes in the lipid bilayers.

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.

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.

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.

Electric pulses induce cylindrical deformations on giant vesicles in salt solutions.

In this article, we report for the first time unusual shape changes of vesicles subjected to strong electric pulses in salt solutions of low concentration. The electric field is created by two parallel electrodes between which the vesicle solution is located. Surprisingly, the vesicles assume cylindrical shapes during the pulse. These deformations are short-lived (their lifetime is approximately 1 ms) and occur only in the presence of salt outside the vesicles, irrespective of their content. When the solution conductivities inside and outside are the same, vesicles with square cross section are observed. Using a fast digital camera, we were able to record these deformations and study the vesicle shape dynamics. The aims of this article are to report the new vesicle morphologies and their dynamics and to provoke theoretical work in this direction.

Reversible photoswitching in a cell-sized vesicle.

A photosensitive amphiphilic molecule can switch the shape of an assembled vesicle as determined by microscopic observation. Photoisomerization induces a change in membrane fluctuation behavior or a morphological transition between ellipsoid and bud shapes, depending on the asymmetrical degree of the initial shape. The mechanism of this reversible photoswitching in the vesicle morphology is interpreted in terms of a change in the effective cross-sectional area of the photosensitive molecule.

Protein electrostriction: a possibility of elastic deformation of the alpha-hemolysin channel by the applied field.

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure-Staphylococcus aureus alpha-hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the alpha-hemolysin pore, we analyze the blockage of the channel by the crown/K(+) complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.

Effect of microwave radiation on the biophysical properties of liposomes.

Steadily growing use of electromagnetic fields, especially in conjunction with wireless communication systems, has led to increasing public concern about possible health effects of electromagnetic radiation. However, besides the well-known thermal effect of electromagnetic fields on biological tissue, there is no clear evidence of further athermal interaction mechanisms with biological systems. The present study was designed to determine the changes in bilayer permeability in egg lecithin multilamellar vesicles after exposure to 900 MHz microwave radiation for a period of 5 h. Specific absorption rate (SAR) of the radiation for the investigated liposome sample was found to be 12 +/- 1 W/kg. Liposomal changes in permeability were monitored using a light scattering technique. Optical anisotropy of the liposome sample decreased dramatically upon exposure to microwave radiation, indicating structural changes in acyl chain packing. IR and NMR ((1)H NMR) studies, which have been employed to reveal structural alterations in microwave, exposed vesicles showed an increased damage upon exposure to microwave. The changes observed in the (1)H NMR spectrum of the microwave exposed sample indicated hydrolysis of carboxylic and phosphoric esters. IR study showed conformational changes in the acyl chains of the lipids upon microwave exposure. However, both IR and (31)P NMR did not show any appreciable changes in the head group part of the lipids.

Photoabsorption study of Bacillus megaterium, DNA and related biological materials in the phosphorus K-shell edge region.

We measured the X-ray transmission spectra of several biologically related samples in the phosphorus K-shell edge absorption region. These include red phosphorus, hydrated sodium phosphate (Na(3)PO(4).12 H(2)O), deoxyribonucleic acid (DNA), adenosine triphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position: Each is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-shell edge absorption thresholds are shifted toward higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B. megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA, ribonucleic acid (RNA) and phosphates within the spore. The B. megaterium spore spectrum provides information for resonant radiation damage studies in the phosphorus K-shell edge absorption region by identifying candidate photoexcitations. In addition, the absorption spectra will be useful in X-ray microscopy and macromolecular crystallography studies at the phosphorus K-shell edge.