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.

Irradiation-induced fusion between giant vesicles and photoresponsive large unilamellar vesicles containing malachite green derivative.

Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG.

Integrity of Membrane Structures in Giant Unilamellar Vesicles as Assay for Antioxidants and Prooxidants.

We have attempted to evaluate, on the basis of optical microscopy for a single giant unilamellar vesicle (GUV), the potency of antioxidants in protecting GUV membranes from oxidative destruction. Photosensitized membrane budding of GUVs prepared from soybean phosphatidylcholine with chlorophyll a (Chl a) and ß-carotene (ß-Car) as photosensitizer and protector, respectively, were followed by microscopic imaging. A dimensionless entropy parameter, ΔE, as derived from the time-resolved microscopic images, was employed to describe the evolution of morphological variation of GUVs. As an indication of membrane instability, the budding process showed three successive temporal regimes as a common feature: a lag phase prior to the initiation of budding characterized by LP (in s), a budding phase when ΔE increased with a rate of kΔE (in s-1), and an ending phase with morphology stabilized at a constant ΔEend (dimensionless). We show that the phase-associated parameters can be objectively obtained by fitting the ΔE-t kinetics curves to a Boltzmann function and that all of the parameters are rather sensitive to ß-Car concentration. As for the efficacy of these parameters in quantifying the protection potency of ß-Car, kΔE is shown to be most sensitive for ß-Car in a concentration regime of biological significance of <1 × 10-7 M, whereas LP and ΔEend are more sensitive for ß-Car concentrations exceeding 1 × 10-7 M. Furthermore, based on the results of GUV imaging and fluorescence and Raman spectroscopies, we have revealed for different phases the mechanistic interplay among 1O2* diffusion, PC-OOH accumulation, Chl a and/or ß-Car consumption, and the morphological variation. The developed assay should be valuable for characterizing the potency of antioxidants or prooxidants in the protection or destruction of the membrane integrity of GUVs.

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.

Astaxanthin Protecting Membrane Integrity against Photosensitized Oxidation through Synergism with Other Carotenoids.

Incorporation of astaxanthin or zeaxanthin in giant unilamellar vesicles (GUVs) of phosphatidylcholine resulted in a longer lag phase than incorporation of ß-carotene or lycopene for the onset of budding induced by chlorophyll a photosensitization and quantified by a dimensionless entropy parameter using optical microscopy and digital image heterogeneity analysis. The lowest initial rate of GUV budding after the lag phase was seen for GUVs with astaxanthin as the least reducing carotenoid, while the lowest final level of entropy appeared for those with lycopene or ß-carotene as a more reducing carotenoid. The combination of astaxanthin and lycopene gave optimal protection against budding with respect to both a longer lag phase and lower final level of entropy by combining good electron acceptance and good electron donation. Quenching of singlet oxygen by carotenoids close to chlorophyll a in the membrane interior in parallel with scavenging of superoxide radicals by astaxanthin anchored in the surface may explain the synergism between carotenoids involving both type I and type II photosensitization by chlorophyll a.

Selective flow-induced vesicle rupture to sort by membrane mechanical properties.

Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells.

Effective protection of biological membranes against photo-oxidative damage: Polymeric antioxidant forming a protecting shield over the membrane.

We have prepared a chitosan polymer modified with gallic acid in order to develop an efficient protection strategy biological membranes against photodamage. Lipid bilayers were challenged with photoinduced damage by photosensitization with methylene blue, which usually causes formation of hydroperoxides, increasing area per lipid, and afterwards allowing leakage of internal materials. The damage was delayed by a solution of gallic acid in a concentration dependent manner, but further suppressed by the polymer at very low concentrations. The membrane of giant unilamellar vesicles was covered with this modified macromolecule leading to a powerful shield against singlet oxygen and thus effectively protecting the lipid membrane from oxidative stress. The results have proven the discovery of a promising strategy for photo protection of biological membranes.

Role of lipid polymorphism in acoustically sensitive liposomes.

Ultrasound (US) triggered drug release is a promising drug delivery method that allows ex vivo modulation of treatment intensity and duration. This method relies on the synergistic interaction between the rupture of sonosensitive particles and enhanced plasma membrane permeability. Conventional liposomal systems where the drug passively diffuses through the membrane show virtually no response to acoustic energy. One method to activate drug transport is to induce a topological restructuring of the lipid membrane (zero intrinsic curvature, H = 0) by puncturing pores (H < 0) through which the drug can readily leak out from the interior of the liposomes. In this work we demonstrate strategies to lower the energy cost of creating such membrane defects by introducing lipid molecules with molecular shapes prone to self-assemble into non-lamellar (negative intrinsic curvature, H < 0) structures. All formulations investigated comprise the relevant components typically required for delivery applications such as stealth moieties, cholesterol, and phospholipids. Small angle X-ray scattering studies of a number of lipid systems at increasing amounts of phosphatidylethanolamine (PE) phospholipids reveal that membranes without PE respond to ultrasound by thinning ca. 10 Å, which concomitantly lowers the bending rigidity quadratically in addition to increasing the passive drug permeability. However, at the appropriate PE content the lipid systems display a classic lamellar structure (H = 0) that undergoes a topological transformation after ultrasound exposure into lipid tubes of the reversed type (H < 0) packed in a 2D hexagonal array. At the dilute regime, Fluorescence Microscopy of giant unilamellar vesicles (GUVs) comprising DOPE also experience ultrasound induced restructuring that can be modulated by DOPE content. In general, smaller vesicles of diverse shape connect and form into a "pearl-necklace" configuration. We argue that the inclusion of DOPE within the GUV membrane may result in curvature-driven lipid sorting, providing the system with local membrane instabilities that drive vesicle pearling when exposed to ultrasound.

Physical damage on giant vesicles membrane as a result of methylene blue photoirradiation.

In this study we pursue a closer analysis of the photodamage promoted on giant unilamellar vesicles membranes made of dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), by irradiating methylene blue present in the giant unilamellar vesicles solution. By means of optical microscopy and electro-deformation experiments, the physical damage on the vesicle membrane was followed and the phospholipids oxidation was evaluated in terms of changes in the membrane surface area and permeability. As expected, oxidation modifies structural characteristics of the phospholipids that lead to remarkable membrane alterations. By comparing DOPC- with POPC-made membranes, we observed that the rate of pore formation and vesicle degradation as a function of methylene blue concentration follows a diffusion law in the case of DOPC and a linear variation in the case of POPC. We attributed this scenario to the nucleation process of oxidized species following a diffusion-limited growth regime for DOPC and in the case of POPC a homogeneous nucleation process. On the basis of these premises, we constructed models based on reaction-diffusion equations that fit well with the experimental data. This information shows that the outcome of the photosensitization reactions is critically dependent on the type of lipid present in the membrane.

The influence of millimeter waves on the physical properties of large and giant unilamellar vesicles.

Exposure of cell membranes to an electromagnetic field (EMF) in the millimeter wave band (30-300 GHz) can produce a variety of responses. Further, many of the vibrational modes in complex biomolecules fall in the 1-100 GHz range. In addition to fundamental scientific interest, this may have applications in the development of diagnostic and therapeutic medical applications. In the present work, lipid vesicles of different size were used to study the effects of exposure to radiation at 52-72 GHz, with incident power densities (IPD) of 0.0035-0.010 mW/cm(2), on the chemical-physical properties of cell membranes. Large unilamellar vesicles (LUVs) were used to study the effect of the radiation on the physical stability of vesicles by dynamic light scattering. An inhibition of the aging processes (Ostwald ripening), which usually occur in these vesicles because of their thermodynamic instability, resulted. Giant unilamellar vesicles (GUVs) were used to study the effect of the radiation on membrane water permeability under osmotic stress by phase contrast microscopy. In this case, a decrease in the water membrane permeability of the irradiated samples was observed. We advance the hypothesis that both the above effects may be explained in terms of a change of the polarization states of water induced by the radiation, which causes a partial dehydration of the membrane and consequently a greater packing density (increased membrane rigidity).

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.

Radiolytic yield of cardiolipin peroxidation by gamma rays in large unilamellar vesicles of phosphatidylcholine.

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1',3'-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min(-1)). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 +/- 0.2) x 10(-7) mol J(-1) for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(-CL) = (2.8 +/- 0.1) x 10(-7) mol J(-1)] and the formation of CL hydroperoxides [G(CLOOH(T)) = (3.1 +/- 0.2) x 10(-7) mol J(-1)]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 +/- 0.1) x 10(-7) mol J(-1) and (2.8 +/- 0.1) x 10(-7) mol J(-1), respectively], their sum [(4.3 +/- 0.2) x 10(-7) mol J(-1)] being higher than G(HO.) (2.8 x 10(-7) mol J(-1)). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH(T)) approximately 0] and the yield of disappearance of PLPC [(1.5 +/- 0.1) x 10(-7) mol J(-1)], likely because lipid fragments (not measured in this work) could be generated from HO(.) reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10-15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO(.)-induced peroxidation of PLPC.

The response of giant phospholipid vesicles to millimeter waves radiation.

Due to the increasing interest in millimeter waves (MMW) applications in medicine and telecommunications, the investigation of their potential biological effects is of utmost importance. Here we report results of the study of interaction between low-intensity radiation at 53.37 GHz and giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Physical changes of vesicles, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles are demonstrated. These effects are reversible and occur only during irradiation with a "switch on" of the effect requiring a short time. Since the average temperature change was very small the effects could not be attributed to thermal mechanisms. We assume that the interaction of MMW with lipid membrane leads to changes at the membrane-water interface, where charged and dipolar residues are located.

Morphological transitions of vesicles induced by alternating electric fields.

When subjected to alternating electric fields in the frequency range 10(2)-10(8) Hz, giant lipid vesicles attain oblate, prolate, and spherical shapes and undergo morphological transitions between these shapes as one varies the field frequency and/or the conductivities lambda(in) and lambda(ex) of the aqueous solution inside and outside the vesicles. Four different transitions are observed with characteristic frequencies that depend primarily on the conductivity ratio lambda(in)/lambda(ex). The theoretical models that have been described in the literature are not able to describe all of these morphological transitions.

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes.

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.

Circumvention of fluorophore photobleaching in fluorescence fluctuation experiments: a beam scanning approach.

Photobleaching is a fluorophore-damaging process that commonly afflicts single-molecule fluorescence studies. It becomes an especially severe problem in fluorescence fluctuation experiments when studying slowly diffusing particles. One way to circumvent this problem is to use beam scanning to decrease the residence time of the fluorophores in the excitation volume. We report a systematic study of the effects of circular beam scanning on the photobleaching of fluorescent particles as observed in single-photon excitation fluorescence fluctuation experiments. We start by deriving a simple expression relating the average detected fluorescence to the photobleaching cross section of the fluorophores. We then perform numerical calculations of the spatial distribution of fluorescent particles in order to understand under which conditions beam scanning can prevent the formation of a photobleaching hole. To support these predictions, we show experimental results obtained for large unilamellar vesicles containing a small amount of the fluorescent lipophilic tracer DiD. We establish the required scanning radius and frequency range in order to obtain sufficient reduction of the photobleaching effect for that system. From the detected increase in fluorescence upon increase in scanning speed, we estimate the photobleaching cross section of DiD.

Spectroscopic studies of D-alpha-tocopherol concentration-induced transformation in egg phosphatidylcholine vesicles.

The effects of embedding up to 60 mol% of alpha-tocopherol (alpha-Toc) on the morphology and structure of the egg phosphatidylcholine (PC) membrane were studied using spectroscopic techniques. The resulting vesicles were subjected to turbidometric and dynamic light scattering measurements to evaluate their size distribution. The alpha-Toc intrinsic fluorescence and its quenching was used to estimate the tocopherol position in the membrane. Optical microscopy was used to visualize morphological changes in the vesicles during the inclusion of tocopherol into the 2 mg/ml PC membrane. The incorporation of up to 15 mol% of tocopherol molecules into PC vesicles is accompanied by a linear increase in the fluorescence intensity and the simultaneous formation of larger, multilamellar vesicles. Increasing the tocopherol concentration above 20 mol% induced structural and morphological changes leading to the disappearance of micrometer-sized vesicles and the formation of small unilamellar vesicles of size ranging from 30 to 120 nm, mixed micelles and non-lamellar structures.