Kinetics of channel formation in bilayer lipid membranes (BLMs) and tethered BLMs: monazomycin and melittin

The kinetics of channel formation by the polyene-like antibiotic monazomycin, both in a bilayer lipid membrane (BLM) and in a tethered BLM (tBLM), and by the peptide melittin in a tBLM, is investigated. Stepping the applied potential from a value at which channels are not formed to one at which they are formed yields current vs time curves that are sigmoidal on a BLM, while they show a maximum on a tBLM; in the latter case, sigmoidal curves are obtained by plotting the charge against time. These curves are interpreted on the basis of a general kinetic model, which accounts for the potential-dependent penetration of adsorbed monomeric molecules into the lipid bilayer, followed by their aggregation with channel formation by a mechanism of nucleation and growth. In the case of monazomycin, which is present in the solution in the form of relatively hydrophilic clusters and is adsorbed as such on top of the lipid bilayer, penetration into the bilayer following a potential jump is assumed to be preceded by a potential-independent disaggregation of the adsorbed clusters into adsorbed monomers.     

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

Kinetics and Yield of Singlet Oxygen Photosensitized by Hypericin in Organic and Biological Media

The spectroscopy and photophysics of the photosensitizer hypericin when in homogeneous solutions and when bound to liposomes were studied. Hypericin was found to partition efficiently into DMPC liposomes, with a binding constant of 58 (mg lipid/mL)^?1. In these liposomes the singlet oxygen production quantum yield was 0.43 ± 0.09. To determine the deactivation constant of singlet oxygen in lipid bilayers for the first time, we calculated extrapolated values from its quenching by DMPC and lecithin in homogeneous solutions and obtained decay times of 36.4 and 12.2 μs, respectively. We also measured the quenching of singlet oxygen, sensitized by hypericin in DMPC liposomes, by NaN₃, diphenyl isobenzofuran and H₂,O: D₂O mixtures and explained the results on the basis of singlet oxygen diffusing rapidly out of the lipid bilayer into the aqueous medium. The observed temperature effect on the lifetime of singlet oxygen of about 50% over a 15°C range in liposome suspension contrasts with a 3% change in a homogeneous solution in 1-nonanol and is explained by the temperature effect on the diffusion out of the liposome. A strong pH effect was observed, indicating that the deprotonated species formed above about pH 10 is a much weaker photosensitizer of singlet oxygen than the native, protonated species.     

Electron transfer between ferrocene-modified Au/octadecanethiol/lipid BLM electrode and redox couples in solution

Bilayers incorporated with ferrocene consisting of self-assembled octadecanethiol and lipid monolayer on gold substrates were fabricated. Its electrochemical behaviors in solutions containing different redox couples were investigated by cyclic voltammetry and ac impedance. The transmembrane electron transfer reaction across octadecanethiol self-assembled film and an adsorbed phospholipid layer mediated by ferrocene have been observed in the solution of Fe(CN)6(3-/4-). The formal potential difference between mediator in bilayer lipid membrane (BLM) and redox couple in solution has a great impact on the transmembrane electron transfer behavior. The ferrocene-modified BLM electrodes might be useful for constructing a bilayer-based electrochemical current rectifying device.     

ELECTRONIC PROCESSES AND PHOTOELECTRIC ASPECTS OF BILAYER LIPID MEMBRANES

Abstract— Owing to the complexity of biological membranes, many model systems have been studied in order to gain insight into the molecular mechanism of specific functions. One such model membrane extensively investigated in the past decade is the so-called bilayer lipid membrane (BLM). With suitable modifications, a BLM less than 100 A thick separating two aqueous solutions has been used as a model for a variety of biological membranes. This paper is devoted to a review of the properties and electronic processes of modified BLM.
Recent experiments using these membranes which contain photosynthetic pigments or dyes have demonstrated that, upon illumination, an EMF and a current can be generated. The connection between the photoelectric BLM and light-sensitive biological membranes and the rationale for this work are described.
Additionally, the effects of physical chemical parameters such as electric field, temperature, light intensity, duration of illumination and chemical agents (electron acceptors, donors, uncouplers, etc.) on the photoresponses of BLM are discussed. Other results indicate that BLM containing photoactive compounds behave similar to that of a silicon solar cell with one side of the membrane reducing and the other side oxidizing. The transverse pathway for the electron across the BLM could be provided by carotenoids such as β-carotene. Photoelectric BLM of this type represents a unique kind of energy transducing system and may well be useful in the conversion of solar energy into electricity and/or other forms of energy.     

LIGHT-INDUCED ELECTRICAL EFFECTS IN A LIQUID CRYSTAL BLM CONTAINING TCNQ

Abstract— The light-induced capacitance changes and also both photovoltage and photocurrent under continuous illumination have been investigated in pigmented liquid crystal bilayer membranes (PBLM)‡ containing TCNQ as photosensitizer with Na₂SO₃ electron donor on one side and methylene blue electron acceptor on the other side. The results have shown that TCNQ in cyanobiphenyl membrane produces a unique photoactive BLM system in which all three main parameters (conductivity, capacity and voltage across the membrane) are in a wide range altered by the light. It is shown that a TCNQ-cyanobiphenyl charge transfer complex is responsible for the observed photochanges. The possible mechanism of photoinduced electrical effects in this type of PBLM is discussed.     

Intracellular Modulation of Excited‐State Dynamics in a Chromophore Dyad: Differential Enhancement of Photocytotoxicity Targeting Cancer Cells

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O₂(a¹Δ_g)], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer‐related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.     

Photosensitizing Antivirals

Antiviral action of various photosensitizers is already summarized in several comprehensive reviews, and various mechanisms have been proposed for it. However, a critical consideration of the matter of the area is complicated, since the exact mechanisms are very difficult to explore and clarify, and most publications are of an empirical and “phenomenological” nature, reporting a dependence of the antiviral action on illumination, or a correlation of activity with the photophysical properties of the substances. Of particular interest is substance-assisted photogeneration of highly reactive singlet oxygen (¹O₂). The damaging action of ¹O₂ on the lipids of the viral envelope can probably lead to a loss of the ability of the lipid bilayer of enveloped viruses to fuse with the lipid membrane of the host cell. Thus, lipid bilayer-affine ¹O₂ photosensitizers have prospects as broad-spectrum antivirals against enveloped viruses. In this short review, we want to point out the main types of antiviral photosensitizers with potential affinity to the lipid bilayer and summarize the data on new compounds over the past three years. Further understanding of the data in the field will spur a targeted search for substances with antiviral activity against enveloped viruses among photosensitizers able to bind to the lipid membranes.     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

Membrane Damage Efficiency of Phenothiazinium Photosensitizers

Structure–activity relationships have been widely reported for porphyrin and phthalocyanine photosensitizers, but not for phenothiazinium derivatives. Here, four phenothiazinium salts (methylene blue, toluidine blue O, 1,9‐dimethyl methylene blue and the pentacyclic derivative DO15) were used to investigate how the ability to damage membranes is affected by membrane/solution partition, photophysical properties and tendency to aggregation of the photosensitizer. These two latter aspects were studied both in isotropic solutions and in membranes. Membrane damage was assessed by leakage of a fluorescent probe entrapped in liposomes and by generation of thiobarbituric acid‐reactive species (TBARS), while structural changes at the lipid bilayer were detected by small‐angle X‐ray scattering. We observed that all compounds had similar singlet‐oxygen quantum yields in ethanol, but only the photosensitizers that had higher membrane/solution partition (1,9‐dimethyl methylene blue and DO15, the latter having the higher value) could permeabilize the lipid bilayer. Moreover, of these two photosensitizers, only DO15 altered membrane structure, a result that was attributed to its destabilization of higher order aggregates, generation of higher amounts of singlet oxygen within the membranes and effective electron‐transfer reaction within its dimers. We concluded that membrane‐based protocols can provide a better insight on the photodynamic efficiency of the photosensitizer.     

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 10¹⁰ ions s⁻¹. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.     

Bilayer Lipid Membrane-Semiconductor Junctions. Spectroscopic and Electrochemical Characterizations and Photoelectron Transfer

Three different systems of glyceryl monooleate (GMO) bilayer lipid membrane (BLM) supported semiconductor particles have been prepared and characterized. A single composition of particulate semiconductor deposited on only one side of the BLM constituted System A, two different compositions of particulate semiconductors sequentially deposited on the same side of the BLM represented System B, and two different compositions of particulate semiconductors deposited on the opposite sides of the BLM made up System C. Effective refractive indices and optical thicknesses of GMO-BLM-supported In₂S₃ and ZnS particles (System A), determined by Brewster-angle and reflection measurements, allowed the assessment of the maximum sizes and the volume fractions of semiconductor particles to be on the order of 1200 Å and 0.3, respectively. Since semiconductor particles are highly porous structures, only the first layer of particulates penetrated into the BLM and were considered in the proposed equivalent circuit and band models. The presence of semiconductors on the BLM surface has been established by voltage-dependent capacitance measurements, absorption spectroscopy, and optical microscopy. Subsequent to the injection of H₂S, the first observable change was the appearance of fairly uniform white dots on the black film. These dots rapidly moved around and grew in size, forming islands which then merged with themselves and with a second generation of dots, which ultimately led to a continuous film which continued to grow in thickness. Cyclic voltammetry established the current rectifying behavior for the semiconductor-particle-coated BLMs. CdS, ZnS, and In₂S₃ (System A) formed an n-type, while Cu_2-(x+y)S (System A) behaved like a p-type, electrolyte-semiconductor (ES) junction. Semiconductor-semiconductor heterojunction (SS') formation was established for System C. Transfer of conduction-band electrons to dissolved oxygen (for the n-type ES junction) and across the membrane was considered to be responsible for the observed dark currents. Steady-state illumination of a CdS-containing BLM resulted in the prompt development of -150 to -200 mV (cis side negative) potential difference in an open circuit across the GMO BLM. This initial photovoltage, V ₁, quickly decayed to a steady value, V _s (- 100 to -150 mV). When the illumination was turned off, the potential difference across the GMO BLM decreased to its dark value in 3-4 minutes.     

Dithiaporphyrin derivatives as photosensitizers in membranes and cells

We synthesized a series of analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (I) as potential photosensitizers for photodynamic therapy (PDT). The photosensitizers differ in the length of the side chains that bind the carboxyl to the phenol at positions 10 and 15 of the thiaporphyrin. The spectroscopic, photophysical, and biophysical properties of these photosensitizers are reported. The structural changes have almost no effect on the excitation/emission spectra with respect to I's spectra or on singlet oxygen generation in MeOH. All of the photosensitizers have a very high, close to 1.00, singlet oxygen quantum yield in MeOH. On the contrary, singlet oxygen generation in liposomes was considerably affected by the structural change in the photosensitizers. The photosensitizers possessing short side chains (one and three carbons) showed high quantum yields of around 0.7, whereas the photosensitizers possessing longer side chains showed smaller quantum yield, down to 0.14 for compound X (possessing side-chain length of 10 carbons), all at 1 microM. Moreover a self-quenching process of singlet oxygen was observed, and the quantum yield decreased as the photosensitizer's concentration increased. We measured the binding constant of I to liposomes and found Kb = 23.3 +/- 1.6 (mg/mL)-1. All the other photosensitizers with longer side chains exhibited very slow binding to liposomes, which prevented us from assessing their Kb's. We carried out fluorescence resonance energy transfer (FRET) measurements to determine the relative depth in which each photosensitizer is intercalated in the liposome bilayer. We found that the longer the side chain the deeper the photosensitizer core is embedded in the bilayer. This finding suggests that the photosensitizers are bound to the bilayer with their acid ends close to the aqueous medium interface and their core inside the bilayer. We performed PDT with the dithiaporphyrins on U937 cells and R3230AC cells. We found that the dark toxicity of the photosensitizers with the longer side chain (X, VI, V) is significantly higher than the dark toxicity of sensitizers with shorter side chains (I, III, IV). Phototoxicity measurements showed the opposite direction; the photosensitizers with shorter side chains were found to be more phototoxic than those with longer side chains. These differences are attributed to the relationship between diffusion and endocytosis in each photosensitizer, which determines the location of the photosensitizer in the cell and hence its phototoxicity.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5－Ditetradecyl－2－ （2－trimethyl－ammonioethyl）－l, 3－dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4^- stimulant with Ru(bpy)3^2 as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4^- anions.     

Bilayer lipid membranes: An experimental system for biomolecular electronic devices development

The lipid bilayer postulated as the basic structural matrix of biological membranes is widely accepted. At present, the planar bilayer lipid membrane (BLM) together with spherical lipid bilayers (liposomes), upon suitable modification, serves as a most appropriate model for biological membranes. In recent years, advances in microelectronics and interest in ultrathin organic films, including BLMs and Langmuir-Blodgett (L-B) films, have resulted in a unique fusion of ideas toward the development of biosensors and transducers. Furthermore, recent trends in interdisciplinary studies in chemistry, electronics, and biology have led to a new field of research: biomolecular electronics. This exciting new field of scientific-technological endeavor is part of a more general approach toward the development of a new, post-semiconductor electronic technology, namely, molecular electronics with a long-term goal of molecular computers.

Recently, it has been demonstrated that BLMs, after suitable modification, can function as electrodes and exhibit nonlinear electronic properties. These and other experimental findings relevant to sensor development and to “biomolecular electronic devices” (BED) will be described in more details in the present review article. Also the potential use of the BLM system together with its modifications in the development of a new class of organic diodes, switches, biosensors, electrochemical photocells, and biofuel cells will be discussed. Additionally, this paper reports also a novel technique for obtaining BLMs (or lipid bilayers) on solid supports. The presence of solid support on one side of the BLM greatly enhances its mechanical stability, while retaining the dynamic properties of the lipid bilayer. Advantages of the new techniques for self-assembling amphiphilic molecules on rigid substrates are discussed in terms of their possible uses. It is evident that the new BLM system (s-BLMs) is potentially useful for technological applications in the area of biosensors and enzyme electrodes as well as molecular electronics.     

Transport of Cesium Ion Across a Bilayer Lipid Membrane and Its Facilitation in the Presence of Iodide Ion

The ion transport current was clearly observed across a bilayer lipid membrane (BLM) between two aqueous phases (W1, W2) containing 0.1 M CsCl. This indicates that Cs⁺ can spontaneously penetrate the BLM in the absence of any kind of transporter. In addition, the current density at a given potential between W1 and W2 increased using I^? instead of Cl^? as the counter ion. Our results strongly suggest that the cell membrane transport is partially responsible for the internal exposure of ¹³⁴Cs and ¹³⁷Cs.     

Effect of acidity on the formation of solvent-free lipid bilayers

Glasses with hydrophobized surface are used to form “dry” bilayer lipid membranes (BLM). The contact angle on the silaned glass plates reaches 125°. Partitions of this kind, as opposed to polymer films, make a system more resistant to heat oscillations and mechanical perturbations generated when studying membranes properties. The specific capacitance of BLM (0.86 ± 0.04 μF cm^?2) testifies to the solvent absence in the bilayer. The dramatic drop of the capacitance (area) of the dry membranes with increasing pH is caused by a greater adsorption of lipid molecules on the hydrophobic substrate, which is probably due to changing conformation of their polar “head.”     

PHOTOELECTRIC EFFECTS IN BILAYER LIPID MEMBRANE CONTAINING METALLO-PORPHYRINS and DYES

The bilayer lipid membrane (BLM) system containing metallo-porphyrins (M-TPP) and dyes as photosensitizers and electron mediators was studied. Cyclic voltammetry was used to determine photoconductivity and photo-emf of the system. The largest photoconductivity was observed for the Mg-TPP containing BLM with methyl viologen (MV²⁺) and iodine (I₂) present in the aqueous solution. Photoactive dyes, due to their redox ability caused photovoltage up to 30 mV to develop, but no conductance change was observed under illumination in absence of Mg-TPP. The relevance of cyclic voltammetry to the photoconductance and the photo-emf observed in the BLM is discussed.     

Voltammetric Elucidation of Ion Transfer Through an Extremely Thin Membrane

Digital simulation of the cyclic voltammogram for the ion transfer through a liquid membrane of thickness from 1 mm to 10 nm was performed. The magnitude of current and the shape of the voltammogram simulated for extremely thin membrane (10 nm thick) were similar to those observed experimentally with a bilayer lipid membrane, BLM, of about 10 nm in thick, when the diffusion coefficient of an ion in the BLM was assumed to be extraordinary small (10^?13 to 10^?14 cm² s^?1).     

PHOTOOXYGENATION OF METHYL LINOLEATE SENSITIZED BY PORPHYRINS AND DYES IN ACETONITRILE SOLUTION AND AQUEOUS EMULSION SYSTEMS

Photooxygenation reaction of an unsaturated fatty acid ester, methyl linoleate (methyl 9- cis. 12- cis -octadecadienoate, ML-H), sensitized by porphyrins and several types of dyes has been studied in aqueous emulsion and acetonitrile solution under air at 40°C. The oxygen (O₂) uptake proceeded slowly in the absence of sensitizers upon irradiation of an aqueous emulsion and an acetonitrile solution of ML-H (20 m M ) at ℷ_ex > 290 nm (11.4 and 6.1 μmol h^-1, respectively). The rate of O₂ uptake was enhanced by a catalytic amount (0.1 m M ) of porphyrins and dyes; hematoporphyrin (HP), zinc tetrakis(N-methyl-4-pyridiniumyi)porphyrin (ZnTMPyP), methylene blue (MB), rose bengal (RB), acridine orange (AO), and acriflavine (AF). In both systems, the sensitized photooxidation of ML-H by O₂ proceeded equimolarly to produce isomeric mixture of C9 and C13 hydroperoxides having the trans,cis and trans,trans conjugated diene configurations, independent of the types of the sensitizers used. The yield ratio of trans,trans/ trans,cis products in the MB-sensitized photooxygenation in acetonitrile and aqueous emulsion were almost equal (0.32 and 0.35. respectively). The sensitizing activity of the sensitizers, as measured by the quantum yield of O₂ uptake, increased in the order: MB (≃ 0) < ZnTMPyP < RB < HP < AF < AO in the aqueous emulsion and AO < AF < HP < RB = MB in the acetonitrile solution. The order in homogeneous acetonitrile solution was interpreted by the sensitizing ability of the dyes to produce singlet oxygen, while that in heterogeneous aqueous emulsion was correlated to the lipophilicity of dyes as well as the singlet-oxygen-producing ability.