Suspension Of Unilamellar Vesicles Research Articles

Medicinal plant constituents interact with membrane-acting local anesthetics

Specific constituents in medicinal plants have been suggested to act on lipid bilayers and modify the physicochemical properties of biomembranes as well as amphiphilic membrane-active drugs. The interactions between selected phytochemicals and local anesthetics were studied at a membrane lipid level to verify the possibility that their concomitant use may influence local anesthesia. Biomimetic lipid bilayer membranes were prepared as unilamellar vesicle suspensions by the injection method using different phospholipids and cholesterol to mimic the lipid composition of neuronal membranes. They were treated at 37 °C for 30 min with apple flavonoid phloretin, chili pepper capsaicinoid capsaicin, harmala alkaloid tetrahydroharman and local anesthetic lidocaine separately or in combination, followed by measuring fluorescence polarization to determine their induced membrane fluidity changes. Lidocaine increased the fluidity of biomimetic membranes at clinically-relevant concentrations. In contrast, phloretin (10 – 50 µM) and tetrahydroharman (10 – 500 nM) decreased the membrane fluidity, but capsaicin (50 – 100 µM) increased. Phloretin (25 µM) and tetrahydroharman (˜15 nM) inhibited or counteracted the membrane-fluidizing effects of lidocaine (0.05 – 1 mg/mL), whereas capsaicin (50 µM) potentiated. Such membrane interactions were also found in bupivacaine. Local anesthetics mechanistically act on neuronal membranes besides voltage-gated sodium channels. The antagonistic or synergistic membrane interactions with medicinal plant constituents suggest the potentially beneficial or adverse effects that the counteraction by phloretin would successfully discontinue anesthesia after the treatment and the cooperation with capsaicin should prolong the duration of nerve block, while the inhibition by tetrahydroharman might reduce the anesthetic efficacy in certain patients. The membrane interactivity will be also an experimental clue to discover the drug leads for anesthetic adjuncts.

Dosimetry of an in vitro exposure system for fluorescence measurements during 2.45 GHz microwave exposure

An in vitro system for 2.45 GHz microwave (MW) exposure with real-time fluorescence measurements is proposed. This system is specifically designed for the measurement of those biophysical parameters of living cells or membrane models which can be quantified by spectrofluorometric methods (e.g. membrane generalized polarization (GP), membrane fluidity, membrane potential, etc.). The novelty of the system consists in the possibility to perform fluorescence measurements on the biological samples simultaneously with their exposure to MW. The MW applicator is an open ended coaxial antenna which is dipped into a cuvette. The distribution of electromagnetic field and specific absorption rate (SAR) in the cuvette are provided from a rigorous electromagnetic numerical analysis performed with a finite difference-time domain (FDTD) based tool. With this system, fluorescence measurements were used to calculate the membrane GP values of giant unilamellar vesicle suspensions that were acquired during exposure to a 1.2 W incident power. For this power, the SAR distribution and mean SAR value for the whole volume were calculated based on temperature measurements made at different positions inside the cuvette.

Perspective and limitations of cryo‐electron microscopy

We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details, More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.

Influences on the phase transition of membranes induced by lipid peroxidation

A homogeneous suspension of unilamellar vesicles of mixed dipalmitoyl lecithin (DPL) and egg lecithin (EL) was prepared by a dialysis technique. The extent of peroxidation was determined by measuring the UV absorption of conjugated dienes and the application of thiobarbituric acid as an indicator of malonaldehyd produced. The changes of the phase transition were detected by fluorescence polarization and ESR spin label techniques.