Lipid Bilayer Breakdown Research Articles

Novel Class of Ultrasound-Triggerable Drug Delivery Systems for the Improved Treatment of Tumors.

The controlled release of anticancer drugs at the tumor site is a central challenge in treating cancer. To achieve this goal, our strategy was based on tumor-specific targeting and ultrasound-triggered release of an anticancer agent from liposomal nanocarriers. To enhance the ultrasound-triggered drug release, we incorporated a lipophilic sonosensitizer, chlorin e6 (Ce6) ester, into the lipid bilayer of liposomes. Additionally, asparagine-glycine-arginine (NGR) that binds to CD13, which is overexpressed in tumor cells, was introduced into these liposomes. Under the navigation effects of the NGR, the novel ultrasound-triggerable NGR-modified liposomal nanocarrier (NGR/UT-L) accumulates in tumor sites. Once irradiated by ultrasound in tumor tissues, the sonodynamic effect produced by Ce6 could create more efficient disruptions of the lipid bilayer of the liposomal nanocarriers. After encapsulating doxorubicin (DOX) as the model drug, the ultrasound triggered lipid bilayer breakdown can spring the immediate release of DOX, making it possible for ultrasound-responsive chemotherapy with great selectivity. By combining tumor-specific targeting and stimuli-responsive controlled release into one system, NGR/UT-L demonstrated a perfect antitumor effect. Moreover, this report provides an example of controlled-release by means of a novel class of ultrasound triggering systems.

Development and characteristics of novel sonosensitive liposomes for vincristine bitartrate

The aim of drug delivery is to increase therapeutic efficacy. Externally triggered drug delivery systems enable site-specific and time-controlled drug release. To achieve this goal, our strategy was based on ultrasound-triggered release of an anticancer agent from sonosensitive liposomes (SL). To realize the ultrasound-triggered drug release, a lipophilic sonosensitizer, hematoporphyrin monomethyl ether (HMME) was incorporated into the lipid bilayer of liposomes. Once irradiated by the ultrasound in tumor tissues, the sonodynamic effect generated by HMME could lead to an efficient disruption of the lipid bilayer in the SL. After encapsulating vincristine bitartrate (VIN) as the model drug, the ultrasound-triggered lipid bilayer breakdown can trigger the instant release of VIN, enabling ultrasound-controlled chemotherapy with great specificity. In the in vitro and in vivo studies, by integrating tumor-specific targeting and stimuli-responsive controlled release into one system, VIN-loaded SL showed excellent antitumor efficacy. The SL could potentially produce viable clinical strategies for improved targeting efficiency of VIN for the treatment of related cancer. More importantly, this report provides an example of controlled release by means of a novel class of ultrasound triggering system.

Lipid-mediated mode of action of local anesthetics on lipid pores induced by polyenes, peptides and lipopeptides.

The effects of local anesthetics (LAs), namely, lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), bupivacaine (BPV), procaine (PC), and tetracaine (TTC), on the steady-state transmembrane conductance induced by the cis-side addition of the antifungal polyene macrolide antibiotic, nystatin (NYS), in planar lipid bilayers were studied. The addition of TTC to model membranes comprising DOPC and cholesterol (33 mol%) led to a nearly twenty-fold increase in the steady-state NYS-induced membrane conductance. BPV slightly enhanced the channel-forming activity of polyene. LDC, PLC, MPV, and PC did not affect the NYS-induced transmembrane current. We concluded that the effects of LAs on the channel-forming activity of NYS were in agreement with their effects on the elastic properties of model membranes. The ability of aminoamide LAs to promote calcein leakage from large unilamellar DOPC-vesicles was decreased in the following order: BPV >> LDC ≈ PLC ≈ MPV. LDC, PLC, and MPV produced a graded leakage of fluorescent marker from liposomes, up to 10-13%. A initial sharp jump in fluorescence after the introduction of BPV was attributed to the solubilization of liposomes and the formation of mixed DOPC:BPV-micelles. Differential scanning microcalorimetry (DSC) of large unilamellar DPPC-vesicles showed that the main transition temperature (Tm) is continuously decreased upon increasing concentrations of TTC. A sharp drop in the enthalpy of the transition at higher TTC concentrations indicated a formation of anesthetic/lipid mixed micelles. In contrast to TTC, PC slightly decreased Tm, broadened the DSC signal and did not provoke vesicle-to-micelle transition. Both the calcein leakage and DSC data together with the results of measurements of threshold voltages that are required to cause the lipid bilayer breakdown might indicate an alteration in the curvature lipid packing stress, induced by BPV and TTC. The data presented here lend support to a lipid-mediated mode of LAs action on NYS pores via an alteration in curvature stress near the trans-mouth. Similar results were obtained for several lipid pores, formed by polyene amphotericin B, lipopeptide syringomycin E, and the peptides magainin and melittin. This finding further developed the concept of non-specific regulation of lipid pores by LAs. In conclusion, the combination of nystatin with LAs could be a novel treatment for efficient therapy of superficial and mucosal candidiasis.

Measurement protocol for planar lipid bilayer viscoelastic properties

This paper describes how to estimate planar lipid bilayer's elasticity module E and surface tension sigma by means of measuring its breakdown voltage and using Dimitrov's viscoelastic model of electric field-induced breakdown of lipid bilayers. Planar lipid bilayers (BLMs) were made of two components: 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl 2-oleoyl phosphatidylserine (POPS) in five different compositions. Folding method for forming planar lipid bilayers in the salt solution of 100 mM KCl was used. Breakdown voltages Ubr and membrane life times tbr were measured by means of applying linear rising voltage signals of seven different slopes. Specific capacitances cBLM of bilayers were measured with charge pulse method. Then Dimitrov's viscoelastic model was fitted to measured data allowing for estimation of elasticity module and surface tension of the lipid bilayer. Our results show that onecomponent bilayers composed from POPS were more stable and thus having higher breakdown voltages and elasticity moduli then bilayers composed of POPC. Surface tension values were similar regardless of the membrane composition. Values of the elasticity (E) and surface tension (sigma) are comparable to those published in the literature. We conclude that the protocol used, though time consuming, is an alternative to other methods used for determination of bilayer's mechanical properties.

A System for the Determination of Planar Lipid Bilayer Breakdown Voltage and Its Applications

In this paper, we focus on measurement principles used in electroporation studies on planar lipid bilayers. In particular, we point out the voltage-clamp measurement principle that has great importance when the breakdown voltage of a planar lipid bilayer is under consideration; however, it is also appropriate for the determination of other planar lipid bilayer electrical properties such as resistance and capacitance. A new experimental system that is based on the voltage-clamp measurement principle is described. With the use of a generator that can generate arbitrary-type signals, many specific shapes of a voltage signal could be generated, and therefore, the experimental system is appropriate for a broad spectrum of measurements.

Determination of the lipid bilayer breakdown voltage by means of linear rising signal

Electroporation is characterized by formation of structural changes within the cell membrane, which are caused by the presence of electrical field. It is believed that “pores” are mostly formed in lipid bilayer structure; if so, planar lipid bilayer represents a suitable model for experimental and theoretical studies of cell membrane electroporation. The breakdown voltage of the lipid bilayer is usually determined by repeatedly applying a rectangular voltage pulse. The amplitude of the voltage pulse is incremented in small steps until the breakdown of the bilayer is obtained. Using such a protocol each bilayer is exposed to a voltage pulse many times and the number of applied voltage pulses is not known in advance. Such a pre-treatment of the lipid bilayer affects its stability and consequently the breakdown voltage of the lipid bilayer. The aim of this study is to examine an alternative approach for determination of the lipid bilayer breakdown voltage by linear rising voltage signal. Different slopes of linear rising signal have been used in our experiments (POPC lipids; folding method for forming in the salt solution of 100 mM KCl). The breakdown voltage depends on the slope of the linear rising signal. Results show that gently sloping voltage signal electroporates the lipid bilayer at a lower voltage then steep voltage signal. Linear rising signal with gentle slope can be considered as having longer pre-treatment of the lipid bilayer; thus, the corresponding breakdown voltage is lower. With decreasing the slope of linear rising signal, minimal breakdown voltage for specific lipid bilayer can be determined. Based on our results, we suggest determination of lipid bilayer breakdown voltage by linear rising signal. Better reproducibility and lower scattering are obtained due to the fact that each bilayer is exposed to electroporation treatment only once. Moreover, minimal breakdown voltage for specific lipid bilayer can be determined.

Vesicle and bi-layer kinetics at surfaces measured by electrical transmission

We present forward transmission measurements of spontaneous lipid bi-layer formation and the effect of enzymatic attack on such bi-layers at frequencies in the 20 kHz–1 GHz range. Measurement of the forward ac transmission parameter at a fixed frequency (1 MHz), on a suitably designed biochip, allows fast and simple real time monitoring of the kinetics of formation of lipid bi-layers on SiO 2 surfaces, through adsorption of vesicles from solution. The bi-layer formation modifies the electrical characteristics in the megahertz frequency regime, by adding an insulating layer on top of the electrode. The observed kinetics is consistent with bi-layer formation observed with, for example, the quartz crystal microbalance with dissipation (QCM-D) and the surface plasmon resonance (SPR) techniques. Measurements on the enzymatic action of phospholipase A 2 (PLA 2) on the breakdown of lipid bi-layers further demonstrates that the method can provide new information on bio-interfacial processes not obtainable by, for example, mass-sensing methods.

Use of irreversible electrical breakdown of lipid bilayers for the study of interaction of membranes with surface active molecules

Lipid bilayers were formed in the presence of different macromolecules and high electrical fields were used to induce mechanical rupture of the membranes. The kinetics of pore formation during irreversible breakdown was studied as a function of the macromolecules. We observed that macromolecules having a strong binding affinity to the membrane alter the time course of pore formation significantly. We propose this method as a simple test for adsorption of macromolecules to membranes.

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.

Hormone-regulated phosphoinositide turnover in permeabilized cells and membranes

This chapter discusses the methods for studying hormone-regulated phosphoinositide turnover in permeable cells and in membranes. There are a large number of methods for rendering animal cells permeable to small- or large-molecular-weight probes. However, for the studies of hormone-regulated phospholipid turnover, there is the stringent requirement that membrane-associated receptors, enzymes, and phospholipid substrates remain functionally intact. Hence, methods that employ detergents (e.g., saponin) might be damaging, as it is found for TRH responsive GH 3 cells (unpublished results) and thrombin-responsive platelets. Electric field-induced permeabilization of cells was extensively characterized for studying Ca 2+ -dependent secretion in adrenal chromaffin cells. Electric field-induced permeabilization is based on the fact that a transmembrane potential difference exceeding 0.5–1 V results in the mechanical breakdown of lipid bilayers leading to a state of high permeability. The chapter describes the measurement of regulated phosphoinositide turnover in permeable cells.

Electric field-induced breakdown of lipid bilayers and cell membranes: a thin viscoelastic film model.

A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982, J. Membrane Biol. 67:165 and U. Zimmermann, 1982, Biochim. Biophys. Acta 694:227). The basic result is a formula for the time tau of membrane breakdown (up to the formation of pores): tau = alpha (mu/G)/(epsilon 2m epsilon 2oU4/24 sigma Gh3 + T2/sigma Gh-1), where alpha is a proportionality coefficient approximately equal to ln(h/2 zeta o), h being the membrane thickness and zeta o the amplitude of the initial membrane surface shape fluctuation (alpha is usually of the order of unity), mu represents the membrane shear viscosity, G the membranes shear elasticity modules, epsilon m the membrane relative permittivity, epsilon o = 8.85 X 10(-12) F/m, U the electric potential across the membrane, sigma the membrane surface tension and T the membrane tension. This formula predicts a critical potential Uc; Uc = (24 sigma Gh3/epsilon 2m epsilon 2o)1/4 (for tau = infinity and T = 0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.

456 — Intramembrane interactions and breakdown of lipid bilayers

456 — Intramembrane interactions and breakdown of lipid bilayers