Spontaneous formation of single component liposomes from a solution.

The diameter of lipid vesicles is generally known to be determined by parameters external to the system, such as fluid shear, electric fields, co-surfactants, etc. We present a mechanism by which a system consisting of a single component lipid can spontaneously assemble from a solution phase to form monodisperse unilamellar vesicles of well-defined diameters dictated only by thermodynamic parameters intrinsic to the system. Here, the lipids self-assemble as vesicles when an aqueous phase diffusively replaces the original solvent in a macroscopically stationary (or quiescent) manner. We demonstrate this using phosphatidyl choline lipid-ethanol-water systems, where the average diameter of the liposomes is shown to be intrinsic, in reasonable agreement with the Helfrich's model of the vesicle free energy. The size depends only on the temperature and the lipid type, eliminating dependence on kinetic effects or external forcing normally observed. The method provides the first pure system to study the self-assembly of vesicle-forming surfactants; and with a natural thermodynamic length scale, it may have an implication for the vesicle size selection under pre-biotic conditions.

Functionalized Vesicles by Microfluidic Device.

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

Study of the influence of ascorbyl palmitate and amiodarone in the stability of unilamellar liposomes.

Amiodarone (AMI) is a low water-solubility drug, which is very useful in the treatment of severe cardiac disease. Its adverse effects are associated with toxicity in different tissues. Several antioxidants have been shown to reduce, and prevent AMI toxicity. The aim of this work was to develop and characterize Dimyristoylphosphatidylcholine (DMPC) liposomal carriers doped with ascorbyl palmitate (Asc16) as antioxidant, in order to either minimize or avoid the adverse effects produced by AMI. The employment of liposomes would avoid the use of cosolvents in AMI formulations, and Asc16 could minimize the adverse effects of AMI. To evaluate the partition and integration of AMI and Asc16 in lipid membranes, penetration studies into DMPC monolayers were carried out. The disturbance of the liposomes membranes was studied by generalized polarization (GP). The stability of liposomes was evaluated experimentally and by means of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The size particle and zeta potential (ζ) values of the liposomes were used for application in calculations for attractive and repulsive forces in DLVO theory. In experimental conditions all of these vesicles showed stability at time 0, but only DMPC + Asc16 10% + AMI 10% liposomes kept their size stable and ζ during 28 days. These results are encouraging and suggest that such systems could be suitable for AMI delivery formulations.

Fiber-optic trap-on-a-chip platform for probing low refractive index contrast biomaterials.

Dual-beam fiber trapping is a versatile technique for manipulating microparticles. We fabricate and evaluate the performance of a compact trap-on-a-chip design and demonstrate, for what we believe is the first time, trapping of low-contrast (m<1.005) lipid vesicles in solution. Counterpropagating fibers are fixed along the chip channel, and we calibrate the trap by optically displacing polystyrene microspheres from the trap center. Measured scattering forces are ~30-49 pN from each beam. Stable trapping and reversible deformation of lipid vesicles is demonstrated under femtonewton trapping forces. This chip has applications in probing a variety of soft biomaterials, such as biological cells, lipid membranes, and protein assemblies.

Two hits are better than one: membrane-active and DNA binding-related double-action mechanism of NK-18, a novel antimicrobial peptide derived from mammalian NK-lysin.

The extensive use and misuse of antibiotics in medicine result in the emergence of multidrug-resistant bacteria, creating an urgent need for the development of new chemotherapeutic agents. Nowadays, antimicrobial peptides are widely recognized as a class of promising candidates with activity against multidrug-resistant bacteria. NK-18 is a truncated peptide derived from NK-Lysin, an effector of cytotoxic T cells and natural killer cells. In this study, we studied the antibacterial mechanism of action of NK-18. The results revealed that NK-18 has potent antibacterial activity against Escherichia coli and Staphylococcus aureus. According to our findings, NK-18 is membrane active and its target of action is not only the bacterial membrane but also the DNA in the cytoplasm. The double targets of NK-18 make it difficult for bacteria to generate resistance, which may present a new strategy to defend against multidrug-resistant bacteria and provide a new lead in the design of potent antimicrobial peptides with therapeutic application in the presence of increasing resistance to conventional antibiotics.

Preparation, characterization and in vitro antimicrobial activity of liposomal ceftazidime and cefepime against Pseudomonas aeruginosa strains

Pseudomonas aeruginosa is an opportunistic microorganism with the ability to respond to a wide variety of environmental changes, exhibiting a high intrinsic resistance to a number of antimicrobial agents. This low susceptibility to antimicrobial substances is primarily due to the low permeability of its outer membrane, efflux mechanisms and the synthesis of enzymes that promote the degradation of these drugs. Cephalosporins, particularty ceftazidime and cefepime are effective against P. aeruginosa, however, its increasing resistance has limited the usage of these antibiotics. Encapsulating antimicrobial drugs into unilamellar liposomes is an approach that has been investigated in order to overcome microorganism resistance. In this study, antimicrobial activity of liposomal ceftazidime and cefepime against P. aeruginosa ATCC 27853 and P. aeruginosa SPM-1 was compared to that of the free drugs. Liposomal characterization included diameter, encapsulation efficiency and stability. Minimum Inhibitory Concentration (MIC) was determined for free and liposomal forms of both drugs. Minimum Bactericidal Concentration (MBC) was determined at concentrations 1, 2 and 4 times MIC. Average diameter of liposomes was 131.88 nm and encapsulation efficiency for cefepime and ceftazidime were 2.29% end 5.77%, respectively. Improved stability was obtained when liposome formulations were prepared with a 50% molar ratio for cholesterol in relation to the phospholipid. MIC for liposomal antibiotics for both drugs were 50% lower than that of the free drug, demonstrating that liposomal drug delivery systems may contribute to increase the antibacterial activity of these drugs.

Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics

Several antihistaminics possess antibacterial activity against a broad spectrum of bacteria. However, the exact mechanism of such activity was unclear. Hence, the aim of this study is to investigate their mechanism of antibacterial activity especially their effect upon the permeability of the bacterial cytoplasmic membrane. The effects of azelastine, cetirizine, cyproheptadine and diphenhydramine were studied using Gram-positive and Gram-negative multiresistant clinical isolates. Leakage of 260 and 280 nm UV-absorbing materials was detected upon treatment with the tested antihistaminics; indicative of membrane alteration. Using an artificial membrane model, cholesterol-free negatively-charged unilamellar liposomes, confirmed the effect of antihistaminics upon the membrane permeability both by showing an apparent membrane damage as observed microscopically and by detection of leakage of preloaded dye from the liposomes colorimatrically. Moreover, examination of the ultrastructure of cells treated with azelastine and cetirizine under the transmission electron microscope substantiated the detected abnormalities in the cell wall and membrane. Furthermore, the effect of pretreating certain isolates for both short and long periods with selected antihistaminics was followed by the viable count technique. Increased vulnerability towards further exposure to azelastine was observed in cells pretreated with azelastine for 2 days and those pretreated with azelastine or cetrizine for 30 days.

Effective line tension and contact angles between membrane domains in biphasic vesicles.

Inhomogeneities in membranes give rise to localized interactions at the interface between domains in two-component vesicles. The corresponding energy is expressed as a line tension between the two phases. In this paper we study the implications of the thickness mismatch between domains which has been experimentally reported to be of order 20-30% and the conditions under which the induced line tension can destabilize the domains in inhomogeneous vesicles. For asymmetric lipidic membranes we prove an increase of the line tension and the existence of a contact angle. Adsorption of impurities is also examined, our scope being the extension of the Canham-Helfrich model to describe elastic deformations and chemical interactions arising at microscopic scales. This mismatch effect may have important consequences for the stability of very small domains.

Using zeta-potential measurements to quantify peptide partition to lipid membranes.

Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (K ( p )) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the K ( p ) from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.

Analysis of lipid vesicle populations from microscopy video sequences.

Conducting research on lipid vesicles is very convenient, since they provide a stable and controllable environment for in vitro observations. Their resemblance to biological cell membranes allows biologists to assess hazardous potential of nanoparticles by exposing the vesicles instead of live organisms. When considering behavior of vesicles during incubation with nanoparticles, majority of existing research focus on observing single vesicles only. Our approach provides an ability to observe thousands of lipid vesicles for more representative behavior estimation. We developed an efficient algorithm to transform video sequences acquired with video microscopy into quantitative data. This includes steps required to filter noise, use multiple frames for more precise content presentation, detection of regions of interest, and segmentation of circular and non-primitively shaped vesicles. Presented work is a crucial step towards the creation of an automated computer analysis for lipid vesicles behavior assessment.

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.

Methods for assessing the structure and function of cationic antimicrobial peptides.

Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic alpha-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of antimicrobial peptides and the production of more potent analogues. To date, however, few of these peptides have been shown to have clinical efficacy, especially for systemic use, in large part due to insufficient selectivity between target and host cells. Recently, we developed a new strategy in the design of antimicrobial peptides. These linear cationic peptides, which form amphipathic beta-sheets rather than alpha-helices, demonstrated superior selectivity in binding to the lipids contained in bacterial vs. mammalian plasma membranes. Here we describe methods to evaluate the structure and function of cationic antimicrobial peptides.

Complex numbers in chemometrics: examples from multivariate impedance measurements on lipid monolayers.

Electrical impedance gives multivariate complex number data as results. Two examples of multivariate electrical impedance data measured on lipid monolayers in different solutions give rise to matrices (16x50 and 38x50) of complex numbers. Multivariate data analysis by principal component analysis (PCA) or singular value decomposition (SVD) can be used for complex data and the necessary equations are given. The scores and loadings obtained are vectors of complex numbers. It is shown that the complex number PCA and SVD are better at concentrating information in a few components than the naïve juxtaposition method and that Argand diagrams can replace score and loading plots. Different concentrations of Magainin and Gramicidin A give different responses and also the role of the electrolyte medium can be studied. An interaction of Gramicidin A in the solution with the monolayer over time can be observed.

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.