Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Emulsification of Liposomes with Incomplete Freund's Adjuvant: Stability of the Liposomes and the Emulsion

Abstract

Emulsification of liposomes with incomplete Freund's adjuvant, a water-in-oil emulsion, resulted in the formation of stable emulsions containing a large fraction of intact liposomes. Although some loss of liposome integrity and loss of emulsion stability did occur at certain concentrations of liposomes, based on the release of trapped glucose, it was determined that formulations of Freund's adjuvant containing liposomes could be produced that still retained a considerable liposomal permeability barrier for at least 7 days.     

Senescence-Dependent Increase in the Permeability of Liposomes Prepared from Bean Cotyledon Membranes

Liposomes containing 79 mM Tris-acetate and 50 mM KCl were preparedfrom the total lipid extracts of smooth microsomal membranesisolated from 2, 4, 7 and 9 d old bean cotyledons. Permeabilityto glycerol was determined by spectrophotometric measurementsof osmotic swelling when the liposomes were placed in eitherisotonic or slightly hypotonic glycerol. For liposomes from2 and 4 d old membrane there was a time-dependent decrease inabsorbance at 450 nm from which initial swelling rates reflectingthe influx of glycerol and water were calculated. At 25 °Cthese rates were not significantly different For liposomes from7 and 9 d old membrane there was no change in absorbance withtime at 450 nm signifying that these older liposomes were equallypermeable and non-electrolytes and non-electrolytes, and thereforeincapable of swelling. Permeability to glucose was determinedby preparing the liposomes in a solution of the sugar, passingthem through a Sephadex column to eliminate unsequestered glucose,and quantifying sugar leaked from the liposomes over time bymeasuring NADPH formation through the tandem actions of hexokinaseand glucose-6-phosphate dehydrogenase. The rate constants forglucose leakage from 2 and 7 d old liposomes were 0.55 and 3.94respectively, again indicating a dramatic increase in permeabilitywith advancing age. These changes in permeability correlatetemporally with the appearance of gel phase lipid in both liposomesand the membranes from which they were derived, suggesting thatthe coexistence of discrete liquid-crystalline and gel phaselipid domains renders membranes leaky and contributes to lossof intracellular compartmentation in senescing tissue.     

Unusual Low Proton Permeability of Liposomes Prepared from the Endogenous Myelin Lipids

Abstract: In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin-induced potassium diffusion potentials as high as -177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [¹⁴C]-methylamine, or the changes in the fluorescence of the probes 9-aminoacridine, acridine orange, and pyranine. The presence of cations (Na⁺, K⁺, Ca²⁺) did not alter this behavior. Voltage clamping did not increase the trans-membrane ApH-driven proton permeability. However, II-posome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H⁺ through the membrane.     

Effect of Formulation and Process Parameters on Chitosan Microparticles Prepared by an Emulsion Crosslinking Technique

There are many studies about the synthesis of chitosan microparticles; however, most of them have very low production rate, have wide size distribution, are difficult to reproduce, and use harsh crosslinking agents. Uniform microparticles are necessary to obtain repeatable drug release behavior. The main focus of this investigation was to study the effect of the process and formulation parameters during the preparation of chitosan microparticles in order to produce particles with narrow size distribution. The technique evaluated during this study was emulsion crosslinking technique. Chitosan is a biocompatible and biodegradable material but lacks good mechanical properties; for that reason, chitosan was ionically crosslinked with sodium tripolyphosphate (TPP) at three different ratios (32, 64, and 100%). The model drug used was acetylsalicylic acid (ASA). During the preparation of the microparticles, chitosan was first mixed with ASA and then dispersed in oil containing an emulsifier. The evaporation of the solvents hardened the hydrophilic droplets forming microparticles with spherical shape. The process and formulation parameters were varied, and the microparticles were characterized by their morphology, particle size, drug loading efficiency, and drug release behavior. The higher drug loading efficiency was achieved by using 32% mass ratio of TPP to chitosan. The average microparticle size was 18.7 μm. The optimum formulation conditions to prepare uniform spherical microparticles were determined and represented by a region in a triangular phase diagram. The drug release analyses were evaluated in phosphate buffer solution at pH 7.4 and were mainly completed at 24 h.     

Calcium Movements Mediated by Proteolipid Protein and Nucleotides in Liposomes Prepared with the Endogenous Lipids from Brain White Matter

A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch-Lees proteolipid apoprotein. Freeze-fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra- and extravesicular spaces. The most significant finding was that Mg-ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin.     

Stability of Liposomes Prepared from the Total Polar Lipids of Methanosarcina Mazei is Affected by the Specific Salt form of the Lipids

Abstract

The total polar lipids (TPL) extracted from the archaeobacterium Metha-nosarcina mazei were predominantly in the form of sodium and potassium salts. Upon passage of this natural salt form of TPL (ns-TPL) through a silica gel G column (s-TPL), the molar ratio of the monovalent cations, sodium plus potassium, to that of the divalent cations, magnesium plus calcium, decreased from about 11:1 to 2:1. Liposomes (archaeosomes) made from ns-TPL were unable to efficiently retain low molecular weight aqueous markers such as 5(6)-carboxyfluorescein (CF). In phosphate buffered saline (PBS, pH 7.14), between 60-90% and 90-100% of entrapped CF leaked out during 21 h storage at room temperature (20-22°) and 3 h at 50°, respectively. However, archaeosomes made with the s-TPL, as well as from TPL converted to the salt forms of predominantly sodium, potassium, calcium or magnesium, were significantly less leaky. The archaeosomes made with s-TPL were the most stable, showing less than 7% leakage after 21 h at room temperature and only 14% leakage of CF after 3 h at 50°. The stability of s-TPL archaeosomes (21 h, 20-22°) was not affected when Tris-HCI buffer (pH 7.4) was used instead of PBS. However, the inclusion of 1 or 10 mM EGTA in the Tris-HCI buffer increased the amount of CF leaked, to about 25% and 100%, respectively. Except for some differences in phosphatidylserine and phosphati-dylglycerol, the lipid compositions of ns-TPL, s-TPL, and the magnesium form of TPL were similar, as determined by thin layer chromatography of labeled lipids. Archaeosomes prepared from s-TPL and ns-TPL had -“P NMR spectra that were similar to each other, but distinct from those of vesicles prepared from the ester lipid dimyristoylphosphatidylcholine. The types and relative proportions of cations associated with the lipids of M. mazei prior to their hydration and vesicle formation have a major influence, although other factors such as lipid composition may have an effect, on the permeability of the bilayer to low molecular weight compounds.     

Uptake of Archaeobacterial Liposomes and Conventional Liposomes by Phagocytic Cells

Abstract

Liposomes in the 200 nm size range were prepared from the ether lipids extracted from various Archaeobacteria (coined archaeosomes), and from conventional lipids. The entrapment of peroxidase or carboxyfluorescein was used to compare the in vitro uptake of various liposomes by murine peritoneal macrophages, J774A.1 macrophages and several non phagocytic cell lines. While liposomes composed of ester lipids dipalmitoylphosphatidylcholine, or dimyristoylphosphatidylcholine: dimyristoylphosphatidylglycerol: cholesterol (1.8:0.2:1.5, molar ratio) were taken up by macrophage species, the uptake of archaeosomes was 3 to 53 times greater. Uptake by non phagocytic HEp-2, HeLa, and EJ/28 cells was considerably less. Evidence from time-course studies using cytochalasins B+D, sub-optimal temperature or formaldehyde treatments of macrophages, indicated that the archaeosomes lost structural integrity following internalization within the J774A. 1 phagocytic cells. No cytotoxicity was observed in viability or growth assays with J774A. 1 cells, using high doses of three representative types of archaeosomes and one type of conventional-liposome. Therefore, archaeosomes may be well suited to applications where phagocytic cells are a target site.     

Quantitative Urine Culture by Surface Drop Method

A simple drop method for quantitative urine culture was developed and tested in comparison with standard methods for bacterial urinary counts. In a group of 452 urines all yielding Escherichia coli, 74 showed counts of more than 100,000 colonies, and 16 showed counts between 10,000 and 100,000 colonies per ml. Of these 90 urines, 3 of the 16 in the doubtful group were false negative with the drop method. Another 7 urines in the total number of 452 showed discrepancies, but, because all would have been repeated, the second urine sample would have corrected the primary result. The ease and cleanliness of the method render it a suitable technique for screening normal and patient populations. The method was applied on a population sample of 1,330 persons from whom unwashed mid-stream urine was collected and yielded figures comparable with results published in the literature. The method discriminates between steps of 10-fold difference, whereas more accurate count methods show a standard error of +/-25% and are reliable in a double dilution series.     

Quantitative Modeling of the High-Throughput Production and In Vivo Kinetics of (Drug-Encapsulating) Liposomes

In developing liposomes for in vivo use, it is important to design the liposomes to have optimal in vivo kinetics, and it is also necessary to identify optimal high-throughput production conditions for these liposomes. Previous work has not definitively established the general relationship between liposomes'' configuration and composition, and their in vivo kinetics. Also, no straightforward method exists to calculate optimal liposome high-throughput production conditions for specific liposome compositions. This work presents first-principles quantitative correlations describing liposomes'' in vivo drug leakage and vascular mass transfer kinetics. This work further presents a simple quantitative model relating specific liposome compositions to ideal high-throughput production parameters. The results have implications for the identification of promising liposome compositions via high-throughput screening methodologies, as well as the design and optimization of high-throughput reactors for liposome production.     

Core-Shell Modeling of Light Scattering by Vesicles: Effect of Size,Contents, and Lamellarity

Having a fast, reliable method for characterizing vesicles is vital for their use as model cell membranes in biophysics, synthetic biology, and origins of life studies. Instead of the traditionally used Rayleigh-Gans-Debye approximation, we use an exact extended Lorenz-Mie solution for how core-shell particles scatter light to model vesicle turbidity. This approach enables accurate interpretations of simple turbidimetric measurements and is able to accurately model highly scattering vesicles, such as larger vesicles, those with multiple layers, and those with encapsulated material. We uncover several surprising features, including that vesicle lamellarity has a larger effect on sample turbidity than vesicle size and that the technique can be used to measure the membrane thickness of vesicles. We also examine potential misinterpretations of turbidimetry and discuss when measurements are limited by forward and multiple scattering and by the geometry of the instrument.     

用微孔板分光光度法测定薯蓣皂苷元的含量

为了对植物样品中薯蓣皂苷元的含量进行高通量快速测定,本研究采用高压酸解制备薯蓣皂苷元,以高氯酸为显色剂,用微孔板分光光度法测定样品中薯蓣皂苷元的含量。合适的分析条件为:反应温度为30℃、高氯酸用量为200μL、振荡时间2 min后静置10 min,在410 nm处测定光吸收值。该方法的线性范围为每孔薯蓣皂苷元2～10μg(R=0.9988),平均回收率为99.9%,精密度的RSD为1.65%。该方法操作简单、准确稳定,可实现大批量样品中薯蓣皂苷元的快速检测。     

Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method

The purpose of the present work was the development and evaluation of stomach-specific controlled release mucoadhesive drug delivery system prepared by ionotropic gelation of gellan beads, containing acid-soluble drug amoxicillin trihydrate, using 3² factorial design with concentration of gellan gum and quantity of drug as variables. The study showed that beads prepared in alkaline cross-linking medium have higher entrapment efficiency than the acidic cross-linking medium. The entrapment efficiency was in the range of 32% to 46% w/w in acidic medium, which increased up to 60% to 90% w/w in alkaline medium. Batches with lowest, medium, and highest drug entrapment were subjected to chitosan coating to form a polyelectrolyte complex film. As polymer concentration increases, entrapment efficiency and particle size increases. Scanning electron microscopy revealed spherical but rough surface due to leaching of drug in acidic cross-linking solution, dense spherical structure in alkaline cross-linking solution, and rough surface of chitosan-coated beads with minor wrinkles. The in vitro drug release up to 7 h in a controlled manner following the Peppas model (r = 0.9998). In vitro and in vivo mucoadhesivity study showed that beads have good mucoadhesivity and more than 85% beads remained adhered to stomach mucosa of albino rat even after 7 h. In vitro growth inhibition study showed complete eradication of Helicobacter pylori. These results indicate that stomach-specific controlled release mucoadhesive system of amoxicillin gellan beads may be useful in H. pylori treatment.     

Quantitative Analysis of Single Particle Trajectories: Mean Maximal Excursion Method

An increasing number of experimental studies employ single particle tracking to probe the physical environment in complex systems. We here propose and discuss what we believe are new methods to analyze the time series of the particle traces, in particular, for subdiffusion phenomena. We discuss the statistical properties of mean maximal excursions (MMEs), i.e., the maximal distance covered by a test particle up to time t. Compared to traditional methods focusing on the mean-squared displacement we show that the MME analysis performs better in the determination of the anomalous diffusion exponent. We also demonstrate that combination of regular moments with moments of the MME method provides additional criteria to determine the exact physical nature of the underlying stochastic subdiffusion processes. We put the methods to test using experimental data as well as simulated time series from different models for normal and anomalous dynamics such as diffusion on fractals, continuous time random walks, and fractional Brownian motion.     

枣疯病植原体实时荧光定量PCR检测方法的研究

目的:建立枣疯病植原体拷贝数检测实时荧光定量PCR方法,为枣疯病植原体定量检测提供技术支持。方法:构建质粒标准品,设计实时荧光PCR探针引物,优化体系,建立标准曲线,并进行重复性验证。结果:制备了枣疯病植原体标准质粒,建立了稳定的质粒标准品检测体系(R2=0.998,检测限10拷贝,定量限100拷贝)。结论:实时荧光定量PCR检测方法重复性好,可用于枣疯病植原体的拷贝数检测,为枣疯病植原体检验检测和病害防治提供了技术支持。