pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells

Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.     

A simple and rapid harvesting method for microalgae by in situ magnetic separation

A simple and rapid harvesting method by in situ magnetic separation with naked Fe₃O₄ nanoparticles has been developed for the microalgal recovery of Botryococcus braunii and Chlorella ellipsoidea. After adding the magnetic particles to the microalgal culture broth, the microalgal cells were adsorbed and then separated by an external magnetic field. The maximal recovery efficiency reached more than 98% for both microalgae at a stirring speed of 120 r/min within 1 min, and the maximal adsorption capacity of these Fe₃O₄ nanoparticles reached 55.9 mg-dry biomass/mg-particles for B. braunii and 5.83 mg-dry biomass/mg-particles for C. ellipsoidea. Appropriate pH value and high nanoparticle dose were favorable to the microalgae recovery, and the adsorption mechanism between the naked Fe₃O₄ nanoparticles and the microalgal cells was mainly due to the electrostatic attraction. The developed in situ magnetic separation technology provides a great potential for saving time and energy associated with improving microalgal harvesting.     

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.     

反义寡核苷酸pH敏前体脂质体的制备及性质分析

为了提高反义寡核苷酸的稳定性和生物利用度及避免在溶酶体内降解,采用旋转蒸发-薄膜水化、超声-挤压、冻干三步法完成pH敏前体脂质体的制备,研究了体外释药规律;以反义寡核苷酸为实验对象,测定了包封率.制得的pH敏前体脂质体复水后形成的pH敏脂质体形态圆整,粒径12.3～389.9 nm范围内,平均粒径为22.7 nm;三批pH敏脂质体的平均包封率为68.3%;体外释药方程为Q=1.8382－2.5186×10^－2T (r=0.9913);结果说明制备的pH敏前体脂质体水合形成的pH敏脂质体粒度适宜,可用于反义寡核苷酸的包封.     

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations — one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP — with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.     

Designing of 'intelligent' liposomes for efficient delivery of drugs

The liposome- vesicles made by a double phospholipidic layers which may encapsulate aqueous solutions- have been introduced as drug delivery vehicles due to their structural flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a large variety of both hydrophilic and hydrophobic compounds. With time the liposome formulations have been perfected so as to serve certain purposes and this lead to the design of "intelligent" liposomes which can stand specifically induced modifications of the bilayers or can be surfaced with different ligands that guide them to the specific target sites. We present here a brief overview of the current strategies in the design of liposomes as drug delivery carriers and the medical applications of liposomes in humans.     

Structural characterization of diC14-amidine, a pH-sensitive cationic lipid used for transfection

The structure of N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC(14)-amidine) cationic vesicles, used for transfection, was investigated at different pH values and ionic strengths, through the analysis of the electron spin resonance (ESR) spectra of spin labels. Phospholipid derivatives, spin labeled at the 5th and 16th C-atoms along the hydrocarbon chain, incorporated in diC(14)-amidine bilayers, show that the bilayer structure is highly sensitive to the pH value of the medium, due to the two titratable groups present in the amphiphile. Compared with samples at higher pH values, the double charged diC(14)-amidine at pH 3 presents a rather non-organized bilayer gel phase, and a much lower gel-fluid temperature transition, in accord with a strong headgroup electrostatic repulsion. In addition, the structure was found to be highly dependent on the ionic strength of the medium. However, pH 3 diC(14)-amidine bilayer, in the fluid phase, was found to be slightly more closely packed than those at pH 7.4 or 9.0, which are less charged. Parallel to that, the larger isotropic hyperfine splitting measured for nitroxides in the center of the pH 3 diC(14)-amidine bilayer suggests a higher membrane polarity for the highly charged low pH sample.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

Phospholipase A2 Sensitive Liposomes for Delivery of Small Interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A₂ (sPLA₂) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA₂ which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA₂-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Effect of dehydration and rehydration of the pH-sensitive liposomes containing chimeric gag-V3 virus like particle on their long-term stability

One of the practical limitations with the use of liposomes for delivery of the pharmaceutical substances such as antigens is that liposomes are relatively unstable in storage. In order to extend the stability of liposome in storage without affecting their functional activity, solution-type liposomes were dehydrated to form a structurally intact dry liposomes. Comparative immunological evaluation was carried out for both dry and solution-type liposomes containing gag-V3 chimera, consequently it was found that dry liposomes elicited both humoral and cellular response as efficiently as solution-type liposomes did against the same gag-V3 antigen. Especially, long-term stability of the liposomes was remarkably enhanced by the dehydration made to liposomes without a significant change in its ability to elicit immune responsein vivo. These results indicate that dry pH-sensitive liposome may become an effective delivery and adjuvant system for general vaccine development.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.     

Rapid PCR-RFLP method for discrimination of imported and domestic mackerel

With the ever-decreasing domestic fishery catch of Japanese mackerel Scomber japonicus, alternative Atlantic mackerel Scomber scombrus has been increasingly imported and currently accounts for approximately 34% of mackerel consumption in Japan. As there is no morphologic difference between the species after removal of their skin, not only fresh and frozen fillets but also processed seafood of S. scombrus are frequently marketed with mislabeling as S. japonicus. In this study, a rapid and reliable polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis was developed to discriminate imported mackerel S. scombrus and domestic mackerel S. japonicus. PCR amplification for the nuclear 5S ribosomal DNA nontranscribed spacer was performed using Scomber-specific primers. Direct digestions of the PCR products using either PvuII or HaeIII restriction enzymes generated species-specific profiles, indicating that both enzymes enable the accurate identification of S. scombrus and S. japonicus. This robust and reproducible method can serve as molecular-based routine food inspection program to enforce labeling regulations.     

Reinvestigation of the mercuration-demercuration reaction on alkylated glycals: an improved method for the preparation of 2,3-dideoxy-alpha,beta-unsaturated carbohydrate enals

Alkyl protected glycals can be easily converted into their corresponding alpha,beta-unsaturated enals (Perlin aldehydes) in good to very good yields by reaction with HgSO4 and aqueous 0.02 N H2SO4 in THF or 1,4-dioxane. While the formation of Perlin aldehydes from benzyl-protected glucal and arabinal was accomplished by refluxing the reaction mixture in 1,4-dioxane, the benzyl-protected galactal and methyl-protected glucal, galactal, and arabinal yielded aldehydes from this reaction at room temperature using THF or 1,4-dioxane as solvent.     

A validated liquid chromatography-mass spectrometry method for the determination of leukotrienes B4 and B5 produced by stimulated human polymorphonuclear leukocytes

A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization-single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M-H](-) and m/z 335.2 for prostaglandin B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 were -7.8 to 1, and -5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B4 and B5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.     

Calorimetric study on pH-responsive block copolymer grafted lipid bilayers: rational design and development of liposomes

This study is focused on chimeric advanced drug delivery nanosystems and specifically on pH-sensitive liposomes, combining lipids and pH-responsive amphiphilic block copolymers. Chimeric liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and two different forms of block copolymers, i.e. poly(n-butylacrylate)-b-poly(acrylic acid) (PnBA-b-PAA) at 70 and 85% content of PAA at six different molar ratios, each form respectively. PAA block exhibits pH-responsiveness, because of the regulative group of –COOH. –COOH is protonated under acidic pH (pK_a ca. 4.2), while remains ionized under basic or neutral pH, leading to liposomes repulse and eventually stability. Lipid bilayers were prepared composed of DPPC and PnBA-b-PAA. Experiments were carried out using differential scanning calorimetry (DSC) in order to investigate their thermotropic properties. DSC indicated disappearance of pre-transition at all chimeric lipid bilayers and slight thermotropic changes of the main transition temperature. Chimeric liposomes have been prepared and their physicochemical characteristics have been explored by measuring the size, size distribution and ζ-potential, owned to the presence of pH-responsive polymer. At percentages containing medium to high amounts of the polymer, chimeric liposomes were found to retain their size during the stability studies. These results were well correlated with those indicated in the DSC measurements of lipid bilayers incorporating polymers in order to explain their physicochemical behavior. The incorporation of the appropriate amount of these novel pH-responsive block copolymers affects thus the cooperativity, the liposomal stabilization and imparts pH-responsiveness.     

Neosporacaninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-γ production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams’ brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered.     

Phospholipase A2 promotes raft budding and fission from giant liposomes

Cellular processes involving membrane vesiculation are related to cellular transport and membrane components trafficking. Endocytosis, formation of caveolae and caveosomes, as well as Golgi membranes traffic have been linked to the existence and dynamics of particular types of lipid/protein membrane domains, enriched in sphingolipids and cholesterol, called rafts [Nature 387 (1997) 569; Trends Cell Biol. 12 (2002) 296; Biochemistry 27 (1988) 6197]. In addition, the participation of phospholipases in the vesiculation of Golgi and other membranes has been already established [Traffic 1 (2000) 504] essentially in their role in the production of second messenger molecules. In this work we illustrate with raft-containing giant lipid vesicles a mechanism for raft-vesicle expulsion from the membrane due to the activity of a single enzyme-phospholipase A(2) (PLA(2)). This leads to the hypothesis that the PLA(2), apart from its role in second messenger generation, might play a direct and general role in the vesiculation processes underlying the intermembrane transport of rafts through purely physicochemical mechanisms. These mechanisms would be: enzyme adsorption leading to membrane curvature generation (budding), and enzyme activity modulation of the line tension at the raft boundaries, which induces vesicle fission.     

A Simple and Rapid Method for the Separation and Characterization of Hypoxanthine-Guanine Phosphoribosyltransferase Isoenzymes

Purinephosphoribosyltransferases catalyze the conversion of purine bases to their nucleotides in the presence of 5-phosphoribosyl-l- pyrophosphate (PRPP) (1). This salvage pathway plays an important role in the regulation of de novo purine synthesis (2). In mammalian cells two distinct phosphoribosyltransferases were demonstrated: the enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyl- transferase; A-PRT; E.C. 2.4.2.7) and the enzyme hypoxanthine-guanine phosphoribosyltransferase (PIP: pyrophosphate phosphoribosyltranferase; HG-PRT; E.C. 2.4.2.8). There has been a great interest in this latter enzyme as the complete absence of this enzyme activity in patients with Lesch-Nyhan syndrome (3) and a partial deficiency in some patients with gout (4) has been demonstrated.     

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.