Manufacture of liposomes by isopropanol injection: characterization of the method

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40–210?nm). Accurate characterization of the particles, by fluorescence, ³¹P-NMR, and cryo–transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.     

pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 °C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.     

Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.     

The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.     

Threshold effects on the lectin-mediated aggregation of liposomes: Influence of the diameter of the liposomes

Summary It had previously been found that small unilamellar liposomes of ca. 0.03 m diameter which bear synthetic cholesterol-containing glycolipids may be aggregated by an appropriate lectin [8]. Where studied, threshold effects have been observed in that the amount of glycolipid incorporated in the liposomes must exceed a certain minimum concentration in order for aggregation to occur [3, 8, 9, 13, 14]. Threshold effects of this type may be important in mediating cell-cell and virus-cell interactions. However, before studies with small unilamellar liposomes are useful as a model for these recognition and binding phenomena, it must be shown that the observed threshold effects are not associated with the very small radius of curvature of these liposomes. This article reports that larger liposomes of average diameter 0.26 and 0.45 m which contain the synthetic glycolipidl also show threshold effects when aggregated with the galactose binding lectin ricin agglutinin. Under conditions where more than 1% (mole) glycolipid is required to support the aggregation of the smallest liposomes, those of intermediate size require only 0.18% (mole) while the largest liposomes examined require between 0.095 and 0.15% (mole) depending on the method of preparation.     

Chemical characterization and in vitro crude protein degradability of thin stillage derived from barley- and wheat-based ethanol production

A study was conducted to characterize the different carbohydrate and protein fractions of wheat- and barley-based thin stillage samples. In vitro crude protein degradability of wheat- and barley-based thin stillage was estimated relative to soyabean (SBM) and canola (CM) meal using a protease enzyme assay. Results of the carbohydrate analysis showed that wheat thin stillage had similar neutral (NDF, average 328.5 g kg⁻¹) and lower (P < 0.05) acid detergent fibre (ADF) than barley-based thin stillage. Relative to barley-based thin stillage, wheat thin stillage had higher (P < 0.05) crude protein (CP) and soluble CP content. However, the amount of CP associated with NDF and ADF was higher (P < 0.05) in barley-based thin stillage than in wheat thin stillage. Fractionation of true protein showed that most of the CP (average 707 g kg⁻¹ of CP) was present in the slowly degradable true protein fraction and was similar in both byproducts. Glutamic acid was the main amino acid in thin stillage and was higher (P < 0.05) in wheat than in barley-based thin stillage. However, barley-based thin stillage had higher (P < 0.05) levels of lysine, methionine, arginine, threonine, leucine and isoleucine than wheat thin stillage. Results of the in vitro trial indicated that effective degradability of CP (g kg⁻¹ of CP) followed the order (P < 0.05): SBM (665.0) > wheat thin stillage (614.0) > CM (531.0) > barley-based thin stillage (493.0). It was concluded that barley-based thin stillage had different chemical characteristics than wheat thin stillage. The reduced CP degradability of barley-based thin stillage relative to wheat thin stillage was attributed to a lower CP and a higher acid detergent in soluble CP level.     

Inhibition of growth of rat hepatoma by local injection of liposomes containing recombinant interleukin-2

Human recombinant interleukin-2 (IL-2) was entrapped in liposome, consisting of egg phosphatidylcholine (PC) and cholesterol.The peri-tumor injections of IL-2 liposome inhibited significantly the growth of solid tumor and prolonged the survival time of rats with solid tumors which were induced by a subcutaneous (s.c.) inoculation of AH-66 cells.Immunohistochemical staining of peritoneal exudate cells and tumor tissues revealed a marked accumulation of activated macrophages in and around the tumor tissues induced by the local injections of IL-2 liposome.     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

The effect of oxytocin and fragment (MIF-I) on the development of tolerance to hypothermic and hypnotic action of ethanol in the rat

The daily pretreatment of rats with oxytocin (OXY) or MIF-I prior to ethanol (Et-OH) administration markedly altered the alcohol tolerance when tested on the fifth day of treatment. OXY (800 and 2400 nmole/kg SC) and MIF (800 nmole/kg SC) inhibited the development of tolerance to the hypnotic effect of Et-OH. MIF at this dose also inhibited the tolerance to the hypothermic effect. Only OXY in the dose of 800 nmole/kg suppressed hypothermia in an acute experiment with Et-OH and produced by itself hypothermia after acute administration (2400 nmole/kg). The tolerance to this last effect developed after four days of peptide treatment. The results indicate that OXY and MIF-I can influence the processes of development of tolerance to some central depressive effects of Et-OH in rats.     

DFT: a dynamic study of the interaction of ethanol and methanol with platinum

The interaction between alcohol molecules and platinum (Pt) was studied using molecular dynamics (MD; Born-Oppenheimer method). Alcohol molecules like ethanol and methanol present a similar molecular structure, with a methyl group (CH₃) at one end and a fragment of hydroxyl (OH) at the other. This fact generates two orientations that are considered in the interaction with Pt. The MD calculation results for these two orientations indicate a preferential orientation due to energy interactions. A plausible reaction mechanism that takes into account the interaction between Pt and alcohol is presented. The charge transference obtained from the Pt–alcohol interaction was also analyzed. The energy for the two orientations was calculated by indicating the preferential orientation. The methyl and hydroxyl groups are involved in heterolytic breakage of hydrogen bonds, joined to a carbon atom in the former and to an oxygen atom in the latter; however, the methyl group reaction seems to be the most important.     

The crossflow injection technique: an improvement of the ethanol injection method

A novel scalable liposome preparation technique for pharmaceutical application is presented. Previous experiments have shown that the concept of continuous crossflow injection is a promising approach. For the characterization of the process, we focus on the influencing parameters like the lipid concentration, the injection hole diameter, the injection pressure, the buffer flow rate, and system performance. These experiments demonstrate that the injection hole diameter and the system performance do not influence the vesicle forming process and that a minimum of buffer flow rate is required to affect batch homogeneity. In contrast, strongly influencing parameters are lipid concentration in combination with increasing injection pressures. After exceeding the upper pressure limit of the linear range, where injection velocities remain constant, the vesicle batches are narrowly distributed, also when injecting higher lipid concentrations. Reproducibility and scalability data show similar results with respect to vesicle size and size distribution and demonstrate the stability and robustness of the novel continuous liposome preparation technique.     

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Manufacture of liposomes by isopropanol injection: characterization of the method

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40-210?nm). Accurate characterization of the particles, by fluorescence, (31)P-NMR, and cryo-transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.     

Effect of chronic ethanol consumption on postnatal development of renal (Na + K)-ATPase in the rat.

Renal (Na + K)-ATPase was studied to ascertain whether it follows the pattern of adaptation of membrane-bound enzymes that are inhibited by acute ethanol exposure and develop greater activity after chronic ethanol treatment. A colony of rats was given 20 per cent (v/v) ethanol as sole drinking solution throughout gestation, lactation and following weaning. (Na + K)-ATPase and ouabain-insensitive Ca(2+)-ATPase activities were determined; regional distribution of these enzymes was assessed in renal cortex and outer medulla. Control rats drank tap water. (Na + K)-ATPase in whole homogenate of kidney increased with age in controls and ethanol-fed rats, but the latter showed higher values at every age studied. Between 15 and 60 days of age, the control group showed 2-fold increases in cortex and 5-fold in outer medulla, whereas ethanol-fed rats reached a 3-fold increase in the enzyme activity in both renal regions. Ca(2+)-ATPase showed the same time course in developing kidney of both groups. Chronic ethanol treatment of adult rats resulted in an increase of (Na + K)-ATPase activity in cortex and outer medulla, but no change in other ATPases. Since an earlier maturational development of renal (Na + K)-ATPase was displayed by ethanol-fed rats, underlying mechanisms that may account for these results are discussed.     

Optimization of solid-phase microextraction conditions for gas chromatographic determination of ethanol and other volatile compounds in blood

A procedure for the determination of acetaldehyde, acetone, methanol, ethanol, 1-propanol and 2-propanol in blood was developed. Separation of analytes was carried out on DB-wax capillary column (l = 30 m, I.D. = 0.32 mm, dF = 0.5 microm) at 40 degrees C, hydrogen was used as a carrier gas (at 30 kPa) and FID as a detector. Quantification was performed with the use of 2-butanol as an internal standard. Headspace solid-phase microextraction was applied as the sample preparation technique. The usefulness of most commercially available fiber coatings was checked and 65 microm Carbowax/DVB proved most effective. Microextraction was carried out from the headspace at 60 degrees C for 10 min. The sample was stirred at 750 rpm. In order to improve the extraction efficiency of analytes, salting-out agents were also applied. Potassium carbonate turned out to be the most efficient. A 1.0-g amount of this salt and 0.1 ml of I.S. were added to 0.5 ml of sample. Validation of the worked-out method was performed. For each analyte, the limits of detection and quantification, linearity, working range, accuracy and precision were determined or tested.     

Clastogenic effect of ethanol in chronic and abstinent alcoholics

Alcoholism is one of the main causes of damage for human health, being relevant to study the induction of chromosomal aberrations (CA) by ethanol, and to investigate the individual susceptibility to diseases caused by alcoholism. A cytogenetic study was performed in human peripheral blood lymphocytes of 29 heavy chronic alcoholics, 11 alcoholics in abstinence, and 10 controls. The values of the chromosomal aberrations, mitotic indexes (MI) and proliferation indexes (PI) were determined. A molecular cytogenetic study was also carried out using fluorescence in situ hybridization (FISH) method with DNA library probes for chromosomes 1, 3 and 6, in lymphocytes from chronic alcoholic individuals in comparison with a control group. The results showed that the CA frequencies for chronic alcoholics (5.15 CA/100 cells) and alcoholics in abstinence (3.87 CA/100 cells) were higher than those obtained for control individuals (1.72 CA/100 cells). The mean translocation frequencies (equivalent to the genome) were calculated for six chronic alcoholics (0.267 translocations/100 cells) and six alcoholics in abstinence (0.167 translocations/100 cells), whose values were significantly higher than those observed for six control individuals (0.067 translocations/100 cells). The CA frequencies were not statistically different when smoker and non-smoker alcoholics were compared, indicating that although the smoking habit had significantly increased (four-fold) the CA frequency in healthy control individuals, a lack of interaction effect was observed within the group of alcoholics when smokers and non-smokers were compared. The CA frequencies presented by alcoholics in abstinence were similar to those obtained for chronic alcoholics. Therefore, chronic ethanol intoxication can lead to chromosome damage and disturbances in the metabolism of endogenous and exogenous compounds, which may persist for a long time, and constitute a relevant factor of risk for the development of neoplasias.     

Clofazimine and liposomes enhance the susceptibility of intracellularMycobacterium avium toward rifabutin

The effects of rifabutin and clofazimine were studied on intracellularly growingMycobacterium avium. Both drugs showed concentration-dependent inhibition of growth but no cidal effect at concentrations that can be reached in human serum. However, a combination of both drugs, even at lesser concentrations, exhibited cidal activity. Liposomes enhanced the susceptibility of the organism towards rifabutin but had no effect on the susceptibility towards clofazimine.     

Effect of silybin dihemisuccinate on the ethanol metabolizing systems of the rat liver

Silybin dihemisuccinate produces a decrease in the ethanol metabolic rate of rats. This effect is ascribed to an inhibition of the microsomal ethanol oxidizing system (MEOS). Alcohol dehydrogenase activity, catalase activity and NADPH cytochrome c reductase activity are not affected by the flavonoid. It is proposed that the inhibition of MEOS by silybin dihemisuccinate is related to its antioxidant properties, acting as a scavenger of the free radicals involved in ethanol metabolism by this enzymatic system. This observation may have therapeutical implications because microsomal lipid peroxidation induced by hydroxyl free radicals has been related to the etiology of hepatic alcoholic disease.     

饮酒小鼠动物模型建立及其对雌性激素的影响

目的建立模拟人类饮酒的小鼠动物模型,并以此动物模型进一步研究酒精对小鼠雌激素水平及乳腺癌的影响。方法 SPF级C57BL/6雌性小鼠,随机分对照组和酒精组两组,酒精组20:00到次日8:00给予含有一定浓度酒精的饮用水,其他时间给予常规饮用水,对照组全天给予常规饮用水。观察两组小鼠的饮水量及体重变化;用ANALOX AM1酒精分析仪检测小鼠凌晨2∶00和8∶00血液的酒精浓度(BEC);酶联免疫法(ELASA)检测两组小鼠血清中雌激素水平的差异。结果饮酒组小鼠血液BEC明显增高,类似人类饮酒水平,饮酒组小鼠的饮水量及体重无明显变化;饮酒组小鼠体内雌激素的水平明显高于对照组。结论成功的建立模拟人类饮酒的动物模型,并通过此动物模型初步证实酒精刺激可以增加小鼠体内血清雌激素的水平。