Solubilization of Multilamellar Liposomes in the Presence of Non-ionized Drug

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37° C by 7.3 mM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 hat pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilizaiton was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.     

Influence of Temperature on Stability of Multilamellar Liposomes in Wool Dyeing

Liposomes are lipid vesicles that are composed of amphiphile molecules and can carry hydrophobic and hydrophilic materials. In this research work liposomes used as carrier for transfer of dye molecules into wool fibers. The preparation and production of multilamellar liposomes (MLV) from Soya lecithin were carried out and the behavior of liposomes at different temperature was studied. The effect of different concentration of liposomes in the dye exhaustion profile of two dyes (Namely, Irgalan Blue FBL and Lanaset Blue 2R) at two different temperatures of 85°C and 95°C on the wool fabric was investigated. The results showed that presence of liposomes in the dye-bath helps to increase the dye absorption on the wool fabric before 80°C. Dyeing at higher temperature and longer time leads to a decrease in the final exhaustion along with increase in the liposomes concentration. Liposomes at high temperature converted to the disperse phospholipids unimers that may deposited on the fabric surface and may produce a hydrophobic barrier against absorption of dye. The presence of 1% o.w.f. (on weight of fabric) of liposomes at 85°C improved the dye exhaustion of Irgalan Blue FBL on the wool fabric. The wash fastness properties of samples which dyed in the dye-bath containing liposomes also improved.     

Structures Suggesting Cell-Wall-Deficient Forms Detected in Circulating Erythrocytes by Fluorochrome Staining

Cell-wall-deficient (CWD) forms of bacteria are associated with certain cases of idiopathic septicemia. In this preliminary study of blood examined immediately after venipuncture, structures with a morphology characteristic of CWD forms were seen parasitizing the erythrocytes. These inclusions were usually circumferential, but in some cases they protruded from the red cells. The CWD forms were detected by staining with Gould's rhodamine-labeled muramidase, which reacted similarly to acridine orange but with greater specificity. A blocking test, employing unlabeled muramidase, indicated the specificity of the reaction between muramidase and the microbial substrate. Reaction of the forms with muramidase indicates their bacterial, rather than mycoplasmal, nature. Thus in vivo CWD forms have a detectable component of muramic acid, at least in certain cases. Sixty-eight individuals with a diagnosis of fever of unknown origin were tested, with 51 nondebilitated individuals serving as controls. More intraerythrocytic forms reacting with muramidase were found in the patients than in the controls. Nearly 40% of the cases had a relatively high incidence of erythrocyte parasitism. In some instances when freshly drawn blood was examined, the structures, which appear to be microbial, extended in rhizoid filaments from the erythrocytes.     

Human Karyotyping by Heat-Giemsa Staining and Comparison with Fluorochrome Techniques

CHARACTERISTIC patterns resembling those revealed by the fluorochrome techniques¹ appear along human mitotic metaphase chromosomes when methanol-acetic acid-fixed preparations are heated to 69° C and stained with Giemsa solution. This procedure is a greatly simplified version of one developed by F. E. Arrighi and T. C. Hsu (personal communication) which heavily stains the centromere regions and produces distinct patterns on the chromosome arms. We chose the conditions for pretreatment of the preparations on the basis of the finding of Marmur and Doty² that the transition temperature, that is, the temperature at which the double helical configuration of DNA changes to a disordered coiling, for adenine-thymine nucleotide pairs, is 69° C. For cytosine-guanine pairs they found that the transition temperature is 110° C. Heating to the lower transition temperature should therefore denature regions of DNA rich in adenine-thymine pairs, leaving regions rich in cytosine-guanine pairs intact; such differences might be reflected in the staining properties, although the differential staining that occurs after heat treatment is obviously open to alternative interpretations.     

Nucleoli Visualized by Silver Staining Combined with A New Rna-Specific Fluorochrome

A technique for distinguishing nucleolar components by light microscopy is described. Silver staining is used for localizing nucleolar-organizing regions. A new RNA-specific fluorochrome, 4'-6-bis(2'-imidazoliny1-4'-5'H)-2-phenyl-4'-phenoxindole, visualizes the RNA-containing “partes-fibrillares et granulares” of the nucleolus. Both staining procedures can be combined to analyze nucleolar structure and function by light microscopy.     

Multilamellar Cationic Liposomes are Efficient Vectors for in Vitro Gene Transfer in Serum

Abstract

Multilamellar vesicles (MLVs) containing the cationic lipid DOTAP were used as vectors to lipofect a number of culture cell lines in the presence of serum. The lipofection efficiency of lipoplexes made of MLVs and the plasmid pSV-β galactosidase are much less sensitive to the lipofection-inhibitory effect of serum than the conventionally used lipoplexes made of sonicated small unilamellar vesicles (SUVs). In order to determine the factors favoring the lipofection efficiency of MLVs, we measured the size, as well as the cellular association and uptake of MLV and SUV lipoplexes containing DOTAP alone or DOTAP:DOPE (1:1). Electron microscope images of these complexes were taken to confirm their structure and size. The single most important factor that correlates with transfection efficiency in serum is the size of the lipoplex. SUV lipoplexes remain smaller than 300 nm in the presence of serum, and the lipofection efficiencies are low. MLV lipoplexes are larger (>300 nm) and the lipofection efficiency, as well as cellular association and uptake, are much higher than those of SUV lipoplexes. Exceptions are those lipoplexes made of MLVs of DOTAP and DOPE (1:1) combined with DNA at higher charge ratios, which form hexagonal structures and show poor lipofection as well as cellular association and uptake, even if their lipoplex size exceeds 300 nm. This finding lends credence to our theory of the serum inhibition effect upon lipofection, and suggests ways to improve the transfection efficiency in the presence of serum, by fabricating lipoplexes of defined sizes.     

黄颡鱼染色体的C-带、Ag-NORs、荧光带和复制带显带的研究

采用Giemas染色、C-带、Ag-NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明, 黄颡鱼只有部分的染色体呈现阳性C-带, 可分为三类, 其中NORs区是染色最深、染色面积最大的区域, 为深染居间C-带。其Ag-NORs位于m     

黄颡鱼染色体的C─带、Ag—NORs、荧光带和复制带显带的研究

采用Giemas染色、C─带、Ag—NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明,黄颡鱼只有部分的染色体呈现阳性C─带,可分为三类,其中NORs区是染色最深、染色面积最大的区域,为深染居间C─带。其Ag－NORs位于m5q末端。CMA3染色显示NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C─带、Ag—NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致,C─带阳性区和中复制带浅染区具有对应性。     

Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Neutral Red as a Lipid Fluorochrome

Aqueous 0.1% neutral red adjusted to pH 6.5, or mixed 1:1 with seawater, produced conspicuous fluorescence in plant and animal fats while acting as a conventional stain for acidic groups including volutin. The water-soluble, nonfluorescent dye predominating in acid solutions became deprotonized, lipophilic and fluorescent under neutral and alkaline conditions. Neutral dye crystals fluoresced dull red, whereas the fluorescence was either bright lemon yellow or blue-green in lipids, apparently dependent upon their composition.     

Selective Staining by the Fluorochrome, 5,5'-Diphenyl-9-ethyl-DiOC2(3). I. Physicochemical Studies of Dye-Dye and Dye-Tissue Interactions

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC₂(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein β-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.     

Multilamellar nuclear inclusions in the horse epididymis

Summary Nuclear inclusions are observed in the columnar epithelial cells of the corpus and cauda epididymidis of horses. They are built of long, straight, regularly spaced, parallel lamellae.     

Dual Fluorochrome Flow Cytometric Assessment of Yeast Viability

A novel staining protocol is reported for the assessment of viability in yeast, specifically the biocontrol yeast, Pichia anomala. Employing both the red fluorescent membrane potential sensitive oxonol stain DiBAC₄(5) (Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol), a structural analog of the commonly used DiBAC₄(3) (Bis-(1,3-dibutylbarbituric acid)trimethine oxonol), with one of the esterase dependent green fluorogenic probes such as CFDA-AM (5-Carboxyfluorescein diacetate, acetoxymethyl ester) or Calcein-AM (Calcein acetoxymethyl ester), a two-color flow cytometric method was developed, which yields rapid quantitative information on the vitality and vigor of yeast cell cultures. The method was validated by cell sorting and analysis of live, heat killed, and UV-treated yeast.     

Mitochondriotropic Liposomes

Mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Increasing pharmacological efforts toward therapeutic interventions have been made leading to the emergence of “Mitochondrial Medicine” as a new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, delivery systems need to be developed, which are able to selectively transport biologically active molecules to and into mitochondria within living human cells. In this study we present the first data demonstrating that conventional liposomes can be rendered mitochondria-specific via the attachment of known mitochondriotropic residues to the liposomal surface.     

Photosensitive Liposomes

Abstract

Photosensitive lipids and liposomes may be designed by a variety of strategies. These include the photochemical modification of individual lipids in the bilayer; the photoinduced change in the association of polyelectrolytes with liposomes; and the photoinitiated polymerization of some or all of the lipids in the liposome. The interaction of light with photosensitive liposomes can cause bilayer reorganization with possible applications in imaging, sensing, as well as therapeutics. The latter is the focus of this review.     

Target-Sensitive Liposomes

Abstract

Target-sensitive liposomes are liposomes which spontaneously destablize when they come into contact with target membrane/surface. The principle lipid in the liposomes ingredient is dioleoyl phosphatidylethanolamine (DOPE) which readily forms inverted micelle at physiological conditions. Earlier design of the liposomes uses acylated antibody as both a bilayer stabilizer and a targeting ligand. Although the immunoliposomes specifically release then-contents upon binding with the target membrane, they are not stable enough for long-term storage. Recent improvement in the design uses a charged phospholipid as a bilayer stabilizer and uses acylated antibody or other ligands at a much lower concentration. The new liposomes are stable for long-term storage, yet still destablize when bound with a target membrane. The rate of destabilization is significantly enhanced at elevated temperatures. The physical and biological properties of these liposomes are reviewed in this paper.     

pH-Sensitive Liposomes

Abstract

pH sensitive liposomes are lipid compositions that can be destabilized when the external pH is changed; usually from a neutral or slightly alkaline pH to an acidic pH. They are designed to circumvent delivery of liposome contents to the lysosomes of cells following internalization of the vesicle via the endocytic pathway. In the majority of compositions, a lipid containing a pH titratable group is mixed with phosphatidylethanolamine containing unsaturated acyl chains in a molar ratio (pH sensitive component/PE) of 1/4 or greater. There are five major groups of phosphatidylethanolamine containing pH-senstive lipid compositions. These can be classified by their acid-titratable component: phospholipids, acylated amino acids, fatty acids, cholesterol derivatives and miscellaneous double chain amphiphiles. The biophysical mechanism of action involves a transition of the lipids from the lamellar phase to the hexagonal phase. In cell culture, pH sensitive vesicles can increase the delivery of fluorescent markers, proteins, cytotoxic compounds, RNA and DNA into the cytoplasm. The mechanism of delivery is suggested to involve the destabilization of the liposome in the endosome as the pH is reduced from 7.4 to 5.0 and subsequent destabilization of, or fusion with, the endosomal membrane; some of the liposome contents are introduced into the cytoplasm. In most cases, the extent of liposome contents delivery into the cytoplasm is less than 1% of the amount that becomes cell associated. However further studies, with more reliable assays to differentiate cytoplasmic from lysosomal delivery, are required to place an exact value on this efficiency. The efficiency of pH sensitive liposomes in vivo is limited by stability of certain of the liposome compositions in serum and targeting to the appropriate cell. Cholesterol hemisuccinate is a particularly attractive component for in vivo use since it stabilizes the liposome when in serum at pH 7.4. The use of pH sensitive liposomes in drug delivery should continue to expand due to the increasing number of macromolecular therapeutic agents with intracellular targets.     

Complexing of Basic Pancreatic Proteinase Inhibitor with Soybean Phospholipid Multilamellar Vesicles

The formation of complexes of basic pancreatic proteinase inhibitor (BPTI) with multilamellar vesicles (MLV) from six preparations of soybean phospholipids of various composition was studied. When BPTI, a non-membrane protein, interacts with MLV, the vesicles aggregate, forming a precipitate of protein–lipid complexes. The BPTI content in the protein–lipid complexes increases with decreasing pH of the medium and on addition of negatively charged components into the lipid mixture. The protein-induced aggregation of the phospholipid vesicles is suggested to be mainly determined by electrostatic forces. The antiproteinase activity of BPTI in the complexes was rather low but increased up to 70% of the initial activity on addition of an ionic detergent (sodium deoxycholate).